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WO1995019987A1 - Nouvelle methode sensible de quantification du facteur de croissance transformant beta actif et compositions utilisees dans cette methode de dosage - Google Patents

Nouvelle methode sensible de quantification du facteur de croissance transformant beta actif et compositions utilisees dans cette methode de dosage Download PDF

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WO1995019987A1
WO1995019987A1 PCT/US1995/001153 US9501153W WO9519987A1 WO 1995019987 A1 WO1995019987 A1 WO 1995019987A1 US 9501153 W US9501153 W US 9501153W WO 9519987 A1 WO9519987 A1 WO 9519987A1
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tgf
plasmid
cells
accordance
cellε
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PCT/US1995/001153
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David J. Lockutoff
Scott A. Curriden
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The Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]

Definitions

  • the present invention relates to a sensitive assay method for quantifying the amount of active transforming growth factor beta (TGF- ⁇ ) and vector compositions for use therein for expressing an indicator molecule in response to TGF- ⁇ 10 activation of a TGF- ⁇ response element in the vector.
  • TGF- ⁇ active transforming growth factor beta
  • Transforming growth factor beta hereinafter referred to as TGF- ⁇
  • TGF- ⁇ is a 25 kilodalton (kD) homodimeric protein that 15 belongs to a family of regulators of cell growth and differentiation that includes activins, inhibins, Mullerian inhibiting substance, the Drosophila decapentaplegic complex and bone morphogenic proteins.
  • kD kilodalton
  • TGF- ⁇ was initially defined by its ability to induce morphological transformation of fibroblastic cells in monolayer culture and stimulation of 25 colony formation in soft agar. Delarco et al. , Proc. Natl.
  • TGF- ⁇ Three distinct molecular isoforms of TGF- ⁇ , the genes of which are located on different chromosomes, have been 30 identified in mammals and are designated TGF- ⁇ l, TGF- ⁇ 2 and
  • the activation process must occur to allow binding of the dimerized TGF- ⁇ to the high affinity TGF- ⁇ receptors expressed on the surfaces of all normal cells and most all neoplastic cells.
  • Tucker et al. Proc. Natl. Acad. Sci.. USA. 81:6757- 6761 (1984) ; Frolik et al . , J. Biol. Che .. 259:10995-11000
  • TGF- ⁇ activation systems generate the mature TGF- ⁇ in nanogram quantities, the majority liberate picogram amounts. These low concentrations, however, are sufficient to induce a variety of biological responses such as macrophage chemotaxis (Wahl et al., Proc. Natl. Acad. Sci., USA. 84:5788- 5792 (1987)), inhibition of endothelial cell migration and proliferation (Heimark et al. , Science. 233:1078-1080 (1986)), stimulation of extracellular matrix deposition (Ignotz et al . , J. Biol . Chem..
  • PA plasminogen activator
  • PAI-1 is the primary inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) , and as such is a potent anti-fibrinolytic molecule.
  • t-PA tissue-type plasminogen activator
  • u-PA urokinase-type plasminogen activator
  • PAI-1 synthesis by cultured cells in vitro is induced by a variety of molecules including cytokines, growth factors, hormones, and other agents such as endotoxin and phorbol myristate acetate. Nuclear transcription run-on assays demonstrate that the regulation of PAI-1 by many of these agents, including TGF- ⁇ , occurs primarily at the level of transcription.
  • TGF- ⁇ released from platelets may be an important negative regulator of the fibrinolytic system of the vessel wall since the TGF- ⁇ in releasates of thrombin-activated platelets causes large increases in PAI-1 synthesis by endothelial cells. This increased PAI-1 synthesis may account for the resistance of platelet-rich thrombi to thrombolytic therapy. The accumulation of PAI-1 in the extracellular matrix in response to TGF- ⁇ protects matrix proteins from proteolytic degradation. Thus, the induction of PAI-1 by TGF- ⁇ may also play a role in both wound healing and fibrotic responses.
  • TGF- ⁇ activity has been used to develop a variety of semiquantitative and quantitative bioassays including those based on chondrogenesis, inhibition -of DNA synthesis and cell growth, differentiation, migration or PA activity.
  • ED 50 0.2 ng/ml; 8 pM
  • An ED 50 represents the half-maximal amount of factor required to produce an effect, activation or inhibition, on differentiation of target cells.
  • the abbreviations ng/ml, pg/ml, nM and pM respectively stand for nanogra s/milliliter, picograms/milliliter, nanomolar and picomolar. These assays are utilized primarily for studying differentiation rather than for quantification of TGF- ⁇ .
  • BAEs can be simultaneously stimulated by endogenously or exogenously supplied bFGF that can abrogate TGF- ⁇ 's inhibitory effect (Sato et al . , J. Cell Biol.. 107:1199-1205 (1988) ) .
  • the PA assay for measurement of TGF- ⁇ concentration is very sensitive and rapid (Flaumenhaft et al . , J. Cell.
  • the assay is based on the ability of TGF- ⁇ to decrease PA activity of BAEs by inhibiting PA synthesis and secretion and by inducing expression of its inhibitor, PAI-1. This assay, however, is also sensitive to other molecules, such as bFGF, that can alter PA activity
  • the ED 50 of the assay varies from 1 to 35 pg/ml (0.04-1.4 pM) of TGF- ⁇ depending on differences in basal PA levels and sensitivity to TGF- ⁇ among primary BAE cultures.
  • TGF- ⁇ The ability of TGF- ⁇ to stimulate PAI-1 expression has recently been used to study TGF- ⁇ receptors.
  • Wrana et al . Cell. 71:1003-1014 (1992) transiently transfected a PAI-1 luciferase construct together with a human type II TGF- ⁇ receptor expression vector into TGF- ⁇ resistant MLE cells .
  • Thi ⁇ luciferase construct contained a short, synthetic TGF- ⁇ response element based on the human PAI-1 promoter and was used to report functional expression of the receptor.
  • this construct appeared to be less sensitive to TGF- ⁇ than the constructs of this invention when transiently transfected into MLE cells, and no information was reported regarding its dose-responsiveness or specificity.
  • transiently transfected TGF- ⁇ responsive cells with constructs containing varying regions of the 5'-flanking domain of the human PAI-1 gene to determine the transcription regulatory mechanism used by TGF- ⁇ .
  • All the constructs contained the gene encoding the enzyme chloramphenicol acetyltransferase to provide for an indirect determination of the transcriptional effect of the various constructs.
  • chloramphenicol acetyltransferase to provide for an indirect determination of the transcriptional effect of the various constructs.
  • a 67 base pair region that contained binding sites for the two proteins, CCAAT-binding transcription factor- nuclear family I family and USF factor. Both sites were necessary to obtain TGF- ⁇ induction.
  • the constructs were not utilized in assays to determine dose-responsiveness nor measure the amount of TGF- ⁇ in a sample.
  • Radioreceptor assays for TGF- ⁇ are the radioreceptor, radioimmumoassay (RIA) , and enzyme-linked immunosorbent assay (ELISA) .
  • Radioreceptor assays using a variety of cell types such as A549 human lung carcinomas and murine AKR-213, have been described and have ranges of 125 pM/ml to 25 ng/ml (5 pM-1 nM) with ED 50 of approximately 0.5 ng/ml (20 pM) . See, Wakefield et al., J. Cell. Biol.. 105:965-975 (1987); Sato et al., J. Cell Biol.. 111:757-763 (1990); Lucas et al .
  • TGF- ⁇ l specific RIAs describe the range of TGF- ⁇ l specific RIAs to be 6.25-200 ng/ml (0.25-8 nM) , with a sensitivity of 2.4 ng/ml (0.1 nM) (Lucas et al . , In Peptide Growth Factors, Barnes et al., Eds, Academic Press Inc. 198:303-316 (1991)) . As demonstrated by the differences in these results, the affinities of the antibodies can greatly alter the sensitivity of the assay.
  • Isoform-specific double antibody or sandwich ELISAs are also very sensitive to the affinities of the antibodies.
  • One such assay using two different monoclonal antibodies specific for TGF- ⁇ l, had a useful range of 0.63 to 40 ng/ml (0.025-16 nM) (Lucas et al. , In Peptide Growth Factors, Barnes et al., Eds, Academic Press Inc. 198:303-316 (1991)) .
  • Using a combination of isoform-specific turkey and ' rabbit antibodies Danielpour et al . , J. Cell Phvsiol ..
  • TGF- ⁇ assays can detect mature TGF- ⁇
  • the low concentrations ( ⁇ 2 pM) generated in various biological systems make many of them impractical without prior concentration of the sample. This can result in large losses of the mature growth factor or more importantly activation of latent TGF- ⁇ .
  • many of the assays are complicated to establish and can be influenced by other factors present in the samples thus reducing their utility for accurating measuring the amount of TGF- ⁇ in the sample. For this reason, a need exists for a relatively simple, sensitive and nonconfounding assay for TGF- ⁇ .
  • TGF- ⁇ assay method of this invention is more rapid, has comparable sensitivity, and has a greater detection range. Specificity of this novel assay was also higher as evidenced by its relative insensitivity to factors such as EGF and bFGF which can greatly affect other assays.
  • the rapid, sensitive, non-radioactive, easily performed assay method of this invention is useful in determining active TGF- ⁇ concentrations in complex solutions.
  • the present invention overcomes the limitations of existing methods used to quantify the amount of TGF- ⁇ in a liquid sample.
  • This invention contemplates a method for quantifying the amount of TGF- ⁇ in a sample using a system comprising a TGF- ⁇ responsive cell containing an expression vector having a regulatory region comprising a TGF- ⁇ response element operatively linked to a promoter and having a structural region encoding an indicator molecule.
  • a method for quantifying the amount of TGF- ⁇ in a liquid sample comprises: (a) incubating the liquid sample together with eucaryotic cells that contain a TGF- ⁇ responsive expression vector having a gene encoding luciferase for a predetermined time period sufficient for the eucaryotic cells to express a detectable amount of the luciferase; (b) measuring the amount of the luciferase expressed during the time period; and
  • (c) determining the amount of TGF- ⁇ present in the sample by comparing the measured amount of the luciferase against a reference curve.
  • the invention further contemplates that the reference curve represents a quantitative relationship derived from a series of measured amounts of luciferase produced from a series of known concentrations of TGF- ⁇ .
  • TGF- ⁇ transforming growth factor- ⁇
  • step (d) comparing the measured amount of the indicator molecule produced in step (c) with the amount of indicator molecule produced in a control assay performed according to steps (a) through (c) by treating the liquid sample with an anti-TGF- ⁇ antibody to obtain a net measured amount of the indicator molecule induced by TGF- ⁇ .
  • plasmids having identifying characteristics of plasmids on deposit with ATCC having the ATCC Accession Numbers 75627, 75628 and 75629. Also contemplated are stably transformed _ Q .
  • eucaryotic cells that contain the TGF- ⁇ response element having the nucleotide sequence in SEQ ID NO 11 where the cells correspond to cells on deposit with ATCC having the ATCC Accession Number CRL 11508.
  • the invention describes plasmids for use in the methods that comprise a nucleotide sequence corresponding to nucleotide sequences listed in SEQ ID NOs 1-10.
  • TGF- ⁇ inducible response elements that comprise a nucleotide sequence corresponding to nucleotide sequences listed in SEQ ID NOs 11-17 are also described.
  • Contemplated promoter nucleotide sequences are listed in SEQ ID NOs 18 and 19.
  • a further embodiment of the methods of the invention are eucaryotic cells that are stably transformed cells containing a plasmid having a gene encoding a selectable marker for the selection of said stably transformed cells.
  • the invention describes such plasmids having nucleotide sequences listed in SEQ ID NOs 1-6.
  • the invention further describes a stably transformed eucaryotic cell on deposit with ATCC having ATCC Accession Number CRL 11508 containing the TGF- ⁇ response element having the nucleotide sequence in SEQ ID NO 11.
  • An additional embodiment are eucaryotic cells that are transiently transformed cells with plasmids corresponding to the nucleotide sequences listed in SEQ ID NOs 7-10.
  • the invention describes quantifying the amount of TGF- ⁇ in a body fluid, in culture medium, and in a tissue extract.
  • a further preferred embodiment is the determination of the amount of a specific isoform of TGF- ⁇ , specifically TGF- ⁇ l, TGF- ⁇ 2 or TGF- ⁇ 3, in a liquid sample.
  • this invention describes the use of mammalian cells.
  • Preferred mammalian cells include mink lung epithelial cells, HeLa cells, Chinese hamster ovary cells, Hep3B cells, GM7373 cells, and NIH 3T3 cells.
  • a preferred indicator molecule also ' described for use with the methods of this invention is a chemiluminescent molecule, preferably luciferase.
  • the invention describes a composition of a plasmid vector in capable of causing expression of an indicator molecule in a eucaryotic cell, where the plasmid contains nucleotide sequences comprising a regulatory region that includes at least one TGF- ⁇ inducible response element operatively linked to a promoter, a structural region downstream of said promoter and coding for said indicator molecule, and a gene encoding a selectable marker for the selection of a stably transformed cell, where the response element is capable of inducing dose- dependent luciferase activity.
  • plasmids with selectable marker genes have the nucleotide sequences corresponding to SEQ ID NOs 1-6.
  • Preferred TGF- ⁇ inducible response elements for use in the expression vectors of this invention have the nucleotide sequences corresponding to SEQ ID NOs 11-17.
  • a further preferred embodiment of the expression vectors of this invention is the use of the neomycin gene for selecting stable transformant ⁇ , the nucleotide sequence of which is listed in SEQ ID NO 20.
  • the invention further describes plasmids lacking a selectable marker gene having the identifying characteristic ⁇ of pla ⁇ mid ATCC Acce ⁇ sion Numbers 75627, 75628, 75629, corresponding to SEQ ID NOs 8-10, respectively.
  • the invention describe ⁇ a eucaryotic cell containing a pla ⁇ mid having a nucleotide ⁇ equence li ⁇ ted in SEQ ID NO ⁇ 1-10.
  • Kits useful in as ⁇ aying the amount of TGF- ⁇ in a liquid sample comprising (a) packaging material; (b) eucaryotic cells capable of expres ⁇ ing an indicator molecule and containing a pla ⁇ mid of this invention and an aliquot of TGF- ⁇ , where the latter is used for generating a reference curve.
  • Figure 1 shows the structure and construction of the p ⁇ OOneoLuc expression vector.
  • p ⁇ OOLuc was digested with AccI and blunt-ended.
  • pMAMneo was then digested with Sal I and Eco RI, blunt-ended, and the fragment containing the neo ycin- re ⁇ i ⁇ tance gene (neo r ) wa ⁇ ligated to the linearized p ⁇ OOLuc to form p ⁇ OOneoLuc.
  • Clones were analyzed via restriction enzyme mapping and one clone with the proper insert was selected. (MCS, multiple cloning site; PA1, 2, 3, polyadenylation regions 1, 2, and 3) . The details of the con ⁇ truction are described in Example 1A.
  • Figure 2A having an inset ( Figure 2B) , ⁇ how ⁇ the do ⁇ e- dependent induction of the pla ⁇ minogen activator inhibitor- 1/luciferase (PAI/L) con ⁇ truct in p ⁇ OOneoLuc expre ⁇ sion vector in stably transformed MLE cells by TGF- ⁇ l, TGF- ⁇ 2, and TGF- ⁇ 3.
  • the TGF- ⁇ as ⁇ ay wa ⁇ performed a ⁇ described in Example 3 with DMEM-BSA containing the indicated concentrations in picomoles
  • FIGS. 2B show ⁇ the treatment of p ⁇ OOneoLuc-tran ⁇ formed MLE cell ⁇ with all three TGF- ⁇ isoforms in a TGF- ⁇ assay that resulted in a linear dose-response over the range of 0 to 4 pM of TGF- ⁇ .
  • the TGF- ⁇ a ⁇ ay wa ⁇ performed with 8 pM rTGF- ⁇ l, TGF- ⁇ 2 or TGF- ⁇ 3 in DMEM-BSA in the presence (cross-hatched bars) or absence (open bars) of 100 ⁇ g/ml of anti-TGF- ⁇ , TGF- ⁇ 2 and TGF- ⁇ 3 monoclonal antibody. Baseline induction is indicated by medium alone (filled bars) .
  • Figures 3A, 3B, 3C and 3D show the effects of medium, cell density and incubation time on sen ⁇ itivity of the TGF- ⁇ assay as described in Example 3B with the amount of TGF- ⁇ l plotted on the X-axi ⁇ in pM against the measured RLU on the Y-axis.
  • the assay was performed with increasing rTGF- ⁇ l concentrations in DMEM (closed ⁇ quares), alpha-MEM (closed circles), CM ⁇ M (closed triangles: Eagle ⁇ MEM supplemented with non-e ⁇ ential amino acids) or RPMI-1640 (closed diamonds: Bio- Whittaker) . All media contained 0.1% BSA.
  • the TGF- ⁇ assays were performed with DMEM-BSA containing the indicated concentrations of rTGF- ⁇ l (closed ⁇ quares), recombinant human bFGF (closed circles), recombinant IL-lalpha (closed triangles) , recombinant PDGF-BB (closed diamonds) , or EGF (open squares) .
  • TGF- ⁇ assays were performed with DMEM-BSA containing 1 pM rTGF- ⁇ l (closed squares) and the indicated concentrations of recombinant human bFGF (closed circles) , recombinant IL-lalpha (closed triangles) , recombinant PDGF (closed triangles) , or EGF (open square ⁇ ) .
  • the assays and results are described in Example 3C.
  • TGF- ⁇ a ⁇ says were performed with DMEM-BSA containing the indicated concentrations of rTGF- ⁇ l alone (closed squares) or with 0.5% (closed circles), 1% (closed triangles), or 2% (closed diamonds) calf ⁇ erum.
  • the assay ⁇ and re ⁇ ult ⁇ are described in Example 3D.
  • FIG. 5 shows the comparison of CMs assayed by the TGF- ⁇ (shown as the PAI/L as ⁇ ay) and MLEC assays.
