Biomedical subjects

C A Reznikoff

Publications and source records attributed to C A Reznikoff.

At least 55 records · Page 3Linked to original sources

PubMed1991

Nonrandom chromosome losses in tumorigenic revertants of hybrids between isogeneic immortal and neoplastic human uroepithelial cells.

Somatic cell hybrid analysis was used to examine the role of recessive cancer genes in tumorigenic transformation in vitro of human uroepithelial cells (HUC). Hybrids between nontumorigenic pseudodiploid SV40-immortalized HUC (SV-HUC) and two aggressive grade III transitional cell carcinomas (TCC) produced in nude mice after in vitro exposure of SV-HUC to 3-methylcholanthrene (MC) were completely suppressed for tumorigenicity at early passage. Tumorigenic reversion occurred after five or more passages in culture and was always accompanied by chromosome losses. Overall, the tumorigenic revertants showed statistically significant losses of chromosomes 1, 4, 5, 9q, 12, 14q, and 17 (all P less than or equal to 0.05) as compared to losses in suppressed hybrids. In addition, hybrid reversion was accompanied by losses that left specific tumors with a single remaining homolog of certain chromosomes (i.e., 3, 5q, 11p, 17p, and 18q). These losses were also considered significant because of the likelihood that genes on these chromosomes were reduced to homozygosity. Many of the significant losses (i.e., 5q, 9q, 11p, and 17p) were of chromosomes that are frequently lost in clinical TCC. Thus, these results support the hypothesis that these chromosomes contain genes whose loss leads to HUC tumorigenesis.

Carcinoma, Transitional Cell↗

PubMed1991

Cyclic AMP response in SV40 immortalized human bladder cells.

Forskolin-mediated increase in cyclic AMP and subsequent activation of protein kinase A were evaluated in SV40-immortalized human urothelial cells. This cell line is being used to evaluate the multistep carcinogenic process. Forskolin elicited a time- and dose-dependent increase in cyclic AMP. Increases in intracellular cyclic AMP preceded media increases in cyclic nucleotide. Large increases in intracellular cyclic AMP occurred within 5 min of forskolin addition. The lowest effective concentration of forskolin was between 0.1 and 1.0 microM. Cyclic AMP increases as large as 20- to 100-fold were observed in cells and media following forskolin addition. A 60 min preincubation with 12-O-tetradecanoylphorbol-13-acetate (TPA) did not reduce the magnitude of the forskolin increase in cyclic AMP. TPA has been shown to affect cyclic AMP metabolism in many types of cells including primary and secondary cultures of urothelial cells. In the latter, preincubation with TPA reduces the magnitude of the forskolin increase. A 4.2-fold increase in protein kinase A activity was observed within 0.5 min of forskolin addition, while only small increases in cyclic AMP (1.6-fold) were detected within 1 min. Much more cyclic AMP is synthesized than is needed to maximally activate protein kinase A. While results demonstrate a forskolin-responsive cyclic AMP system, this system does not appear to be regulated by TPA. Lack of regulation of this second messenger system by TPA may be part of the immortalization process.

Cell Line, Transformed↗

PubMed1990

EJ/ras neoplastic transformation of simian virus 40-immortalized human uroepithelial cells: a rare event.

