CA1298220C - Immortalized human cell lines - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Immortalized human bronchial epithelial and human mesothelial cell lines have been obtained. Various uses of these cell lines have been described.

Description

1 IM~ORTALIZED RUMAN CELL LINES

2 BAC~GROUND OF THE INVENTION

3 The present invention is related to immortalized 4 cell lines. More particularly, the present invention is related to immortalized human bronchial epithelial and 6 human mesothelial cell lines or eell lines derived 7 therefrom.
8 Lung eancer is one of the more common forms of 9 eaneer and the eell type in which the ma~ority of these eaneers arise is the bronchial epithelial eells.
11 Meaothelial eells are a less eommon, but important, site 12 of origin of lung eaneer. Both, normal human bronehlal 13 epithelial eells and normal human mesothelial eells eould 14 be eultured in vitro, but only for a limited period of time before eellular replication eeases. When 16 transformed by transfeetion of the vlral Harvey ras 17 oncogene (Yoakum, et al., Science, 227:1174, 1985), human 18 bronchial epithelial cells replicate for longer periods l9 of time, but these cells are tumorigenie, grow in serum-eontaining media as do earcinoma cell lines~ and 21 have been constructed to contain an oncogene closely 22 related to oncogenes sometimes found in human 23 carcinomes. Similarly, human bronchial carcinoma cell lZ98ZZO

1 lines and mesothelioma cell lines are tumorigenic.
2 Clearly, such tumorigenic cell lines are undesi~able, 3 inter alia, for carcinogenic studies.

4 SUMMA~Y OF INVENTION

It is, therefore, an object o~ the present 6 invention ~o provide non-tumorigenic human cell lines of 7 bxonchial epithelial and of mesothelial cell origin with 8 unlimited proliferative potential and capable of growing 9 in the same serum-free media as their normal counterpart cells, and which do not contain an oncogene found in 11 naturally occurring tumors.

12 The present invention provides a non-tumorigenic, human bronchial 13 epithelial or mesothelial cell line or derivative thereof growing without 14 senescence when cultured in vitro in growth medium, said cell lines being selected from the group consisting of those having the identifying 16 characteristics of ATCC CRL 9608, 9609, 9442, 9443, 9444, 9482 and 9483.
17 In another aspect, the invention provides a kit for screening 18 carcinogenic or chemotherapeutic agent comprising a container containing 19 a non-tumorigenic, human bronchial epithelial or mesothelial cell line or derivative thereof as described above growing without senescence when 21 cultured in vitro in growth medium.
22 Also disclosed is a method for testing carcinogenicity of an 23 agent, comprising culturing the cell line as descirbed above with an 24 agent suspected of being carcinogenic and determining formation of abnormal cellular mass by said cell line, the formation of abnormal 26 cellular mass being indicative of carcinogenicity of said agent.
27 Finally, there is described a method for testing antineoplastic 28 activity of an agent, comprising culturing the cell line as described 29 above with a potential antineoplastic agent and determining growth of said cell line, a lack of growth of said cell line being indicative of 31 antineoplastic potency of said agent.
.

~:98~Z() - 2a -1 Other objects and advantages of the present ~ invention would become apparent from the Detailed 3 Description of Invention.

4 DETaILED DESC~IPTION OF INVENTION

~he above and other objects and advantages of the ~ present invention are achieved by non-tumorigenic, human 7 bronchial epithelial cell line continually growing when 8 cultured in vitro in suitable growth medium.
9 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as 11 commonly understood by one of ordinary skill in the art 12 to which this invention belongs. Although any methods 13 and materials similar or equivalent to those described 14 herein can be used in the practice or testing of the present invention, the preferred methods and materials 16 are now described.

~,,-"' ~29~3Z20 1 The terrn "immortalized" as used herein means that 2 the cell line grows continually without senescence when 3 cultured in vitro in a suitable growth medium.