  • DMEM BSA closed squares
  • COS X-marked lines
  • BSM closed triangles
  • BAE closed circles
  • cell conditioned medium (CM) with the indicated concentrations of rTGF- ⁇ l were a ⁇ ayed by PAI/L (TGF- ⁇ ) assay (broken line) as measured by RLU on the right-hand Y- axis and MLEC (unbroken line) as ⁇ ay as measured by tritiated thymidine ( 3 H-thymidine) incorporation percent of controls described in Example 3E.
  • the data points were normalized to DMEM-BSA.
  • Figure 6 shows the effects of growth factor ⁇ on DNA ⁇ ynthe ⁇ is as measured by 3 H-thymidine incorporation percent of control.
  • DMEM-BSA containing rTGF- ⁇ l (closed squares), TGF- ⁇ 2 (closed circles), TGF- ⁇ 3 (closed triangles), recombinant human bFGF (closed diamonds), recombinant IL-lalpha (open squares), EGF (open circles), or recombinant PDGF-BB (open triangles) were ⁇ eparately a ⁇ sayed using the MLEC assay as described Example 3C.
  • Recombinant DNA (rDNA) Molecule A DNA molecule produced by operatively linking two DNA segment ⁇ . Thu ⁇ , a recombinant DNA molecule i ⁇ a hybrid DNA molecule compri ⁇ ing at least two nucleotide sequence ⁇ not normally found together in nature. rDNA's not having a common biological origin, i.e., evolutionarily different, are said to be "heterologous" .
  • Vector A rDNA molecule capable of autonomous replication in a cell and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment.
  • Vector ⁇ capable of directing the expression of genes encoding for one or more polypeptides are referred to herein a ⁇ "expression vector ⁇ ".
  • Upstream In the direction opposite to the direction of DNA transcription, and therefore going from 5' to 3 ' on the non-coding strand, or 3' to 5' on the mRNA.
  • Downstream Further along a DNA sequence in the direction of sequence transcription or read out, that is traveling in a 3 ' - to 5 ' -direction along the non-coding strand of the DNA or 5 ' - to 3 '-direction along the RNA transcript.
  • Reading Frame Particular sequence of contiguous nucleotide triplets (codons) employed in tran ⁇ lation that define the ⁇ tructural protein encoding-portion of a gene, or structural gene. The reading frame depends on the location of the translation initiation codon.
  • Re ⁇ pon ⁇ e element also referred to as an enhancer element, is a short DNA sequence that occur ⁇ further up ⁇ tream than the up ⁇ tream promoter element.
  • Re ⁇ pon ⁇ e element ⁇ contain specific nucleotide sequences recognized by transcription factors that are DNA-binding proteins.
  • Promoter A region on a DNA molecule, generally from 100 to 200 base pairs long ⁇ , up ⁇ tream from the coding ⁇ equence; an area to which the RNA polymera ⁇ e initially bind ⁇ prior to the initiation of trancription.
  • the nucleotide ⁇ equence of the promoter, or at least part of it, determines the nature of the polymerase that associates with it. Certain consen ⁇ us sequences, CAT and TATA boxes, with the promoter region are important for binding of RNA polymerase.
  • Re ⁇ ulatorv Region A DNA control module upstream from the coding sequence containing an upstream promoter element and response elements, the latter of which is also referred to as enhancer elements.
  • Growth Factor A small protein that binds to a receptor for controlling cell proliferation.
  • Receptor A molecule, ⁇ uch as a protein, glycoprotein and the like, that can specifically (non-randomly) bind to another molecule.
  • Receptors of one type are plasma membrane proteins that bind ⁇ pecific molecule ⁇ including growth factor ⁇ , hormones, or neurotransmitter ⁇ , re ⁇ ulting in the transmis ⁇ ion of a signal to the cell's interior causing the cell to respond in a specific manner.
  • Sense Strand A nucleotide sequence referred to as a sense ⁇ trand of a double- ⁇ tranded deoxyribonucleic acid sequence i ⁇ the nucleotide ⁇ equence that when read in the 5 ' to 3 ' direction by the genetic code define ⁇ an amino acid ⁇ equence of interest.
  • sen ⁇ e ⁇ trand is referred to as a coding strand.
  • TGF- ⁇ Transforming Growth Factor- ⁇
  • TGF- ⁇ Transforming growth factor- ⁇ , hereinafter referred to as TGF- ⁇ , i ⁇ a growth inhibitor that exhibit ⁇ a diver ⁇ ity of biological activities in addition to its effects on cellular proliferation.
  • TGF- ⁇ belongs to a large family of related molecules with a wide range of regulatory activities as described in the Background. For review, see Barnard et al. , Biochi . Bionhvs. Acta.. 1032:79-67 (1990), the disclosure of which is hereby incorporated by reference.
  • TGF- ⁇ i ⁇ produced and secreted from cells in three distinct molecular i ⁇ oform ⁇ of TGF- ⁇ , the gene ⁇ of which are located on different chromosomes, have been identified in mammals and are designated ' TGF- ⁇ l, TGF- ⁇ 2 and TGF- ⁇ 3.
  • Derynck et al. Nature. 316:701-705 (1985); Hank ⁇ et al., Proc. Natl. Acad. Sci.. USA. 85:71-72 (1988); and Madi ⁇ en et al., DNA. 7:1-8 (19 ⁇ ) .
  • Each of the i ⁇ oform ⁇ are synthesized as high molecular weight latent or inactive precur ⁇ or polypeptides that are then proces ⁇ ed to 12.5 kD monomers that then dimerize to form biologically active, also referred to a ⁇ mature, TGF- ⁇ .
  • the activation process must occur to allow binding of the dimerized TGF- ⁇ to the high affinity TGF- ⁇ receptors expres ⁇ ed on the ⁇ urface ⁇ of all normal cells and most all neopla ⁇ tic cells.
  • Tucker et al. Proc. Natl. Acad. Sci.. USA. 81:6757- 6761 (1964) ; Frolik et al. , J. Biol. Chem.. 259:10995-11000 (1984) ; Pircher et al . , Biochem. Biophys . Res. Co mun.. 136:30- 37 (1986) .
  • TGF- ⁇ has been shown to induce the increase secretion of the inhibitor, plasminogen activator inhibitor-1 (PAI-1) (Laiho et al., -T. Biol. Chem.. 262:17467-17474 (1987)) .
  • PAI-1 i ⁇ the primary inhibitor of both ti ⁇ ue-type plasminogen activator (t- PA) and urokinase-type pla ⁇ minogen activator (u-PA) , and as such i ⁇ a potent anti-fibrinolytic molecule.
  • t- PA ti ⁇ ue-type plasminogen activator
  • u-PA urokinase-type pla ⁇ minogen activator
  • a ⁇ a con ⁇ equence of PAI-1 induction by TGF- ⁇ , the activity of pla ⁇ minogen activator (PA) i ⁇ decreased.
  • the resulting cascade of activation of plasminogen to pla ⁇ min i ⁇ thereby inhibited re ⁇
  • PAI-1 synthe ⁇ is by TGF- ⁇ has been shown to occur primarily at the level of transcription following the TGF- ⁇ receptor-ligand interaction, the mechani ⁇ m of activation of the PAI-1 promoter resulting in the transcription of the PAI-1 gene is less well understood. Studie ⁇ of PAI-1 gene tran ⁇ cription have shown that the signal tran ⁇ duction mechani ⁇ m ⁇ are independent of de novo protein synthesi ⁇ a ⁇ determined by the lack of inhibition by cycloheximide and rapid on ⁇ et of induction a ⁇ de ⁇ cribed by Sawdey et al. , J. Biol . Chem.. 264:10396-10401 (1989), the disclosure of which is hereby incorporated by reference.
  • TGF- ⁇ -induced enhancement of promoter activity for the alpha 2 collagen gene ha ⁇ been ⁇ hown to be mediated by a binding ⁇ ite for nuclear factor I as described by Sporn et al., J. Cell Biol.. 105:1039-1045 (1987) .
  • the PAI-1 promoter contains AP-1- like nucleotide ⁇ equence ⁇ which i ⁇ bound by the AP-1 heterodimeric transcription factor complex of Fos and Jun protein ⁇ ubunit ⁇ .
  • AP-1-like DNA enhancer ⁇ ite ⁇ are pre ⁇ ent in PAI-1, as shown in Example 4, activation of these site ⁇ by the AP-1 heterodimeric complex wa ⁇ independent of the TGF- ⁇ -mediated induction of PAI-1 synthe ⁇ is.
  • TGF- ⁇ -induced activation Since the TGF- ⁇ respon ⁇ e element i ⁇ activated by TGF- ⁇ resulting in the induction of indicator protein expression, the TGF- ⁇ re ⁇ pon ⁇ e element is also referred to a ⁇ a TGF- ⁇ inducible response element
  • the present invention is based on the discovery that when eucaryotic cells, transformed with a TGF- ⁇ -respon ⁇ ive expre ⁇ sion vector of this invention, were exposed to liquid samples of TGF- ⁇ , the resulting expression of an indicator molecule was dose-dependent in relationship to the amount of TGF- ⁇ present in the sample.
  • the present invention provides for a method to quantify the amount of TGF- ⁇ in an liquid sample by measuring the amount of indicator molecules expres ⁇ ed.
  • the induced expression of the indicator molecules was the result of activation of TGF- ⁇ respon ⁇ e element ⁇ present in the regulatory region of the TGF- ⁇ responsive expression vectors, the latter of which are described in Section D.
  • the regulation of transcription in the TGF- ⁇ responsive expression vector- tran ⁇ formed eucaryotic cell ⁇ is dependent TGF- ⁇ .
  • the TGF- ⁇ occupation of the TGF- ⁇ receptor expressed on the surface of cells results in the activation of a TGF- ⁇ - related transcription factor.
  • tran ⁇ cription factors are site- ⁇ pecific DNA-binding protein ⁇ . Typically, usually positioned 5 ' to a structural gene i ⁇ a region of nucleotide sequences that are responsible for controlling transcription. This region has been coined the "control module" .
  • the control module compri ⁇ es two categories of regulatory sequences, the promoter element and the enhancer elements.
  • the promoter i ⁇ referred to a ⁇ an upstream promoter as it lies upstream of the structural gene ⁇ .
  • Promoter elements are u ⁇ ually 100 to 200 ba ⁇ e pairs long and the ⁇ egment of DNA i ⁇ relatively close to the site of initiation of transcription.
  • a particular sequence recognized by one of several tran ⁇ cription factor ⁇ that are known to bind to the promoter region is the TATA box, a region that is rich in A-T base pairs.
  • the enhancer regions are also referred to as response regions or respon ⁇ e elements.
  • TGF- ⁇ response element can also be designated "TGF- ⁇ enhancer", “TGF- ⁇ enhancer region”, or “TGF- ⁇ respon ⁇ e region”, and the like.
  • the enhancer region i ⁇ hereinafter referred to as a re ⁇ pon ⁇ e element . They are ⁇ hort DNA ⁇ egment ⁇ that occur further up ⁇ tream from the initiator site than the upstream promoter element.
  • Response elements contain specific ⁇ equences that are recognized by transcription factor ⁇ .
  • the response element ⁇ are often a few 1000 base pairs 5' to the promoter but may even be 20,000 base pairs or more distant.
  • RNA molecule serves as a template for synthe ⁇ i ⁇ of a ⁇ pecific molecule, such as the indicator molecule of this invention.
  • activation of a TGF- ⁇ response element refers to a proces ⁇ whereby the functional ⁇ tate of the TGF- ⁇ re ⁇ pon ⁇ e element i ⁇ altered.
  • the re ⁇ ult of the TGF- ⁇ activation of the TGF- ⁇ re ⁇ pon ⁇ e element is an increase in the tran ⁇ criptional efficiency of the ⁇ tructural gene driven from the promoter.
  • a further embodiment of a TGF- ⁇ response element is that it is inducible.
  • the term "inducible" refers to a an enhancement of a particular function.
  • the functional activity of a TGF- ⁇ response element is increased or induced following activation by the TGF- ⁇ -related transcription factor. Thu ⁇ , the TGF- ⁇ re ⁇ pon ⁇ e element i ⁇ also referred to a ⁇ a TGF- ⁇ inducible re ⁇ pon ⁇ e element.
  • the re ⁇ ult of TGF- ⁇ respon ⁇ e element activation is the coordinate transcription and translation of the structural region containing a gene encoding an indicator protein of this invention a ⁇ de ⁇ cribed in Section D.
  • the term "do ⁇ e-dependent” refer ⁇ to the functional relation ⁇ hip between the amount of TGF- ⁇ activating the TGF- ⁇ re ⁇ ponse element and the resulting expression of the indicator molecule.
  • the functional relation ⁇ hip between TGF- ⁇ activation and expression of an indicator molecule can be referred to as a linear relationship. Because of the dose- dependent expres ⁇ ion of an indicator molecule, such as luciferase, in respon ⁇ e to TGF- ⁇ expo ⁇ ure, the amount of TGF- ⁇ re ⁇ pon ⁇ ible for the activation of the expre ⁇ ion can be readily determined using the methods of this invention.
  • a TGF- ⁇ response element nucleotide sequence i ⁇ characterized by it ⁇ ability to be re ⁇ pon ⁇ ive to TGF- ⁇ -induced activation.
  • a TGF- ⁇ respon ⁇ e element of thi ⁇ invention compri ⁇ e ⁇ any nucleotide ⁇ equence that is activated by TGF- ⁇ , the process of which is as described in Section B.
  • nucleotide sequence refers to a plurality of joined nucleotide units formed from naturally- or non-naturally occurring base ⁇ and cyclofurano ⁇ yl groups joined by phosphodie ⁇ ter bond ⁇ . Thu ⁇ , the nucleotide sequence includes the use of nucleotide analogs .
  • TGF- ⁇ response element of this invention is an isolated double-stranded deoxyribonucleic acid molecule comprising a ⁇ equence of nucleotide ba ⁇ es that defines a TGF- ⁇ re ⁇ ponse element.
  • the TGF- ⁇ response element may be found in DNA or RNA, in regulatory sequence ⁇ , exon ⁇ , or intron ⁇ .
  • Preferred TGF- ⁇ re ⁇ pon ⁇ e element ⁇ are derived from selected regions of the promoter regions of the plasminogen activator inhibitor type 1 gene, hereinafter referred to as PAI-1, as described by Loskutoff et al. , Biochem.. 26:3763-3768 (1987), the di ⁇ clo ⁇ ure of which i ⁇ hereby incorporated by reference.
  • Bo ⁇ ma et al. J. Biol. Chem.. 263:9129-9141 (1966), have de ⁇ cribed the entire 15,867 bp PAI-1 gene ⁇ equence including significant stretches of DNA that extend into its 5'- and 3 '-flanking DNA regions, the nucleotide sequence of which is available in the GenBank' IM /EMBL Data Bank with Acces ⁇ ion Number J03764.
  • PAI-1 promoter-derived TGF- ⁇ response elements for use in this invention are identified by the nucleotide po ⁇ ition ⁇ corre ⁇ ponding to the region in the PAI-1 promoter a ⁇ li ⁇ ted in the GenBankTM/EMBL Data Bank Acce ⁇ ion Number J03836.
  • Exemplary TGF- ⁇ re ⁇ pon ⁇ e elements derived from the PAI-1 promoter have the nucleotide sequences listed in the Sequence Listing in SEQ ID NOs 11-17. The nucleotide ⁇ equence ⁇ are listed showing only the sense strand in the 5' to 3 ' direction of a double-stranded isolated TGF- ⁇ respon ⁇ e element nucleotide ⁇ equence.
  • a TGF- ⁇ respon ⁇ e element u ⁇ eful for practicing the present invention may be derived from any promoter nucleotide sequence.
  • a TGF- ⁇ response element may be designed to contain pre ⁇ elected nucleotide ba ⁇ es.
  • a subject TGF- ⁇ respon ⁇ e element need not be identical to the nucleotide ⁇ equence of the PAI-1-derived TGF- ⁇ re ⁇ pon ⁇ e element ⁇ de ⁇ cribed herein, ⁇ o long as the nucleotide sequence is activatable by TGF- ⁇ .
  • a TGF- ⁇ response element of this invention thus may contain a variety of nucleotide units of any length, typically from about 5 to about 2000 nucleotides in length. More preferably, a TGF- ⁇ response element comprises nucleotide units from about 15 to about 1500 nucleotides in length.
  • a preferred embodiment is a TGF- ⁇ response element having nucleotide sequence ⁇ that is greater than 50 base pairs in length.
  • Exemplary long TGF- ⁇ re ⁇ pon ⁇ e element ⁇ derived from PAI-1 are li ⁇ ting in the Sequence Li ⁇ ting in SEQ ID -NO ⁇ 11-13.
  • a preferred embodiment is a TGF- ⁇ response element having nucleotide sequence ⁇ that i ⁇ le ⁇ than 50 ba ⁇ e pairs in length.
  • Exemplary short TGF- ⁇ respon ⁇ e element ⁇ derived from PAI-1 are li ⁇ ting in the Sequence Listing in SEQ ID NOs 14-17.
  • the invention contemplates the presence of at least one TGF- ⁇ response element present in the regulatory region of the expre ⁇ ion vector ⁇ a ⁇ de ⁇ cribed in Section D.
  • one or more stretches of a nucleotide sequence comprising a TGF- ⁇ respon ⁇ e element may be present within a regulatory region. If more than one TGF- ⁇ respon ⁇ e element i ⁇ pre ⁇ ent, they are not required to be identical.
  • TGF- ⁇ response elements having different nucleotide sequences as well as different lengths can be combined in a regulatory region of an expres ⁇ ion vector of thi ⁇ invention.
  • TGF- ⁇ re ⁇ ponse elements can be derived or produced from the PAI-1 promoter by truncation or expan ⁇ ion of the native or wild-type PAI-1 promoter nucleotide ⁇ equence or a ⁇ a variant of the native PAI-1 promoter by ⁇ ite-directed ⁇ ub ⁇ titution of a preselected nucleotide base or bases.
