Establishment and characterization of SV40-transformed human breast epithelial cell lines

Human breast epithelial cells cultured from milk have been transformed with SV40. Indirect immunofluorescence tests using monoclonal antibodies show that cells from clones grown in soft agar have SV40 large T-antigen in their nuclei and epithelia-specific tonofilament antigens on their intermediate filaments. In primary cultures of milk epithelial cells, the tonofilaments form a characteristic delicate basketwork throughout the cytoplasm, but in the SV40-transformed epithelial cell strains, the filament network is grossly distorted. Insulin, hydrocortisone, and serum stimulate the growth of the cell strains. At passages 8 to 11, the cell strains become quiescent and usually die. One cell strain survived this crisis period and gave rise to the fR series of cell lines. Most cell lines have a cuboidal morphology and react with a monoclonal antibody that recognizes a differentiation antigen on the membranes of breast epithelia. Line fR2 expressed the highest level of this antigen whereas fR5, the only fR line isolated with fusiform morphology, had relatively little. The in vitro-transformed lines may be related to the two dominant epithelial cell types seen in primary milk cultures and could be useful for studying the relationship between transformation and differentiation in human mammary epithelial cells.     

Differential susceptibility to carcinogen-induced amplification of SV40 and dhfr sequences in SV40-transformed human keratinocytes.

Gene amplification contributes to carcinogenesis by enhancing proto-oncogene activity and causing chromosomal instability. The ease of detecting amplified tumor-virus sequences has encouraged use of this system as a surrogate for studying the molecular events involved in endogenous gene amplification. We report here a new system for studying carcinogen-induced amplification of both endogenous and viral sequences in the SV40-transformed human keratinocyte line AG06. Treatment with carcinogens induced a transient dose-dependent amplification of the integrated SV40 sequences. The amplified sequences appeared in the extrachromosomal fraction. Treatment of these cells with carcinogens prior to methotrexate (MTX) selection increased the frequency of MTX-resistant colonies, 67% of which exhibited dihydrofolate reductase (dhfr) amplification. The abilities of five carcinogens with different DNA-damaging activities (the DNA-damaging agents N-methyl-N-nitro-N-nitrosoguanidine, mitomycin C (MMC), ultraviolet light C, and X-rays and the non-DNA-damaging agent arsenite) to induce SV40 and dhfr amplification at concentrations that result in 50% clonal survival were compared. All four DNA-damaging carcinogens (as well as growth arrest) were able to elicit some SV40 amplification, but responses varied markedly, from 1.8-fold for X-rays to sevenfold to eightfold for MMC. There was no correlation between the ability to elicit the two amplification responses. Arsenite, which did not induce SV40 amplification, was the best inducer of MTX resistance. These results point to different controls involved in the induction of viral and dhfr amplification. The signal for amplification of viral genes may be triggered by DNA damage and growth arrest, whereas amplification of dhfr, and perhaps other endogenous sequences, seems to be triggered by other signals as well.     

Simian virus 40 (SV40) production from SV40-transformed human amnion cells of established lines.

Sixteen established cell lines of simian virus 40 (SV40)-transformed human amnion cells were examined for SV40 production. Many of these lines produced SV40 for extensive periods. Virus production had not ceased for 2 lines after 18 months, for 3 lines after 12 months, and for 3 lines at 3 months after recovery from "crisis". Three lines became virus-free in the first month, 1 line in the second month, 1 in the third month, and 1 in the fourth month, and 2 lines stopped virus production between 6 and 11 months after recovery. The virus titers were relatively low. Inclusion body-containing cells were infrequent. In contrast, in most cultures of SV40-transformed human fibroblasts rescued from crisis, no infectious virus was demonstrated, although exceptions have been reported. Virus was produced after heterokaryon formation of cells of the virus-free amnion lines with CV-1 cells in the presence of inactivated Sendai virus, as observed for SV40-transformed human fibroblasts. During the crisis period, some of the SV40-transformed amnion cells produced substantial amounts of virus. Titers decreased during the later periods of crisis. The most pronounced decrease in titers was in cultures from which established lines were recovered.     

