EP0929661A4 - Novel method of culturing human epithelial cells for the identification of cancer therapeutics and diagnostics - Google Patents

Abstract

Methods are provided for obtaining primary cultures of human breast, colonic and prostate epithelial cells using a fibroblast co-culture tissue culture system. The methods can be used to obtain normal, precancerous and malignant epithelial cell cultures. Methods are also provided for deriving substantially pure epithelial cell strains from the primary cultures, and for immortalizing the cell strains to provide cell lines. The primary cultures, cell strains and cell lines are used in biological and biochemical in vitro screening and/or diagnostic assays.

Description

NOVEL METHOD OF CULTURING HUMAN EPITHELIAL

CELLS FOR THE IDENTIFICATION OF

CANCER THERAPEUTICS AND DIAGNOSTICS

Technical Field

The present invention relates to novel methods of obtaining human breast, colonic and prostate epithelial cell cultures, the cultures and/or cell lines thereby obtained and their biological and biochemical applications.

Background

Neoplastic disease (cancer) , constitutes a complex illness of multifactorial origin that defies simple categorization. Depending on the nature of the population at risk and the environment, the initiation of the cancer process may involve interactions between various factors, such as: environmental carcinogens or pollutants; diet; radiation; oncogenic viruses; chronic mechanical or thermal trauma; infection; genetic predisposition and the aging process.

Cancer remains a leading cause of death in Western societies. Cancer can occur in any organ of the body, in tissues of ectoder al, mesodermal, or endodermal origin. Despite the identification of a number of relatively effective therapies such as surgery, radiotherapy and chemotherapy in the treatment of neoplastic disease, the annual morbidity and mortality caused by the most common cancers, such as mammary (breast) cancer, colon cancer and prostate cancer is staggering. Thus a considerable amount of

-l- bio-medical research continues to probe the molecular basis of these diseases, and a majority of this research relies on investigation of cells or tissue growing in culture. It is generally accepted that cancer cells develop from normal tissue cells through a series of events which lead to malignancies. Such malignant cells grow without the restraints that regulate normal tissue growth (e.g., differentiation, organ size limitation or hormonal regulation) . In this manner, cancer can occur as a result of genetic mutation in a cell's regulatory genes which normally encode proteins that control cell division, differentiation or initiate cell death. There is a significant overlap in the pattern of mutated genes carried by different tumor cell types, such as between bladder and colon cancer cells, and among individual tumors of the same type. Mutations in the p53 gene, for example, are found in over half of all human tumors. See, e.g., Harris,

CC. (1994) Science 262:1980. Harris et al. (1993) N . Engl . J . Med . 329:1315, Harper et al. (1992) Cell 7_5:805, Vogelstein et al. (1992) Cell 7_.0:523, and Hollstein et al. (1991) Science 253:49. However, there are also differences in the genetic changes carried among individual tumors, such that some tumors will carry a specific mutation while others will not. Even within the same tumor, there may be several genetically distinct clonal variants. Such variance causes the exact molecular mechanisms involved in the development of cancer to remain largely unknown.

Tissue and/or cell culture techniques offer a way to explore the molecular basis of cancer, to identify potential genotypic and/or phenotypic cell markers which can be used as indicators of cancerous cells in clinical cancer screening, and to assay the effect of cancer therapeutics against neoplastic tissue or cells. In this regard, it would be useful to provide epithelial cell cultures derived from normal tissue, pre-cancerous tissue and cancerous tissue—all obtained from the same tissue source. These cultures could be manipulated to provide

"genetically altered cells" to mimic various genetic events thought to be associated with the cancer process. Further, it would be useful if a "conditional" immortalized epithelial cell culture system could be established to investigate cells in three distinct stages of the immortalization process: before immortalization; while immortalized; and when the immortalization system is turned off.

A number of techniques exist for the growth of breast epithelial cells in culture. See, e.g.,

Taylor-Papadiraitriou et al., "Culture of Human Mammary Epithelial Cells," Cul ture of Epithelial Cells , Eds. Wiley-Liss (1992) . Normal cells from skin (e.g., keratinocytes and melanocytes) are also capable of growth in cell culture.

For example, U.S. Patent No. 4,423,145, issued 27 December 1983 to Stampfer et al., describes a method of isolating and culturing human mammary epithelial cells to provide a clonal growth of cells suitable for quantitative assessment of the cytotoxicity of selected compounds.

International Publication No. WO 90/15862, published 27 December 1990, describes a method of in vitro cultivation of epithelial cells using a population of fibroblast cells as a feeder layer for the epithelial cells wherein the cultivation is sufficient to obtain differentiation in the epithelial cells.

Band et al. (1989) Proc . Natl . Acad . Sci . USA ___: 1249-1253 describe a medium that supports long- term growth in culture of human primary mammary tumor cells, normal epithelial cells and of mammary tumor cell lines.

Band et al. (1990) Proc . Natl . Acad . Sci .

USA JT7:463-467 describe the immortalization of normal human mammary epithelial cells by a plasmid containing the linearized genome of Human papilloma virus types

16 or 18.

Halbert et al. (1991) J . Virol . 65(1) :473-

478 describe the contribution of the E6 and E7 open reading frames of human papilloma virus type 6b

(HPV6b) and HPV16 in the immortalization of primary human foreskin epithelial cells.

Taylor-Papadimitriou et al., "Culture of

Human Mammary Epithelial Cells," Cul ture of Epithelial Cells , Eds. Wiley-Liss (1992) describe materials and methods for the culture of human mammary epithelial cells, and criteria for identification of epithelial cells in culture.

However, none of the above-described art discloses cell culture methods for establishing human breast, colonic and prostate epithelial cell cultures comprising normal, precancerous or malignant cells.

Further, the art fails to disclose methods of providing a series of normal, precancerous and immortalized epithelial cell cultures wherein the cultures are all derived from the same tissue source or individual.

Summary of the Invention Accordingly, the present invention provides novel cell culturing systems that allow for the establishment of human breast, colonic and prostate epithelial cell cultures comprising normal, precancerous, immortalized or malignant cells. These systems are used to provide cell lines useful for screening agents which are specifically toxic to precancerous or malignant cells relative to normal cells, and for identifying agents capable of differentiating between precancerous or malignant cells and normal cells in clinical diagnosis of tissue or biological fluid samples. In one embodiment of the invention, a method is provided for establishing a primary culture of human epithelial cells using a fibroblast co-culture system. The method entails first isolating epithelial cell masses from a tissue sample obtained from a human subject. The cell masses are then generally physically and enzymatically treated to yield epithelial cell clusters substantially free from stromal and other cellular material. Thus, after a feeder cell population has been cultured onto a substrate in a suitable medium, the epithelial cell clusters are applied to a membranous support arranged over the feeder cell population such that the membrane prevents co-mingling of the two cell types while allowing for the free diffusion of soluble moieties between the cell populations such as growth factors produced by the feeder cells and the like. Outgrowth from the cell clusters onto the support provides a cell culture system capable of supporting the continued in vitro growth of the epithelial cells. In another embodiment of the invention, a method is provided for passaging human epithelial cells from a primary co-culture system as described above to obtain a substantially pure epithelial cell culture. The method entails initiating a fresh feeder cell population of human skin fibroblasts on a second substrate and in a suitable medium. The epithelial cells from the primary co-culture are then passaged by first selectively detaching non-epithelial cells from the membranous support and then transferring the support having the outgrowth of epithelial cells from the primary co-culture by arranging the substrate over the fresh feeder cell population whereby a substantially pure epithelial cell culture is maintained on the support. This process may be repeated a number of times as needed in order to obtain a substantially pure epithelial cell culture. in further related embodiments of the invention, a passaged epithelial cell strain can be genetically manipulated to construct precursor cells of human cancers. Particularly, passaged cell strains can be subjected to homologous recombination in order to introduce mutations into target genes such as tumor suppressor genes or the like. Transfection techniques are used to introduce various exogenous nucleotide sequences into the cells which can further carry any number of distinct mutations. Introduction of DNA tumor virus genes, or portions thereof, can be used to selectively inactivate gene products of tumor suppressors, or to immortalize the cells to provide cell lines.

In yet a further embodiment of the invention, a method is provided whereby in vitro assays can be conveniently carried out to determine the toxicity of an agent toward human epithelial cells of normal, precancerous, immortalized and malignant phenotype. The method initially entails providing a population of human epithelial cells grown in a co- culture system constructed according to the invention. Thus, healthy, growing epithelial cells are removed from the support and re-suspended in a volume of fresh medium. After the cells are counted using known techniques, a volume corresponding to a particular cell population is repetitively plated on fresh supports in a suitable substrate. A portion of the freshly plated cells are then exposed to an agent of interest for a selected period of time. The exposure can generally be terminated by aspirating off the medium containing the agent, washing the cells and adding fresh media. After a sufficient time has passed to allow the cells to proliferate to a point where it is possible to distinguish those cells which continue to grow from those which have ceased to grow, agent toxicity is calculated by comparing the number of viable cells in a treated group with a control group of untreated cells.

In additional related embodiments, the above described assay method is carried out using genetically altered cells having mutations in selected genes such as tumor suppressor genes or the like.

Such cells are constructed by transfecting DNA into the cells to provide mutations in target genes (tumor suppressors) by homologous recombination events, or by other transfection techniques wherein various exogenous nucleotide sequences carrying genetic mutations are introduced into the cells (e.g., inactivating mutations or selected mutations corresponding to those found in human tumors) .

In one particular embodiment, a conditional immortalization system is constructed wherein portions of a population of epithelial cells cultured under the invention are assayed as described above prior to immortalization, while actively immortalized and when the immortalization system is silent or inactivated. Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or can be learned by practice of the invention.

Detailed Description of the Invention

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual , Second Edition (1989) ; Oligonucleotide Synthesis (M.J. Gait ed. 1984); Immo- bilized Cells and Enzymes (IRL press, 1986) ; the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and CC. Blackwell eds., 1986, Blackwell Scientific Publications) ; Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition) ; and Fundamental Virology, 2nd Edition, vol. I & II (B.N. Fields and D.M. Knipe, eds.).

A. Definitions:

Before the invention is described in detail, it is to be understood that this invention is not limited to the specific embodiments described herein as such may vary while remaining within the spirit of the invention. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

Under the invention, the terms "cell culture" and "tissue culture" may be used interchangeably and denote the maintenance of cells in vitro , in suspension culture in a liquid medium or on a surface such as glass, plastic or agar provided with a liquid medium. In general, "cell culture" necessitates a medium that is buffered to maintain a conεtant suitable pH. Media used in cell culture are generally formulated to include an adequate supply of necessary nutrients and can be osmotically tailored to the particular cells being maintained, with temperature and gas phase also being controlled within suitable limits. Cell culture techniques are well known in the art. See, e . g . , Morgan et al., Animal Cell Culture , BIOS Scientific Publishers, Oxford, UK (1993) , and Adams, R.L.P. Cell Culture for

Biochemists , Second Edition, Elsevier (1990) .

