EP0494225A1 - Human esophageal epithelial cell lines - Google Patents

Human esophageal epithelial cell lines

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Publication number
EP0494225A1
EP0494225A1 EP90914817A EP90914817A EP0494225A1 EP 0494225 A1 EP0494225 A1 EP 0494225A1 EP 90914817 A EP90914817 A EP 90914817A EP 90914817 A EP90914817 A EP 90914817A EP 0494225 A1 EP0494225 A1 EP 0494225A1
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EP
European Patent Office
Prior art keywords
cells
cell line
het
esophageal
cell
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EP90914817A
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German (de)
French (fr)
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EP0494225A4 (en
Inventor
Gary David Stoner
Roger Robert Reddel
Curtis Craig Harris
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US Department of Health and Human Services
US Department of Commerce
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US Department of Health and Human Services
US Department of Commerce
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Publication of EP0494225A1 publication Critical patent/EP0494225A1/en
Publication of EP0494225A4 publication Critical patent/EP0494225A4/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G01N33/5017—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity for testing neoplastic activity

Definitions

  • the present invention relates to human epithelial cells that originate from the esophagus and are immortal- ized in culture, particularly to such cells genetically transformed with genes from tumor viruses, especially,
  • the present invention also relates to methods of using such cells for detection of various types of agents, for instance, chemicals that can cause cancer.
  • esophageal cancer is associated with exposure to chemical carcinogens in the environment and in the diet.
  • Factors associated with an increased risk of developing esophageal cancer include tobacco, alcoholic beverages, and moldy foods, all of which contain nitrosamines; and asbestos.
  • esophageal neoplasms probably are the result of a sequence of genetic and phenotypic changes in the esophageal epithelium.
  • the phenotypic keratin patterns of squamous cell carcinomas of the esophagus are consistently different from those of the normal tissue (S.P. Banks- Schlegal and C.C. Harris, Cancer Res. 1984, 44: 1153- 1157.) (M.P. Grace, K.H. Kim, L.D. True and Fuhs, 1985, Cancer Res. 45: 841-846).
  • the esophageal epithelial cell is able to form crosslinked envelopes when it terminally differentiates (S.P. Banks- Shlegal and C.C. Harris, Cancer Res. 1986, 250-258. This function is variably expressed in esophageal carcinomas (S.P.
  • N- nitrosobenzylmethylamine led to the establishment of epithelial cell lines that, after prolonged subculture, produced well-differentiated squamous cell carcinomas following transplantation in vivo.
  • human esophage ⁇ al epithelial cells that are neoplastic have an unlimited ability to replicate in culture, whether neoplastic by virtue of derivation from a cancer or by neoplastic transformation with a tumor virus in culture, such trans ⁇ formed cells are, of course, useless for applications requiring normal cells to detect carcinogenic activities.
  • neoplastic transformation of normal cells into cells that can cause tumors is thought to result from multiple cellular changes.
  • Evidence supporting this concept comes from studies which compared the neoplastic transformation of normal cells and cell lines that were immortalized by genetic transformation with viral and cellular oncogenes. Unlike normal cells, such as those in primary cultures of epithelial cells which are prepared directly from tissues, immortalized cells have the ability to replicate in culture for an indefinite number of generations. The results of the comparative studies indicate that certain oncogenes induce immortalization in normal cells but, by themselves, cannot induce frank neoplasia as evidenced by tumorigenicity.
  • an immortalization gene can cooperate with a second class of oncogene to induce tumorigenic potential.
  • at least some cells that have become immortalized without addition of an exogenous immortalizing oncogene nevertheless require only the second type of oncogene for complete neoplastic transformation into tumor forming cells.
  • a problem with the use of infectious SV40 virus to immortalize cells is that, although the infection with SV40 is not lethal and tends to be self-limiting, the potential for resurgence of more active viral replication persists. Such replication may alter the physiology of the cells and thus render them unreliable for long term culture and chemical testing purposes. Accordingly, the present inventors have been involved in research efforts to extend the replicative potential of various human epithelial cells without introducing genes allowing complete viral synthesis. Within the past year they have demonstrated that genetic transformation with a plasmid containing only certain SV40 genes, namely the so-called early region genes, leads to immortalization of human cells derived from tissues other than esophageal epitheli ⁇ um.
  • epithelial cells from the bronchus include epithelial cells from the bronchus (R.R. Reddel, et al., 1988, Cancer Res., 48:1904-1909; see also U.S. Patent Application Ser. No. 07/114,508 filed October 30, 1987) mesothelium (Y. Ke, et al., 1989, J. Pathol. 134:979-991; see also U.S. Patent Application Ser. No. 07/114,508), and neonatal prostate (M.E. Kaighn, et al., 1989, Cancer Res. 49:3050-3056).
  • pRSV-T The plasmid used to immortalize these cells, which is called pRSV-T, is known in the art (D.E. Brash, R.R. Reddel, M. Quanrad, K. Yang, M.P. Farrell, and C.C. Harris, 1987, Mol. Cell. Biol., 7:2031-2034). It also comprises certain genetic elements from another tumor virus, the Rous sarcoma virus. (Since this plasmid comprises viral genes, the process of genetic transforma- tion in this instance is referred to as "transfection", a term of the art that is a hybrid of the words "transforma ⁇ tion” and "infection".
  • transfection Even though the viral genes of the plasmid are insufficient to produce infectious virus, the use of the term transfection here is convenient for distinguishing the process from that of neoplastic trans ⁇ formation. Accordingly, hereinafter, the term transforma ⁇ tion will be used exclusively in connection with the latter process of neoplasia.
  • transfection procedure using strontium phosphate has yielded stable trans- fectants (i.e., transfected cells) in the several human epithelial cell types cotransfected with the plasmid pRSV- T (as cited above) , and that this strontium procedure is more effective than the traditional calcium phosphate method for insertion of DNA into epithelial cells that are sensitive to high levels of calcium.
  • the present invention contemplates the application of methods of recombinant DNA technology to fulfill the above needs for esophageal cell lines for chemical testing and other purposes. More specifically, it is an object of the present invention to provide a line of human esophage ⁇ al epithelial cells or a derivative thereof having a replicative capacity in cell culture that is enhanced compared to normal cells, and is unable to produce tumors. Further, it is an object of the present invention to provide a cell line of this type which replicates continu ⁇ ously in cell culture.
  • the normal human esophageal epithelial cells can be made to grow continuously by transfecting normal esophageal epithelial cells with the T antigen gene of SV40 virus.
  • the plasmid containing the SV40 early region genes further comprises a genetic element derived from another tumor virus, the long terminal repeat (LTR) or Rous sarcoma virus, which serves to stimulate transcription of the SV40 DNA sequences.
  • LTR long terminal repeat
  • Rous sarcoma virus Rous sarcoma virus
  • Transfection or infection can be accomplished by use of a virus or a plasmid obtaining the T antigen gene of the SV40 virus. Either transfection or infection may lead to transformation of the cell line.
  • Other transfor ⁇ mation vectors may be useful, such as papilloma virus or Epstein Barr virus. Techniques for making continuous human cell lines are described in the following referenc ⁇ es: Grahm, F.L., Smiley J. , Russell, W.C. and Nairn, R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol., 36: 59-72, 1977; Zur Hausen, H. Oncogenic herpes viruses In: J.
  • the formation of a focus or foci is indicative of carcinogenicity of an agent.
  • a lack of growth of the cell line is indicative of antineoplastic potency of an agent, particularly for esophageal epithelial cells.
