WO1995021918A1

WO1995021918A1 - Rat osteosarcoma cell lines

Info

Publication number: WO1995021918A1
Application number: PCT/US1994/002096
Authority: WO
Inventors: Claus-Jens Walter Doersen; Robert Joseph Isfort
Original assignee: The Procter & Gamble Company
Priority date: 1992-06-24
Filing date: 1994-02-14
Publication date: 1995-08-17

Abstract

The present invention relates to a novel cell line isolated from a rat osteosarcoma, wherein the novel cell line has all the identifying characteristics of American Type Culture Collection Accession No. CRL 11066, CRL 11067, CRL 11068, CRL 11069, or CRL 11070. The present invention also relates to growth factors produced by the novel cell line. This invention also relates to a process for producing such growth factors.

Description

RAT OSTEOSARCOMA CELL LINES

TECHNICAL FIELD This invention relates generally to a novel cell line, and specifically to a novel rat osteosarcoma cell line, as well as to certain growth factors produced by the cell line.

BACKGROUND Polypeptide growth factors play a key role in regulating the development of multicellular organisms and in the processes of tissue maintenance and repair, (see Cross and Dexter (1991) CeJL Vol. 64, pp. 271-280; Aaronson (1991) Science. Vol. 254, pp. 1146-1153.) At the cellular level, growth factors are involved in regulating proliferation and the progressive acquisition of the differentiated phenotype. Growth factors are capable of stimulating cellular proliferation as well as inhibiting cellular proliferation and many growth factors have been found to be multifunctional (Sporn and Roberts (1988) Nature. Vol. 332, pp. 217-219). The highly coordinated functions of growth factors is perhaps best exemplified in the development of the hematopoiectic cell system (Metcalf (1989) Nature. Vol. 339, pp. 27-30) where a limited number of stem cells give rise to a larger population of developmentally restricted progenitor cells. These progenitors cells are further stimulated to proliferate and differentiate into the mature lymphoid, erythroid and myeloid cells. A balance between cell types and numbers of cells must be maintained throughout the developmental cascade. This requires the concerted actions of growth factors which commit a cell (now developmentally restricted) along a particular cell lineage, of growth factors which stimulate the proliferation of committed cells, and finally, of growth factors which promote the differentiation of the committed cells and inhibit the proliferation of the mature, fully differentiated cells.

Tumor cells represent naturally occurring examples of cells where the processes that control cellular proliferation and differentiation have been uncoupled (Cross and Dexter (1991) Cell, Vol. 64, pp. 271-280; Aaronson (1991) Science. Vol. 254, pp. 1146-1153). The observation that many types of tumor cells secrete growth factors suggests that these factors can contribute to the tumorigenic process as well as normal cellular processes. Tumor cells have been found to secrete autocrine growth factors which stimulate the proliferation of the tumor cells themselves and paracrine growth factors which stimulate surrounding cells to secrete factors promoting the proliferation of the tumor cells. Paracrine factors can also stimulate the surrounding cells to provide a cellular environment promoting the survival of the tumor cells. For example, many types of tumor cells secrete growth factors that recruit endothelial cells and stimulate their proliferation and differentiation resulting in a new vasculature supplying nutrients for the tumor cells (Liotta et al., (1991) Cell. Vol. 64, pp. 327-336). Objects of the Present Invention

It is an object of the present invention to provide a novel rat osteosarcoma cell line.

It is also an object of the present invention to provide novel growth factors produced by a rat osteosarcoma cell line of the present invention.

It is also an object of the present invention to provide a process for producing novel growth factors from a cell line of the present invention. SUMMARY

The present invention relates to a novel cell line isolated from a rat osteosarcoma, wherein the cell line has all the identifying characteristics of ATCC Accession No. CRL 11066, CRL 11067, CRL 11068, CRL 11069, or CRL 11070.

The present invention also relates to growth factors produced by a cell line having all the identifying characteristics of ATCC Accession No. CRL 11066, CRL

11067, CRL 11068, CRL 11069, or CRL 11070.

The present invention also relates to a process for producing a growth factor comprising: a) cultivating a cell line having all the identifying characteristics of ATCC Accession No. CRL 11066, CRL 11067, CRL 11068, CRL 11069, or CRL 11070, and b) harvesting the growth factor.

The present invention also relates to a process for producing a growth factor comprising; a) isolating mRNA coding for the growth factor from a cell line having all the identifying characteristics of ATCC Accession No. CRL 11066, CRL 11067, CRL

11068, CRL 11069, or CRL 11070., b) making cDNA of the mRNA, c) expressing the cDNA in an expression vector, and d) recovering the growth factor.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB show results of the heparin agarose chromatography of OSR3TR1 conditioned medium.

Figures 2A and 2B show results of the DEAE-Sephacel chromatography of the heparin agarose flow through fraction for OSR3TR1.

Figure 3 shows the mitogenic activity of the mitogenic growth factors isolated from OSR4TR1 conditioned medium.

Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, and 41 show the MATRIGEL growth characteristics of the C3H10T1/2 cell line in the presence of various growth factors and OSR4TR1 conditioned media.

Figure 5 shows the mitogenic activity of the mitogenic growth factors isolated from OSR5TR2 conditioned medium.

Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, 6H, and 61 show the MATRIGEL growth characteristics of the C3H10T1/2 cell line in the presence of various growth factors and OSR5TR2 conditioned media.

Figures 7 A, 7B and 7C relate to heparin agarose chromatography of OSR-6 conditioned medium. Figure 7A indicates absorbance at 280 nm of the pooled fractions was recorded, and NaCl concentration was determined by conductivity measurements of selected fractions. Figure 7B indicates mitogenic activity of the pooled fractions using OSR-2 cells. Figure 7C indicates mitogenic activity of the pooled fractions using NIH-3T3 cells.

Figure 8A indicates mitogenic response of OSR-2 cells to heparin agarose fractionated conditioned medium from OSR-8 cells. Figure 8B indicates mitogenic response of MC3T3-E1 cells to heparin agarose fractionated conditioned medium from OSR-8 cells.

Figure 8C indicates mitogenic response of NIH-3T3 cells to heparin agarose conditioned medium from OSR-8 cells.

DETAILED DESCRIPTION As used herein "growth factor" means a protein which has an ability to stimulate proliferation of cells or to inhibit proliferation of cells.

As used herein "inhibitory growth factor" means a protein which has an ability to inhibit proliferation of cells.

As used herein "mitogenic growth factor" means a protein which has an ability to stimulate proliferation of cells.

As used herein, "tumorigenic" means an ability to form tumors in a host animal.

As used herein, "heparin agarose elution property of X M NaCl" means the molarity of NaCl used to achieve elution of a protein from heparin agarose column when a conditioned media comprising the protein is subjected to the heparin agarose column procedure described below, wherein X is a numerical value.

As used herein, "diethylaminoethyl elution property of X M NaCl" means the molarity of NaCl used to achieve elution of a protein from DEAE

(diethylaminoethyl)-Sephacel column when flow through fraction from heparin agarose column comprising the protein is subjected to the DEAE-Sephacel column procedure described below, wherein X is numerical value.

As used herein, "ATCC" means American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland, 10852, U.S.A.).

As used herein, "OSR-2" means cell line ATCC CRL 11065. As used herein, "OSR3TR1" refers to a novel cell line of the present invention, ATCC No. CRL 11066.

As used herein, "OSR4TR1" refers to a novel cell line of the present invention, ATCC No. CRL 11067.

As used herein, "OSR5TR2" refers to a novel cell line of the present invention, ATCC No. CRL 11068.

As used herein, "OSR-6" refers to a novel cell line of the present invention, ATCC No. CRL 11069.

As used herein, "OSR-8" refers to a novel cell line of the present invention, ATCC No. CRL 11070.

As used herein, "w/v" and "v/v" mean percent by weight and percent by volume, respectively.

As used herein, "DEAE" means diethylaminoethyl.

The present invention relates to a novel cell line isolated from a rat osteosarcoma, the cell line having all the identifying characteristics of OSR3TR1 (ATCC No. CRL 11066), OSR4TR1 (ATCC No. CRL 11067), OSR5TR2 (ATCC No. CRL 11068), OSR-6 (ATCC No. CRL 11069), or OSR-8 (ATCC No. 11070).

The present invention also relates to growth factors isolated from a rat osteosarcoma cell line having all the identifying characteristics of OSR3TR1, OSR4TR1, OSR5TR2, OSR-6, or OSR-8.

The present invention also relates to a method for producing one or more of the above-identified growth factors comprising: a) cultivating a cell line isolated from a rat osteosarcoma, and b) harvesting the growth factor, wherein the cell line has all the identifying characteristics of OSR3TR1, OSR4TR1, OSR5TR2, OSR-6, or OSR- 8.

An alternative way of obtaining the growth factors produced by a cell line of the present invention is by isolation of the growth factor mRNA for use by those skilled in the art for expression of the protein of interest. Protein expression from mRNA covers a wide variety of techniques including PCR methodologies using a number of organisms for the final expression of the protein including bacteria, fungus, animal cells, insect cells and plant cells as well as noncellular protein expression methodologies (for an overall review of molecular biology techniques used in protein expression cloning see Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Briefly, this method comprises isolating RNA from an osteosarcoma cell line which codes for the growth factor, making cDNA of the mRNA, expressing the cDNA in an expression vector, and recovering the growth factor. More preferably, this procedure involves isolation of mRNA from the osteosarcoma cell, making cDNA from the mRNA, cloning of the cDNA into an appropriate expression source, expression of the protein of interest from the cDNA inserted into the expression vector, screening the expression source for the protein of interest, purifying the expression clone containing the cDNA coding for the protein of interest, and expression of this purified cDNA in an expression vector in an appropriate expression source for large scale synthesis of the protein of interest. For example, the cDNA sequence coding for the mature form of the protein of interest can be fused in-frame to the E.coli la B signal sequence at the 5' end in the plasmid pMON2690 and the recombinant protein of interest can be expressed in stably transformed bacterial cells of the strain E. coli JM101 (Wong et al. (1988) Gene. Vol. 68, pp. 193-203; Obukowicz et al. (1988) Mol. Gen. Genet.. Vol 215, pp. 19-25). Alternatively, the cDNA coding for the mature form of the protein of interest can be inserted into the yeast Pichia pastoris expression vector pHIL-Sl and the recombinant protein of interest can be expressed in stably transformed cells of P. pastoris strain GS115 (Phillip Petroleum Company). As a further alternative, the cDNA coding for the protein of interest can be inserted into the mammalian expression vector pDSRα, which is a derivative of pcDL-SRα296 (Takebe et al. (1988 Mol. Cell. Bio Vol. 8, pp. 466-472) containing the mouse dihydrofolate reductase minigene (Crouse et al. ⁽19831 Mol. Cell. BioL Vol. 3, pp. 257-266), and the recombinant protein of interest can be expressed in stably transfected CHO cells, clone DG44 (Urlaub et al. (1986) Somat Cell Mol. Genet.. Vol. 12, pp. 555-566). In a further example of a means for performing this aspect of the invention, the cDNA coding for the protein of interest can be inserted into the baculovirus transfer vector pVL1392 and recombinant baculovirus can be isolated by cotransfection of Sf9 insect cells with the recombinant transfer vector and AcMNPV viral DNA (Invitrogen Corporation). The recombinant virus stock can then be used to infect Sf21 insect cells (Invitrogen Corporation) for the high level production of the protein of interest. As a yet further example, the cDNAsequence coding for the mature form of the protein interest can be fused in- frame to the sequences coding for the presequence of the extracellular PR-S protein of tobacco at the 5' end (Sijmons et al. (1990) Biotechnology. Vol. 8, pp. 217-221) in the plant expression cassette vector pMOG18 (Mogen International NV). The resulting plasmid can be mobilized into Agorbacterium for the transformation of potato tuber discs and the recombinant protein of interest can be produced in transgenic potato plants (Sijmons et al. (1990) Biotechnology. Vol. 8, pp. 217-221).

Multiple technical variations of this general expression schemes are well understood by those skilled in the art and all would apply.