The term "co-culture" is used herein to refer to a cell culture system wherein a population of cells, termed "feeder cells," is established in a cell culture in order to facilitate the growth of a subsequently added cell population of interest. Feeder cells can be previously irradiated or otherwise treated to prevent their division in the culture. Not being limited by any particular theory, mechanisms by which feeder cells assist in the growth of the cells of interest include: metabolic cooperation, wherein nucleotides, nutrients and hormonal moieties such as growth factors are passed to the cell culture via gap junctions; digestion of potentially toxic cell debris; provision of initial stimulus to cells via cell-to- cell contact; or combinations thereof. Under the invention, fibroblast cells or other mesenchymal cells, either syngeneic, allogeneic or xenogeneic, are used as feeder cells to maintain an epithelial cell culture. A number of epithelial co-culture systems have been described in the art. See, e . g. , Taylor- Papadi itriou et al. (1977) Int . J . Cancer 20:903-908 , Armstrong et al. (1978) Cancer Res . 3_8:984-998, and Kirkland et al. (1979) J . of Nat . Cancer Inst . 63:29- 42.

The term "epithelial cell" is used herein to denote differentiated cells derived from epithelial tissue such as breast, colon and prostate tissue. Major epithelial cell types include basal and luminal cells and are isolated from the continuous layers of cells forming both internal and external tissue surfaces. Epithelial cells derive from both the ectodermal and endodermal embryonic cell layers. Cultured epithelial cells can be grown on glass or plastic substrates to which they adhere due to the secretion of proteins such as laminin and collagen. The terms "fibroblast" and "fibroblast cell" denote a type of stellate connective cell found in fibrous tissue that is responsible for collagen synthesis in cartilage, tendon, cornea, etc. Cultured fibroblast cells are adherent cells and are normally grown on glass or plastic substrates. Cultured fibroblasts have the general morphology of tissue fibroblasts (flat, elongated or triangular-shaped cells) , however they are not as differentiated as true fibroblasts. Fibroblasts derive from embryonic mesoderm and the cells that grow in culture appear to be mesodermal stem cells. A number of continuous murine fibroblast cell lines have been described. Todaro et al. (1963) J . Cell Biol . r7:299, Aaronson et al. (1968) J. Cell Physiol . 72_.: 141, and Jainchill et al. (1969) J . Virol . 4_:549. A number of finite human fibroblast cell strains have also been described. Jacobs et al. (1970) Wature 227: 168, Nichols et al. (1977) Science 196:60, and Hay et al. (eds) (1992) American Tissue Type Cul ture Collection , 7th Edition ATCC, Rockville, MD.

As used herein the term "normal cell" intends to refer to any cell directly derived from essentially normal cells or tissue. Thus, under the invention, "normal epithelial cells" are epithelial cells which are not of tumor cell origin, are not transformed in any detectable way and have not been i morta1ized. A "malignant cell" refers to a cell directly derived from cancerous cells (or tissue) which have undergone phenotypic transformation, such as but not limited to, transformation by oncogenes, protooncogenes, TS mutations, or by other such mechanisms. "Malignant cells" are generally characterized by their capacity for invasive, unregulated growth. Such cells have one or more phenotypic derangements which can be expressed as alterations in cellular membranes, in the levels of certain cellular enzymes (e.g., enzymes involved in nucleic acid synthesis and metabolism) , or by the appearance of inappropriate gene products.

A "precancerous cell" refers to a cell directly derived from cells or tissue having a pathological condition which is likely to develop into cancer. "Precancerous cells" have undergone genetic damage but are not yet capable of invasive, unregulated growth, metastasis or the like. Under the invention, such cells can exhibit a precancerous phenotype or relate to an inherited (genetic) cancer predisposition such as genetic polymorphism of enzymes involved in activation and detoxification of carcinogens, or germline mutations in tumor suppressor genes (e.g., tumor suppressor genes which are defective in retinoblastoma (Rb) and adenomatous polyposis coli (APC) ) .

As used herein, the term "primary culture" refers to a culture of cells that are directly derived from cells or tissue taken from an organism without intermediate culture. The cells or tissue from which a primary culture is derived is termed an explant. Cells are generally considered to be "primary cells" until they are subcultured. "Primary cells" will grow for a variable but finite length of time in culture, after which time they senesce and eventually die. Under the invention, primary cultures can be derived from a variety of tissue sources and a number of techniques for their isolation from human tissue are known in the art. See, e . g . , Whitley et al. (1987) Mol . Cell . Endocrinol . 52.:279 and Fickling et al. (1992) Exp . Cell Res . 201:517.

"Passage" refers to the act of subculturing a cell population. A "subculture" intends to refer to a cell culture established by the inoculation of fresh sterile medium with a sample from a previous culture. Each repeated subculture is counted as one passaging event.

A "cell strain" is a population of cells derived from a primary culture using subcultivation techniques. Thus, a primary culture can be subcultured into two or more new cultures and the subculturing repeated at periodic intervals for several months to maintain the cell strain.

A "cell line" refers to a population of cells derived from a single explant which are characterized as having the potential for unlimited growth in vitro . Under the invention, a cell line can be isolated from a primary culture based on its ability to survive and continue to grow in culture. Cell lines are frequently aneuploid due to an in vitro transformation event, and the capacity of cell lines to grow and divide indefinitely in culture is generally associated with an aneuploid karyotype. Cell lines which have been derived originally from tumor tissue may have been transformed in vivo , although not all neoplastic cell populations have the capacity to grow indefinitely in vitro . Further, cell lines generally retain their differentiated character through many rounds of division.

A "clonal cell line" denotes a population of apparently genetically identical cells grown in culture from a single, isolated cell—provided that the cells have not undergone further differentiation in culture. Thus, a "clonal cell line" refers to the propagation of apparently identical daughter cells from a single parent cell.

An "immortalized cell" is a cell which by virtue of a transformation event, e.g. , infection with virus, becomes capable of indefinite growth and division. Immortalized cell lines are genetically altered cells which have been derived from a primary culture or cell strain to produce a continuously growing cell line. Under the invention, a cell line can be immortalized using various transfection techniques known in the art. Exemplary techniques are described further below.

A "transformed cell," or a "transformed cell line" is a cell or cell line, respectively, which is either derived from a tumor cell or has been manipulated in some way (e.g., by transfection with oncogenes or treatment with carcinogens) to produce a cell or cell line that expresses a novel transformed phenotype. See, e . g . , Moore et al. (1966) J. Natl . Cancer Inst . , 3_6:405. The transformed phenotype may manifest itself in a number of ways, such as an acquired capacity for unregulated growth (resembling the growth of cancer cells) , a newly acquired anchorage independence in adherent cells or by reduced serum and growth factor requirements. A cell can be transformed following infection with a virus such as by SV40 or polyoma virus. A transformed cell also has the capacity for unlimited growth in culture. "Transfection" refers to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. In this manner, the exogenous DNA may or may not be integrated (covalently linked) to chromosomal DNA making up the genome of the cell. In procaryotes and yeasts, for example, the exogenous DNA can be maintained on an episomal element, such as a plasmid. With respect to the invention, a eucaryotic cell is "stably transfected" when exogenous DNA has become integrated into the cellular genome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA. "Transient transfection" refers to cases where exogenous DNA does not remain in the cells for an extended period of time, e.g., where plasmid DNA is transcribed into mRNA and translated into protein without integration into the host cell genome. A number of transfection techniques are known in the art. See, e.g., Graham et al. (1973) Virology,

5_2:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual , Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.

A "host cell" is a cell which has been transfected, or is capable of transfection, by an exogenous DNA sequence.

DNA "control sequences" refer collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell. Not all of these control sequences need always be present so long as the desired gene is capable of being replicated, transcribed and translated in an appropriate packaging cell. A control sequence "directs the transcription" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.

Two DNA or polypeptide sequences are "substantially homologous" when at least about 80% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, volε J & TJ, supra; Nucleic Acid Hybridization , supra. In one embodiment of the invention, a novel method of establishing a primary culture of human epithelial cellε using a fibroblast co-culture system is provided. An explant taken from a human breast, colon or prostate tissue sample is initially treated to isolate epithelial cell clumps substantially free of stromal and other cellular material. Treatment of the tissue sample to remove epithelial cell clumps from the stromal matrix and other tissue cells involves physical separation techniques such as laceration and scraping, enzymatic separation techniques such aε uεing a digeεtion medium containing proteolytic enzymes, and combinations thereof.

A feeder cell population is initiated by plating a population of human skin fibroblasts onto a suitable substrate and growing those cellε in a tiεsue culture medium under suitable tissue culture conditions. The feeder cells can be selected from an established cell strain of human or murine fibroblasts such as, but not limited to cell types Detroit 551, MRC-5, PH140SK, 3T3 or NIH/3T3. See, e.g., U.S.

Patent No. 4,423,145 to Stampfer et al., and Rickwood et al. (eds) Animal Cells Cul ture and Media , BIOS Scientific Publishers Ltd, Oxford, UK (1994) . In one particular embodiment of the invention, the fibroblasts are obtained from the same tissue sample as the epithelial cells of interest. Thus, an explant of connective tissue derived from the same tissue sample used to isolate the epithelial clumps can be plated under suitable tissue culture conditions to provide a primary culture of human fibroblasts using techniques well known in the art. Suitable cell culture substrates are generally a container that can be sterilized, does not leach toxic factors and does not distort microscopy images. Thus plates formed from glass and plastic are suitable substrates under the invention. Plastic containers may further be treated to encourage cell attachment using techniques known in the art. Ramsey et al. (1984) In Vitro ^0:802. Suitable tissue culture media generally consist of an isotonic, buffered, basal nutrient medium which provides an energy source, coupled with inorganic salts, amino acids, vitaminε and various supplements. Supplements may include serum (e.g. , fetal calf serum, or the like) various antibiotics to prevent contamination or to provide selective conditions, attachment and growth factors, or the like. A number of media formulations are known in the art, such as, but not limited to, minimal essential medium (MEM) , Rosewell Park Memorial Institute (RPMI) 1640 or Dulbecco's modified Eagle's medium (DMEM) . Suitable tissue culture conditions are also known in the art. See, e.g., Morgan et al.,

Animal Cell Culture , BIOS Scientific Publishers Ltd., Oxford, UK (1993) , and Adams, R.L.P. Cell Cul ture for Biochemists , Second Edition, Elsevier (1990).

After the fibroblast feeder cells have been plated as described above, allowed to attach to the substrate, and grown for at least about 12 hours, a removable membranous support iε arranged over the fibroblast culture. Suitable membranous supports under the invention include microporous, permeable films developed for tissue culture which generally permit the free permeation of substances such as soluble nutrients, metabolites and hormonal factors through the membrane while preventing cell migration therethrough. Such supports are available from, for example, Costar® (Cambridge, MA) and include Transwell™ supports (e.g., Transwell-Col membranes having a membrane thickness of 25-50μm, pore sizes from 0.4-3.0μm and which are treated with Types I and II collagen derived from bovine placentae) . Other suitable supports include fibronectin-, collagen Type I- and laminin-treated membranes, Matragel™ membranes, or the like.