  • HE human esophagus
  • NHE normal human esophagus
  • HBS Hepes buffered saline
  • BPE bovine pituitary extract
  • SV40 Simian virus 40
  • CFE colony forming efficiency
  • PD population doublings
  • EM electron microscopy.
  • DESCRIPTION means that the cell line grows continually without senescence when cultured in a suitable growth medium.
  • continuous cell line means that the cell line grows continually without senes ⁇ cence when cultured in a suitable growth medium.
  • the "derivatives" of the esophageal cell line(s) of the present invention include cells which have been further genetically altered by adding, for example, genes for drug metabolizing enzymes, other oncogenes, anti- oxidant genes, or other genes, thereby creating a continu ⁇ ous derivative of the cell line.
  • the present invention relates to a line of human esophageal epithelial cells or a derivative thereof having a replicative capacity in cell culture that is enhanced compared to normal cells and being unable to produce tumors.
  • the general method for obtaining immor ⁇ talized human esophageal epithelial cells is as follows.
  • NHE cells Normal human esophageal (NHE) cells were obtained from explant outgrowths of autopsy specimens from noncan- cerous males. Dispersed cell suspensions were plated at 3-5 x 10 5 cells/dish and transfected with 10 ⁇ g of plasmid pRSV-T coprecipitated with strontium phosphate. After 4 hrs, the cells were shocked with glycerol, as described in Example 1, below. After the appearance of foci of trans ⁇ formed cells, control and transfected cultures were subcultured (2.5 x 10 5 /100-mm dish) . Control strains could be subcultured for no more than 20 PDs, after which they senesced.
  • pRSV-T-transfected cells e.g., lines designated HE-451, HE-457, see below
  • HE-451, HE-457 grew exponentially for approxi- mately 50 PDs, after which they went into crisis.
  • this crisis period which lasted for several months, the majority of cells in both strains senesced. Recovery from this state depended upon their continued maintenance at the highest possible density.
  • two separate immortalized cell lines, designated HET-IA and HET-2A were developed, on each from the HE-451 and HE-457 cultures, respectively.
  • a deposit of the cell lines of the present inven ⁇ tion has been made at the American Type Culture Collec- tion, 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A., on August 25, 1989, under accession numbers CRL- 10209 (HET-IA) and CRL-10210 (HET-2A) , in accordance with the Budapest Treaty.
  • the deposits shall be viably main ⁇ tained, replaced if they become non-viable, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and made available to the public without restriction in accordance with the provisions of the law.
  • the Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
  • the pRSV-T transfected cell lines expressed T- antigen by immunoperoxidase nuclear staining. This is consistent with the Southern blotting data indicating that all lines contain integrated SV40 early region DNA. All transfected lines were confirmed as epithelial by virtue of their positive reaction with antibodies to keratin and the presence of desmosomal junctions and cytoplasmic microfilaments in EM preparations.
  • HET-IA a hypodiploid line
  • HET-IA has a chromosome complement similar to that of pRSV-T immortalized human bronchial epithelial cells (R.R. Reddel, et al., 1988, supra) and prostatic epithelial cells (M.E. Kaighn et al. 1989, supra) .
  • HET-2A cells are larger than those of HET-IA. This is consistent with the observation that the chromosome number of HET-2A cells is in the hypotetraploi ⁇ range whereas HET-IA is hypodiploid.
  • HET-2A cells originated from a male specimen, they had lost the Y chromosome by passage 6. Chromosome analysis indicated that the precursor strain to HET-2A, i.e., HE-451 cells, contain the Y chromosome. Loss of the Y chromosome in transformed cells is frequently observed. In general, instability of the karyotype is a characteris ⁇ tic of virally-transformed cells.
  • UTILITY OF THE CELL LINES Identification of potential carcinogens, tumor promoters and antagonists thereof. These cells are useful for screening chemicals or other agents in the human environment for the potential to neoplastically transform the cells.
  • Putative carcinogens or tumor promoters may be added to the growth medium of the cells and the state of transformation of the cells as a function of time and dose of exposure may be ascertained using anchorage indepen ⁇ dence growth, matrix invasion, cells to cell communication assays, and/or nude mice tumorigenicity assays.
  • Anti- carcinogenic or anti-promoting agents may be added to the medium with carcinogens or promoters and the state of transformation as a function of time may be ascertained as for carcinogens or promoters alone.
  • Such screening could be used to identify specific esophageal toxins in the diet or in other substances and to identify dietary or other compounds that could prevent chemically-induced esophageal cancer. Identification of potential chemotherapeutic drugs. These cells are also useful, either before or after further alteration by other oncogenes or carcino ⁇ gens, for screening chemicals particularly suitable for the treatment of esophageal cancer and related diseases, by growing them in culture medium containing the chemical to be tested and then, after a suitable period of expo ⁇ sure, determining whether and to what extent cytotoxicity has occurred, e.g., by trypan blue exclusion assay or related assays (Paterson, Methods Enzymol., 58:141, 1979), or by growth assays such as colony forming efficiency as described in the examples, herein, all of which are standard techniques well known in the art.
  • Identification of anti-esophageal cancer drugs which act bv inducing terminal cell differentiation Chemical and biological substances are screened for their ability to induce terminal differentiation by adding them to the growth medium of these esophageal cells and then after a suitable period of time, determining whether a complex of changes occurs, including for example, the phenotypic keratin patterns of the normal esophagus (S.P. Banks-Schlegel and C.C. Harris, 1984, Cancer Res. 44:1153- 1157; M.P. Grace, K.H. Kim, L.D. True and E. Fuchs, 1985, Cancer Res. 45:841-846). Induction of terminal differen ⁇ tiation may be an effective way of controlling the growth of cancer.
  • Carcinogens and other xenobiotics may be added to the growth medium of these cells and the appear ⁇ ance of metabolic products of these compounds may be monitored by techniques such as thin layer chromatography or high performance liquid chromatography and the like, and the interaction of the compounds and/or their metabo ⁇ lites with DNA is determined.
  • Studies of DNA mutagenesis Substances known or suspected to be mutagens may be added to the growth medium of the cells and then mutations may be assayed, e.g., by detection of the appearance of drug resistant mutant cell colonies (Thompson, Methods Enzymol., 58:308, 1979).
  • chromosome damaging agents Substances known or suspected to cause DNA or chromosomal damage may be added to the culture medium of these cells lines, and then the extent of chromosomal damage may be measured by techniques such as measurement of the frequency of sister chromatic exchange (Latt et al.. In: Tice, R.R. and Hollander, A., Sister Chromatid Exchanges, New York: Planum Press, pp. 11 ff., 1984), and DNA damage can be determined by the measurement of unscheduled DNA synthesis (Mirsalis, J.C., Banbury Report vol. 13, pp. 83-99, 1982). Studies of malignant transformation bv additional oncogenes.
  • viral agents and genes trans ⁇ ferred by genetic transformation including oncogenes and high molecular weight genomic DNA from tumors, may be tested using standard assays such as anchorage independent growth or tumor formation in athymic nude mice.
  • such cells transformed by an additional oncogene can be used to screen for potential chemotherapeutic agents by the techniques described above, especially those which may be specific for cells transformed by the activation of particular oncogenes or combination of oncogenes.
  • HET-IA cell line Development of the HET-IA cell line.