To determine if expression of mutant p21 ras could convert Simian Virus 40-immortalized human uroepithelial cell line (SV-HUC) to tumorigenicity, SV-HUC cells were transfected with pSV2-neo (a neomycin-resistant gene) or PREJ/ras (c-HA-ras-1 with the 12th codon mutation and neo). Seven independent G418-resistant clones (A----G) were isolated from each group (SV-HUC/ras and SV-HUC/neo). SV-HUC/ras clones were morphologically altered, while SV-HUC/neo clones retained a typical SV-HUC epithelial morphology. Electrophoretic analysis of immunoprecipitated ras proteins detected altered p21 ras protein in four of seven SV-HUC/ras clones at passage (P)2 and in five of seven clones at P12 posttransfection. The relative levels of ras p21 differed among the clones and appeared to increase with passage in culture. RNA and DNA dot blot analyses showed that clones with more abundant mutant p21 also had higher ras RNA levels and, in one case, increased ras gene copy number. No altered ras protein was detected in any SV-HUC/neo clones. ras- and neo-transfected clones were tested for tumorigenicity at P2 posttransfection and again at P12 by four s.c. inoculations each into athymic nude mice. None of 56 inoculations of SV-HUC/neo clones was tumorigenic. None of the SV-HUC/ras clones at P2 gave rise to tumors at all four injection sites. However, two ras-transfected clones, SV-HUC/ras-B and SV-HUC/ras-F, produced one tumor each. One clone, SV-HUC/ras-D which produced abundant mutant p21, was negative when inoculated at P2, but produced tumors in four of four sites when reinoculated after ten passages in vitro. All tumorigenic clones had detectable levels of mutant ras p21. However, the relative levels of altered p21 ras protein among the SV-HUC/ras clones did not directly predict their tumorigenic potential, as several nontumorigenic SV-HUC/ras clones had protein levels equal to or higher than the most tumorigenic clone (SV-HUC/ras-D at P12). Cell lines established from the tumor explants exhibited higher ras gene copy numbers, higher RNA levels, and more abundant p21 than was seen in the clones at the time of inoculation. Therefore, increases in ras protein abundance occurred during tumor formation in vivo, as well as during passage of cells in culture, and such cells apparently had a selective growth advantage. However, expression of abundant mutant ras protein was not in itself sufficient for neoplastic transformation of SV-HUC.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Adherence of uropathogenic E. coli to differentiated human uroepithelial cells grown in vitro.

A quantitative in vitro model to measure E. coli adherence to differentiated human uroepithelial cells has been developed. Primary cultures of uroepithelial cells were initiated from normal ureteral epithelium. Adherence of uropathogenic 3H-labelled Escherichia coli to postconfluent human uroepithelial cells was directly related to the bacteria:epithelial cell ratio during incubation. Bacterial attachment was inhibited either by mannose or by urine containing anti-E. coli antibodies. Transmission electron microscopy showed that epithelial cells differentiated in vitro to resemble normal uroepithelium in vivo. Furthermore, electron microscopy showed specific adherence of bacteria to the glycocalyx of microvilli of the superficial uroepithelial cells in vitro in a manner which closely mimics the in vivo interaction. This model of bacterial adherence permits in vitro analysis of adhesin-receptor interactions between uropathogenic E. coli and a layer of viable uroepithelial cells similar to those lining the bladder.

Animals↗

PubMed1989

Basis for the differential modulation of the uptake of 5-iododeoxyuridine by 5'-aminothymidine among various cell types.

We have previously reported that 5'-aminothymidine (5'-AdThd), an antagonist of the feedback inhibition exerted by dTTP that regulates thymidine kinase, enhances the uptake and cytotoxicity of 5-iododeoxyuridine in various human bladder cancer cell lines but not in normal human urothelial cells (HU) propagated in vitro. In this work we have analyzed the factors that could potentially account for the differential effect of 5'-AdThd among various cell types: 647V (a human bladder cancer cell line); HU; SV-HU (a SV40-transformed human urothelial cell line), and C3H/10T1/2 mouse embryo fibroblasts (10T1/2) cells. 5'-AdThd enhanced the uptake of IdUrd in SV-HU cells (greater than 400%), similar to what we have observed before for 647V cells. However, in 10T1/2 and HU cells, 5'-AdThd only minimally increased the uptake of 5-iododeoxyuridine (about 160%). Thymidine kinases purified from the different sources were similarly sensitive to inhibition by dTTP or 5'-AdThd and to deinhibition of the dTTP-induced regulation of enzyme activity by 5'-AdThd. Furthermore, [3H]-5'-AdThd permeated and accumulated intracellularly in all cell types. In none of these cultures was nucleoside phosphorylase activity detected, as indicated by the inability of the cells to produce thymine or iodouracil after exposure to the appropriate nucleosides. Also, 5'-AdThd did not affect the breakdown of dTMP by crude preparations of cytosolic 5'-nucleotidase from the different cells. We found that intracellular dTTP pools in the various cell types were substantially high (15-26 microM) compared to the sensitivity of thymidine kinase to inhibition by dTTP (IC50 2-4 microM). This suggests that thymidine kinase is in a strongly inhibited state in situ. To test the sensitivity of thymidine kinase (in situ) to regulation by dTTP we investigated: (a) the effect of depleting intracellular dTTP pools with methotrexate on the uptake of thymidine (dThd); and (b) the effect of pH on the uptake of dThd and its perturbation by 5'-AdThd, since the inhibition of thymidine kinase activity by dTTP is known to be pH dependent. We found that a 47% reduction of dTTP pools by methotrexate in 10T1/2 and HU cells did not result in an increase in thymidine kinase activity, as indicated by the lack of an effect on the uptake of dThd. However, we have previously shown that, under similar conditions, 647V cells show a substantial increase in dThd uptake.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells, Cultured↗

