WO1999031505A1

WO1999031505A1 - Neurotrophic factor assay

Info

Publication number: WO1999031505A1
Application number: PCT/US1998/025445
Authority: WO
Inventors: Charlotte M. Mcguinness; Marthajoy Spano; George W. Belenduik
Original assignee: Shire Laboratories Inc.
Priority date: 1997-12-15
Filing date: 1998-12-01
Publication date: 1999-06-24
Also published as: WO1999031505A8

Abstract

The present invention provides a cultured transformed glial cell based assay e.g. using cell line T98G for screening neurotrophic factors in physiologic sample. The assay is quantative and rapid, and is useful for screening columns fractions generated during the purification of growth factors.

Description

NEUROTROPHIC FACTOR ASSAY This application is based on United States provisional application serial no.

60/069,468 filed on December 15, 1997.

The present invention relates to a method of detecting neuroregulatory factors.

BACKGROUND OF THE INVENTION

Late stage post-mitotic neural development is influenced by two groups of polypeptide factors, neurotrophic factors and neuronal differentiation factors. Neurotrophic factors promote neuronal survival both in vivo and in vitro. Neurotrophic factors are proteins that are made locally in the nervous system or are transported in the blood or cerebrospinal fluid to their targets. Most neurotrophic factors are closely related to a group of growth factors that interact with their tyrosine kinase receptor (tyrosine phosphorylation). The nerve growth factor (NGF) family of "target-derived" neurotrophic factors are believed to have complementary and sequential roles in neuron development. Non target-derived neurotrophic factors, which family includes growth factors such as acidic fibroblast growth factor (aFGF) and basic FGF (bFGF) have biological activities in the nervous system. Basic FGF may influence precursor cell differentiation in a neuronal direction and may act as a neurotrophic molecule on non-dividing mature neurons. The neurotrophic activities of some growth factors induce tyrosine phosphorylation which has a role in promoting neuronal survival. Ciliary neurotrophic factor (CNTF), identified as a neuronal differentiation factor, and choline acetyltransferase development factor (CDF) also have neurotrophic activity. Neurotrophic factors interact synergistically as well as qualitatively at different activities (Yamamori, T., Neuroscience Research, 12(1992) 545-582). Neurotrophic factors may be used to treat nervous system deficits by controlling normal processes that tightly regulate growth and survival of neurons. Identifying, purifying and characterizing neurotrophic factors is therefore important in the development of treatments of central nervous system diseases.

A critical part of neurotrophic factor research is the necessity for a quick, easy and accurate assay that can quantitatively screen large numbers of samples for desired characteristics. Since growth factors are present in minute amounts, it is important to have a sensitive assay. Growth factors are often unstable, thus it is important that the assay be rapid, especially so that purification of the factors can proceed before the factors degrade.

An obstacle in the development of a quick assay to screen for neurotrophic factors has been that neurons cannot be propagated in culture because they do not divide. To date, the most common assay for neurotrophic factors involves the measurement of increased neurite outgrowth from primary cultures of rat neurons in the presence of neurotrophic factors (Laerum, O.D., Ada Neurol Seand.,

1985:72:529-549). An alternate assay is the PC12 differentiation/neurite outgrowth assay Fujita, K., et al. Environmental Health Perspectives, 1989:80:127-142). PC12 cells are a rat adrenal medullary cell line that demonstrates outgrowth of neurites in response to some neurotrophic factors. However, neurite outgrowth assays are not suitable for screening purposes as the cells take up to one week to differentiate. In addition the measurement of neurite outgrowth is subjective and evaluations will vary between observers. Moreover, the assays are labor intensive as they require examination of large numbers of cultures.

Thus, there exists a need for an objective, quantitative and quick cell based assay to serve as an effective screen for neurotrophic factors. More particularly, there exists a need for a quantitative assay which can be used to quickly screen samples, generated during the purification of tissues or fluids suspected of containing neuroregulatory factors. SUMMARY OF THE INVENTION

The invention provides a method of detecting the presence of a neurotrophic factor in a sample by measuring the amount of mitotic growth stimulation of glioblastoma cells which have been contacted with the sample. The presence or absence of a neurotrophic factor in the sample is correlatable to the presence or absence of glioblastoma cell mitotic growth stimulation.

The invention also provides a method for quantitating the amount of a neurotrophic factor in a sample comprising measuring the amount of mitotic growth stimulation of glioblastoma cells by the neurotrophic factor wherein the amount of neurotrophic factor in the sample is correlatable to the amount of mitotic growth stimulation.

The method of the invention provides an assay which is rapid enough to be conveniently used as a screen in a neurotrophic factor purification scheme and which is accurate enough to quantitate neurotrophic factors in individual column fractions. In one aspect, the present invention provides a thymidine uptake based assay for mitosis growth stimulation to detect or, or to quantitate the amount of, neurotrophic factors in a sample.