  • NHE normal human esophageal
  • the outgrowths were suspended with PET [1% polyvi- nylpyrrolidone, 0.02% ethylenebis-(oxyethylenenitrilo) tetraacetic acid, 0.2% crystalline tryspin in HBS, pH 7.4] at room temperature and subcultured into tissue culture dishes or T flasks which had been coated with a mixture of 100 ⁇ g/ml bovine serum albumin, 10 ⁇ g/ml bovine fibro- nectin (both from Calbiochem) , and 20 ⁇ g/ml type 1 colla- gen (Vitrogen 100, Collagen Corp., Palo Alto, CA) to promote cell attachment (J.F. Lechner and M.A. LaVeck, 1985, J. Tissue Culture Meth. 9:43-48).
  • PET 1% polyvi- nylpyrrolidone, 0.02% ethylenebis-(oxyethylenenitrilo) tetraacetic acid, 0.2% crystalline tryspin in HBS, pH 7.4
  • This formulation consisting of modified MCDB 153 supplemented with growth factors (KGM) is available from Clonetics Corporation, San Diego, CA.
  • the basal medium (cKBM) used in these studies and purchased from Clonetics Corporation was a customized modification of KBM without phenol red.
  • other components omitted from cKBM were added just before use CaCl 2 , FeS0 4 , ZnCl 2 , trace element concentrate, thymidine, phospho- ethanolamine, ethanolamine, glutamine and antibiotics) .
  • the growth medium (cKGM-AE consisted of cKBM supplemented with 5 ng/ml epidermal growth factor, 1.4 ⁇ M hydrocortisone, 0.1 mM ethanolamine and phosphoethanol- amine, 5 ⁇ g/ml insulin, 40 ⁇ g/ml bovine pituitary extract (BPE) , 250 ⁇ g/ml bovine serum albumin, and 0.5 ⁇ g/ml epinephrine.
  • Antibiotics were added as needed (100 units/ml penicillin G, 100 ⁇ g/ml kanamycin, 50 ⁇ g/ml gentamicin) .
  • plasmid obtained from Dr. Bruce Howard, NCI, is an ori construct (i.e., lacks the SV40 site for origin of DNA replication) containing the SV40 early region genes and the Rous sarcoma virus long terminal repeat (R.R. Reddel, Y. Ke, B.I. Gerwin, M.G. McMenamin, J.F.
  • strain HE-457 formed two discreet colonies in a single flask. One of these contin ⁇ ued to grow following isolation; the other did not sur- make. At passage 14, this strain (HET-IA) was switched to cKGM-AE medium. Its growth has accelerated, and it has doubled at least 143 times thus far.
  • HET-2A Development of the HET-2A cell line. Based on the observation that the switch to a different growth medium appeared to enhance growth of the few surviving cells of the pRSV-T-transfected cells designated HE-457, a frozen ampoule of the other strain, HE-451, was recovered and cultured in the same medium (cKGM-AE) . In this case, there was a much shorter lag before consistent exponential growth was seen.
  • This line (HET-2A) has now achieved at least 122 PDs.
  • Pre-crisis cultures and immor ⁇ talized cell lines were characterized by immunohistochem- istry. Keratin staining was intensely positive in both pre-crisis strains (HE-451 and HE-457) , especially in closely apposed foci of cells. Vimentin was also posi ⁇ tive, although to a lesser extent than keratin. Numerous atypical nuclear features were observed including dyspla- sia and para-nuclear clearing (koilocytosis) . Keratin stained positively in all cells of both immortalized lines. Both lines stained heterogeneously for vimentin with more intense staining in the smaller cells. Keratin stained much more intensely than vimentin in both cell lines.
  • Chromosome and isozyme analyses Chromosome and isozyme analyses. Chromosome studies were performed by Dr. Ward D. Peterson, Children's Hospital of Michigan, Detroit, MI, using standard methods. Metaphases were stained with Giemsa and counted at low power for ploidy determination. Exact counts on 30 metaphases were made on banded chromosomes, and at least 8 karyotypes per cell line were prepared. Analyses of 8 isozymes were carried out using standard procedures.
  • HET-IA and HET-2A The chromosomal profiles of SV40-T antigen immor ⁇ talized cell lines, HET-IA and HET-2A showed that both lines are aneuploid and each has its own complement of marker chromosomes.
  • HET-IA is hypodiploid with only about 5% of metaphases examined in the hypotetraploid range.
  • Cell line HET-2A is hypotriploid with 28% of metaphases having 100+ chromosomes.
  • DNA was isolated by SDS proteinase-K incuba ⁇ tion followed by phenol/chloroform extraction and ethanol precipitation. The purity of the DNA was assessed spec- trophoto etrically using the 260/280 nm ratio.
  • DNAs (5 ⁇ g each) from cell strains HE-451, HE-457, HOC-517, and HB- 56B, and cell lines RE-149, BEAS-2B, HET-IA and HET-2A were loaded into individual wells of a S&S Slot Blot apparatus (Schleicher and Schuell, Inc., Keene, New Hampshire) . DNA was blotted onto Hybond-N (Amersham) nylon membrane.
  • the samples were probed with a nick- translated EcoRI-Hindlll fragment of the plasmid, pRSV-T in 2x SSC-0.1% SDS at 65°C for 16 hours.
  • the membrane was washed to 0.2x SSC-0.1% SDS at 65°C and autoradiographed at -75°C.
  • a sample (1 ⁇ g) of the plasmid DNA was used as a control.
  • cell strain HOC-517 appears to have incorporated only a few gene copies, longer autoradio- graphic exposures indicated that it is definitely posi ⁇ tive, and that the negative controls, HB-56B and RE149, have no detectable copies of the SV40-T gene.

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Abstract

Cellules épithéliales et oesophagiennes humaines possédant une capacité de réplication dans la culture cellulaire qui est augmentée par rapport aux cellules normales, ces cellules étant incapables de provoquer des tumeurs. Du tissu de l'oesophage humain normal provenant de deux pièces d'autopsie a été explanté dans un milieu dépourvu de sérum. Les excroissances épithéliales ont été repiquées, puis transfectées par la coprécipitation au phosphate des strontium au moyen du pRSV-T plasmidal constitué du promoteur RSV-LTR et du grand gène SV40 de l'antigène-T. Les cellules transfectées ont formé des colonies à plusieurs couches après 3 à 4 semaines, et ont été transférées et développées pour en faire des souches cellulaires (HE-451 et HE-457). Les deux souches ont subi une croissance exponentielle pendant 8 à 10 semaines puis ont subi une sénescence. Après une ''crise'' de 6 à 8 mois, des colonies isolées se sont développées en deux lignées, les HET-1A à partir du HE-457 et HET-2A à partir du HE-451. Les populations de ces lignées ont doublé respectivement 143 et 122 fois. Elles ont toutes les deux une morphologie épithéliale, elles se colorent lors du contact avec la cytokératine et l'antigène-T SV40 au moyen de l'immunofluorescence, et sont restées non tumorgènes dans des souris athymiques pendant 12 mois. Les lignées oesophagiennes immortalisées dans le système dépourvu de sérum sont utiles aux recherches concernant l'action des concérogènes oesophagiens putatifs.Human epithelial and esophageal cells with an increased replication capacity in cell culture compared to normal cells, these cells being unable to cause tumors. Normal human esophagus tissue from two autopsy pieces was explanted in a serum-free medium. The epithelial growths were subcultured, then transfected with strontium phosphate coprecipitation using the plasmid pRSV-T consisting of the RSV-LTR promoter and the large SV40 gene of the T-antigen. The transfected cells formed multi-layer colonies after 3 to 4 weeks, and were transferred and grown to make cell strains (HE-451 and HE-457). Both strains grew exponentially for 8-10 weeks and then underwent senescence. After a 6 to 8 month `` crisis '', isolated colonies developed into two lines, HET-1A from HE-457 and HET-2A from HE-451. The populations of these lines doubled 143 and 122 times respectively. They both have an epithelial morphology, they stain on contact with cytokeratin and the SV40 T-antigen by means of immunofluorescence, and remained non-tumorgenic in athymic mice for 12 months. Esophageal lines immortalized in the serum-free system are useful for research on the action of putative esophageal concerogens.