Once the isolated epithelial cell clumps are subεtantially freed from other tissue cells, the clumps are finely divided to provide epithelial cell clusters that are as small as possible. The cell clusters (explants) are then brought into εuspension in a suitable medium and applied to the membranous support arranged over the culture of human skin fibroblast feeder cells. The co-culture system thus provided is incubated under suitable tissue culture conditions. After a suitable time (generally about 2- 3 days) , outgrowth from the explant to the support occurs, whereby a primary culture of human epithelial cells is formed capable of further in vitro growth. Under the invention, the above described methods can be used to produce primary cultures of normal human epithelial cells derived from samples of human breast, colon or prostate tissue. In like manner, primary cell culture systems derived from excised cancerous or malignant breast, colon or prostate tissue can also be produced. These systemε have, for the most part, not been obtainable in the prior art, primarily due to the lack of suitable in vitro tissue culture methods for establishing well- defined, malignant epithelial cells.

In further related embodiments of the invention, human subjects are identified which expresε precancerous phenotypes, such as those individualε carrying mutated tumor suppressor genes or the like. Tissue samples can be biopsied from appropriate organs from those individuals, and the above described methods used to establish primary epithelial cell cultures of precancerous cells.

More particularly, it is generally recognized that one of the earliest genetic events which can initiate tumorigenesis occurs within a specific class of genes known as "tumor suppressorε. " Proteins which are encoded by these genes are essential in the control of normal cell growth. Thus, inherited mutation of a tumor suppressor gene may render an individual at high risk for certain types of cancer. As a consequence of mutational damage to such genes, the internal metabolism of the cell may be altered, leading to abnormal growth characteristics. Not being limited by any particular theory, genes which encode growth-stimulating products can be induced by the inherited mutation, leading to a condition in which a higher incidence of genetic mutation is likely to occur ("hypermutable state") . The hypermutable state accelerates emergence of mutationally damaged cells that are capable of unregulated growth, leading to cancer. One of the most common dominantly inherited human diseases, neurofibromatosis NF1, or von Recklinghausen NF, affectε one in 4000 children, half of whom carry germ line mutations in the NF1 gene. NF1 is generally recognized as a tumor suppressor gene that may be involved in the regulation of the p21ras signal transduction pathway. Inactivating mutations in NF1 have been connected with increased intracellular levels of p21ras-GTP, suggesting that inactivation of NF1 leads to abnormal cell growth. Neurofibromatosis is characterized by the variable expression of features such as neurofibromas, cafe-au- lait mascules, predisposition to certain malignant tumors such as schwannoma, glioma, pheochromocytoma, and, at low frequency, leukemia and rhabdomyosarcoma.

For breast cancer, several early acting tumor suppressor genes have been identified, including the p53, Rb, and BRCA1 genes. See, e.g., Harris, CC (1994) Science 262:1980, Harris et al. (1993) N . Engl . J . Med . 329:1315, Harper et al. (1992) Cell 75:805, Vogelstein et al. (1992) Cell 7_.0.523, Hollstein et al. (1991) Science 253 :49, Lee et al. (1988) Science 241:218-221. and Miki et al. (1994) Science 266:66. Thuε, it iε a particular object of the invention to provide breast epithelial cell cultures derived from tissue samples taken from individuals expressing mutations in one or more of these genes. Such culture systemε provide valuable in vitro models for the analysis and characterization of tumor suppressor genes and their participation in the cancer process.

In colon cancer, the APC, MSH₂ and MSH₃ genes are thought to be involved in tumor suppresεion. See, e . g. , Spirio et al. (1993) Cell 7^:951-957; Fishel et al. (1993) Cell 75:1027-1038; Joslyn et al. (1991) Cell 6^:601-613; and Gorden et al. (1991) Cell 66:589- 600. Particularly, germline mutations in the APC gene, which predisposes individuals to adenomatous polyposis coli, dramatically increases the risk of colon cancer, and the APC gene is frequently inactivated in the majority of sporadic colon tumors. Miyoshi et al. (1992) Hum . Mol . Genet . 1:229. Thuε, in a further embodiment of the invention, human epithelial cell cultureε are initiated from colon tissue samples taken from individuals expressing mutations in those genes. Once primary cultures have been obtained as described above, epithelial cells from those cultures can be passaged using tissue culture techniques known in the art. After several pasεages, an outgrowth of epithelial cells capable of many population doublings, and even growth as individual clones is obtainable.

Thus, in a further embodiment of the invention, a method is provided for passaging human breast, colon and prostate epithelial cells from the primary cultures establiεhed above. In the maintenance of the primary cultureε, epithelial cells are not permitted to reach confluence on the membranous supports in order to avoid diminished growth potential upon subculture. Thus, when the cells approach confluence, they are pasεaged to a fresh co-culture system. Pasεaging techniqueε are used under the invention to obtain substantially pure epithelial cell cultures and to establish subcultures thereof. In order to obtain a substantially pure epithelial cell culture from the primary cultures initiated above, a fresh culture of fibroblast feeder cells is establiεhed uεing techniqueε aε previously described. The feeder cells are allowed to adhere to the fresh substrate and grow for at least about 12 hours. In this regard, it is generally preferable to use the same fibroblast line with which the co-culture εystem was started. However, if a fibroblast line has also been established from the same source (e.g., the same tissue sample) from which the primary epithelial culture was derived, those cells can be used in place of an existing fibroblast cell line.

In passaging the cells, a selective detaching step is performed whereby non-epithelial cells are detached from the support. One particular technique, which iε not limiting in the invention, entails differential trypsinization techniques that are known in the art. Taylor-Papadimitriou et al., "Culture of Human Mammary Epithelial Cells," in Culture of Epithelial Cells , Eds. Wiley-Lisε (1992) . More specifically, after tisεue culture growth media iε removed or aεpirated, the εupport-bound tissue explant and any cell outgrowth can be washed in a saline-trypsin-versene (STV) εolution and allowed sit for about 1-2 minutes. Once fibroblast cells are observed to detach from the support while the epithelial cells remain adherent, the STV medium is removed, the cells waεhed and fed with freεh tissue culture medium.

After selectively detaching non-epithelial cells from the support, the support is positioned over the fresh fibroblast feeder cells and the epithelial culture maintained on the support. The process can be repeated a number of times to provide a substantially pure epithelial cell culture. Generally, the epithelial cells will grow actively through several passages, after which the cell population will gradually change morphology. At this time, breast epithelial cells begin to expresε fibrillar fibronectin, high levels of keratins and vimentin. Taylor-Papadimitriou et al. (1989) J . Cell . Sci . 94_.:403-413. After the epithelial cells undergo their morphological change, actively growing cells can be selected from the background of senescing cells. Such cells are then subcultured to establish an epithelial cell strain. Subculturing under the invention can be carried out using established tissue culture techniques. Generally, a fresh fibroblast cell culture is established as described above. The support is removed from the co-culture system and the remaining "conditioned" media is transferred to the fresh fibroblast culture. Epithelial cells are detached from the support using techniques known in the art. At this point, the cells may be counted and known populations seeded onto fresh εupportε arranged over a fresh fibroblast feeder culture to provide epithelial cell strains. In yet a further embodiment of the invention, precursor cells of human cancers can be constructed from the breast, colon and prostate epithelial cell strainε described above. Mutations to inactivate the gene products of suspected tumor suppressors can be introduced into those cell strains using homologous recombination or transfection techniques (e.g., using DNA tumor virus genes or potions thereof) .

Particularly, the tumor suppreεsor genes p53 and Rb, and the putative antioncogene BRCA1 are each believed to play a role in human breast cancer carcinogenesiε. Family studies indicate that p53 can be rate-limiting in breast cancer formation. Martin et al. (1990) Science 250: 1233-1238. In sporadic breast tumors, mutations in the p53 gene have either been found directly, or implicitly e.g., by εpecific loεε of heterozygosity. See e . g . , Harris, CC. (1994) Science 262: 1980, Harris et al. (1993) N . Engl . J . Med . 329:1315, Harper et al. (1992) Cell 75:805, Vogelstein et al. (1992) Cell 7J).523, Callahan et al. (1992) Cancer [Suppl ] .69:1582-1588, and Hollstein et al. (1991) Science 253:49-53. Although familial studies suggest that mutations in the Rb gene may not be strongly rate-limiting with respect to formation of breast tumors, such mutations may somewhat increase the risk of breast cancer. Somatic mutations in the Rb gene can be found in breast carcinomas, suggesting that aberrant alleles of this gene do have etiologic significance. Lee et al. (1988) Science 241:218-221. Additionally, mutations in the putative antioncogene BRCA1 is indicated in approximately 45% of families with high breast cancer incidence, and at least about 80% of families with increased incidence of both early-onset breast cancer and ovarian cancer. Miki et al. (1994) Science 266: 66-71; Ford et al. (1994) Lancet 343 : 692; and Easton et al. (1993) Breast Cancer linkage Consortium 52 : 678.

Precursor cells of human cancers are constructed from the human breast, colon and proεtate epithelial cell εtrains under the invention using transfection techniques to introduce exogenous DNA capable of inactivating tumor suppressor genes into the cells. There are generally two major steps in transfection: first, the exogenous DNA must traverse the recipient cell plasma membrane in order to be exposed to the cell's transcription and replication machinery; and second, the DNA must either become stably integrated into the host cell genome, or exist as an episomal moiety that is capable of extra- chromosomal replication at a sufficient rate. A number of transfection methods have been described in the art, such as calcium phosphate co-precipitation

(Graham et al. (1973) Virol . 5_2:456-467) , direct micro-injection into cultured cells (Capecchi, M.R. (1980) Cell .22:479-488) , eiectroporation (Shigekawa et al. (1988) BioTeα nigues 6_:742-751) , liposome mediated gene transfer (Mannino et al. (1988) BioTechniques _.6: 682-690), lipid-mediated transfection (Feigner et al. (1987) Proc . Natl . Acad . Sci . USA 84:7413-7417) , and nucleic acid delivery using high-velocity icroprojectiles (Klein et al. (1987) Nature 327:70- 73).

Aε explained above, in one embodiment of the invention, homologous recombination can be used to inactivate cellular genes such as p53 or Rb in the epithelial cell strains of the invention. Generally, a nucleotide sequence (or a gene) homologous to a gene of interest, or portion thereof, is introduced to the cell using transfection techniques. In this regard, the sequences of p53 and Rb have been described. Lamb et al. (1986) Mol . Cell . Biol . 6 : 1379-1385, Ewen et al. (1992) Science 255:85-87 _f Ewen et al. (1991) Cell j56_: 1155-1164, and Hu et al. (1990) EMBO J . 9_:1147- 1155. The introduced gene or nucleotide sequence can then combine into the identical sequence in the host genome by homologous recombination. A number of homologous recombination techniques have been described in the art. See, e .g. , Bollag et al. (1989) "Homologous Recombination in Mammalian Cells," in Ann. Rev . Gen . 2J3: 199-225, Capecchi, M.R. (1989) Science 244:1288-1292, Mansour et al. (1988) Nature 336:348- 352, and Smithies et al. (1985) Nature 317:230-234. Cellε in which successful homologous recombination events have occurred can be enriched for using selectable marker genes. Not being bound by any particular technique, epithelial cells can be transfected with a cloned p53 gene which has been disrupted by insertion of an aph gene (encoding resistance to geneticin) , rendering it nonfunctional. The aph gene lacks a promoter of its own such that random integration events do not lead to transcription of the gene, rendering those cells geneticin- sensitive. In targeted integrations, the aph genes becomes part of the targeted gene's mRNA, leading to a geneticin-resistant cell in conjunction with the disruption of the targeted gene. Culture of the cells may thus be carried out in media containing geneticin to select successful recombinants having a disrupted p53 gene.