  • regulatory regions • containing multiple TGF- ⁇ respon ⁇ e element ⁇ that can be eithe ' r longer, ⁇ horter, tandemly arranged, reversed in orientation, and permutations thereof.
  • the design and construction of ⁇ uch arrangement ⁇ are well known to one of ordinary ⁇ kill in the art of oligonucleotide de ⁇ ign and ⁇ ynthesis and are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory, pp 390-401 (1982) .
  • nucleotide base modifications can be made resulting in nucleotide analogs to provide certain advantages to the TGF- ⁇ response elements of this invention.
  • a nucleotide analog refers to moietie ⁇ that function ⁇ imilarly to nucleotide ⁇ equence ⁇ in a TGF- ⁇ re ⁇ pon ⁇ e element of thi ⁇ invention but which have non-naturally occurring portion ⁇ . Thu ⁇ , nucleotide analog ⁇ can have altered ⁇ ugar moietie ⁇ or inter- ⁇ ugar linkage ⁇ . Exemplary are the pho ⁇ phorothioate and other ⁇ ulfur-containing ⁇ pecies, analogs having altered base units, or other modifications consistent with the ⁇ pirit of thi ⁇ invention.
  • Preferred modi ications include, but are not limited to, the ethyl or methyl phosphonate modifications disclo ⁇ ed in the U.S. Patent No., 4,469,863 and the pho ⁇ phorothioate modified deoxyribonucleotides described by LaPlanche et al. , Nucl . Acids Res.. 14:9081 (1986) and Stec et al . , J. Am. Chem. Soc.. 106:6077 (1984), the disclosure ⁇ of which are hereby incorporated by reference. These modifications provide re ⁇ i ⁇ tance to nucleolytic degradation. Preferred modifications are the modifications of the 3 ' -terminus using phosphothionate (PS) sulfurization modification de ⁇ cribed by Stein et al. , Nucl . Acids Res .. 16:3209 (1988) .
  • PS phosphothionate
  • TGF- ⁇ response elements comprising nucleotide sequences can be obtained by a variety of procedures well known in the art, including de novo chemical synthe ⁇ i ⁇ of complementary oligonucleotide ⁇ and derivation of nucleic acid fragment ⁇ from native nucleic acid sequences existing as genes, or parts of genes, in a genome, plasmid, or other vector, such as by re ⁇ triction endonuclea ⁇ e dige ⁇ tion of larger nucleic acid fragment ⁇ and strand separation or by enzymatic synthesi ⁇ using a nucleic acid template.
  • oligonucleotide ⁇ can be purified after ⁇ ynthe ⁇ i ⁇ u ⁇ ing published procedures a ⁇ de ⁇ cribed by Miller et al . , J.
  • oligonucleotide ⁇ are hybridized to form double- ⁇ tranded DNA segments that are TGF- ⁇ respon ⁇ e element ⁇ .
  • Particularly preferred chemically-synthesized oligonucleotides are described in Example 1C and the sen ⁇ e ⁇ trand ⁇ of which are li ⁇ ted in SEQ ID NO ⁇ 14-17, a ⁇ described above.
  • TGF- ⁇ response element Derivation of a TGF- ⁇ response element from nucleic acids involves the cloning of a nucleic acid into an appropriate host by means of a cloning vector, replication of the vector and therefore multiplication of the amount of the cloned nucleic acid followed by isolation of ⁇ ubfragment ⁇ of the cloned nucleic acids .
  • ⁇ ubcloning nucleic acid fragment ⁇ see Sambrook et al. , Molecular Cloning: A
  • TGF- ⁇ re ⁇ pon ⁇ e element ⁇ are obtained by re ⁇ triction dige ⁇ tion of cloned vector ⁇ containing the PAI-1 promoter as described in Example 1A and 1C.
  • Particularly preferred nucleotide sequence ⁇ containing TGF- ⁇ response elements as well as the minimal promoter ⁇ equence obtained in thi ⁇ manner include nucleotide sequences corresponding to the nucleotide positions in the PAI-1 promoter sequence from -1481 to +76, specifically a Kpn I/Eco RI digest and -800 to +76, specifically a Hind III/Eco RI dige ⁇ t.
  • TGF- ⁇ respon ⁇ e element nucleotide ⁇ equence be known in order to obtain a TGF- ⁇ response element capable of being activated by TGF- ⁇ .
  • contemplated for use in thi ⁇ invention are TGF- ⁇ re ⁇ pon ⁇ e element ⁇ obtained from promoter regions of other genes that can be determined to contain TGF- ⁇ respon ⁇ e element ⁇ u ⁇ ing the method ⁇ of this invention.
  • the present invention contemplates TGF- ⁇ responsive plasmid expression vectors in sub ⁇ tantially pure form capable of causing expression of an indicator molecule in a eucaryotic cell.
  • TGF- ⁇ respon ⁇ ive identifie ⁇ an expre ⁇ ion vector of this invention that by its composition contains TGF- ⁇ response elements that are activated by TGF- ⁇ mediated through a TGF- ⁇ response element specific transcription factor as described in Section C.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein a ⁇ "expression vectors".
  • vector refer ⁇ to a nucleic acid molecule capable of tran ⁇ porting between different genetic environments another nucleic acid to which it has been operatively linked.
  • One type of preferred vector i ⁇ an epi ⁇ ome, i.e., a nucleic acid capable of extra-chromo ⁇ omal replication.
  • Preferred vector ⁇ are tho ⁇ e capable of autonomou ⁇ replication and/or expre ⁇ ion of nucleic acid ⁇ to which they are linked.
  • a TGF- ⁇ expre ⁇ ion vector of thi ⁇ invention is a circular double-stranded plasmid that contains at least the following elements: 1) a regulatory region having at least one TGF- ⁇ re ⁇ pon ⁇ e element a ⁇ defined in Section C, where the regulatory region i ⁇ operatively linked to a promoter; and 2) a ⁇ tructural region down ⁇ tream of the promoter that contain ⁇ a gene coding for an indicator molecule of thi ⁇ invention.
  • a TGF- ⁇ expre ⁇ ion vector al ⁇ o contain ⁇ a gene, the expre ⁇ ion of which confers a selective advantage, such as a drug re ⁇ i ⁇ tance, to the eucaryotic ho ⁇ t cell when introduced or tran ⁇ formed into tho ⁇ e cell ⁇ .
  • a typical eucaryotic drug re ⁇ istance genes confer ⁇ re ⁇ i ⁇ tance to neomycin, also referred to as G416 or Geneticin.
  • the choice of vector to which the regulatory region, promoter, and ⁇ tructural region of the pre ⁇ ent invention is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g., replication or protein expression, and the host cell to be transformed, these being limitations inherit in the art of constructing recombinant DNA molecules.
  • the vector utilized include ⁇ procaryotic sequences that facilitate the propagation of the vector in bacteria, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally when introduced into a bacterial host cell. Such replicons are well known in the art.
  • the TGF- ⁇ expre ⁇ ion vector of thi ⁇ invention include ⁇ one or more tran ⁇ cription units that are expressed only in eucaryotic cell ⁇ .
  • the expre ⁇ ion vector ⁇ of thi ⁇ invention al ⁇ o contain distinct sequence elements that are required for accurate and efficient polyadenylation, referred to a ⁇ PAl, 2 and 3 and a ⁇ shown in Figure 1.
  • splicing signals for generating mature mRNA are included in the vector.
  • the eucaryotic TGF- ⁇ responsive expression vector ⁇ contain viral replicons, the presence of which provides for the increase in the level of expres ⁇ ion of cloned genes.
  • a preferred replication sequence is provided by the simian viru ⁇ 40 or SV40 papovavirus.
  • Operatively linking refers to the covalent joining of nucleotide sequences, preferably by conventional phosphodie ⁇ ter bond ⁇ , into one ⁇ trand of DNA, whether in single- or double- stranded form.
  • nucleotide ⁇ equence ⁇ re ⁇ ult ⁇ in the joining of functional element ⁇ such as response elements in regulatory regions with promoters and down ⁇ tream structural regions as described herein.
  • a preferred eucaryotic expres ⁇ ion vector of thi ⁇ invention a ⁇ prepared in Example 1 contain ⁇ a regulatory region having TGF- ⁇ re ⁇ pon ⁇ e element ⁇ derived from the 5 ' promoter end of the human pla ⁇ minogen activator inhibitor type 1 (PAI-1) gene operatively linked to PAI-1 minimal promoter and a down ⁇ tream ⁇ tructural region containing a gene coding for an indicator polypeptide, preferably lucifera ⁇ e.
  • PAI-1 human pla ⁇ minogen activator inhibitor type 1
  • Exemplary TGF- ⁇ re ⁇ pon ⁇ ive expression vectors include the following expres ⁇ ion vectors, the designations of which are indicated along with the corresponding SEQ ID NO in which the sense strand of the expres ⁇ ion vector i ⁇ listed where the first nucleotide of the double-stranded circular vector is the middle "T" nucleotide present in the Eco RI restriction site as de ⁇ cribed in Example 1: 1) p ⁇ OOneoLuc (SEQ ID NO 1); 2) p ⁇ 00/636neoLuc (SEQ ID NO 2); 3) p56neoLuc (SEQ ID NO 3); 4) p674neoLuc (SEQ ID NO 4); 5) p743neoLuc (SEQ ID NO 5); 6) p732neoLuc (SEQ ID NO 6); 7) p56Luc (SEQ ID NO 7); 8) p674Luc (SEQ ID NO 8); 9) p743Luc (SEQ ID NO 9); and 10) p732Luc
  • the exemplary TGF- ⁇ expression vectors of this invention are derived from the starting cloning expression vector, designated pl9Luc, as described in Example 1.
  • the nucleotide sequence of the sen ⁇ e ⁇ trand of an Eco Rl-linearized pl9LUC vector is listed in the Sequence Listing as SEQ ID NO 21.
  • a further embodiment of this invention is the preparation of TGF- ⁇ respon ⁇ ive expression vectors having altered arrangements of and selected types of TGF- ⁇ re ⁇ ponse element ⁇ in the regulatory region.
  • pl9Luc and the pl9Luc- derived p39Luc expression cloning vectors are vectors that allow for the construction of TGF- ⁇ respon ⁇ ive vector ⁇ having any ⁇ elected regulatory region operatively ligated to a ⁇ elected promoter.
  • any regulatory region of any length containing one or more TGF- ⁇ re ⁇ ponse elements can be paired with any promoter, a non-TGF- ⁇ respon ⁇ ive PAI-1 or heterologou ⁇ HBV promoter a ⁇ u ⁇ ed herein but not limited to that, to prepare TGF- ⁇ re ⁇ pon ⁇ ive expression vector ⁇ that provide for the quantitation of inducing TGF- ⁇ .
  • other method ⁇ of preparing pl9Luc- derived expre ⁇ ion vector ⁇ having TGF- ⁇ re ⁇ pon ⁇ e element ⁇ and promoter ⁇ are familiar to one of ordinary ⁇ kill in the art of vector con ⁇ truction and are described by Au ⁇ ebel, et al. , In Current Protocol ⁇ in Molecular Biology, Wiley and Son ⁇ , New York (1993) and by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1989.
  • a preferred embodiment i ⁇ a TGF- ⁇ re ⁇ pon ⁇ ive expre ⁇ sion vector having a gene for encoding a selectable marker providing for stably transformed cell ⁇ .
  • Stably tran ⁇ formed cell ⁇ confer the ability to utilize a reproducible ⁇ ource for practicing the method ⁇ of this invention over a course of time.
  • a preferred selectable marker gene i ⁇ the gene conferring neomycin- re ⁇ istance.
  • Such a gene for encoding the ⁇ electable marker was derived from an expres ⁇ ion vector, designated pMAMneo, as described in Example 1.
  • a TGF- ⁇ respon ⁇ ive expre ⁇ sion vector contains a first nucleotide sequence comprising a regulatory region that includes at least one TGF- ⁇ inducible response element operatively linked to a promoter, a second nucleotide sequence comprising a structural region downstream of the promoter and coding for an indicator molecule, and a third nucleotide sequence comprising a gene encoding a ⁇ electable marker for the ⁇ election of a ⁇ tably tran ⁇ formed cell, where the re ⁇ ponse element i ⁇ capable of inducing do ⁇ e-dependent lucifera ⁇ e activity and the ⁇ tructural region codes for luciferase.
  • Preferred expression vectors containing the neomycin- resi ⁇ tance conferring gene include the following designations followed in parenthesis by the corresponding SEQ ID NO in which the sense strand of each Eco Rl-linearized vector is listed according to the convention adopted in this invention for listing vector sequences: 1) p ⁇ OOneoLuc (SEQ ID NO 1); 2) p800/636neoLuc (SEQ ID NO 2); 3) p56neoLuc (SEQ ID NO 3); 4) p674neoLuc (SEQ ID NO 4); 5) p743neoLuc (SEQ ID NO 5); 6) p732neoLuc (SEQ ID NO 6) .
  • the pla ⁇ mid expre ⁇ ion vectors of this invention contain TGF- ⁇ inducible response elements that correspond to a nucleotide sequence listed in SEQ ID NOs 11-17 as described in Section C.
  • Preferred promoters for use in the TGF- ⁇ expres ⁇ ion vector ⁇ of this invention for stably transforming cells a ⁇ well as for transient transformation are the PAI-1 minimal promoter ⁇ equence and the hepatiti ⁇ B viru ⁇ minimal promoter ⁇ equence, the sense ⁇ equence ⁇ of which are re ⁇ pectively li ⁇ ted in SEQ ID NO ⁇ 18 and 19.
  • Contemplated for u ⁇ e in thi ⁇ invention are promoter ⁇ that are not re ⁇ pon ⁇ ive to TGF- ⁇ . The selection of alternative promoter ⁇ i ⁇ within the ⁇ cope of one having ordinary skill in the art.
  • This invention contemplates additional TGF- ⁇ expression vector ⁇ for stably transforming cells that can be designed to have regulatory region ⁇ that contain alternative TGF- ⁇ re ⁇ ponse elements and promoter ⁇ .
  • the regulatory region of a TGF- ⁇ expression vector of this invention contains at lea ⁇ t one TGF- ⁇ re ⁇ pon ⁇ e element a ⁇ described herein and in Section C of this invention.
  • the regulatory region of a TGF- ⁇ expre ⁇ ion vector can range in length from 5 to 2000 ba ⁇ e pair ⁇ , preferably 15 to 1500 ba ⁇ e pair ⁇ , and can contain more than one TGF- ⁇ response element in any orientation and arrangement. Thu ⁇ , if two or more TGF- ⁇ re ⁇ pon ⁇ e elements are present in a regulatory region, they may be contiguous with one another or separated by an intervening nucleotide sequence.
  • TGF- ⁇ re ⁇ pon ⁇ e element ⁇ pre ⁇ ent in the regulatory region of a TGF- ⁇ expre ⁇ ion vector are derived from the PAI-1 promoter and have the nucleotide ⁇ equences listed in the Sequence Listing in SEQ ID NOs 11-17.
  • a plasmid vector of the present invention contain a structural region having a nucleotide sequence that encodes an indicator molecule.
  • the structural region is operatively linked to the regulatory region such that the inducible promoter of the regulatory region, under the inducible control of the TGF- ⁇ respon ⁇ e element, control ⁇ tran ⁇ cription and expre ⁇ ion of the indicator molecule. Thu ⁇ , upon induction of the TGF- ⁇ re ⁇ pon ⁇ e element, the regulatory region transcribes and thereby expres ⁇ e ⁇ the indicator molecule re ⁇ ulting in a detectable event in the cell, which event can be measured by detection of the amount of the expressed indicator molecule.
  • the respon ⁇ e element is capable of inducing the expression of the indicator molecule by virtue of it ' s controlling expression of the indicator through the promoter to which the response element is operatively linked.
  • Other configurations can be utilized so long as the induction of the TGF- ⁇ respon ⁇ e element re ⁇ ult ⁇ in the expression of the indicator polypeptide. Exemplary and preferred configurations are described in Examples.
  • indicator molecule refers ⁇ to a molecule encoded by a reporter gene, the expre ⁇ ion of which in the expre ⁇ ion vector ⁇ of this invention, re ⁇ ult ⁇ in a detectable measurable protein, polypeptide, enzyme and the like.
  • Alternative expres ⁇ ion ⁇ for indicator molecule are reporter molecule, reporter polypeptide, indicator protein, indicator polypeptide and the like.
  • the indicator molecule is a protein.
  • indicator polypeptide ⁇ suitable for use in the present invention, and the invention need not be so limited to any particular indicator.
  • a preferred indicator polypeptide is luciferase encoded by the firefly luciferase gene. Use of the lucifera ⁇ e gene for expre ⁇ ion of lucifera ⁇ e ha ⁇ been de ⁇ cribed by Gould et al . , Anal. Biochem.. 7:5-13 (1986) and Bra ⁇ ier et al. , Bio- Techniques. 7:1116-1122 (1989) .
  • a preferred structural region includes a nucleotide sequence having the sequence characteristics of the luciferase gene shown in SEQ ID NO 21.
  • Alternative embodiments include indicator proteins ⁇ uch a ⁇ -galactosidase and chloramphenicol acetyltransfera ⁇ e (CAT) .
  • CAT chloramphenicol acetyltransfera ⁇ e
  • a ⁇ sociated with the use of an indicator molecule in the quantifying TGF- ⁇ are means for measuring the indicator molecule.
  • a preferred method for detecting the luciferase indicator molecule is the use of a luminometer commercially available from Dynatech Laboratories Inc., Chantilly, VA a ⁇ described in Example 3A and analyzed according to manufacturer's instructions.
  • a simple-phase extraction assay has been developed and described by Seed et al., Gene. 67:271-277 (1986), the disclosure of which is hereby incorporated by reference.
  • Alternative preferred methods for detecting CAT activity are described in Current Protocols in Molecular Biology, Eds, Ausebel et al., Unit 9.0, John Wiley & Son ⁇ (1993).