Description

HUMAN ESOPHAGEAL EPITHELIAL CELL LINES
FIELD OF THE INVENTION
The present invention relates to human epithelial cells that originate from the esophagus and are immortal- ized in culture, particularly to such cells genetically transformed with genes from tumor viruses, especially,
SV40 early region genes. The present invention also relates to methods of using such cells for detection of various types of agents, for instance, chemicals that can cause cancer.
BACKGROUND OF THE INVENTION
Epidemiological evidence indicates that esophageal cancer is associated with exposure to chemical carcinogens in the environment and in the diet. Factors associated with an increased risk of developing esophageal cancer include tobacco, alcoholic beverages, and moldy foods, all of which contain nitrosamines; and asbestos. As in most forms of cancer, esophageal neoplasms probably are the result of a sequence of genetic and phenotypic changes in the esophageal epithelium.
For example, the phenotypic keratin patterns of squamous cell carcinomas of the esophagus are consistently different from those of the normal tissue (S.P. Banks- Schlegal and C.C. Harris, Cancer Res. 1984, 44: 1153- 1157.) (M.P. Grace, K.H. Kim, L.D. True and Fuhs, 1985, Cancer Res. 45: 841-846). Like epidermal keratinocytes, the esophageal epithelial cell is able to form crosslinked envelopes when it terminally differentiates (S.P. Banks- Shlegal and C.C. Harris, Cancer Res. 1986, 250-258. This function is variably expressed in esophageal carcinomas (S.P. Banks-Shlegal and C.C. Harris, Cancer Res. 1984, 44:1153-1157 and carcinoma cell lines (S.P. Banks-Shlegal and C.C. Harris Cancer Res. 1986, 250-258). In addition, a number of tumor associated antigens have been found in esophageal neoplasms that are not present in normal cells, including such antigens as human chorionic gonadotropin, placental lactogen, α-fetoprotein, carcinoembryonic antigen, and nonspecific crossreacting antigen (C.L. Burg- Kurland, D.M. Purnell, J.W. Combs, E.A. Hillman, C.C. Harris and B.F. Trump, 1986, 46: 2936-2943) .
To facilitate discovery of agents that represent important causes of esophageal cancer in the human envi- ronment, as well as development of preventive measures and treatments, there is a need for cell culture systems suitable for investigating chemical carcinogenesis in esophageal epithelial cells. These cells are the precur¬ sors of esophageal tumors, most of which are classified as squamous cell carcinomas (S.F. Stinson and G. Reznik Cancer of the Esophagus, Vol. 2., pp. 139-168, Boca Raton, CRC Press, Inc. 1982) . In investigations with the rat esophagus, treatment of explanted tissue cultures with N- nitrosobenzylmethylamine (NBMA) led to the establishment of epithelial cell lines that, after prolonged subculture, produced well-differentiated squamous cell carcinomas following transplantation in vivo. (G.D. Stoner, M.S. Babcock, G.A. Cothern, J.E. Klaunig, .T. Gunning III, and S.M. Knipe, 1982, Carcinogenesis 3: 629-634). There was a positive correlation between transformation of these cells and alterations in: (a) cytoskeletal microfilaments, (b) response of the cells to extracellular calcium ion concen¬ tration and serum-induced terminal differentiation (G.D. Stoner, M.S. Babcock, M.M. McCorquodale, W.T. Gunning III, R. Jamasbi, N. Budd, and B. Hukku, Comparative properties of untreated and N-nitrosobenzylmethylamine-transformed rat esophageal epithelial cell lines (in press) ; and, (c) the tendency of the cells to accumulate cholesterol sulfate when grown in medium containing high calcium (J.E. Rearick, G.D. Stoner, M.A. George, and A.M. Jetten, 1988, Cancer Res. 48:5289-5295). However, comparisons between NBMA transformed cells and normal cells were frequently compromised by the marked tendency of rat esophageal epithelial cells to undergo spontaneous neoplastic trans- formation. Moreover, there are distinct histological differences between rat and human esophageal epithelium.
In view of these difficulties with rat cells, there is a need to develop cultures of human esophageal epithelial cells for transformation studies. Normal human cells in general are known to be stable with respect to neoplastic transformation in culture, and have never been convincingly demonstrated to undergo spontaneous transfor- mation. (G.H. Sack, Jr., 1981, In Vitro, 17: 1-19). Therefore, normal human esophageal epithelial cells appear to offer an advantageous system, for instance, for detec¬ tion of substances with carcinogenic potential for the human esophagus. On the other hand, human esophageal epithelial cells are known to have a limited lifespan and replicative capacity in culture (C.C. Harris, 1987, Cancer Res. 47:1- 10; K. Sasajima, J.C. Willey, S.P. Banks-Schlegel, and C.C. Harris, 1987, J. Nat. Can. Inst. 78:419-423), which severely limits the number of cells that can be produced from a single human tissue specimen. To provide such cells in quantities and with the reproducible quality needed for extensive screening of chemicals for carcinoge- nicity or potential for protection against such carcino- gens, for instance, the replicative potential of these cells would need to be extended. Although human esophage¬ al epithelial cells that are neoplastic have an unlimited ability to replicate in culture, whether neoplastic by virtue of derivation from a cancer or by neoplastic transformation with a tumor virus in culture, such trans¬ formed cells are, of course, useless for applications requiring normal cells to detect carcinogenic activities.
The neoplastic transformation of normal cells into cells that can cause tumors (for example, upon injection into rodents) is thought to result from multiple cellular changes. Evidence supporting this concept comes from studies which compared the neoplastic transformation of normal cells and cell lines that were immortalized by genetic transformation with viral and cellular oncogenes. Unlike normal cells, such as those in primary cultures of epithelial cells which are prepared directly from tissues, immortalized cells have the ability to replicate in culture for an indefinite number of generations. The results of the comparative studies indicate that certain oncogenes induce immortalization in normal cells but, by themselves, cannot induce frank neoplasia as evidenced by tumorigenicity. However, an immortalization gene can cooperate with a second class of oncogene to induce tumorigenic potential. On the other hand, at least some cells that have become immortalized without addition of an exogenous immortalizing oncogene, nevertheless require only the second type of oncogene for complete neoplastic transformation into tumor forming cells.
It is known that normal human epithelial cells have a limited lifespan in culture (C.C. Harris, 1987, Cancer Res. 47:1-10), even when optimal serum-free media are used (J.F. Lechner, and M.A. LaVeck, 1985, Meth. 9:43- 48) . It is also known that certain human cells infected with the SV40 tumor virus have extended lifespans in culture (V. Defendi, P. Naimski and M.L. Steinberg, 1982, J. Cell Physiol. Suppl., 2:131-140). However, in many cases, SV40-infected cells undergo a condition called crisis during which the rate of proliferation markedly decreases or ceases entirely (G.H. Sack, Jr., 1981, In Vitro, 17:1-19). Following crisis, a culture of any given type of human cells infected with SV40 may eventually die; or, in some cases, a few individual cells will eventually replicate sufficiently to produce a visible colony of descendants. This phenomenon of crisis is poorly under¬ stood and may be equivalent to senescence in non- transfected cells, in which the cells appear to "age" and lose the ability to replicate. Crisis is not a universal phenomenon, however, since there are reports that SV40- transformed amnion epithelial cell lines (E. Gaffney, J. Fogh, L. Ramos, J.D. Loveless, H. Fogh, and A.M. Dowling, 1970, Cancer Res. 30:1668-1676), and SV40-transformed foreskin keratinocytes (M.L. Steinberg and V. Defendi, 1979, Proc. Nat. Acad. Sci. USA, 76:801-805), became immortalized without the intervention of a crisis.
