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DNA repair efficiency may play a significant role in individual susceptibility to bladder cancer, the third most common cancer in Europe. Bladder cancer arises from the urothelial cell layer which lines the urinary tract. As DNA repair gene expression levels should reflect DNA repair capacity, we investigated the expression of genes from the base excision, nucleotide excision and mismatch repair pathways in normal human urothelial (NHU) cells in vitro. RNA was extracted from six independent NHU cell lines and expression of 26 DNA repair genes was determined by ribonuclease protection assay. The results show that all the genes analysed were detected in NHU cells in vitro with a similar expression pattern in most cell lines. However, there was some variation between cell lines, with one expressing base excision repair genes very strongly, but another having weak expression of mismatch repair genes. These results suggest that DNA repair genes are constitutively expressed by NHU cells and that there is some inter-individual variation. Prospective studies are required to determine whether these differences in gene expression may play a role in susceptibility to bladder cancer.

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The molecular basis of how rodent nongenotoxic hepatocarcinogens such as phenobarbitone cause liver-tumor formation is poorly understood. An early effect of phenobarbitone exposure is to induce hepatocyte proliferation transiently, and there is evidence that this may be important for subsequent tumor development. In this investigation, we have used the differential display reverse transcriptase polymerase chain reaction technique to analyze differential gene expression in male C57B1/10J mouse liver during the mitogenic phase of the phenobarbitone response. Seventy-seven putative differentially expressed cDNAs were isolated by differential display, and 13 of them were subsequently confirmed as being differentially expressed (both increased and decreased by phenobarbitone). Seven of the cDNAs were homologous to known mouse or human genes (carboxylesterase, coagulation factor X, amine N-sulphotransferase, human protein disulphide isomerase-related protein, cytochrome c oxidase subunit IV, golgin-245, thioredoxin reductase, betaine-homocysteine methyl transferase) and the remainder were novel. The expression pattern of the sulphotransferase was further characterized, and in mouse liver it was found to be significantly induced by phenobarbitone and not by five other rodent nongenotoxic hepatocarcinogens. In summary, the technique has enabled the identification of previously uncharacterized genes whose expression patterns are differentially altered by phenobarbitone in the mouse liver.

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PURPOSE: Recent advances in combinatorial chemistry and high throughput screens for pharmacologic activity have created an increasing demand for in vitro high throughput screens for toxicological evaluation in the early phases of drug discovery. METHODS: To develop a strategy for such a screen, we have conducted a data mining study of the National Cancer Institute's Developmental Therapeutics Program (DTP) cytotoxicity database. RESULTS: Using hierarchical cluster analysis, we confirmed that the different tissues of origin and individual cell lines showed differential sensitivity to compounds in the DTP Standard Agents database. Surprisingly, however, approaching the data globally, linear regression analysis showed that the differences were relatively minor. Comparison with the literature on acute toxicity in mice showed that the predictive power of growth inhibition was marginally superior to that of cell death. CONCLUSIONS: This datamining study suggests that in designing a strategy for high throughput cytotoxicity screening: a single cell line, the choice of which may not be critical, can be used as a primary screen; a single end point may be an adequate measure and a cut off value for 50% growth inhibition between 10(-6) and 10(-8) M may be a reasonable starting point for accepting a cytotoxic compound for scale up and further study.

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Phenobarbitone (PB) treatment of mice causes a decrease in the growth factor responsiveness of hepatocytes. Here, epidermal growth factor receptor (EGFR) expression and receptor autophosphorylation was determined in hepatocytes isolated from control and PB-treated mice. There was a decrease in the level of EGFR expression in hepatocytes isolated from mice following PB administration when compared to controls. EGF caused an approximate 20-fold increase of the 170 kD phosphotyrosine band in control hepatocytes, which was inhibited by the EGFR specific tyrosine kinase inhibitor 4, 5-dianilinopthalamide. Following PB treatment, the degree of basal receptor phosphorylation (in the absence of EGF) was significantly greater and therefore the fold rise in EGFR phosphorylation in isolated hepatocytes was lower than in controls. However, the overall extent of EGF-induced receptor phosphorylation was not diminished in hepatocytes isolated from PB-treated mice. Therefore the reduction in responsiveness to growth factors seen in hepatocytes ex vivo or the cessation of proliferation observed in vivo following PB administration is unlikely to be attributed to a decrease in ligand binding and subsequent receptor autophosphorylation.

