Tissue culture model of transitional cell carcinoma: characterization of twenty-two human urothelial cell lines.

Twenty-two continuous cell lines derived from normal and neoplastic urothelium, maintained under identical culture conditions, were characterized in terms of isozyme phenotype, tumorigenicity, and xenograft morphology following xenotransplantation to nude mice, cytological appearance, in vitro growth rate, labelling index, and colony-forming efficiency, in parallel with separate studies of in vitro drug sensitivities and monoclonal antibody reactivities. Three groups were identified: (a) distinct lines with differing isozyme patterns, a broad spectrum of growth characteristics, and xenograft morphologies similar to the histopathology of the parent tumors after periods of up to 17 yr following establishment in vitro; (b) cross-contaminated sublines (maintained separately in different laboratories for periods of up to 10 yr), with identical isozyme patterns and similar growth characteristics, but differing markedly in tumorigenicity and xenograft morphology; and (c) lines derived from normal urothelium which were nontumorigenic and had an isozyme pattern usually only encountered in untransformed cells. These data indicate that cell lines representative of human transitional cell carcinomas can be selected on the basis of xenograft morphology and isozyme patterns, and that a panel of lines derived from normal and neoplastic urothelium could provide a model system to study the biology and treatment of this disease.

Immortalized human pituitary cells express glycoprotein alpha-subunit and thyrotropin beta (TSH beta).

A major problem in the study of human pituitary cells is their lack of proliferative capacity in vitro. To address this issue, we have infected normal human, postmortem pituitary cells in monolayer culture with a temperature-sensitive (tsA58) mutant of SV40 large T antigen. Several epithelial-like colonies were isolated; and one, designated CHP2, has been studied in detail to identify its functional characteristics. CHP2 cells have undergone more than 150 culture passages and retain an epithelial morphology. They exhibit tight temperature-dependent growth, in the presence and absence of serum, with cell division at 33 C and growth inhibition at 39 C. CHP2 cells, at both temperatures, showed diffuse immunostaining for human alpha-subunit and focal staining for TSH beta. Gene expression was confirmed by RT-PCR and sequencing. TRH and GnRH receptors were not detectable, and their absence was confirmed by their lack of effects on intracellular calcium and inositol phospholipids. Cytogenetic analysis showed that the cells had a modal peak in the diploid range and a smaller peak in the tetraploid range. There was also a consistent loss of chromosome 22 and a normal chromosome 2 homologue, the latter being replaced by one of two chromosome 2 markers, M2A or M2B. In conclusion, we have immortalized human pituitary cells using SV40 tsT, from which we have cloned a cell line expressing alpha-subunit and TSH beta.

Epidermal growth factor protects GH3 cells from adenosine induced growth arrest.

We have demonstrated that high doses of adenosine (0.1 mM) inhibit the growth of the rat pituitary GH(3) cell line. This effect was not mediated via cell surface adenosine receptors as the adenosine agonists N(6)-(2-phenylisopropyl)adenosine (PIA, 0.1 mM) and 5'-N- ethylcarboxamidoadenosine (NECA, 0.1 mM) were unable to reproduce the growth inhibitory effect of adenosine. The adenosine uptake inhibitor dipyridamole completely blocked the growth inhibitory effect of adenosine suggesting that its site of action is intracellular. Epidermal growth factor (EGF) was able to completely reverse the growth inhibitory effect, restoring the growth rate of cultures treated with adenosine and EGF to that of control cultures. The ratio of cells in G(0)/G(1):S:G(2)/M phases of the cell cycle was unaltered in adenosine treated compared with control cultures suggesting that there was no change in the rate of cell division, however the degree of apoptosis was markedly increased in adenosine treated cultures. EGF was able to reduce the adenosine induced apoptosis almost to levels seen in the control cultures. Thus the mechanism of the growth inhibitory effect of adenosine does not appear to be by reducing the rate of cell division but rather by increasing the rate of cell death and EGF restores the growth rate of adenosine treated cells to that of untreated cells by preventing adenosine induced apoptosis.

Loss of ACTH expression in cultured human corticotroph macroadenoma cells is consistent with loss of the POMC gene signal sequence.