4 General Method for Construction of Cell Lines Normal human bronchial epithelial (NHBE) cells 6 were cultuxed from explants of necropsy tracheobronchial 7 specimens from noncancerous individuals as described by 8 Lechner, et al., J. Tissue Culture Methods 9:43-48, 9 1985. Normal human mesothelial (NHM) cells were cultured from pleural effusions or ascites fluids as described by 11 Lechner, et al, (Proc. Natl. Acad. Sci. USA 82:3884-3888, 12 1985). The cells were infected with SV40 virus or with 13 adenovirus-12 SV40 hybrid virus, or transfected with a 14 recombinant plasmid containing the Rous sarcoma virus long terminal repeat and the ori-SV40 early region by 16 strontium phosphate coprecipitation (Brash, et al., 17 Molec. Cell. Biol.~, 1987). Colonies of cells transformed 18 by each of these three methods were easily recognizable 19 morphologically using phase contrast microscopy and were individually trypsinized and serially passaged. In all 21 cases the lifespan of these cultures was extended 22 compared to NHBE or NHM; most of the cultures underwent a 23 prolonged period of senescence referred to as "crisis".
24 With continued culture~ in some cases colonies of cells which had escaped senescence arose; such surviving 26 colonies were subsequently passaged for extended period 27 of time and showed unlimited growth potential. Like NHBE
28 cells, but unlike bronchial carcinoma cells, some of the 29 cell lines thus derived retained the capacity to undergo squamous differentiation in response to serum exposure.
31 Injection of these cells into irradiated athymic nude 32 mice did not result in formation of tumors after periods 33 of up to nine months. Furthermore, these cell lines were 1;~9~3Z~V

1 found to be suitable recipients for transfection of 2 additional oncogenes and useful for testing the 3 cytotoxicity potential of chemical and physical agents, 4 the growth inhibition or promoting capability of biological agents, and squamous differentiating potential 6 of chemical and biological agents.

8 Development of BES-lAl.6 Cell Line 9 Normal human bronchial epithelial (NHBE) cells were cultured from explants of autopsy specimens from 11 noncancerous individuals as described by Lechner, et al., 12 J. Tissue Culture Methods 9: 43-48, 1985. The calls were 13 cultured in a serum-free medium, LHC-9, consisting of LHC
14 basal nutrient medium with calcium 0.08 mM, L-glutamine lmM, trace elements, gentamicin 50 ~g/ml, insulin 5 16 ~g/ml, transferrin 10 ~g/ml, hydrocortisone 200 nM, 17 epidermal growth factor 5 ng/ml, phosphoethanolamine 0.5 18 ~M, ethanolamine 0.5 ~M, epinephrine 0.5 ~g/ml, retinoic 19 acid 0.33 nM, trilodothyronine 10 nM, and bovine pltuitary extract (Lechner, et al., supra). In the 21 initial stages of the development of this cell llne LHC-8 22 medium which contains the ingredients listed for LHC-9, 23 with the exception of epinephrine and retinoic acid, was 24 used.
NHBE cells were harvested by trypsinization and 26 seeded in 10 ml growth medium into 100 mm culture dishes 27 (Lux, Miles Scientific, Naperville, IL) whose growth 28 surfaces had been coated with a solution of bovine serum 29 albumin, fibronectin and collagen (Lechner, et al., supra).
31 SV40 virus was prepared in CV-l cells as described 32 by Su, et al., J. Virol. 28: 53-65, 1978. NHBE cells ~298Z~O

1 were exposed at 37C for 90 min. at a multiplicity of 2 infection of approximately 1. The cells were subcultured 3 twice in LHC-8 medium, and exposed to 1% fetal calf serum 4 (FCS) in LHC-8 medium for 47 days. Sixty-one day~ after infection three colonies of transformed cells were 6 individually subcultured by trypsinization. All 7 subsequent culture of these cells was in serum-free LHC-8 8 medium. The cell strains thus derived were designated as 9 BES-lA. Two of these strains (BES-lAl and BES-lA2) were subcloned by limiting dilution.
11 All of these clonal isolates continued to 12 proliferate for about 18 weeks at which time the cultures 13 senesced (i.e., entered culture "crisis"). After a 14 further period of ll weeks, proliferating cells appeared ln a subcloned culture designated BES-lAl.6. From these 16 cells a line was established which remains in culture 17 more than one year from the time of the initial SV40 18 infection. These cells are non-tumorigenic.
19 The BES-lA1.6 line has the special property of being resistant to the squamous differentiation-inducing 21 effects of serum. Whereas NHBR cells are able to be 22 induced to undergo s~uamous differentiation when exposed 23 to serum, bronchlal carcinomas are resistant to this 24 effect (Lechner, et al, Cancer Res. 43:5915-5921, 1983).
TBE cells lines (human bronchial epithelial cells 26 transformed by the v-Ha-ras oncogene are tumorigenic and 27 are resistant to this effect of serum. The 28 non-tumorigenic LES-lA1.6 cell line is, therefore, 29 intermediate between normal and fully malignant bronchial epithelial cells in this respect.