The cell lines of the present invention are useful as a biological source for the growth factors of the present invention and/or the mRNA coding for such growth factors. The growth factors of the present invention are useful for one or more of the following: 1) treating diseases affecting the bone and cartilage (e.g., those growth factors demonstrating an ability to stimulate osteoblast cells), 2) wound healing (e.g., those growth factors demonstrating an ability to stimulate fibroblast cells) and/or 3) cancer treatment (e.g., those growth factors demonstrating an ability to inhibit the growth of osteoblast, fibroblast and/or multipotent cells). I. ESTABLISHMENT OF CELL LINES FROM RAT OSTEOSARCOMAS A. OSR3TR1

1. Establishment of the OSR3TR1 Cell Line

A rat tumor cell line is established from a tumor excised from a Sprague- Dawley rat with osteosarcoma. The site of the excised tissue is the right tibia. The tumor tissue is aseptically removed from the euthanized animal and carefully trimmed of connective tissues. The tumor is minced in a culture dish (100 mm diameter) containing 15 ml of growth medium (10% fetal bovine serum, 90% RPMI 1640 medium (GIBCO)) further supplemented with penicillin (100 units/ml, GIBCO), Fungizone (0.25 microgram/ml, GIBCO) and streptomycin (100 micrograms/ml, GIBCO), and incubated at 37°C in an atmosphere of 5% CO2 and 95% air with a relative humidity of approximately 95%. The adherent cells which migrate from the minced tumor pieces onto the surface of the culture dish are further expanded as a cell population using standard tissue culture techniques. Once the rat tumor cell line reaches confluency in a tissue culture flask (150 cm^ surface area), the cell line is designated as having a mean population doubling of 1. At this stage, the cells are subcultured in growth medium (10% fetal bovine serum, 90% RPMI 1640 medium) with no antibiotic or antimycotic supplements. The rat tumor cell line is routinely tested for mycoplasma contamination by DAPI assay (Stanbridge (1981) Isr. J. Med. ScL Vol. 1 , pp. 563-568) and is found to be negative. The rat tumor cell line is found to be poorly tumorigenic when assayed for tumor formation in congenitally athymic mice (a nude mouse, a product of Harlan Sprague Dawley, Inc.). The rat tumor cells at a mean population doubling of 8, are injected subcutaneously in the mid-flank region of the rat at an inoculum of approximately 1 x 10^ cells per site. A total of eight sites (two per rat) are injected. Only one tumor arose at the site of injection. This tumor is aseptically excised from the euthanized rat and the tumor cell population is re-established in culture according to the methods described above for the establishment of the original tumor cell population. Once the cells reached confluency in a 150 cm^ tissue culture flask, the cells are designated as OSR3TR1 at a mean population doubling of 1. These cells are found to be free of mycoplasma contamination by the DAPI assay (Stanbridge (1981) Isr. J. Med ScL Vol. 17, pp. 563-568).

2. Characteristics of the OSR3TR1 Cell Line

The OSR3TR1 cell line can be serially propagated in culture with no known limited life span. The OSR3TR1 cell line displays a limited osteoblast-like phenotype in vitro. The cells show intense staining for the presence of alkaline phosphatase (Rodan and Rodan (1983) Bone and Mineral Research. Annual 2 (Peck, ed.) pp. 244- 285) as detected by a cytochemical assay (Sigma Chemical Company, Procedure 85). It has been reported (Vukicevic et al., (1990) CeJ Vol. 63, pp. 437-445) that osteoblastic cells are capable of forming cell clusters exhibiting networks of canalicular cell processes when cultured on reconstituted basement membrane extracts such as MATRIGEL (a solubilized basement membrane from Collaborative Biomedical Products, Bedford, Massachusetts, U.S.A., comprising laminin, collagen type IV, heparin sulfate, proteoglycan and entactin). Fibroblasts, chondrocytes and embryonic stem cells did not demonstrate the canalicular cell processes. OSR3TR1 cells when cultured on MATRIGEL at a density of approximately 50,000 cells per well of a standard 24-well tissue culture plate formed a monolayer of individual cells with no network of canalicular cell processes. This growth pattern is very different from the cell clusters with the characteristic canalicular processes that are formed by the osteoblastic MC3T3-E1 cells plated at an equivalent density (Sudo et al., (1983) J. Cell BioL Vol. 96, pp. 191-198; Vukicevic et al., (1990) Cel Vol. 63, pp. 437- 445). The inability to form the canalicular network may be a property of the tumorigenicity of the OSR3TR1 cells.

The OSR3TR1 cell line is found to be highly tumorigenic when assayed for tumor formation in congenitally athymic mice (a nude mouse, a product of Harlan Sprague Dawley, Inc.). OSR3TR1 cells are injected subcutaneously in the mid-flank region of the animals at an inoculum of approximately 1 x 10^ cells per site. A total of eight sites (two per animal) are injected, and tumors arose at 100% of the sites. Histologically, the tumors appeared to be fully differentiated osteosarcomas producing a boney mineralized extracellular matrix.

B. OSR4TR1

1. Establishment of the OSR4TR1 Cell Line

A second cell line is established from a tumor excised from a Sprague-Dawley rat with osteosarcoma. The site of the excised tissue is the right tibia. The tumor tissue is aseptically removed from the euthanized animal and carefully trimmed of connective tissues. The tumor is minced in a culture dish (100 mm diameter) containing 15 ml of growth medium (10% fetal bovine serum, 90% RPMI 1640 medium (GIBCO)) further supplemented with penicillin (100 units/ml, GIBCO), Fungizone (0.25 microgram/ml, GIBCO) and streptomycin (100 micrograms/ml, GIBCO), and incubated at 37°C in an atmosphere of 5% CO2 and 95% air with a relative humidity of approximately 95%. The adherent cells which migrate from the minced tumor pieces onto the surface of the culture dish are further expanded as a cell population using standard tissue culture techniques. Once the rat osteosarcoma cell line reaches confluency in a tissue culture flask (150 cm² surface area), the cell line is designated as having a mean population doubling of 1. At this stage, the cells are subcultured in growth medium (10% fetal bovine serum, 90% RPMI 1640 medium) with no antibiotic or antimycotic supplements. The rat osteosarcoma cell line is routinely tested for mycoplasma contamination by DAPI assay (Stanbridge (1981) Isr. J. Med. ScL Vol. 17, pp. 563-568) and is found to be negative. The rat osteosarcoma cell line is found to be highly tumorigenic when assayed for tumor formation in congenitally athymic mice (a nude mouse, product of Harlan Sprague Dawley, Inc.). The rat osteosarcoma cells at a mean population doubling of 9, are injected subcutaneously in the mid-flank region of the animals at an inoculum of approximately 1 x 10' cells per site. A total of eight sites (two per animal) are injected, and tumors arose at 100% of the sites. One of these tumors is aseptically excised from the euthanized animal and the tumor cell population is re-established in culture according to the methods described above for the establishment of the original tumor cell population. Once the cells reach confluency in a 150 cm² tissue culture flask, the cells are designated as OSR4TR1 at a mean population doubling of 1. These cells are found to be free of mycoplasma contamination by the DAPI assay (Stanbridge (1981) Isr. J. Med ScL Vol. 17, pp. 563-568).

2. Characteristics of the OSR4TR1 Cell Line

The OSR4TR1 cell line can be serially propagated in culture with no known limited life span. The OSR4TR1 cells remain tumorigenic when assayed in congenitally athymic mice (nude mouse, product of Harlan Sprague Dawley, Inc.) as described above for the rat osteosarcoma cell line. The subcutaneous injection of

OSR4TR1 cell are 1 x 10 cells per site resulted in a 100% tumor incidence.

The OSR4TR1 cell line displays a limited, immature osteoblast-like phenotype in vitro. The cells show no staining for the presence of alkaline phosphatase (Rodan and Rodan (1983) Bone and Mineral Research. Annual 2 (Peck, ed.) pp. 244-285) as detected by a cytochemical assay (Sigma Chemical Company, Procedure 85). It has been reported (Vukicevic et al., (1990) Cell. Vol. 63, pp. 437-445) that osteoblastic cells are capable of forming cell clusters exhibiting networks of canalicular cell processes when cultured on reconstituted basement membrane extracts such as MATRIGEL (Collaborative Biomedical Products). Fibroblasts, chondrocytes and embryonic stem cells do not demonstrate the canalicular cell processes. OSR4TR1 cells when cultured on MATRIGEL at a density of approximately 50,000 cells per well of a standard 24-well tissue culture plate demonstrate a large mass of cells with a network of canalicular cell processes. This growth pattern is distinct from smaller cell clusters with the characteristic canalicular processes that are formed by the osteoblastic MC3T3-E1 cells plated at an equivalent density (Sudo et al., (1983) I Cell BioL Vol. 96, pp. 191-198; Vukicevic et al., (1990) CeH, Vol. 63, pp. 437-445). The differences in the size of the cell masses displaying the canalicular processes may be due to the tumorigenic properties of the OSR4TR1 cells. C. OSR5TR2 1. Establishment of the OSR5TR2 Cell Line

A third cell line is established from a tumor excised from a Sprague-Dawley rat with osteosarcoma. The site of the excised tissue is the right tibia. The tumor tissue is aseptically removed from the euthanized animal and carefully trimmed of connective tissues. The tumor is minced in a culture dish (100 mm diameter) containing 15 ml of growth medium (10% fetal bovine serum, 90% RPMI 1640 medium (GIBCO)) further supplemented with penicillin (100 units/ml, GIBCO), Fungizone (0.25 microgram/ml, GIBCO) and streptomycin (100 micrograms/ml, GIBCO), and incubated at 37°C in an atmosphere of 5% CO2 and 95% air with a relative humidity of approximately 95%. The adherent cells which migrate from the minced tumor pieces onto the surface of the culture dish are further expanded as a cell population using standard tissue culture techniques. Once the rat osteosarcoma cell line reaches confluency in a tissue culture flask (150 cm² surface area), the cell line is designated as having a mean population doubling of 1. At this stage, the cells are subcultured in growth medium (10% fetal bovine serum, 90% RPMI 1640 medium) with no antibiotic or antimycotic supplements. The rat osteosarcoma cell line is routinely tested for mycoplasma contamination by DAPI assay (Stanbridge (1981) Isr. J. Med. ScL Vol. 17, pp. 563-568) and is found to be negative. The rat osteosarcoma cell line is found to be highly tumorigenic when assayed for tumor formation in congenitally athymic mice (a nude mouse, product of Harlan Srague Dawley, Inc.). The rat osteosarcoma cells at a mean population doubling of 9, are injected subcutaneously in the mid-flank region of the animals at an inoculum of approximately 1 x 10^ cells per site. A total of eight sites (two per animal) are injected, and tumors arose at 100% of the sites. One of these tumors is aseptically excised from the euthanized animal and the tumor cell population is re-established in culture according to the methods described above for the establishment of the original tumor cell population. Once the cells reach confluency in a 150 cm² tissue culture flask, the cells are designated as OSR5TR2 at a mean population doubling of 1. These cells are found to be free of mycoplasma contamination by the DAPI assay (Stanbridge (1981) Isr. J. Med ScL Vol. 17, pp. 563-568). 2. Characteristics of the OSR5TR2 Cell Line

The OSR5TR2 cell line can be serially propagated in culture with no known limited life span. The OSR5TR2 cell line displays a limited, immature osteoblast-like phenotype in vitro. The cells show no staining for the presence of alkaline phosphatase (Rodan and Rodan (1983) Bone and Mineral Research. Annual 2 (Peck, ed.) pp. 244-285) as detected by a cytochemical assay (Sigma Chemical Company, Procedure 85). It has been reported (Vukicevic et al., (1990) Cell. Vol. 63, pp. 437- 445) that osteoblastic cells are capable of forming cell clusters exhibiting networks of canalicular cell processes when cultured on reconstituted basement membrane extracts such as MATRIGEL (Collaborative Biomedical Products). Fibroblasts, chondrocytes and embryonic stem cells do not demonstrate the canalicular cell processes. OSR5TR2 cells when cultured on MATRIGEL at a density of approximately 50,000 cells per well of a standard 24-well tissue culture plate demonstrate a large mass of cells with a network of canalicular cell processes. This growth pattern is distinct from smaller cell clusters with the characteristic canalicular processes that are formed by the osteoblastic MC3T3-E1 cells plated at an equivalent density (Sudo et al., (1983) J. Cell BioL Vol. 96, pp. 191-198; Vukicevic et al., (1990) Cell. Vol. 63, pp. 437-445). The differences in the size of the cell masses displaying the canalicular processes may be due to the tumorigenic properties of the OSR5TR2 cells. D. OSR-6 1. Establishment of the OSR-6 Cell Line A fourth cell line, OSR-6, is established from a tumor excised from a Sprague-

Dawley rat with osteosarcoma. The site of the excised tissue is the right tibia. The tumor tissue is aseptically removed from the euthanized animal and carefully trimmed of connective tissues. The tumor is minced in a culture dish (100 mm diameter) containing 15 ml of growth medium (10% fetal bovine serum, 90% RPMI 1640 medium (GIBCO)) further supplemented with penicillin (100 units/ml, GIBCO), Fungizone (0.25 microgram/ml, GIBCO) and streptomycin (100 micrograms/ml, GIBCO), and incubated at 37°C in an atmosphere of 5% CO2 and 95% air with a relative humidity of approximately 95%. The adherent cells which migrate from the minced tumor pieces onto the surface of the culture dish are further expanded as a cell population using standard tissue culture techniques. Once the OSR-6 cell line reaches confluency in a tissue culture flask (150 cm² surface area), the cell line is designated as having a mean population doubling of 1. At this stage, the cells are subcultured in growth medium (10% fetal bovine serum, 90% RPMI 1640 medium) with no antibiotic or antimycotic supplements. The OSR-6 cell line is routinely tested for mycoplasma contamination by DAPI assay (Stanbridge (1981) Isr. J. Med. ScL Vol. 17, pp. 563-568) and is found to be negative.