The present invention also provides an assay for determining the mitogenic properties of identified growth factors.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates the time course of ³H-Thymidine uptake by T98G cells after stimulation with FCS.

Fig. 2 illustrates thymidine uptake in T98G cells after growth factor stimulation with FCS, EGF, bFGF, NGF and insulin.

Fig. 3 illustrates thymidine uptake in T98G cells after growth factor stimulation at various concentrations with FCS, transferrin, PDGF and bFGF.

Fig. 4 illustrates thymidine uptake in T98G cells after growth factor stimulation with FCS, prothrombin and thrombin.

Fig. 5 illustrates T98G uptake of ³H-thymidine after stimulation with FCS after 6 hours and 24 hours.

Fig. 6 illustrates T98G uptake of ³H-thymidine after growth factor stimulation with FCS, Cohn fraction IN/one and Cohn fraction IN/4.

Fig. 7 illustrates the time course of ³H-thymidine uptake by T98G cells after stimulation of mitotic growth by growth factors.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery that glial cells respond to many of the same factors shown to stimulate neurite outgrowth in neurons. The inventors have discovered that mitotic growth of glial cells can be measurably stimulated by a variety of neurotrophic factors. Therefore, glial cells can be used to accurately and quantitatively detect known and as yet unknown neurotrophic factors by measuring the mitotic growth of the glial cells stimulated by the factors.

Accordingly, the present invention provides a method of screening samples for neurotrophic factors employing a glioblastoma cell line, T98G. The use of the T98G cells enables effective screening for neurotrophic factors.

In one aspect, the invention provides a method for detecting the presence of a neurotrophic factor in a sample suspected of containing said neurotrophic factor comprising contacting quiescent cultured human glioblastoma cells with the sample; and determining the amount of mitotic growth stimulation of the cells after contact with the sample, wherein the amount of mitotic growth stimulation is correlatable to the amount of neurotrophic factor in the sample.

In another aspect, the invention provides a method for quantitating the amount of a neurotrophic factor in a sample suspected of containing said neurotrophic factor.

As used herein, the term "neurotrophic factor" means a factor that is required for proper function and survival of a neuron, either in vivo, in vitro or both, either alone or in combination with other neurotrophic factors. Neurotrophic factors can include natural or chemically synthesized members of the nerve growth factor (NGF) family which include NGF, brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3 and NT-4; neuronal differentiation factors such as CNTF; cytokines such as interleukin (IL)-2, IL-3, IL-4, IL-5 and IL-6; members of a supergene family of calcium-binding proteins, for example calbindin-D9, whose neurotrophic activity may be mediated through regulation of calcium and protein kinase activities; heparin-binding growth factors including acidic FGF (aFGF) and basic FGF (bFGF), members of the epidermal growth factor (EGF) family, insulin-like growth factor (IGF)-I and IGF-II, and platelet derived growth factor (PDGF).

Neurotrophic activity as used herein signifies mitotic growth stimulation.

As used herein, a "sample" can be a physiological sample such as body fluid sample, plasma, aliquots of column fraction eluents, conditioned media, placenta, cerebrospinal fluid, tissue extracts e.g., pituitary and adrenal extracts, and the like.

Growth factors are polypeptides which regulate the processes of cell proliferation and differentiation and cellular function (Growth Factors: A Practical

Approach, McKay, LA. ed, IRL Press, Oxford 1993). Polypeptide growth factors can be defined as mitogenic factors and can include mitogens for fibroblasts, neurons, myoblasts, endothelial cells, epithelial cells and hematopoietic cells.

The term "mitogenic" means inducing the process of mitosis, or cell division, in cells, including DNA synthesis.

Various aspects of the invention are described in more detail in the following subsections.

Glial cells

Unlike neuronal cells, glial cells are successfully propagated in vitro cultures. (McKeever, P.E. et al, Critical Reviews in Neurobiology, 6: 119-147 1991). We have discovered that T98G cells have the unusual property among transformed cells of being both contact inhibited and quiescent (G-l arrestable) in serum depleted media. These cells have provided a significant advantage for designing a quantitative assay for growth factors since all the cells in culture can be arrested or stimulated in the same cell life cycle and DNA synthesis cycle. Mitotic growth stimulation measurement

For the purposes of the present invention a quick non labor intensive method was required for measuring differences in mitotic growth stimulation levels by the growth factors in large numbers of samples. Autoradiography and histology, both time consuming and labor intensive, have been used to measure differences in mitotic level (Stein, 1979 #1). In accordance with this aspect of the invention, the method further provides labeling newly synthesized DNA with ³H-thymidine, followed by precipitation of proteins and scintillation counting.