Thus, the art of immortalizing human cells is highly unpredictable, not only with respect to the course of the process in those cases that are successful, but also in the matter of achieving eventual success with any given new tissue source.
It has been reported (S.E. Chang, 1986, Biophys. Acta, 823:161-194, 1986; P. Kahn, W.C. Topp, and S.I. Shin, 1983, Virology, 126:348-360; Y. Ohnuki, J.F. Lechner, S.E. Bates, L.W. Jones and M.E. Kaighn, 1982, Cell Genet., 33:170-178; B.J. Christian, L.J. Loretz, T.D. Oberley and A. Reznikoff, 1987, Cancer Res., 47:6066- 6443) , that human and other primate cells immortalized by SV40 virus are nontumorigenic in certain mice which are athymic, having an immune system disorder that allows transplantation of tissues from other species without rejection. (Since these creatures also happen to be hairless due to a genetic defect, they are generally known as "nude" mice.) Therefore, infection with SV40 appears to offer a means for extending the replicative potential of at least some types of cultured human epithelial cells.
A problem with the use of infectious SV40 virus to immortalize cells is that, although the infection with SV40 is not lethal and tends to be self-limiting, the potential for resurgence of more active viral replication persists. Such replication may alter the physiology of the cells and thus render them unreliable for long term culture and chemical testing purposes. Accordingly, the present inventors have been involved in research efforts to extend the replicative potential of various human epithelial cells without introducing genes allowing complete viral synthesis. Within the past year they have demonstrated that genetic transformation with a plasmid containing only certain SV40 genes, namely the so-called early region genes, leads to immortalization of human cells derived from tissues other than esophageal epitheli¬ um. These include: epithelial cells from the bronchus (R.R. Reddel, et al., 1988, Cancer Res., 48:1904-1909; see also U.S. Patent Application Ser. No. 07/114,508 filed October 30, 1987) mesothelium (Y. Ke, et al., 1989, J. Pathol. 134:979-991; see also U.S. Patent Application Ser. No. 07/114,508), and neonatal prostate (M.E. Kaighn, et al., 1989, Cancer Res. 49:3050-3056).
The plasmid used to immortalize these cells, which is called pRSV-T, is known in the art (D.E. Brash, R.R. Reddel, M. Quanrad, K. Yang, M.P. Farrell, and C.C. Harris, 1987, Mol. Cell. Biol., 7:2031-2034). It also comprises certain genetic elements from another tumor virus, the Rous sarcoma virus. (Since this plasmid comprises viral genes, the process of genetic transforma- tion in this instance is referred to as "transfection", a term of the art that is a hybrid of the words "transforma¬ tion" and "infection". Even though the viral genes of the plasmid are insufficient to produce infectious virus, the use of the term transfection here is convenient for distinguishing the process from that of neoplastic trans¬ formation. Accordingly, hereinafter, the term transforma¬ tion will be used exclusively in connection with the latter process of neoplasia.)
Further, the present inventors have shown that a transfection procedure using strontium phosphate (D.E. Brash, et al., 1987, supra) has yielded stable trans- fectants (i.e., transfected cells) in the several human epithelial cell types cotransfected with the plasmid pRSV- T (as cited above) , and that this strontium procedure is more effective than the traditional calcium phosphate method for insertion of DNA into epithelial cells that are sensitive to high levels of calcium.
SUMMARY OF THE INVENTION The present invention contemplates the application of methods of recombinant DNA technology to fulfill the above needs for esophageal cell lines for chemical testing and other purposes. More specifically, it is an object of the present invention to provide a line of human esophage¬ al epithelial cells or a derivative thereof having a replicative capacity in cell culture that is enhanced compared to normal cells, and is unable to produce tumors. Further, it is an object of the present invention to provide a cell line of this type which replicates continu¬ ously in cell culture.
As described in the following Examples, the normal human esophageal epithelial cells can be made to grow continuously by transfecting normal esophageal epithelial cells with the T antigen gene of SV40 virus. The plasmid containing the SV40 early region genes further comprises a genetic element derived from another tumor virus, the long terminal repeat (LTR) or Rous sarcoma virus, which serves to stimulate transcription of the SV40 DNA sequences. Other constructs can be used for the same purpose, accord¬ ing to the methods well known in the art of genetic engineering.
Transfection or infection can be accomplished by use of a virus or a plasmid obtaining the T antigen gene of the SV40 virus. Either transfection or infection may lead to transformation of the cell line. Other transfor¬ mation vectors may be useful, such as papilloma virus or Epstein Barr virus. Techniques for making continuous human cell lines are described in the following referenc¬ es: Grahm, F.L., Smiley J. , Russell, W.C. and Nairn, R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J. Gen. Virol., 36: 59-72, 1977; Zur Hausen, H. Oncogenic herpes viruses In: J. Tooze (ed.), DNA tumor viruses, Rev. Ed. 2, pp 747-798. Cold Spring Harbor, New York, Cold Spring Press, 1981; DiPaolo, J.A. Pirisi, I., Popeseu, N., Yasumoto, S., Poniger, J. Progressive changes induced in human and mouse cells by human Papillomavirus Type-16 DNA. Cancer Cells 5:253-257, 1987.
Further, it is an object of this invention to provide a method for testing carcinogenicity of an agent, comprising culturing the cells line of this invention with an agent suspected of being carcinogenic; and determining formation of an abnormal cellular mass (i.e., a trans¬ formed "focus") by the cell line. The formation of a focus or foci is indicative of carcinogenicity of an agent. Further, it is an object of the present invention to provide a method for testing antineoplastic activity of an agent, comprising culturing a cell line of this inven¬ tion with a potential anti-neoplastiσ agent; and determin- ing growth of the cell line. A lack of growth of the cell line is indicative of antineoplastic potency of an agent, particularly for esophageal epithelial cells.
The present invention may be understood more readily by reference to the following detailed description of specific embodiments and the Examples included therein.
DESCRIPTION OF SPECIFIC EMBODIMENTS Unless defined otherwise, all technical and scientific terms used herein have the same meanings commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to this described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. ABBREVIATIONS
The abbreviations used are: HE, human esophagus;
NHE, normal human esophagus; HBS, Hepes buffered saline;
BPE, bovine pituitary extract; SV40, Simian virus 40; CFE, colony forming efficiency; PD, population doublings; EM, electron microscopy.
DESCRIPTION The term "immortalized" as used herein means that the cell line grows continually without senescence when cultured in a suitable growth medium. The term "continuous cell line" as used herein means that the cell line grows continually without senes¬ cence when cultured in a suitable growth medium.
The "derivatives" of the esophageal cell line(s) of the present invention include cells which have been further genetically altered by adding, for example, genes for drug metabolizing enzymes, other oncogenes, anti- oxidant genes, or other genes, thereby creating a continu¬ ous derivative of the cell line. In one aspect, the present invention relates to a line of human esophageal epithelial cells or a derivative thereof having a replicative capacity in cell culture that is enhanced compared to normal cells and being unable to produce tumors. The general method for obtaining immor¬ talized human esophageal epithelial cells is as follows.