The OSR-6 cell line is tumorigenic when assayed for tumor formation in congenitally athymic (nu/nu) mice (Harlan Sprague Dawley, Inc.). OSR-6 cells at a mean population doubling 13, are injected subcutaneously in the mid-flank region of the animals at an inoculum of approximately 1 x 10^ cells per site. A total of eight sites (two per animal) are injected. Tumors arose at 100% of the sites injected with the OSR-6 cells. 2. Characteristics of the OSR-6 Cell Line The OSR-6 cell line can be serially propagated in culture with no known limited life span. The OSR-6 cell line displays a limited, immature osteoblast-like phenotype in vitro. The cells show faint staining for the presence of alkaline phosphatase (Rodan and Rodan (1983) Bone and Mineral Research, Annual 2 (Peck, ed.) pp. 244-285) as detected by a cytochemical assay (Sigma Chemical Company, Procedure 85). It has been reported (Vukicevic et al., (1990) Cell. Vol. 63, pp. 437- 445) that osteoblast cells are capable of forming cell clusters exhibiting networks of canalicular cell processes when cultured on reconstituted basement membrane extracts such as MATRIGEL (Collaborative Biomedical Products). Fibroblasts, chondrocytes and embryonic stem cells do not demonstrate the canalicular cell processes. OSR-6 cells when cultured on MATRIGEL at a density of approximately 50,000 cells per well of a standard 24-well tissue culture plate form many clusters of cells with a network of canalicular cell processes. This growth pattern is very similar to the cell clusters with the characteristic canalicular processes that are formed by the osteoblastic MC3T3-E1 cells plated at an equivalent density (Sudo et al., (1983) I Cell BioL Vol. 96, pp. 191-198; Vukicevic et al., (1990) Cell, Vol. 63, pp. 437-445). E. OSR-8 1. Establishment of the OSR-8 Cell Line

A fifth cell line, OSR-8, is established from a tumor excised from a Sprague- Dawley rat with osteosarcoma. The site of the excised tissue is the right tibia. The tumor tissue is aseptically removed from the euthanized animal and carefully trimmed of connective tissues. The tumor is minced in a culture dish (100 mm diameter) containing 15 ml of growth medium (10% fetal bovine serum, 90% RPMI 1640 medium (GIBCO)) further supplemented with penicillin (100 units/ml, GIBCO), Fungizone (0.25 microgram/ml, GIBCO) and streptomycin (100 micrograms/ml, GIBCO), and incubated at 37°C in an atmosphere of 5% CO2 and 95% air with a relative humidity of approximately 95%. The adherent cells which migrate from the minced tumor pieces onto the surface of the culture dish are further expanded as a cell population using standard tissue culture techniques. Once the OSR-8 cell line reaches confluency in a tissue culture flask (150 cm² surface area), the cell line is designated as having a mean population doubling of 1. At this stage, the cells are subcultured in growth medium (10% fetal bovine serum, 90% RPMI 1640 medium) with no antibiotic or antimycotic supplements. The OSR-8 cell line is routinely tested for mycoplasma contamination by DAPI assay (Stanbridge (1981 Isr. J. Med. ScL Vol. 17, pp. 563-568) and is found to be negative.

The OSR-8 cell line is found to be poorly tumorigenic when assayed for tumor formation in congenitally athymic (nu/nu) mice (Harlan Srague Dawley, Inc.). OSR-8 cells at a mean population doublings of 7 and 14, are injected subcutaneously in the mid-flank region of the animals at an inoculum of approximately 1 x 10^ cells per site. For each population doubling, a total of eight sites (two per animal) are injected. Only one tumor arose at the site of injection with a latent period of approximately eleven months. 2. Characteristics of the OSR-8 Cell Line

The OSR-8 cell line can be serially propagated in culture with no known limited life span. The OSR-8 cell line displays a limited, immature osteoblast-like phenotype in vitro. The cells show no staining for the presence of alkaline phosphatase (Rodan and Rodan (1983) Bone and Mineral Research. Annual 2 (Peck, ed.) pp. 244-285) as detected by a cytochemical assay (Sigma Chemical Company, Procedure 85). It has been reported (Vukicevic et al., (1990) Cell. Vol. 63, pp. 437- 445) that osteoblastic cells are capable of forming cell clusters exhibiting networks of canalicular cell processes when cultured on reconstituted basement membrane extracts such as MATRIGEL (Collaborative Biomedical Products). Fibroblasts, chondrocytes and embryonic stem cells do not demonstrate the canalicular cell processes. OSR-8 cells when cultured on MATRIGEL at a density of approximately 50,000 cells per well of a standard 24-well tissue culture plate formed many clusters of cells with a network of canalicular cell processes. This growth pattern is very similar to the cell clusters with the characteristic canalicular processes that are formed by the osteoblastic MC3T3-E1 cells plated at an equivalent density (Sudo et al., (1983) J. Cell Biol. Vol. 96, pp. 191-198; Vukicevic et al., (1990) Cell, Vol. 63, pp. 437-445). π. MOLECULAR CHARACTERIZATION OF THE RAT OSTEOSARCOMA CELL LINES

In order to characterize the cell lines in terms of unique mutations which can identify a particular cell line we have screened the cell line for mutations in genes which are commonly mutated in osteosarcomas. This molecular fingerprint of the cell provides a convenient and useful way to identify the cell line because these mutations are critical for the growth of these cells and is not expected to change.

In this respect human osteosarcomas have been extensively studied with respect to mutations in the retinoblastoma (RB-1) and p53 tumor suppressor genes (Iavarone et al., (1992) Proc. Natl. Acad. Sci. USA Vol. 89, pp. 4207-4209; Diller et al., (1990) Mol. Cell. BioL Vol. 10, pp. 5772-5781; Masuda et al., (1987) Proc. Natl. Acad. Sci. USA. Vol. 84, pp. 7716-7719; Levine and Momand (1990) Biochem. et Bvophvs. Acta. Vol. 1032, pp. 119-136; Miller et al., (1990) Cancer Res.. Vol. 50, pp. 7950-7954; Friend et al., (1987) Proc. Natl. Acad. Sci. USA Vol. 84, pp. 9059- 9063; Mulligan et al., (1990) Proc. Natl. Acad. Sci. USA Vol. 87, pp. 5863-5867; Hansen et al., (1985) Proc. Natl. Acad. Sci. USA Vol. 82, pp. 6216-6220; Horowitz et al., (1989) Science. Vol. 243, pp. 937-940; Toguchida et al., (1989) Nature. Vol. 338, pp. 156-158). RB-1 mutations include point mutations, deletions, and splicing mutations (Friend et al., (1987) Proc. Natl. Acad. Sci. USA Vol. 84, pp. 9059-9063; Levine and Momand, (1990) Biochem. et Biophvs. Acta. Vol. 1032, pp. 119-136; Mori et al., (1990) Oncoeene. Vol. 5, pp. 1713-1717; Horowitz et al., (1989) Science. Vol. 243, pp. 937-940). All of these mutations result in the production of a non-functional RB-1 protein. RB-1 is believed to function by acting as a "brake" on cellular proliferation at the appropriate time in the cell cycle . Control of the RB-1 function is achieved by selective and timely phosphorylation of RB-1 protein; phosphorylated RB-1 allows cellular proliferation while unphosphorylated RB-1 protein inhibits cellular proliferation (Laiho et al., (1990) Cell. Vol. 62, pp. 175-185; DeCaprio et al., (1989) CelL Vol. 58, pp. 1085-1095; Buchkovich et al., (1989) CelL Vol. 58, pp. 1097-1105; Chen et al., (1989) CelL Vol. 58, pp. 1193-1198; Furukawa et al., (1990) Proc. Natl. Acad. Sci. USA. Vol. 87, pp. 2770-2774). RB-1 control of cellular proliferation is one part of a closely regulated network of cell cycle controls, which include the cell cycle control proteins c-myc, c-fos and TGF-B (Laiho et al., (1990) CelL Vol. 62, pp. 175-185; Moses et al., (1990) Cell Vol. 63, pp. 245-247; Robbins et al., (1990) Nature. Vol. 346, pp. 668-671).

The p53 tumor suppressor gene is originally identified as an SV40 large T antigen binding protein whose expression is greatly increased (approximately 5-100 fold at the protein level) during SV40 cellular transformation (Levine and Momand, (1990) Biochem. et Biophvs. Acta. Vol. 1032, pp. 119-136; Levine et al., (1991) Nature. Vol. 351, pp. 453-456). The p53 gene has since been found to be mutated in a variety of tumor types (Hollstein et al., (1991) Science. Vol. 253, pp. 49-53) including human osteosarcoma cells (Mulligan et al., (1990) Proc. Natl. Acad. Sci. USA Vol. 87, pp. 5863-5867; Miller et al., (1990) Cancer Res.. Vol. 50, pp. 7950- 7954; Masuda et al., (1987) Proc. Natl. Acad. Sci. USA Vol. 84, pp. 7716-7719; Diller et al., (1990) Mol. Cell. BioL Vol. 10, pp. 5772-5781). Mutation of the p53 gene either results in an altered protein which does not function normally or a complete loss of protein. Both of these mechanisms result in the absence of a functional p53 protein (Halevy et al., (1990) Science. Vol. 250, pp. 113-116; Chen et al., (1990) Science. Vol. 250, pp. 1576-1580; Milner and Medcalf, (1991) CelL Vol. 65, pp. 765-774) and cellular transformation. The p53 protein is believed to function in several ways. The p53 protein is a transcriptional activator since p53 protein binds to specific DNA sequences (Raycroft et al., (1990) Science. Vol. 249, pp. 1049-1051; Kern et al., (1991) Science. Vol. 252, pp. 1708-1711) and contains an NH2-terminal acidic domain which efficiently activates the transcription of genes in both yeast and mammalian cells (Fields and Jang, (1990) Science. Vol. 249, pp. 1046-1049). The protein produced from mutated p53 genes does not transcriptionally activate responsive genes (Raycroft et al., (1990) Science. Vol. 249, pp. 1049-1051). In addition, p53 regulates DNA replication since wild-type protein but not protein from mutated p53 genes associates with replication complexes (Levine et al., (1991) Nature. Vol. 351, pp. 453-456; Levine and Momand, (1990) Biochem. et Biophvs. Acta. Vol. 1032, pp. 119-136) and is phosphorylated by p34(cdc2)-p60 and p34(cdc2)-cyclin B complexes (Bischoff et al., (1990) Proc. Natl. Acad. Sci. USA Vol. 87, pp. 4766-4770; Milner et al., (1990) EMBO J. Vol. 9, pp. 2885-2889). Two additional transforming genes which have been implicated in human and non-human osteosarcoma formation are the fos and myc oncogenes. The oncogenic variant of c-fos, v-fos, is first isolated as the transforming gene in retroviruses which resulted in murine osteosarcoma formation (Varmus (1984) Ann. Rev. Genet.. Vol. 18, pp. 553-612). Interestingly, transgenic mice carrying the v-fos oncogene display both cartilage and osteosarcoma tumors (Wang et al., (1991) EMBO J. Vol. 10, pp. 2437-2450). Oncogenic mutation of c-fos results when the fos gene is either over expressed or inappropriately expressed as a result of either viral transduction or mutation. C-fos functions in the cell, in conjunction with c-jun as the complex which binds the AP-1 transcriptional activation site (Abate et al., (1990) Science. Vol. 249, pp. 1157-1161; Sassone-Corsi et al., (1988) Cold Spring Harbor Symposia on Quantitative Biology LIU pp. 749-760).