~2982~:0 2 Development of the BEAS-2B Cell Line 3 NHBE cells were cultured from explants of autopsy 4 specimens from noncancerous individuals as describe-d by Lechner, et al., supra. The cells were cultured in a 6 serum-free medium, LHC-9, harvested by trypsinization and 7 seeded in 10 ml growth medium into 100 mm culture dishes 8 (Lux, Miles Scientific, Naperville, IL) whose growth 9 surfaces had been coated with a solution of bovine serum albumin, fibronectin and collagen (Lechner, et al., 11 supra).
12 Adenovirus 12-SV40 (Adl2SV40) hybrid virus 13 (Schell, et al., Proc. Natl. Acad. Sci. USA 55:81-88, 14 1966) was grown in Vero cells as described by Rhim, et al., Proc. Natl. Sci, USA 78: 313-317, 1981. NHBE cells 16 were exposed to the virus at 37C for four hours at a 17 multiplicity of infection of approximately 100. When the 18 cultures reached confluence, each dish was subcultured 19 into two 75 cm2 flasks, the cells were allowed to reach confluence again and then were re-fed twice weekly until 21 transformed colonies appeared and the normal cells 22 senesced. Senescence of the normal cells was accelerated 23 by exposing the cultures to 1% FCS in LHC-9 for 28 days 24 (Lechner, et al., Differentiation 25: 229-237, 1984); all subseguent culture of these cells was in serum-free LHC-9 26 medium. Individual colonies were subcultured 41 days 27 after the viral infection and cell strains thus derived 28 from this experiment were designated BEAS-2.
29 One of the clonal cultures thus derived, BEAS-28, has proliferated continuously for more than a year and 31 appears to be permanently established. Cells from this 32 cell line injected as passage 18 into athymic nude mice Z98ZZ~) 1 have not formed tumors after one year. This cell line 2 retains the ability to undergo squamous differentiation 3 in response to serum; of the cell lines developed BEAS-2B
4 was the most sensitive to this effect and is thus particularly useful for studies of differentiation-6 inducing agents. It is able to form an epithelium in 7 de-epithelialized rat tracheas implanted subcutaneously 8 in athymic nude mice and is thus particularly suitable 9 for in vivo studies, especially of chemical carcinogenesis. In assays of invasiveness using matrigel 11 coated filters and Boyden chambers (Albini, et al., 12 Cancer 47:3239, 1987) BEAS-2B cells were similar to NHBE
13 cells and 100 times less invasive than TBE-l cells.

Development of the BET-lA Cell Line 16 NHBE cells were cultured from explants of autopsy 17 specimens from noncancerous individuals as described by 18 Lechner, et al., 1985, ~E~ The cells were cultured in 19 a serum-free medium, LHC-9, harvested by trypsinization and seeded in 10 ml growth medium into 100 mm culture 21 dishes (Lux, Miles Sclentific, Naperville, IL) whose 22 growth surfaces had been coated with a solution of bovine 23 serum albumin, fibronectin and collagen (Lechner, et al., 24 1985, supra).
The cells were transfected with a plasmid, pRSV-T
26 (obtained from National Cancer Institute), which is an 27 SV40 ori- construct containing the SV40 early region 28 genes and the Rous sarcoma virus long terminal repeat 29 (LTR).
Transfection was by DNA strontium phosphate 31 coprecipitation as described by Brash, et al., Molec.
32 Cell. Biol. 7: 2031-2034, 1987. 5 x 105 NHBE cells *Trade Mark ~z~zz~

