JP2004509612A - Oncogene determination and therapeutic screening using characteristic genes - Google Patents

Abstract

A process for the analysis of a potential anti-tumor factor based on the regulation of the expression of a specific gene or group of genes of a cell suspected of having cancer, the cancer or the potential for expression of said gene or group of genes, A method for diagnosing a severe cancer condition is disclosed. A method for determining a functionally related gene or genes and a method for treating cancer based on targeting the expression product of said gene or genes and determining genes involved in the cancer process are also provided. Also disclosed.

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
This application is related to US Provisional Patent Application No. 60/209473 filed on Jun. 5, 2000, US Provisional Patent Application No. 60 / 209,531 filed on Jun. 5, 2000, and filed on Sep. 18, 2000. U.S. Provisional Patent Application No. 60/233133 filed, U.S. Provisional Patent Application No. 60/233617 filed September 18, 2000, and U.S. Provisional Patent Application No. 60/233 filed September 20, 2000. U.S. Provisional Patent Application No. 60 / 234,034, filed Sep. 20, 2000, U.S. Provisional Patent Application No. 60 / 234,052, filed Sep. 20, 2000, filed Sep. 22, 2000. U.S. Provisional Patent Application No. 60/234509, filed Sep. 22, 2000, U.S. Provisional Patent Application No. No. 0/234567, U.S. Provisional Patent Application No. 60/234923, filed Sep. 25, 2000, U.S. Provisional Patent Application No. 60/234924, filed Sep. 25, 2000, September 25, 2000. U.S. Provisional Patent Application No. 60/235077, filed September 25, 2000, and U.S. Provisional Patent Application No. 60 / 235,082 filed September 25, 2000; U.S. Provisional Patent Application No. 60 / 235,280, filed Sep. 25, 2000; U.S. Provisional Patent Application No. 60 / 235,637, filed Sep. 26, 2000; U.S. Provisional Patent Application No. 60 / 235,638, filed September 27, 2000, filed Sep. 27, 2000 Provisional Patent Application No. 60/235711; U.S. Provisional Patent Application No. 60/235720 filed on Sep. 27, 2000; U.S. Provisional Patent Application No. 60/235840, filed on Sep. 27, 2000; U.S. Provisional Patent Application No. 60 / 235,863 filed on September 27, 2000; U.S. Provisional Patent Application No. 60/236028, filed on September 28, 2000; Provisional Patent Application No. 60/236032, U.S. Provisional Patent Application No. 60/236603 filed on Sep. 28, 2000, and U.S. Provisional Patent Application No. 60/236034, filed on Sep. 28, 2000. US Provisional Patent Application No. 60/236109 filed Sep. 28, 2000, Sep. 28, 2000 U.S. Provisional Patent Application No. 60/236111, filed Sep. 29, 2000, U.S. Provisional Patent Application No. 60/236842, and U.S. Provisional Patent Application No. 60 filed September 29, 2000. No./2386891, U.S. Provisional Patent Application No. 60/237172 filed Oct. 2, 2000, and U.S. Provisional Patent Application No. 60/237173 filed Oct. 2, 2000, Oct. 2, 2000. U.S. Provisional Patent Application No. 60/237278 filed on Oct. 2, 2000, U.S. Provisional Patent Application No. 60/237294 filed on Oct. 2, 2000, and U.S. Provisional Patent Application No. 60 filed on Oct. 2, 2000. No./237295, U.S. Provisional Patent Application No. 60/237316, filed Oct. 2, 2000, U.S. Provisional Patent Application No. 60/237425, filed October 3, 2000, U.S. Provisional Patent Application No. 60/237598, filed October 3, 2000, filed October 3, 2000 U.S. Provisional Patent Application No. 60/237604, U.S. Provisional Patent Application No. 60/237606, filed October 3, 2000, U.S. Provisional Patent Application No. 60/237608, filed October 3, 2000, Claims priority of US Provisional Patent Application No. 60/244867 filed on November 1, 2000 and US Provisional Patent Application No. 60/245084 filed on November 1, 2000.
[0002]
The present invention relates to a method for analyzing potential anti-tumor factors based on the regulation of the expression of a specific group of genes, and a method for diagnosing a cancer state or a potential cancer state as a result of the tendency of expression of said group of genes. About the method.
[0003]
BACKGROUND OF THE INVENTION
Screening assays for new drugs are based on the response of an in vitro model cell-based system for treatment with specific compounds. A variety of measures of cellular response are utilized, including cytokine release, denaturation in cell surface labeling, activation of specific enzymes and changes in ion flux density and / or pH. Some of these tests rely on specific genes, such as oncogenes (or gene mutations).
[0004]
BRIEF SUMMARY OF THE INVENTION
According to the present invention, there is provided a unique gene sequence group in which expression, non-expression, change in expression, and increase or decrease thereof indicate a cancer state or a non-cancer state of a specific cell. More particularly, said gene from a specific tissue (especially any disclosed herein) whose expression is altered in cancer cells as compared to non-cancer cells may be the nucleotide sequence of SEQ ID NOS: 1-8447 or the aforementioned A gene containing one of the sequences substantially identical to the sequence. Said alteration of expression may be an increase or decrease in the expression or activity of a gene or gene sequence disclosed herein.
[0005]
It is another object of the present invention to provide a method for analyzing the potential of a selected chemical that modulates the expression of said unique or characteristic gene group upward or downward as said basis. To provide a method using a specific gene group.
[0006]
It is a further object of the present invention to provide a method for determining the cancerous or non-cancerous state (or potential cancerous state) of cells grown in culture or selected as cells maintained in situ. Another object of the present invention is to provide a method for detecting the expression or non-expression or expression level of a characteristic gene group and its site.
[0007]
A still further object of the present invention is to utilize a selected chemical entity determined based on its ability to modulate (ie, increase or decrease) the selected unique or characteristic genes disclosed herein. , To provide a method for treating a cancer condition. Here, the gene comprises or comprises the sequence of SEQ ID NOs: 1-8447 or one of the sequences substantially identical to the sequence.
[0008]
In another aspect, the present invention relates to a process for determining a gene that induces, promotes, or suppresses cancer, the process comprising contacting a cancer cell with a cancer regulatory factor and a group comprising the gene sequence of SEQ ID NOS: 1-8447. Measuring the change in the expression of a gene selected from the above, and confirming the gene as a cancer-inducing or promoting gene. The gene can be, for example, a cancer gene, a cancer promoting gene or a cancer suppressor gene. The factor may increase or decrease gene expression.
[0009]
The present invention also relates to a process for treating cancer comprising contacting a cancer cell with an agent having activity against an expression product encoded by a gene sequence selected from the group consisting of SEQ ID NOs: 1-84747, The process can be performed ex vivo or in vivo.
[0010]
In another aspect, the present invention further relates to a process for determining a cancer-inducing, promoting or repressing gene in a cancer cell, said process comprising a gene sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-8447. Measuring the change in expression of Said change in expression is in particular a change in the number of copies, said difference being an increase or decrease thereof. Here, the change is observed or otherwise measured as a change in messenger RNA production. The changes can be readily used to diagnose a cancer condition, in vivo or ex vivo.
[0011]
The present invention still further relates to a process for treating cancer, said process comprising inserting a gene construct comprising an anti-oncogene operably linked to a promoter or enhancer element into a cancer cell, whereby the anti-cancer agent is treated. Gene expression comprises causing suppression of said cancer. Here, the promoter or enhancer element is a promoter or enhancer element that regulates a gene sequence selected from the group consisting of the sequences of SEQ ID NOS: 1 to 8447, wherein the gene may be a cancer suppressor gene. Alternatively, the gene here encodes a polypeptide having anti-cancer activity, such as one having apoptotic activity.
[0012]
In an additional aspect, the invention relates to a process for determining a functionally related gene, said process comprising one or more gene sequences selected from the group consisting of the sequences of SEQ ID NOs: 1-8447. Is contacted with an agent that regulates the expression of two or more genes in said group, thereby determining a portion of said group of genes. The functionally related gene includes a gene that regulates the same metabolic pathway or a gene that encodes a functionally related polypeptide, wherein the expression is regulated by the same transcription activator or enhancer sequence. .