Normal human esophageal (NHE) cells were obtained from explant outgrowths of autopsy specimens from noncan- cerous males. Dispersed cell suspensions were plated at 3-5 x 105 cells/dish and transfected with 10 μg of plasmid pRSV-T coprecipitated with strontium phosphate. After 4 hrs, the cells were shocked with glycerol, as described in Example 1, below. After the appearance of foci of trans¬ formed cells, control and transfected cultures were subcultured (2.5 x 105/100-mm dish) . Control strains could be subcultured for no more than 20 PDs, after which they senesced. pRSV-T-transfected cells (e.g., lines designated HE-451, HE-457, see below) grew exponentially for approxi- mately 50 PDs, after which they went into crisis. During this crisis period which lasted for several months, the majority of cells in both strains senesced. Recovery from this state depended upon their continued maintenance at the highest possible density. Eventually, as described in Examples 1 and 2 below, two separate immortalized cell lines, designated HET-IA and HET-2A, were developed, on each from the HE-451 and HE-457 cultures, respectively.
A deposit of the cell lines of the present inven¬ tion has been made at the American Type Culture Collec- tion, 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A., on August 25, 1989, under accession numbers CRL- 10209 (HET-IA) and CRL-10210 (HET-2A) , in accordance with the Budapest Treaty. The deposits shall be viably main¬ tained, replaced if they become non-viable, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and made available to the public without restriction in accordance with the provisions of the law. The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
The pRSV-T transfected cell lines expressed T- antigen by immunoperoxidase nuclear staining. This is consistent with the Southern blotting data indicating that all lines contain integrated SV40 early region DNA. All transfected lines were confirmed as epithelial by virtue of their positive reaction with antibodies to keratin and the presence of desmosomal junctions and cytoplasmic microfilaments in EM preparations.
The chromosomal alterations in the transfected human esophageal epithelial cells are similar to those previously reported after infection with SV40 virus (M.E. Kaighn, K.S. Narayan, Y. Ohnuki, L.W. Jones, and J.F. Lechner, 1980, Carcinogenesis, 1:635-645). HET-IA, a hypodiploid line, has a chromosome complement similar to that of pRSV-T immortalized human bronchial epithelial cells (R.R. Reddel, et al., 1988, supra) and prostatic epithelial cells (M.E. Kaighn et al. 1989, supra) . The chromosome number and structural aberrations of HET-IA cells will probably increase with continued subculture, since this has occurred with pRSV-T immortalized bronchial and prostatic epithelial cell lines. HET-2A cells are larger than those of HET-IA. This is consistent with the observation that the chromosome number of HET-2A cells is in the hypotetraploiά range whereas HET-IA is hypodiploid. Although HET-2A cells originated from a male specimen, they had lost the Y chromosome by passage 6. Chromosome analysis indicated that the precursor strain to HET-2A, i.e., HE-451 cells, contain the Y chromosome. Loss of the Y chromosome in transformed cells is frequently observed. In general, instability of the karyotype is a characteris¬ tic of virally-transformed cells.
None of the pRSV-T transfected cells tested, including the HET-IA and HET-2A cell lines, were tumori- genic in athymic nude mice. In addition, they did not induce transient nodules of carcinoma-like cells at the injection site as had been observed with carcinogen- treated rat esophageal epithelial cells and human neonatal prostatic epithelial cells (M.E. Kaighn, R.R. Reddel, J.F. Lechner, D.M. Peehl, R.F. Camalier, D.E. Brash, U. Saffio- tti and C.C. Harris, 1989, Cancer Res. 49:3050-3056). These immortalized lines may represent an intermediate between normal and neoplastic in which altered growth control is offset by the capability of undergoing differ¬ entiation when injected into mice (M.E. Kaighn, et al., 1989, supra; E.J. Stanbridge, C.J. Der, C-J. Doersen, R.Y. Nishimi, D.M. Peehl, B.E. Weissman and J.E. Wilkinson, 1982, Science, 215:252-259).
Since both calcium and fetal bovine serum are known to have profound effects on growth and differentia¬ tion of epithelial cells, the effects of these factors were assessed by clonal titration experiments, as de¬ scribed in Example 2. A dose-dependent stimulation of CFE was observed with maximal CFE at 0.3 mM Ca++ in both HET- IA and HET-2A cell lines. In contrast, fetal bovine serum inhibited the CFE at all concentrations tested. Half maximal inhibition was seen at 1% (HET-IA) and 3% (HET-2A) serum. There was no significant effect of either Ca++ or serum on the clonal growth rate (PDs/day) of HET-IA or HET-2A cell lines at all concentrations tested.
UTILITY OF THE CELL LINES Identification of potential carcinogens, tumor promoters and antagonists thereof. These cells are useful for screening chemicals or other agents in the human environment for the potential to neoplastically transform the cells. Putative carcinogens or tumor promoters may be added to the growth medium of the cells and the state of transformation of the cells as a function of time and dose of exposure may be ascertained using anchorage indepen¬ dence growth, matrix invasion, cells to cell communication assays, and/or nude mice tumorigenicity assays. Anti- carcinogenic or anti-promoting agents may be added to the medium with carcinogens or promoters and the state of transformation as a function of time may be ascertained as for carcinogens or promoters alone. Such screening could be used to identify specific esophageal toxins in the diet or in other substances and to identify dietary or other compounds that could prevent chemically-induced esophageal cancer. Identification of potential chemotherapeutic drugs. These cells are also useful, either before or after further alteration by other oncogenes or carcino¬ gens, for screening chemicals particularly suitable for the treatment of esophageal cancer and related diseases, by growing them in culture medium containing the chemical to be tested and then, after a suitable period of expo¬ sure, determining whether and to what extent cytotoxicity has occurred, e.g., by trypan blue exclusion assay or related assays (Paterson, Methods Enzymol., 58:141, 1979), or by growth assays such as colony forming efficiency as described in the examples, herein, all of which are standard techniques well known in the art.
Identification of anti-esophageal cancer drugs which act bv inducing terminal cell differentiation. Chemical and biological substances are screened for their ability to induce terminal differentiation by adding them to the growth medium of these esophageal cells and then after a suitable period of time, determining whether a complex of changes occurs, including for example, the phenotypic keratin patterns of the normal esophagus (S.P. Banks-Schlegel and C.C. Harris, 1984, Cancer Res. 44:1153- 1157; M.P. Grace, K.H. Kim, L.D. True and E. Fuchs, 1985, Cancer Res. 45:841-846). Induction of terminal differen¬ tiation may be an effective way of controlling the growth of cancer.
Studies on the metabolism of carcinogens and other xenobiotics. Carcinogens and other xenobiotics may be added to the growth medium of these cells and the appear¬ ance of metabolic products of these compounds may be monitored by techniques such as thin layer chromatography or high performance liquid chromatography and the like, and the interaction of the compounds and/or their metabo¬ lites with DNA is determined. Studies of DNA mutagenesis. Substances known or suspected to be mutagens may be added to the growth medium of the cells and then mutations may be assayed, e.g., by detection of the appearance of drug resistant mutant cell colonies (Thompson, Methods Enzymol., 58:308, 1979).