C-myc has been found to be mutated both in radiation induced murine osteosarcomas (Sturm et al., (1990) Cancer Res.. Vol. 50, pp. 4146-4153) and in primary human osteosarcoma tumors (Bogenmann et al., (1987) Cancer Res.. Vol. 47, pp. 3808-3814). The myc oncogene is originally identified as the transforming gene in several retroviruses (v-myc) and c-myc mutations in a variety of tumor types are later identified (DePinho et al., (1987) J. Cell Biochem.. Vol. 33, pp. 257-266; Varmus (1984) Ann. Rev. Genet.. Vol. 18, pp. 553-612). C-myc is most commonly amplified or translocated, both of which, along with retroviral transduction, result in the inappropriate expression of the myc gene (Varmus (1984) Ann. Rev. Genet.. Vol. 18, pp. 553-612; DePinho et al., (1987) J. Cell. Biochem.. Vol. 33, pp. 257-266). Myc protein and the Max protein form a complex which binds the regulatory region of genes, via a unique sequence, to control their expression (Blackwood and Eisenman, (1991) Science. Vol. 251, pp. 1211-1217; Cole (1991) Cell, Vol. 65, pp. 715-716). C-myc, along with c-fos are immediate early genes and are believed to play a central role in mitogenic signaling in the cell (Rozengurt (1986) Science. Vol. 234, pp. 161-166).

An additional way to assay c-fos and c-myc functional activity is to determine if theses genes are transcriptionally activated following mitogenic stimulation. C-fos and c-myc are both members of a class of genes called the immediate early genes(Sassone-Corsi et al., (1988) Cold Spring Harbor Symposia on Quantitative Biology LIII. pp. 749-760; Depinho et al., (1987) J. Cell. Biochem.. Vol. 33, pp. 257- 266). Genes in this class are transcriptionally activated following mitogenic stimuli usually within 1 hour and without the need of protein synthesis (Almendral et al., (1988) Mol. Cell. BioL Vol. 8, pp. 2140-2148; Greenberg et al., (1986) Mol. Cell. BioL Vol. 6, pp. 1050-1057). Since this is a complex pathway, alterations in immediate early gene transcription is indicative of either a mutation in the cellular pathway which leads from the receptor/plasma membrane to the gene/nucleus of the cell or a mutation which leads to the autocrine expression of a growth factor. Either of the above mutated phenotypes lead to a down regulation and subsequent inhibition of expression of the c-myc and c-fos genes following serum starvation and mitogen stimulation. A. Characterization of Tumor Suppressor Genes and Oncogenes High molecular weight DNA is isolated from the cell line as follows.

Approximately 1 x 10^ cells are cultured as described above until confluent. These cells are then scraped off the tissue culture flask into culture media, collected by centrifugation at 3300 x g for 5 minutes, the culture media is removed from the cell pellet and the pellet is resuspended in 9.5 ml of NET buffer (100 mM sodium chloride, 10 mM Tris - pH 8.0, 1 mM EDTA). To the resuspended DNA is added 50 ul of 10 mg/ml of proteinase K (Boehringer Mannheim) and 0.5 ml of 10% (w/v) sodium dodecyl sulphate/water. This mixture is mixed well and incubated at 55°C for one hour followed by extraction twice with an equal volume of a 50:50 mixture of phenolxhloroform. The aqueous phase is collected by centrifugation at 3300 x g for 5 minutes and reextracted with an equal volume of chloroform. The aqueous phase is collected by centrifugation at 3300 x g for 5 minutes, removed to a new tube and to it is added 1 ml of 3M sodium acetate pH 5.2 and 20 ml of 100% ethanol. This solution is mixed well and incubated at -20°C for 2 hours followed by the collection of high molecular weight DNA by centrifugation at 3300 x g for 30 minutes. The high molecular weight DNA is washed once with 5 ml of 70 v/v.% (ethanol/water) dried and resuspended in TE (10 mM Tris-pH 8.0 and 1 mM EDTA) at a concentration of 1 mg/ml.

For restriction enzyme analysis, 20 ug of the above isolated high molecular weight DNA in 20 ul TE is added to 2.5 ul of 10X restriction enzyme buffer (IX restriction enzyme buffer consist of 50 mM Tris - pH 8.0, 10 mM magnesium chloride, and 100 mM sodium chloride) and 2 ul of restriction enzyme (either Eco Rl or Hind III at 10 units/ml both from New England Biolabs) and this mixture is incubated at 37°C for 16 hours. Following incubation, the above solution is added to 3 ul of 10X gel loading buffer (10X gel loading buffer is 0.25% bromophenol blue, 0.25% xylene cyanol, 25% Ficoll type 400, 10 mM EDTA in water) and the restriction endonuclease generated fragments are separated by agarose gel electro- phoresis. Agarose gel electrophoresis is performed as follows. A 0.6% (w/v) agarose (Bethesda Research Laboratories) gel in IX TBE (IX TBE consist of 0.089 M Tris, 0.089 M boric acid, and 0.002 M EDTA) is poured in a BioRad horizontal gel electrophoresis apparatus, the above DNA solution is loaded into a well and electrophoresis is performed in a IX TBE solution for 16 hours at 20 volts. Following electrophoresis, the gel is stained for 1 hour in 0.5 ug/ml ethidium bromide (Sigma Chemical Company)/water solution followed by photography under 302 nm ultraviolet light irradiation. The gel is then prepared for Southern blotting as follows. The gel is soaked for 1 hour in a solution of 1.5M sodium chloride and 0.5M sodium hydroxide with constant shaking followed by an incubation for 1 hour in a solution of 1.5M sodium chloride and 1M Tris - pH 8.0 with constant shaking. The gel is then transferred to an LKB VacuBlot apparatus (LKB Scientific) prefitted with a sheet of BAS-NC nitrocellulose (Schleiser and Schuel) and the transfer of DNA from the agarose gel to the nitrocellulose membrane is performed under 40 cm. H2O of pressure using 10X SSC (10X SSC is 1.5M sodium chloride and 0.15M sodium citrate, pH 7.0) as the transfer medium. The DNA Southern blot is then used in a hybridization analysis as follows. The nitrocellulose filter is first wet in 6X SSC followed by prehybridization in hybridization buffer [50% formamide (molecular biology grade, Bethesda Research Laboratories), 5X Denhardt's solution (Denhardt's solution is 0.1% Ficoll, 0.1% polyvinylpyrrolidone, and 0.1% bovine serum albumin - pentax fraction V - all from Sigma Chemical Company), 5X SSPE (20X SSPE is 3M sodium chloride, 0.2M sodium phosphate, and 0.02M EDTA - pH 7.4), 0.1% sodium dodecyl sulphate, and 100 ug/ml of denatured salmon sperm DNA (Sigma Chemical Company)] for 4 hours at 42°C with constant agitation. Molecular probes for the tumor suppressor genes RB-1 (ATCC #57450) and p53 (Levine and Momand (1990) Biochemical et Biophysical Acta.. Vol. 1032, pp. 119-136) and the oncogenes c-myc (ATCC #41008) and c-fos (ATCC #41040) are radiolabelled using an Amersham nick translation kit and [^ P]dCTP (ICN) to a specific activity of 1 x 10^ cpm/ug DNA by following the manufacturers recommendations. These radiolabelled probes are then added to the hybridization solution/nitrocellulose filter of the prehybridization step and incubated at 42°C for 40 hours with constant agitation. Following hybridization, the nitrocellulose filters (blots) are first incubated in 2X SSC and 0.1% SDS at room temperature for 1 hour followed by an incubation in 0.2X SSC and 0.1% SDS at 65° C for 1 hour. The results of the hybridization experiment are visualized by autoradiography at -70°C. Following autoradiography, the films are developed and used in data analysis. 1. OSR3TR1

The molecular analysis of the OSR3TR1 cell line for RB-1, p53, c-myc and c- fos genes indicated that the RB-1, p53 and c-fos genes appeared normal (non- mutated, wild-type) as compared to a non-tumorigenic Sprague-Dawley rat cell control at the above described level of analysis (restriction enzyme analysis and Southern blotting). The c-myc gene is amplified 50 fold as compared to a non- tumorigenic Sprague-Dawley rat cell control.

2. OSR4TR1

The molecular analysis of the OSR-4TR cell line for RB-1, p53, c-myc and c- fos genes indicated that all these genes appeared normal (non-mutated, wild-type) as compared to a non-tumorigenic Sprague-Dawley rat cell control at the above described level of analysis (restriction enzyme digestion and Southern blotting).

3. OSR5TR2

The molecular analysis of the OSR5TR2 cell line for RB-1, p53, c-myc and c- fos genes indicated that the RB-1, p53 and c-fos genes appeared normal (non- mutated, wild-type) as compared to a non-tumorigenic Sprague-Dawley rat cell control at the above described level of analysis (restriction enzyme analysis and Southern blotting). The c-myc gene was amplified 10 fold as compared to a non- tumorigenic Sprague-Dawley rat cell control.

4. OSR-6

The molecular analysis of the OSR-6 cell line for RB-1, p53, c-myc and c-fos genes indicated that all these genes appeared normal (non-mutated, wild-type) as compared to a non-tumorigenic Sprague-Dawley rat cell control at the above described level of analysis (restriction enzyme digestion and Southern blotting).

5. OSR-8

The molecular analysis of the OSR-8 cell line for RB-1, p53, c-myc and c-fos genes indicated that all these genes appeared normal (non-mutated, wild-type) as compared to a non-tumorigenic Sprague-Dawley rat cell control at the above described level of analysis (restriction enzyme digestion and Southern blotting). B. RB-1 and p53 Protein Analysis.

Immunological identification of the p53 and RB-1 proteins are performed as follows. Approximately 1 x 10^ cells are labelled for 4 hours with 100 uCi/ml of [³⁵S]methionine (Tran³⁵S-Label - ICN) in methionine-free RPMI-1640 (GIBCO) media containing 10% fetal bovine serum for 4 hours at 37°C. Following labeling, the cells are scraped off the tissue culture plastic into the labeling media, the cells are collected by centrifugation at 3300 x g for 5 minutes, followed by removal of the labeling media and the snap freezing of the cell pellet in liquid nitrogen. The frozen cell pellets are dissolved in 1 ml of ice-cold lysis buffer (50 mM Tris - pH 8.0, 5 mM EDTA, 150 mM sodium chloride, 0.5% Nonidet P-40, and 1 mM phenylmethylsulfonylfluoride) by vigorous vortexing and incubated on ice with intermediate vortexing for 30 minutes. The lysates are clarified of nonsoluable material by centrifugation at 10,000 x g for 30 minutes, the supernate is removed to a new tube to which is added 10 ul of antibodies specific for either mutant p53 (Oncogene Science p53 Ab-3), normal and mutant p53 (Oncogene Science p53 Ab- 1), or RB-1 (a 50:50 mixture of Oncogene Science RB Ab-2 and Ab-3), and 50 ul of a Protein A/G agarose (Boehringer Mannheim):lysis buffer (50:50). The above mixture is incubated overnight at 4°C with constant shaking. The cell lysate is aspirated off and the antigen/antibody/protein A-G pellet is washed one time in 1 ml of lysis buffer, one time in 1 ml of SNTE buffer (50 mM Tris-pH 7.4, 5 mM EDTA 5% sucrose - w/v, 1% Nonidet P-40, and 0.5 M sodium chloride), and one time in 1 ml of RIP A buffer (50 mM Tris - pH 7.4, 150 mM sodium chloride, 1% Triton X- 100, 0.1% sodium dodecyl sulphate and 1% sodium deoxycholate). Following washing, the antigen/antibody/protein A-G agarose pellet is redissolved in 25 ul of sample buffer (62.5 mM Tris - pH 6.8, 2% sodium dodecyl sulphate, 10% glycerol, 5% 2-mercaptoethanol), boiled at 100°C for 2 - 3 minutes, and applied to a well of a 10% SDS-PAGE gel. SDS-PAGE is performed as described by Laemmli (Laemmli, U.K. (1970) Nature. Vol. 227, pp. 680-685) for 6-8 hours at 30 mA constant current per gel. Following electrophoresis, the SDS-PAGE gels are incubated in 30% methanol and 10 % acetic acid for at least 1 hours, impregnated with ENHANCE (NEN-DuPont) according to the manufacturers recommendations, dried onto Whatman 3 mm paper using a BioRad gel dryer set at 60°C for 2 hours and autoradiography at -70°C is performed. 1. OSR3TR1

The results of the p53 and RB-1 protein analysis of the OSR3TR1 cell line indicated that the p53 protein is deleted because we failed to detect p53 protein with either of the above described p53 antibodies. These results indicate that while the gene suffered no major alteration (deletion, duplication, etc) it had suffered a point mutation or a minor deletion which resulted in the loss of protein expression. The deletion of p53 protein in transformed cells has been observed previously (Levine et al., (1991) Nature. Vol. 351, pp. 453-456; Hollstein et al., (1991) Science. Vol. 253, pp. 49-53; Gannon et al., (1990) EMBO J.. Vol. 9, pp. 1595-1602; Diller et al., (1990) Mol. Cell. BioL Vol. 10, pp. 5772-5781). The RB-1 protein is normal both in its level of expression and its physical characteristics, indicative of a wild-type gene.