Monoclonal antibodies against two human lung carcinoma cell lines

Monoclonal antibodies against 2 human lung carcinoma cell lines (E14 and BEN) were prepared by production and cloning of somatic cell hybrids between the murine myeloma NS1, and spleens from E14- and BEN-immune BALB/c mice. Approximately 2000 hybrid culture supernatants were screened for antibody simultaneously against the immunizing cell line and lung fibroblasts (573 Lu) using a radiolabelled Protein A binding assay. Although the vast majority secreted antibodies which recognized species-specific antigens, a few supernatants showed marked differential reactivity against E14 or BEN. These were cloned and subsequently tested against a panel of up to 25 human cell lines originating from different neoplastic and non-neoplastic tissues. Two anti-E14 clones (3E19.8 and 4EAB3.7) displayed preferential activity against lung cancer cell lines, but a low level of reactivity was also detectable with cell lines of different tissue provenance. The antibodies of 3 anti-BEN clones (7B3.5, 7B5.4, 7B17.7) likewise recognized antigens present to a higher density on lung cancer cell lines but were also reactive (to a variable extent for the different clones) with a diversity of other tumour cell lines. The antibodies of 2 further clones were exceptional in so far as one (7BC9.1) reacted only with BEN and WIDR (colorectal cancer) cells, while another (7B24.4) reacted, with apparent exclusivity, against BEN cells. With the exception of the latter, the distinction in antigen expression between many of the cell lines was quantitative rather than qualitative and the emergent picture is one of random expression of individual determinants on several disparate types of cancer cells, rather than restriction to cells of a given morphological type or histogenic derivation.     

Differential sensitivity of paclitaxel-induced apoptosis in human esophageal squamous cell carcinoma cell lines

Purpose: Paclitaxel is a highly effective chemotherapy agent against adenocarcinomas and squamous cell carcinomas of the esophagus. However, its precise effects in human esophageal cancer cells are not well understood. This study was designed to examine the relationship between cell-cycle phases of paclitaxel-activated checkpoints and to elucidate the molecular pathway of the effect of paclitaxel in human esophageal squamous cell carcinoma (ESCC) cell lines. Methods: The three human ESCC cell lines—TE-2, TE-13 and TE-14—were examined for their response to paclitaxel. ESCC cells were treated with various concentrations of paclitaxel for 1–3 days using MTT assay. The cell-cycle progression and apoptosis were examined by flow cytometry. DNA fragmentation assay was carried out to confirm the fragmented cells as hallmark for apoptotic cells. In additional, the expression of apoptosis-related proteins in ESCC-treated cells was then examined by Western blot analysis. Results: TE-14 cells demonstrated the highest sensitivity among all cells. G2/M cell-cycle arrest occurs prior to paclitaxel-induced apoptosis in ESCC cells. The fragmentation of chromatin was observed in drug treated TE-13 and TE-14 cells by flow cytometry and DNA ladder formation. In contrast, the measurement for TE-2 cells was more suggestive of phenotype a resistant in response to paclitaxel treatment. Western blot analysis results showed that the mitochondrial pathway might be involved in paclitaxel-induced apoptosis in ESCC cell lines. Conclusion: Differential sensitivity was observed in human ESCC cell lines in response to paclitaxel treatment. G2/M arrest occurs with a prior to paclitaxel-induced apoptosis and might be mediated by the mitochondrial (intrinsic) apoptosis pathway in human ESCC cells. A. Faried and L.S. Faried contributed equally to this project and are considered co-first authors.     

Establishment of human fibroma cell lines from a MEN1 patient by introduction of either hTERT or SV40 early region

Establishment of tumor cell lines as model systems for studying tumor biology or as a part of immunotherapeutic anti-cancer strategies is of high importance, whereby the highest possible preservation of the original tumor cell phenotype is a prerequisite for these aims. Since overexpression of the catalytic subunit of human telomerase (hTERT) is known to minimally alter the cellular phenotype, we focused on the establishment of cell lines derived from human fibroma from a MEN1 patient by ectopic expression of hTERT. Additionally, a cell line was generated by introduction of the early region of SV40 (SV40 ER). Both approaches resulted in continuous cell lines, and neither T1-LOHG (hTERT) nor SV1-LOHG (SV40 ER) showed a transformed phenotype. While SV40 ER-transfected cells underwent dramatic changes in morphology and growth characteristics, hTERT-expressing cells indeed retained a phenotype highly similar to the parental cells. Nevertheless, hTERT overexpression resulted in increased growth rates after about 70 population doublings (PD) and alterations of mRNA levels of genes associated with tumor pathogenesis. Thus, our data suggest that ectopic hTERT expression leads to immortalization of LOHG-F, sustaining many characteristics of the non-transfected counterparts, but continuous growth in vitro is associated with changes of the cellular phenotype.     