  • ⁇ - galacto ⁇ idase activity is performed in activity as ⁇ ays performed essentially as described by Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, New York, (1972), the di ⁇ clo ⁇ ure of which is hereby incorporated by reference.
  • additional reagent ⁇ are required to visualize its presence following induced expression.
  • additional reagent ⁇ for ⁇ -galactosidase include o-nitrophenyl- ⁇ -D-galactopyran ⁇ o ⁇ ide and the like for the development of a color reaction by ab ⁇ orbance at wavelengths of 500 and 420.
  • the plasmid vector of the present invention include ⁇ a gene that encodes a selectable marker that is effective in a eucaryotic cell, preferably a drug resi ⁇ tance ⁇ election marker.
  • a preferred drug resistance ⁇ election marker i ⁇ a gene whose expres ⁇ ion re ⁇ ults in neomycin resi ⁇ tance, i.e., the neomycin phosphotransfera ⁇ e (neo) gene [Southern et al. , J. Mol . APPI . Gene ..
  • kanamycin resistance i.e., the chimeric gene containing nopaline syntheta ⁇ e promoter, Tn5 neomycin pho ⁇ photran ⁇ fera ⁇ e II and nopaline synthetase 3 ' non-translated region de ⁇ cribed by Roger ⁇ et al . , Method ⁇ for Plant Molecular Biology, A.
  • the invention contemplates the use of a nucleotide ⁇ equence which confer ⁇ a eucaryotic ⁇ election mean ⁇ , including but not limited to gene ⁇ for resistance to neomycin and kanamycin.
  • a preferred nucleotide sequence defining a selectable marker gene is a nucleotide sequence having the ⁇ equence characteristics of the neomycin resi ⁇ tance gene ⁇ hown in SEQ ID NO 20.
  • a selectable marker for eucaryotic cells provides the advantage of producing stably transformed cells, as di ⁇ cu ⁇ ed herein.
  • a selectable marker for eucaryotic cells provides the advantage of producing stably transformed cells, as di ⁇ cu ⁇ ed herein.
  • preferred embodiments that include a procaryotic replicon also include a gene whose expre ⁇ ion confer ⁇ a selective advantage, such as a drug resi ⁇ tance, to the bacterial ho ⁇ t cell when introduced into those transformed cells.
  • Typical bacterial drug re ⁇ i ⁇ tance gene ⁇ are tho ⁇ e that confer re ⁇ i ⁇ tance to ampicillin or tetracycline.
  • Tho ⁇ e vector ⁇ that include a procaryotic replicon al ⁇ o typically include convenient re ⁇ triction ⁇ ite ⁇ for in ⁇ ertion of a recombinant DNA molecule of the pre ⁇ ent invention.
  • Typical of ⁇ uch vector pla ⁇ mid ⁇ are pUC8, pUC9, pBR322, and PBR329 available from BioRad Laboratorie ⁇ , (Richmond, CA) and pPL, pK and K223 available from Pharmacia, (Pi ⁇ cataway, NJ) , and pBLUESCRIPT and pBS available from Stratagene, (La Jolla, CA) .
  • a vector of the present invention may also be a Lambda phage vector including tho ⁇ e Lambda vector ⁇ described in Molecular Cloning: A Laboratory Manual. Second Edition, Maniatis et al . , eds., Cold Spring Harbor, NY (1989) .
  • Plasmid vector ⁇ for use in the present invention are also compatible with eukaryotic cells.
  • Eucaryotic cell expres ⁇ ion vector ⁇ are well known in the art and are available from ⁇ everal commercial sources. Typically, such vectors provide convenient re ⁇ triction ⁇ ite ⁇ for in ⁇ ertion of the desired recombinant DNA molecule, and further contain promoter ⁇ for expre ⁇ ion of the encoded gene ⁇ which are capable of expre ⁇ ion in the eucaryotic cell, a ⁇ di ⁇ cu ⁇ sed earlier.
  • Typical of such vectors are pSVO and pKSV-10 (Pharmacia) , and pPW-l/PML2d (International Biotechnology, Inc.), and pTDTl (ATCC, No. 31255) .
  • TGF- ⁇ respon ⁇ ive expre ⁇ ion vector ⁇ having regulatory, promoter and structural regions but lacking a gene for encoding a ⁇ electable marker.
  • TGF- ⁇ expre ⁇ ion vector ⁇ for tran ⁇ ient tran ⁇ formation of eucaryotic cells are contemplated.
  • This embodiment allow ⁇ for an alternative to ⁇ table transformation of cell ⁇ for use practicing the methods of this invention.
  • Transiently tran ⁇ formed cell ⁇ produced a ⁇ de ⁇ cribed in Example 2D. are useful for performing TGF- ⁇ as ⁇ ay ⁇ when having stably transformed cells i ⁇ not required or neces ⁇ itated.
  • transiently transformed cells are useful for determining the nucleotide ⁇ equence of TGF- ⁇ re ⁇ pon ⁇ e element ⁇ a ⁇ well as quantifying the amount of TGF- ⁇ present in a heterogeneous or homogeneous liquid sample.
  • Preferred TGF- ⁇ expres ⁇ ion vector ⁇ used for transiently transforming eucaryotic cells include the following vectors ⁇ hown with their de ⁇ ignation ⁇ and SEQ ID NO ⁇ in which the ⁇ en ⁇ e strand of the double-stranded Eco Rl-linearized vectors i ⁇ listed: 1) p56Luc (SEQ ID NO 7); 2) p674Luc (SEQ ID NO 8); 3) p743Luc (SEQ ID NO 9); and 4) p732Luc (SEQ ID NO 10) .
  • the invention further describe ⁇ TGF- ⁇ re ⁇ pon ⁇ ive pla ⁇ mid ⁇ lacking a selectable marker gene having the identifying characteristic ⁇ of pla ⁇ mids that have been deposited with the American Type Culture Collection, Rockville, MD having the assigned ATCC Acce ⁇ ion Number ⁇ 75627, 75628, 75629., the pla ⁇ mid ⁇ of which re ⁇ pectively corre ⁇ pond to the Eco RI- linearized ⁇ en ⁇ e strand nucleotide sequences listed SEQ ID NOs 8-10.
  • this invention describe ⁇ the co-transformation of TGF- ⁇ expression vectors for transient transformation in conjunction with a second expres ⁇ ion vector from which a ⁇ electable marker i ⁇ expre ⁇ sed.
  • the advantage thi ⁇ approach provide ⁇ is that further vector constructions for inserting selectable marker gene ⁇ can be avoided, thereby providing ⁇ tably tran ⁇ formed cell ⁇ for u ⁇ e in practicing thi ⁇ invention when necessitated.
  • eucaryotic cells that have been co-transformed with a transient TGF- ⁇ expres ⁇ ion vector and a ⁇ econd plasmid such a ⁇ RSVneo provide for an alternative approach to create ⁇ tably transformed eucaryotic cells.
  • Any transient TGF- ⁇ expres ⁇ ion vector of thi ⁇ invention can be u ⁇ ed in thi ⁇ context.
  • a preferred co-tran ⁇ formed eucaryotic cell is the cell line Hep3B that has been co- transformed with RSVneo and the pl500Luc expre ⁇ ion vector having the TGF- ⁇ re ⁇ ponse element in SEQ ID NO 11. This stably transformed cell line has been deposited with the American Type Culture Collection, Rockville, MD and has been a ⁇ igned ATCC having ATCC Acce ⁇ ion Number CRL 11508.
  • additional TGF- ⁇ expre ⁇ ion vector ⁇ for tran ⁇ iently tran ⁇ forming cell ⁇ can be de ⁇ igned to have regulatory region ⁇ that contain alternative TGF- ⁇ response elements and promoter ⁇ .
  • these additional vectors can be used to prepare stably transformed cells through the use of the co- transformation approach.
  • the invention also contemplates a eucaryotic cell containing a plasmid vector of the present invention.
  • a eucaryotic cell suitable for use can be any eucaryotic cell which expresse ⁇ a TGF- ⁇ receptor on it ⁇ cell ⁇ urface and LO to to l-> o L ⁇ o L ⁇ L ⁇
  • eucaryotic cell ⁇ that contain a pla ⁇ mid vector having a nucleotide sequence with the nucleotide sequence characteri ⁇ tic ⁇ of the TGF- ⁇ re ⁇ pon ⁇ e element ⁇ elected from the group con ⁇ i ⁇ ting of the ⁇ equences shown in SEQ ID NO ⁇ 11-17.
  • a particularly preferred eucaryotic cell contain ⁇ a pla ⁇ mid vector having a nucleotide sequence with the nucleotide sequence characteristics of the plasmid vector ⁇ elected from the group consi ⁇ ting of the ⁇ equence ⁇ ⁇ hown in SEQ ID NOs 1-10.
  • a preferred eucaryotic cell described further herein is Hep3B stably transformed with the plasmid vector pl500Luc, referred to as LUCI, and having the ATCC acces ⁇ ion No. CRL 11508.
  • the present invention de ⁇ cribe ⁇ methods for detecting the presence, and preferably quantifying the amount, of TGF- ⁇ in a liquid sample, either containing purified TGF- ⁇ or TGF- ⁇ in a heterogeneous admixture, and is al ⁇ o referred to herein a ⁇ a TGF- ⁇ a ⁇ say.
  • the as ⁇ ay system provides for the quantification of TGF- ⁇ through the expres ⁇ ion of an indicator polypeptide which is expres ⁇ ed in level ⁇ proportional to the amount of TGF- ⁇ being detected.
  • the a ⁇ ay is a highly sen ⁇ itive and ⁇ pecific, non- radioactive assay, for detecting mature (active) TGF- ⁇ .
  • the TGF- ⁇ as ⁇ ay method of thi ⁇ invention i ⁇ more rapid, ha ⁇ comparable ⁇ en ⁇ itivity, and ha ⁇ a greater detection range.
  • EGF epidermal growth factor
  • bFGF basic fibroblast growth factor
  • TGF- ⁇ re ⁇ pon ⁇ ive expression vector having a gene encoding an indicator polypeptide for a predetermined time period sufficient for the eucaryotic cells to express a detectable amount of the indicator polypeptide; 5 (b) measuring the amount of the indicator polypeptide expres ⁇ ed during the time period;
  • the reference curve represents a quantitative relationship derived from a ⁇ erie ⁇ of mea ⁇ ured amount ⁇ of indicator polypeptide produced from a series of known concentration ⁇ of TGF- ⁇ .
  • the standardized reference curve is obtained from parallel
  • TGF- ⁇ allows for the measurement of TGF- ⁇ from the expres ⁇ ion and subsequent detection of an indicator polypeptide from a concentration range from le ⁇ than 5 picogram ⁇ /ml (pg/ml) equivalent to 0.2 pM up to 10 ng/ml equivalent to 40 pM (or 0.4 nM) .
  • the do ⁇ e-dependent response to TGF- ⁇ is linear
  • the analysi ⁇ of comparative data (comparing) produced by conducting the pre ⁇ ent method both with and without anti-TGF- ⁇ antibody for the purpo ⁇ e of determining the level of TGF- ⁇ in a liquid sample can be conducted by a variety of statistical method ⁇ that are not to be construed as limiting to the invention.
  • Exemplary comparative analyse ⁇ are de ⁇ cribed in the Example ⁇ .
  • TGF- ⁇ as ⁇ ay method ⁇ of this invention are plasmids having identifying characteristic ⁇ of pla ⁇ mid ⁇ on depo ⁇ it with ATCC having the ATCC Acce ⁇ ion Number ⁇ 75627, 75628 and 75629.
  • eucaryotic cells that contain the TGF- ⁇ re ⁇ pon ⁇ e element having the nucleotide ⁇ equence in SEQ ID NO 11 where the cell ⁇ corre ⁇ pond to cell ⁇ on deposit with ATCC -having the ATCC Accession Number CRL 11508.
  • the use of stably transformed eucaryotic cells are contemplated.
  • the invention de ⁇ cribes plasmid ⁇ for u ⁇ e in the methods that comprise a nucleotide sequence corresponding to nucleotide sequences listed in SEQ ID NOs 1-10.
  • TGF- ⁇ inducible response elements that comprise a nucleotide ⁇ equence corresponding to nucleotide sequences listed in SEQ ID NOs 11-17 are also described.
  • Contemplated promoter nucleotide sequence ⁇ are li ⁇ ted in SEQ ID NOs 18 and 19.
  • a further embodiment of the methods of the invention are eucaryotic cells that are stably tran ⁇ formed cell ⁇ containing a plasmid having a gene encoding a selectable marker for the selection of ⁇ aid ⁇ tably tran ⁇ formed cell ⁇ .
  • the invention describes such plasmid ⁇ having nucleotide ⁇ equence ⁇ li ⁇ ted in SEQ ID NO ⁇ 1-6.
  • the invention further describes a stably transformed eucaryotic cell on deposit with ATCC having ATCC Acce ⁇ ion Number CRL 11508 containing the TGF- ⁇ re ⁇ ponse element having the nucleotide sequence in SEQ ID NO 11.
  • An additional embodiment are eucaryotic cells that are transiently transformed cells with plasmids corresponding to the nucleotide sequence ⁇ listed in SEQ ID NOs 7-10.
  • the use of stably transformed cells is particularly preferred because it provides uniformity and reproducibility to the cell based assay without the need for additional controls for the efficiency of transformation typically as ⁇ ociated with method ⁇ u ⁇ ing tran ⁇ ient tran ⁇ formation.
  • Stably transformed cells do not require the u ⁇ e of an internal ⁇ tandard for tran ⁇ formation efficiency, and all of the cells utilized are typically uniformly transformed.
  • the methods do not require the additional step of transforming the cell ⁇ transiently because the stably transformed cell line is already available.
  • the invention describes quantifying the amount of TGF- ⁇ in a body fluid, in culture medium, in a tis ⁇ ue extract, and in the like liquid ⁇ amples.
  • a further preferred embodiment i ⁇ the determination of the amount of a specific i ⁇ oform of TGF- ⁇ , specifically TGF- ⁇ l, TGF- ⁇ 2 or TGF- ⁇ 3, in a liquid ⁇ a ple.
  • this invention describes the use of any eucaryotic host cell that contain ⁇ a TGF- ⁇ receptor and i ⁇ capable of inducing a TGF- ⁇ re ⁇ pon ⁇ e element upon activation by TGF- ⁇ .
  • Exemplary a ⁇ ay ⁇ for measuring activation by TGF- ⁇ and induction of a TGF- ⁇ response element are described herein and can be used to identify candidate host cells ⁇ uitable for u ⁇ e in the pre ⁇ ent diagno ⁇ tic method ⁇ .
  • a preferred ho ⁇ t cell is a mammalian cell.
  • Preferred mammalian cells include mink lung epithelial (MLE) cell ⁇ , particularly clone C32 from MLE cell ⁇ , HeLa cell ⁇ , Chine ⁇ e ham ⁇ ter ovary
  • Conditions for incubating a eucaryotic cell in the present methods are the same as general cell culture methods.
  • Typical cell culture media for culturing and incubating eucaryotic cell ⁇ include alpha-MEM, Eagle' ⁇ MEM (having non-e ⁇ ential amino acid ⁇ ), RPMI 1640 and Dulbecco' ⁇ modified MEM (DMEM), all which are well known in the art.
  • the culture medium preferably contain ⁇ 0.5 to 2 % (v/v) ⁇ eru , preferably a fetal calf or fetal bovine ⁇ erum (FCS or FBS) .
  • Cell culture condition ⁇ include the u ⁇ e of cell ⁇ plated at a density of about 0.8 to about 3.2 x 10 4 cells per well of a 96-well tissue culture plate, preferably about 1.6 x 10 4 cell ⁇ per well.
  • Cell ⁇ are typically plated at the indicated density, and allowed to grow until they reach a confluence density of from about 70% confluent to about 1 day post-confluent, but should preferably be allowed to grow after plating for a time period sufficient for the cells to expres ⁇ detectable level ⁇ of TGF- ⁇ receptor, which time period i ⁇ typically about 0.5-24 hour ⁇ , preferably about 1-5 hour ⁇ , and preferably is about 3 hours.
  • the eucaryotic cells are incubated under culturing condition ⁇ with culture medium that include ⁇ a predetermined volume of a liquid ⁇ ample believed to contain TGF- ⁇ .
  • the incubation time period i ⁇ a time ⁇ ufficient for any TGF- ⁇ pre ⁇ ent in the liquid sample to interact with the eucaryotic cell TGF- ⁇ receptor and thereby induce the TGF- ⁇ respon ⁇ e element and express the indicator polypeptide.
  • the time required for the expressed indicator polypeptide to accumulate to detectable levels will vary with the choice of indicator and method of detection, and can be predetermined.
  • typical incubation times for contacting the cell with the liquid sample can range from 2 to 24 hours, preferably about 6 to 22 hours, more preferably 10 to 20 hours, and particularly about 14 hours.
  • Particularly preferred culturing and incubation conditions for use in the present methods are described in the Examples.
  • TGF- ⁇ level ⁇ are significant in disease ⁇ characterized by excessive fibrosis such as hepatic fibro ⁇ i ⁇ and the like, in proliferative and in condition ⁇ where there i ⁇ an increase in collagen expres ⁇ ion, and the like condition ⁇ where TGF- ⁇ i ⁇ believed to participate.
  • the present assay methods are useful for mea ⁇ uring the therapeutic fate of admini ⁇ tered TGF- ⁇ in patients being treated therapeutically with TGF- ⁇ .
  • the present invention also contemplates a diagnostic system in kit form for as ⁇ aying the amount of TGF- ⁇ in a liquid sample according to the pre ⁇ ent method ⁇ .
  • the diagno ⁇ tic kit contains, in an amount sufficient for at least one assay, a eucaryotic cell of this invention useful for practicing the diagnostic methods for detection of TGF- ⁇ .
  • the kit can further contain a packaging material.
  • Packaging material can include container(s) for ⁇ torage of the materials of the kit, and can include a label or instruction ⁇ for use.
