Transformation of Human Bronchial Epithelial Cells by Infection With

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(CANCER RESEARCH 48, 1904-1909, April 1, 1988] Transformation of Human Bronchial Epithelial Cells by Infection with SV40 or Adenovirus-12 SV40 Hybrid Virus, or Transfection via Strontium Phosphate Coprecipitation with a Plasmid Containing SV40 Early Region Genes Roger R. Reddel,1 Yang Ke,2 Brenda I. Gerwin, Mary G. McMenamin, John F. Lechner, Robert T. Su,3 Douglas E. Brash, Joo-Bae Park,4 Johng S. Rhim, and Curtis C. Harris5

Laboratory of Human Carcinogenesis [R. R. R., Y. K., B. 1. C., M. G. M., J. F. L., R. T. S., D. E. B., C. C. H.J, and Laboratory of Cellular and Molecular Biology fJ-B. P., J. S. R.], Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20892

ABSTRACT tumor virus genes introduced by infection or transfection. The cells were infected with SV40 virus or with Adl2-SV40 hybrid Normal human bronchial epithelial cells were infected with SV40 virus virus or transfected with plasmids containing the SV40 virus or an adenovirus 12-SV40 hybrid virus, or transfected via strontium early region genes. This latter approach avoided the necessity phosphate coprecipitation with plasmids containing the SV40 early region of screening for viral nonproducer colonies. Clonal cultures genes. Colonies of morphologically altered cells were isolated and cul tured; these cells had extended culture lifespans compared to normal derived by each of these methods have been characterized and human bronchial epithelial cells. All cultures eventually underwent se shown to be nontumorigenic and to retain features of human nescence, with the exception of one which appears to have unlimited bronchial epithelial cells. proliferativi; potential. Colonies arising after viral infection were screened for virus production by cocultivation with Vero cells; only viral nonpro- MATERIALS AND METHODS ducer cultures were analyzed further. The cells retained electron micro scopic features of epithelial cells, and keratin and SV40 T-antigen were Cell Culture. NHBE cells were cultured from expiants of autopsy detected by indirect Â¡mmunofluorescence.All of the cultures were aneu- specimens from noncancerous individuals as described previously (1). ploid with karyotypic abnormalities characteristic of SV40-transformed The cells were cultured in a serum-free medium, LHC-9. In some cases cells. No tumors formed after s.c. injection of the cells in nude mice. where indicated, LHC-8 medium was used. These cells should be useful for studies of multistage bronchial epithelial NHBE cells were harvested by trypsinization and seeded in 10 ml carcinogenesis. growth medium into 100-mm culture dishes (Lux; Miles Scientific, Naperville, IL) whose growth surfaces had been coated with a solution of bovine serum albumin, fibronectin, and collagen (1). INTRODUCTION COS-1 cells, African green monkey CV-1 cells which contain a single integrated copy of the SV40 early region and which were transformed Lung cancer is currently the most common cause of cancer by an origin-defective SV40 mutant that encodes wild-type T-antigen deaths in many Western countries, and most lung cancers arise (20); Vero cells, derived from an African green monkey kidney (21, in the epithelium of the bronchial tree. The study of bronchial 22); and C3AT1, a C3H mouse cell line stably transformed by adeno epithelial cells is therefore of considerable interest. Procedures virus-12, obtained from Dr. Gilbert Jay, National Cancer Institute, have been devised in this (1) and other laboratories (2, 3) for were all grown in Dulbecco's modified Eagle's minimal essential me culturing NHBE6 cells grown out from expiants of bronchial dium with glutamine, gentamicin, and 10% FCS. tissue obtained at autopsy. This has allowed the investigation Cell cultures were monitored for mycoplasma contamination by R. of many aspects of bronchial epithelial cell biology in vitro (for A. Del Guidice, Litton Bionetics, Frederick, MD, using culture on review see Ref. 4) including control of growth (5-9) and squa axenic agar medium and DNA fluorochrome staining of an indicator cell culture (23); no contamination was detected. mo us differentiation (3,7,10-13), the metabolic activation (14) Viral Infection. SV40 virus was prepared in CV-1 cells as previously and effects of carcinogens and tumor promoters (2, 12-16), and described (24). NHBE cells were exposed at 37Â°Cfor 90 min at a DNA repair (17-19). NHBE cells can be maintained as repli multiplicity of infection of approximately 1. The cells were subcultured cative cultures for relatively short times before cellular senes twice in LHC-8 medium and exposed to 1% FCS in LHC-8 medium cence occurs; however, in order to be able to study multistage for 47 days. Sixty-one days after infection three colonies of transformed bronchial carcinogenesis in vitro, an important objective was to cells were individually subcultured by trypsinization. AH subsequent develop cultures with extended lifespans. culture of these cells was in serum-free LHC-8 medium. The cell strains In this study, culture longevity has been increased using DNA thus derived were designated as BES-1A. Two of these strains (BES- 1A1 and BES-1A2) were subcloned by limiting dilution. Received 6/23/87; revised 12/15/87; accepted 12/28/87. Ad 12-SV40 virus (25) was grown in Vero cells as described previously The costs of publication of this article were defrayed in part by the payment (26). In two consecutive experiments, NHBE cells were exposed to the of page charges. This article must therefore be hereby marked advertisement in virus at 37Â°Cfor2 to 4 h at a multiplicity of infection of approximately accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by a C. J. Martin Fellowship of National Health and Medical 100. In one experiment, the NHBE cells infected were the outgrowths Research Council of Australia. from bronchial expiants; the cells were infected after the expiants had 2Supported by an NIH Visiting Fellowship. 3 Present address: Department of Microbiology, University of Kansas, Law been transferred from the dish. The culture was passaged twice after which time most of the normal cells were senescent, and two colonies rence, KS 66045. 4 Present address: Department of Biochemistry, College of Medicine, Seoul of transformed cells were evident. Transformed cells from this experi National University, Seoul, Korea. ment were designated BEAS-1. In a second experiment, cells growing *To whom requests for reprints should be addressed. out from bronchial expiants were subcultured, 2.5 x 10* cells were 6The abbreviations used are: NHBE, normal human bronchial epithelial cells; Adl2-SV40, adenovirus 12-SV40 hybrid virus; El, early region 1; PCS, fetal calf seeded in each of three 100-mm-diameter culture dishes, and the cells serum; PD, population doubling; LHC-9, Laboratory of Human Carcinogenesis were then infected with the hybrid virus. When the cultures reached basal nutrient medium with 0.08 DIMcalcium, 1 niM L-glutamine, trace elements, confluence each dish was subcultured into two 75-cm2 flasks, the cells 50 Â«g/mlgentamicin, 5 Mg/ml insulin, 10 ng/ml transfer rin. 20 n\i hydrocorti- were allowed to reach confluence again, and then were refed twice sone, 5 ng/ml epidermal growth factor, 0.5 >i\i phosphoethanolamine, 0.5 JIM ethanolamine, 0.5 pg/ml epinephrine, 0.33 n\i retinole acid, 10 nM triiodothy- weekly until transformed colonies appeared and the normal cells se- ronine, and bovine pituitary extract; UK'S, medium which contains the ingre nesced. Senescence of the normal cells was accelerated by exposing the dients listed for LHC-9 with the exception of epinephrine and reti noie acid. cultures to 1% FCS in LHC-9 for 28 days (8); all subsequent culture of 1904