Nuclear matrix protein composition of human lung carcinoma cell lines

The nuclear matrix is the RNA-protein skeleton within the nucleus that contributes to the structural and functional organization of DNA. Differences in the nuclear matrix protein composition between cancer and normal cells have been reported in various cell lines and tissues, suggesting altered gene expression. This study examined the nuclear matrix protein composition of various human lung cell lines. Using high resolution two-dimensional electrophoresis, at least ten common proteins, as well as specific differences, were identified in each category of lung cell lines. These protein differences may be responsible, at least in part, for the different phenotypes of human lung cancer.     

Differential characteristics of two newly established human breast carcinoma cell lines

Two human breast carcinoma cell lines, EP and MW, were established in culture from malignant pleural effusions. In addition to producing tumors in antithymocyte serum-immunosuppressed mice, both cell lines showed epithelial characteristics and anchorage-independent growth in soft agar. EP and MW differed in morphology (spindle-shaped versus round), chromosomal mode (hyperdiploid versus near triploid), estrogen receptor content (43.8 versus 5.1 fmol/mg protein), cloning efficiency (0.24 versus 15%), and activities (milliunits/10(6) cells) of creatine phosphokinase (25.7 versus 62.6) and lactate dehydrogenase (346.7 versus 778.5). Electron microscopy revealed that MW cells had more perinuclear filamentous material and more frequent intracytoplasmic vacuole formation than did EP cells. While having no effect on MW cells at the concentrations studied (10(-5) to 10(-11) M), beta-estradiol (10(-7) M) stimulated the growth of EP cells by 106% over the hormone-depleted control. In a variety of systems, EP was consistently the more drug sensitive of the two lines. In vitro, EP was significantly (p less than 0.001) more sensitive to methotrexate, vincristine, and 5-fluorouracil, respectively. In antithymocyte serum-mouse xenografts, EP displayed a greater response to three different dosages of a combination of cyclophosphamide, methotrexate, and 5-fluorouracil. One such dosage (cyclophosphamide, 32.0 mg/kg/day; methotrexate, 13.0 mg/kg/day; 5-fluorouracil, 190.0 mg/kg/day; for 1 day) reduced EP and MW tumor weights to 5.9 and 41% of controls, respectively. These results correlated well with the clinical responses.     

Differential growth inhibition by 5-fluorouracil in human colorectal carcinoma cell lines

The effects of 5-fluorouracil (5-FU) on cell growth were investigated using a primary culture of human fibroblasts, MRC-5, and three established human colon cancer cell lines, DLD-1, LoVo and SW620. Detailed flow cytometric analyses revealed differential growth inhibition among these cell lines including three modes of cell growth modulation: (a) loss or accumulation of S phase cells; (b) G2/M block; and (c) G1-S arrest. From analyses on the amount of 5-FU incorporated into cellular RNA and the activity of thymidylate synthase (TS), suppression of TS and depletion of dTTP, a possible consequence of the former, was considered to be the major action of 5-FU in these cells. Differences in the cellular responses to the nucleotide pool imbalance appeared to make the cell growth modulation diverse. Loss of S phase cells and G1-S phase arrest were evident in p53 wild-type cells, MRC-5 and LoVo. Cells proficient in DNA mismatch repair, SW620 and MRC-5, showed marked modulations in S-G2/M progression. These findings suggest that multiple factors, including p53 and DNA mismatch repair, participate in diverse cell growth modulations in cells treated with 5-FU. Cellular resistance to 5-FU correlated well with a loss of modulations in S-G2/M progression, rather than with a defect of G1-S arrest, which suggests the significance of DNA mismatch repair as a factor affecting the sensitivity of cells to 5-FU.     