[0013]
The present invention also relates to a method for producing a product, comprising identifying an agent according to the process of claim 1, wherein the product is obtained in association with the agent as a result of the process. Where the data is sufficient to present the chemical structure and / or properties of the factor.
[0014]
DESCRIPTION OF THE INVENTION
The present invention provides a method for analyzing a potential anti-tumor factor based on the regulation of a specific gene group and a method for diagnosing cancer or a potential cancer state as a result of a tendency to express the gene group, The present invention also relates to a method for determining a cancer-inducing or regulatory gene based on normal expression or regulation of the gene or genes.
[0015]
In accordance with the present invention, model cell systems using cell lines, primary cells or tissue samples can be maintained in growth medium and treated with compounds that can be at a single concentration or a range of concentrations. At a specific time after treatment, cellular RNAs are isolated from the treated cells, primary cells or tumors, and the RNAs exhibit expression of a selected gene. Cellular RNA is then split and subjected to analysis to detect the presence and / or amount of specific RNA transcripts. The transcript can then be amplified for detection purposes using, for example, reverse transcription polymerase chain reaction (RT-PCR), or other standard methodology. The presence or absence or level of a specific RNA transcript is determined from measurements and metrics obtained from the type and degree of response of the sample to the treated compound, as compared to a control sample.
[0016]
Also in accordance with the present invention, there is provided herein a group of unique or characteristic genes whose expression as a result of the method of the present invention is associated or used to characterize the cancerous or non-cancer state of the cell or tissue under test. And gene sequences are disclosed. Thus, the methods of the present invention identify novel anti-tumor agents based on their ability to convert the expression of a unique or indicative or characteristic subgroup of genes in a specific model system. Thus, the methods of the present invention may be performed using various cell lines or in vitro under appropriate culture conditions for various periods of time, or using primary samples from tumors maintained in situ in appropriate animal models. Can be used.
[0017]
More particularly, certain genes have been identified that are expressed at different levels in cancer cells as compared to those in non-cancer cells. In one example, the identified gene is expressed at higher levels in cancer cells than in normal cells. In another example, the identified gene is expressed at lower levels in cancer cells as compared to normal cells.
[0018]
In accordance with the foregoing, the present invention relates to a process for screening for an antitumor agent, said process comprising the following steps:
(A) exposure of the cells to a chemical agent to be tested for antineoplastic activity, and
(B) measuring the change in expression of at least one gene containing one of the sequences of SEQ ID NOs: 1-847 or at least 95% identical thereto;
Here, the change in expression serves as an indicator of antitumor activity.
[0019]
In certain embodiments, the change in expression may be an increase or decrease in expression or activity.
[0020]
More particularly, the present invention relates to a process for screening for an antitumor agent, said process comprising the following steps:
(A) exposure of known cancer cells to a chemical agent to be tested for antineoplastic activity;
(B) regulation of the activity of one or more genes present in the cell by the chemical agent, wherein the genes have the sequence of SEQ ID NOs: 1-8447, a sequence substantially identical to the sequences, or Comprise or consist of one of the sequences selected from the group consisting of any of the foregoing supplements;
(C) measuring the expression of one or more genes in step (b);
(D) comparing the expression of the gene with or without exposure to the chemical agent;
Here, the difference in expression is an indicator of the ability of the antitumor activity.
[0021]
In a specific embodiment of the invention, the chemical agent tested modulates the expression of two or more of the genes, particularly where the chemical agent modulates at least two of the genes, more particularly At least 3 or at least 5 of said genes in said characteristic group, or even 10 or more of said genes are regulated. In a preferred embodiment, all of the genes are regulated.
[0022]
In one embodiment of the invention, the gene regulation is a down regulation, so that as a result of the exposure to the chemical agent being tested, one or more genes of the cancer cell are exposed to the factor. May be expressed at lower levels (or not at all) as compared to expression when not exposed to the factor.
[0023]
In a preferred embodiment, the selected genes are expressed in a reference cell, but are not expressed in the tested cell as a result of exposure of the cell to the chemical agent. Thus, here, due to said chemical factor, the gene of the tested cell is expressed at a lower level than the same gene of the reference cell, which is indicative of a down-regulation and that the tested chemical factor is It has oncogenic activity.
[0024]
In another embodiment, exposure of said test cell to a chemical agent, especially one suspected of having antitumor activity, may result in an upregulation of said gene in said cell being tested. The upregulation is that the gene that was not expressed prior to exposure is expressed or otherwise the gene is more abundant when exposed to the factor compared to when the factor was not exposed. It is determined that it means that it is expressed in the amount of Said upregulation can be taken as an indication that the antitumor activity of the tested chemical agent whose gene has been modulated has caused low neoplastic activity in some of the cells. Here, an increase in the expression of the gene results in a decrease in the proliferation and / or an increase in the differentiation of said cells far from the cancer state.
[0025]
The genes utilized in the analysis process each include, as a part thereof, at least one of the sequences of SEQ ID NOs: 1-8447 or a sequence selected from the group consisting of sequences substantially identical thereto. The sequences also include sequences that are complementary to any of the sequences disclosed herein.
[0026]
At least about 90% identical to the sequence selected from SEQ ID NO: 1-8447, preferably at least about 95% identical to the sequence, more preferably at least about 98% identical to the sequence, and most preferably to the sequence. Genes containing sequences that are 100% identical are specifically contemplated by all processes of the present invention. In addition, sequences that encode proteins similar to any of these sequences may also be independent of the percent identity of the sequences, regardless of how differently they are described or limited. It is specifically contemplated by any method of the invention depending on any or all of the above sequences. Thus, any of the above sequences can be utilized for use in performing any of the methods disclosed in accordance with the present invention. The sequence may also include any open reading frame as defined herein, occurring within any of the sequences of SEQ ID NOs: 1-8447.
[0027]
Further in accordance with the present invention, the term "percent identity" or "proportion identical" when referring to a sequence is disclosed or claimed as the compared sequence ("comparison sequence"). It means that after alignment of the sequence ("reference sequence"), the sequence is compared to the claimed or disclosed sequence. The percent identity is then determined according to the following formula:
Identity ratio = 100 [1- (C / R)]
Where C is the number of differences between the reference sequence and the comparison sequence over the entire length of the alignment between the reference sequence and the comparison sequence, wherein (i) the comparison sequence has no complementary aligned bases or amino acids Bases or amino acids in the reference sequence and (ii) gaps in the reference sequence and (iii) aligned bases or amino acids in the reference sequence that differ from the aligned bases or amino acids in the comparison sequence constitute differences. R is the number of bases or amino acids in the reference sequence over the entire length of the alignment with the comparison sequence, and any gap formed in the reference sequence is also considered as a base or amino acid.
[0028]
If there is an alignment between the reference sequence and the reference sequence, the percent identity calculated above for that alignment is equal to or greater than the specified minimum percent identity. As a result, even if the comparison sequence has a specific minimum percentage identity to the reference sequence and an alignment exists, the above calculated percentage identity will be less than or equal to the specific percentage identity.
[0029]
As used herein, the terms "site,""segment," and "fragment," when used in reference to a polypeptide, refer to a contiguous sequence of residues, such as amino acid residues, wherein the sequence Forms part of a larger array. For example, if the polypeptide is treated with any common endopeptidase, such as trypsin or chymotrypsin, the resulting oligopeptide will correspond to the first polypeptide site, segment or fragment. When used in reference to a polynucleotide, the term refers to the product produced by treatment of the polynucleotide with any common endonuclease or extension of a polynucleotide that can be synthesized synthetically.
[0030]
As used herein, and unless otherwise specified, all terms are defined as given below.
[0031]
According to the present invention, the term "DNA segment" or "DNA sequence" refers to a DNA polymer in the form of discrete fragments or as a component of a larger DNA construct, and is in substantially pure form, ie, contaminating endogenous. Obtained from the DNA isolated at least once in the absence of the substance and in an amount or concentration that allows the determination, manipulation and regeneration of the segment and component nucleotide sequences, for example by standard biochemical methods using cloning vectors. The segments are provided in the form of open reading frames contiguous with internal untranslated sequences or introns usually present in eukaryotic genes. The sequence of the untranslated DNA is located downstream of the open reading frame, which does not prevent manipulation or expression of the coding region.