  • a diagnostic kit includes, in an amount sufficient for at least one as ⁇ ay, the following: (a) packaging material; (b) eucaryotic cells contained within the packaging material, where the cells are capable of expres ⁇ ing an indicator molecule and containing a pla ⁇ mid compri ⁇ ing, in the direction of tran ⁇ cription, a regulatory region that include ⁇ at lea ⁇ t one TGF- ⁇ inducible response element that is operatively linked to a promoter, and a structural region downstream of ⁇ aid promoter, where the TGF- ⁇ response element is capable of inducing dose-dependent indicator molecule activity and the structural region coding for ⁇ aid indicator molecule; and (c) an aliquot of TGF- ⁇ contained within ⁇ aid packaging material, where the TGF- ⁇ i ⁇ used for generating a reference curve as described herein representing a measured amount of the indicator molecule produced
  • packaging material refers to a solid matrix or material such as glass, plastic, paper, foil and the like capable of holding within fixed limits eucaryotic cells and an aliquot of TGF- ⁇ .
  • packaging, material can be a plastic vial used to contain eucaryotic cells in growth medium to which liquid ⁇ ample ⁇ can be added for activating the TGF- ⁇ re ⁇ ponsive plasmid within the cells.
  • Packaging material can al ⁇ o be a gla ⁇ vial in which an aliquot of TGF- ⁇ i ⁇ contained for use in generating a reference curve, the latter of which i ⁇ described in Section E.
  • an "aliquot" of TGF- ⁇ refer ⁇ to an amount of TGF- ⁇ ⁇ ufficient to generate a reference curve of this invention.
  • the aliquot of TGF- ⁇ is provided in the form of a substantially dry powder, i.e., in lyophilized form, for subsequent recon ⁇ titution or in the form of a ⁇ olution, i.e., a liquid di ⁇ per ⁇ ion.
  • the amount of powdered TGF- ⁇ i ⁇ in the range of 25 nanogram ⁇ (ng) , more preferably 125 ng to 625 ng, and mo ⁇ t preferably 250 ng.
  • the amount of TGF- ⁇ in liquid solution is in the range of 1 to 50 nanomolar (nM) , more preferably 5 to 25 nM and mo ⁇ t preferably 10 nM.
  • Preferred ⁇ erial dilution ⁇ of TGF- ⁇ u ⁇ ed in generating the reference curve are described in Section E.
  • the TGF- ⁇ provided in the kit preferably includes each of the three TGF- ⁇ isoforms as described in Section B.
  • indicator molecule or indicator polypeptide refers to a molecule encoded by a reporter gene, the expres ⁇ ion of which in the expre ⁇ ion vector ⁇ of thi ⁇ invention, re ⁇ ults in a detectable measurable protein, polypeptide, enzyme and the like.
  • the packaging material includes a label indicating that eucaryotic cells containing TGF- ⁇ respon ⁇ ive expre ⁇ ion vector ⁇ can be u ⁇ ed for determining the amount of TGF- ⁇ in a liquid ⁇ ample that includes the steps of (a) incubating the cells with the selected liquid sample; (b) measuring the amount of the induced indicator molecule; and (c) comparing the amount of measured indicator molecule with a reference curve.
  • the packaging material contain ⁇ a label that i ⁇ a tangible expre ⁇ sion describing the method ⁇ of thi ⁇ invention a ⁇ de ⁇ cribed in Section E. of u ⁇ ing pla ⁇ mid- transformed eucaryotic cells for quantifying the amount of TGF- ⁇ in a test liquid sample.
  • kits or diagnostic sy ⁇ tem ⁇ are those customarily utilized in kits or diagnostic sy ⁇ tem ⁇ .
  • Such material ⁇ include gla ⁇ and pla ⁇ tic, the latter of which include polyethylene, polypropylene and polycarbonate, bottles, vials, plastic and plastic-foil laminated envelopes and the like.
  • the eucaryotic cells transformed with the TGF- ⁇ responsive expression vectors of this invention are cells that express
  • TGF- ⁇ receptor on their cell ⁇ urface a ⁇ described in Section E. All normal cells and most all neoplastic cell ⁇ have cell surface membrane receptors al ⁇ o referred to a binding protein ⁇ for TGF- ⁇ .
  • ⁇ ee Tucker et al . Proc. Natl. Acad. Sci.. USA. 81:6757-6761 (1984) and Frolik et al. , J. Biol.
  • Preferred cells for use with the TGF- ⁇ as ⁇ ay kit include mink lung epithelial cell ⁇ (MLE cell ⁇ ) , HeLa cell ⁇ , Chine ⁇ e Hamster Ovary cells, Hep3B cells, GM7373 cells and NIH 3T3 cells, with the C32 clone from the mink lung epithelial cells being the mo ⁇ t preferred cell line.
  • MLE cell ⁇ mink lung epithelial cell ⁇
  • HeLa cell ⁇ HeLa cell ⁇
  • Chine ⁇ e Hamster Ovary cells Hep3B cells
  • GM7373 cells GM7373 cells
  • NIH 3T3 cells NIH 3T3 cells
  • the eucaryotic cell ⁇ are transformed with the expre ⁇ sion vector plasmids described in Section D have a nucleotide sequence that corresponds to a sequence in SEQ ID NOs 1-10.
  • Contemplated for u ⁇ e in the kit are stably and tran ⁇ iently transformed eucaryotic cells.
  • the plasmids corresponding to SEQ ID NO ⁇ 1-6 are preferred for u ⁇ e.
  • the plasmids corresponding to SEQ ID NOs 7-10 are preferred for use.
  • eucaryotic cells for use with the kit contain a plasmid having the identifying characteristics of a plasmid on deposit with ATCC having the Accession Numbers 75627, 74628 and 75629 as described in Section C.
  • the kit of this invention further includes an anti-TGF- ⁇ antibody for use in a parallel control assay for determining the amount of indicator molecule produced other than by TGF- ⁇ induction.
  • Preferred anti-TGF- ⁇ antibodies are anti-TGF- ⁇ l, anti-TGF- ⁇ 2 or anti-TGF- ⁇ 3 monoclonal antibodies commercially available from Genzyme Corp., Cambridge, MA.
  • a liquid sample can include an i ⁇ oform of TGF- ⁇ , ⁇ pecifically TGF- ⁇ l, TGF- ⁇ 2 or TGF- ⁇ 3.
  • a liquid ⁇ ample further includes any body fluid, culture medium and a tis ⁇ ue extract that may contain unknown quantities of TGF- ⁇ .
  • the liquid sample include ⁇ the body fluids, serum, plasma, whole blood, lymph fluid, synovial fluid, follicular fluid, ⁇ eminal fluid, amniotic fluid, urine, spinal fluid, saliva, sputum, tears, perspiration, mucus and the like.
  • Culture medium includes culture supernatant, also referred to a ⁇ conditioned medium, collected from cell ⁇ maintained in ti ⁇ ue culture as de ⁇ cribed in Example 3B.
  • Ti ⁇ ue extract ⁇ al ⁇ o encompa ⁇ extract ⁇ of cell ⁇ referred to a ⁇ cellular extract ⁇ .
  • organs such as placentas can be obtained and extracted with well known procedure ⁇ to prepare placental extract ⁇ .
  • Extract ⁇ can al ⁇ o be obtained of any body organ or portion thereof, ti ⁇ sue or cell ⁇ , including normal, tumorigenic, and malignant cell ⁇ .
  • Thi ⁇ is generally accomplished by surgical mean ⁇ , i.e., by biopsy sample ⁇ including needle aspirate ⁇ , tissue scraping ⁇ , or freshly dissected tis ⁇ ue ⁇ and the like.
  • Extract ⁇ are the collected ⁇ ample ⁇ are then prepared by mean ⁇ including homogenization in ly ⁇ i ⁇ buffer ⁇ , including detergent ⁇ ⁇ uch as NP-40, Triton X- 100, and the like. Common methods include using potters, blenders, ultrasound generators, and dounce homogenizers.
  • Operatively linking refers to the covalent joining of nucleotide sequences, preferably by conventional phosphodiester bonds, into one strand of DNA, whether in single- or double-stranded form.
  • the expres ⁇ ion vector con ⁇ truct ⁇ of thi ⁇ invention were then used for preparing stably transformed cell ⁇ for use in the quantitative TGF- ⁇ as ⁇ ays of thi ⁇ invention.
  • the expre ⁇ ion vectors were designed to contain varying lengths and arrangements of the TGF- ⁇ response elements from the PAI-1 promoter, a neomycin-resi ⁇ tance conferring gene for selection and a gene encoding an indicator polypeptide, preferably luciferase. Two starting vectors were required to prepare the expression vectors having a neomycin-resi ⁇ tance conferring gene.
  • pl9Luc One of these starting cloning plasmid vectors, designated pl9Luc, was previously described by van Zonneveld et al., Proc. Natl. Acad. Sci.. USA. 85:5525-5529 (1988), the disclosure of which is hereby incorporated by reference.
  • the promoter-les ⁇ reporter gene pl9Luc pla ⁇ mid wa ⁇ originally designed by van Zonneveld et al . , Proc. Natl. Acad. Sci.. USA. 85:5525-5529 (1988) to monitor promoter activity with a ⁇ tructural region, having the firefly luciferase gene to function as a reporter gene, fused to a SV40 splice and polyadenylation site.
  • the pl9Luc plasmid al ⁇ o contained a multiple cloning site preceded by two SV-40-derived polyadenylation site ⁇ .
  • the pl9Luc pla ⁇ mid wa ⁇ constructed from PSVOAL-A ⁇ 5 ' , a vector described by De Wet et al. , __________________________
  • a second portion of the Hind Ill-cleaved pSV0AL-A ⁇ 5 ' was ligated to a 55 bp polylinker and cleaved with Eco RI.
  • the resulting 2831 bp fragment containing the multiple cloning site and the pBR322-derived ampicillin re ⁇ i ⁇ tance-conferring gene wa ⁇ i ⁇ olated.
  • the ⁇ e fragment ⁇ were ligated to create the circular double-stranded pl9Luc plasmid that contained the three fragments in their original orientation but with the multiple cloning ⁇ ite in the original Hind III ⁇ ite.
  • the continuou ⁇ 6170 bp sen ⁇ e ⁇ trand, also referred to as the coding strand, nucleotide sequence of an Eco Rl-linearized pl9LUC vector is li ⁇ ted in the Sequence Li ⁇ ting as SEQ ID NO 21.
  • the Eco Rl-linearized pl9Luc vector contained the following list of element ⁇ and re ⁇ triction ⁇ ite ⁇ beginning with the 5' middle Eco RI "T" nucleotide po ⁇ ition 1 and extending to the 3 ' end of the vector ending with the middle Eco RI "A" nucleotide position 6170 (nucleotide positions a ⁇ listed in SEQ ID NO 21 are indicated in parentheses) : a P ⁇ t I re ⁇ triction ⁇ ite (750-755) within the pBR322-derived ampicillin re ⁇ i ⁇ tance- conferring gene (amp) ; an Ace I re ⁇ triction site downstream of the amp gene (2113-2118) ; two tandem polyadenylation sites immediately upstream of the multiple cloning site beginning with Bam HI (2771-2776) and Hind III (2778-2783), continuing with adjacent Sph I, PstI, Hinc II/Acc T/Sal I, Xba I, Bam HI, X
  • pl9Luc-derived expre ⁇ ion vector ⁇ having TGF- ⁇ re ⁇ ponse element ⁇ and promoter ⁇ are familiar to one of ordinary ⁇ kill in the art of vector con ⁇ truction and are de ⁇ cribed by Au ⁇ ebel, et al., In Current Protocol ⁇ in Molecular Biology, Wiley and Son ⁇ , New York (1993) and by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1989.
  • pl500Luc One expre ⁇ ion vector of this invention, designated pl500Luc, was constructed from pl9Luc and a cosmid containing the PAI-1 promoter in which TGF- ⁇ respon ⁇ e element ⁇ are located.
  • pl500Luc a 1547 ba ⁇ e pair (bp) Kpn I- Eco RI fragment of the PAI-1 promoter was obtained from a * cosmid containing the entire PAI-1 gene (Loskutoff et al. , Bioche ..
  • the fragment contained the 1442 bp TGF- ⁇ re ⁇ pon ⁇ e element (SEQ ID NO 11) from the PAI-1 promoter that corre ⁇ ponded to nucleotide position -1481 and extended to the nucleotide po ⁇ ition -40 continuou ⁇ with a 115 bp minimal (non-TGF- ⁇ re ⁇ pon ⁇ ive) PAI-1 promoter sense strand sequence (SEQ ID NO 18) corresponding to nucleotide po ⁇ ition -39 ending with an E. coli DNA polymerase filled-in Eco RI site at nucleotide position at +76 as described by Bosma et al., J. Biol. Chem.. 263:9129-9141 (1988) .
  • the PUCEK19 plasmid prepared above was then dige ⁇ ted with Kpn I and Eco RI and the i ⁇ olated fragment wa ⁇ then ligated into the multiple cloning ⁇ ite of a ⁇ imilarly dige ⁇ ted pl9Luc.
  • the resulting vector was designated pl500Luc.
  • p ⁇ OOLuc wa ⁇ prepared for ⁇ ubsequent constructon of p ⁇ OOneoLuc a ⁇ described below.
  • the p ⁇ OOLuc plasmid having a deletion in the 5' end of the PAI-1 construct so that the 5' end began with the -800 nucleotide in the native PAI-1 promoter, was prepared by digesting the PAI-1-gene-containing cosmid described above with Hind III and Eco RI .
  • the actual Hind III-Eco RI digest of the PAI-1 promoter resulted in a fragment that corresponded to nucleotides -799 to +71 bp in the PAI-1 promoter that wa ⁇ subsequently ligated into a similarly dige ⁇ ted pl9Luc vector forming a PAI-1 region extending from nucleotide -800 to +76.
  • the resulting p ⁇ OOLuc plasmid retained all the features of pl9Luc with the exception of the insertion of the PAI-1-derived regulatory region having a TGF- ⁇ response element and a promoter.
  • the restriction fragments described to prepare pl500Luc and p ⁇ OOLuc had an identical 3' end (an Eco RI site at +71 nucleotide of the PAI-1 promoter) and a different 5' end.
  • the vectors, pl500Luc and p ⁇ OOLuc, were used for transient transformations as they lacked a selectable marker gene.
  • the pl500Luc plasmid wa ⁇ also used to'prepare stable transformations with a second vector as described in Example 1C.
  • the p ⁇ OOLuc served as the starting cloning construct for the preparation of p ⁇ OOneoLuc as described below.
  • the TGF- ⁇ respon ⁇ e element in the -800 to +76 PAI-1 promoter region began at -800 and ended at -40, the nucleotide ⁇ equence of which i ⁇ li ⁇ ted in SEQ ID NO 12.
  • the remaining nucleotides comprised the non-TGF- ⁇ respon ⁇ ive minimal promoter in thi ⁇ PAI-1 fragment are listed in SEQ ID NO 18.
  • expres ⁇ ion vector designated p39Luc, having a promoter for activating transcription of the lucifera ⁇ e gene while lacking TGF- ⁇ re ⁇ ponse element ⁇ , thereby lacking re ⁇ pon ⁇ iveness to TGF- ⁇ , wa ⁇ prepared as described by Keeton et al., J. Biol. Chem.. 266:23046-23052 (1991) .
  • a fragment of the PAI-1 promoter (i.e., between -39 and +76, which had been determined in the TGF- ⁇ assay as de ⁇ cribed in Example 3A to have low ba ⁇ al activity and only minimal response to TGF- ⁇ (average induction of 2.7-fold), was used a ⁇ a minimal promoter in the con ⁇ truct ⁇ for u ⁇ e in quantifying the amount of TGF- ⁇ in a te ⁇ t liquid sample. Since the minimal promoter sequence conferred only a minimal background respon ⁇ e to TGF- ⁇ a ⁇ ⁇ hown in Example 3A, the minimal PAI-1-derived promoter is also referred to as being "non-TGF- ⁇ responsive" .
  • the p ⁇ OOLuc vector was linearized by digestion with Hind III followed by 5' dige ⁇ tion of PAI-1 promoter with Bal-31 ⁇ low exonuclea ⁇ e (International Biotechnologie ⁇ , New Haven, CT) a ⁇ de ⁇ cribed by Keeton et al., J. Biol . Chem.. 266:23048-23052 (1991) .
  • the dige ⁇ tion wa ⁇ allowed to proceed until the -39 nucleotide po ⁇ ition of the PAI-1 promoter wa ⁇ reached.
  • the linearized and Bal-31 digested plasmid was ligated with T4 ligase forming a double- ⁇ tranded circular vector de ⁇ ignated p39Luc.
  • the re ⁇ ultant expre ⁇ ion vector into which TGF- ⁇ re ⁇ pon ⁇ e element ⁇ were subsequently ligated as described in Example 1C, contained the PAI-1 minimal promoter nucleotide sequence corresponding to -39 to +76 of the promoter as listed in SEQ ID NO 18. This minimal promoter was operatively linked to and continuous with the structural region that contained the firefly luciferase gene present in the vector. Since the p39Luc cloning vector was derived from p ⁇ OOLuc which itself was derived from pl9Luc, the remaining elements and features of the vector were retained unchanged from pl9Luc. The 6229 bp ⁇ ense strand nucleotide sequence of the Eco Rl-linearized p39Luc vector is li ⁇ ted in the SEQ ID NO 23.
  • the p39Luc cloning expres ⁇ ion vector i ⁇ al ⁇ o obtained by preparing a double- ⁇ tranded olignucleotide sequence corresponding to the ⁇ equence in SEQ ID NO 16 and ligating it into the Hind I I/Eco RI multiple cloning site of pl9Luc.
  • Other con ⁇ truction methods are well known to and easily accomplished by one of ordinary skill in the art.
  • the p39Luc vector was useful for operatively ligating regulatory regions that contained TGF- ⁇ response elements resulting in an expression vector that was re ⁇ pon ⁇ ive to DNA- binding proteins, the result of which was induction of the transcription and translation of the indicator molecule, luciferase.