1 plated in 100 mm dishes were transfected with 10 ug DNA
2 precipitated at pH 7.8. The cells were exposed to the 3 precipitate for 4 hr before glycerol shock (Brash, et 4 al., supra). Three days after transfection the cells were passaged; thereafter the cell culture medium was 6 changed twice weekly until subculturing of transformed 7 colonies. Upon confluence the cells were passaged a 8 second time, and senescence of normal cells was hastened 9 b~-exposure to LHC-9 medium with-1~ FCS for 46 days. One colony only was subcultured at day 61 after 11 transfection. All subseguent culture of these cells was 12 in serum-free LHC-9 medium, and the cell strain thus 13 obtained was designated BET-lA.
14 These cells continued to proliferate for about 16 weeks at which time the culture senesced (i.e., entered 16 "crisis"). After a further 13 weeks, colonies of 17 dividing cells appeared from which a cell line has become 18 established; BET-lA cells have been in culture for more 19 than a year from the time of initial transfection. These cells are non-tumorigenic, and retain the ability to 21 undergo s~uamous differentiation in response to serum.

23 Development of the BET-2A Cell Line 24 NHBE cells were cultured from explants of autopsy specimens from noncancerous individuals as described 26 previously herein above. The cells were cultured in a 27 serum-free medium, LHC-9, harvested by trypsinization and 28 seeded in 10 ml growth medium into 100 mm culture dishes 29 (Lux, Miles Scientific, Naperville, IL) whose growth surfaces had been coated with a solution of bovine serum 31 albumin, fibronectin and collagen (Lechner, et al., 1985, 32 supra).

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1 The cells were transfected with a plasmid, pRSV-T, 2 (obtained from National Cancer Institute) which is an 3 SV40 ori- construct containing the SV40 early region 4 genes and the Rous sarcoma virus lony terminal repeat ~LTR).
6 Transfection was by DNA strontium phosphate 7 coprecipitation as described previously (Brash, et al., 8 supra). 5 x 105 NHBE cells plated in 100 mm dishes were 9 transfected with 10 ~g DNA precipitated at pH 7.8. The cells were exposed to the precipitate for 4 hr before 11 glycerol shock (Brash, et al., supr_). Three days after 12 transfection the cel~s were passaged. Thereafter, the 13 cell culture medium was changed twice weekly. Three 14 transformed colonies were subcultured individually at 28 days following transfection, and the clonal cell strains 16 thus derived continued to proliferate in culture for 11 17 weeks after which time the cultures senesced (i.e.
18 entered "crisis"). After a further 36 weeks, colonies of 19 dividing cells appeared in culture BET-2A from which a cell line has become established; BET-2A cells have been 21 in culture for more than a year from the time of initial 22 transfection. The BET-2A cell line, like the BES-lA1.6 23 cell line, appears to be resistant to the squamous 24 differentiation-inducing effects of serum. Whereas NH~E
cells are able to be induced to undergo squamous 26 differentiation when exposed to serum, bronchial 27 carcinomas are resistant to this effect (Lechner, et al., 28 Cancer Res., 43:5915-5921, 1983). TBE cell lines (human 29 bronchial epithelial cells transformed by the v-Ha-ras oncogene) are tumorigenic and are resistant to this 31 effect of serum. The non-tumorigenic BET-2A cell line 32 is, therefore, intermediate between normal and fully 33 malignant bronchial epithelial cells in this respect.

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2 Development of MeT-5A Cell Line 3 Human mesothelial cells were cultured as described 4 by Lechner, et al., Proc. Natl. Acad. Sci. USA, 82:
3884-3888, 1985, and were transformed at a frequency of 6 2 x 10-4 by transfection using strontium phosphate 7 coprecipitation (Brash, et al., supra) of a recombinant 8 plasmid, pRSV-T, containing the SV40 virus early region.
9 Colonies of cells transformed by the plasmid, pRSV-T, were isolated and propagated by serial passaging for 11 periods of up to 140 days and 60 - 70 population 12 doublings from the time of transfection, before cellular 13 senescence occurred. This contrasts with the usual 14 culture lifespan of normal mesothelial cells of 30 days and 15 population doublings. Colonies of dividing cells 16 arose from one such senescent culture, and from these 17 colonies an immortalized cell line, MeT-5A, has been 18 established by continued passaging. This cell line is 19 non-tumorigenic. Although it has been maintained routinely in the serum-containing LHC-MM medium, it also 21 grows well in a serum-free medium.