PubMed1988

Cytogenetic instability with balanced chromosome changes in an SV40 transformed human uroepithelial cell line.

Cytogenetic analysis at the 15th, 34th, 50th, and 56th passages of an SV40 immortalized human uroepithelial cell line (SV-HUC-1) showed continuous chromosome change and marker formation. Throughout these passages the transformed cells maintained their epithelial morphology, were SV40 T antigen positive, did not shed infectious SV40 virus, and were repeatedly found to be nontumorigenic when innoculated into athymic nude mice. Each of the passages studied was characterized by extensive karyotypic changes due to formation, rearrangement, and disappearance of different markers. A marker involving chromosome 1 was stable at three of the passages studied, whereas markers involving the X chromosome changed at each passage studied. Because of the incorporation of several chromosomes or chromosome arms into markers, the karyotype was genetically balanced in the first passage studied, with no net loss or gain of chromosomal material despite a modal number of 44. In subsequent passages, despite continued instability and generation of new markers, there was a slight but additive loss of genomic balance which increased with time in culture. Since continued karyotypic rearrangements did not lead to tumorigenic conversion, it is probable that genetic instability coupled with selection for the most balanced genome may be important for the immortalization of this cell line.

Cell Line, Transformed↗

PubMed1988

Normal and malignant human urothelium: in vitro response to blockade of polyamine synthesis and interconversion.

To assess the effect of polyamine blockade on urinary epithelium, growth of normal human urothelial cell cultures and human transitional cell carcinoma (TCC) cell lines in the presence of various inhibitors of polyamine synthesis was evaluated. All four human TCC cell lines tested were quite sensitive to the specific inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine (DFMO), requiring less than or equal to 1.0 mM DFMO to double generation time. Alternatively, all three human urothelial cultures tested required 5-20-fold higher DFMO concentrations to achieve similar growth inhibition. The inhibitory effect of DFMO on TCC was found to act synergistically with that of the inhibitor of S-adenosylmethionine decarboxylase, methylglyoxal bis(guanylhydrazone), and additively with that of recombinant beta-serine interferon. Addition of individual polyamines entirely prevented the inhibitory effects of DFMO and/or methylglyoxal bis(guanylhydrazone) but not that of beta-serine interferon. It appears that inhibition of polyamine synthesis and/or interconversion holds promise in the management of TCC and that the in vitro model described will be of value in investigating such clinical applications.

Carcinoma, Transitional Cell↗

PubMed1988

Clonal growth of normal human uroepithelial cells.

We report the development of culture conditions which routinely support clonal growth of normal human uroepithelial cells (HUC). Secondary cultures seeded at clonal densities and grown under conditions described herein have a colony-forming efficiency (CFE) and colony size that will be useful for in vitro experiments. Primary cultures were dispersed to single cells and seeded in a supplemented Ham's F12 medium containing 1% fetal bovine serum together with 3 X 10(5) lethally irradiated Swiss 3T3 feeder cells on plastic substrates preequilibrated with F12 medium containing 5 or 10% serum. Using these conditions, the average CFE was 16.1 +/- 2.5%. A cloning efficiency of 4.9 +/- 1.5% was obtained under the same conditions in serum-free F12+ when supplemented with a mixture of trace elements or 0.1 mM ethanolamine. The epithelial nature of the cloned cells was confirmed by morphology and by positive immunofluorescent staining for human epithelial keratin proteins. To make this system useful for mutagenesis experiments, a clone of Swiss 3T3 feeder cells resistant to 5 micrograms/ml 6-thioguanine (6TG) was derived from the parental cell line. This 6-TG-resistant Swiss 3T3 clone supports HUC clonal growth with a CFE of 17.9 +/- 2.0% CFE. We also report clonal growth of HUC without feeder cells using supplemented MCDB 170 medium containing 70 micrograms/ml bovine pituitary extract. The average cloning efficiency using these conditions was 5.7 +/- 1.7%.