[0032]
The term "coding region" refers to a site in a gene that naturally or normally encodes the expression product of a gene in the natural genomic environment, ie, the region that encodes the natural expression product of the gene in vivo. The coding region can be from a normal, mutated or transgene. Still further, it may be derived from a DNA sequence or gene completely synthesized in a laboratory using methods well known to those having ordinary knowledge in the field of DNA synthesis.
[0033]
According to the present invention, the term "nucleotide sequence" refers to a heteropolymer or a deoxyribonucleotide. Generally, a DNA segment encoding a protein provided by the present invention is used to provide a synthetic gene that can be expressed in a recombinant transcription unit consisting of a regulatory element obtained from a microorganism or viral operon. , Assembled from cDNA fragments and short oligonucleotide linkers, or from contiguous oligonucleotides.
[0034]
The term "expression product" refers to a polypeptide or protein that is the natural translation of a gene and nucleic acid sequence that encodes the substantial amount resulting from the degeneracy of the genetic code and thereby the encoding of the same amino acid.
[0035]
The term "fragment", when referring to a coding sequence, refers to a DNA whose expression product consists of less than the entire coding region that essentially retains the same biological function or activity as the expression product of the complete coding sequence. Means the site.
[0036]
The term "primer" refers to a short nucleic acid sequence that pairs with a single strand of DNA and provides a free 3'-OH terminus at which DNA polymerase initiates synthesis of a deoxyribonucleotide chain.
[0037]
The term "promoter" refers to a region of DNA involved in binding RNA polymerase to initiate transcription. The term "enhancer" refers to a DNA which, when present and activated, has the effect of increasing the expression of a different DNA sequence to be expressed, thereby increasing the amount of expression product formed from said different DNA sequence. Indicates the area.
[0038]
The term "open reading frame (ORF)" refers to a contiguous triplet encoding an amino acid that does not include a stop codon, and is a (potentially) translatable protein sequence.
[0039]
As used herein, references to DNA sequences include both single-stranded and double-stranded DNA. Thus, a specific sequence, unless otherwise specified, refers to a single-stranded DNA of the sequence, a double-stranded (double-stranded DNA) of the sequence with a complementary strand, and a complementary strand of the sequence.
[0040]
In performing the analysis, the relative anti-tumor activity can be ascertained to the extent that certain chemical factors regulate the expression of genes present in cancer cells. Thus, the expression of a gene associated with a cancer condition (ie, a gene that includes one of the sequences disclosed herein and that is present in a cancer cell) is determined by the second chemical agent tested by the assay of the present invention. A first chemical agent that modulates to a greater extent would then have a higher or more desirable or more favorable antitumor activity than the second chemical agent. Alternatively, the first and second chemical factors regulate the expression of two or more of said genes, wherein the second chemical factor regulates the expression of, for example, five of said genes, while the first chemical factor modulates the expression of said genes Regulates the expression of only three, in particular, here three of the five. As a result, the second chemical factor appears to be a more effective anti-tumor factor than the first chemical factor. The antitumor activity identified using the assays of the present invention may necessarily include a combination of the possibilities, which is not to be considered limiting in any way.
[0041]
In utilizing these gene sequences for analysis in accordance with the present invention, those genes whose activity is determined with or without the compound being evaluated for anti-tumor activity, are disclosed herein as SEQ ID NOs: 1-8447. One or more or a combination of the gene sequences. However, how the gene sequences are used in the analysis depends on the disclosed gene expression trends for characteristic groups for various organs and tissues. For example, sequences that are expressed in cancer cells but not in normal cells may be associated with potential anti-cancer factors by the ability of the factors to reduce expression of the sequences in tumor cells. Conversely, sequences that are expressed in normal cells but not in tumor cells may be associated with potential anti-tumor factors by their ability to increase expression in tumor cells. A similar relationship holds that the sequence is expressed in both cancer and normal cells, but at a higher level than the other. The same is true for the opposite case. Based on the gene sequences disclosed herein and the expression trends disclosed for the characteristic groups, methods for performing an analysis of potential anti-tumor factors using the characteristic genes are well-known in the art. Will be readily apparent to those having The same is true for sequences or characteristic genes being used to determine the effect of a cell on cancer condition or an agent for treating the cancer condition.
[0042]
Thus, in one aspect, the invention relates to a process for screening for an anti-tumor agent, wherein the process comprises the steps of (a) exposing the cell to a chemical agent to be tested for anti-tumor activity and (b) characterizing the cell. Measuring the change in expression of at least one gene of the generic gene group or a sequence at least 95% identical thereto, wherein the change in expression is indicative of antitumor activity. The change in expression is intended to mean that the change involves any activity of the gene, which may be an increase or decrease in expression. In addition, the change in activity may be at least one of the genes, five or ten of the genes or more of a distinct group of genes, or even half of the genes or even all of the particular genes. It may be a change in gene expression or other activity.
[0043]
The measured gene expression is usually analyzed using RNA expression as an indicator. Therefore, the higher the level of detected RNA (messenger RNA), the higher the expression level of the corresponding gene. Thus, the absolute or relative gene expression, such as several different genes being quantitatively quantified and compared, is a chemical factor, for example, where the chemical factor is the expression of two or more genes, such as 3, 4, 5, or more. Regulate, but is determined by the relative expression of RNAs encoded by the gene.
[0044]
RNA is dissolved and denatured with a phenol solution containing a chaotropic agent (eg, triazole), followed by isopropanol precipitation, ethanol washing and resuspension in aqueous solution; or lysis and denaturation, followed by solid support such as Qiagen resin. Isolation from a sample can be accomplished by a variety of methods, including isolation above and reconstitution in aqueous solution; or lysis and denaturation in non-phenolic aqueous solutions, followed by enzymatic conversion of RNA to DNA template replication.
[0045]
Generally, prior to application of the process of the present invention, steady state RNA expression levels for the genes and genes disclosed herein have been obtained. What is affected by the antitumor agent as identified herein is the steady state level of said expression. The steady state level of expression is readily determined by any method that is sensitive, specific and accurate. The method can be based on any of a variety of commercially available software packages such as Primer Express software, solid support-based hybridization array technology or microchips using appropriate internal controls for quantification, including filters, beads and the like. For example, a Perkin-Elmer 7700 with a combination of gene-specific primer probes designed to use a solid support-based hybridization array using chemiluminescence, fluorescence, or an electrochemical reaction-based detection system. Including but not limited to real-time quantitative polymerase chain reaction (PCR) using a sequence detection system.
[0046]
In one embodiment of the present invention, a group of genes useful for the evaluation or screening or otherwise analysis of one or more chemical agents for anti-tumor activity in the assays disclosed herein may be normal or non- Genes disclosed herein have already been shown to differ in the proportion of steady state RNA levels in cancer cells or tissues compared to tumor cells or tissues, or in tumor cells compared to normal cells. Show a difference in the expression rate between certain genes of the gene, or have increased gene expression from an undetectable level to a detectable level, or in some cases, especially where a sensitive detection method is used And vice versa, i.e. conversely having a reduced gene expression from a detectable level to an undetectable level using said method, especially a sensitive method
[0047]
Genes and gene sequences useful in the practice of the methods of the invention may be selectively expressed or not found to be expressed in cancer cells as compared to non-cancer cells, or may be cancer cells as compared to non-cancer cells. A gene whose expression is down-regulated or up-regulated in some cases. Thus, genes or genes that are expressed in cancer cells but are not absent or activated in non-cancer cells may be included, or genes or genes that are expressed in non-cancer cells but not expressed in cancer cells. May be included. Alternatively, genes useful in the practice of the present invention may be more or less expressed in cancer cells as compared to non-cancerous cells. Said gene usually consists of the sequence of SEQ ID NO: 1-8447.
[0048]
All of these sequences are provided here on CD-ROM only.
[0049]
In accordance with the foregoing, the present invention further relates to a process for determining the cancer status of a cell to be tested, said process comprising at least 95% identical to one of the nucleotide sequences selected from the sequence SEQ ID NO: 1-8447 or at least 95% identical thereto. Measuring the expression in the test cell of at least one gene comprising a nucleotide sequence, and then comparing the expression to the expression of the at least one gene in at least one cell known not to be cancerous, The difference in expression comprises indicating that the cell is a cancer.