2. OSR4TR1

The results of the p53 and RB-1 protein analysis of the OSR4TR1 cell line indicated that the OSR4TR1 p53 protein was a mutant protein because of its reaction with the mutant specific p53 antibody. These results indicate that while the gene suffered no major alteration (deletion, duplication, etc) it had suffered a point mutation which results in a protein with a mutant profile. This mutant p53 profile has been observed in other transformed cells (Levine et al., (1991) Nature. Vol. 351, pp. 453-456; Hollstein et al., (1991) Science. Vol. 253, pp. 49-53; Gannon et al., (1990) EMBO J.. Vol. 9, pp. 1595-1602; Diller et al., (1990) Mol. Cell. BioL Vol. 10, pp. 5772-5781). The RB-1 protein was normal both in its level of expression and its physical characteristics, indicative of a wild-type gene.

3. OSR5TR2

The results of the p53 and RB-1 protein analysis of the OSR5TR2 cell line indicated that the OSR5TR2 p53 protein was deleted because we failed to detect p53 protein with either of the above described p53 antibodies. These results indicate that while the gene suffered no major alteration (deletion, duplication, etc) it had suffered a point mutation or a minor deletion which resulted in the loss of protein expression. The deletion of p53 protein in transformed cells has been observed previously (Levine et al., (1991) Nature. Vol. 351, pp. 453-456; Hollstein et al., (1991) Science. Vol. 253, pp. 49-53; Gannon et al., (1990) EMBO J.. Vol. 9, pp. 1595-1602; Diller et al., (1990) Mol. Cell. BioL Vol. 10, pp. 5772-5781). The RB-1 protein was normal both in its level of expression and its physical characteristics, indicative of a wild-type gene.

4. OSR-6 The results of the p53 and RB-1 gene analysis of the OSR-6 cell line indicated that both p53 and RB-1 genes were normal both in their level of expression and the gene's physical characteristics. 5. OSR-8 The results of the p53 and RB-1 protein analysis of the OSR-8 cell line indicated that both p53 and RB-1 protein were normal both in their level of expression and the protein's physical characteristics. C. Immediate Early Gene Expression Following Mitogen Stimulation

In order to characterize the immediate early gene (c-myc and c-fos) transcription following mitogen stimulation in the cell lines the following experiments are performed. Approximately 1 x 10** cells are grown to 70% confluence in a tissue culture flask in 25 ml of standard growth media. The cells are washed 2 times with serum-free media followed by the addition of 25 ml of serum-free culture media and incubated for 12-16 hours at 37°C under an atmosphere of 10% carbon dioxide in an incubator. To the serum-starved cells is added 3 ml (10v/v.%) of fetal bovine serum and 10 ug/ml of cycloheximide and the cells are incubated as above for 3 hours. One group of cells is not mitogen stimulated and remained serum-starved. RNA from the mitogen stimulated and non-stimulated cells is isolated using the RNAzol (Cinna Biotecx Inc.) methodology according to the manufacturer's recommendation. Briefly, 1 x 10** cells are lysed in situ with 10 ml of RNAzol, the lysate is collected, 1 ml of chloroform is added to the lysate, the samples are vortexed vigorously for 15 seconds, and the mixture is then centrifuged at 12,000 x g (4°C) for 15 minutes. The upper (aqueous) phase is transferred to a new tube, an equal volume of isopropanol is added to it, the samples are cooled to -20°C for 45 minutes, followed by pelleting of the RNA by centrifugation at 12,000 x g (4°C) for 15 minutes. The pelleted RNA is washed once with ice-cold 70% ethanol/water, dried, and resuspended in RNAse-free water at 20 ug/4.5 ul. RNA formaldehyde agarose gel electrophoresis is performed as described (Sambrook et al., (1989) Molecular Cloning. Cold Spring Harbor Press, Cold Spring Harbor, New York). Briefly, 20 ug of total cellular RNA is denatured by heating to 55°C for 15 minutes in denaturation buffer [4.5 ul RNA solution, 2.0 ul 10X RNA gel buffer (0.2M MOPS - pH 7.0, 50 mM sodium acetate, and 10 mM EDTA), 3.5 ul formaldehyde and 10.0 ul formamide] followed by the addition of 2 ul of loading buffer (50% glycerol, 1 mM EDTA, 0.4% bromophenol blue, and 0.4% xylene cyanol) and loading of the sample into a well of the formaldehyde gel (1% agarose, 20 mM MOPS - pH 7.0, 5 mM sodium acetate, 1 mM EDTA and 2.2 M formaldehyde). Electrophoresis is performed at 30 volts (constant voltage) for 16 hours. Following electrophoresis the gel is stained with ethidium bromide (0.5 ug/ml in water) for 1 hour, destained in water for 1 hour, and photographed under 300 nm ultraviolet light using a Foto/Prep I (Fotodyne) transilluminator. Following photography, the gel is transferred to nitrocellulose (Schleicher & Schuell, BA-S NC) using a LKB Vacugene vacublotting apparatus operating at 50 cm H2O with a 20X SSC (3 M sodium chloride and 0.3 M sodium citrate - pH 7.0) fluid transfer medium. Following transfer, the RNA is fixed to the nitrocellulose filter by UV irradiation using a Stratalinker (Stratagene Inc.) UV crosslinker at 0.12 Joules/cm². Following RNA fixation, the Northern blots are used in probe hydridization studies following previously described procedures (Sambrook et al., (1989) Molecular Cloning. Cold Spring Harbor Press, Cold Spring Harbor, New York) that are modified as follows. The probes of interest, c-fos (ATCC #41040) or c-myc (ATCC #41008) are radioactively labelled by using an Amersham nick translation kit following the manufacturer's recommendations. Briefly, 1 ug of probe DNA is incubated with IX nick translation buffer, 50 uCi alpha [³²P]-dCTP (NEN), and polymerase mix in a total volume of 20 ul at 15°C for 2 hours followed by the addition of 80 ul of IX STE (100 mM sodium chloride, 10 mM Tris - pH 8.0, and 1 mM EDTA). Separation of the incorporated from non-incorporated nucleotides is achieved using a Biospin column (BioRad). Typically 1 ug of probe had a specific activity of greater than 1 x 10⁸ dpm. Following nick translation, the probe is boiled for 10 minutes and added to a prehybridized filter (4 hours in hybridization solution at 42°C) in 10 ml of hybridization solution [6X SSPE (3M sodium chloride, 0.2 M sodium phosphate - pH 7.4 and 20 mM EDTA), 5X Denhardt's solution (1% Ficoll, 1% polyvinyl-pyrro- lidone, and 1% BSA - Pentax fraction V), 0.5% sodium dodecyl sulfate, 100 ug/ml denatured - sonicated salmon sperm DNA, and 50% formamide] and incubated for 48 hours at 42°C. Following hybridization the filters are washed at room temperature in 2X SSC - 0.1% SDS followed by a wash at 65°C with 0.2X SSC - 0.1% SDS. The filters are then dried, mounted to 3MM paper (Whatman) and autoradiography at -80 °C is performed using intensifying screens (NEN).

1. OSR3TR1

The OSR3TR1 cell line displayed an altered immediate early gene response with a constitutive expression of c-myc, even in the absence of added growth factor, and an absence of transcriptional upregulation of the c-fos gene in response to mitogen stimulation.

2. OSR4TR1

The OSR4TR1 cell line displayed an altered immediate early gene response with a constitutive expression of c-myc, even in the absence of added growth factor, and an absence of transcriptional upregulation of the c-fos gene in response to mitogen stimulation.

3. OSR5TR2

The OSR5TR2 cell line displayed an altered immediate early gene response with a constitutive expression of c-myc, even in the absence of added growth factor, and an absence of transcriptional upregulation of the c-fos gene in response to mitogen stimulation.

4. OSR-6

The OSR-6 cell line displayed an altered immediate early gene response with a constitutive expression of c-myc, even in the absence of added growth factor, and an absence of transcriptional upregulation of the c-fos gene in response to mitogen stimulation.

5. OSR-8

The OSR-8 cell line displayed an altered immediate early gene response which was exemplified by an absence of transcriptional upregulation of the c-myc and c-fos gene in response to mitogen stimulation. πi USE OF THE RAT OSTEOSARCOMA CELL LINES AS A SOURCE OF GROWTH FACTORS FROM OSR4TR1. OSR5TR2. OSR-6 AND OSR-8 The ability of a growth factor to stimulate, or inhibit DNA synthesis in a serum-starved quiescent cell is perhaps the most widely studied response to growth factors. This ability to stimulate, or inhibit the mitogenic response of a target cell is an indicator of the growth factor's ability to stimulate, or inhibit cellular proliferation. The mitogenic response of cells can be quantitated using a [³H]thymidine incorporation assay as previously described (Isfort (1990) Somatic Cell Molec. Genet.. Vol. 16, pp. 109-121). Briefly, target cells are plated at 2 x 10³ cells/well in a 96 well microtiter plate and incubated overnight in growth medium to allow the cells to attach. The growth medium is removed and the cells are washed three times with phosphate buffered saline (GIBCO) followed by an 8 hour incubation in 50 ul/well of serum-free medium. Mitogens such as serum and various growth factors are added to the serum starved cells in a volume of 50 ul of medium, with serum-free medium serving as a control. After 16 hours incubation, the cells are exposed to 1 uCi [³H] thymidine for 6 additional hours of incubation. The cells are then harvested and lysed on glass fiber filters using a PHD cell harvester (Cambridge Technology, Inc.) and the radioactivity in the samples is assayed by scintillation spectrophotometry. To test for the secretion of mitogenic growth factors by the rat osteosarcoma cells, serum-free conditioned medium is harvested from cell cultures. The cells are grown to confluency in a 150 cm² tissue culture flask. The growth medium is removed and the cell monolayer is rinsed three times with approximately 15 ml of phosphate buffered saline. The cells are then incubated in 25 ml of serum free medium for approximately 24 hours, and the conditioned medium is harvested and clarified by centrifugation for 15 minutes at 2000 x g to remove cells and cellular debris.

To gain some information on the types of growth factors produced by the rat osteosarcoma cells, the clarified conditioned medium is fractionated by heparin agarose chromatography (type I heparin agarose, purchased from Sigma Chemical Company). The binding properties of a variety of growth factors to heparin has been reported (Shing et al., (1984) Science. Vol. 223, pp. 1296-1298; Klagsbrun and Shing (1985) Proc. Natl. Acad. Sci. USA Vol. 82, pp. 805-809; Hauschka et al., (1986) I Biol. Chem.. Vol. 261, pp. 12665-12674), thus allowing an empirical classification of the types of growth factors. After collecting the flow through fraction of the conditioned medium, heparin binding factors are eluted in a stepwise application of 0.2M NaCl, 1.0M NaCl, and 2.0M NaCl, all in 25 mM Tris buffer, pH8.0. All fractions are dialyzed versus water and lyophilized. For analysis of mitogenic activity, the lyophilized fractions are resuspended in 2.0 ml of serum free medium and sterilized by centrifugal filtration (Centrex filters, Schleicher and Schuell).