Cells, Cultured↗

PubMed1988

Marker chromosome stability associated with neoplastic transformation of human uroepithelial cells.

Chromosome studies were performed on three independently derived tumor cell lines established from carcinomas induced in nude mice after innoculation of SV40 immortalized human uroepithelial cells that had been treated with methylcholanthrene. Tumor 1 was an undifferentiated carcinoma, while tumors 7 and 9 were both squamous carcinomas. After six to eight passages in vitro the tumor cells were each reinoculated into other nude mice to yield secondary tumors (1.1 and 7.1). Chromosome studies on both primary and secondary tumors demonstrated the same distinctive chromosome markers. Tumors 1 and 1.1 shared the same histopathology in addition to the same modal chromosome number and identical chromosomal duplications and deficiencies; the same was true of tumors 7 and 7.1. Tumor 9, which did not yield a secondary tumor, nevertheless showed the same chromosome pattern in different passages. The stability of the characteristic marker chromosomes in the three tumor cell lines distinguishes these malignant lines from the nonmalignant SV40 transformed parent line from which the three tumors derived because the parent line was characterized by extreme marker instability. This suggests that the stable marker chromosomes that characterize the tumor cell lines may be critical for their tumorigenicity, and that evolution of an adaptive neoplastic genome may select for cytogenetic stability as long as there are no new selective pressures.

Animals↗

PubMed1988

Neoplastic transformation of SV40-immortalized human urinary tract epithelial cells by in vitro exposure to 3-methylcholanthrene.

Normal human urinary tract epithelial cells (HUC) were neoplastically transformed in vitro using a step-wise strategy. First, a partially transformed non-virus-producing cell line was obtained after infection of HUC with simian virus 40 (SV40). This cell line (SV-HUC-1) was demonstrated to be clonal in origin, as 100% of cells contained at least five of seven marker chromosomes. Marker chromosomes were formed by balanced translocations resulting in a 'pseudodiploid' cell line. SV-HUC-1 showed altered growth properties in vitro (e.g. anchorage independent growth) but failed to form tumors in athymic nude mice, even after 3 years in culture (80 passages). In the studies reported here, SV-HUC-1 at early passages (P15-P19) were exposed to 3-methylcholanthrene (MCA) in three separate experiments. After a six-week post-treatment period of cell culture, cells were inoculated s.c. into athymic nude mice. In all experiments, MCA-treated SV-HUC-1 formed carcinomas in mice usually with a latent period of 5-8 weeks. These carcinomas showed heterogeneity with respect to histopathologies and growth properties in the mice and karyotypes. All the tumors retained SV-HUC-1 chromosome markers, but each independent transformant was aneuploid and contained unique new marker chromosomes. Chromosomes usually altered in tumor cells included numbers 3, 5, 6, 9, 11 and 13. Mutations in the ras family of cellular proto-oncogenes resulting in altered mobility of the p21 protein product were not detected in six cell lines established from independently derived tumors. It is not yet known whether other cellular proto-oncogenes are activated in these tumorigenic transformants. Neither control SV-HUC-1 (which were not exposed to MCA), nor early passage HUC exposed to MCA formed tumors when inoculated into mice. Thus, the tumorigenic transformation of HUC resulted from the combined actions of SV40 and MCA.

Animals↗

PubMed1987

Characterization of human uroepithelial cells immortalized in vitro by simian virus 40.