  • TGF- ⁇ re ⁇ pon ⁇ ive expre ⁇ ion vector ⁇ for u ⁇ e in practicing thi ⁇ invention having TGF- ⁇ re ⁇ ponse element ⁇ other than tho ⁇ e ⁇ pecified herein are readily con ⁇ tructed through the use of either pl9Luc or p39Luc ⁇ tarting cloning expression vectors.
  • This promoter corresponded to the nucleotide sequence from -188 to +145 of the Hepatitis B promoter and showed only 4-fold induction in response to TGF- ⁇ .
  • the 6464 bp ⁇ en ⁇ e ⁇ trand nucleotide sequence of the Eco Rl- linearized pHBVLuc vector is listed in the SEQ ID NO 25.
  • pMAMneo (Clontech, Palo Alto, CA) was in ⁇ erted into the p800Luc vector containing - ⁇ OO to +76 of the 5' end of the human PAI-1 gene followed by the firefly lucifera ⁇ e gene.
  • p ⁇ OOLuc prepared above wa ⁇ fir ⁇ t digested with Ace I, repaired to blunt ends with the Klenow fragment of DNA polymerase I, and then was isolated.
  • the pMAMneo plasmid was digested with Sal I and Eco RI then blunt-ended with Klenow.
  • the entire Eco Rl-linearized 11293 bp nucleotide ⁇ equence of the sense strand of the double-stranded p ⁇ OOneoLuc vector is listed in the Sequence Li ⁇ ting in SEQ ID NO 1.
  • DNA sequencing was performed by a modification of the dideoxy chain-termination procedure with a Sequenase kit (United State ⁇ Biochemical; Cleveland, OH) . This clone, purified from large scale plasmid preparations via CsCl2 gradient ⁇ , wa ⁇ u ⁇ ed for subsequent transfections.
  • the p ⁇ OOneoLuc cloning vector wa ⁇ derived from p ⁇ OOLuc which itself was derived from pl9Luc, the remaining elements and features of the vector were retained unchanged from ' pl9Luc.
  • the p ⁇ OOneoLuc vector thus contained the neomycin-re ⁇ i ⁇ tance conferring gene providing for stable transformants .
  • the p ⁇ OOneoLuc vector also contained an operatively ligated regulatory region that contained TGF- ⁇ re ⁇ pon ⁇ e element in the ⁇ equence corre ⁇ ponding to -600 to -40 of the PAI-1 promoter resulting in an expression vector that was respon ⁇ ive to TGF- ⁇ .
  • the induced activation of the transcription and translation of the indicator molecule, luciferase was obtained further allowing for the quantitation of the amount of TGF- ⁇ responsible for activating gene expres ⁇ ion.
  • TGF- ⁇ respon ⁇ e elements were operatively ligated 5' to the minimal promoter sequence of the p39neoLuc as de ⁇ cribed in Example 1C for the preparation of plasmids for transient tran ⁇ formation.
  • the pl500Luc vector prepared above i ⁇ similarly ligated with the neomycin-resistance gene from pMAMneo to form pl500neoLuc.
  • Other PAI-1-promoter containing expre ⁇ ion vector ⁇ lacking the neomycin re ⁇ i ⁇ tance gene, p800/636Luc, p56Luc, p674Luc, p743Luc and p732Luc, containing ⁇ maller TGF- ⁇ response elements were prepared as de ⁇ cribed in Example 1C.
  • the neomycin-re ⁇ i ⁇ tance gene from pMAMneo is separately ligated with each of these five vector ⁇ to form expre ⁇ ion vector ⁇ u ⁇ ed for generating ⁇ table cell tran ⁇ formation ⁇ .
  • the five re ⁇ ultant vector ⁇ having the neomycin-re ⁇ i ⁇ tance gene in ⁇ erted are de ⁇ ignated p ⁇ 00/63 ⁇ neoLuc (10697 bp) , p56neoLuc (10549 bp) , p674neoLuc (10558 bp) , p743neoLuc (10569 bp) and p732neoLuc (10558 bp) and have the respective complete nucleotide sequence ⁇ of the ⁇ en ⁇ e ⁇ trand from the Eco Rl-linearized double- ⁇ tranded vector ⁇ in SEQ ID NO ⁇ 2-6.
  • the designated names either had "Luc” alone or "neoLuc” respectively for vectors lacking the neomycin (neo) selectable marker gene or containing it.
  • the pla ⁇ mids were further designated by the 5' end of the PAI-1 TGF- ⁇ respon ⁇ e element. For example, five pla ⁇ mids with shorter TGF- ⁇ respon ⁇ e element ⁇ were thu ⁇ named p800/636neoLuc, p56Luc, p674Luc, p743Luc and p732Luc.
  • TGF- ⁇ Responsive Vectors and a Selectable Marker Vector for Stable Transforma ion Stably transformed Hep3B cells were also obtained as described in Example 2B below through the use of co- transfections of a TGF- ⁇ respon ⁇ ive vector lacking a ⁇ electable marker gene of thi ⁇ invention, specifically the pl500Luc prepared in Example 1A3), with a selectable marker vector, RSVneo, available from American Type Culture Collection (ATCC) , Rockville, MD, ATCC Accession Number 37198.
  • ATCC American Type Culture Collection
  • the stably transformed cell line containing plasmid pl500Luc, designated LUCI was depo ⁇ ited with the ATCC on or before December 16, 1993 and wa ⁇ assigned the ATCC Accession Number CRL 11508.
  • TGF- ⁇ respon ⁇ ive expre ⁇ ion vectors were prepared for use in this invention.
  • the TGF- ⁇ response elements having a smaller length, thereby providing re ⁇ pon ⁇ ivene ⁇ to TGF- ⁇ with reduced or ab ⁇ ent re ⁇ pon ⁇ iveness to other growth modulator ⁇ were made by either re ⁇ triction dige ⁇ tion of the PAI-1 promoter or ⁇ ynthe ⁇ izing double- ⁇ tranded blunt-end oligonucleotide ⁇ .
  • the oligonucleotide ⁇ equence ⁇ corre ⁇ ponded to pre ⁇ elected regions of the PAI-1 promoter ⁇ equence.
  • the re ⁇ ultant TGF- ⁇ re ⁇ pon ⁇ e element ⁇ pre ⁇ ent within a regulatory region were then directionally ligated into p39Luc or p39HBV.
  • the regulatory region from the PAI-1 promoter corresponding to nucleotide position -800 up to and including -636 wa ⁇ obtained by restriction digestion and had the following sense strand sequence: 5 'AAGCTTACCATGGTAACCCCTGGTCCCGTTCAGCCACCACCACCACCCAGCACACCTCC AACCTCAGCCAGACAAGGTTGTTGACACAAGAGAGCCCTCAGGGGCACAGAGAGTCTGGAC ACGTGGGGAGTCAGCCGTGTATCATCGGAGGCGGCCGGGCA3 ' (SEQ ID NO 13) .
  • the additional ⁇ elected regions for preparing oligonucleotides included the following sense strand nucleotide sequence ⁇ with the indicated nucleotide po ⁇ ition ⁇ as present in the intact
  • PAI-1 promoter 1) promoter nucleotide position -56 up to and including -41: 5 'AGTTCATCTATTTCCT3 ' (SEQ ID NO 14); 3) promoter nucleotide position -674 up to and including -650: 5 'GTGGGGAGTCAGCCGTGTATCATCG3 ' (SEQ ID NO 15) ; 4) nucleotide position -743 up to and including -708:
  • the resulting double-stranded oligonucleotides were then separately operatively linked to the -39 position of this minimal promoter sen ⁇ e strand sequence listed in SEQ ID NO 16 present in the expre ⁇ ion vector, p39Luc, prepared a ⁇ de ⁇ cribed in Example 1A4) .
  • the ⁇ equence ⁇ were confirmed by double- stranded sequencing methods.
  • the resulting five plasmids with shorter TGF- ⁇ re ⁇ pon ⁇ e elements were thus named p800/636Luc, p56Luc, p674Luc, p743Luc and p732Luc.
  • the plasmid ⁇ , p56Luc, p674Luc, p743Luc and p732Luc have the re ⁇ pective complete ⁇ en ⁇ e ⁇ trand nucleotide sequence ⁇ beginning with the middle T of the Eco RI site as previously described listed in SEQ ID NOs 7-10.
  • the plasmid ⁇ , p674Luc, p743Luc and p732Luc were depo ⁇ ited with ATCC a ⁇ described in Example 5 and respectively assigned the ATCC Accession Numbers 75627, 75628 and 75629.
  • TGF- ⁇ re ⁇ pon ⁇ e elements having a heterologous hepatitis B viral promoter, HBV, instead of the PAI-1 minimal promoter were prepared with the shorter TGF- ⁇ re ⁇ pon ⁇ e elements, p800/636Luc, p56Luc, p674Luc, p743Luc and p732Luc.
  • the HBVLuc cloning expres ⁇ ion vector wa ⁇ prepared a ⁇ de ⁇ cribed in Example 1A4) .
  • the TGF- ⁇ re ⁇ ponse element ⁇ were ligated into linearized HBVLuc, prepared a ⁇ described in Example 1A5) , to form TGF- ⁇ response element-containing plasmid ⁇ lacking the neomycin-resistance-conferring gene.
  • the cloning vector construct ⁇ , pl9Luc and p39Luc provide for the operative linking of pre ⁇ elected regulatory region ⁇ with pre ⁇ elected promoters, both of which are not limited to the ⁇ pecific con ⁇ tructs described herein and above.
  • Additional TGF- ⁇ respon ⁇ e element ⁇ in varied lengths and arrangement ⁇ along with promoters that provide for the tran ⁇ cription of the reporter gene are contemplated for u ⁇ e in thi ⁇ invention.
  • Example 1 To identify the cell types mo ⁇ t re ⁇ ponsive to TGF- ⁇ in which to transfect the TGF- ⁇ responsive expres ⁇ ion vectors for use in as ⁇ aying the amount of TGF- ⁇ , the vector ⁇ prepared in Example 1 were tran ⁇ fected a ⁇ de ⁇ cribed in Example 2B and 2C into recipient cell line ⁇ including mink lung epithelial cell ⁇ (MLE cell ⁇ ) (ATCC CCL 64), HeLa cell ⁇ (ATCC CCL 2), Chine ⁇ e hamster ovary (CHO cell ⁇ ) (ATCC CCL 61), GM7373 (chemically tran ⁇ formed metal bovine aortic endothelial cell ⁇ or BAEs) (NIGMS Human Genetic Mutant Cell Repository, Ca den, NJ) , Hep3B (ATCC HB 8064) and NIH 3T3 cell ⁇ (ATCC CRL 1658) .
  • MLE cell ⁇ mink lung epithelial cell ⁇
  • HeLa cell ⁇ ATCC CCL 2
  • CHO cell ⁇
  • tran ⁇ fection ⁇ of mink lung epithelial cell ⁇ (hereinafter referred to as MLE cells to distinguish from the TGF- ⁇ proliferation assay called MLEC) were performed.
  • MLE cells tran ⁇ fection ⁇ of mink lung epithelial cell ⁇
  • the MLE cell ⁇ were ⁇ eeded at 7 x 10 5 cells/100 mm di ⁇ h for 24 hours at which point they were transfected with the PAI/L con ⁇ truct, p ⁇ OOneoLuc, by calcium pho ⁇ phate precipitation a ⁇ described by Wigler et al . , Proc. Natl. Acad. Sci .. USA.
  • tran ⁇ fected MLE cells were maintained in DMEM containing 10% fetal calf serum and 250 ⁇ g/ml Geneticin (G-418 sulfate) (Gibco BRL, Grand Island, NY) .
  • Stable transformations are also performed as described above with the expression vectors, p800/636neoLuc, p56neoLuc, p674neoLuc, p743neoLuc and with p732neoLuc, all of which are prepared as described in Example 1A.
  • Hep3B cell ⁇ at a concentration of 6 X 10 5 cell ⁇ /well were ⁇ eeded a ⁇ de ⁇ cribed above in Example IB for 24 hours at which point they were transfected with the PAI/L con ⁇ truct, pl500Luc, by calcium phosphate precipitation followed by selection with Geneticin.
  • the re ⁇ ultant cell line stably transformed with pl500Luc, designated LUCI was deposited with ATCC on December 16, 1993 and was assigned the ATCC Accession Number CRL 11508.
  • D. Transient Transformation For preparing transiently tran ⁇ formed cell ⁇ containing TGF- ⁇ responsive expres ⁇ ion vectors lacking the neomycin resi ⁇ tance gene prepared a ⁇ described in Example 1C, Hep3B human hepatoma cells obtained from ATCC (ATCC Accession Number HB8064) were maintained in DMEM/HAMs F-12 (Whittaker Bioproduct ⁇ , Walker ⁇ ville, MD) ⁇ upplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) , glutamine, sodium pyruvate, non-es ⁇ ential amino acids and penicillin/ ⁇ treptomycin (Whittaker) .
  • ATCC Accession Number HB8064 Hep3B human hepatoma cells obtained from ATCC (ATCC Accession Number HB8064) were maintained in DMEM/HAMs F-12 (Whittaker Bioproduct ⁇ , Walker ⁇ ville, MD) ⁇ upplemented with 10% fetal bovine serum (Hyclon
  • tissue culture plates (Corning Inc., Corning, NY) were washed twice with ⁇ erum free media (DMEM/F-12) then incubated in ⁇ erum free media.
  • DMEM/F-12 ⁇ erum free media
  • TGF- ⁇ mea ⁇ urement a ⁇ says performed with cells transiently transformed with the remaining expres ⁇ ion vector ⁇ containing TGF- ⁇ response element ⁇ are presented in Example 4.
  • the TGF- ⁇ as ⁇ ay allows for the quantification of the amount of TGF- ⁇ in a liquid sample, either containing purified TGF- ⁇ or TGF- ⁇ in a heterogeou ⁇ admixture.
  • the assay ⁇ y ⁇ tem provide ⁇ for the quantification of TGF- ⁇ through the expression of an indicator polypeptide, such as luciferase.
  • an indicator polypeptide such as luciferase.
  • This reference curve i ⁇ obtained from parallel a ⁇ ays performed by exposing similarly transfected cells to a range of known measured amounts of TGF- ⁇ , one or more of the known TGF- ⁇ isoforms. The resulting expressed luciferase is then determined in a luminometer. A reference curve is then generated by plotting the measured amount of expre ⁇ ed lucifera ⁇ e again ⁇ t the known range of inducing amount ⁇ of TGF- ⁇ . The amount of unknown TGF- ⁇ in the te ⁇ t liquid sample i ⁇ then determined by extrapolating the measured amount of test luciferase to the reference curve.
  • the TGF- ⁇ a ⁇ ay of thi ⁇ invention allows for the measurement of TGF- ⁇ from the expres ⁇ ion and ⁇ ub ⁇ equent detection of an indicator polypeptide from a concentration range from less than 5 picogram ⁇ /ml (pg/ml) equivalent to 0.2 pM to 10 ng/ml equivalent to 0.4 nM.
  • a ⁇ ay for quantifying TGF- ⁇ in complex ⁇ olutions was the use of neutralizing anti-TGF- ⁇ monoclonal antibodies admixed with the test liquid sample in as ⁇ ay ⁇ run in parallel to untreated te ⁇ t liquid samples as described in Example 3B.
  • These control as ⁇ ay ⁇ are u ⁇ ed to determine if other molecule ⁇ are pre ⁇ ent in the te ⁇ t ⁇ ample 5 that can affect the a ⁇ ay through either inhibition or activation of other region ⁇ of the truncated PAI-1 promoter.
  • conditioned medium obtained from cell culture ⁇ and body fluid ⁇ contain growth factor ⁇ and DNA binding protein ⁇ that function a ⁇ tran ⁇ criptional activator ⁇ or inhibitor ⁇ .
  • TGF- ⁇ response elements used in the expre ⁇ ion vector ⁇ y ⁇ tem ⁇ of thi ⁇ invention are less likely to have non-TGF- ⁇ respon ⁇ e element ⁇
  • Example ⁇ 3C-3F the u ⁇ e of parallel antibody control a ⁇ ay ⁇ to allow for a determination of the amount of lucifera ⁇ e produced from only TGF- ⁇ activation is preferred when expres ⁇ ion vector ⁇ having longer re ⁇ pon ⁇ e element ⁇ are u ⁇ ed.
  • TGF- ⁇ a ⁇ say is not i ⁇ oform ⁇ pecific, u ⁇ ing the appropriate ⁇ tandard reference curve ⁇ and parallel a ⁇ says with neutralizing antibodies to the various TGF- ⁇ specie ⁇ allow ⁇ for quantification of unique TGF- ⁇ i ⁇ oforms.
  • reagent ⁇ including their ⁇ ource ⁇ are li ⁇ ted: recombinant human TGF- ⁇ l (rTGF- ⁇ l) (gift from Berlix Bio ⁇ cience ⁇ , South San Franci ⁇ co, CA) ; rTGF- ⁇ 2 and neutralizing monoclonal antibodie ⁇ again ⁇ t TGF- ⁇ l, TGF- ⁇ 2 and TGF- ⁇ 3 (Genzyme, Cambridge, MA); rTGF- ⁇ 3, recombinant human interleukin-lalpha (rIL-lalpha) and 5 recombinant human platelet-derived growth factor-BB (PDGF-BB) (R&D Sy ⁇ tem ⁇ , Minneapoli ⁇ , MN) ; recombinant human ba ⁇ ic fibrobla ⁇ t growth factor (bFGF) (Synergen Inc., Boulder, CO); epidermal growth factor (EGF) from mou ⁇ e ⁇ ubmaxillary gland ⁇ (Boehringer Mannheim Biochemical ⁇ , Indianapoli ⁇ , IN) ; dex
  • thrombin Armour Pharmaceutical Co., Kankakee, ID ; and hematopoetic factor ⁇ granulocyte-colony ⁇ timulating factor (GCSF) , granulocyte-macrophage-colony ⁇ timulating factor (GMCSF) , ⁇ tem cell factor, and IL-3 (Amgen, Thou ⁇ and Oak ⁇ , CA) .