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1988 American Association for Cancer Research. T-ANTIGEN TRANSFORMED HUMAN BRONCHIAL EPITHELIAL CELLS these cells was in serum-free LHC-9 medium. The mean number of cein isothiocyanate-labeled anti-mouse immunoglobulin antibody colonies per flask was 18. Individual colonies were subcultured from (Miles). one flask 41 days after the viral infection as described below and cell Detection of adenovirus 12 El proteins was carried out by fixing the strains thus derived from this experiment were designated BEAS-2. monolayers in 2% formaldehyde, permeabilizing in 80% acetone, and Transfection. The plasmids used were as follows. pRSV-T7 (a gift incubating with antisera to E l a (rabbit antiserum against the (-terminal from Dr. J. Brady and Dr. B. Howard, National Cancer Institute) is an peptide) or Elb (mouse monoclonal; Oncogene) followed by a fluores- SV40 ori" construct containing the SV40 early region genes and the cein conjugated second antibody. Rous sarcoma virus long terminal repeat. pRSV.2 (a gift from Dr. E. Northern Analysis. Cells were lysed in guanidine isothiocyanate, and Long, National Institute of Allergy and Infectious Diseases, NIH) is the lysate was centrifuged through a CsCl gradient (28). RNA was equivalent to pRSV-T without the SV40 early region genes. pBRSV further purified by extraction with 1:1 phenolxhloroform. RNA was (obtained from Dr. J. Brady) contains the entire SV40 genome cloned electrophoresed on 1% agarose 17% formaldehyde gels, and transferred into pBR322 at the BamH\ restriction site. The pBR322 cloning vector to Gene Screen Plus (New England Nuclear Research Products, Boston, was also used for transfection. MA). The SV40 probe used was the 1.17-kilobase ///millI fragment of Transfection was by DNA strontium phosphate coprecipitation as the SV40 early region. The adenovirus 12 probes were derived from a described previously (27). 5x10* NHBE cells plated in 100-mm dishes plasmid, pNAR, constructed by subcloning the adenovirus 12 early were transfected with 10 MgDNA precipitated at pH 7.8. The cells were region EcoRl insert of pAdl2RlC (29) into the EcoRl site of the exposed to the precipitate for 4 h before glycerol shock (27). Three plasmid, pSV2 neo (30). The 0.42-kilobase Kpnl/Scal fragment and the days after transfection the cells were passaged; thereafter the cell culture 1.1-kilobase Ieri fragment were used as a probe for the adenovirus 12 medium was changed twice weekly until subculturing of transformed Eia and Elb gene transcripts, respectively. A probe for the gene colonies. Cell strains designated BET-1 and BET-2 were obtained from encoding glyceraldehyde-3-phosphate dehydrogenase (31) was used to indicate the relative quantity of a "constitutive" transcript. Each of two consecutive experiments in which NHBE cells were transfected with the plasmili pRSV-T. In the first of these two experiments, the these DNA fragments was labeled by nick translation (32) and used to cells were passaged a second time, and senescence of normal cells was probe the membrane according to the procedure recommended for hastened by exposure to LHC-9 medium with 1% PCS for 46 days. Gene Screen Plus. Karyotype Analysis. Karyotypic analyses were carried out by Dr. One colony only was subcultured at day 61 after transfection. All Ward D. Petersen, Jr., Children's Hospital of Michigan, Detroit, MI. subsequent culture of these cells was in serum-free LHC-9 medium. In For each cell strain, chromosomes were counted in 40 to 145 meta- the second experiment, the cells were passaged once after transfection and were then maintained by refeeding with LHC-9 alone until colonies phases, and a minimum of 7 Giemsa-banded karyotypes were examined. appeared. Colonies were subcultured individually 28 days after trans Oncogenicity of Transformed Cells. Adult athymic nude mice were fection. inoculated s.c. with freshly trypsinized cells in order to determine The donor from whom BET-1 cells were derived was the same as for tumorigenicity of the transformed cells. In the case of BES-1A1, BEAS- 2B, BET-1A, BET-2A cultures, 5 x 10" cells were injected into mice BEAS-2. irradiated with 350 rads 24 h previously; in the case of four BEAS-1 Selection of