In another embodiment, transfection techniques are employed to introduce genes which encode proteins that act in trans to inactivate tumor suppressor genes. Particularly, proteins encoded by the E6 and E7 genes of human papilloma virus 16

(HPV16) have been shown to bind to the p53 and Rb gene products, respectively. Howley, P.M. (1991) Cancer Res . [Suppl] 51:5019-5022. The E6/E7 open reading frames have been sequenced and cloned. Kaur et al. (1989) J . Gen . Virol . 7J).1261-1266. Binding of E6 to p53 and of E7 to Rb results in the loss of their normal growth inhibitory functions. Thus, transient transfection of epithelial cell strains uεing plasmid constructs containing HPV16 E6 and/or E7 can be used herein to construct precursor cells of human cancers from the above-described epithelial cell strains. Such cells can also be stably transformed using known techniques. In this manner, transcription and translation of the transfected E6 and/or E7 genes provides a basis for inactivation of either or both of the p53 or Rb gene products in transfected epithelial cellε.

In yet a further embodiment of the invention, epithelial cell strains derived from prostate, breast or colon tisεue are immortalized to provide cell lineε. The uεe of tranεfection techniques to introduce "immortalization genes" into primary cells in vitro has been described. See, e . g . , Fickling et al. (1992) Exp . Cell Res . 201:517, and Ambesi-Impiombato et al. (1980) Proc . Natl . Acad . Sci . USA 22:3455. These techniques allow cells to proliferate as immortal cell lines without suffering a concomitant loss of function. Use of immortalized cells constructed under the invention in, for example, cancer research avoids the problems associated with having to use cell lineε derived from malignant tiεεue which express a transformed phenotype.

One immortalization technique involves the introduction of the SV40 Large T gene (or T-antigen) into an epithelial cell strain. SV40 is a small (5kb genome) DNA virus of the Papovavirus family which iε capable of amphotropic infection. It is generally recognized that infection of quieεcent cellε by SV40 induceε synthesis of cellular DNA and cell replication. When cloned into plasmids and transfected into cells, SV40 T-antigen can induce proliferation in cells without changing their morphology, thereby possibly inducing immortality rather than transformation. Plasmids expressing SV40 large T protein have been used to immortalize a number of different primary cell types. See, e.g., Fickling et al. (1992) Exp . Cell Res . 201: 517 , and Ambesi- Impiombato et al. (1980) Proc . Natl . Acad . Sci . USA 77:3455.

However, certain problems have come to be associated with SV40 T-antigen immortalized cells. Cells which retain many of their in vivo characteristics in the quiescent state appear to lose such characteristicε when they proliferate. Many immortal cell lineε become genotypically abnormal, either losing genetic material--sometimes a number of entire chromosome —or gaining genetic material—in εome cases almost doubling chromosome number. These aneuploid cells have a reduced utility in cancer modeling, and such aberrant cells may become well adapted to cell culture conditions and aε εuch will grow fastest in culture and be naturally selected for by growth in culture. Additionally, the inappropriate growth of an immortalized cell that is of a type not normally required to traverse a large number of cell cycles may lead to inaccuracy in newly synthesized DNA, resulting in genetic mutation and a consequent change in cell characteristicε. In order to overcome some of these problems, temperature-sensitive mutants of SV40 Large T protein have been described. Jat et al. (1989) Mol . Cell Biol . 9_:1672. The mutant large T protein is fully functional at lower than normal temperatures (e.g., 33°C) and not functional when exposed to elevated temperatures (e.g., 37°C) . In this manner, transfected cells are able to proliferate at 33°C, but withdraw from the cell cycle at 37°C.

In a particular embodiment of the invention, human breast, colon and prostate epithelial cell strains are immortalized using amphotropic recombinant virus tranεfection to introduce the E6 and/or E7 genes of human papilloma virus 16 (HPV16) into the cells. Recombinant retrovirus vectors containing the E6 and/or E7 genes from HPV16 can be transfected into an ecotropic packaging cell line, and viruses produced by those cells used to infect an amphotropic packaging cell line as previously described. Miller et al. (1993) Methods in Enzymol . 2T7: 581-599, Halbert et al. (1991) J. Virol . 65_.(1) :473-478, and Miller et al. (1988) J . Virol . 62_.: 4337-4345. The retrovirus vectors are constructed to contain a selectable marker, whereby succesεfully immortalized cells can be selected for using an appropriate medium.

Under the invention, the primary cell cultures, passaged cell strainε, genetically altered cell strains and immortalized cell lines can be characterized by their expression of specific functional markers such as keratinocytes, hormonal and growth factor receptors and the like. Particularly, human breast epithelial cells may be phenotypically characterized by their expresεion of keratin 19 marker (luminal cell phenotype) , keratin 14 marker (baεal cell phenotype), keratin 18, Vimentin and Fibronectin uεing immunoaffinity and detection techniqueε known in the art. See, e.g., Hermanson et al. Immobilized Affinity Ligand Techniques (Academic Press, Inc., 1992) , and Handbook of Experimental Immunology , Vols. I-IV (D.M. Weir and CC Blackwell eds., 1986, Blackwell Scientific Publications) . Anti-keratin 19 monoclonal antibodies (e.g. BA16 and BA17, Bartek et al. (1985) J. Cell Sci . 2_.5:17-33), anti-keratin 14 monoclonal antibodies (e.g., LL001, Taylor- Papadimitriou et al. (1989) J . Cell Sci . 94:403-413 , and 12C8-1, Dairkee et al. (1985) Proc . Natl . Acad . Sci . USA .82_.:7409-7413) , anti-keratin 18 monoclonal antibodies (e.g., C04, Bartek et al. (1989) : in "Monoclonal Antibodies to Tumor Associated Antigens and Their Clinical Application , " Abelev G.I. (ed) , Akademai Kiado, Budapest, DA7, Lauerova et al. (1988) Hybridoma 2:495-504, and LE61, Lane, (1982) J . Cell . Biol . 9_2.:665-673) , anti-Vimentin monoclonal antibodies (e.g., V9, Osborn et al. (1984) Eur . J . Cell . Biol . 3_4: 137-143) , and anti-Fibronectin monoclonal antibodies (e.g., FN-3, Keen et al. (1984) Mol . Biol . Med. 2 : 15-27) have been described.

Thus, monoclonal antibodies specific for a selected cell marker (or a second antibody directed against the monoclonal antibody) , can be labeled with a suitable detectable chemical group, such as an enzyme, radioisotope, fluoreεcer, chromophore, luminescer or ligand for rapid and sensitive detection using techniques known in the art. Depending on the nature of the label, a number of techniques to detect the presence of the label are known, e.g., fluorometric, spectrophotometric, autoradiography, scintillation counting, and visual (e.g., colorimetric or chemiluminescence) techniques.

Further, estrogen is important in vivo in the regulation of growth and function of the mammary gland, and is indicated in promotion of breaεt epithelial cell proliferation. Haslam et al. (1980) J . Cell . Biol . _.86:730. Hormone receptors for estrogen (ER) are present in breast epithelial cells, and ER concentration varies depending on the developmental state of the mammary gland. Haslam et al. (1981) Endocrinology 108:825. Thus, breast epithelial cells can be further characterized by assay of ER wherein the cells are incubated with [³H] estradiol with or without radioinert estradiol, processed as previously described (Haslam, S.Z. (1986) Cancer Res . 46:310) , and the binding data analyzed by known methods (Scatchard, G. (1947) Ann . New York Acad . Sci . .51:660) . Other selected phenotypic markers, or fragments thereof, can be used as an antigen to produce antibodieε, either polyclonal, monoclonal, or both, by methods which are well known in the art. If polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horεe, pig, etc.) is immunized with a selected antigen or a fragment thereof. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures.

Monoclonal antibodies to a selected cell marker can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies using hybridoma technology is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniqueε εuch aε direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al . , Hybridoma Techniques (1980); Hammerling et al . , Monoclonal Antibodies and T-cell Hybridomas (1981) ; Kennett et al . , Monoclonal Antibodies (1980); see also U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890.

In yet another embodiment of the invention, a method is provided whereby in vitro assays are carried out using human breast, colon or prostate epithelial cell cultures prepared according to the invention. Thus, phenotypically normal, precancerous, and/or malignant cells can be screened to identify compounds with potential diagnostic efficacy. Such compounds include, but are not limited to, differential stains which allow detection of cancerous cells in tissue or fluid samples, or signal enhancers for radiographic images (e.g. , X-ray or MRI) .

Further, in vitro asεays are carried out under the invention using human breast, colon or prostate epithelial cell cultures, wherein the effect of noxious agents (e.g., as chemicals, chemotherapy drugs, radiation and the like) on phenotypically normal, precancerous and malignant cells can be determined. In this regard, agents which are capable of selectively killing, inhibiting the growth of, or inducing apoptosiε in cancerous cells or precancerous cells can be identified by screening agents in tandem against normal, precancerous and/or cancerous cells derived from the same tissue source.

In one particular aspect of the invention, a method is provided wherein a population of human epithelial cellε is established using the co-culture syεtem aε deεcribed above. The cells are prepared by detaching growing epithelial cellε from a co-culture support using techniques known in the art. The detached cells are then resuspended in a suitable medium and counted. A particular volume of the cell εuspension corresponding to a particular cell population is then repetitively plated on fresh supports in a suitable subεtrate, e.g. , a multiwell tissue culture plate. In this regard, it is generally desired to establiεh a number of identical co-cultureε from a single epithelial cell culture so as to provide an experimental serieε with control cultureε (non- treatment groups) , or the like.

After the fresh co-cultures have been plated and allowed to suitably adjust, a portion of the cellε are exposed to an agent or treatment of interest for a selected period of time. In this manner, a chemical or drug in solution with tissue culture medium is added to the co-culture system in an effective amount. After a sufficient period of time, the exposure to the agent or treatment is terminated by removing culture media containing the agent, washing with a suitable buffered wash solution (e.g. basal salts medium) and adding fresh growth media.

After the cellε have had a chance to proliferate to a point where it iε possible to distinguish between those cells which continue to grow from those which have ceased to grow, agent toxicity or treatment effectiveness can be calculated by comparing the number of viable cells in a treated group with a control group of untreated cells using techniques known in the art.

In another aεpect, the εubject in vitro assay method is practiced using the aforementioned genetically altered cells. Thus, transfected epithelial cells having inactivating mutations in tumor εuppressor genes such as p53, Rb, BRCA1, APC,

MSH₂ and MSH₃ (e.g., which have been introduced using homologous recombination events as described herein) are used to screen for differential stains, chemicals or chemotherapeutic agents effective in the diagnosis or selective killing of precancerous cells.