23 Development of BBM Cell Line:
24 BEAS-2B cells were transfected via strontium phosphate co-precipitation (Brash, et al., Molec. Cell 26 Biol., 7:2031-2034, 1987) with a recombinant plasmid, 27 B-mys/pSV2neo, which had been constructed by ligating a 28 BanHl/EcoRl fragment of the c-myc gene from the Burkitt's 29 lymphoma cell line CA46 (Showe, et al., Mol. Cell Biol., 5:501-509, 1985) to a BamH1/EcoR1 fragment of the pSV2neo 31 vector (Southern, et al., Mol. Appl.

~Z98;~2() 1 Genet., 1:327-341, 1982). BEAS-2B cells so transfected 2 were selected in LCH-9 medium with G418 (Geneticin), and 3 colonies resistant to G418 were isolated individually and 4 subcultured. The cell line arising from one such colony has been designated BBM.

7 Development of BZR Cell Line:

8 This cell llne has been derived by infecting the 9 BEAS-2B cell line with a recombinant containing the viral Harvey-ras (v-Ha-ras) oncogene. The cell line so derived 11 is highly tumorigenic in athymic nude mice.
12 The details of the construction are as follows.
13 Zip-neo-v-Ha-ras recombinant retrovirus was constructed 14 by recombining the pZipNeoSV(X) retrovirus (Cepko, et al., Cell, 37:1053-1062, 1984) at its unique Bam Hl 16 restriction enzyme site with a Bam H1-linkered 1.3 Kb 17 fragment of the Hl clone (Ellis, et al., J. Virol., 18 36:408-420, 1980) containing the v-~a-ras oncogene.
19 Recombinant DNA molecules containing the v-Ha-ras DNA in sense orientation with respect to the pZipNeoSV(X) 21 retrovirus 5' long terminal repeat, were identified by 22 standard DNA manipulation techni~ues and were used to 23 transfect the psi2 packaging mutant cell line (Mann, et 24 al., Cell, 33:153-159, 1983). Supernatants from these cells were shown to contain infectious retrovirus, and 26 were used to infect the amphotrophic packagaing mutant 27 cell line, psiAM (Cone, et al., Proc. Natl. Acad. Sci.
28 USA, 81:6349-6353, 1984). Supernants from this cell line 29 were titered and used to infect BEAS-2B cells.
Following infection of BEAS-2B cells with this 31 virus, G418 resistant cells were selected (Southern, et 32 al., J. Mol. Appln. Genet., 1:327-341, 1982) and 1~9~

1 serially subcultured; the cell line so derived was 2 designated ~ZR. This cell line is highly tumorigenic, 3 forming tumors with a latency period of 2 weeks in 12/15 4 athymic nude mice each inJected with 5 x 106 cells subcutaneously.
6 A deposit of the cell lines of the present 7 invention has been made at the ATCC, Rockville, Maryland, 8 on June 12, 1987 and July 14, 1987 under the accession 9 numbers CRL 9608, 9609, 9442~ 9443, 9444, 9482 and 9483, 10` corresponding to cell lines BES-lAl-6, BEAS-2B, BET-lA, 11 BET-2A, MeT-5A, BBM and BZR, respectively. The deposits 12 shall be viably maintained, replacing if it became 13 non-viable, for a period of 30 years from the date of the 14 deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and made 16 available to the public without restriction in accordance 17 with the provisions of the law. The Commissioner of 18 Patents and Trademarks, upon request, shall have access 19 to the deposit.

UTILITY OF CELL LINES

21 (1) Identification of ~otential chemotherapeutic 22 drugs: These cells are useful for screening chemicals 23 suitable for the treatment of cancer and related 24 diseases, by growing them in vitro in medium containing the chemical to be tested and then, after a suitable 26 period of exposure, determining whether and to what 27 extent cytotoxicity has occurred, e.g. by trypan blue 28 exclusion assay or related assays (Paterson, Methods 29 Enzymol., 58:141, 1979), or by growth assays such as colony forming efficiency (MacDonald, et al., Exp. Cell.
31 Res., 50: 417, 1968), all of which are standard 32 techniques well known in the art.