The proopiomelanocortin (POMC) gene is highly expressed in the pituitary gland where the resulting mRNA of 1200 base pairs (bp) gives rise to a full-length protein sequence. In peripheral tissues however both shorter and longer POMC variants have been described, these include for example placental tissue which contain 800 (truncated at the 5' end) and 1500 as well as the 1200 bp transcripts. The importance of the 800 bp transcript is unclear as the lack of a signal sequence renders the molecule to be non-functional. This transcript has not been previously demonstrated in the pituitary gland. In this report we show evidence of a 5' truncated POMC gene in human pituitary corticotroph macroadenoma cells (JE) maintained in primary culture for >1 year. The original tumour tissue and the derived cells during early passage (up to passage 4-5) immunostained for ACTH and in situ hybridisation confirmed the presence of the POMC gene in the cultured cells. These cells also secreted 15-40 pg/10(5) cells/24 h ACTH. In addition, as expected RT-PCR demonstrated the presence of all three POMC gene exons and is thus indicative of a full-length POMC gene. In late culture passages (passages 8-15) JE cells ceased to express ACTH and cell growth became very slow due presumably to cells reaching their Hayflick limit. ACTH immunostaining in these cells was undetectable and ACTH secretion was also at the detection limits of the assay and no greater than 10 pg/10(5) cells/24 h. ACTH precursor molecules were also undetectable. RT-PCR for the POMC gene in these late passage cells showed that only exon 3 was detectable, in contrast to early passage cells where all three exons were present. In summary we isolated in culture, human pituitary cells that possessed initially all three exons of the POMC gene and immunostained for ACTH. On further passaging these cells showed a loss of exons 1 and 2 in the POMC gene and a loss of ACTH immunostaining and secretion. We would like to suggest that the loss of ACTH peptide expression in these late passage cells is in part due to the loss of the POMC signal sequence. An alternative explanation for our findings is that there were originally two populations of corticotrophs in the cultures, one of which possessed the full-length POMC gene and the other only the 5' truncated POMC transcript and it is these latter cells which survived in culture. In either scenario this is the first report of the 5' truncated POMC gene occurring in pituitary cells.

Influence of clonogenic assay methodology on measurement of drug sensitivities in vitro.

To assess the influence of clonogenic assay methodology on measurement of reproductive cell kill, the in vitro sensitivities of a human bladder cancer cell line, RT112, to methotrexate and adriamycin were determined using ten different procedures. Marked differences in dose-response to methotrexate, but not adriamycin, were observed.

Angiogenic factors expressed by human prostatic cell lines: effect on endothelial cell growth in vitro.

BACKGROUND: Antiangiogenic therapy for prostatic cancer should offer additional ways of combating tumor progression. Knowledge of the possible angiogenic factors expressed by prostate cancer cell lines would therefore assist in the design and testing of such potential treatments. METHODS: Changes in the proliferation and morphology of several endothelial cell lines (BAEC, HUVEC, and BACE) in response to either coculturing with human prostatic cell lines or culturing with conditioned medium derived from these lines were assessed. RESULTS: Proliferation of BAEC cells was significantly stimulated by conditioned media from DU145, LNCaP, and DuPro-1, and also by coculture with LNCaP and DuPro-1. Growth of HUVEC cells was significantly increased with conditioned media from LNCaP, Ten12, and PC3, and by coculture with DU145 and DuPro-1. FGF2 supplementation is required for BACE growth in vitro, and only conditioned medium from Ten12 cells, which produce the highest levels of this growth factor, significantly increased cell numbers. BACE growth, however, was stimulated in coculture experiments with DU145, DuPro-1, PC3, and LNCaP. Morphological changes were only observed in the BAEC and BACE cells when cultured with conditioned media. CONCLUSIONS: Prostatic carcinoma cell lines express a variety of angiogenic substances, including FGF2, which can stimulate endothelial cell proliferation in vitro, but this response may be modified by the prostatic-cell expression of other factors such as TGF alpha and TGF beta.

Cell proliferation in prostatic carcinoma: comparative analysis of Ki-67, MIB-1 and PCNA.

Antibodies to assess the proliferative index of tumours are being increasingly employed together with established markers for prognostic evaluation. This study set out to compare three cell proliferation markers, Ki-67, MIB-1 and PCNA, utilizing a semi-quantitative method of assessment, in 20 human prostatic carcinomas. The streptavidin-biotin immunostaining system was used for the monoclonal antibodies MIB-1 and PCNA and an indirect immunoperoxidase assay for the monoclonal antibody Ki-67. Significant correlations were found between the expression of Ki-67 in frozen tissues and MIB-1 in formal saline-fixed wax-embedded tissues (p = 0.0003); between Ki-67 and PCNA expression in Bouin's-fixed tissues (p < or = 0.0001); and MIB-1 (formalin-saline-fixed tissues) and PCNA (Bouin's-fixed tissues) (p < or = 0.0001). A more intense nuclear staining pattern with less heterogeneity was observed for MIB-1 compared with PCNA, suggesting the antibody of choice, on formal saline-fixed tissues, is MIB-1, which closely correlated with Ki-67, a marker we have previously shown to be of prognostic value in prostatic carcinoma.