  • GCSF granulocyte-colony ⁇ timulating factor
  • GMCSF granulocyte-macrophage-colony ⁇ timulating factor
  • IL-3 Amgen, Thou ⁇ and Oak ⁇ , CA
  • the medium was replaced with the test ⁇ ample containing unknown quantities of TGF- ⁇ , DMEM, 0.1% BSA (DMEM-BSA) containing rTGF- ⁇ l, rTGF- ⁇ 2, rTGF- ⁇ 3, IL-lalpha, PDGF-BB, bFGF, or EGF for 14 hours at 37°C.
  • Time course ⁇ of exposure to the sample ⁇ were performed a ⁇ shown for optimizing the assay as shown below. However, in general, approximately 24 hour ⁇ after addition ⁇ of the ⁇ ample to the tran ⁇ fected cells, the cells were observed under phase contrast microscopy.
  • the pre ⁇ ence of TGF- ⁇ in quantitie ⁇ at least or greater than 0.1 ng/ml TGF- ⁇ in the sample was detected visually by the change of morphology and density of the cell population.
  • the untreated cells remained organized with cell size decreasing upon confluence until the cell borders were no longer visible.
  • the untreated cell den ⁇ ity wa ⁇ never attained and the cell ⁇ were larger, flatter and le ⁇ organized.
  • cell extracts were prepared and as ⁇ ayed for luciferase activity u ⁇ ing the enhanced lucifera ⁇ e a ⁇ ay kit (Analytical Lumine ⁇ cence, San Diego, CA) as per the manufacturer's illustructions .
  • Treated cells were first washed twice with 2 ml phosphate-buffered saline (PBS) without Ca ++ and Mg ++ and then extracted with 100 ul of 0.25% Triton-X 100 (cell ly ⁇ i ⁇ buffer, Analytical Lumine ⁇ cence) . The plate ⁇ were gently ⁇ haken until the monolayer detached from the pla ⁇ tic. The plate ⁇ were then placed on a rotator at room temperature for 20 minute ⁇ .
  • PBS ml phosphate-buffered saline
  • Triton-X 100 cell ly ⁇ i ⁇ buffer, Analytical Lumine ⁇ cence
  • reference curve ⁇ were prepared from parallel a ⁇ ay ⁇ performed by expo ⁇ ing ⁇ imilarly tran ⁇ fected cell ⁇ to a range of known mea ⁇ ured amounts of TGF- ⁇ , one or more of the known TGF- ⁇ isoform ⁇ .
  • Serial dilution ⁇ of the control TGF- ⁇ concentration ⁇ were prepared from a 1 nanomolar (nM) concentration down to 0.078 picomolar (pM) .
  • the TGF- ⁇ a ⁇ ay wa ⁇ performed for each ⁇ erial dilution and the re ⁇ ulting expre ⁇ ed lucifera ⁇ e was then determined in a luminometer.
  • a reference (standard) curve was then generated by plotting the mea ⁇ ured amount of expressed luciferase against each of the known concentrations of inducing amounts of TGF- ⁇ .
  • the amount of unknown TGF- ⁇ in the test liquid sample was then determined by extrapolating the mea ⁇ ured amount of te ⁇ t lucifera ⁇ e to the reference curve.
  • the p ⁇ OOneoLuc construct prepared in Example 1A was stably tran ⁇ fected a ⁇ described in Example 2B into a variety of cell lines including MLE cells, HeLa, Chinese hamster ovary (CHO) , GM7373 cells (chemically transformed fetal bovine aortic endothelial cells obtained from the NIGMS Human Genetic Mutant Cell Repo ⁇ itory, Ca den, NJ) and NIH 3T3 cell ⁇ .
  • the cell ly ⁇ ate ⁇ were a ⁇ ayed for luciferase activity and protein content. There was a linear relationship between the luciferase activity and the protein content of the cell ly ⁇ ate ⁇ between 0.7 and 14 ⁇ g for all of the cell lines. Nontransfected parental cell ⁇ demonstrated no detectable lucifera ⁇ e activity. Of the variou ⁇ cell line ⁇ , the tran ⁇ fected MLE cell ⁇ demon ⁇ trated the greate ⁇ t ⁇ en ⁇ itivity to TGF- ⁇ .
  • C32 cell ⁇ from clone 32
  • pM picomolar
  • nM nanomolar
  • the TGF- ⁇ as ⁇ ay wa ⁇ performed with 8 pM of rTGF- ⁇ l, rTGF- ⁇ 2 or rTGF- ⁇ 3 in DMEM-BSA in the pre ⁇ ence (partially filled ⁇ quare ⁇ ) or ab ⁇ ence (open squares) of 100 ⁇ g/ml of anti-TGF- ⁇ l, anti-TGF- ⁇ 2 or anti-TGF- ⁇ 3 monoclonal antibodies (Genzyme Corp., Cambridge, MA) .
  • TGF- ⁇ assay was performed using increasing concentration of rTGF- ⁇ l in DMEM (closed squares), alpha-MEM (closed circles), CMEM (Eagles medium supplemented with nones ⁇ ential amino acids; closed triangles), or RPMI-1640 (closed diamonds) . All media contained 0.1% BSA.
  • Cell den ⁇ itie ⁇ greater than 1.6 x 10 4 cell ⁇ /well decreased the sen ⁇ itivity of the a ⁇ ay at low TGF- ⁇ concentration ⁇ and did not ⁇ ignificantly increase sensitivity at higher TGF- ⁇ 3 levels. Decreasing the concentration of cell ⁇ to 0.8 x 10 4 cells/well increased the ⁇ ensitivity at low TGF- ⁇ 3 levels ( Figure 3D (inset in Figure 3C) but decreased ⁇ en ⁇ itivity at higher TGF- ⁇ concentration ⁇ .
  • Incubation time with the ⁇ ample al ⁇ o affected the a ⁇ ay.
  • 1.6 X 10 4 C32 cell ⁇ were incubated with variou ⁇ concentration ⁇ of rTGF- ⁇ l ranging from 0 to 50 pM for 6 (clo ⁇ ed ⁇ quare ⁇ ), 14 (clo ⁇ ed circle ⁇ ) or 22 hour ⁇ (closed triangles) prior to as ⁇ aying for lucifera ⁇ e activity a ⁇ ⁇ hown in Figure 3C.
  • Incubation times of 12-14 hours were found to give the best results over the wide ⁇ t concentration range.
  • TGF- ⁇ as ⁇ ay wa ⁇ After examining the sen ⁇ itivity of the a ⁇ ay, specificity of the TGF- ⁇ as ⁇ ay wa ⁇ then examined.
  • bFGF fibroblast growth factor
  • PDGF-BB platelet-derived growth factor
  • rIL-lalpha interleukin-1 alpha
  • the assay was performed as described in Example 3A with DMEM-BSA containing rTGF- ⁇ l (closed squares), recombinant human bFGF (clo ⁇ ed circle ⁇ ), recombinant IL-lalpha (closed triangles), recombinant PDGF-BB (clo ⁇ ed triangle ⁇ ) or EGF (open squares) ranging in concentration from 0.1 to 500 pM.
  • rTGF- ⁇ l closed squares
  • recombinant human bFGF clo ⁇ ed circle ⁇
  • recombinant IL-lalpha closed triangles
  • recombinant PDGF-BB clo ⁇ ed triangle ⁇
  • EGF open squares
  • a ⁇ ay ⁇ were performed with C32 cells maintained in DMEM/BSA containing 1 pM rTGF- ⁇ l (closed square ⁇ ) ⁇ eparately admixed with each of the growth factor ⁇ , bFGF (clo ⁇ ed circle ⁇ ) , recombinant IL-lalpha (clo ⁇ ed triangle ⁇ ), recombinant PDGF (clo ⁇ ed diamond ⁇ ) or EGF (open squares), ranging in concentration from 0.2 to 500 pM.
  • thi ⁇ enhancement may have resulted from a bFGF- mediated increase in total cell number and/or protein
  • crystal violet staining of parallel cultures and protein as ⁇ ay ⁇ of the cell ly ⁇ ate ⁇ wa ⁇ performed.
  • the normalization of the amount of protein using these value ⁇ did not reduce the lucifera ⁇ e activity in the bFGF plu ⁇ rTGF- ⁇ l-treated culture ⁇ to that of cell ⁇ treated with rTGF- ⁇ l alone.
  • 5 uncloned transfected MLE cells were les ⁇ ⁇ en ⁇ itive to bFGF and other factors including TGF- ⁇ .
  • TGF- ⁇ as ⁇ ay ⁇ were performed u ⁇ ing the ATCC depo ⁇ ited LUCI cell line containing the pl500Luc expre ⁇ ion vector co-tran ⁇ fected with RSVneo as described in Example 2C to
  • TGF- ⁇ assays were performed as de ⁇ cribed in Example 3A with the exception that the pl500Luc vector wa ⁇ u ⁇ ed in ⁇ tead of the p ⁇ OOneoLuc vector. Controls in these as ⁇ ay ⁇ included the u ⁇ e of two
  • the a ⁇ says were performed a ⁇ indicated in Table 1 in which the fold induction i ⁇ indicated a ⁇ mea ⁇ ured by relative light
  • the 1500 bp PAI-1 promoter present in the pl500Luc vector was slightly responsive to IL-6, LPS and a mixture of IL-6 plus dexamethasome.
  • IL-6 and IL-6 plus dexamethasone were effective activating agents when u ⁇ ed in the presence of a vitronectin promoter. None of the agents were ⁇ ignificantly effective at inducing expre ⁇ ion from the RSV promoter.
  • TGF- ⁇ i ⁇ the predominant activator of the PAI-1 promoter and that the TGF- ⁇ a ⁇ ay of thi ⁇ invention exhibit ⁇ remarkable ⁇ pecificity.
  • the a ⁇ say is valuable in that the mea ⁇ urement of TGF- ⁇ that ha ⁇ been purified or even TGF- ⁇ pre ⁇ ent in unknown quantitie ⁇ in a complex ⁇ olution containing many promoter-specific molecules can be readily determined without confounding by con.taminant ⁇ .
  • the absolute amounts of TGF- ⁇ as well as isomer type can be determined.
  • TGF- ⁇ a ⁇ ays were performed in the pre ⁇ ence of DMEM- BSA containing rTGF- ⁇ l alone (clo ⁇ ed ⁇ quare ⁇ ), or with 0.5% (clo ⁇ ed circle ⁇ ), 1% (clo ⁇ ed triangle ⁇ ), or 2% (clo ⁇ ed diamonds) calf serum.
  • the rTGF- ⁇ l concentrations in the as ⁇ ay ⁇ ranged from 0 to 8 pM.
  • TGF- ⁇ a ⁇ ay ⁇ were also performed in COS, BSM and BAE cell conditioned medium (CM) , all of which normally contain latent but little, if any, active TGF- ⁇ . These ⁇ amples were tested u ⁇ ing the TGF- ⁇ a ⁇ ay method of thi ⁇ invention in compari ⁇ on with the MLEC (mink lung epithelial cell tritiated thymidine uptake cell as ⁇ ay) .
  • MLEC mink lung epithelial cell tritiated thymidine uptake cell as ⁇ ay
  • the TGF- ⁇ assay was performed a ⁇ de ⁇ cribed in Example 3A with rTGF- ⁇ l ranging in concentration from 0 to 40 pM in the pre ⁇ ence of either DMEM-BSA (clo ⁇ ed ⁇ quare ⁇ ) , COS CM (cro ⁇ ses) , BSM CM (closed triangles) or BAE CM (closed circles) .
  • DMEM-BSA clo ⁇ ed ⁇ quare ⁇
  • COS CM cro ⁇ ses
  • BSM CM closed triangles
  • BAE CM closed circles
  • BAE cell ⁇ were cultured in alphaMEM medium (Bio-Whittaker, Walkersville, MD) containing 5% fetal calf ⁇ erum.
  • BSM and COS cell ⁇ were cultured in DMEM supplemented with 10% calf serum (Bio-Whittaker) .
  • Conditioned medium was prepared by a 24 hour incubation of the indicated cells with DMEM containing 0.1% pyrogen-poor BSA (weight/volume) (Pierce, Rockford, ID . All media were supplemented with L-glutamine (2 mM) , penicillin G (100 U/ml) and streptomycin sulfate (100 ⁇ g/ml) (Irvine Scientific, Santa Ana, CA) .
  • the MLEC a ⁇ ay was performed es ⁇ entially a ⁇ de ⁇ cribed by Luca ⁇ et al. , In Peptide Growth Factor ⁇ , Barne ⁇ et al . , Ed ⁇ , Academic Pre ⁇ Inc. 198:303-316 (1991) .
  • TGF- ⁇ i ⁇ oform ⁇ e ⁇ pecially TGF- ⁇ 3, decrea ⁇ ed ⁇ H-thymidine incorporation a ⁇ expected.
  • IL-lalpha and PDGF-BB had little effect, but bFGF and EGF had ⁇ trong dose-dependent stimulatory effects on ⁇ H-thymidine incorporation. Such effects can make the MLEC as ⁇ ay ⁇ inaccurate and difficult to analyze.
  • TGF- ⁇ concentrations of 23.4+3.4 pM (12 hour CM) and 122.1+16 pM (24 hours CM) were determined via comparison with a rTGF- ⁇ ⁇ tandard reference curve generated from plotting the detected amounts of luciferase activity that resulted from a range of predetermined amounts of TGF- ⁇ as described in Example 3A.
  • CM The heat-activated CM were al ⁇ o a ⁇ ayed u ⁇ ing the highly ⁇ pecific radioreceptor a ⁇ ay as described by Kojima et al., J. Cell. Phvsiol.. 155:323-332 (1993), the disclo ⁇ ure of which i ⁇ hereby incorporated by reference.
  • murine AKR-2B fibrobla ⁇ ts at 1 X 10 5 cells/well were plated in a 24-well plate in McCoy' ⁇ 5A medium (Gibco BRL) ⁇ upplemented with 5% fetal calf ⁇ erum.
  • the cell ⁇ were wa ⁇ hed 3 times with binding buffer (McCoy's 5A, 0.1% BSA, 25 mM HEPES at pH 7.4) and were pre-incubated in 250 ul of binding buffer for 1 hour at room temperature. The medium was removed, and the cells were incubated for 2 hour ⁇ at room temperature in a mixture of 125 ul of binding buffer containing 50 pM 125 I-rTGF- ⁇ l and an equal volume of heat-activated (80°C for 10 minutes) BAE CM or ⁇ erial dilutions of cold rTGF- ⁇ l.
  • binding buffer McCoy's 5A, 0.1% BSA, 25 mM HEPES at pH 7.4
  • the medium was removed, and the cells were incubated for 2 hour ⁇ at room temperature in a mixture of 125 ul of binding buffer containing 50 pM 125 I-rTGF- ⁇ l and an equal volume of heat-activated (80°C for 10 minutes) BAE CM or ⁇ erial
  • the cells were washed 3 time ⁇ with binding buffer, and the bound radioactivity wa ⁇ ⁇ olubilized in cell ly ⁇ is buffer (Analytical Luminescence) and was measured in a Packard Multi-PRIASl gamma counter (Meriden, CT) .
  • TGF- ⁇ a ⁇ say of this invention In addition to the TGF- ⁇ a ⁇ say of this invention and the MLEC and radioreceptor a ⁇ ay ⁇ de ⁇ cribed herein, other a ⁇ ay ⁇ have been used to detect mature TGF- ⁇ including anchorage- independent growth assays, differentiation-based as ⁇ ays, cell migration and plasminogen activity as ⁇ ay ⁇ , radioimmunoa ⁇ ay ⁇ and enzyme-linked immuno ⁇ orbent a ⁇ ay ⁇ .
  • TGF- ⁇ as ⁇ ay of thi ⁇ invention overcome ⁇ the ⁇ e deficiencie ⁇ by being highly ⁇ en ⁇ itive and ⁇ pecific a ⁇ well a ⁇ nonradioactive.
  • the ⁇ pecificity and sensitivity of the assay are the result of u ⁇ ing a truncated PAI-1 promoter beginning at -800 and extending through 76 of the PAI-1 5' promoter that retain ⁇ two region ⁇ re ⁇ pon ⁇ ible for maximal re ⁇ ponse to TGF- ⁇ a ⁇ de ⁇ cribed by Keeton et al . , J. Biol. Chem.. 266:23048-23052 (1991) .
  • U ⁇ e of the complete PAI-1 promoter and up ⁇ tream element ⁇ re ⁇ ult in decrea ⁇ ed ⁇ pecificity a ⁇ re ⁇ pon ⁇ ive element ⁇ for other molecule ⁇ pre ⁇ ent in complex ⁇ olution ⁇ may be activated or inhibited deleteriously effecting the ability to quantify TGF- ⁇ .
  • the truncated PAI-1 promoter used above has been further fragmented to smaller more specific TGF- ⁇ response elements as de ⁇ cribed in Example 4 to enhance specificity and increase the sen ⁇ itivity of the TGF- ⁇ a ⁇ ay method.
  • the TGF- ⁇ assay When the TGF- ⁇ assay is compared to the sen ⁇ itive and widely u ⁇ ed MLEC a ⁇ ay for quantifying TGF- ⁇ concentration ⁇ , the TGF- ⁇ a ⁇ say was more rapid, had comparable ⁇ en ⁇ itivity but with a greater detection range. Specificity of the a ⁇ ay wa ⁇ al ⁇ o higher a ⁇ evidenced by the TGF- ⁇ ' ⁇ a ⁇ ay insensitivity to growth factor ⁇ ⁇ uch a ⁇ EGF and bFGF that have been ⁇ hown to greatly effect other a ⁇ ay ⁇ .
  • TGF- ⁇ a ⁇ ay i ⁇ not i ⁇ oform specific
  • use of the appropriate standard reference curves and addition of neutralizing antibodies to the various TGF- ⁇ specie ⁇ allow ⁇ for quantification of unique i ⁇ oforms.