A panel of target cells is used which incorporated osteoblastic, fibroblastic and multipotential mesenchymal cell types. These include the osteoblastic cells OSR-2 (ATCC No. CRL 11065) and MC3T3-E1 (Sudo et al., (1983) J. Cell BioL Vol. 96, pp. 191-198); the fibroblastic cell line NIH-3T3 (American Type Culture Collection, CRL 1658); the fibroblastic cell line MRC-5 (American Type Culture Collection, CCL 171); the multipotential cell line C3H10T1/2, clone 8 (American Type Culture Collection, CCL 226); and the multipotential cell population isolated from neonatal rat muscle (designated NRM) according to slight modifications of the procedure describe by Sampath et al., (1984) Proc. Natl. Acad. Sci. USA Vol. 81, pp. 3419- 3423. Briefly, the tricep muscles are aseptically isolated from euthanized newborn Sprague-Dawley rats and cleaned of connective and vascular tissue. The muscles are minced and cultured in 15 ml of 10% fetal bovine serum, 90% CMRL-1066 medium (GIBCO) supplemented with antibiotics and antimycotic mixture (penicillin, 100 units/ml; streptomycin, 100 ug/ml; Fungizone, 0.25 ug/ml, GIBCO). Once the explant cultures reached confluency in a 150 cm² tissue culture flask, the NRM cell line is designated as being at mean population doubling of 1. The NRM cell line is capable of forming multinucleate myotubes in confluent cultures and can be stimulated by TGF-βl to differentiate into chondrocyte-like cells (Seyedin et al., (1985) Proc. Natl. Acad. Sci. USA Vol. 82, pp. 2267-2271).

The mitogenic responses elicited by the heparin agarose fractionated conditioned medium from OSR3TR1, OSR4TR1, OSR5TR2, OSR-6 and OSR-8 cells are compared to a number of known growth factors. All growth factors are purchased from GIBCO BRL, Life Technologies, Inc., and are tested over a 3-4 log concentration range incorporating the effective concentrations suggested by the supplier. The growth factors are human recombinant platelet derived growth factor- AB heterodimer (PDGF-AB); human recombinant platelet derived growth factor-AA homodimer (PDGF-AA); human recombinant platelet derived growth factor-BB homodimer (PDGF-BB); human recombinant acidic fibroblast growth factor (aFGF); human recombinant basic fibroblast growth factor (bFGF); human recombinant epidermal growth factor (EGF); human recombinant insulin-like growth factor I (IGF- I); human recombinant insulin-like growth factor II (IGF-II); human recombinant transforming growth factor beta, type 1 (TGF-βl); human recombinant interleukin 1- beta (IL-lβ); recombinant murine leukemia inhibitory factor (LIF); recombinant murine tumor necrosis factor alpha (TNF-α). In addition, a mixture of bone morphogenic proteins (BMP-2, BMP-3, BMP-4, and BMP-7) as isolated from bovine bone by Koenig et al., (1991) J. Bone Mineral Res.. Vol. 6, pp. 206 is included in the analysis. The BMPs have been shown to induce the formation of cartilage and bone in vivo (reviewed by Wozney (1989) Progress in Growth Factor Research. Vol. 1, pp. 267-280) and heparin affinity chromatography is used in the purification of the bone derived BMP mixture. The results of this analysis is shown in Table 2 (below). IV. GROWTH FACTORS PRODUCED BY THE CELL LINES A. Growth Factors Produced Bv OSR3TR1 Cell Line The results of the mitogenicity assay (Table 2) indicate that OSR3TR1 cells produce an inhibitory growth factor and three mitogenic growth factors that can be fractionated from the serum-free conditioned medium by heparin agarose chromatography. The inhibitory growth factor present in the heparin agarose flow through fraction appears to be a novel factor since none of the known inhibitory cytokines are reported to have such broad target cell specificity (Wang and Hsu (1986) Trends Biochem. Sci.. Vol. 11, pp. 24-26). 1. Mitogenic Growth Factors Produced bv OSR3TR1 Cell line

For the larger scale generation of serum-free conditioned medium from OSR3TR1 cell cultures, the cell population is expanded to twenty 150 cm² flasks. After the cells reached confluency, the growth medium is decanted from the flasks, the attached cells are rinsed three times with approximately 15 ml of phosphate buffered saline, and 25 ml of serum-free medium per flask is added. The cell cultures are incubated at 37°C for 24 hours in an atmosphere of 5% CO2/95% air with a relative humidity of about 95%. The conditioned serum-free medium is harvested, clarified by centrifugation for 15 minutes at 2000 x g, and stored frozen at -20°C. The cell cultures are replenished with 25 ml of serum containing growth medium and cultured for one or two days. This recovery period is followed by another cycle of incubation in serum-free medium, as described above. Twelve cycles of serum-free medium incubation followed by serum containing growth medium recovery are employed and a total of about 6 liters of serum-free conditioned medium is collected.

The frozen serum-free conditioned media harvested from OSR3TR1 cells is thawed and applied to a heparin agarose column (Type I, Sigma) with a bed volume of approximately 90 ml. After collecting the flow through fraction as successive one liter batches, proteins binding to the heparin agarose are eluted with a 1000 ml linear gradient of 25 mM Tris buffer, pH8.0, to 2.0 M NaCl, 25 mM Tris buffer, pH8.0. Approximately 15 ml fractions are collected. Aliquots of 10 ml from the flow through and 1.0 ml from three consecutive fractions of the gradient are dialyzed against H2O, and lyophilized. The lyophilized fractions are resuspended in 2.0 ml of serum-free medium, sterilized by centrifugal filtration (Centrex filters, Schleicher and Schuell), and assayed for mitogenic activity as described above. The osteoblastic OSR-2 cells (ATCC No. CRL 11065), the osteoblastic MC3T3-E1 cells (Sudo et al., (1983) I Cell BioL Vol. 96, pp. 181-193) and the fibroblastic NIH-3T3 cells (American Type Culture Collection, CRL 1658) are used as target cells. The mitogenic response of cells is quantitated using a [ H]thymidine incorporation assay as previously described (Isfort (1990) Somatic Cell Molec. Genet.. Vol. 16, pp. 109-121). Briefly, target cells are plated at 2 x 10³ cells/well in a 96 well microtiter plate and incubated overnight in growth medium to allow the cells to attach. The growth medium is removed and the cells are washed three times with phosphate buffered saline (GIBCO) followed by an 8 hour incubation in 50 ul/well of serum-free medium. The heparin agarose fractions are added to the serum starved cells in a volume of 50 ul of medium, with serum-free medium serving as a control and 20% FBS (10% FBS final concentration) serving as a positive mitogenic control. After 16 hours incubation, 10 ul of serum-free medium containing 1 uCi of [³H]thymidine (Amersham, 5 Ci/mmol, 185 MBq/mmol) are added to each well and the cells are incubated for 6 additional hours. The cells are then harvested and lysed on glass fiber filters using a PHD cell harvester (Cambridge Technology, Inc.) and the radioactivity in the samples is assayed by scintillation spectrophotometry. The data are calculated from the mean dpms of quadruplicate samples of the experimental groups and expressed as the fold incorporation relative to the serum-free treated cells. The heparin agarose fractionation of mitogenic growth factors from the serum-free conditioned medium of OSR3TR1 cells is shown in Figures 1A and IB. The fractions containing the various heparin binding growth factors are indicated by a horizontal line. The fractions indicated by "#1", "#2", "#3", and "#4" indicate the first, second, third, and fourth heparin binding growth factors, respectively (Figure lb).

Figure 1 shows the heparin agarose chromatography of OSR3TR1 conditioned medium. Panel A; absorbance at 280 nm of the pooled fractions is recorded, and NaCl concentration is determined by conductivity measurements of selected fractions. Panel B; mitogenic activity of the pooled fractions using OSR-2 cells, MC3T3-E1 cells, and NIH-3T3 cells (note, data for fractions 10-12, 13-15, 16- 18, are not obtained for NIH-3T3). 2. Non-Heparin Binding Growth Factor Produced by OSR3TR1 Cell Line

The heparin agarose flow through fraction in aforementioned process is concentrated from approximately 6 liters to 200 ml using an Amicon RU2000 spiral concentrator with a nominal molecular weight cut off of about 10 kDa. The concentrated flow through is diluted with 400 ml of 25 mM Tris buffer, pH8.0, and applied to a DEAE-Sephacel column (purchased from Sigma Chemical Company) with a bed volume of approximately 90 ml. After collecting the flow through fraction, the proteins bound to the DEAE-Sephacel column are eluted with a 1000 ml linear gradient from 25 mM Tris buffer, pH8.0, to 2.0 M NaCl, 25 mM Tris buffer, pH8.0. Aliquots of 10 ml from the flow through and 1.0 ml from three consecutive fractions of the gradient are dialyzed against H2O, and lyophilized. The lyophilized fractions are resuspended in 2.0 ml of serum-free medium, sterilized by centrifugal filtration (Centrex filters, Schleicher and Schuell), and assayed for mitogenic activity as described below. The osteoblastic OSR-2 cells (ATCC No. CRL 11065), the osteoblastic MC3T3-E1 cells (Sudo et al., (1983) J. Cell BioL Vol. 96, pp. 181-193), the multipotential NRM cell population (described above), and the fibroblastic NIH- 3T3 cells (American Type Culture Collection, CRL 1658) are used as target cells. The mitogenic response of cells is quantitated using a [ H]thymidine incorporation assay as previously described (Isfort (1990) Somatic Cell Molec. Genet.. Vol. 16, pp. 109-121). Briefly, target cells are plated at 2 x 10³ cells/well in a 96 well microtiter plate and incubated overnight in growth medium to allow the cells to attach. The growth medium is removed and the cells are washed three times with phosphate buffered saline (GIBCO) followed by an 8 hour incubation in 50 ul/well of serum-free medium. The DEAE-Sephacel fractions are added to the serum starved cells in a volume of 50 ul of medium, with serum-free medium serving as a control and 20% FBS (10% FBS final concentration) serving as a positive mitogenic control. After 16 hours incubation, 10 ul of serum-free medium containing 1 uCi of [ H]thymidine (Amersham, 5 Ci mmol, 185 MBq/mmol) are added to each well and the cells are incubated for 6 additional hours. The cells are then harvested and lysed on glass fiber filters using a PHD cell harvester (Cambridge Technology, Inc.) and the radioactivity in the samples is assayed by scintillation spectrophotometry. The data are calculated from the mean dpms of quadruplicate samples of the experimental groups and expressed as the fold incorporation relative to the serum-free treated cells. The results of DEAE-Sephacel fractionation are shown in Figures 2A and 2B. Figure 2A illustrates mitogenic activity of the pooled fractions using OSR-2 and MC3T3-E1 as target cells. Figure 2B illustrates mitogenic activity of the pooled DEAE-Sephacel flow through fractions using NIH-3T3 and NRM as target cells. The fraction containing the mesenchymal growth inhibitory factor is indicated as #1. The fractions eluting from DEAE-Sephacel from more than 0 to about 0.2M NaCl containing the osteoblast/fibroblast growth factor are indicated as #2.

The results of these analyses demonstrate that OSR3TR1 cells produce the following inhibitory and growth factors: i. A mesenchymal inhibitory growth factor which has an ability to inhibit the proliferation of cells of mesenchymal origin. The mesenchymal inhibitory growth factor does not bind to heparin agarose and does not bind to DEAE- Sephacel. ii. A non-heparin binding growth factor that preferentially stimulates the proliferation of osteoblastic cells and fibroblasts, but not multipotent cells of mesenchymal origin. The non-heparin binding growth factor binds to DEAE- Sephacel and elutes in the 0-0.2M NaCl range, iii. A first heparin binding growth factor present in the 0-0.2M NaCl eluate that preferentially stimulates the proliferation of fibroblasts. iv. A second heparin binding growth factor present in the 0.3-0.7 M NaCl eluate that stimulates the proliferation of some osteoblastic cells (OSR-2 cells, ATCC No. 11065) and fibroblasts (NIH-3T3). This factor may be a member of the TGF-B/BMP family of growth factors. v. A third heparin binding growth factor present in the 1.0-1.3M NaCl eluate that stimulates the proliferation of osteoblastic cells and fibroblasts. This factor may be a member of the FGF family of growth factors, vi. A fourth heparin binding growth factor present in the 1.5-1.7M NaCl eluate the stimulates the proliferation of osteoblastic cells and fibroblasts. This factor may be a member of the FGF family of growth factors.