Transformed Colonies. Colonies of transformed cells cultures, 1 x IO7BEAS-1 cells were injected into non irradiated mice. were easily identifiable by cellular morphology on phase microscopy, and by the time colonies approximately 1 cm in diameter had appeared (4-8 wk), most of the remaining NHBE had senesced (Fig. 1). Individ RESULTS ual colonies therefore could readily be subcultured by trypsinization. In one experiment in which NHBE were transfected with the plasmids Colonies of transformed cells were easily distinguished from pRSV-T, pRSV.2, pBRSV, and pBR322, the number of transformed nontransformed cells by phase contrast microscopy (Fig. 1). colonies at 28 days was counted. Morphological Studies. Phase-contrast microscopy was carried out The transformed cells were more tightly packed than normal with an Olympus IM microscope equipped with an Olympus OM-2 cells, and by the time colonies had reached a diameter of about camera. Cells were fixed and examined by transmission electron mi 1 cm (4 to 8 wk from the time of infection or transfection) most croscopy using procedures described previously (5, 6). of the nontransformed cells were clearly senescent. This facili Rescue of Virus. To assess whether cultures derived from transformed tated selection of colonies which were individually trypsinized colonies were shedding virus, cocultivation of these cells with Vero cells and then serially passaged as clonally derived cell strains. was carried out as follows. Equal numbers of transformed BE cells and The resulting bronchial epithelial cell strains were designated Vero cells were plated in 25-cm2 flasks in LHC-9 medium and incubated by the transforming agent. Cells transformed by Adl2-SV40 for 10 days. Medium was collected from these cultures and added to a virus infection were referred to as BEAS, those transformed by fresh culture of Vero cells. The latter culture was maintained for 3-4 SV40 as BES, and those transformed by transfection of the T- wk and observed for appearance of characteristic cytopathic effects. antigen-containing plasmid, pRSV-T, as BET. The experiment Clonogenic Assays. For clonogenic assays 1500 cells were plated in and colony from which the strain was derived were identified 60-mm dishes in LHC-8 medium and colonies after 7 days of incubation by a suffixed number and letter, respectively, e.g., BET-2C were scored following fixation with 10% formalin and staining with refers to the third colony chosen from the second pRSV-T crystal violet. The cells tested were BEAS-2A, BEAS-2B, BEAS-2C, BES-1A1, BES-1A2, BES-1A3, BET-1A, BET-2A, BET-2B, BET-2C, transfection experiment. and NHBE cells; BEAS-1 series cells did not form colonies under these Cells were derived from four individuals. The BEAS-1, BES- assay conditions. Cells per colony were enumerated using an Artek 1, and BET-2 series of cells were from three separate donors image analyzer; a minimum of 18 colonies were analyzed per cell line. and the BET-1 and BEAS-2 series were from an additional Indirect Immunofluorescence. Cells (BEAS-ID, BEAS-2 A, HI AS donor. Three colonies were identified in the BES-1 experiment, 2B, BEAS-2C, BES-1A1, BES-1A2, BES-1A3, BET-1A, BET-2A, but karyotypic analysis indicates that these probably arose from BET-2B, AND BET-2C) were grown on collagen/fibronectin-coated a single transformed cell (see below); the resulting cell strains Chamber-Tek chamber slides (Miles) and fixed in ice-cold absolute have therefore been designated BES-1 Al, -1A2, and -1A3 to methanol. Indirect immunofluorescence was obtained for SV40 early reflect this. Two colonies were trypsinized together in the region proteins using a mouse monoclonal antibody to SV40 large and BEAS-1 experiment, and limiting dilution subcloning of the small T-antigens (Oncogene Science, MineÃ³la, NY). Similarly, keratin resulting combined culture was used to derive clonal cell strains; was detected with a mouse monoclonal antibody to cytokeratin (Lab- the 8 BEAS-1 strains (BEAS-1 A through -IH) therefore prob systems, Chicago, IL). In each case the second antibody was a (Inores ably originate from no more than two transformed cells. One 7N. Z. Xu, and B. Howard, unpublished. colony only was chosen for characterization in experiment 1905