Functional characterization of the GDEP promoter and three enhancer elements in retinoblastoma and prostate cell lines

GDEP (gene differentially expressed in prostate cancer aka. PCAN1), a newly discovered gene with remarkable tissue specificity, is a promising candidate for regulatory analysis because it exhibits a high level of expression that is limited to two tissues, the retina and the prostate. As these two tissues have different origins and disparate functions it is likely that the regulatory mechanisms responsible for expression are not shared in their entirety. In addition, both the retina and prostate are prime targets for gene therapy. To date there have been no functional studies of the GDEP promoter. Therefore to understand tissue-specific expression of GDEP we constructed promoter expression constructs. To further characterize functional regulatory regions within the GDEP gene, we investigated potential regulatory components for tissue-specific expression in the 40 kb intron of this gene. We have identified a 1.5 kb prostate-specific promoter from the proximal region of the GDEP gene. A smaller 0.5 kb promoter exhibited minimal activity in the retinoblastoma cell line Y79, but not in the prostate cells tested. In addition we have investigated three enhancer elements located in the 40 kb intron of the GDEP gene. We identified two enhancer elements that increase reporter gene expression in prostate cell line LNCaP and one additional enhancer element that increases expression in the Y79 cell line approximately 8-fold making it a strong retinal-specific enhancer.     

In vivo and in vitro invasiveness of human lung carcinoma cell lines.

Eight cell lines derived from human non-small cell lung carcinomas were used to compare their in vivo invasiveness, in vitro chemoinvasive abilities and type IV collagenase activity. For the evaluation of the in vivo invasive potential, the tumor cells were seeded into deepithelialized rat tracheas and transplanted subcutaneously into nude mice. The invasive behavior of the cells was observed at 4, 8 and 12 weeks and assessed histologically by determination of the levels of penetration of tumor cells into the different layers of the tracheal wall. Except for two cell lines that did not grow at all in vivo, there was a very good correspondence between the levels of in vivo tracheal wall penetration and the in vitro chemoinvasion assay using fibronectin as chemoattractant and Matrigel as barrier. This also correlated very well with the capacity of the cells to secrete type IV collagenase. The in vivo evaluation of invasion using tracheal transplants, although requiring several weeks of experimentation, proved to be very reliable, yielding homogeneous results with little internal variation, and is proposed as a dependable in vivo invasion assay that closely mimics the in vivo human conditions in which most carcinomas develop and eventually invade neighboring tissues.     

Differential sensitivity of human mammary epithelial and breast carcinoma cell lines to curcumin

Curcumin has anti-inflammatory, antiproliferative, and antitumor effects. To understand the chemopreventive mechanism of curcumin against human malignancies, the cellular and molecular changes induced by this agent in human mammary epithelial (MCF-10A) and breast carcinoma (MCF- 7/TH) cell lines were investigated. The human multidrug- resistant breast cancer cell line was 3.5 fold more sensitive to curcumin than the mammary epithelial cell line. Even though both cell lines accumulated a similar amount of curcumin, a significantly higher percentage of apoptotic cells was induced in breast cancer cells compared to a very low percentage of apoptosis in mammary epithelial cells. Incubation of breast cancer cells with 20 and 40 microM curcumin for 24 h induced G2 block and sub-G0/G1 cell population, respectively. Curcumin treatment caused a reduction in the expression of Ki67, PCNA, and p53 mRNAs in breast cancer cells. The human mammary epithelial cell line showed a down-regulation of p21 mRNA and an up-regulation of Bax mRNA expression with curcumin treatment. The results suggest that apoptosis is involved in the curcumin-induced inhibition of tumor cell growth, and genes associated with cell proliferation and apoptosis may be playing a role in the chemopreventive action of curcumin.     

Detection and quantification of SV40 large T-antigen DNA in mesothelioma tissues and cell lines

SV40 viral DNA sequences, particularly large T-antigen (T-ag) DNA, have been reported in malignant pleural mesothelioma (MPM) and suggested to play a role in the tumorigenesis of this cancer. These results remain somewhat controversial owing to variability among a number of laboratories in reported SV40 DNA and protein detection in MPM tissues. This could be explained in part if SV40 DNA is present in relatively low abundance in many MPM tissues determined to contain viral sequences and is therefore difficult to detect. To this end, we investigated the efficacy of real time quantitative PCR in detecting low copy number SV40 DNA sequences, then we quantified SV40 copy number in MPM tissues and cell lines at our institution. Quantitative PCR demonstrated consistent precision and accuracy in detecting SV40 DNA sequences over a large logarithmic range of viral copy number. In addition, SV40 sequences were found in 2/35 tissues and 3/7 cell lines in relatively low abundance. We conclude that SV40 is not a contributing factor in the pathobiology of the majority of MPM tumors from patients at our institution.     