[0050]
In certain embodiments, the present invention is directed to a process for determining the cancer status of a cell being tested, said process comprising one of a nucleotide sequence selected from the sequence of SEQ ID NOs: 1-8847, wherein Identifying the presence in the cell of a sequence having substantial identity to the sequence, or a unique fragment thereof, or any of the aforementioned supplements, and then determining the presence and / or absence of the gene. Comparing the propensity to a cell known or considered to be non-cancerous or normal with respect to at least the gene supplement.
[0051]
For a gene comprising at least one of the sequences of SEQ ID NOs: 1-8447, the up-regulation of expression in cancer cells (as compared to the gene or the non-cancer cells that may lack the gene expression, both) Indicator of cancer or potential cancer status.
[0052]
In a specific embodiment, the present invention provides a gene propensity to modulate the expression of two or more genes, preferably 3, 4 or 5 genes, and even a propensity to modulate the expression of 10 or more genes. Embodiments including: Thus, here the genetic tendency is a non-cancer cell from the same tissue type, such as a colon cancer cell or any other tissue cancer cell such as any of the organs or tissues disclosed herein in connection with the specifically listed SEQ ID NOs. Is the regulation of the expression of five genes in cancer cells as compared to non-cancer cells of the same tissue or organ, the tendency being that the gene (ie, regulation of expression of the five genes) is an indicator of cancer status Showing the possibility of providing a method for diagnosing cancer or a potential cancer condition. The absence of a specific gene group from a cancer cell is also an indicator of the association with a cancer condition, since the gene is present in normal cells, especially normal cells of the same type, and is therefore also cancer. It can be used as a method for diagnosing a cancer condition in other cells that are thought to be present.
[0053]
For example, with respect to the colon, especially colon adenocarcinoma, this can be SEQ ID NOs: 1-333 (expressed in normal colon cells but not in colon cancer cells), SEQ ID NOs: 334-522 (in colon adenocarcinoma). Is expressed at a high level but is not expressed in normal colon cells), SEQ ID NO: 523-837 (expressed in colon adenocarcinoma at a reduced level of 2.09 times or more, but expressed in normal colon cells) No) and SEQ ID NOs: 838-1067 (expressed at high (minimum 2.1 fold) levels in colon adenocarcinoma, but not high levels in normal colon cells). Thus, the above sequence classification for the colon implies four distinct groups of genes in the colon. For example, SEQ ID NOs: 334-522 refers to a characteristic group or characteristic gene group of the colon. The same is true for the organs and tissues described below with their respective characteristic groups or characteristic gene groups.
[0054]
As in the colon, other gene sequences are indicative of cancer or normal state of other organs and tissues. Accordingly, as disclosed herein, SEQ ID NOs: 1068-2459 are included for breasts, wherein SEQ ID NOs: 1068-1255 are not expressed at detectable levels in normal breasts, but in invasive ductal carcinomas of the breasts Indicates a gene that is expressed, wherein SEQ ID NO: 1256-159 indicates a gene that is not expressed at detectable levels in normal breast but is expressed in breast cancer, where SEQ ID NO: 1459-1664 is normal breast Shows genes that are not expressed at detectable levels but are expressed in invasive lobular carcinoma of the breast, where SEQ ID NO: 1665--2067 is absent or not expressed in invasive ductal carcinoma of the breast Indicates a gene that is expressed in normal breast, where SEQ ID NO: 2068-2459 is expressed in normal breast cells but is detectable in normal breast. The invasive lobular glands in cancer of the breast that does not show the gene that do not express or is absent.
[0055]
In addition, SEQ ID NOs: 2460-3027 are included for the stomach, wherein SEQ ID NOs: 2460-2773 represent genes or gene sequences that are not expressed at detectable levels in normal gastric cells but are expressed in gastric cancer; Here, SEQ ID NOs: 2774-3027 represent genes or gene sequences that are not expressed at detectable levels in gastric cancer cells but are expressed in normal gastric cells.
[0056]
In addition, SEQ ID NOs: 3028-5303 are included for lung, where SEQ ID NOs: 3028-3119 represent genes or gene sequences that are not expressed at appreciable levels in normal lung cells but are expressed in lung adenocarcinoma. Wherein SEQ ID NOs: 3120-3322 represent genes or gene sequences that are not expressed at appreciable levels in lung adenocarcinoma but are expressed in normal lung cells, where SEQ ID NOs: 3323-3570 are present in malignant lung samples. Indicates a gene or gene sequence that is not expressed at a detectable level but is expressed in non-cancerous lung tissues, and SEQ ID NOs: 3571-3777 are not expressed at detectable levels in non-malignant lung cells but are present in malignant lung samples. Shows a gene or gene sequence to be expressed, wherein SEQ ID NO: 3778-3836 is expressed in both normal and malignant lung adenocarcinoma In lung adenocarcinoma, it shows a gene or gene sequence that is up-regulated with a difference of at least about two-fold, wherein SEQ ID NO: 3837-3980 is expressed in appreciable levels in normal lung samples but not in lung squamous cell carcinoma. Shows a gene or gene sequence that is not normally expressed, wherein SEQ ID NO: 3981-4215 indicates a gene or gene sequence that is expressed in normal lung tissue but not normally expressed in lung neuroendocrine cancer. And SEQ ID NOs: 4216-4634 represent genes or gene sequences that are not expressed at detectable levels in normal lung but are expressed at detectable levels in pulmonary neuroendocrine cancer, where SEQ ID NOs: 4635-4877 are Shows a gene or gene sequence that is not expressed at detectable levels but is expressed in squamous cell carcinoma. 878-5303 shows a down-regulation or poorly expressed genes or gene sequences compared to lung adenocarcinoma in that although the normal lung tissue expression in normal lung and Haisen in cancer.
[0057]
In addition, SEQ ID NOs: 5304-5886 are included for the thyroid, where SEQ ID NOs: 5304-5408 represent genes or gene sequences that are not found in normal thyroid tissue but are expressed in papillary thyroid cancer. No. 5409-5602 indicates a gene or gene sequence which is not expressed in thyroid papillary carcinoma but is expressed in normal thyroid cells, and SEQ ID No. 5603-5886 indicates thyroid papillary carcinoma as compared to normal thyroid cells Genes or gene sequences that are expressed at a level at least about 5 times higher are shown.
[0058]
In addition, SEQ ID NOs: 5887-6147 are included for the esophagus, wherein SEQ ID NOs: 5887-6015 represent genes or gene sequences that are expressed in esophageal adenocarcinoma but are not expressed in normal esophagus from the same patient; Here, SEQ ID NOs: 6016-6147 represent genes or gene sequences that are expressed in normal esophagus but not expressed in esophageal adenocarcinoma samples derived from the same patient.
[0059]
Furthermore, SEQ ID NOs: 6148-6472 are included for ovaries, where SEQ ID NOs: 6148-6371 indicate genes or gene sequences that are expressed only in malignant ovarian cancer, where SEQ ID NOs: 6372-6424 are in normal ovarian tissue And SEQ ID NOs: 6425-6472 represent genes or gene sequences expressed only in metastatic ovarian cancer.
[0060]
In addition, SEQ ID NOs: 6473-7473 are included for the kidney, where SEQ ID NOs: 6473-6615 represent genes or gene sequences that are expressed in normal kidney but not in renal clear cell carcinoma, where SEQ ID NO: : 6616-6885 shows a gene or gene sequence that is expressed in clear cell cancer cells but not normal kidney cells, wherein SEQ ID NO: 6666-6973 is expressed in normal kidney cells but not in kidney kidney SEQ ID NOs: 6974-7156 represent genes or gene sequences that are not expressed in renal cell carcinoma but are not expressed in normal kidney, wherein the genes or gene sequences are not expressed in cell cancer. : A gene or gene sequence that is expressed in normal kidney but not in Wilms tumor cells Shows, also wherein SEQ ID NO: 7230-7473 indicates the gene or gene sequence not expressed in normal kidney cells express during Wilms' tumor.