â€¢BEAS-2B â€¢BEAS-2A >Ad12SV40 A BEAS2C D BET-2A o BET-2B A BET-2C SV40 TAg v BET-1A D BES-1A1 C BES-1A2 SV40 * BES-1A3

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 WEEKS Fig. 2. Growth of transformed colonies of bronchial epithelial cells. At time 0 normal human bronchial epithelial cells were infected with SV40 virus or Adl2- SV40 hybrid virus or transfected with the plasmid, pRSV-T, as described in "Materials and Methods." Each cell strain was subcultured from a single trans formed colony. Calculation of population doublings is based on the assumptions that each colony arose from a single cell and that plating efficiency at each passage was approximately 50%. BEAS-2A, BEAS-2B, and BEAS-2C were from an Adi2- SV40 infected culture, BES-1A1, BES-1A2, and BES-1A3 were from an SV40 infected culture, and BET-1 A, BET-2A, BET-2B, and BET-2C were from pRSV- T transfected cells. Only BEAS-2B has proliferated continuously since transfor mation. BEAS-2A was found to produce virus and culture was therefore discon tinued. For all other cell strains, data are shown until culture senescence (//) occurred. HI:AS-1 cells (not shown) proliferated for 45-60 population doublings and 10-15 wk before cultures were discontinued. Under the conditions of these experiments, control NHBE cultures had mostly senesced by 4 to 6 wk.

