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  • C12N2510/00—Genetically modified cells
  • C12N2510/04—Immortalised cells

Definitions

• 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.
• a passaged epithelial cell strain can be genetically manipulated to construct precursor cells of human cancers.
• 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.
• 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) .
• 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.
• 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 "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 .
• 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.
• 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.
• 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.
• the exogenous DNA may or may not be integrated (covalently linked) to chromosomal DNA making up the genome of the cell.
• the exogenous DNA can be maintained on an episomal element, such as a plasmid.
• 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,
• the tumor suppre ⁇ sor genes p53 and Rb, and the putative antioncogene BRCA1 are each believed to play a role in human breast cancer carcinogenesi ⁇ .
• Family studies indicate that p53 can be rate-limiting in breast cancer formation. Martin et al. (1990) Science 250: 1233-1238.
• mutations in the p53 gene have either been found directly, or implicitly e.g., by ⁇ pecific lo ⁇ of heterozygosity. See e . g . , Harris, CC. (1994) Science 262: 1980, Harris et al. (1993) N . Engl . J . Med . 329:1315, Harper et al.
• 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.
• a method whereby in vitro assays are carried out using human breast, colon or prostate epithelial cell cultures prepared according to the invention.
• 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) .
• MSH 2 and MSH 3 are used to screen for differential stains, chemicals or chemotherapeutic agents effective in the diagnosis or selective killing of precancerous cells.
• an in vitro a ⁇ ay method whereby a conditional immortalization system i ⁇ con ⁇ tructed in order to allow assay of a cell population prior to immortalization, while actively immortalized and when the immortalization system is inactive.
• a human breast, colon or prostate epithelial cell strain is selected and portions of the strain subjected to immortalization using the temperature- ⁇ en ⁇ itive mutant SV40 Large T ⁇ ystem 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 culture ⁇ 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.
• 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.
• expression of E6 and/or E7 in transfected cells may be regulated using a tetracycline inducible transactivator (tTA) regulation sy ⁇ tem containing control element ⁇ of the tetracycline-re ⁇ istance operon encoded in TnIO of Escherichia coli fused with the activating domain of virion protein 16 of herpes simplex virus as previously described.
• tTA tetracycline inducible transactivator
• the tTA system allows differential control over expression of the subject genes, as well as reversible "on/off" ⁇ witching. 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 concentration ⁇ by assay of luciferase activity. Gossen et al., supra .
• 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.
• Apoptosi ⁇ or programmed cell death
• 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 .
• 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 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®) .
• 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.
• a cell line can be established using methods known in the art.
• 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 po ⁇ itive after it ⁇ fourth passage.
• Example 3 In order to provide immortalized human mammary epithelial cell lines, the E6 and E7 genes of
• the colon epithelial cells grow very slowly, doubling every week or so and di ⁇ play 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.
• fibroblast co-culture system of the present invention Using the fibroblast co-culture system of the present invention, a number of primary cultures of human prostate epithelial cell ⁇ were successive ⁇ fully established using the above-described procedures.
• the cell cultures are depicted in Table 3 wherein the source of the prostate tissue, tis ⁇ ue type and co- culture conditions are reported.
• a deposit of biologically pure cultures of the following strains was made with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland.
• ATCC American Type Culture Collection
• the accession number indicated was a ⁇ igned after successful viability testing, and the requisite fees were paid.
• the deposit ⁇ 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 depo ⁇ it and at lea ⁇ t five (5) years after the most recent request for the furnishing of a sample of the deposit by the depository.

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