B. Growth Factors Produced bv OSR4TR1 Cell Line

The results of the mitogenicity assay shown in Table 2 and Figure 3, indicate that OSR4TR1 cells produce four mitogenic growth factors. The non-heparin binding mitogenic growth factor has an ability to stimulate osteoblast cells and multipotent cells. The first heparin binding mitogenic growth factor having a heparin agarose elution property of from greater than 0 to less than or equal to 0.2M NaCl, has an ability to stimulate osteoblast cells and fibroblast cells. The second heparin binding mitogenic growth factor having a heparin agarose elution property of from greater than 0.2 to less than or equal to 1.0M NaCl, has an ability to stimulate the prolifera- tion of osteoblast cell, multipotent cell and fibroblast cell. The third heparin binding mitogenic growth factor having a heparin agarose elution property of from greater than 1.0 to about 2.0M NaCl, has an ability to stimulate the proliferation of osteoblast cells and fibroblast cells.

1. Osteoblastic Differentiation Growth Factor Produced by OSR4TR1 Cells The C3H10T1/2 cell line (American Type Culture Collection, CCL 226) has been shown to be capable of expressing multiple new phenotypes when stimulated in an appropriate manner (Taylor and Jones (1979) Cell. Vol. 17, pp. 771-779). In order to determine if the serum-free conditioned medium from OSR4TR1 cells are able to stimulate the osteoblastic differentiation of C3H10T1/2 cells a MATRIGEL growth assay is performed. Osteoblastic cells show a characteristic canalicular cell processes which form a network when cultured on MATRIGEL (Vukicevic et al., (1990) CelL Vol. 63, pp. 437-445). Briefly, C3H10T1/2 cells are plated in 24 well culture dishes at a seeding density of approximately 50,000 cells per well in 1 ml of growth medium and allowed to attach overnight. The next day, the media is removed from the wells, the cells are washed three times with phosphate buffered saline and 1 ml of OSR4TR1 conditioned serum-free medium is applied. The OSR4TR1 conditioned media is prepared as described above for the detection of mitogenic growth factors. In addition, various growth factors are added in 1 ml of serum-free medium. Serum-free medium and growth medium are added to cells and served as experimental controls. The cells are further cultured for 48 hours under standard conditions 37°C under 5% carbon dioxide/95% air atmosphere. After 48 hours, the cells are transferred to 24 well culture plates containing gelled MATRIGEL along with 0.5 ml of the experimentally conditioned medium to which the cells are exposed. In the cases of serum-free media, fetal bovine serum is added in 0.5 ml of fresh medium such that the final concentration is 2.5%. After 3 days of additional culture, the growth pattern on MATRIGEL is photographed.

As can be seen in Figure 4, the MATRIGEL growth characteristics of the C3H10T1/2 cells in the presence of either C3H10T1/2 conditioned media alone or C3H10T1/2 conditioned media and 5% fetal bovine serum resembles a characteristic "endothelial-Iike" pattern previously described for human umbilical vein endothelial cells (Grant et al., (1989) CeH, Vol. 58, pp. 933-943). In comparison, the OSR-4TR1 conditioned media resulted in a change in the MATRIGEL growth characteristic of the C3H10T1/2 cell line from the "endothelial-like" pattern to the "canalicular" pattern which is typical for osteoblastic cells (for example, see MC3T3E1 growth pattern in Figure 4. None of the other growth factors tested, neither the bone morphogenic proteins, TGF-β, PDGF-AB, EGF nor aFGF affect the MATRIGEL growth pattern of the C3H10T1/2 cells (Figure 4). Accordingly, the OSR4TR1 cell line has an ability to produce a differentiation growth factor. Figure 4 shows the MATRIGEL growth characteristics of the C3H10T1/2 cell line in the presence of various growth factors and OSR4TR1 conditioned media. MATRIGEL growth is performed as described in the text. A. MATRIGEL growth of MC3T3E1 cells showing typical canalicular growth of an osteoblastic cell line. B. MATRIGEL growth of C3H10T1/2 cells in the presence of C3H10T1/2 cell conditioned media showing typical "endothelial-like" growth pattern. C. MATRIGEL growth of C3H10T1/2 cells in the presence of C3H10T1/2 cell conditioned media and 5% fetal bovine serum. D. MATRIGEL growth of C3H10T1/2 cells in the presence of OSR4TR1 conditioned media showing canalicular growth pattern. E. MATRIGEL growth characteristics of the C3H10T1/2 cells in the presence of bone morphogenic proteins. F. MATRIGEL growth characteristics of the C3H10T1/2 cells in the presence of TGF-β. G. MATRIGEL growth characteristics of the C3H10T1/2 cells in the presence of PDGF-AB. H. MATRIGEL growth characteristics of the C3H10T1/2 cells in the presence of EGF. I. MATRIGEL growth characteristics of the C3H10T1/2 cells in the presence of aFGF.

The results of these analyses demonstrate that OSR4TR1 cells produce the following growth and differentiation factors: i. An osteoblastic differentiation growth factor which has an ability to stimulate the differentiation of multipotent cells to osteoblastic cells; ii. A non-heparin binding mitogenic growth factor which has an inability to stimulate the proliferation of osteoblast cells and multipotent cells, iii. A first heparin binding growth factor which has a heparin agarose elution property of from greater than 0 to less than or equal to 0.2M NaCl, and an ability to stimulate the proliferation of osteoblast cells and fibroblast cells. iv. A second heparin binding grow factor which has a heparin agarose elution property of from greater than 0.2 to less than or equal to 1.0M NaCl, and an ability to stimulate the proliferation of osteoblast cells, fibroblast cells, and multipotent cells, v. A third heparin binding growth factor which has a heparin agarose elution property of from greater than 1.0 to about 2.0M NaCl, and an ability to stimulate the proliferation of osteoblast cells and fibroblast cells. C. Growth Factors Produced bv OSR5TR2 Cell Line

The results of the mitogenicity assay (Table 2 and Figure 5) indicate that OSR5TR2 cells produce 4 mitogenic growth factors that are secreted into the serum- free conditioned medium. The non-heparin binding mitogenic growth factor has an ability to stimulate the proliferation of OSR-2 cells. The first heparin binding mitogenic growth factor eluting from 0 to not greater than 0.2M from heparin, has an ability to stimulate the proliferation of OSR-2 cells. The second heparin binding mitogenic growth factor eluting from more than 0.2 to not greater than 1.0M NaCl from heparin, has an ability to stimulate the proliferation of OSR-2 and MC3T3-E1 cells. The third heparin binding mitogenic growth factor eluting from about more than 1.0 to not greater than 2.0M NaCl from heparin, has an ability to stimulate the proliferation of OSR-2 and MC3T3-E1 cells.

1. Osteoblastic Differentiation Growth Factor produced by OSR5TR2 Cells The C3H10T1/2 cell line (American Type Culture Collection, CCL 226) has been shown to be capable of expressing multiple new phenotypes when stimulated in an appropriate manner (Taylor and Jones (1979) Cell. Vol. 17, pp. 771-779). In order to determine if the serum-free conditioned medium from OSR5TR2 cells is able to stimulate the osteoblastic differentiation of C3H10T1/2 cells a MATRIGEL growth assay is performed. Osteoblastic cells show a characteristic canalicular cell processes which form a network when cultured on MATRIGEL (Vukicevic et al., (1990) Cell, Vol. 63:437-445). Briefly, C3H10T1/2 cells are plated in 24 well culture dishes at a seeding density of approximately 50,000 cells per well in 1 ml of growth medium and allowed to attach overnight. The next day, the media is removed from the wells, the cells are washed three times with phosphate buffered saline and 1 ml of OSR5TR2 conditioned serum-free medium is applied. The OSR5TR2 conditioned media is prepared as described above for the detection of mitogenic growth factors. In addition, various growth factors are added in 1 ml of serum-free medium. Serum- free medium and growth medium are added to cells and served as experimental controls. The cells are further cultured for 48 hours under standard conditions 37°C under 5% carbon dioxide/95% air atmosphere. After 48 hours, the cells are transferred to 24 well culture plates containing gelled MATRIGEL along with 0.5 ml of the experimentally conditioned medium to which the cells are exposed. In the cases of serum-free media, fetal bovine serum is added in 0.5 ml of fresh medium such that the final concentration was 2.5%. After 3 days of additional culture, the growth pattern on MATRIGEL is photographed (Figure 6).

As can be seen in Figure 6, the MATRIGEL growth characteristics of the C3H10T1/2 cells in the presence of either C3H10T1/2 conditioned media alone or C3H10T1/2 conditioned media and 5% fetal bovine serum resembles a characteristic "endothelial-like" pattern previously described for human umbilical vein endothelial cells (Grant et al., (1989) Cell, Vol. 58, pp. 933-943). In comparison, the OSR5TR2 conditioned media results in a change in the MATRIGEL growth characteristic of the C3H10T1/2 cell line from the "endothelial-like" pattern to the "canalicular" pattern which is typical for osteoblastic cells (for example, see the MC3T3-E1 growth pattern in Figure 6). None of the other growth factors tested, neither the bone morphogenic proteins, TGF-β, PDGF-AB, EGF nor aFGF affect the MATRIGEL growth pattern of the C3H10T1/2 cells (Figure 6). Accordingly, the OSR5TR2 cell line produces a differentiation growth factor stimulating the differentiation of C3H10T1/2 cells. Figure 6 shows that MATRIGEL growth characteristics of the C3H10T1/2 cell line in the presence of various growth factors and OSR5TR2 conditioned media. MATRIGEL growth was performed as described in the text. A. MATRIGEL growth of MC3T3-E1 cells showing typical canalicular growth of a osteoblastic cell line. B. MATRIGEL growth of C3H10T1/2 cells in the presence of C3H10T1/2 cell conditioned media showing typical "endothelial-like" growth pattern. C. MATRIGEL growth of C3H10T1/2 cells in the presence of C3H10T1/2 cell conditioned media and 5% fetal bovine serum. D. MATRIGEL growth of C3H10T1/2 cells in the presence of OSR5TR2 conditioned media showing canalicular growth pattern. E. MATRIGEL growth characteristics of the C3H10T1/2 cells in the presence of bone morphogenic proteins. F. MATRIGEL growth characteristics of the C3H10T1/2 cells in the presence of TGF-β. G. MATRIGEL growth characteristics of the C3H10T1/2 cells in the presence of PDGF-AB. H. MATRIGEL growth characteristics of the C3H10T1/2 cells in the presence of EGF. I. MATRIGEL growth characteristics of the C3H10T1/2 cells in the presence of aFGF.

D. Growth Factors Produced Bv OSR-6 Cell Line

The results of the mitogenicity assay (Table 2) indicate that OSR-6 cells produce a number of growth factors that can be fractionated from the serum-free conditioned medium by heparin agarose chromatography. Based on the selective stimulation of the 0.2M NaCl, l.OM NaCl, and 2.0M NaCl fractions, the identity of the growth factors present in these fractions is not readily apparent, as none of the known growth factors tested show a similar profile of mitogenic activity for the target cells in the panel. In particular, the mitogenic growth factor, or growth factors, present in the 0-0.2M NaCl elute preferentially stimulated the proliferation of osteoblastic cells and not multipotential cells and fibroblasts.

1. Mitogenic Growth Factors Produced bv OSR-6 Cell Line

OSR-6 cells are cultured in roller bottles (Corning, 850 cm² surface area) for the large scale generation of serum-free conditioned medium. Briefly, OSR-6 cells from a confluent 150 cm² flask are transferred to a roller bottle containing approximately 100 ml of growth medium and the roller bottle is sealed after introducing at atmosphere of 5% CO2/95% air. A total of 40 roller bottles are seeded with OSR-6 cells. The roller bottle cell cultures are incubated at 37°C using a roller apparatus (Wheaton) adjusted to approximately 2 rpm of the roller bottles. After the cells reaches confluency, the growth medium is decanted from the roller bottle, the attached cells are rinsed twice with approximately 50 ml of phosphate buffered saline, and 150 ml of serum-free medium per roller bottle is added. The cell cultures are gassed with the 5% CO2/95% air mixture and cultured with rotation at 37o for 48 hours. The conditioned serum-free medium is harvested, clarified by centrifugation, and stored frozen at -20°C. The cell cultures are replenished with serum containing growth medium and cultured for two days. This recovery period is followed by another cycle of incubation in serum-free medium, as described above. After the second recovery period, the volume of serum-free medium is reduced to 100 ml per roller bottle and the cell cultures are incubated for 24 hours, followed by a two-day recovery period in serum containing growth medium. Four additional cycles of serum-free medium incubation followed by serum containing growth medium recovery are employed. The last three cycles use 30 roller bottle cultures. A total of 29 liters of serum-free conditioned media is collected.