In yet another aspect of the invention, epithelial cell lines which have been immortalized by transfection with SV40 T-antigen, or HPV16 E6 and/or E7 genes as described above are used in the in vitro asεay method. Such cell lines can be selected to screen cells of a particular phenotype. In particular embodiments of the invention, the in vitro assay method is practiced using immortalized human breast epithelial cells expresεing either the keratin 14 or keratin 19 phenotype.

In another embodiment, an in vitro aεεay method is provided whereby a conditional immortalization system iε conεtructed in order to allow assay of a cell population prior to immortalization, while actively immortalized and when the immortalization system is inactive. Particularly, a human breast, colon or prostate epithelial cell strain is selected and portions of the strain subjected to immortalization using the temperature- εenεitive mutant SV40 Large T εystem described herein. The assay is carried out by subjecting cultures of the cell strain which have not been immortalized to the same experimental protocol as employed on cultures of the cell strain which have been immortalized. One of the immortalized cultureε is incubated at 33°C, the other at 37°C in order to provide cell populations under the effect of both active and inactive immortalization systems.

In one aspect of the invention, a conditional immortalization system is constructed using a tetracycline inducible transactivator (tTA) to control expression of transfected E6 and/or E7 genes in immortalized human breast, colon or prostate epithelial cells. Particularly, expression of E6 and/or E7 in transfected cells may be regulated using a tetracycline inducible transactivator (tTA) regulation syεtem containing control elementε of the tetracycline-reεistance operon encoded in TnIO of Escherichia coli fused with the activating domain of virion protein 16 of herpes simplex virus as previously described. Gosεen et al. (1992) Proc . Natl . Acad . Sci . USA 89_.:5547-5551. The tTA system allows differential control over expression of the subject genes, as well as reversible "on/off" εwitching. Integration of a luciferase reporter gene controlled by a tTA-dependent promoter in the above- described tTA system further allows efficient monitoring of E6 and/or E7 expression in response to various tetracycline concentrationε by assay of luciferase activity. Gossen et al., supra .

Thus, cultures of a cell strain which have not been immortalized are treated under the same experimental protocol which immortalized cells are subjected to, wherein the immortalized cells are divided into two groups and incubated in media with or without tetracycline to provide immortalized cell populations under the effect of both active and inactive immortalization systems.

The human breast, colon and prostate epithelial cell cultures of the invention can also be used in the context of in vitro asεayε to screen for potential carcinogens. Epithelial cells provide a useful model syεtem in carcinogen teεting εince they mimic more closely the usual situation in humans and animals, where carcinomas are typically derived from epithelial, not mesenchymal cells. Epithelial cell cultures of normal or precancerous phenotypes that are produced using the co-culture system of the present invention can be plated, allowed to adjust, and a portion thereof exposed to an agent suspected of being a carcinogen for a suitable period of time. Exposure to the agent is terminated by removal of culture media containing the agent, washing with a suitable buffered wash solution and adding fresh growth media. Transformation events which occur as a consequence of exposure to the agent can be identified by development of foci of transformed cells. Alternatively, transformation events can be monitored by injection of treated cellε (either sub-cutaneously or intraperitoneally) , into immunocompromised animal models (e.g., nude or SCID mice), wherein the ability of the treated cells to form solid tumors can be readily ascertained.

In yet another aspect of the invention, the epithelial cell cultures formed herein can be used in the context of in vitro assays to screen for agents capable of at least participating in inducing apoptotic events. Apoptosiε (or programmed cell death) , is distinguishable, both morphologically and functionally, from other types of cell death. Particularly, apoptosis can be ascertained morphologically by appearance of membrane blebbing, cytoplasmic shrinking, chromatin condensation, and digestion of the genomic DNA into fragments (e.g., formation of a "DNA ladder") . See, e . g . , Ellis et al. (1991) Annu . Rev . Cell Biol . 2:663-698; and Tomei et al., Eds. (1991) Apoptosis : The Molecular Basis of Cell Death , Cold Spring Harbor Laboratory Press, Plainview, NY. Disruption of the ability of a cell to enter apoptotic pathways may have serious consequences. For example, the survival of cells that normally should die can lead to cancer or autoimmune disease. Recently, it has been suggested that mutations in the human p53 gene can disable or derail the apoptotic pathway induced by DNA damage. Kastan et al. (1992) Cell 21:587; and Yin et al. (1992) Cell 20:937. Thuε, the ability to identify agents that may trigger apoptotic events in cells that are refractive to an apoptosis-inducing signal (e.g., DNA damage) provides a unique and promising approach to the identification of chemotherapeutics.

Epithelial cell strains comprising human cancer precursor cells that are formed aε described above can be provided, wherein the p53 gene has been mutated to yield cells that are refractive to DNA damage-induced apoptosis. Such cell strains can be cultured, exposed to ultraviolet light, exposed to agents suspected of being able to trigger apoptosis, and then examined for signs of apoptosis. In this regard, one of the hallmarks of apoptosis is the digestion of the genomic DNA of the dying cell into small fragments of about 180 base pairs (bps) , or multiples thereof (e.g., formation of DNA ladders) . Thus, the treated cells can be readily assayed for positive apoptotic events using gel electrophoresis techniques that are known in the art. It is to be underεtood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the description above as well aε the exampleε which follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

Examples

Materials and Methods A. General Cell Co-Culture Methods: A.l. Preparation of MCDB 170 complete medium.

The following were added to 485 mis of sterile MCDB 170 media (Cloneticε, San Diego, CA) : Tranεferrin (Sigma, St Louiε, MO) , 5 ug/ml final concentration; Hydrocortiεone (Sigma) , 0.5 ug/ final concentration; Bovine Pituitary Extract (Hammond Cell Technology, Alameda, CA) , 70 ug/ml final concentration; Insulin (Sigma) , 10 ug/ml final concentration; and EGF (Sigma) , 5 ng/ml final concentration.

A.2 Preparation of the enzyme solution.

Collagenase (Sigma, St Louis, MO) (1500 U/ml) was disεolved in appropriate amountε of MCDB 170 complete medium at 37°C and εterilized by filtering through a 0.22 μm filter. Hyaluronidaεe (Sigma) (1000 U/ml) waε diεεolved in an appropriate amount of MCDB 170 complete medium at 37°C and sterilized by filtering through a 0.22 μm filter. Equal volumes of the sterile collagenase and hyaluronidase solutions were then combined to yield a 5X solution. The enzyme solution may be stored in aliquots at 80°C for up to one year.

A.3. Preparation of the tissue digestion media. To make 50 mis of sterile tissue digestion media, 10 mis of 5X Collagenase and Hyaluronidase solution were added under εterile conditionε to 5 mis of fetal calf serum (Hyclone, Logan, UT) and brought to volume with 35 mis of sterile complete MCDB 170 media.

A.4. Preparation of fibroblast cells for co-culture system.

Early fibroblast cells were plated into sterile six well diεhes containing DMEM (Sigma, St Louis, MO) plus 10% fetal calf serum at a concentration of at leaεt 5 X IO⁴ cellε per well. The cellε were then allowed to attach to the plates and grow 12 to 24 hours before use thereof in a co-culture system.

Under the invention, fibroblast cells for the primary co-culture system do not have to be from the same individual; however, for long term co-culture growth it is beεt to use fibroblasts derived from the same individual as the epithelial culture.

A.5. Tissue processing.

Human mammary tissue discarded from either mastectomies, reduction mammoplasties or biopsies was collected by placing the tissue in sterile containers containing tissue culture medium plus 10% fetal calf serum and antibiotics. Human colon and prostate tissue were obtained as surgical sampleε and handled in an analogouε manner. If the tissue is not to be used immediately (e.g. , within about 5 hours of surgery), it can be stored for up to 72 hours at 4°C without significant losε of epithelial cell viability. The breast tisεue εample was prepared by first cutting the tissue into small pieces in a sterile reaction vesεel such as a petri diεh. The epithelial areas of the cut tissue were separated from the adipose tissue and stromal matrix by manipulating the tissue sample with sterile scalpel blades, scissors, forceps, or any combination thereof. The remaining tissue was finely minced using opposing scalpels and transferred to a sterile 15 ml centrifuge tube. Again, colon and prostate tissue samples were handled in an analogous manner.

The minced tiεεue was then incubated for about 12 to 18 hours at 37°C in tisεue digestion media, preferably using a tube rotator for gentle mixing. After incubation, the tube containing the digested tisεue waε εpun down at 1000 rpm for 10 minutes, and the supernatant (along with any floating adipose tissue) removed and discarded leaving a pellet of minced digested tissue. Digestion may be monitored at this point and is complete when microscope examination reveals clumpε of cells with ductal, alveolar, or ductal-alveolar structures free from attached stroma. If necessary, further digestion may be carried out by resuspending the pellet in fresh tissue digestion media and reincubating as above.

The pellet of tisεue thus obtained was then decanted into a sterile 100 mm petri dish where the larger pieces of tissue were further divided using opposing scalpels until all of the tissue was aε εmall as possible. The tissue was then resuspended in 5 ml complete MCDB 170 media, and centrifuged quickly to pellet any large pieces of tissue in the bottom of the tube.

A.6. Setting up the co-culture system. Six well disheε containing fibroblast cells which were plated 12 to 24 hours earlier were checked to ensure that the fibroblastε had adhered to the plateε and were growing. The cells should be about 40 to 60% confluent in the six well dishes. The fibroblast growth media was aspirated off and discarded, and 3 is of fresh MCDB 170 complete media added to each well. A collagen-coated microporous membrane, such as a Transwell-COL 0.4μm support (available from COSTAR, Cambridge, MA) was placed within each well using sterile technique. After placing the supports in the wells, all air bubbles that became trapped under the support were removed, and about 0.5 mis of MCDB 170 complete medium added to each support to ensure that the entire surface area of the support was sufficiently moist.

Media containing small pieces of tissue that were floating in the 15 ml centrifuge tube after the quick spin were added dropwise to each support, carefully avoiding the large pieces of tisεue that had pelleted in the bottom of the tube. It is generally best to use the smallest pieces of tissue to plate onto the support, as, smaller tissue fragments are capable of rapid attachment to the support which results in earlier cell migration from the tissue. The co-culture system thus provided was incubated at 37°C in 5% C0₂. After two to three dayε, the co-cultureε were checked under a microεcope to determine whether cells had begun to migrate off the pieces of tissue and attach to the support. A.7. Maintaining the co-culture.

In order to maintain the co-culture, the growth media waε partially replaced with fresh MCDB 170 complete media at least about every six to seven days. However, the media was never completely removed from the wells, as it is important that conditioned media always remain on the co-culture system to maintain constant cell growth. Thus when new media was to be added, spent media on top of the support waε carefully aspirated without removing any media present under the support. Approximately 2 is of fresh MCDB 170 complete media was then added to each support and the co-culture reincubated.