Studies of chromosome damaging agents. Substances known or suspected to cause DNA or chromosomal damage may be added to the culture medium of these cells lines, and then the extent of chromosomal damage may be measured by techniques such as measurement of the frequency of sister chromatic exchange (Latt et al.. In: Tice, R.R. and Hollander, A., Sister Chromatid Exchanges, New York: Planum Press, pp. 11 ff., 1984), and DNA damage can be determined by the measurement of unscheduled DNA synthesis (Mirsalis, J.C., Banbury Report vol. 13, pp. 83-99, 1982). Studies of malignant transformation bv additional oncogenes. The effects of viral agents and genes trans¬ ferred by genetic transformation, including oncogenes and high molecular weight genomic DNA from tumors, may be tested using standard assays such as anchorage independent growth or tumor formation in athymic nude mice. Further, such cells transformed by an additional oncogene can be used to screen for potential chemotherapeutic agents by the techniques described above, especially those which may be specific for cells transformed by the activation of particular oncogenes or combination of oncogenes.
Studies of cellular responses to growth factors and production of growth factors. These cells are partic¬ ularly useful for identification and purification of growth factors important for growth and differentiation of human esophageal epithelial cells, since they grow in serum-free media. Therefore, responses to added growth factors or isolation or produced growth factors can be readily accomplished without interference from serum and its complexities.
It will be readily appreciated by one skilled in the art that a kit for screening carcinogenic or antineo¬ plastic agents, for instance, or for any other usage described herein, is easily assembled, comprising contain¬ er(s) containing cells of the present invention. Other components routinely found in such kits may also be included with instructions for performing the test. EXAMPLE 1
Development of the HET-IA cell line. Cell Culture. Normal human esophageal (NHE) cells were obtained from outgrowths of autopsy tissue from noncancer- ous individuals as previously described (G.D. Stoner and J. Klaunig, In: T.G. Pretlow III and T.P. Pretlow (eds.) Cell Separation: Methods and Selected Applications, Vol. 2, pp. 8192. New York, Harcourt, Brace and Jovanovich, 1983) . The outgrowths were suspended with PET [1% polyvi- nylpyrrolidone, 0.02% ethylenebis-(oxyethylenenitrilo) tetraacetic acid, 0.2% crystalline tryspin in HBS, pH 7.4] at room temperature and subcultured into tissue culture dishes or T flasks which had been coated with a mixture of 100 μg/ml bovine serum albumin, 10 μg/ml bovine fibro- nectin (both from Calbiochem) , and 20 μg/ml type 1 colla- gen (Vitrogen 100, Collagen Corp., Palo Alto, CA) to promote cell attachment (J.F. Lechner and M.A. LaVeck, 1985, J. Tissue Culture Meth. 9:43-48).
A serum-free medium, LHC-9, purchased from Biofluids, Inc. , was used in the early phase of this research (J.F. Lechner and M.A. LaVeck, 1985, supra) . It was found later that a serum-free formulation developed for human epidermal keratinocytes (S.T. Boyce and R.G. Ham, In: M. Webber and L. Sekely (eds.) In Vitro Models for Cancer Research, Vol. 3, pp. 245-274. Boca Raton, Florida, CRC Press, 1985) was more appropriate for growth of these esophageal epithelial cells. This formulation, consisting of modified MCDB 153 supplemented with growth factors (KGM) is available from Clonetics Corporation, San Diego, CA. The basal medium (cKBM) used in these studies and purchased from Clonetics Corporation was a customized modification of KBM without phenol red. other components omitted from cKBM were added just before use CaCl2, FeS04, ZnCl2, trace element concentrate, thymidine, phospho- ethanolamine, ethanolamine, glutamine and antibiotics) .
The growth medium (cKGM-AE consisted of cKBM supplemented with 5 ng/ml epidermal growth factor, 1.4 μM hydrocortisone, 0.1 mM ethanolamine and phosphoethanol- amine, 5 μg/ml insulin, 40 μg/ml bovine pituitary extract (BPE) , 250 μg/ml bovine serum albumin, and 0.5 μg/ml epinephrine. Antibiotics were added as needed (100 units/ml penicillin G, 100 μg/ml kanamycin, 50 μg/ml gentamicin) .
Cultures were monitored for mycoplasma contamina¬ tion by culture on anexic agar and by DNA fluorochrome staining of an indicator culture (R. DelGiudice and H.E. Hopps, In: G.J. McGarrity, D.G. Murphy, and W.W. Nicols (eds.), Mycoplasma Infection of Cell Cultures, pp. 57-69. New York, Plenum Publishers, 1978) . No contamination was detecte .
Transfection. Subcultures of NHE cells were used for transfection. Cells were plated at 3-5 x 105/100-mm coated dish and transfected the next day with 10 μg of plasmid DNA coprecipitated with strontium phosphate as previously described (D.E. Brash et al. , 1987, supra). The plasmid, pRSV-T, obtained from Dr. Bruce Howard, NCI, is an ori construct (i.e., lacks the SV40 site for origin of DNA replication) containing the SV40 early region genes and the Rous sarcoma virus long terminal repeat (R.R. Reddel, Y. Ke, B.I. Gerwin, M.G. McMenamin, J.F. Lechner, R.T. Su, D.E. Brash, J.B. Park, J.S. Rhim and C.C. Harris, 1988, Cancer Res., 48:1904-1909). Four hrs after trans- fection, the cells were shocked with 15% glycerol in HBS, washed 3 times with LHC basal medium and incubated in LHC- 9 medium. After the appearance of foci of transformed cells (3-4 weeks) the cells were subcultured (5 x 105/100- mm dish) . The cultures were fed 3 times per week with fresh LHC-9 and transferred at weekly intervals at a seeding density of 5 x 105 cells per 100-mm dish or T75 flask. In this and the following Example, the newly developed strontium phosphate procedure for transfection (D.E. Brash, et al., 1987, supra) was used, and yielded stable transfectants with an efficiency of 2-4 x 10~5. This frequency is 5 to 10-fold lower than that reported for human cells from the bronchial epithelium (R.R. Reddel, et al., 1988, supra) mesothelium (Y. Ke et al., 1989, supra) and neonatal prostate (M.E. Kaighn et al, 1989, supra) transfected with the same plasmid. The reason for the observed lower incidence of transfection in HE cells is unknown.
The surviving cells of strain HE-457 formed two discreet colonies in a single flask. One of these contin¬ ued to grow following isolation; the other did not sur- vive. At passage 14, this strain (HET-IA) was switched to cKGM-AE medium. Its growth has accelerated, and it has doubled at least 143 times thus far.
EXAMPLE 2 Development of the HET-2A cell line. Based on the observation that the switch to a different growth medium appeared to enhance growth of the few surviving cells of the pRSV-T-transfected cells designated HE-457, a frozen ampoule of the other strain, HE-451, was recovered and cultured in the same medium (cKGM-AE) . In this case, there was a much shorter lag before consistent exponential growth was seen. This line (HET-2A) has now achieved at least 122 PDs.
Characterization. Pre-crisis cultures and immor¬ talized cell lines were characterized by immunohistochem- istry. Keratin staining was intensely positive in both pre-crisis strains (HE-451 and HE-457) , especially in closely apposed foci of cells. Vimentin was also posi¬ tive, although to a lesser extent than keratin. Numerous atypical nuclear features were observed including dyspla- sia and para-nuclear clearing (koilocytosis) . Keratin stained positively in all cells of both immortalized lines. Both lines stained heterogeneously for vimentin with more intense staining in the smaller cells. Keratin stained much more intensely than vimentin in both cell lines. The overall staining pattern of both pre- and post-crisis cells was consistent with their epithelial origin. Transmission electron microscopy also confirmed that all pre- and post-crisis cells are of epithelial origin since they contained tonofilaments and were joined by desmosomal junctions.