lZ982;20 1 (2) Studies of the control of squamous 2 differentiation, and identification of chemical and 3 biological agents which induce squamous differentiation:
4 This is accomplished by assays previously described for normal human bronchial epithelial cells (Masui, Proc.
6 Natl. Acad. Sci. USA, 83:2438, 1986). As noted in the 7 cell line specification, some retain ability to undergo 8 sguamous differentiation in response to serum. Induction 9 of terminal differentiation may be an effective way of controlli`ng the growth of cancer. Chemical and 11 biological substances are screened for their ability to 12 induce differentiation by adding them to the growth 13 medium of these cells and then after a suitable time 14 interval determining whether a complex oi changes including cessation of DNA synthesis and the appearance 16 of squamous morphology has occured. The cells are also 17 useful for studies of the biological mechanisms of 18 squamous differentiation, and the existence of both 19 serum-resistant and serum-sensitive cell lines enables comparisons and identification of genes of their protein 21 products involved in the process of differentiation.
22 (3) Studies of metabolism of carcinogens and 23 other xenobiotics: Carcinogens and other xenoblotics may 24 be added to the growth medium of these cells and then the appearance of metabolic products of these compounds may 26 be monitored by techniques such as thin layer 27 chromatography or high performance liquid chromatography 28 and the like, and the interaction of the compounds and/or 29 their metabolltes wlth DNA ls determined.
(4) Studies _ of DNA mutagenesis: Substances 31 known or suspected to be mutagens may be added to the 32 growth medium of the cells and then mutations may be 33 assayed, e.g., by detection of the appearance of drug 34 resistant mutant cell colonies (Thompson, Methods 129~3~2() 1 Enzymol., 58:308, 1979). Similarly, cell-mediated DNA
2 mutagenesis, by cocultivating the cells with cell types 3 known or suspected to be capable of secreting mutagenic 4 compounds (Hsu, et al., Proc. Natl. Acad. Sci. USA, 75:2003, 1978).
6 (5) Studies of chromosome damagin~ agents:
7 Substances known or suspected to cause chromosomal damage 8 may be added to the culture medium of these cell lines, 9 and then the extent of chromosomal damage may be measured by techniques such as measurement of the frequency of 11 sister chromatid exchange (Latt, et al., In: Tice, R.R.
12 and Hollaender, A., Sister Chromatid Exchanges. New York:
13 Plenum Press, pp. 11 ff., 1984).
14 ~ (6) Studies of malignant transformation by chemical, physical and viral agents, and transferred 16 genes including oncogenes and high molecular weight 17 genomic DNA from tumors, using standard assays such as 18 anchorage independent growth or tumor formation in 19 athymic nude mice. For example, a cloned cellular oncogene from a human tumor has been transferred into the 21 BEAS-2B cell line; the cell line thus derived is BBM.
22 This cell line has been shown to be resistant to the 23 squamous differentiation inducing effects of serum. In a 24 second example, a cloned viral oncogene, v-Ha-ras, has been introduced into the BEAS-2~ cell line; the cell line 26 thus derived is BZR. Thls cell line has been shown to be 27 able to form tumors in nude mice with a latency period of 28 two weeks, and is able to grow in an anchorage-29 independent fashion in soft agar.
(7) Use of cells altered by transfer of 31 oncogenes as in paragraph 6 above to screen for potential 32 chemotherapeutic agents (by the techniques described in 33 paragraph 1 above) especially those which may be specific lZ~8ZZC~

1 for cells transformed by the activation of particular 2 oncogenes or combination of oncogenes.
3 (8) Studies of cellular biochemistry, including 4 changes in intracellular pH and calcium levels, as correlated with cell growth and action of exogenous 6 asents including but not limited to those described in 7 paragraphs- 1 through 7 above. To study intracellular pH
8 and calcium levels, cells in suitable culture vessels are 9 exposed to fluorescent indicator dyes and then fluorescence emissions are detected with a fluorescence 11 spectrophotometer (Gryn~iewicz, et al., J. Biol. Chem., 12 260:3~40-3450, 1985).
13 (9) Studies of cellular responses to growth 14 factors and production of growth factors: Identification and purification of growth factors important for growth 16 and differentiation of human bronchial epithelial cells.