of the cells to low concentrations of serum. The transformed cells continued to proliferate for a minimum of 7 wk and 20 PDs beyond this time. Most of the cultures eventually underwent a marked slowing in proliferation rate and culture senescence ("crisis") at 30-50 Fig. 1. Morphological transformation of human bronchial epithelial cells. PDs and 10-19 wk following transfection or infection (Fig. 2). Phase-contrast micrographs of bronchial epithelial cells 35 days after transfection with the plasmid, pRSV-T, as described in "Materials and Methods." The cells BEAS-2B cells have continued to proliferate for more than a were passaged once 3 days after transfection, then refed twice weekly. Top, year and appear to be established. senescing normal cells. Bottom, center of a tightly packed colony of transformed Morphological Characterization. Under phase-contrast mi cells. croscopy all of the transformed BE cell strains had a predomi nantly polygonal appearance typical of epithelial cells (data not BET-1 and three each in experiments BET-2 and BEAS-2. shown). Electron microscopy revealed desmosomes and tight Transfection Results. The transformation efficiency for trans junctions. The epithelial nature of the cells was also supported fection of the plasmid, pRSV-T, was 0.025% of the cells trans- by evidence of keratin upon indirect immunofluorescence (Fig. fected. The results with pBRSV were not significantly different 3) in every cell strain tested; human bronchial fibroblasts did (0.017%). No transformed colonies were seen in dishes of cells not stain with this antibody. transfected with a plasmid containing the Rous sarcoma virus Viral Production. Viral particles were seen on electron mi LTR (pRSV.2), or with pBR322 DNA. Cell strains were devel croscopy of BEAS-2A cells (passage 4), and viral cytopathology oped only from NHBE transfected with pRSV-T to avoid the was evident on cocultivation of these cells (passage 11) with potential problem of excision of viral DNA and virion forma Vero cells. Culture of this cell strain was therefore discontinued tion following transfection with pBRSV. at passage 12. There was no evidence of viral persistence by Increase in Culture Lifespan. All of the transformed cell either of these criteria in BES-1A1, BES-1A2, BES-1A3, strains studied had increased culture lifespans compared to BEAS-ID, BEAS-2B, and BEAS-2C cells. their nontransformed counterparts (Fig. 2). The culture lifespan Growth Characteristics. Colony-forming efficiencies of trans of BET strains was less than the lifespan of strains derived by formed cell cultures in LHC-8 medium were 7-12% and growth viral infection, but it is not possible to draw conclusions from rate was 0.92 Â±0.14 PD/D (mean Â±SD, 10 cell strains) neither this observation because of the small numbers of cell strains in of which parameters was significantly different from NHBE each group and because several different donors' cells were cells. used. Transformed colonies reached a diameter of about 1 cm Expression of Transforming Viral Genes. Greater than 99% in 4 to 6 wk, except in the SV40 infection experiment in which of cells in every cell strain exhibited indirect immunofluores the cells took 8 to 10 wk to reach this stage. In all experiments, cence with anti-SV40 T-antigen antibodies, as shown previously the control cultures had ceased replication by the time of colony for BET-1A cells (27). The abundance of T-antigen mRNA was selection; in some experiments this was expedited by exposure very similar for the cell strains studied and slightly greater than 1906