  • TGF- ⁇ as ⁇ ay of thi ⁇ invention i ⁇ highly specific the use of highly specific neutralizing antibodies to TGF- ⁇ was used to verify that no other molecules were pre ⁇ ent in te ⁇ t liquid ⁇ ample ⁇ that may have affected the quantitation of TGF- ⁇ in the a ⁇ say.
  • TGF- ⁇ The regulation of PAI-1 by TGF- ⁇ appears to affect a number of biological systems and the mechanism of transcriptional regulation by TGF- ⁇ has been studied by a number of groups.
  • the autoinduction of the TGF- ⁇ l promoter suggest ⁇ a feedback loop de ⁇ igned to amplify the re ⁇ pon ⁇ e to TGF- ⁇ under certain condition ⁇ . This respon ⁇ e wa ⁇ ⁇ hown to involve specific AP-1 site ⁇ .
  • AP-1 i ⁇ a heterodimeric complex of Fo ⁇ and Jun protein ⁇ ubunits that binds to specific DNA enhancer sites which have the consen ⁇ u ⁇ ⁇ equence TGASTCA
  • SEQ ID NO 26 S can be either G or C.
  • AP-1 i ⁇ believed to mediate the transcriptional effects of the tumor promoting phorbol ester ⁇ .
  • the TGF- ⁇ response sequence in the promoter for type 1 collagen has been localized to a sequence with homology to a nuclear factor 1 (NF-1) binding site.
  • NF-1 nuclear factor 1
  • a number of different consen ⁇ u ⁇ ⁇ equence ⁇ for NF-1 have been described and these include the sequences TGGN 7 GCCAA (SEQ ID NO 27), where N can be either A, C, G or T, and TGGCA (SEQ ID NO 28) .
  • the effect of TGF- ⁇ on the PAI-1 promoter has been ⁇ tudied resulting in the demonstration that the re ⁇ pon ⁇ ive region ⁇ contain sequences with homology to the AP-1 consen ⁇ us sequence.
  • AP-1 like site ⁇ are located within each of three region ⁇ of the 5' flanking region of the-PAI-1 promoter from -87 to -49, from -674 to -636 and from -740 to -703.
  • Oligonucleotide ⁇ having portion ⁇ or all of the ⁇ e region ⁇ were ⁇ ynthe ⁇ ized and cloned into a pUC-lucifera ⁇ e expre ⁇ ing pla ⁇ mid containing the minimal promoter a ⁇ de ⁇ cribed in Example 1C.
  • the resultant plasmid ⁇ were tran ⁇ iently tran ⁇ fected into recipient Hep3B cell ⁇ a ⁇ de ⁇ cribed in Example 2C and evaluated for their re ⁇ pon ⁇ e to TGF- ⁇ a ⁇ mea ⁇ ured by luciferase expres ⁇ ion as described in Example 3A.
  • the plasmid de ⁇ ignated p56Luc contained an oligonucleotide ⁇ equence that corre ⁇ ponded to -56 to -41 of the PAI-1 promoter gene (al ⁇ o referred to a ⁇ region A) and conferred a 10-fold induction of measurable.
  • TGF- ⁇ as compared to a 3-fold induction obtained with a plasmid expression vector only containing the minimal promoter sequence.
  • This nucleotide sequence conferred a 70-fold induction on the minimal promoter.
  • the pla ⁇ mid de ⁇ ignated p743Luc contained an oligonucleotide ⁇ equence 35 bp in length that corresponded to -743 to -708 of the PAI-1 promoter (al ⁇ o referred to a ⁇ region C) .
  • Thi ⁇ nucleotide ⁇ equence conferred a 35-fold induction in the promoter.
  • the plasmid designated p732Luc exhibited 62-fold induction while the plasmid, p732HBV, having the hepatitis B virus (HBV) minimal promoter sequence instead of the PAI-1 ⁇ equence exhibited 47-fold induction.
  • HBV hepatitis B virus
  • the stably transformed p!500Luc similarly resulted in approximately 150-fold induction.
  • the ⁇ e re ⁇ ult ⁇ a ⁇ well a ⁇ the other ⁇ pre ⁇ ented in the Example ⁇ repre ⁇ ent the average of at least 4 independent experiments, each performed in duplicate.
  • Regions A and C contained only a single AP-1 like sequence whereas region B contained 2 AP-1 like binding ⁇ equence ⁇ . Thu ⁇ , oligonucleotide ⁇ containing AP-1 like ⁇ equence ⁇ from each region were able to confer TGF- ⁇ re ⁇ pon ⁇ ivene ⁇ to a non- re ⁇ pon ⁇ ive minimal promoter.
  • Nucleotide Sequence in the PAI-1 Promoter from Nucleotide -743 to -708 (Region C) To find the minimal TGF- ⁇ re ⁇ pon ⁇ ive ⁇ equence in the PAI-1 promoter region from nucleotide po ⁇ ition -743 to -706, the sequence of which is li ⁇ ted in SEQ ID NO 16, two oligonucleotide ⁇ were made, the fir ⁇ t from the 3' ⁇ ide of region C which contained the AP-1 like ⁇ equence (C2 : -723 to -708 corresponding to the sequence in SEQ ID NO 16 from 21 to 36) and the second from the remaining 5' sequence (C3: -743 to -727 corre ⁇ ponding to the ⁇ equence in SEQ ID NO 16 from 1 to 17) .
  • the AP-1 site was therefore mutated to produce a consen ⁇ us AP-1 sequence (TGACACA to TGAGTCA, SEQ ID NOs 29 and 30, respectively) and the response of mutant to both c-fos/c-jun and TGF- ⁇ was compared.
  • Thi ⁇ mutation increa ⁇ ed the AP-1 re ⁇ pon ⁇ e from 19- fold to 105-fold but did not improve the TGF- ⁇ re ⁇ pon ⁇ e.
  • a con ⁇ i ⁇ tent decrease was seen in the TGF- ⁇ respon ⁇ e following this mutation (63-fold induction with TGF- ⁇ for the wild type AP-1 like site to 30-fold for the consen ⁇ u ⁇ AP-1 ⁇ ite) .
  • the AP-1 like ⁇ ite was then mutated by changing the critical TGA base ⁇ , a change ⁇ hown by other ⁇ to decrea ⁇ e the activity of the AP-1 binding ⁇ ite. Although this mutation had the expected effect of abolishing the AP-1 response, it did not completely abolish the respon ⁇ e of thi ⁇ con ⁇ truct to TGF- ⁇ (10- fold induction with c-fo ⁇ /c-jun [i.e., vector background] but a 13-fold induction with TGF- ⁇ [i.e., 5-fold above vector background] ) .
  • the 25 bp PAI-1 fragment wa ⁇ able to dramatically increase the TGF- ⁇ respon ⁇ e of the viral promoter from 4-fold to 47-fold but did not alter the AP-1 response (25-fold compared to 26-fold) .
  • mutation of base ⁇ between -732 and -728 of the PAI-1 promoter oligonucleotide dramatically reduced the TGF- ⁇ induction of this fragment but did not lower the response to AP-1.
  • a 15 bp oligonucleotide containing bases between -732 and -718, corresponding to the nucleotide se ⁇ uence from position 1 to 15 in SEQ ID NO 17) (which excludes the AP-1 like site) was cloned into a pUC- lucifera ⁇ e expression vector having the minimal PAI-1 promoter.
  • Thi ⁇ 15 bp ⁇ equence wa ⁇ able to confer 20-fold induction with TGF- ⁇ with the minimal PAI-1 promoter and did not ⁇ how any AP-1 activity.
  • thi ⁇ region appeared to be more critical than the AP-1 ⁇ equence ⁇ ince mutation of thi ⁇ region almo ⁇ t completely aboli ⁇ hed the TGF- ⁇ re ⁇ pon ⁇ e even though the AP-1 region was intact.
  • this sequence alone was evaluated, it was able to act independently of the AP-1 site and promote ⁇ trong TGF- ⁇ induction of the normally unresponsive minimal promoter.
  • the full TGF- ⁇ respon ⁇ e was dependent on the functional activity of both the AP-1 like site and the 5' site.
  • the TGF- ⁇ respon ⁇ e of the PAI-1 promoter ha ⁇ been localized to specific AP-1 like site ⁇ .
  • the full TGF- ⁇ re ⁇ pon ⁇ e of this region of the PAI-1 promoter is dependent on the interaction of two binding site ⁇ .
  • thi ⁇ ⁇ ite i ⁇ not e ⁇ ential it is required for the full TGF- ⁇ induction from this region.
  • the second site located 5' to the AP-1 site, appears to be critical in the TGF- ⁇ respon ⁇ e.
  • Thi ⁇ ⁇ ite is 15 bp in size and contain ⁇ a motif that i ⁇ pre ⁇ ent in both active region ⁇ of the PAI-1 promoter as well as in the mo ⁇ t re ⁇ pon ⁇ ive region of the TGF- ⁇ promoter.
  • Thi ⁇ novel sequence does not appear to match any previously described tran ⁇ cription factor binding ⁇ ite ⁇ and may repre ⁇ ent a new and ⁇ pecific binding ⁇ ite which i ⁇ critical for a ⁇ trong TGF- ⁇ re ⁇ pon ⁇ e.
  • the plasmid ⁇ , p674Luc, p743Luc and p732Luc were depo ⁇ ited on or before December 16, 1993, with the American Type Culture Collection, 1301 Parklawn Drive, Rockville, MD, USA (ATCC) and a ⁇ igned the re ⁇ pective ATCC Acce ⁇ ion Number ⁇ ATCC 75627, ATCC 75626 and ATCC 75629.
  • the cell line, Hep3B, ⁇ tably transfected with plasmid pl500Luc for a transformed cell line designated LUCI was also deposited on or before December 16, 1993 with ATCC and as ⁇ igned the ATCC Acce ⁇ ion Number CRL 1150 ⁇ .
  • the deposit thus provides plasmids and a stably transfected cell line containing pla ⁇ mid pl500Luc.
  • the ⁇ e depo ⁇ it ⁇ were made under the provi ⁇ ion ⁇ of the Budape ⁇ t Treaty on the International Recognition of the Depo ⁇ it of Microorgani ⁇ ms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty) .
  • the plasmids and cell line will be made available by ATCC under the terms of the Budapest Treaty which as ⁇ ure ⁇ permanent and unrestricted availability of the progeny of the culture to the public upon is ⁇ uance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever come ⁇ first, and as ⁇ ure ⁇ availability of the progeny to one determined by the U.S. Co mi ⁇ ioner of
  • Patent ⁇ and Trademark ⁇ to be entitled thereto according to 35 U.S.C. ⁇ 122 and the Commi ⁇ ioner' ⁇ rule ⁇ pursuant thereto (including 37 CFR ⁇ 1.14 with particular reference to 886 OG 638) .
  • the assignee of the present application ha ⁇ agreed that if the pla ⁇ mid or cell line depo ⁇ its should die or be lost or de ⁇ troyed when cultivated under ⁇ uitable condition ⁇ , they will be promptly replaced on notification with a viable ⁇ pecimen of the ⁇ ame pla ⁇ mid or cell culture.
  • Availability of the depo ⁇ ited plasmids is not to be construed a ⁇ a licen ⁇ e to practice the invention in contravention of the right ⁇ granted under the authority of any government in accordance with it ⁇ patent laws.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO:l:
  • ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC 5640
  • ATCAGGAACC CAGCACTCCA CTGGATAAGC ATTATCCTTA TCCAAAACAG CCTTGTGGTC 6120 AGTGTTCATC TGCTGACTGT CAACTGTAGC ATTTTTTGGG GTTACAGTTT GAGCAGGATA 6180
  • CTGACGCGCC CTGACGGGCT TGTCTGCTCC CGGCATCCGC TTACAGACAA GCTGTGACCG 6540
  • ACATTTCGCA GCCTACCGTA GTGTTTGTTT CCAAAAAGGG GTTGCAAAAA ATTTTGAACG 8400
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC 5640
  • CTGACGCGCC CTGACGGGCT TGTCTGCTCC CGGCATCCGC TTACAGACAA GCTGTGACCG 6540
  • CTCTGTGTCC TCATAAACCC TAACCTCCTC TACTTGAGAG GACATTCCAA TCATAGGCTG 10020
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC 5640
  • CTGACGCGCC CTGACGGGCT TGTCTGCTCC CGGCATCCGC TTACAGACAA GCTGTGACCG 6540
  • CAAGTTCATC TATTTCCTCC CACATCTGGT ATAAAAGGAG GCAGTGGCCC ACAGAGGAGC 7140
  • AGAAGACCCC AAGGACTTTC CTTCAGAATT GCTAAGTTTT TTGAGTCATG CTGTGTTTAG 9300
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID N0:4:
  • TACATACCTC GCTCTGCTAA TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG 1500 TCTTACCGGG TTGGACTCAA GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC 1560

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Abstract

La présente invention concerne une méthode de dosage quantitative, non radioactive, spécifique et très sensible servant à quantifier le facteur de croissance transformant bêta (TGF-β) dans un échantillon liquide. L'invention concerne également des vecteurs d'expression sensibles au TGF-β qui expriment la molécule indicatrice, la luciférase, dans une réponse, reliée à la dose, à l'activation du TGF-β. L'invention concerne en outre des cellules eucaryotes transformées par les vecteurs d'expression décrits, et des systèmes de diagnostic se présentant sous la forme de kits permettant de déterminer la quantité de TGF-β dans un échantillon liquide à l'aide des procédés et des vecteurs d'expression décrits.
PCT/US1995/001153 1994-01-25 1995-01-25 Nouvelle methode sensible de quantification du facteur de croissance transformant beta actif et compositions utilisees dans cette methode de dosage WO1995019987A1 (fr)

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US18822794A 1994-01-25 1994-01-25
US08/188,227 1994-01-25

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997037678A1 (fr) * 1996-04-05 1997-10-16 Boehringer Ingelheim International Gmbh Medicament pour le traitement de tumeurs
WO2000000641A1 (fr) * 1998-06-26 2000-01-06 Renovo Limited Procede de mesure quantitative du tgf-beta
EP0998936A1 (fr) * 1998-10-01 2000-05-10 Giovanni Gambaro Composés pour prévenir la surexpression du facteur de croissance transformant beta et méthodes pour les déterminer
US6333408B1 (en) * 1999-03-08 2001-12-25 Kureha Chemical Industry Co., Ltd. Oligonucleotides inhibitors of PAI-1 MRNA
US6855505B2 (en) 1998-06-26 2005-02-15 Renovo Limited Method for quantifying TGF-β
US7094550B2 (en) 1993-05-13 2006-08-22 Neorx Corporation Method to determine TGF-beta
US7511070B2 (en) 1997-04-11 2009-03-31 Poniard Pharmaceuticals, Inc. Compounds and therapies for the prevention of vascular and non-vascular pathologies
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
US7625410B2 (en) 2001-05-02 2009-12-01 Boston Scientific Scimed, Inc. Stent device and method

Citations (2)

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Publication number Priority date Publication date Assignee Title
US5216126A (en) * 1991-06-19 1993-06-01 Genentech, Inc. Receptor polypeptides and their production and uses
US5268295A (en) * 1991-05-31 1993-12-07 W. Alton Jones Cell Science Center, Inc. Mammalian adipocyte protein p154, nucleic acids coding therefor and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5268295A (en) * 1991-05-31 1993-12-07 W. Alton Jones Cell Science Center, Inc. Mammalian adipocyte protein p154, nucleic acids coding therefor and uses thereof
US5216126A (en) * 1991-06-19 1993-06-01 Genentech, Inc. Receptor polypeptides and their production and uses

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CELL, Volume 71, issued 11 December 1992, J.L. WRANA et al., "TGFbeta Signals Through a Heteromeric Protein Kinase Receptor Complex", pages 1003-1014. *
JOURNAL OF CELLULAR PHYSIOLOGY, Volume 152, issued 1992, R. FLAUMENHAFT et al., "Cell Density Dependent Effects of TGF-beta Demonstrated by a Plasminogen Activator-Based Assay for TGF-beta", pages 48-55. *
MOLECULAR AND CELLULAR BIOLOGY, Volume 12, No. 4, issued April 1992, A. RICCIO et al., "Transforming Growth Factor beta1-Responsive Element: Closely Associated Binding Sites for USF and CCAAT-Binding Transcription Factor-Nuclear Factor I in the Type 1 Plasminogen Activator Inhibitor Gene", pages 1846-1855. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 193, issued 1951, O.H. LOWRY et al., "Protein Measurement With the Folin Phenol Reagent", pages 265-275. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7094550B2 (en) 1993-05-13 2006-08-22 Neorx Corporation Method to determine TGF-beta
WO1997037678A1 (fr) * 1996-04-05 1997-10-16 Boehringer Ingelheim International Gmbh Medicament pour le traitement de tumeurs
US6383733B1 (en) 1996-04-05 2002-05-07 Boehringer Ingelheim International Gmbh Methods of screening for pharmacologically active compounds for the treatment of tumour diseases
US7511070B2 (en) 1997-04-11 2009-03-31 Poniard Pharmaceuticals, Inc. Compounds and therapies for the prevention of vascular and non-vascular pathologies
WO2000000641A1 (fr) * 1998-06-26 2000-01-06 Renovo Limited Procede de mesure quantitative du tgf-beta
US6855505B2 (en) 1998-06-26 2005-02-15 Renovo Limited Method for quantifying TGF-β
EP0998936A1 (fr) * 1998-10-01 2000-05-10 Giovanni Gambaro Composés pour prévenir la surexpression du facteur de croissance transformant beta et méthodes pour les déterminer
US6333408B1 (en) * 1999-03-08 2001-12-25 Kureha Chemical Industry Co., Ltd. Oligonucleotides inhibitors of PAI-1 MRNA
US7625410B2 (en) 2001-05-02 2009-12-01 Boston Scientific Scimed, Inc. Stent device and method
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies

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