Intravesical chemotherapy: in vitro studies on the relationship between dose and cytotoxicity.

The relative importance of two variables, drug concentration and period of exposure, in relation to the therapeutic potential of intravesical chemotherapy was examined in an experimental system. A human bladder cancer cell line was exposed to a range of concentrations of the four drugs commonly used to treat superficial bladder cancer (adriamycin, epodyl, mitomycin-c, thiotepa) for periods of 30, 60 and 120 min. An exponential relationship was observed between clonogenic cell kill and both drug concentration and period of exposure. Thus, under the experimental conditions employed, cytotoxicity is proportional to dose (i.e. concentration X period of exposure). These two variables are of equal importance in relation to tumor cell kill, indicating that maximum therapeutic benefit may be obtained by using the highest concentration achievable for as long as the patient can retain the instillate, bearing in mind the potential increase in toxicity to the patient and the cost.

Monoclonal antibodies to human urothelial cell lines and hybrids: production and characterization.

Eleven independent monoclonal antibodies, the LBS series, were isolated after immunization of mice with RT112 cells, a continuous cell line derived from a transitional cell carcinoma of the human bladder. These antibodies were tested by indirect immunofluorescence on a panel of 28 human cell lines, of which 17 were urothelial carcinoma-derived, 4 of non-urothelial carcinoma origin, 3 fibroblast cell lines, 4 lymphoblastoid lines and 7 murine cell lines. Also tested were 7 somatic cell hybrid clones derived by fusion of human RT112 cells with murine bladder carcinoma MB63T/H cells. None of the LBS antibodies reacted with mesenchyme-derived cells, although all reacted with RT112 cells. On the basis of reactivity with the cell line panel, the antibodies were divided into 3 groups. Group I (LBS-1 and 19) reacted with all human epithelium-derived cell lines. Group II (LBS-2, 8, 15 and 17) reacted only with human urothelium-derived cells, tending to recognise the least anaplastic types. Group III antibodies (LBS-10, 20A, 20B, 21 and 34) were urothelium-specific on the human continuous cell line panel, but additionally reacted with murine urothelial and epithelial cell lines. The 6 human-specific antibodies (Group I and II) were used for preliminary analysis of human gene expression in a series of 7 mouse X human urothelial somatic cell hybrids. Each hybrid reacted with at least 1 LBS antibody, although there were changes in gene expression with time in culture, indicating both loss and unmasking of human genes. These data suggest the LBS-series antibodies recognise different determinants associated with epithelial and urothelial cell differentiation, and thus may be valuable probes in the study of normal differentiation and malignant transformation in human urothelial cells.

Comparison of the cytotoxic activities of chemotherapeutic drugs using a human bladder cancer cell line.

Many chemotherapeutic drugs have been used to treat patients with advanced bladder cancer, but few of these have been evaluated adequately in phase II clinical trials. Continuous cell lines provide one means for comparing the in vitro cytotoxicities of anticancer agents. In this study, a continuous cell line derived from a transitional cell cancer of the human bladder, which still produces tumours histologically similar to the tumour of origin on xenotransplantation, was used to measure the in vitro cytotoxicities of twelve chemotherapeutic drugs by clonogenic assay. The most cytotoxic agents tested were methotrexate, mitoxantrone, adriamycin, mitomycin C and cisplatin. These in vitro findings are compatible with the activity of these drugs given systemically as single agents in phase II clinical trials in patients with advanced bladder cancer.

Identification of synergistic combinations of spirogermanium with 5-fluorouracil or cisplatin using a range of human tumour cell lines in vitro.

A series of continuous human tumour cell lines, derived from various tumour types, were used to establish whether the combination of spirogermanium (SP) with other 'standard' antitumour drugs proved superior to these as single agents in reducing cell survival in vitro. A non-cytotoxic concentration of SP was selected and when combined with a range of concentrations of cisplatin or 5-fluorouracil (5-FU), definite synergistic cell kill was noted in all lines tested. In contrast, the combination of SP with various other antitumor drugs, including adriamycin, methotrexate and the vinca alkaloids and with X-irradiation did not enhance cytotoxicity. These pre-clinical in vitro studies suggest that benefit may accrue from combining SP with either 5-FU or cisplatin and provide a basis for their clinical evaluation in colo-rectal tumours or transitional cell cancer of the bladder, respectively.