normal chromosomes, and apparently random translocations and deletions. While most of the karyotypes examined were aneuploid, the Adl2-SV40 hybrid virus transformed cell strains had predominantly near-diploid (42-50) chromosome counts (Table 1). For two of these strains, BEAS-2B and BEAS-2C, karyotypes were examined on more than one occasion to assess stability. Whereas at passage four 68% of BEAS-2C cells were near-diploid, shortly before crisis (passage 12) 95% had 70-80 chromosomes and 5% had 115+ chromosomes. In contrast, for the BEAS-2B cell line (which had no overt culture crisis) 72% of the cells at passage four were near-diploid, and at passage 29 the proportion remained similar (81%). A common marker chromosome was observed in the cultures derived from each of the three colonies in the BES-1 experi ment. It is likely, therefore, that these colonies were the result of a single transformation event. The cell strains transformed by SV40 virus infection (BES- Fig. 3. Indirect immunofluorescence for keratin. All of the cell strains tested 1A1, -1A2, and -1A3) and one cell strain transformed by (BEAS-ID, BEAS-2A, BEAS-2B, BEAS-2C, BES-1AI, BES-1A2, BES-1A3, transfection with pRSV-T plasmid DNA (BET-1A) had no BET-1 A, BET-2A, BET-2B, and BET-2C) exhibited indirect immunofluorescence for cytokeratins. The example shown is BES-1A1. near-diploid cells and were predominantly tetraploid when ex amined at early passages. In addition, these cells had a higher incidence of homogeneous staining regions and double minutes co (O tn f-: m r- than the BEAS strains. In contrast, BET-2A, -2B, and -2C had _ < ^ < < CM <Ã UJ V CN ih *- mostly near-diploid cell populations. ^ co T ? W h- < CO m b< co I O LU LU LU x III II I Tumorigenicity. None of the cell strains tested formed tumors Z u m m m CO CO CO in athymic nude mice. The mice injected with BEAS-1A (pas sage 5), BEAS-1C (passage 8), BEAS-ID (passage 13), or BEAS-IF (passage 11) cells were sacrificed 6 mo after injection; the mice injected with BEAS-2B (passage 18), BET-1A (passage 8), BET-2A (passage 7), or BES-1 Al (passage 6) cells were sacrificed a minimum of 12 mo after injection. â€” 2.2 Kb DISCUSSION â€” 1.4 Kb The object of this study, extension of the in vitro lifespan of human bronchial epithelial cells, was achieved by a variety of techniques involving expression of SV40 T-antigen in the cells, namely infection with SV40 virus or Adl2-SV40 hybrid virus, SV40 T Antigen GAPDH or transfection via strontium phosphate coprecipitation with a Fig. 4. Northern analysis of SV40 early region mRNA in transformed bron recombinant plasmid containing the SV40 early region. A num chial epithelial cells. Total cellular RNA was prepared from selected transformed BE cell lines as described in "Materials and Methods," and 20-<*gsamples were ber of other human epithelial cell types have previously been electrophoresed on 1% agarose 17% formaldehyde gels and transferred to Gene transformed by SV40 genes (for reviews see Refs. 34-36). Screen Plus membrane. The probe was the nick-translated 1.17-kilobase ///'millI Expression of SV40 T-antigen correlated with transforma fragment of pRSV-T. To indicate the amount of mRNA loaded per lane, the radioactive probe was removed from the membrane by boiling, and the membrane tion. In all cell strains, T-antigen was detectable in more than was rehybridized with a probe for the gene, glyceraldehyde phosphate dehydro- 99% of the cell population, suggesting a selective pressure for genase (GAPDH). the continued retention of this phenotype. The karyotypic changes seen in the transformed bronchial in the COS-1 cell line which has a single copy of the SV40 epithelial cultures are consistent with those seen in other trans early region (20) (Fig. 4). formed cell types. An extensive body of literature on this subject All of the cells infected with Adl2-SV40 hybrid virus had has been reviewed (33), but much of it predates the use of undetectable levels of Elb protein on indirect immunofluores currently used chromosomal banding techniques. Chromatid cence, but presence of this protein at a low level could not be breaks, abnormal chromosomes such as diet-nines, and abnor excluded. The probable absence of this protein was supported mal numbers of chromosomes are considered characteristic of by the lack of detectable Elb mRNA; abundant Elb mRNA SV40-induced transformation (33). was detected in the C3AT1 cells used as a positive control (data All of the transformed BE cell strains appeared to be epithe not shown). One cell strain, BEAS-ID, appeared positive for lial in origin based on light and electron microscopic criteria Eia on indirect immunofluorescence. BEAS-2B and BEAS-2C and the presence of keratin. A hallmark of NHBE cells is their were negative for Eia immunofluorescence and no Eia mRNA sensitivity to the growth-inhibiting and differentiation-inducing could be detected in either of these strains, whereas there was effects of serum and transforming growth factor-/3, and many abundant Eia mRNA in the C3AT1 controls (data not shown). of the transformed BE cell strains retained this feature to a Karyotypes. Typical SV40 effects (33) were observed in many greater or lesser extent.8 In addition, BEAS-2B and BEAS-2C of the karyotypes including chromosome gaps, chromatid gaps, 8 Y. Ke, R. Reddel, B. I. Gerwin. et al. Human bronchial epithelial cells with chromosome breaks, endoreduplication, pulverized chromo integrated SV40 virus T antigen genes retain the ability to undergo squamous somes, acentric fragments, apparently random loss and gain of differentiation, submitted, 1987. 1907

Table 1 Karyolypes of transformed bronchial epithelial cells number42-50708879851009681726873"87718980-10017122115415183220*88758480132911150+of metaphases by chromosome CelltypeBEAS- age"333455104442424787% observations*1320q*300+ 600+ Other 1ABEAS- IBBEAS- 1CBEAS-1DBEAS-