Further, each individual primary epithelial culture generally grows at a different rate. Thus the cultures were carefully monitored to avoid allowing an εupport to reach confluence which can cause cell death. When the cells reach 80% to 90% confluence, the cells were pasεaged to a new co-culture system. When passing the cellε to a new co-culture εystem, a fresh culture of fibroblast cells was established using techniqueε aε deεcribed above. Thiε waε carried out at leaεt about 12 hourε in advance of the paεεaging so that the fresh fibroblaεt cellε were able to properly adhere to the new plateε. It iε generally beεt to continue to use the same fibroblast line that the co-culture system waε started with, however, if a fibroblast line waε initiated from the same individual from which the primary epithelial cultures were derived, thoεe cellε can be used in place of an existing fibroblast line. In this regard, a fibroblast cell line which has been established from the same individual is preferred under the invention, but this is not always possible when primary cultures are initiated.

To passage the cellε, the epithelial cells were selectively transferred to a new co-culture system containing fresh fibroblaεt cells. Thus two steps were generally carried out at the same time, selective trypsinization of the epithelial cells from the support and preparation of the new fibroblast culture. To trypsinize the co-culture support, the media on top of the support was aspirated and discarded, and the support gently transferred under sterile conditions to a fresh, sterile dish that did not contain any fibroblast cells. After removing the fibroblast growth media from the wells of the new plate containing the freshly plated fibroblast cellε, any remaining media from the old co-culture dish was transferred to the wells in the new plate (containing the freshly plated fibroblasts) . Use of the remaining media in this manner is considered important since it is "conditioned" media which is thought to aid in the promotion of cell growth in the newly passaged cells.

Approximately 2 mis of trypsin-EDTA(10X) (Sigma, St Louis, MO) was added to each support, wherein about 1.5 mis was placed under, and about 0.5 mis placed on top of each support. After incubation for about 3 to 5 minutes at 37°C, the supportε were checked to see if the cellε had detached. If the cellε were still attached, the cells were reincubated for a few more minutes. If most of the cells had detached, the cells were gently rinsed off of the support with media and moved to a centrifuge tube. The support should be rinsed in this manner several times to remove all the cells from the support. Once the cells were thus collected, they were centrifuged for 10 minutes at 1000 rpm to form a pellet.

After placing a new support in the dish containing the new fibroblast cells, 1 ml of fresh culture media was gently added to the top of the support such that there were no bubbleε formed under the εupport and the entire support was sufficiently moist. Thus, after the epithelial cells were pelleted, the supernatant was removed and the cells resuspended in MCDB 170 complete media and counted using standard techniques. The epithelial cells were then passaged to the new co-culture wells. In this regard, for the first passage, the cellε may be split at a ratio of 1:2 or as high as 1:5 depending on the desired final cell density, however, they εhould not be split at too high of a ratio or they will not recover.

Once the supports were seeded with the desired number of cells and reincubated, the cultures were generally checked for attachment and growth after 24 hours. The passaged cellε were fed by partial replacement of the growth media at leaεt about every 4 to 5 days. Partial replacement was carried out by removing the media from above the support and replacing it with new media such that the media was never completely removed from the wells. Leaving conditioned media in the cultures helps to maintain constant growth in the co-culture syεtem. The new media defuses through the support and mixes with the old media so that nutrients were constantly being replenished in the wells.

A.8. Freezing the digeεted tissue. An appropriate cell-preservative media is MCDB 170 complete media with 20% fetal calf serum pluε 20% glycerin (Sigma, St Louiε, MO) . Any remaining digeεted tiεsue prepared as described above may be preserved by freezing. Thus, remaining tissue was centrifuged at 1000 rpm for 10 minutes to form a pellet, all supernatant removed and discarded, and the remaining pellet resuspended in cell-preservative media. The pellet was generally resuspended to provide a 1:2 ratio of tissue to media and about 1.2 mis of the resuspended tissue aliquoted into appropriate vials (e.g., 1.8 ml Nunc Cryovials®) . The vials containing the tissue were frozen slowly (e.g. , at 1 degree per minute) and then transferred to storage at -135°C for future use.

B. General Immortalization Methods:

Once a primary human epithelial cell culture, or cell strain has been established using the techniques described above, a cell line can be established using methods known in the art.

Retroviral vector syεtemε for gene transfer comprise two major components, the retroviral vector and the packaging cells. The vector can be manipulated in its DNA form as part of a bacterial plasmid. The vector does not encode viral proteins. The retrovirus- packaging cells provide the viral proteins necessary for encapsidation of the vector RNA into virions and for subsequent infection, reverse transcription, and integration of the vector into the genomic DNA of the host cells.

B.l. Construction of the Vector.

In the cloning of DNA fragments, except where noted, all DNA manipulations were done according to standard procedures. See e.g., Sambrook et al., supra . Restriction enzymes, exonuclease III, T. DNA ligaεe, E . coli , DNA polymeraεe I, Klenow fragment, and other biological reagents were purchased from commercial supplierε and uεed according to the manufacturers' directions.

Retrovirus vector pLXSN (available from Fred Hutchinson Cancer Research Center, Seattle, WA) , was obtained. pLXSN is characterized as having the following major elements: L (long terminal repeat) ; X (cloning site) ; S (simian virus 40 (SV40) early promoter); and N (neo gene) . See, e.g., Miller et al. (1989) BioTechniques 2:980, and Stockschlaeder et al. (1991) Hum . Gene Ther . 2:33. The vector pLXSN contains two promoters, one driving expression of the selectable marker (neo) and the other driving expreεεion of the inserted DNA. DNA fragments containing the E6 or E7 gene or the contiguous region encoding E6 and E7 from HPV16 (Seedorf, et al. (1985) Virology 145:181-185) were isolated using appropriate restriction enzymes and exonuclease III digestions. The fragments were cloned into the pLXSN vector to produce the following constructε: pLXSN16E6 (containing the E6 gene) ; pLXSN16E7 (containing the E7 gene) ; and pLXSN16E6E7 (containing the E6/E7 contiguouε gene region) using techniques known in the art. See, e . g. , Sambrook, supra .

B.2. Construction of the Recombinant Viruses.

The constructs pLXSN16E6, pLXSN16E7 and pLXSN16E6E7 were transfected by calcium phosphate precipitation into the Psi-2 ecotropic packaging cell line (Mann et al. (1983) Cell 3_3:153) . Viruses produced from the Psi-2 cellε were then used to infect the amphotropic packaging line PA317 (available from the American Type Culture Collection, Rockville, MD, ATCC No. CRL 9078) which provide the following recombinant amphotropic viruseε: LXSN16E6, LXSN16E7 and LXSN16E6E7.

B.3. Infection of the Epithelial Cells.

Recombinant amphotropic viruses LXSN16E6, LXSN16E7 and LXSN16E6E7 were then used to infect the human breast, colon and prostate epithelial cell strainε produced under the invention using techniques known in the art.

Generally, the epithelial cells were used when in the logarithmic growth phase. Cell concentration waε adjuεted to about 2X10⁵/ml, and 2ml placed in a suitable tissue culture flask. Approximately 32 μl DMSO (Sigma, St Louis, MO) and 24 μl of 1 mg/ml Polybrene® (Abbott Labs, North Chicago, IL) which had been filtered through a 0.2 μm filter was added to 2 ml of the supernatant from the virally infected PA317 cells, and the resultant mixture added to the epithelial cells in the culture flask.

After about 2 hours of incubation, a further 2 ml of PA317 supernatant was supplemented with 16 μl DMSO and 12 μl of 1 mg/ml Polybrene®. The flask was then gently removed from the incubator and 3 ml of the medium removed and replaced with the fresh 2 ml aliquot of PA317 supernatant. This process may be repeated at 2-hour intervals as required (generally 5 to 6 infection cycles were sufficient) .

When infection was complete, the cells were centrifuged for 5 min at 200 rpm and resuspended in MCDB 170 + 20% (v/v) fetal calf serum (Hyclone, Logan, UT) before cloning by limiting dilution in the presence of geneticin G418® (GIBCO-BRL, Gaithersburg, MD) at an effective concentration of from about 50-300 μg/ml.

Example 1

In order to test the ability to establiεh primary cultureε using the co-culturing system of the invention, a number of primary cultures of human breast epithelial cells were produced using the above- described cell co-culture methods. Breast tisεue samples were obtained from biopsy, cosmetic breast surgery, lumpectomy or mastectomy. The tissue sources, explant type (e.g., normal or cancerous) , cell type, co-culture conditions and the amount of time to obtain a primary cell outgrowth from the explant are depicted in Table 1. l-J ro t P> o o (Jl

TABLE 1

Type of

Tissue Type of Primary Cell

Cell ID Source Explant Cell Culture Outgrowth Conditions

BE-20 40+ year old 9 - normal epithelial co-culture using 2-3 days biopsy (NF-1 DFCl-1 medium carrier) pluε collagen support

BE-23 33 year old 9 - normal epithelial co-culture using 3 days in radical mastectomy either DFCl-1 or DFCl-1; (infiltrating MCDB 170 medium 5 days in

I

4*. ducta1 cancer) plus collagen MCDB 170 Ul I support

BE-27 normal epithelial co-culture using 3 days in either DFCl-1 or DFCl-1; MCDB 170 medium 5 days in plus collagen MCDB 170 support

BE-28 47 year old 9 - normal epithelial co-culture using 2-3 days in radical mastectomy either DFCl-1 or DFCl-1; (infiltrating MCDB 170 medium 5-7 days in ductal cancer) plus collagen MCDB 170 support

TABLE 1

Type of

Tissue Type of Primary Cell

Cell ID Source Explant Cell Culture Outgrowth Conditions

BE-29-N 38 year old 9 - normal epithelial co-culture using 2-3 days in radical mastectomy either DFCl-1 or DFCl-1; (multifocal ductal MCDB 170 medium 5-7 days in cancer) plus collagen MCDB 170 support

BE-29-T 38 year old 9 - tumor mixed co-culture using 2-3 days

I radical mastectomy tissue epithelial DFCl-1 medium

*-

Cs (multifocal ductal and plus collagen I cancer) fibroblast support

BE-30-PC 30-40 year old 9 - possible epithelial co-culture using 2-3 days in biopsy (possible pre¬ either MCDB 170 DFCl-1; BRCA-1 carrier) cancerous or DFCl-1 medium 5-7 days in plus collagen MCDB 170 type-1 support

BE-30-PC 30-40 year old 9 - possible epithelial co-culture using 2-3 days in biopsy (possible pre¬ either MCDB 170 DFCl-1; BRCA-1 carrier) cancerous or DFCl-l medium 5-7 days in plus fibronectin MCDB 170 support

TABLE 1

Type of

Tissue Type of Primary Cell

Cell ID Source Explant Cell Culture Outgrowth Conditions

BE-30-PC 30-40 year old 9 - posεible epithelial co-culture using 2-3 days in biopsy (possible pre¬ either MCDB 170 DFCl-1; BRCA-1 carrier) cancerous or DFCl-1 medium 5-7 days in plus collagen MCDB 170 type I & III support