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Liver enlargement is a common feature of non-genotoxic rodent hepatocarcinogens administered at high doses. In the present study, the expression of growth factors and growth factor receptors was investigated in the C57BL/1OJ mouse during liver enlargement induced by the non-genotoxic rodent hepatocarcinogen, sodium phenobarbitone (PB). Male mice were dosed 0-2500 p.p.m. PB in the diet for 1, 4 and 13 weeks. There was a dose and time dependent increase in liver weight. Hepatocyte replication, assessed by incorporation of bromodeoxyuridine, was increased in a dose-dependent manner at week 1 only (18-fold increase at 2000 p.p.m.) and was predominantly localized in the centrilobular region. At week 1, PB (2500 p.p.m.) caused transient increases in transforming growth factor alpha (TGFalpha) and epidermal growth factor receptor (EGFR) and decreases in transforming growth factor beta1 (TGF-beta1) and mannose-6-phosphate receptor (M6PR) in centrilobular hepatocytes which correlated with the replication in this region. At week 1, there was an increase in both hepatocyte growth factor (HGF) and hepatocyte growth factor receptor (HGFR) which colocalized in centrilobular hepatocytes; in some mice or periportal hepatocytes in other mice. After 13 weeks, HGF and HGFR were localized in the cytoplasm of centrilobular hepatocytes of all mice but exhibited a differential intracellular distribution across the lobule. At 2500 p.p.m. PB, EGFR and HGFR mRNA were essentially unchanged over the 13 week dosing period whilst M6PR mRNA was increased 2- to 4-fold. At 2500 p.p.m. PB, EGFR protein levels from immunoblots showed a consistent decrease over the 13 weeks whilst M6PR and HGFR protein levels were essentially unchanged. The protein level and mRNA data for EGFR suggest post-transcriptional modification. Thus, phenobarbitone caused transient replication of hepatocytes and modulation of growth stimulatory and inhibitory factors and their associated receptors in terms of overall levels and regional distribution in the liver.

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The majority of genotoxic carcinogen-induced liver tumours of the sensitive B6C3F1 mouse contain activated H-ras oncogenes. Such mutations also occur in hepatocarcinogenesis-resistant strains. In order to determine whether this is true of non-genotoxic carcinogen-induced tumours, liver tumours induced in B6C3F1 and C57BL/10J mice by methylclofenapate (MCP) were compared. Polymerase chain reaction (PCR) analysis revealed H-ras codon 61 mutations in 11/46 B6C3F1 and 4/31 C57BL/10J liver tumours. The nude mouse tumorigenicity (NMT) assay was used to analyse tumours without codon 61 mutations. Of the 12 B6C3F1 liver tumour DNAs subjected to this assay, one contained a H-ras codon 117 mutation. Further PCR analysis on frozen tumour samples (46 B6C3F1 and 15 C57BL/10J) revealed no codon 12 mutations; one additional codon 117 mutation was identified in a B6C3F1 tumour. Overall, then, H-ras codon 61 mutations were detected in MCP-induced B6C3F1 tumours less frequently than in genotoxin-induced tumours. Two B6C3F1 tumours contained codon 117 mutations similar to those previously found in tumours induced by ciprofibrate, furan and furfural, and in at least one spontaneous tumour. Ras mutations were also detected in some C57BL/10J tumours, providing further evidence that ras oncogenes can participate in hepatocarcinogenesis in resistant mice.

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Activation of the ras family of oncogenes occurs frequently in liver tumors of the B6C3F1 mouse, a strain which is highly sensitive to hepatocarcinogenesis. Many other mouse strains are much more resistant to hepatocarcinogenesis; the aim of this study was to determine the frequency and pattern of oncogene activation in spontaneous and chemically induced liver tumors of three such strains, the C57BL/6J, the C57BL/6 x DBA/2 F1 hybrid (B6D2F1) and the C57BL/6 x Balb/c F1 hybrid (B6BCF1). The C57BL/6, DBA/2 and Balb/c strains are all relatively resistant to spontaneous hepatocarcinogenesis (1.5-3.6% of animals develop liver tumors in 2 years); with regard to chemically induced hepatocarcinogenesis the Balb/c is highly resistant, the C57BL/6 has low susceptibility and the DBA/2 has low to moderate susceptibility. The nude mouse tumorigenicity assay was used to search for activated oncogenes in 15 C57BL/6J liver tumors induced by a single neonatal dose of vinyl carbamate (VC, 0.15 mumol/g body weight). Three tumors contained H-ras genes activated by point mutations at codon 61 and one contained a non-ras oncogene. The polymerase chain reaction and allele-specific oligonucleotide hybridization were used to study H-ras mutations in spontaneous and VC-induced tumors from all three strains of mice. The frequency of H-ras codon 61 mutations in tumors induced by 0.15 mumol/g body weight VC in the C57BL/6J mouse (5/37) was similar to that in spontaneous tumors (2/9); surprisingly, tumors induced by a lower dose of VC (0.03 mumol/g body weight) had a higher frequency of H-ras mutations (12/28). The frequencies of H-ras activation detected in VC (0.03 mumol/g body weight)-induced tumors from the two F1 hybrids studied differed markedly. Only one VC-induced B6BCF1 tumor contained a mutated H-ras gene (1/10), whereas the majority of B6D2F1 tumors contained such mutations (23/33). Several spontaneous B6D2F1 liver tumors contained H-ras codon 61 mutations (6/15). Thus, H-ras activation frequency does not determine susceptibility to hepatocarcinogenesis in inbred mice and their F1 hybrids, since a relatively high frequency of H-ras mutations was observed in two resistant strains and a low frequency was found in the other strain.