[0061]
Also included for the prostate is SEQ ID NO: 7474-8131, wherein SEQ ID NO: 7475833 represents a gene or gene sequence that is expressed in prostate cancer but not detectably expressed in normal prostate cells. SEQ ID NOs: 7834-8071 represent genes or gene sequences that are expressed in normal prostate cells but are not expressed at appreciable levels in prostate cancer, and wherein SEQ ID NOs: 8072-8131 are more highly expressed in prostate cancer Shows genes or gene sequences for ribosomal proteins that are highly expressed but not expressed at appreciable levels in normal prostate cells.
[0062]
Furthermore, for the pancreas, SEQ ID NOs: 8132-847 are included, wherein SEQ ID NOs: 8132-8358 represent genes or gene sequences that are expressed in normal pancreas but not in pancreatic adenocarcinoma, and wherein SEQ ID NO: : 8359-8447 represent genes or gene sequences that are expressed in pancreatic adenocarcinoma but not in normal pancreas.
[0063]
Genetic propensity, an indicator of cancer status, need not be unique to all cells that are found to be cancerous. Thus, the methods disclosed herein are useful for detecting the presence of a cancerous condition in tissues where not all cells exhibiting a complete tendency are present. For example, selected genes consisting of sequences homologous under stringent conditions or at least 90%, and preferably 95%, identical to at least one of the sequences of SEQ ID NOs: 1-847 are herein described in the practice of the invention. As disclosed above, the characteristic group used in the above-described characteristic group is composed of genes that are expressed and / or up-regulated in cancer cells as compared to normal cells, and using an appropriate probe DNA or RNA. Thus, it is present in 60% of cells obtained from a sample of tumor or malignant tissue, whereas it is absent in 60% of cells obtained from the corresponding non-cancerous or otherwise normal tissue (and therefore Present in 40% of the normal tissue cells). In a preferred embodiment, the genetic predisposition is present in at least 70% of cells obtained from cancer tissue and appears to be absent in at least 70% of the corresponding normal, non-cancerous tissue sample. In a particularly preferred embodiment, the genetic predisposition is present in at least 80% of cells obtained from cancer tissue and appears to be absent in at least 80% of the corresponding normal, non-cancerous tissue sample. In a most preferred embodiment, the genetic predisposition is present in at least 90% of cells obtained from cancer tissue and appears to be absent in at least 90% of the corresponding normal, non-cancerous tissue sample. In further or additional embodiments, the genetic predisposition is present in at least 100% of cells obtained from the cancerous tissue and appears to be absent in at least 100% of the corresponding normal, non-cancerous tissue sample. However, the latter embodiment is rare.
[0064]
Conversely, the characteristic group herein is expressed or up-regulated in cancer cells relative to normal cells, and as disclosed herein, expression in normal cells not expressed in cells suspected of being cancerous. The status of the cancer in the sample suspected of being a cancer can be confirmed. The same is true for the assays disclosed herein for potential anti-tumor factors.
[0065]
The presence or absence of expression of one or more selected gene sequences can be an indicator of cancer status for a particular cell, but the mere presence or absence of the genetic propensity alone is sufficient to understand the malignant status. Rather, the expression level of the genetic propensity can therefore also be an important factor in determining a grasp of the cancer status. Thus, while gene trends may be present in both cancer and non-cancer cells, expression levels may differ between cancer and non-cancer cells. This is measured by any of the methods disclosed herein, all of which are well known in the art. Therefore, it is also essential to measure the expression levels of one or more of the above genes as a separate method of diagnosing the presence of a cancer condition in a particular cell, group of cells or tissue, culture or in situ.
[0066]
In accordance with the invention disclosed herein, the determination of anti-cancer factors using characteristic genes for the various organs and tissues described above is based on the propensity to regulate the genes, and thus the presence of the potential factors Increasing or decreasing the expression of a gene may or may not be important. Thus, the more genes in the group of genes affected by the factor, the greater the likelihood that the factor is an effective therapeutic factor.
[0067]
In addition, different factors may have different abilities to affect genes of a characteristic group of genes. For example, if a potential therapeutic factor, such as factor A, causes a decrease in expression of a gene or group of genes of a unique or distinct gene cluster, or even all genes of said gene cluster, A low level of mRNA is expressed from or a low level of protein is produced from the mRNA, but a second potential factor, such as factor B, modulates the activity of the gene or related genes, and It causes a reduction to half the amount for A and only half the mRNA for factor A is transcribed or only half the protein is translated from said mRNA. Thus, factor B appears to have twice the therapeutic potential of factor A.
[0068]
Modulating or altering the activity measured using the assays disclosed herein above may include increasing or decreasing the activity of the gene or gene sequence. Thus, the gene here is expressed in cancer cells of the same organ or tissue type but not in normal cells, or is up-regulated in cancer cells compared to normal cells of the same organ or tissue type. You. Factors that down regulate the gene or gene sequence or completely suppress expression are considered potential anti-tumor factors in the present disclosure. Conversely, here the factor causes expression of the gene or gene sequence and is expressed in normal cells but not in cancer cells, or where the factor up regulates the gene or gene sequence and Alternatively, it is expressed in normal cells of tissue type but not in cancer cells, or is up-regulated in normal cells of the same organ or tissue type but not in cancer cells. Said factors are considered potential anti-tumor factors in the present disclosure.
[0069]
The present invention also relates to a method for producing a product comprising identifying an agent according to one of the processes for identifying the agent disclosed herein, wherein the product is the result of the identification process. Data collected for said factor as said, wherein said data is sufficient to present the chemical characteristics and / or structure and / or properties of said factor.
[0070]
In accordance with the foregoing, the present invention further relates to a process for determining the cancer status of a cell to be tested, said process comprising the sequence of SEQ ID NO: 1-8447, a sequence substantially identical to said sequence or a unique fragment thereof. Or determining the level of expression in the cell of at least one gene comprising one nucleotide sequence selected from any of the foregoing supplements, and then comparing the expression to the expression of a cell known not to be cancer, The difference in expression thereby comprises that the cell being tested is a cancer.
[0071]
In specific embodiments of the invention, the expression is for a gene that is considered to be in 2, 3, 4, 5, or more groups, or even two or more of the genes, such as a group of ten of the genes. It is determined. A group of genes, such as five of the above genes, may appear to be expressed at a particular level in cancer cells, but at a lower level (or not at all) in non-cancerous or normal cells. Conversely, a group of, for example, five such genes may appear to be expressed in normal (ie, non-cancer) cells but at lower levels (or not at all) in cancer cells. Therefore, to determine a group or trend of genes that are expressed in cancer cells but expressed at lower levels (or not at all) in non-cancer cells, or vice versa, to diagnose a cancer condition Wherein the gene is selected from those comprising a sequence comprising a complementary sequence or one of the fragments of the sequence selected from SEQ ID NOs: 1-8447.
[0072]
In a specific embodiment, the process of the invention is a method wherein the expression is the expression of two or more of said genes or at least three of said genes, preferably at least five of said genes or even ten or more of said genes. Including examples.
[0073]
In other embodiments, the process of the invention comprises the step of expressing the gene more in the tested cells than in the non-cancerous cells or in expressing the gene in the tested cells more than the non-cancerous cells. For less state.
[0074]
Using the methods disclosed herein, the cancer or non-cancerous state of a cell or tissue sample can be readily determined. For example, a colon or other cancer can be easily detected using the method of the present invention.
[0075]
In accordance with the present invention, the gene expression of a gene that includes, in part, one of the nucleotide sequences of SEQ ID NOS: 1-8447 is identified by use of a probe that is a fragment of said nucleotide sequence, wherein the probe is It should be understood that they can be formed from different sites. Thus, for each gene in the characteristic group of the invention, the nucleotide sequence disclosed for a specific SEQ ID NO is only a portion of the nucleotide sequence encoding the expression of the gene. As a result, gene expression is measured by use of a nucleotide probe that hybridizes to messenger RNA (mRNA) transcribed from a portion of the gene other than the specific nucleotide sequences disclosed with respect to the SEQ ID NOs listed herein. be able to.