17 These cells are particularly useful for such an 18 application since they grow in serum-free media.
l9 Therefore, responses to growth factors can be studied in precisely defined growth media and any factors produced 21 by the cells may be identified and pur$fied without the 22 complication of the presence of serum.
23 (10) Use of recomblnant DNA expression vectors to 24 produce proteins of lnterest. For example, the gene encoding a protein of therapeutic value may be recombined 26 with controlling DNA segments (i.e. containing a promoter 27 with or without an enhancer sequence), transferred into 28 the cell (e.g., by strontium phosphate transfection) and 29 then the protein produced may be harvested from the culture supernatant or a cellular extract by routine 31 procedures well known in the art.
32 (11) Studies of intracellular communication e.g., 33 by dye scrape loading assays. To determine whether the 34 cells growing in vitro have the ability to communicate 12982~3 1 via gap junctions, the cultures may be scraped, e.g., 2 with a scalpel, in the presence of a fluorescent dye in 3 the growth medium. Cells at the edge of the wound are 4 mechanically disrupted and therefore take up dye; whether intercellular communication has occurred may be 6 ascertained by determining whether cells distant from the 7 wound also contain dye.
8 (12) Characterization of cell surface antigens:
9 The cells are incubated with an antibody against the cell surface antigen of interest, and then reacted with a ll second antibody which is conjugated to a fluorescent 12 dye. The cells are then evaluated using a fluorescence 13 activated cell sorter to determine whether they are 14 fluorescent and therefore possess the cell surface antigen.
16 (13) Cell-cell hybrid studies for identification 17 of tumor suppressor activity (Stanbridge, et al., 18 Science, 215:252-259, 1982). To determine whether these 19 cell lines contain tumor suppressor genes, they are fused to malignant tumor cells. The presence of tumor 21 suppressor genes is indicated by loss of malignancy e.g., 22 as detected by loss of ability to form tumors in athymic 23 nude mice, in the hybrid cells.
24 (14) Identification of novel genes, including transforming genes in naturally occurring cancers 26 dsscribed in paragraph 6 above, growth factor genes as 27 described in paragraph 9 above, tumor suppressor genes as 28 described in paragraph 13 above, using standard molecular 29 biological techniques (Davis, et al., Methods in Molecular Biology, New York: Elsevier, 1986) and 31 techniques such as cDNA subtraction cloning and the like.
32 Of course, a kit for screening carcinogenic or 33 antineoplastic agents and for any other usage as 34 described herein supra, is easily assembled, comprising 1298~i~V

1 container(s) containing the cell line(s) of the present 2 invention. Other components routinely found in such kits 3 may also be included with instructions for performing the 4 test.
It is understood that the examples and embodiments 6 described herein are for illustrative purposes only and 7 that various modifications or changes in light thereof 8 will be suggested to persons skilled in the art and are 9 to be included within the spirit and purview of this application and scope of the appended claims.

Claims (12)

1. Non-tumorigenic, human bronchial epithelial or mesothelial cell line or derivative thereof growing without senescence when cultured in vitro in growth medium, said cell lines being selected from the group consisting of those having the identifying characteristics of ATCC CRL 9608, 9609, 9442, 9443, 9444, 9482 and 9483.

2. The cell of claim 1 having the identifying characteristics of ATCC
CRL 9608.

3. The cell of claim 1 having the identifying characteristics of ATCC
CRL 9609.

4. The cell of claim 1 having the identifying characteristics of ATCC
CRL 9442.

5. The cell of claim 1 having the identifying characteristics of ATCC
CRL 9443.

6. The cell of claim 1 having the identifying characteristics of ATCC
CRL 9444.

7. The cell of claim 1 having the identifying characteristics of ATCC
CRL 9482.

8. The cell of claim 1 having the identifying characteristics of ATCC
CRL 9483.

9. The cell of claim 1 further comprising an oncogene.

10. A kit for screening carcinogenic or chemotherapeutic agent comprising a container containing a non-tumorigenic, human bronchial epithelial or mesothelial cell line or derivative thereof according to claim 1 growing without senescence when cultured in vitro in growth medium.

11. A method for testing carcinogenicity of an agent, comprising culturing the cell line of claim 1 with an agent suspected of being carcinogenic and determining formation of abnormal cellular mass by said cell line, the formation of abnormal cellular mass being indicative of carcinogenicity of said agent.

12. A method for testing antineoplastic activity of an agent, comprising culturing the cell 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.