Normal human uroepithelial cells (HUC) were transformed with simian virus 40 (SV40) in vitro. SV40-transformed HUC (SV-HUC) were selected by their ability to survive senescence which normally occurs in HUC between passages 4 and 6. At passage 6, 100% of SV-HUC stained positive for SV40 T-antigen. The epithelial nature of SV-HUC was confirmed by positive staining for human cytoplasmic keratins in all cells. SV-HUC have altered growth characteristics compared to HUC including the capacity to grow on plastic, independent of a collagen-gel substrate; loss of the dependence on medium supplements for optimal growth, loss of the dependence on feeder cells for growth at clonal density, and an apparently unlimited lifespan in culture (greater than 2 years). Although SV-HUC have an increased percentage of viable cells and increased saturation density compared to HUC, the generation time of SV-HUC during log phase is similar to that of HUC. Cultures of SV-HUC are epithelial in appearance and show some morphological heterogeneity in cell size and shape. At the ultrastructural level, SV-HUC have numerous alterations such as, irregularly shaped nuclei and nucleoli, pleomorphic microvilli, and the lack of a glycocalyx on the cell surface. In addition, SV-HUC does not stratify in culture, suggesting an inability to differentiate. Unlike HUC, SV-HUC are capable of growth in soft agarose, a property which increased with serial passage. Yet, through at least P50, SV-HUC remained nontumorigenic as determined by the inability to form tumors in athymic nude mice. This cell line of human epithelial origin may be suitable for studying the conversion of cells to tumorigenicity by subsequent treatment with another oncogenic agent.

Cell Survival↗

PubMed1987

Normal and malignant human urothelium: in vitro effects of epidermal growth factor.

Cultures of human normal urothelial (HU) cells and transitional cell carcinoma (TCC) cell lines were tested for their ability to specifically bind and respond to epidermal growth factor (EGF). The criteria of response investigated were stimulation of growth and induction of ornithine decarboxylase (ODC) activity. All four human TCC cell lines tested were stimulated to grow by EGF (1-100 ng/ml) in a dose related fashion while only one of four HU cell cultures was similarly stimulated, and growth stimulation of this cell line occurred only at early passage. TCC and HU cells bound EGF to similar degrees with Kds of 0.86 nM for TCC and 2.54 nM for HU (P greater than 0.05) and 91637 +/- 9816 (SD) (high affinity) receptors/cell for TCC and 124275 +/- 16841 for HU (P greater than 0.10). Baseline ODC activity was statistically similar in the two TCC and the three HU cell cultures tested. However, EGF induced dose related ODC activity only in the TCC cell lines and not in any of the HU cell cultures. Thus, while both malignant and normal urothelium bind EGF equally well in vitro, only TCC responds to EGF in two parameters relevant to neoplasia: growth and induction of ODC activity.

Carcinoma, Transitional Cell↗

PubMed1987

Growth kinetics and differentiation in vitro of normal human uroepithelial cells on collagen gel substrates in defined medium.

Conditions have been described for the selective growth, serial cultivation, and postconfluent morphological differentiation in vitro of normal adult human uroepithelial cells (HUC) on collagen gel substrates in a serum-free medium without the deliberate addition of undefined components and without a requirement for a polypeptide growth factor. The culture medium used (F12) was the standard Ham's F12 medium (0.3 mM calcium) supplemented with 1 microgram/ml hydrocortisone, 5 micrograms/ml transferrin, 10 micrograms/ml insulin, 0.1 mM nonessential amino acids, 2.0 mM L-glutamine, 2.7 mg/ml D-glucose, 10(-4) M ethanolamine or 10(-4) M phosphoethanolamine, and 5 X 10(-8) M selenium. HUC grown in F12 on Type I collagen gel substrates had a generation time of 33 hours and could be serially passed 3-5 times during log phase of growth (20-25 population doublings) before spontaneously senescing. Transmission electron microscopy showed that cultures of HUC grown entirely in serum-free F12 on collagen gel substrates morphologically differentiate postconfluence to resemble in some respects the stratified uroepithelium in vivo, although neither a basal lamina nor an asymmetric unit membrane develop. The addition of epidermal growth factor (EGF) to the F12 did not improve either the growth rate or the lifespan in vitro of HUC. In contrast, the addition of fetal bovine serum (FBS) to F12 was mitogenic to HUC in a dose-dependent manner in the concentration range 0.01-1.00% (4-400 micrograms/ml protein), but higher concentrations of FBS did not improve growth further. The generation time of HUC in 1% FBS-F12 decreased to 21 hours, and the potential population doublings in vitro increased to 31-36. Small amounts (140 micrograms/ml) of bovine pituitary extract (BPE) were similarly mitogenic to HUC in F12. Altering the calcium concentration in the standard Ham's F12 medium (0.3 mM), however, did not improve the growth of HUC in serum-containing or serum-free medium. Higher calcium concentrations (0.30-0.90 mM) were neither mitogenic nor inhibitory to HUC growth, but resulted in decreasing viability of HUC in growing cultures, suggesting an accelerating rate of cellular differentiation. In contrast HUC in low calcium, serum-free F12 (0.1 mM) failed to stratify and morphologically differentiate even in postconfluent cultures. This failure of HUC to differentiate in low calcium F12 medium did not confer a long-term growth advantage.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1987