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The high incidence and profile of ras gene mutations reported in spontaneous and chemically induced liver tumours of the B6C3F1 mouse provides a potential means of determining in vivo genotoxicity and its relevance to carcinogenicity. We analysed spontaneous and chemically induced [with 4-amino-biphenyl (ABP), 2-acetylaminofluorene (AAF) and diethylnitrosamine (DEN)] hepatocellular tumours of the C57Bl/10J mouse for H-ras, K-ras and N-ras gene mutations to see if mutational analysis of the ras genes could be useful for such a determination in this strain. Regions of DNA spanning codons 12, 13 and 61 of the ras genes were amplified from formalin fixed liver tumour sections using the polymerase chain reaction. Mutations were detected using allele specific oligonucleotide probing and confirmed by sequencing. We have found that there are few ras mutations in either spontaneous or chemically induced liver tumours in the C57Bl/10J mouse. Out of 25 spontaneous tumours two contained an A to T transversion and one contained an A to G transition in base 2 of H-ras codon 61 and two contained a G to A transition in base 2 of K-ras codon 13 (the K-ras mutations were only faintly detectable and may be present in a subpopulation of the tumour cells). In the case of the 18 ABP induced tumours one contained a C to A transversion in base 1 of H-ras codon 61, and one contained an A to T transversion in base 2 of H-ras codon 61 and one contained a G to C transversion in base 1 of K-ras codon 13. One C to A transversion in base 1 of H-ras codon 61 was detected out of eight AAF induced tumours. Of the 25 DEN induced tumours, one contained an A to G transition and one contained an A to C transversion in base 2 of H-ras codon 61. The data indicate that at least in hepatocellular tumours of the C57Bl/10J strain and using chronic dosing regimes the ras genes do not represent markers for in vivo genotoxic activity.

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The cell volume increase in individual clones of cells of the yeast Saccharomyces cerevisiae has been measured using time lapse cinematography in populations showing steady state balanced exponential growth. There were significant differences in clonal specific growth rates within the population in each of 10 experiments using different strains on different media supporting different growth rates. The results suggest that specific growth rates of cells which are either genetically identical or very closely related can be different and this difference can be propagated over at least three generations. Since the proliferation rate in yeast is determined by growth rate, these observed differences provide an additional source of cell cycle variability for yeast cells that has not been considered before. The implications for the theoretical analysis of cell cycle kinetics are examined.

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Administration of butylated hydroxyanisole (BHA) orally at either 0.5 g or 1 g/kg daily for 14 days to rats did not produce any DNA adducts in the forestomach as measured by the 32P-postlabeling method using (1) limiting concentrations of 32P-ATP; (2) nuclease P1 enhancement; or (3) butanol extraction. Experiments were conducted to establish the effects of BHA administration on aristolochic acid (AA) DNA adduct formation in the forestomach and liver, when BHA was administered prior to, together with or after AA administration. Adduct levels per 10(9) nucleotides in the liver after oral dosing daily for 5 days with 1 mg/kg AA and BHA (1 g/kg) or corn oil (5 ml/kg) for 7 days were as follows: (a) BHA and AA given simultaneously; 235 +/- 71, (b) AA + corn oil; 63 +/- 39, (c) AA followed by BHA; 57 +/- 13, (d) AA followed by corn oil; 91 +/- 38, (e) BHA followed by AA; 90 +/- 12, (f) corn oil followed by AA; 83 +/- 24. For the forestomach the values were: (a) 236 +/- 86, (b) 77 +/- 25, (c) 367 +/- 97, (d) 296 +/- 47, (e) 217 +/- 81, (f) 70 +/- 64. These data suggest that BHA could have an enhancing effect on AA-induced lesions in the forestomach if dosed together with, or prior to, AA as adduct levels are significantly higher than in controls.