Other, non-radioactive methods of measuring mitotic stimulation can include, for example, bromodeoxyuridine-horseradish peroxidase-(BRDU-HRP), and BRDU- alkaline phosphatase or other methods of labeling DNA during synthesis.

Mitotic growth stimulation is determined by measuring the levels of H-

Thymidine uptake by the cells after contact with the sample, the ³H-thymidine levels being indicative of the amount of mitotic growth stimulation of the cells by the neurotrophic factor. Greater DNA synthesis induces increased ³H-thymidine uptake and is reflected in increased levels of radioactive label in the samples.

By comparing to controls the amount of mitotic growth stimulation measured for a sample, the presence or absence of a neurotrophic factor in a sample can be determined. In another aspect, the amount of neurotrophic factor can be quantitated, or the amount of neurotrophic activity of a factor can be quantitated, by comparing to standards the amount of mitotic growth stimulation measured for a sample.

In the practice of the method of the present invention, various aspects of the method can be easily modified by those skilled in the art to obtain optimum assay conditions and will vary with the different factors being tested. Peaks of activity for stimulation differ with different factors and can easily be determined by using the methods described herein. Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention. Unless otherwisely indicated, reagents and materials are readily available from commercial sources. Samples were counted in either a Beckman or Wallac scintillation counter.

EXAMPLES Example I

T98G Assay T98G human glioblastoma cells (ATCC (Rockville, MD) were maintained in

DMEM with 10% fetal calf serum (FCS)(BioFluids, Rockville, MD) and pen/strep (GIBCO, Rockville, MD). Cells were subcultured at 37°C in 5% CO2 and subcultured at 80% confluence.

For thymidine uptake assays, cells were trypsinized, centrifuged, washed in fresh media, counted, re-centrifuged, and resuspended at l-2xl0⁵ /ml in DMEM with 10% FCS. 0.5 ml of the cell suspension was added to each well of a 24-well plate and cells allowed to attach to the plate for 6 hours to overnight. Media was then changed to 0.3% FCS in DMEM/F12, and cells were allowed to become quiescent for 24-48 hrs.

Quiescent cells were stimulated by replacing the media containing 0.3% FCS with fresh serum- free media containing either 0.3 to 10% fetal calf serum, or sample. After 18-24 hours, 50μl of lm Ci/ml ³H-thymidine (Dupont, NEN, Brewster, MA) was added to each well. After a labeling period of 4 hours, cells were washed 3 times with phosphate buffered saline (PBS) at 4°C and the protein was precipitated with 10% trichloroacetic acid (TCA) at 4°C for at least 30 min. Cells were then washed again in cold PBS and solubilized by the addition of 0.5 ml/well of 0.2 N NaOH/0.1% SDS. The solubilized protein was pipetted into scintillation vials and acidified with lOOμl 1MHC1. Aquasol scintillation fluid was added (10 mls)(Dupont, NEN) and samples were counted in a scintillation counter. The foregoing procedure was utilized, with alterations made as indicated and necessary, in the following examples, Experiments were set up according to a protocol similar to the following:

Well# DMEM vol FCS vol Sample vol Sample ³H- thymidine

(mL) (mL) (mL) ^" vol (mL)

1,2,3 0.5 0 0 FCS 0.05

4,5,6 0.475 0.025 0 standard 0.05

7,8,9 0.450 0.050 0 controls 0.05

10,11,12, etc. 0.475 0 0.025 sample 0.05

EXAMPLE 2

Different concentrations of FCS and different labeling times were evaluated to establish the optimal labeling schedule. Optimal labeling produced the largest differential in thymidine uptake between low and high serum levels and allowed greater distinctions to be made between the mitotic capacity of different protein fractions.

0, 0.5%, 1%, 5% and 10% FCS were tested at 6 hours and 24 hours, according to the method described in Example 1.

The T98G thymidine uptake results are shown in Table 1 and Figure 1. At 6 hours, 0.5% FCS uptake levels were 13.6% of 10% FCS uptake; while at 24 hours, 0.5% FCS uptake was 66% of 10% FCS. These results indicated that 6 hour labeling was preferred since it produced greater differences between low and high levels of stimulation than 24 hour labeling. Table 1

In experiments performed in a similar manner, it was determined that a concentration of isotope of 0.5μCi. per ml was preferred for the present method over lμCi/ml.

Example 3

The mitogenic capacity of total plasma proteins in Cohn fractions IV/one and IV/4 (courtesy American Red Cross)(Cohn, E.J., et.al., J.Am.Chem.Soc. 68:459, 1946) were compared to that of FCS using the method in Example 1. The results of this experiment, in Table 2 and Figure 2, show that both Cohn fractions stimulated cell growth, and even demonstrated greater growth stimulation than FCS. Fraction IV/one stimulated the cells by a greater than ten fold increase.