Effect of interferon alpha, interferon beta, and interferon gamma on the in vitro growth of human renal adenocarcinoma cells.

Interferon-alpha, interferon-beta, and interferon-gamma differ in their antiproliferative effects for several cell lines. Interferons were thus assessed for their activity in inhibiting proliferation of three renal cell carcinoma cell lines. The malignant epithelial phenotype of each of these cell lines was confirmed by electron microscopy, histology, karyotype and tumorigenicity. When compared on an anti-viral unit basis, naturally produced interferon-beta was more effective than natural interferon-alpha for all cell lines and clones. Proliferation of each of the cell lines was inhibited by interferon-gamma. In all cases, removal of interferons from culture media resulted in resumption of the rate of cell growth after a variable delay of 6-10 days. If the antiproliferative effects of interferons predominate in mediating tumor regression, clinical response may depend upon the type of interferon to which the tumor is exposed.

Adenocarcinoma↗

PubMed1986

Perturbation of thymidine kinase regulation: a novel chemotherapeutic approach.

The design of compounds which disrupt the regulation of key enzymes is a new strategy for drug development. 5'-AdThd, which can antagonize the feedback inhibition that normally regulates thymidine kinase, is representative of this novel class of agents. The ability of 5'-AdThd to deinhibit thymidine kinase markedly increases the capacity of some cells to phosphorylate thymidine and IdUrd and, thereby, enhance their cytotoxicity. This effect is evident in three human bladder cancer cell lines, but not in normal human urothelial cells. Thus, 5'-AdThd may be able to increase the therapeutic selectivity of IdUrd. Intracellular dTTP pools are critically involved in mediating the effects of 5'-AdThd, but differences in these pools from cell type of cell type may not account for the selective stimulation of IdUrd uptake produced in the cancer cells.

Cell Division↗

PubMed1986

Quantitative assessments of the cytotoxicity of bladder carcinogens towards cultured normal human uroepithelial cells.

Chemical carcinogens are known to exert cytotoxic effects on cells. The survival of cultured human uroepithelial cells (HUC) after exposure to several important classes of human and experimental animal bladder carcinogens has been quantitatively assessed in vitro using reduction in cell number and/or colony forming efficiency as the endpoint(s). Cells were treated with different carcinogens or various metabolites of a procarcinogen and the responses were analyzed with respect to the cell type used and to the donor source of the cells. The cytotoxic responses of HUC to the stable bladder procarcinogens tested [4-aminobiphenyl (ABP), 4-nitrobiphenyl, N-[4-(5-nitro-2-furyl)-2-thiazole]formamide and 2-amino-4-(5-nitro-2-furyl)thiazole] were dependent on both the concentration of chemical used and the duration of exposure. The survival of HUC after exposure to several metabolites of ABP differed. The N-hydroxylated derivatives of ABP (N-hydroxy-4-amino-biphenyl and N-hydroxy-4-acetylaminobiphenyl) were considerably more cytotoxic toward HUC than ABP or 4-acetylaminobiphenyl. The survival of HUC from different individuals after treatment with the direct acting carcinogen N-nitro-N-methylurea was very similar. In contrast, the survival of HUC from different donors varied considerably after treatment with the procarcinogen 3-methylcholanthrene which requires metabolic activation. However, significant heterogeneity in the survival of HUC from five donors after exposure to the human bladder procarcinogen ABP was not observed in this study. Cultures of normal human fibroblasts from four donors showed an unexpected heterogeneous response to the cytotoxic effects of ABP. These results demonstrate that many variables affect the cytotoxic response of normal cells to bladder carcinogens.