IFBEAS- 1GBEAS-2ABEAS-2BBEAS-2CBEAS-2FBES-lAlBES-1A2BES-1A3BET-1ABET-2ABET-2BBET-2CPass in8/15kr22q+ 5k4 in 5/1 16p*in7/15k22q*in4/l5k10

16p*in5/12k5q*in Ip*in9/14kI4q*in5/I4k7 12/l4k,

Ip*copies/klp~ in 6/7k, 2-3 2k3q~in4/7k,in 4/7k, 2 copies in Ilp+in5/7k16q+ 2k19q+in 4/7k, 2 copies in 2k7 in 6/7k, 2 copies in 5 del(l)(p21:)in5/9kRearranged 6x/9k18 chromosome 3 5 2 del(l)(p21:)in3/10kRearranged 6x/10k16 chromosome 3 del(l)(p21:)in2/10kRearranged Ok12 chromosome 3 in 4/1 8 20q*in7/8k19q*in5/8k2p*

2222p+,in 4/12k, monosomy 19, 2219q+or 22p+q+, monosomy in 4/8k, monosomy 19 " Metaphases were prepared at the passages indicated. Chromosomes were counted in 40 to 145 metaphases. A minimum of 7 Giemsa-banded karyotypes were examined. * For each of the cell cultures, many of the following features were observed: chromosome gaps, chromatid gaps, chromosome breaks, minutes, double minutes, endoreduplication, pulverized chromosomes, acentric fragments, random loss and gain of normal chromosomes, and random translocations and deletions. The incidence of homogeneous staining regions and double minutes was higher in polyploid (BES, BET-1A, and BEAS-2F) than in near-diploid karyotypes. Only frequent aberrations are listed for each cell strain. ' k, karyotype. d 55-80 chromosomes. '110+ chromosomes. were able to form an epithelium in deepithelialized rat tracheas strains characterized in each group, it is not clear whether these implanted s.c. in athymic nude mice,9 and BEAS-2B cells apparent differences are related to the transforming agent. elaborate mucin when inoculated onto a collagen matrix.10 The Adl2-SV40 virus, originally isolated from copropaga- These cells should therefore be useful for studies of differen tion of adenovirus type 12 and SV40 virus in African green tiated functions. monkey kidney cultures, was found to be more oncogenic than The phenomenon of cellular "senescence" (37) appears to be SV40 virus in newborn hamsters. The tumors formed in new characteristic of all normal human cells in culture. SV40- born hamsters phenotypically resembled tumors produced by transformed human cells typically have extended in vitro life- adenovirus alone but contained both adenovirus and SV40 spans but eventually reach a stage referred to as crisis (38) tumor antigens (25). In vitro transformation of hamster and where net proliferation is greatly reduced or ceases and the chimpanzee skin and human skin fibroblasts has been described remaining cells often appear morphologically bizarre. This (26, 39, 40). The Adl2-SV40 hybrid virus has recently been phenomenon of crisis has been observed following SV40 trans used to transform human foreskin epidermal keratinocytes (41). formation of human fibroblasts and epithelial cells, but in a A nonproducer culture of transformed keratinocytes so derived recent review of this subject it was concluded that keratinocytes appeared to have unlimited proliferation potential but was are unique among human epithelial cells in that crisis does not nontumorigenic when injected into 129J nude mice. In contrast always occur and is transient when it does occur (35). In the to the hamster transformants, however, no transcripts were present study the transformed bronchial epithelial cell strains detected from adenovirus Eia or Elb regions in these trans which remained in continuous culture all eventually underwent formed human cells (41). Although one Adl2-SV40 trans prolonged culture crisis, with the exception of BEAS-2B which formed bronchial epithelial cell culture, BEAS-ID, appeared to has continued to proliferate for more than 1 yr (Fig. 2). Of the express adenovirus 12 Eia protein, neither Eia protein nor strains which senesced, some subsequently appear to have re adenovirus early region 1 mRNA was detected in the BEAS- commenced proliferation; these include BET-1A, BET-2A, 2B and BEAS-2C cultures. It thus appears unlikely that the BES-lAl, and a subclone of BES-1 Al designated BES-1A 1.6 adenovirus early region accounts for the differences seen be (data not shown). tween BEAS-2B and the other cultures. It can be concluded There appeared to be some differences between the cells tentatively that for the human bronchial epithelial cells in this transformed by the Adl2-SV40 hybrid virus (BEAS) and those study, the three modes of transformation, Adl2SV40 infection, transformed by SV40 virus (BES) or SV40 early region-con SV40 infection, and SV40 early region plasmid transfection, taining plasmici DNA (BET). In addition to the absence of overt merely represented different means of delivery of the transform crisis in the BEAS-2B culture, a higher proportion of the BEAS ing region of SV40 DNA. However, whether the SV40 se strains were near-diploid. In view of the small number of cell quences in the Adl2-SV40 hybrid virus have similar transform " A. J. P. Klein-Szanto, et al., unpublished data. ing potential to those of wild type virus and the pRSV-T 10R. Wu, et al., unpublished data. plasmid is unknown. None of the cells tested formed tumors in 1908