I

*- BE-30-PC 30-40 year old 9 - possible epithelial co-culture uεing 2-3 days in

I biopsy (possible pre¬ either MCDB 170 DFCl-1; BRCA-1 carrier) cancerous or DFCl-1 medium 5-7 days in pluε matragel MCDB 170 εupport

BE-30-T 30-40 year old 9 - tumor mixed co-culture uεing 2-3 days biopsy (possible tissue epithelial DFCl-1 medium BRCA-1 carrier) and pluε collagen fibroblaεt support

BE-31-E 29 year old 9 - normal epithelial co-culture using 2-3 days in reduction either MCDB 170 DFCl-1; mammoplasty or DFCl-1 medium 5-7 days in plus collagen MCDB 170 support

TABLE 1

Type of

Tissue Type of Primary Cell

Cell ID Source Explant Cell Culture Outgrowth Conditions

BE-31-F 29 year old 9 - normal fibroblast DMEM or MCDB 170 10-12 days reduction medium in MCDB 170; ma moplaεty 10-14 days in DMEM

BE-32 37 year old 9 - normal epithelial co-culture using 5-7 days

•ta. reduction MCDB 170 medium

OS

I mammoplasty plus collagen support

BE-33 36 year old 9 - non- epithelial co-culture using 5-7 days lumpectomy (family cancerous MCDB 170 medium history of breast plus collagen cancer) support

BE-40 72 year old 9 - pre¬ epithelial co-culture using 2-3 days mamotest sample cancerous DFCl-1 medium (BRCA-1 carrier) plus collagen support

The results thus obtained indicate that the present co-culture methods are useful to provide primary cultures of human breast epithelial cells from a variety of sources. Particularly, primary cultures of normal epithelial cells were readily establiεhed within 2-7 days from normal breast tisεue explantε (see, e.g., BE-20, BE-23, BE-27, BE-28, BE-29-N, BE- 31-E, BE-31-F and BE-32) . Primary cultures of potentially precancerous cells were established (BE- 30-PC) from a subject identified as a possible BRCA-l carrier. In this regard, four primary cultures were obtained in co-culture systems using collagen type I, collagen type I & III, fibronectin and Matragel® membranous supports. A primary culture of precancerous cells was established from a positively identified BRCA-l carrier. A cell strain (BE-40) was derived from the primary culture, and has been deposited with the ATCC (ATCC Accession No. ATCC CRL 11981) . Several primary cultures of cancerous cells were also obtained within about 2-3 days from cancerous tissue explants (see, e . g . , BE-29-T and BE- 30-T) .

Primary culture outgrowth was generally obtained more rapidly in co-culture systems using DFCl-1 medium (Band et al. (1989) Proc . Natl . Acad . Sci . USA 8^:1249-1253) than in the complex MCDB 170 medium (Clonetics, San Diego, CA) which is formulated for clonal growth of epithelial cellε.

Example 2

A number of the primary cultures obtained in Example 1 were passaged using the methods of the invention to obtain substantially pure human breast epithelial cell strains. The cells were further characterized to monitor differentiation of those cells after each passage. Particularly, keratins 14, 18 and 19, as well as estrogen receptor and epidermal growth factor receptor markers were assayed using the characterization methods described above.

The results of the characterization of the cell strainε are depicted in Table 2 wherein PO indicateε the primary culture, Pl indicateε the firεt passage, P2 indicates the second passage, P6 indicates the sixth passage and the like.

TABLE 2

Cell ID Passage Number Characterization

P5 Keratin 14 : 54%

Keratin 18 : 74%

Keratin 19 : 69%

EGFR : 19%

BE-23 ER : —

P6 Keratin 14 : 15%

Keratin 18 : 97%

Keratin 19 : 90%

EGFR : —

ER : 65%

BE-27 Pl Keratin 14 : 57% Keratin 18 : — Keratin 19 : 62% ER : 43%

TABLE 2

Cell ID Passage Number Characterization

Pl Keratin 14 : 74%

Keratin 18 : 99%

Keratin 19 : 77%

ER 7%

P2 Keratin 14 71%

Keratin 18 96%

Keratin 19 75%

ER 1%

BE-28 P3 Keratin 14 73%

Keratin 18 —

Keratin 19 84%

ER 29%

P4 Keratin 14 85%

Keratin 18 —

Keratin 19 87%

ER 59%

PO Keratin 14 22%

Keratin 19 . 84%

ER 26%

Pl Keratin 14 50%

Keratin 19 64%

ER 46%

BE-30-N P2 Keratin 14

Keratin 19 25%

ER 37%

P3 Keratin 14 —

Keratin 19 16%

ER 26%

PO Keratin 14 58%

Keratin 19 95%

ER 35%

Pl Keratin 14 . 82%

BE-31-E Keratin 19 94%

ER 65%

P2 Keratin 14 :

Keratin 19 87%

ER 21%

As can be seen by the resultε of Table 2, several cell strainε retained the luminal cell phenotype (which is keratin 19 positive cells) after multiple passageε. This is considered important under the invention, as breast cancer is generally considered to originate in the luminal cells. Taylor- Papadimitriou et al. (1989) J . Cell . Sci . 94:403-413. Particularly, at the sixth passage, the BE-23 (normal breast epithelial) cell strain was characterized as 90% keratin 19 positive. Both the BE-30-PC and the BE-31-E (precancerous and normal breast epithelial, respectively) cell strains lost the basal cell phenotype (keratin 14 positive cells) after their second passage. The BE-31-E and BE-30-PC cells are further characterized as 87% and 16% keratin 19 positive, respectively, after their second passage. The BE-28 (normal breast epithelial) cell strain is characterized as both keratin 14 and keratin 19 poεitive after itε fourth passage.

Example 3 In order to provide immortalized human mammary epithelial cell lines, the E6 and E7 genes of

HPV16 were introduced separately or in combination into the human mammary epithelial cell strain BE-31-E by transfection with amphotropic recombinant retroviruses using the methods described herein. Halbert et al (1991) J . Virology 65:473-478.

Following infection with the amphotropic viruses and selection in geneticin G418® (50μg/ml) , human mammary epithelial cell colonies (5X10⁴ colonies) were pooled and passaged weekly. The surprising resultε thus obtained are that pooled cultures of BE-31-E cells containing E6, E7 and E6E7 each exhibit extended life spans (greater than 4 months) , whereas pooled cultures of BE-31-E cells containing vector alone senesce within a month. The E6, E7 and E6E7 infected BE-31-E cellε exhibit differences both in morphology and in developmental markers. The E6 infected cells are 100% keratin 14 positive, whereas E7 and E6E7 infected cultures contain keratin 14 positive cells and keratin 19 positive cells.

The human mammary epithelial cell strain BE- 20 waε immortalized by tranεfection with amphotropic recombinant retroviruses using the methods described herein. Halbert et al (1991) J. Virology 65:473-478. Particularly, at the first passage, BE-20 cells were infected with amphotropic viruses containing the E6/E7 genes of HPV16, and selected in geneticin G418®

(50μg/ml) . Band et al. (1991) J . Virol . 65: 6671-6676. An immortalized human mammary epithelial cell line, BE-20 E6/E7, was obtained and has been deposited with the ATCC (ATCC Accession No. ATCC CRL 11980) . The BE- 20 E6/E7 colonies thus obtained were pooled and paεεaged weekly into flasks. The E6/E7-immortalized cell line exhibits differences in cell morphology, and has been passaged more than 55 times over the span of over one year.

Example 4 Using the fibroblast co-culture system of the present invention, primary cultures of human colonic epithelial cells were succeεεfully eεtablished and maintained for more than four months using the following procedure: normal human colon tissue was obtained immediately following surgery and placed into ice-cold culture media containing the following: 10% fetal calf serum (Hyclone, Logan, UT) , 2mM glutamine (Sigma, St Louis, MO) , 10 μg/L EGF (Sigma) , 5 μg/L hydrocortisone (Sigma) , 0.5 μg/L transferrin (Sigma) , 0.5 μg/L insulin (Sigma), 4.2 g/L bovine pituitary extract (Sigma) , and 200 mg/L gentamicin (Sigma) in Dulbecco's Modified Eagle's Media (D-MEM, GIBCO-BRL, Gaithersburg, MD) .

Tissue was fragmented and digested aε previously described to obtain suitable tissue explants. Initially, small tissue pieces were placed on collagen-coated membraneε seated over human skin fibroblast cells. After approximately two weekε, there waε viεible outgrowth from the tissue pieces which were subsequently removed. One-half volume of media was removed from the cells and replaced with fresh media every week.

Epithelial cellε were selected from the other cell types that initially grew from the colon tissue by selective trypsin digestions (to provide a substantially pure epithelial cell strain) . Since epithelial cells are more firmly attached to the collagen substrate than the fibroblast cells, it was possible to remove the majority of contaminating fibroblasts while leaving the epithelial cells undisturbed. This selective trypsinization was performed approximately eight times, each time saving the resulting colon fibroblastε to culture independently. As soon as possible, these fibroblasts were used as the feeder layer below the colon epithelial cells.

The colon epithelial cells grow very slowly, doubling every week or so and diεplay a rounded "cobblestone" morphology characteristic of epithelial cells. The human colonic epithelial cell strain thus obtained was immortalized by transfection with an amphotropic recombinant retrovirus expressing the HPV16 E6 or E7 gene as described above.

Example 5

Using the fibroblast co-culture system of the present invention, a number of primary cultures of human prostate epithelial cellε were succesεfully established using the above-described procedures. The cell cultures are depicted in Table 3 wherein the source of the prostate tissue, tisεue type and co- culture conditions are reported.

TABLE 3

Type of

Cell Tissue Type of Culture Comments ID Source Explant Cell Conditions

PC-59 59 year old cf, tumor epithelial MCDB 170 plus adenocarcinoma prostectomy, left tissue collagen support εide

PC-63 69 year old cf, normal epithelial MCDB 170 plus no pathological biopsy, left side collagen support diagnosis

I

PC-63 69 year old cf, normal epithelial MCDB 170 plus no pathological biopsy, right side collagen εupport diagnosis

PC-64 76 year old cf, tumor epithelial MCDB 170 plus glandular prostectomy, right tissue collagen support hypoplastic side moderately differentiated adenocarcinoma

PC-65 65 year old cf, normal epithelial MCDB 170 plus no tumor biopsy, left side collagen support tissue, prostate gland only

TABLE 3

Type of

Cell Tiεsue Type of Culture Comments ID Source Explant Cell Conditions

PC-65 65 year old cf, normal epithelial MCDB 170 plus no tumor biopsy, right side collagen support tisεue, prostate gland only

PC-66 70 year old cf, normal epithelial MCDB 170 plus prostate biopsy, left side collagen support tisεue, no

I neoplaεm

Ui cn

I PC-66 70 year old cf, normal epithelial MCDB 170 plus prostate biopsy, right side collagen support tisεue, no neoplasm

PC-67 58 year old cf, tumor epithelial MCDB 170 plus differentiated prostectomy, left tissue collagen support adenocarcinoma side 50% of tissue was tumorous

PC-67 58 year old cf, tumor epithelial MCDB 170 plus glandular prostectomy, right tissue collagen support hyperplasia side immortalized with E6/E7 at first passage |

Cells from the PC-59, PC-63, PC-64 , PC-65 and PC-66 primary cultures were passaged using the methods described above to provide cell εtrains. Cells from the PC-67, PC-68 and PC-69 primary cultures were immortalized at the time of first passage by transfection with an amphotropic recombinant retrovirus expresεing the HPV16 E6 or E7 gene as described above.