2. Heparin Agarose Elution Procedure

Ten liters of the frozen serum-free conditioned media harvested from roller bottle cultures of OSR-6 cells are thawed and two liters were directly applied to a heparin agarose column (Type I, Sigma) with a bed volume of approximately 500 ml. Because the column became clogged, the remaining 8 liters of conditioned media is mixed with the heparin agarose in two successive rounds of batch binding 4 x 1 liter of conditioned media per 100 ml of heparin agarose. The conditioned media and heparin agarose are mixed by gently rocking the slurry for 2 hours at 4°C. The heparin agarose is allowed to settle and the supernatant carefully decanted. After the second round of batch binding, the heparin agarose is resuspended in approximately 100 ml of 25 mM tris buffer, pH 8.0, per 100 ml of heparin agarose and combined with the remaining heparin agarose from the original column. A new column is poured and equilibrated with 25 mM Tris buffer, pH 8.0. Proteins binding to the heparin agarose are eluted with a 2 liter linear gradient of 25 mM Tris buffer, pH 8.0, to 2.0 M NaCl, 25 mM tris buffer, pH 8.0. Approximately 15 ml fractions are collected. Aliquots of 10 ml from the flow through and 1.0 ml from three consecutive fractions of the gradient are dialyzed against H2O, and lyophilized. The lyophilized fractions are resuspended in 2.0 ml of serum-free medium, sterilized by centrifugal filtration (Centrex filters, Schleicher and Schuell), and assayed for mitogenic activity. The osteoblastic OSR-2 cells (American Type Culture Collection No. CRL 11065) and the fibroblastic NIH-3T3 cells (American Type Culture Collection No. CRL 1658) are used as target cells. The mitogenic response of cells is quantitated using a [³H]thymidine incorporated assay as previously described (Isfort (1990) Somatic Cell Molec. Genet. 16:109-121). Briefly, target cells are plated at 2 x 10³ cells/well in a 96 well microtiter plate and incubated overnight in growth medium to allow the cells to attach. The growth medium is removed and the cells are washed three times with phosphate buffered saline (GIBCO) followed by an 8 hour incubation in 50 ul well of serum-free medium. The heparin agarose fractions are added to the serum starved cells in a volume of 50 ul of medium, with serum-free medium serving as a control and 20% FBS (10% FBS final concentration) serving as a positive mitogenic control. After 16 hours incubation, 10 ul of serum-free medium containing 1 uCi of [³H]thymidine (Amersham, 5 Ci/mmol, 185 MBq/mmol) are added to each well and the cells are incubated for 6 additional hours. The cells are then harvested and lysed on glass fiber filters using a PHD cell harvester (Cambridge Technology, Inc.) and the radioactivity in the samples is assayed by scintillation spectrophotometry. The data is calculated from the mean dpms of quadruplicate samples of the experimental groups and expressed as the fold incorporation relative to the serum-free treated cells. The heparin agarose fractionation of mitogenic growth factors from the serum-free conditioned medium of OSR-6 cells is shown in Figure 7. The fractions containing the various heparin binding growth factors are indicated in Figure 7B and Figure 7C wherein "#1", "#2", "#3", "#4", "#5" and "#6" refer to the first, second, third, fourth, fifth and sixth heparin binding growth factors, respectively. The results of these analyses demonstrate that OSR-6 cells produce the following growth factors: i. A non-heparin binding growth factor that stimulates the proliferation of fibroblasts and osteoblast cells. ii. A first heparin binding, osteoblast specific growth factor present in the 0-0.2M NaCl eluate from heparin agarose chromatography which preferentially stimulates the proliferation of osteoblast cells, iii. A second heparin binding growth factor present in the 0.2-0.4M NaCl eluate that stimulates the proliferation of fibroblasts and osteoblast cells. iv. A third heparin binding growth factor present in the 0.5-0.7M NaCl eluate that stimulates the proliferation of osteoblast cells and fibroblasts. v. A fourth heparin binding growth factor present in the 0.7-0.9M NaCl eluate which stimulates the proliferation of osteoblast cells. vi. A fifth heparin binding growth factor present in the 1.0-1.2M NaCl eluate which stimulates the proliferation of osteoblast cells and fibroblasts. vii. A sixth heparin binding growth factor present in the 1.5-1.8M NaCl eluate which stimulates the proliferation of osteoblast cells and fibroblasts.

E. Growth Factors Produced bv OSR-8 Cell Line The results of the mitogenicity assay (Table 2 and Figure 8) indicate that

OSR-8 cells produce a number of growth factors that can be fractionated from the serum-free conditioned medium by heparin agarose chromatography. Based on the selective stimulation of the flow through, 0.2M NaCl, and l.OM NaCl fractions, the identity of the growth factors present in these fractions is not readily apparent, as none of the known growth factors tested show a similar profile of mitogenic activity for the target cells in the panel. The mitogenic growth factors present in the flow through fraction and the 0.2M NaCl elute preferentially stimulated the proliferation of fibroblasts. The mitogenic growth factor, or growth factors, present in the l.OM

NaCl fraction preferentially stimulated the proliferation of osteoblastic cells. The mitogenic activity present in the 2.0M NaCl fraction may be attributed to a member of the FGF family of growth factors (Burgess and Maciag (1989) Annu. Rev. Biochem.

Vol. 58, pp. 575-606), or a novel growth factor.

The result of this analysis demonstrates that OSR-8 cells produce the following growth factors: i. A non-heparin binding growth factor that preferentially stimulates fibroblasts and multipotent cells of mesenchymal origin, ii. A first heparin binding growth factor present in the 0-0.2M NaCl eluate that stimulates fibroblasts and preferentially stimulates some osteoblastic cells, iii. A second heparin binding, osteoblast specific growth factor present in the 0.2- l.OM NaCl eluate from heparin agarose chromatography that preferentially stimulates osteoblastic cells, iv. A third heparin binding growth factor present in the 1.0-2.0 M NaCl eluate that stimulates osteoblastic cells and fibroblasts which may be a member of the FGF family of growth factors.

The characteristics of the novel cell lines of the present invention and the results of the mitogenesis analysis are shown in Tables 1 and 2, respectively.

TABLE 1 Characterizations of Cell Lines

OSR3TR1 OSR4TR1 OSR5TR2 OSR-6 OSR-8 p53 protein non- mutated non- normal normal expressed expressed

RB-1 gene* normal normal normal normal normal c-fos gene* normal normal normal normal normal c-myc gene* amplified 50x normal amplified lOx normal normal

MATRIGEL growth monolayer canalicular canalicular canalicular canalicular pattern tumorigenic in yes yes yes yes poor congenitally athymic mice alkaline phosphatase normal no no low no activity ability to be serially propagated greater than yes yes yes yes yes

60 population doublings deregulated immediate yes yes yes yes early gene yes response heparin binding yes yes yes yes yes growth factors osteoblastic differentiation no yes yes no no growth factor mesenchymal inhibitory yes no no no no growth factor

"as compared to a non-tumorigenic Sprague-Dawley rat cell control TABLE 2 Mitogenesis Results

OSR3TR1

HepAg FT (-) (-) (-) (-) (-) ND

0.2M NaCl ND 0 + 0 + ND l.OM NaCl + 0 + 0 + ND

2.0M NaCl + + + + + ND

OSR4TR1

HepAg FT + 0 + 0 0 ND

0.2M NaCl + 0 0 0 + ND l.OM NaCl + + + + + ND

2.0M NaCl + + 0 0 + ND

OSR5TR2 Cond. Med. + + + + + ND

HepAg FT + 0 ND ND ND ND

0.2M NaCl + 0 ND ND ND ND l.OM NaCl + + ND ND ND ND

2.0M NaCl + + ND ND ND ND

OSR-6

HepAg FT + 0 0 0 0 +

0.2M NaCl + + 0 0 0 ND l.OM NaCl 0 + 0 0 + ND

2.0M NaCl + 0 0 0 0 ND

OSR-8

Cond. Med. + + + ND ND +

HepAg FT 0 0 ND ND ND +

0.2M NaCl 0 0 ND ND ND + l .OM NaCl + + ND ND ND 0

2.0M NaCl 0 + ND ND ND 0

Table Legend: Comparison of proliferative effects of a variety of mitogens on target cells. 0 = no stimulation (equivalent to 0% serum control), + = mitogenic stimulation at least 50% above the 0% serum control, - = inhibition of proliferation at least 50% of 0% serum control. ND = not determined. Deposit of Cell Lines

OSR3TR1 was deposited at the ATCC on June 5, 1992. The deposited strain was assigned Accession No. CRL 11066.

OSR4TR1 was deposited at the ATCC on June 5, 1992. The deposited strain was assigned Accession No. CRL 11067.

OSR5TR2 was deposited at the ATCC on June 5, 1992. The deposited strain was assigned Accession No. CRL 11068.

OSR-6 was deposited at the ATCC on June 5, 1992. The deposited strain was assigned Accession No. CRL 11069.

OSR-8 was deposited at the ATCC on June 5, 1992. The deposited strain was assigned Accession No. CRL 11070.

The subject cells have been deposited under conditions that assure access to the cultures will be available as required by patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that Applicants' granting of permission to the depository to distribute samples of the deposit does not constitute an express or implied license to practice the invention claimed in any patent issuing on the subject application or any other patent.

The subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposits, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The Applicants' acknowledge the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.

As is recognized in the art, there are occasionally errors in DNA and amino acid sequencing methods. As a result, the sequences encoded in the deposited material are incorporated herein by reference and controlling in the event of an error in any of the sequences found in the written description of the present invention. It is further noted that one of ordinary skill in the art reproducing Applicants' work from the written disclosure can discover any sequencing errors using routine skill. The deposit of ATCC Accession No. CRL 11066, CRL 11067, CRL 11068, CRL 11069, and CRL 11070 is not to be considered as an admission that the deposited material is essential to the practice of the present invention.

All publications mentioned hereinabove are hereby incorporated in their entirety by reference. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to one skilled in the art and are to be included in the spirit and purview of this application and scope of the appended claims.

Claims

1. A rat osteosarcoma cell characterized in that the cell has all the identifying characteristics of American Type Culture Collection Accession No. CRL 11066, CRL 11067, CRL 11068, CRL 11069, or CRL 11070.

2. A growth factor characterized in that the growth factor has the characteristics of a growth factor isolated from a rat osteosarcoma cell, wherein the rat osteosarcoma cell has all the identifying characteristics of American Type Culture Collection Accession No. CRL 11066, CRL 11067, CRL 11068, CRL 11069, or CRL 11070.

3. A process for producing a growth factor characterized in that the process comprises growing a rat osteosarcoma cell line in a culture medium and recovering the growth factor from the medium, wherein the rat osteosarcoma cell line has all the identifying characteristics of American Type Culture Collection Accession No. CRL 11066, CRL 11067, CRL 11068, CRL 11069, or CRL 11070.

4. A process for producing a growth factor characterized in that the process comprises a. expressing the cDNA in host, and b. recovering a recombinant form of the growth factor wherein the cDNA is obtained by or is the same as that obtained by c. isolating mRNA from a rat osteosarcoma cell line which codes for the growth factor, d. making cDNA of the mRNA and the rat osteosarcoma cell line has all the identifying characteristics of American Type Culture Collection Accession No. CRL 11066, CRL 11067, CRL 11068, CRL 11069 or

CRL 11070.

5. A growth factor characterized in that it is produced by a rat osteosarcoma cell line having all the identifying characteristics of American Type Culture Collection Accession No. CRL 11066, CRL 11067, CRL 11068, CRL 11069, or CRL 11070.

6. The growth factor of Claim 5 characterized in that the growth factor is an osteoblastic differentiation growth factor and the cell line has all the identifying characteristics of American Type Culture Collection Accession No. CRL 11067 or CRL 11068.

7. The growth factor of Claim 5 characterized in that the growth factor is a mesenchymal inhibitory growth factor and the cell line has all the identifying characteristics of American Type Culture Collection Accession No. CRL 11066.