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1988 American Association for Cancer Research. T-ANTIGEN TRANSFORMED HUMAN BRONCHIAL EPITHELIAL CELLS athymic nude mice. This is in agreement with many other and Harris, C. C. Biochemical and morphological effects of cigarette smoke condensate and its fractions on normal human bronchial epithelial cells in studies which have failed to show tumorigenicity of human and vitro. Cancer Res., 47:2045-2049, 1987. other primate cells transformed with SV40 virus. Although 17. Fornace, A. J., Jr., Lechner, J. F., Grafstrom. R. C., and Harris, C. C. DNA repair in human bronchial epithelial cells. Carcinogenesis (Lond.), ÃŒ:1373- there may be some evidence that this is partly due to tumor 1377, 1982. suppression via a T-cell-independent mechanism, it seems that 18. Grafstrom, R. C., Fornace, A. J., Jr., Autrup, H., Lechner, J. F., and Harris, the interaction of the SV40 primate virus with primate cells is C. C. Formaldehyde damage to DNA and inhibition of DNA repair in human bronchial cells. Science (Wash. DC). 220: 216-218, 1983. intrinsically less oncogenic than its effect on rodent cells (42). 19. Krokan, H., Lechner, J., Krokan, R. H., and Harris, C. C. Normal human The nontumorigenic phenotype of these SV40-transformed hu bronchial epithelial cells do not show an adaptive response after treatment with /V-methyl-iV'-nitro-A'-nitrosoguanidine. Mutation Res., 146: 205-209, man bronchial epithelial cell cultures should make them suita 1985. ble model systems for the study of multistage carcinogenesis. 20. Gluzman, Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell, 23: 175-182, 1981. 21. Yasamura, Y., and Kawakita. Y. Research into SV40 by tissue culture. ACKNOWLEDGMENTS Nippon Rinsho, 21: 1201-1205, 1963. 22. Rhim, J. S, Schell, K., Creasy, B., and Case, W. Biological characteristics The authors thank Dr. Bruce Howard, Dr. John Brady, and Dr. Eric and viral susceptibility of an African green monkey kidney cell line (Vero). Long for plasmids. Dr. Ward Peterson, Jr., Children's Hospital of Proc. Soc. Exp. Biol. Med., 132: 670-678, 1969. 23. Del Guidice, R. A., and Gardella. R. S. Mycoplasma infection of cell culture: Michigan, for karyotyping, Dr. M. Moore and B. Sheatz, Hazleton effects, incidence, and detection, /n: M. K. Patterson, Jr. (Ã©d.).Uses and Laboratories, Rockville, MD, and Dr. P. Arnstein, Laboratory of Standardization of Vertebrate Cell Culture, Tissue Culture Association Mon Cellular and Molecular Biology, for care of nude mice, and Dr. Gary ographs Vol. 5, pp. 104-115. Gaithersburg, MD: Tissue Culture Association. Stoner for comments on the manuscript. 1984. 24. Su, R. T., and DePamphilis, M. L. Simian virus 40 DNA replication in isolated replicating viral chromosomes. J. Virol., 28: 53-65, 1978. 25. Schell, K., Lane, W. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1988 American Association for Cancer Research. Transformation of Human Bronchial Epithelial Cells by Infection with SV40 or Adenovirus-12 SV40 Hybrid Virus, or Transfection via Strontium Phosphate Coprecipitation with a Plasmid Containing SV40 Early Region Genes

Roger R. Reddel, Yang Ke, Brenda I. Gerwin, et al.

Cancer Res 1988;48:1904-1909.

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