[0076]
The invention further relates to a process for determining a gene that induces, promotes or suppresses cancer, wherein said process is selected from the group consisting of contacting a cell with a cancer regulatory factor, the gene sequence of SEQ ID NOS: 1-847. Measuring the altered expression of the gene and determining that said gene is a cancer-inducing or promoting gene.
[0077]
Thus, some or all of the distinctive genes disclosed herein as SEQ ID NOs: 1-8447 directly affect cancer induction or progression or even other diseases and disease processes. I think that the. Since altered expression (either up-regulated or down-regulated) of these genes is associated with the disease state (ie, cancer), altered expression may contribute to disease induction or progression. For example, when the gene that is up-regulated is an oncogene or the gene that is down-regulated is a tumor suppressor gene, a screening method for small molecule therapy that is superior to a test based only on the expression level of the gene is used. provide. For example, a gene that exhibits an up-regulation in cancer and whose increased expression contributes to the induction or progression of a disease represents a target in a test for a small molecule that suppresses or inhibits its function. Examples include, but are not limited to, kinase inhibition, cell proliferation, substrate analogs that inhibit the active site of a protein target, and the like. Similarly, genes that exhibit down-regulation in cancer and whose absence results in the induction or progression of the disease are valuable therapeutic factors for gene therapy.
[0078]
It should be noted that there are a variety of different situations where genes are determined to be involved in the cancer process. Thus, some genes are oncogenes, which encode proteins directly involved in the cancer process, and may thereby promote the development of cancer in animals. In addition, other genes may contribute to the suppression of the cancer state in certain cells or cell types, thereby adversely affecting the cancer state that occurs in animals. Other genes are directly or indirectly involved in the cancer process or condition and may serve as auxiliary abilities to the cancer condition. It is believed that all of the genotypes are to be determined according to the invention disclosed herein. Thus, the gene determined by the process of the present invention may be an oncogene. Alternatively, the gene determined by the process can be a cancer promoting gene. The latter include genes that directly or indirectly affect cancer processes, either in vivo or ex vivo, in promoting a cancer state or promoting the progression of cancerous growth or otherwise regulating the growth of cancer cells. In addition, the gene determined by the process can be a cancer suppressor gene. Here, the gene acts directly or indirectly to suppress the induction or progression of a cancer state. The gene can act indirectly, where its expression alters the activity of another gene or gene expression product that is directly involved in inducing or promoting the progression of the cancer condition. For example, a gene that encodes a wild-type or mutated polypeptide that acts to suppress a tumor suppressor gene or its expression product will therefore serve to indirectly promote tumor growth.
[0079]
In accordance with the foregoing, the processes of the present invention include cancer modulators that are polypeptides or small chemicals that affect cancer processes, including inducing or inhibiting or promoting tumor growth, in vivo or ex vivo. As disclosed herein, the cancer modulator may have an effect of increasing gene expression or the cancer modulator may have an effect of decreasing gene expression.
[0080]
In accordance with the disclosure herein, the present invention also relates to a process for treating cancer, the process comprising the step of treating cancer cells against an expression product encoded by a gene sequence selected from the group consisting of SEQ ID NOs: 1-8847. Contacting the agent with
[0081]
Thus, some or all of the genes in these characteristic genes represent, at least in part, specific targets for therapeutic intervention based on their propensity to express. For example, genes in a distinct group of genes encode cell surface molecules and are up-regulated in cancer cells as compared to normal cells. The protein encoded by the gene may be highly susceptible to “targeted therapy” utilizing affinity structures, such as antibodies conjugated to cytotoxic factors, as a result of increased expression of the gene in cancer cells. Is a useful therapeutic target. Advantageously, in the methodology, the endogenous ligand or binding partner for the cell surface molecule need not be known. Rather, antibodies or equivalent molecules (which can include artificial peptides, alternative ligands, and the like) that specifically recognize cell surface molecules that bind to factors that can cause cell death or cell cycle inhibition Show that these proteins are promising therapeutically. Thus, the method involves the use of so-called self-death "bullets" for intracellular proteins.
[0082]
The process of the invention comprises an example of the above process, wherein the cancer cells are contacted in vivo and ex vivo, preferably wherein the factor is a molecular structure having an affinity for a site, ie, the expression product. Consists of a part of the whole. In one such embodiment, the site having an affinity for the expression product is an antibody, particularly where the expression product is a polypeptide or an oligopeptide or consists of an oligopeptide site or consists of a polypeptide.
[0083]
Thus, the factor may be a short molecule structure consisting of both an affinity site and an anti-cancer active site. Wherein the site is obtained from a separate molecule or molecular structure that, when separated, has the activity, wherein the factor is such that the site is bound in a type of addition product. It is formed by linking sites into one larger molecular structure. The anti-cancer and affinity sites may be covalently linked, such as in the form of a single polypeptide or polypeptide-like structure, or may be non-covalently linked, such as by hydrophobic or electrostatic interactions. The structure is formed by a method well known in the chemical arts. Alternatively, anti-cancer and affinity sites can be formed from separate domains of a single molecule that exhibit more than one activity as part of the same chemical structure. Here one of the activities is for cancer cells or tumorigenesis or tumor growth, and another activity has an affinity for an expression product produced by the expression of a gene associated with a cancer process or condition.
[0084]
In one embodiment of the invention, the chemical agent, such as a protein or other polypeptide, is encoded by a gene associated with a cancer process, particularly a gene sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-847. Binds to factors such as antibodies having an affinity for cancer cell expression products such as polypeptides or proteins. In a specific embodiment, the expression product is a protein or cell surface receptor such as a glycoprotein or lipoprotein present on the surface of a cancer cell, wherein the expression product is part of the cell membrane of the cancer cell. And serves as a therapeutic target for the affinity site of the anti-cancer factor, wherein, after binding of the affinity site of the factor to the expression product, the anti-cancer site of the factor It acts to neutralize the effects on the induction or promotion of formation and / or proliferation. In another embodiment of the present invention, binding of the factor to the expression product may have the effect of promoting cancer or suppressing growth. In particular here, the presence of said expression product is closely or simply related to the progression and growth of the tumor. Thus, here the presence of the expression product is essential for tumor initiation and / or growth, and the binding of the factor to the expression product has the effect of counteracting the tumor promoting activity. In one such embodiment, the factor is an apoptosis-inducing factor that induces cell self-death, thereby destroying cancer cells and stopping tumor growth.
[0085]
As disclosed herein, the invention further relates to a process for determining a cancer-inducing, promoting or repressing gene in a cancer cell, said process comprising measuring a change in expression of a gene sequence, Wherein the sequence is one selected from the group consisting of the sequences of SEQ ID NOs: 1-8447.
[0086]
Thus, the process of the present invention correlates changes in mRNA expression characteristics of these characteristic genes with potential (cancer morphology dependent) changes in the number of DNA copies in the chromosomal region where these genes are located. Use Of course, the distinct nature of alterations in mRNA expression (e.g., characteristic genes up-regulated at the transcriptional level) can also be attributed to alterations in RNA copy number in the genomic region where these genes are located (e.g., genomic DNA containing related genes). Region amplification).
[0087]
All cancers typically contain a chromosomal rearrangement that corresponds to a translocation, amplification or deletion of a specific region of genomic DNA. Repetitive chromosomal rearrangements associated with specific stages and types of cancer always affect one or more genes that play a direct and important role in the development or progression of disease. Many of the known oncogenes or tumor suppressor genes that play a direct role in cancer were first identified based on positional cloning from repetitive chromosomal rearrangements. Alternatively, other methods have shown a reduction in reconstitution following its cloning. In all cases, the gene shows amplification both at the level of DNA copy number and at the level of transcriptional expression at the mRNA level.
[0088]
At least some of the genes contained within the characteristic genes disclosed herein (SEQ ID NOS: 1-8447), some of which have DNA copy numbers resulting from specific chromosomal rearrangements in cancer cells 2 shows changes in mRNA expression characteristics (depending on the relevant distinct reading frame) in cancer samples due to changes in. The utility obtained therefrom provides (i) a time-saving method for identifying novel chromosomal rearrangements, amplifications or deletions associated with cancer in which the genes contained in these characteristic genes are; Further or (ii) indicates a major gene affected by the chromosomal rearrangement, amplification or deletion, thereby playing an important role in the development or progression of disease. Genes in the distinctive group identify changes in DNA copy number (based on changes in expression at the mRNA level), and consequently (eg, when gene X identifies a novel chromosomal copy associated with cancer) The specific chromosomal region defined by gene X serves as the basis for diagnostic analysis for cancer, where genomic DNA is extracted from the tissue sample and measured for the presence of specific amplification) Provide entry points to other methods of diagnostic analysis of cancer development, mid-stage or progression, performed at the DNA level, and rapid positional cloning of genes that play an essential and direct role in the initiation or progression of cancer. provide.