Table 2

Example 4 The results of example 3 demonstrated that FCS did not stimulate mitosis to the same levels as previously shown. We considered the possibility FCS may stimulate DNA synthesis with a different latency than plasma proteins. We therefore tested whether slightly altered timing of ³H labeling relative to mitosis-induction might have an effect on relative increases in thymidine uptake by growth factors. Published accounts of FCS stimulation of DNA synthesis in T98G cells give the peak of stimulation as 18 hours. Our labeling point was longer than this, 20-22 hours. We ran a time course of DNA synthesis comparing FCS with IV/one and IV/4. The results are shown in Table 3 and Figure 3. The peak of activity for FCS occurred at about 18 hours and a peak of activity for IV/one occurred at 24 hours. Thus, the most likely reason for greater labeling by IV/one at 22 hours compared to FCS is that IV/one is at the peak of activity at that time, while DNA synthesis following FCS stimulation is beginning to decline. This data further indicates that the mechanism of mitosis-stimulation activated by IV/one plasma fraction may be different then the mechanism activated by FCS.

Table 3

Example 5

Unless indicated otherwise, growth factors/reagents used in this and the following examples were obtained from SIGMA Chemicals (St. Louis, Mo.). To further characterize mitosis stimulation in the T98G cells, we assayed a series of known growth factors for their ability to stimulate mitotic growth as shown in Table 4 and Figure 4, FCS, EGF Receptor Grade, Calbiochem #324856) and bFGF (Calbiochem #341595) stimulated thymidine uptake and NGF (Calbiochem #480354) did not stimulate uptake. NGF and bFGF are growth factors that are known to affect neurons. (Laerum, O.D., Ada Neurol Seand. 1985:72:529-549) The results confirmed the theory that this pathway may not be involved in mitosis in T98G cells, but could be involved in differentiation. Table 4 Thymidine Uptake in T98G Cells

Example 6 We tested additional concentrations of bFGF and 3 concentrations of PDGF. PDGF, like bFGF, has neuronal activity as well as more general wound healing responses (Deuel, T.F. et al., Annu.Rev.Med. 1991. 42:567-84). It is released from platelets in the clotting response, and stimulates regrowth at wound sites. We also tested transferrin a blood protein that transports iron into red blood cells. The results are shown in Table 5 and Figure 5. Cells respond very strongly to PDGF, in the range used. Transferrin has essentially no activity in this assay.

Table 5 Thymidine Uptake in T98G Cells

Example 7 We further compared the effect of FCS on growth stimulation with different concentrations of thrombin and prothrombin, vitamin K dependent plasma proteases (Enzyme Research Labs, South Bend, Indiana). We found that thrombin, a plasma protein associated with clotting and therefore present at sites of wounds or tissue damage presumably to stimulate wound healing, surprisingly has many of the properties of a growth/neurotrophic factor. The results are shown in Table 6 and Figure 6. Both prothrombin and thrombin, the activated form of prothrombin, induce a saturable increase in thymidine uptake, indicating an increase in DNA synthesis. Thrombin is a very effective mitogenic factor in this assay. None of these is as effective as FCS, reflecting the fact that FCS contains a combination of different growth factors activating multiple mitotic pathways and producing a synergistic or additive effect.

Table 6 Thymidine Uptake in T98G Cells

Claims

1. A method of detecting the presence of a neurotrophic factor in a sample suspected of containing the neurotrophic factor comprising measuring the amount of mitotic growth stimulation of glioblastoma cells by the sample, wherein the amount of mitotic growth stimulation is correlatable to the amount of neurotrophic factor in the sample.

2. A method for detecting the presence of a neurotrophic factor in a sample suspected of containing said neurotrophic factor comprising

(a) contacting quiescent cultured transformed human glioblast cells with the sample;and

(b) determining the amount of mitotic growth stimulation of the cells after contact with the sample, wherein the amount of mitotic growth stimulation is correlatable to the amount of neurotrophic factor in the sample.

3. The method of claim 2, wherein mitotic growth stimulation is determined by measuring levels of ³H-Thymidine uptake by the cells after contact with the sample, said levels being indicative of the amount of mitotic growth stimulation of the cells by the neurotrophic factor.

4. The method of claim 1 wherein the cells are from cell line T98G ATCC #CRL 1690.

5. A method for measuring the neurotrophic activity of a growth factor comprising,

(a) contacting quiescent cultured T98G cells with the growth factor;

(b) measuring the level of ³H-Thymidine uptake by the cells after contact with the growth factor; and (c) determining the neurotrophic activity of the growth factor.

6. The method of claim 5 wherein the growth factor is selected from the group consisting of NGF, BDNF, NT-3, NT-4, CNTF, cytokines, FGF, IGF-I, IGF- II, PDGF, prothrombin and thrombin.