Clonal growth assays. Response to growth factors and inhibitors was assessed by a clonal growth assay (J.F. Lechner and M.E. Kaighn, 1979, J. Cell Physiol. 100:519- 529) . Subconfluent cultures were suspended with PET, and plated at 500-1000 cells/60 mm-coated dish containing 4 ml of cKGM-AE from which the factor under consideration was omitted. After overnight incubation, the medium was removed and experimental medium was added. Plates were fixed in 10% neutral buffered formalin and stained with Giemsa after 6-8 days incubation. Both colony forming efficiency (CFE) and clonal growth rate, (population doublings/day (PD/d) were determined. Four replicate dishes per variable were used for CFE assay, and at least 18 colony counts were averaged for determination of PD/day.
Chromosome and isozyme analyses. Chromosome studies were performed by Dr. Ward D. Peterson, Children's Hospital of Michigan, Detroit, MI, using standard methods. Metaphases were stained with Giemsa and counted at low power for ploidy determination. Exact counts on 30 metaphases were made on banded chromosomes, and at least 8 karyotypes per cell line were prepared. Analyses of 8 isozymes were carried out using standard procedures.
The chromosomal profiles of SV40-T antigen immor¬ talized cell lines, HET-IA and HET-2A showed that both lines are aneuploid and each has its own complement of marker chromosomes. HET-IA is hypodiploid with only about 5% of metaphases examined in the hypotetraploid range. Cell line HET-2A is hypotriploid with 28% of metaphases having 100+ chromosomes. There are 2 normal X chromosomes in all metaphases examined but no Y. DNA hybridization blot analysis. Cells were grown to 80-85% confluence, trypsinized and collected by cen- trifugation. DNA was isolated by SDS proteinase-K incuba¬ tion followed by phenol/chloroform extraction and ethanol precipitation. The purity of the DNA was assessed spec- trophoto etrically using the 260/280 nm ratio. DNAs (5 μg each) from cell strains HE-451, HE-457, HOC-517, and HB- 56B, and cell lines RE-149, BEAS-2B, HET-IA and HET-2A were loaded into individual wells of a S&S Slot Blot apparatus (Schleicher and Schuell, Inc., Keene, New Hampshire) . DNA was blotted onto Hybond-N (Amersham) nylon membrane. The samples were probed with a nick- translated EcoRI-Hindlll fragment of the plasmid, pRSV-T in 2x SSC-0.1% SDS at 65°C for 16 hours. The membrane was washed to 0.2x SSC-0.1% SDS at 65°C and autoradiographed at -75°C. A sample (1 μg) of the plasmid DNA was used as a control.
The results of DNA blot hybridization analyses of SV40-T antigen gene showed that cell lines HET-IA and HET- 2A, strains HE-451 and HE-457, as well as positive control cell lines, HOC-517 and BEAS-2B (R.R. Reddel, et al., 1988, supra) , were all positive for pRSV-T plasmid DNA. HET-2 and HE-451 appeared to have incorporated approxi¬ mately the same number of copies of the SV40-T antigen gene. HET-IA had slightly fewer copies, although more than HE-457. Although cell strain HOC-517 appears to have incorporated only a few gene copies, longer autoradio- graphic exposures indicated that it is definitely posi¬ tive, and that the negative controls, HB-56B and RE149, have no detectable copies of the SV40-T gene.
Immunofluorescence and electron microscopy. Cells were fixed with 3% buffered glutaraldehyde for transmis¬ sion electron microscopy or in absolute methanol for immunofluorescence. Alternatively, cell suspensions were fixed and attached to slides by cytocentrifugation. The cells were stained with antibodies to cytokeratin and vimentin by the immunoperoxidase technique (Y. Katoh, G.D. Stoner, C.C. Harris, K.R. Mclntire, T. Hill, R. Anthony, E. McDowell and B.F. Trump, 1979, J. Nat. Cancer Inst. 62:1177-1185), and with a monoclonal antibody to SV40 large T-antigen (Oncogene Science, Inc., Mineola, NY) by immunofluorescence. Tumorigenicity assay. HE-451 (passage 16) , HE-457
(Passage 14) , HET-IA (passage 10) and HET-2A (passage 7) cells were tested for tumorigenic potential in athymic nude mice. Cells were suspended with PET and injected s.c. into both the left and right flanks of each animal. A total of 5 x 106 cells in 0.1 ml of medium were injected into each flank, and at least ten animals per cell line were used. Animals were observed weekly for tumor devel¬ opment up to 12 months.
For purposes of completing the background descrip- tion and present disclosure, each of the published arti¬ cles, patents and patent applications heretofore identi¬ fied in this specification are hereby incorporated by reference into the specification.
The foregoing invention has been described in some detail for purposes of clarity and understanding. It will also be obvious that various combinations in form and detail can be made without departing from the scope of the invention.

Claims

WHAT IS CLAIMED IS:
1. A line of human esophageal epithelial cells or a derivative thereof having a replicative capacity in cell culture that is enhanced compared to normal cells and being unable to produce tumors.
2. The cell line according to claim 1, which replicates continuously in cell culture.
3. The cell line according to claim 1, which is infected or transfected with a virus or plasmid which causes immortalization of normal human esophageal epithe¬ lial cells.
4. The cell line according to claim 3, which is transfected with the plasmid pRSV-T.
5. The cell line according to claim 1, which contains an oncogene.
6. The cell line according to claim 1, which is either HET-IA (ATCC CRL-10209) or is HET-2A (ATCC CRL- 10210) .
7. A kit for screening carcinogenic or chemo- therapeutic agents comprising: a container containing cells of a human esophageal epithelial cell line or derivative thereof which replicates continuously in cell culture and is unable to produce tumors.
8. A method for testing carcinogenicity of an agent, comprising culturing the cells line of claim 1 with an agent suspected of being carcinogenic; and determining formation of an abnormal cellular mass by said cell line, the formation of abnormal cellular mass being indicative of carcinogenicity of said agent.
9. A method for testing antineoplastic activity of an agent, comprising culturing the cells line of claim 1 with a potential antineoplastic agent; and determining growth of said cell line, a lack of growth of said cell line being indicative of antineoplastic potency of said agent.
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US5585237A (en) * 1993-10-25 1996-12-17 Creative Biomolecules, Inc. Methods and compositions for high protein production from recombinant DNA
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WO1989003994A1 (en) * 1987-10-30 1989-05-05 The United States Of America, As Represented By Th Immortalized human cell lines

Non-Patent Citations (4)

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Title
CANCER RESEARCH vol. 45, no. 2, February 1985, PHILADELPHIA pages 841 - 846 GRACE M.P. ET AL 'Keratin expression in normal esophageal epithelium and squamous cell carcinoma of the esophagus' *
IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY vol. 26, no. 3, March 1990, GAITHERSBURG, MD page 23A STONER G.D. ET AL 'Immortalized human esophageal epithelial cell lines for studies of carcinogen-induced cell transformation' *
PROCEEDINGS OF THE EIGHTY-FIRST ANNUAL MEETING OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH vol. 31, no. 0, March 1990, WASHINGTON page 136 LIGHT, B. ET AL 'Hst-1 induces tumorigenicity in SV40 T antigen immortalized non-tumorigenic, human esophageal epithelial cells' *
See also references of WO9105062A1 *

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