[0089]
In one embodiment of the invention, the change in expression can be measured by measuring a change in the number of gene copies, wherein the change in the number of copies is an increase in the number of copies, or A change in the number of copies is a decrease in the number of copies.
[0090]
The change in the number of copies of the gene can be measured by measuring the change in the expression of a messenger RNA encoded by a specific gene sequence. In particular, the sequence is a sequence represented by SEQ ID NO: 1-8447. One selected from the group consisting of: Also, according to the present invention, the gene may be a cancer-inducing gene, a cancer-promoting gene or a cancer-suppressing gene. In the practice of the method of the present invention, a cancer-promoting gene is a gene that does not directly induce or suppress tumor formation or proliferation, and the gene directly promotes the progression of a cancer state, such as through the action of its expression product. Work to enhance or otherwise facilitate. In addition, here the gene acts on a gene or gene expression product that would otherwise have the effect of reducing tumor formation and / or growth.
[0091]
Accordingly, the present invention also provides a process for diagnosing cancer cells, said process comprising determining a cancer inducing, promoting or repressing gene according to the method described above.
[0092]
The present invention also relates to a process for treating cancer, said process comprising inserting into a cancer cell a genetic construct comprising an anti-oncogene operably linked to a promoter or enhancer element, thereby producing a gene for the anti-oncogene. Expression comprises causing the suppression of the cancer, wherein the promoter or enhancer element is a promoter or enhancer element that regulates a gene sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-8447.
[0093]
Thus, the characteristic groups or characteristic genes disclosed herein are useful in identifying gene regulatory elements in the promoters of genes contained in normal tissues and / or characteristic cancer-specific characteristic groups. It is. Accordingly, each characteristic group is a collection of genes that share the overall general tendency of cancer transcriptional regulation to normal (eg, characteristical groups whose transcription is up-regulated in cancer).
[0094]
In one of the foregoing examples, analysis and comparison of the DNA sequences of the promoter regions of all genes included in the characteristic group specifically promotes the formation of gene expression (eg, increased transcriptional expression in cancer). It is useful for conservative extension of a part of the sequence of a gene corresponding to a cis-acting factor or identification of a motif. As a result, the identification of the cis-acting regulator can be used for enhancing cancer-specific expression of an autologous gene or toxin by gene therapy using techniques already well known in the art.
[0095]
In another embodiment, the anti-oncogene is a tumor suppressor gene or encodes a polypeptide having anti-cancer activity, particularly where the polypeptide has apoptotic activity.
[0096]
In a further embodiment, the insertion of the gene construct of the invention into cancer cells is performed in vivo, for example, using a viral or plasmid vector. The method can also be used for in vitro use. Thus, the methods of the present invention are readily applicable to different methods of gene therapy, wherein cells are genetically regulated ex vivo and then introduced into a host. Alternatively here gene regulation includes adeno-associated viruses, adenoviruses and retroviruses, as well as vectors particularly suitable for said treatment, such as the use of specific viral vectors, including plasmids specially constructed for carrying out said treatment. Performed in vivo using any of a number of suitable methods. The use of these and other vectors is well known to those of ordinary skill in the art and need not be further disclosed.
[0097]
The present invention also relates to a process for determining a functionally related gene, said process comprising the step of inserting one or more gene sequences selected from the group consisting of the sequences of SEQ ID NOs: 1-8447 into said group. Contacting an agent that regulates the expression of two or more genes of the group, thereby determining a portion of the genes of the group.
[0098]
According to the present invention, said functionally related gene is a gene that regulates the same metabolic pathway, or said gene is a gene encoding a functionally related polypeptide. In one such embodiment, the gene is a gene whose expression is regulated by the same transcriptional activator or enhancer sequence, particularly wherein the transcriptional activator or enhancer comprises SEQ ID NOS: 1-8447. Increase or otherwise modulate the activity of a gene sequence selected from the group.
[0099]
Thus, the characteristic genes disclosed herein also find use as a basis for small molecule analysis for therapy based on changes in expression characteristics. In one such embodiment, small molecule testing is useful for measuring changes in gene expression in characteristic populations, thereby providing a means for identifying specific functional pathways and methods for conferring distinct functions on novel genes. provide.
[0100]
In accordance with the foregoing, monitoring the transcriptional expression of the genes contained in the characteristic groups disclosed herein is the basis for analysis for small molecule therapy. For example, in situations in which transcription of a characteristic group of genes is up-regulated in cancer cells as compared to normal cells, the test may identify small molecules that down-regulate gene expression in characteristic groups within the cancer cells. To facilitate. Although the treatment makes cancer cells appear more normal based on the expression of genes in the characteristic group, what actually happens when the test is performed is that all genes in the characteristic group Does not react similarly with each small molecule in the compound library. For example, the average characteristic group has 200 different genes, the expression of all 200 genes is monitored for response to a library of 50,000 compounds, and some of the genes in the characteristic group Uniformly changes its expression tendency in response to a particular compound (for example, 10 genes constantly change expression in a similar manner, such as down-regulation of one gene in 10 groups) ), Always results in down regulation of the other nine specific genes as well.
[0101]
A portion or subgroup of said genes in a characteristic group that reacts with a compound to alter its expression in a similar manner are genes located in a common metabolic, signaling, physiological or functional pathway; Thus, by analyzing and identifying said parts, (a) assigning known and novel genes to specific pathways, and (b) classifying them into pathways with genes whose function has already been characterized or disclosed. Specific functions and functional roles of novel genes can be identified. For example, 10 in a characteristic group that alter expression in a similar manner, with 5 known genes associated with apoptosis, thereby linking the other 5 genes to play a role in apoptotic cell processes. Subgroups (5 known genes and 5 novel genes) can be identified. Thus, the process disclosed in accordance with the present invention simultaneously provides a novel method of conferring function on genes, a novel method of functional genomics and a method for identifying compounds with potential therapeutic effects on specific cellular pathways. provide. The compounds may have a similar therapeutic relevance to various diseases other than cancer. Here, there is an effect if the disease is known or shown to be associated with a specific cellular pathway.
[0102]
It should be noted that in practicing the methods of the invention disclosed herein, references to particular buffers, media, reagents, cells, culture conditions and the like are not intended to be limiting. That is, one of ordinary skill in the art should be construed to include all the relevant material that we find of interest or value in the particular situation in which the discussion is presented. For example, one buffer system or culture medium can be replaced with another and still obtain similar, if not identical, results. Persons of ordinary skill in the art will have sufficient knowledge of the systems and methodologies to make substitutions that best serve their purpose in the use of the methods disclosed herein without undue experimentation. Can be.
[0103]
The present invention is now further disclosed as the following non-limiting examples, but other different embodiments of the disclosed method according to the present invention will be shown to those of ordinary skill in the relevant art. Should be clearly noted.
[0104]
BEST MODE FOR CARRYING OUT THE INVENTION
(Example)
SW480 cells were cultured in Leibovitz's L-15 medium supplemented with 2 mM L-glutamine (90%) and 10% fetal calf serum. ⁵ Pieces / cm ² To a concentration of. Cells are harvested after treatment with 0.25% trypsin, 0.02% EDTA at 37 ° C for 2-5 minutes. The trypsinized cells are then diluted in 30 ml of growth medium and seeded in 96-well plates at a concentration of 50,000 cells per well (200 μl / well). The next day, cells are treated with the complex buffer alone or with the complex buffer containing the chemical agent to be tested for 24 hours. The media is then removed, the cells are lysed and the RNA is recovered using RNAeasy reagents and protocols purchased from Qiagen. RNA was quantified and 10 ng of sample in 1 μl was sampled from 1 × PCR buffer, RNAsin, reverse transcriptase, nucleoside triphosphate, amplitaq gold, Tween 20, glycerol, bovine serum albumin (BSA) and reference gene (18S RNA) Is added to a 24 μl Taqman reaction mixture containing the specific PCR primers and probe, and the gene to be tested (gene X). Thereafter, reverse transcription was performed at 48 ° C. for 30 minutes. The sample is then applied to a Perlin Elmer 7700 sequence detector and heat denatured at 95 ° C for 10 minutes. Amplification is performed for 40 cycles of 15 seconds of annealing at 60 ° C., followed by 60 seconds of extension at 72 ° C. and 30 seconds of denaturation at 95 ° C. The data file is then recorded and the data is analyzed based on the appropriate baseline display and threshold.