Aminobiphenyl Compounds↗

PubMed1986

Preferential stimulation of iododeoxyuridine phosphorylation by 5'-aminothymidine in human bladder cancer cells in vitro.

5'-Aminothymidine represents a novel class of compounds capable of antagonizing the feedback inhibition which normally regulates thymidine kinase. As a consequence, the uptake of iododeoxyuridine, a substrate of thymidine kinase, can be substantially increased by 5'-aminothymidine. in this study the phosphorylation, uptake, and cytotoxicity of iododeoxyuridine was markedly enhanced by 5'-aminothymidine in three human bladder cancer cell lines, T24, HT1197 and 647V. In contrast, neither the uptake nor the toxicity of iododeoxyuridine was increased by 5'-aminothymidine in normal human urothelial cells propagated in vitro. Although 30 microM 5'-aminothymidine increased the uptake of 3 microM iododeoxyuridine 4- to 5-fold in the HT1197 and 647V cells and 2.5-fold in the T24 cells, no stimulation was produced in the normal urothelial cells. The modulation of iododeoxyuridine uptake was associated with parallel changes in the inhibition of cellular replication. The cytotoxicity of iododeoxyuridine was strongly augmented by 5'-aminothymidine in the HT1197 and 647V cells, modestly increased in the T24 cells, and unchanged in the normal urothelial cells. The degree to which iododeoxyuridine phosphorylation was stimulated did not correlate with cellular replication rates or with intracellular thymidine triphosphate concentrations. Iododeoxyuridine uptake was markedly increased in the HT1197 (doubling time = 52 h) and 647V (doubling time = 24 h) cells, moderately in the rapidly growing cells T24 (doubling time = 20 h), and minimally in the normal urothelial cells which doubled every 32 h. In exponentially growing cells the thymidine triphosphate pools were approximately 18 microM in the normal cells and about 21, 24, and 35 microM in the HT1197, T24, and 647V cells, respectively. The use of 5'-aminothymidine and other compounds capable of antagonizing feedback inhibition may provide a new means of increasing the efficacy of cytotoxic nucleosides.

Antimetabolites, Antineoplastic↗

PubMed1985

Serial cultivation of normal rat bladder epithelial cells in vitro.

Recent advances in culture techniques for human urothelial cells have led to the development of an improved method for growing primary rat bladder epithelial cells. We report here the conditions developed for large-scale in vitro growth and serial cultivation of normal diploid rat bladder epithelial cells. Primary cultures were initiated by attachment of bladder mucosal explants to type I collagen gels. A rapid outgrowth of epithelial cells from the explants occurred when cultured in a hormone-supplemented medium with epidermal growth factor. These primary outgrowths were passaged by nonenzymatic dispersion with 0.1 per cent ethylenediaminetetracetic acid and replating onto new gels. The capacity for routine serial passaging and maintenance of rat bladder epithelial cells required the presence of epidermal growth factor, a requirement not observed with human urothelial cells. The characteristics of the cultured rat bladder epithelial cells were similar to human urothelial cells in: ultrastructural and phase-contrast morphologic properties, showing junctional complexes, desmosomes, stratification and an apical glycocalyx; the absence of stromal cell contamination; and the ability to be serially passaged. Spontaneous cell-line formation was observed with the rat bladder epithelial cells, but has not been found with the human urothelial cells. With the method that we have developed, the number of rat bladder epithelial cells generated from a single bladder of a 4 to 6 week old rat was increased 100-fold from about 7 X 10(5) cells to 7 X 10(7) viable cells within 3 weeks of culture. The capability of culturing normal, primary rat bladder epithelial cells on this scale has not been reported previously and will facilitate comparative studies of the biological and molecular characteristics of the mammalian urothelium. Furthermore, this culture system will be useful for carcinogenesis studies, including metabolic activation of carcinogens and cellular transformation in vitro.

Animals↗