Depositε of Strains Useful in Practicing the Invention

A deposit of biologically pure cultures of the following strains was made with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland. The accession number indicated was aεεigned after successful viability testing, and the requisite fees were paid. The depositε were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty) . This assures maintenance of viable cultures for a period of thirty (30) years from the date of depoεit and at leaεt five (5) years after the most recent request for the furnishing of a sample of the deposit by the depository. The organisms will be made available by the ATCC under the terms of the Budapest Treaty, which assures permanent and unrestricted availability of the cultures to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.12) .

These deposits are provided merely as convenience to those of skill in the art, and are not an admission that a deposit iε required under 35 USC §112. The nucleic acid εequenceε of these plasmids, as well as the amino acid sequences of the polypeptides encoded thereby, are controlling in the event of any conflict with the deεcription herein. A license may be required to make, use, or sell the depoεited aterialε, and no such license is hereby granted.

Strain Deposit Date ATCC No.

BE-20 E6/E7 September 15, 1995 ATCC CRL 11980

BE-40 September 15, 1995 ATCC CRL 11981

Claims

Claims :

1. A method of producing a primary culture of human epithelial cells using a fibroblaεt co- culture system, comprising:

(a) plating a first cell population comprising a culture of human fibroblasts onto a substrate in a suitable medium;

(b) providing a membranous support upon which to grow human epithelial cells, εaid εupport being arranged over the fibroblast culture;

(c) applying a suspension comprising a εecond, heterogeneous cell population including epithelial cellε obtained from a human tiεεue εample to the support, said tisεue εample being substantially free of stromal and other cellular material; and

(d) allowing outgrowth of epithelial cells from the tissue sample to the support whereby a co- culture is formed capable of continued in vitro growth of the human epithelial cells.

2. The method of claim 1, wherein the human fibroblasts and the human epithelial cells are both obtained from an individual human source.

3. The method of claim 2 , wherein the human fibroblasts and the human epithelial cells are obtained from a single tisεue sample.

4. The method of claim 1, wherein the human epithelial cells are normal cells selected from the group consisting of mammary, colonic and prostate epithelial cells.

5. The method of claim 1, wherein the human epithelial cells are precancerous cells selected from the group conεisting of mammary, colonic and prostate epithelial cells.

6. The method of claim 5, wherein the precancerous cells comprise breast epithelial cells derived from an individual expressing mutations in one or more of the genes selected from the group consisting of p53, Rb and BRCA1.

7. The method of claim 5, wherein the precancerous cells comprise colonic cells derived from an individual expressing mutations in one or more of the genes selected from the group consisting of APC, MSH₂ and MSH₃.

8. The method of claim 1, wherein the human epithelial cellε are malignant cells selected from the group consisting of mammary, colonic and prostate epithelial cells.

9. The method of claim 1, wherein the εupport comprises a collagen-coated, permeable membrane.

10. A method of pasεaging epithelial cellε from a primary culture to obtain a εubεtantially pure epithelial cell εtrain, compriεing:

(a) providing a primary culture obtained by the εteps of claim 1; (b) plating a third cell population comprising a culture of human fibroblastε onto a second substrate in a suitable medium;

(c) selectively detaching non-epithelial cells from the substrate by incubating the co-culture with a suitable detaching agent for a period of time sufficient to detach non-epithelial cells from the substrate without detaching a substantial amount of epithelial cells; and

(d) arranging the support over the fibroblast culture obtained in step (b) whereby a substantially pure epithelial cell strain is maintained on the εupport.

11. A method of passaging epithelial cells from a primary culture to obtain a substantially pure epithelial cell strain, comprising:

(a) providing a primary culture obtained by the steps of claim 4;

(b) plating a third cell population comprising a culture of human fibroblasts onto a second substrate in a suitable medium;

(c) selectively detaching non-epithelial cells from the substrate by incubating the co-culture with a suitable detaching agent for a period of time sufficient to detach non-epithelial cells from the substrate without detaching a substantial amount of epithelial cells; and

(d) arranging the support over the fibroblast culture obtained in step (b) whereby a substantially pure epithelial cell strain iε maintained on the support.

12. A method of pasεaging epithelial cellε from a primary culture to obtain a substantially pure epithelial cell strain, comprising: (a) providing a primary culture obtained by the steps of claim 5;

(b) plating a third cell population comprising a culture of human fibroblasts onto a second substrate in a suitable medium; (c) selectively detaching non-epithelial cells from the substrate by incubating the co-culture with a suitable detaching agent for a period of time sufficient to detach non-epithelial cellε from the εubεtrate without detaching a εubstantial amount of epithelial cells; and

(d) arranging the support over the fibroblast culture obtained in step (b) whereby a substantially pure epithelial cell strain is maintained on the support.

13. A method of passaging epithelial cells from a primary culture to obtain a substantially pure epithelial cell strain, comprising:

(a) providing a primary culture obtained by the εtepε of claim 8;

(b) plating a third cell population compriεing a culture of human fibroblasts onto a second εubεtrate in a suitable medium;

(c) selectively detaching non-epithelial cells from the substrate by incubating the co-culture with a suitable detaching agent for a period of time sufficient to detach non-epithelial cells from the substrate without detaching a subεtantial amount of epithelial cells; and

(d) arranging the support over the fibroblast culture obtained in step (b) whereby a substantially pure epithelial cell strain is maintained on the support.

14. The method of claim 12, wherein the precancerous cell strain comprises breast epithelial cells derived from an individual expresεing mutations in one or more of the geneε εelected from the group conεiεting of p53, Rb and BRCA1.

15. The method of claim 14, wherein the precancerous cell strain comprises subεtantially pure, iεolated BE-40 cellε (ATCC Accession No. ATCC CRL 11981) . 16. The method of claim 12, wherein the precancerous cell strain comprises human breast epithelial cells stably transfected with a nucleic acid construct comprising a p53 gene having a disabling disruption therein, whereby integration of the nucleic acid sequence with the host genome by homologous recombination introduces a disruption in the host p53 gene.

17. The method of claim 12, wherein the precancerous cell strain comprises colonic cells derived from an individual expressing mutations in one or more of the genes selected from the group consisting of APC, MSH₂ and MSH₃.

18. The method of any one of claims 10, 11, 12 and 13, comprising the additional step of immortalizing the subεtantially pure epithelial cell strain to provide a cell line.

19. The method of claim 18, wherein the immortalizing step compriseε transfecting the cell strain with at least one expression cassette comprised of a nucleic acid molecule encoding a papillomaviruε polypeptide εelected from the group consisting of E6, E7 and combinations thereof, and control sequences that direct the transcription of εaid nucleic acid molecule whereby εaid sequence can be transcribed and translated in the transfected cell line.

20. The method of claim 18, wherein the immortalizing step comprises transforming the cell strain with at least one retroviral vector including an expression cassette comprised of a nucleic acid molecule encoding a papillomavirus polypeptide selected from the group consiεting of E6, E7 and combinations thereof, and control sequences that direct the transcription of the nucleic acid molecule whereby the sequence can be transcribed and translated in the transfected cell line.

21. An immortalized cell line produced by the method of claim 20, wherein the cell line is of human mammary epithelial cell origin.

22. The immortalized cell line of claim 21 characterized by the expresεion of the keratin 14 phenotype.

23. The immortalized cell line of claim 21 characterized by the expresεion of the keratin 19 phenotype.

24. The immortalized human mammary epithelial cell line BE-20 E6/E7 (ATCC Accession No. ATCC CRL 11980) .

25. An asεay method for determining the toxicity of an agent to human epithelial cells, comprising:

(a) providing the cell strain obtained by the steps of claim 10;

(b) exposing the cells to the agent;

(c) terminating the exposure of the cells to the agent;

(d) maintaining the cells on the support; and

(e) determining the toxicity of the agent by comparing the number of viable cells with an appropriate control.

26. An assay method for determining the toxicity of an agent to human epithelial cells, comprising: (a) providing the cell strain obtained by the steps of claim 11;

(b) exposing the cells to the agent;

(c) terminating the exposure of the cells to the agent;

(d) maintaining the cells on the support; and

(e) determining the toxicity of the agent by comparing the number of viable cells with an appropriate control.

27. An aεsay method for determining the toxicity of an agent to human epithelial cells, comprising: (a) providing the cell strain obtained by the εteps of claim 12 ;

(b) exposing the cells to the agent;

(c) terminating the exposure of the cells to the agent; (d) maintaining the cells on the support; and

(e) determining the toxicity of the agent by comparing the number of viable cells with an appropriate control.

28. An assay method for determining the toxicity of an agent to human epithelial cells, comprising:

(a) providing the cell strain obtained by the εtepε of claim 13;

(b) expoεing the cellε to the agent;

(c) terminating the exposure of the cells to the agent;

(d) maintaining the cells on the support; and (e) determining the toxicity of the agent by comparing the number of viable cells with an appropriate control.

29. The assay method of claim 27, wherein the precancerous cell strain comprises breast epithelial cells derived from an individual expressing mutations in one or more of the genes selected from the group consiεting of p53, Rb and BRCA1.

30. The aεεay method of claim 29, wherein the precancerouε cell strain is BE-40 (ATCC Acceεεion No. ATCC CRL 11981) .

31. The assay method of claim 27, wherein the precancerous cell strain comprises colonic cells derived from an individual expressing mutations in one or more of the genes selected from the group conεiεting of APC, MSH₂ and MSH₃.

32. The assay method of any one of claims 25, 26, 27 and 28, wherein the substantially pure epithelial cell strain has been immortalized to provide a cell line.

33. The asεay method of claim 32, wherein the cell εtrain haε been stably tranεfected with at leaεt one expreεεion caεsette comprised of a nucleic acid molecule encoding a papillomaviruε polypeptide εelected from the group conεiεting of E6, E7 and combinations thereof, and control sequences that direct the transcription of said nucleic acid molecule whereby said sequence can be transcribed and translated in the transfected cell line.

34. The asεay method of claim 32, wherein the cell εtrain has been stably transfected with at least one retroviral vector including an expression cassette comprised of a nucleic acid molecule encoding a papillomavirus polypeptide selected from the group conεisting of E6, E7 and combinations thereof, and control sequences that direct the transcription of the nucleic acid molecule whereby the sequence can be transcribed and translated in the transfected cell line.

35. The assay method of claim 34, wherein the cell line is of human mammary epithelial cell origin.

36. The assay method of claim 35, wherein the cell line is characterized by expresεion of the keratin 14 phenotype.

37. The assay method of claim 35, wherein the cell line is characterized by expression of the keratin 19 phenotype.

38. The assay method of claim 35, wherein the cell line is BE-20 E6/E7 (ATCC Accession No. ATCC CRL 11980) .