[0105]
The quantitative difference between the target and reference genes is then calculated and the relative expression values are measured for all samples used. This procedure is then repeated for each target gene in a particular characteristic or unique group, and the relative expression rate for each gene pair is determined (ie, the expression rate is determined by the other genes whose expression is measured). For each target gene for each, the absolute expression of each gene is measured relative to a reference gene for the compound or chemical agent being tested). The sample is then recorded and evaluated according to the degree of conversion of the expression characteristics of the treated sample relative to the control. The overall expression of a group of genes relative to a control is also determined if it is regulated by one chemical factor associated with another. Chemical agents that have the greatest effect on a particular gene or group of genes are considered to be the most antitumor.
[0106]
In practicing the methods of the present invention, rather than all cells of a particular sample of cells suspected of being cancer expressing all or most of their genes, a significant amount of expression in a significant number of said cells is indicative of cancer or cancer. It should be considered that it is sufficient to guarantee the determination of the potential cancer condition. The sequences disclosed herein are shown in numerical order from SEQ ID NO: 1 to 8447, but other genes are more or less related to other organs or tissues, some of which are up-regulated in cancer. It is not up-regulated in normal cells, while others are up-regulated in normal cells but not in cancer cells. The sequences presented here are genomic or cDNA sequences and can be represented as RNA sequences.

Claims (49)

One process for screening for an antitumor agent, wherein the process comprises:
(A) exposure of the cells to the chemical agent to be tested for antineoplastic activity and (b) expression of at least one gene comprising one of the sequences SEQ ID NO: 1-847 or at least 95% identical thereto. A process wherein the change in expression is indicative of an antitumor activity. The process of claim 1, wherein said expression is measured for more than one of said genes. 2. The process of claim 1, wherein said expression is measured for at least five of said genes. 2. The process of claim 1, wherein said expression is measured for at least 10 of said genes. The process of claim 1, wherein said expression is measured for all said genes in a particular characteristic group of genes. A process for determining the cancer status of a test cell, wherein the process is one of a nucleotide sequence selected from the sequences of SEQ ID NOs: 1-847 or at least 95% identical thereto. Measuring the expression of the at least one gene in the test cell, and then comparing the expression with the expression of the at least one gene in the at least one cell known not to be cancerous, thereby providing a difference in the expression Indicating that the cell is a cancer. 7. The process of claim 6, wherein said expression is expression of two or more said genes. 7. The process of claim 6, wherein said expression is expression of at least five of said genes. 7. The process of claim 6, wherein said expression is expression of at least 10 of said genes. 7. The process of claim 6, wherein said expression is expression of all said genes. A process for determining a cancer-inducing, promoting or repressing gene, wherein said process contacts a cell with a cancer modulator and is selected from the group consisting of the gene sequences SEQ ID NOS: 1-8447. Measuring a change in the expression of the gene, thereby identifying the gene as a cancer-inducing or promoting gene. The process according to claim 11, wherein the gene determined by the process is an oncogene. The process of claim 11, wherein the gene determined by the process is a cancer promoting gene. The process according to claim 11, wherein the gene determined by the process is a cancer suppressor gene. 12. The process of claim 11, wherein said cancer modulator has an effect of increasing gene expression. 12. The process of claim 11, wherein the cancer modulator has an effect of reducing gene expression. A process for treating cancer, wherein the process contacts a cancer cell with an agent having activity on an expression product encoded by a gene sequence selected from the group consisting of SEQ ID NOs: 1-847. A process comprising: 18. The process of claim 17, wherein the cancer cells are contacted in vivo. 18. The process of claim 17, wherein said factor comprises a site having affinity for said expression product. 20. The process according to claim 19, wherein said site having affinity for said expression product is an antibody. 18. The process according to claim 17, wherein said factor is an apoptosis-inducing factor. A process for determining a cancer-inducing, promoting or repressing gene in a cancer cell, wherein the process comprises altering the expression of a gene sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-8447. A process comprising measuring. 23. The process of claim 22, wherein said change in expression is measured by measuring a change in gene copy number. 24. The process according to claim 23, wherein the change in the number of copies is an increase in the number of copies. 24. The process of claim 23, wherein said change in copy number is a decrease in copy number. 24. The process of claim 23, wherein said change in gene copy number is measured by measuring a change in expression of a messenger RNA encoded by said gene sequence. 24. The process of claim 23, wherein said gene is a cancer-inducing gene. 24. The process according to claim 23, wherein said gene is a cancer promoting gene. 24. The process according to claim 23, wherein said gene is a cancer suppressor gene. 24. A process for diagnosing cancer cells, wherein the process comprises determining a cancer inducing, promoting or suppressing gene according to claim 23. A process for treating cancer wherein the process inserts a gene construct comprising an anti-oncogene operably linked to a promoter or enhancer element into a cancer cell, whereby the anti-oncogene is Comprises causing the suppression of the cancer, wherein the promoter or enhancer element is a promoter or enhancer that regulates a gene sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-8447. And the process. 32. The process of claim 31, wherein said anti-oncogene is a tumor suppressor gene. 32. The process of claim 31, wherein the anti-oncogene encodes a polypeptide with anti-cancer activity. 34. The process of claim 33, wherein said polypeptide has apoptotic activity. 32. The process of claim 31, wherein said inserting into said cancer cells occurs in vivo. 32. The process of claim 31, wherein the insertion into the cancer cell further comprises using a viral or plasmid factor, and is performed in vitro or in vivo. 32. The process of claim 31, wherein said cancer is selected from the group consisting of colon, breast, stomach, lung, thyroid, esophagus, ovary, kidney, prostate and pancreatic cancer. A process characterized by that: 32. The process of claim 31, wherein the cancer is an adenocarcinoma, malignant tumor, clear cell carcinoma, invasive ductal carcinoma, invasive lobular carcinoma, squamous cell carcinoma, neuroendocrine carcinoma, papillary carcinoma and Wilms tumor. A process selected from the group consisting of: A process for determining a functionally related gene, wherein the process comprises combining one or more gene sequences selected from the group consisting of the sequences of SEQ ID NOs: 1-8847 into the group. Contacting with a factor that regulates the expression of two or more genes therein, thereby determining a portion of said group of genes. 40. The process of claim 39, wherein said functionally related genes are genes that regulate the same metabolic pathway. 40. The process of claim 39, wherein said gene is a gene encoding a functionally related polypeptide. 40. The process of claim 39, wherein all of said genes are genes whose expression is regulated by the same transcriptional activator or enhancer sequence. 2. A method for producing a product comprising identifying a factor according to the process of claim 1, wherein the product is data collected for the factor resulting from the process; Wherein the data is sufficient to present the chemical structure and / or properties of the agent. One process for screening for an anti-tumor agent, said process comprising:
From measuring (a) exposure of cells to a chemical agent to be tested for antineoplastic activity and (b) changes in the expression of characteristic genes or at least one gene having a sequence at least 95% identical thereto. A process characterized in that the altered expression is indicative of an antitumor activity. 46. The process of claim 44, wherein said change in expression is an increase in expression. 46. The process of claim 44, wherein said change in expression is a decrease in expression. 45. The process of claim 44, wherein said change in expression is a change in expression of at least five genes of said characteristic group of genes. 47. The process of claim 44, wherein said change in expression is a change in expression of at least 10 genes of said characteristic group of genes. 45. The process of claim 44, wherein said change in expression is a change in expression of at least half of said characteristic group of genes.