Differential expression of nuclear envelope lamins A and C in human lung cancer cell lines

The lamins, an intranuclear class of intermediate filament proteins, are major structural proteins of the nuclear envelope. In the present study, the three abundant mammalian lamins (lamins A, B, and C) were observed to be present in roughly equivalent amounts in the Calu-1, Calu-3, H157, and SK-MES-1 non-small cell lung cancer lines. In the small cell lung cancer lines OH-1, OH-3, NCI-H82, NCI-H209, and NCI-H249, levels of lamin B were similar to those observed in the non-small cell lines, but the levels of lamins A and C were diminished by greater than or equal to 80%. The relationship between lung cancer phenotype and lamin expression was explored further in the NCI-H249 small cell line. Introduction of the v-rasH oncogene into this line gives rise to a cell line (NCI-H249rasH) with many features of large cell carcinoma of the lung (Falco, J. P., Baylin, S. B., Lupu, R., et al. J. Clin. Invest., 85: 1740-1745, 1990). Concomitant with the v-rasH-induced change in phenotype, a greater than 10-fold increase in the amounts of lamins A and C was observed. Levels of the cytoplasmic intermediate filament protein vimentin also increased. In contrast, levels of a variety of nonlamin nuclear polypeptides including topoisomerase I, topoisomerase II, poly(ADP-ribose) polymerase, and the nucleolar protein B23/nucleophosmin did not change. Comparison of polyadenylated RNA from NCI-H249 and NCI-H249rasH cells on Northern blots revealed similar levels of the mRNA for lamin B but higher levels of the mRNAs for lamins A and C in the v-rasH-expressing cell line. These observations provide evidence for differences in nuclear envelope structure in histologically different neoplastic cells derived from the same epithelial cell system and suggest that differences in lamina structure result from phenotype-specific differences in lamin gene expression.     

Differential cytotoxicity of tumour promoter TPA for EBV-negative human lymphoma cell lines and their EBV-converted sublines

The tumour-promoting phorbol diester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is toxic at nanomolar concentrations for Epstein-Barr virus (EBV)-negative human lymphoma cell lines BJAB and Ramos. Sublines that have been converted to Epstein-Barr nuclear antigen (EBNA)-positivity by infection with EBV survive and grow in TPA concentrations that are lethal for their non-converted parental lines. Resistance to the growth-inhibiting effect of TPA is higher in sublines of BJAB and Ramos that have been converted by the transforming B95-8 strain of EBV than in those converted by the non-transforming P3-HRI strain. The loss of TPA-sensitivity may reflect a change in the transformation or differentiation state of lymphoma cells as a result of EBV-conversion.     

Differential characteristics of two new tumorigenic cell lines of human breast carcinoma origin

Permanent human tumor cell lines are an important tool for the study of breast cancer. Two new breast cancer cell lines (BrCa-MZ-01 and BrCa-MZ-02) were isolated from a solid tumor and a pleural effusion, respectively. One cell line was established from a medullary carcinoma, the other from a ductal carcinoma. These cells exhibit ultrastructural and immunohistochemical features of epithelial cells of mammary origin. Intermediate filament and cytokeratin typing showed a clear predominance of the simple-epithelial cytokeratins CK 8, CK 18 and CK 19, although the expression was reduced in comparison to the hormone receptor-positive reference cell lines MCF-7 and ZR-75-1. Both cell lines produced slow-growing tumors after subcutaneous (s.c.) transplantation of 1 × 10⁷ viable tumor cells into nude mice. The cell line BrCa-MZ-01 expresses the estrogen and progesterone receptor, whereas the cell line BrCa-MZ-02 remains negative. Both cell lines are positive for secretion of platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), whereas interleukin-6 (IL-6) is only secreted by the cell line BrCa-MZ-02. Int. J. Cancer 77:415–423, 1998. © 1998 Wiley-Liss, Inc.