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A cDNA library has been constructed from the poly(A)+ mRNA of oestrogen-stimulated ZR-75-1 human breast cancer cells. Screening by differential hybridization has identified eight clones which are stimulated between 4- and 16-fold by oestrogen. Two clones (pLIV-1) that are stimulated 4-fold, hybridize to three different mRNA species. A further five recombinants encode for a mRNA 600 bp long which is induced greater than 16-fold and have been shown to cross-hybridize to the oestrogen-responsive clone, pS2, isolated from the MCF-7 breast cancer cell line. Oestradiol was shown to be without detectable effect upon the expression of mRNA for dihydrofolate reductase, which is reported to be oestrogen regulated in MCF-7 cells. Actin gene expression is also unresponsive to oestradiol in ZR-75-1 cells. These results suggest that pLIV-1 represents a previously unidentified mRNA that may be involved in the oestrogen-regulated growth of ZR-75-1 human breast cancer cells.

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The control of cell proliferation under steady-state conditions in the budding yeast, Saccharomyces cerevisiae, is well described by either the tandem or sloppy size control models, both of which suggest that differences in cycle time between individual cells or between parents and daughters is largely due to differences in birth size. These models have been investigated further under conditions in which cell size has not been a rate-determining factor for cell cycle initiation. Two approaches have been used. The first involves the growth of cells in low concentrations of hydroxyurea (HU), which has the effect of prolonging the duration of DNA synthesis. This leads to a lengthening of the budded period, which in turn leads to daughter cells being larger at division than the normal cell cycle initiation size of daughters in steady-state populations. The second approach involves the accumulation of cells at the key control point of the cycle, called start, using the pheromone alpha-factor. Since growth is unaffected, all cells eventually become larger than the volume at which they would normally initiate the cell cycle. The kinetics of proliferation were followed after release from alpha-factor arrest. The results from both approaches were broadly consistent with the predictions of both models. However, abolition of birth-size differences between parents and daughters in the presence of HU did not lead to a complete disappearance of differences in either cycle time or proliferation kinetics. Furthermore, following release from alpha-factor arrest, the rate of cell cycle initiation of parent cells was slower than in steady-state culture and the daughters' cells behaved as if comprising two separate populations. These discrepancies suggest that besides a size difference, there are additional physiological differences between parent and daughter cells.

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The kinetics of cell proliferation of Saccharomyces cerevisiae were studied at 4 growth rates using time-lapse cinephotomicrography. Cells were grown on media with a high refractive index to reveal greater intracellular detail under the phase-contrast microscope. The morphological cell-cycle events scored were: bud emergence, nuclear migration, nuclear division, onset of cytokinesis and cell separation. Cell size was measured at cell separation and at bud emergence. The daughter-cycle time was always longer than the parent-cycle time mainly due to the large difference in the lengths of the unbudded phases. Parent cells had a shorter budded period than daughter cells. The large variance in daughter-cycle times was accounted for by the large variance in the lengths of the unbudded phase of daughter cells. The duration and variability of the periods in the cyclc from nuclear migration onwards were equivalent for parent and daughter cells. Daughter cells were always smaller than parent cells at division. There was wide variation in cell size at both division and bud emergence. The results indicated that a modified deterministic model could best explain cell proliferation kinetics in yeast. The data were used to evaluate 2 different models. The 'sloppy size control' model of Wheals (1981 a) was more consistent with the data than the 'tandem' model of Shilo, Shilo & Simchen (1976). The distribution of unbudded periods of daughter cells suggested that there was an additional incompressible period not present in parent cells.

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The unequal division model proposed for budding yeast (L. H. Hartwell and M. W. Unger, J. Cell Biol. 75:422-435, 1977) was tested by bud scar analyses of steady-state exponential batch cultures of Saccharomyces cerevisiae growing at 30 degrees C at 19 different rates, which were obtained by altering the carbon source. The analyses involved counting the number of bud scars, determining the presence or absence of buds on at least 1,000 cells, and independently measuring the doubling times (gamma) by cell number increase. A number of assumptions in the model were tested and found to be in good agreement with the model. Maximum likelihood estimates of daughter cycle time (D), parent cycle time (P), and the budded phase (B) were obtained, and we concluded that asymmetrical division occurred at all growth rates tested (gamma, 75 to 250 min). D, P, and B are all linearly related to gamma, and D, P, and gamma converge to equality (symmetrical division) at gamma = 65 min. Expressions for the genealogical age distribution for asymmetrically dividing yeast cells were derived. The fraction of daughter cells in steady-state populations is e-alpha P, and the fraction of parent cells of age n (where n is the number of buds that a cell has produced) is (e-alpha P)n-1(1-e-alpha P)2, where alpha = IN2/gamma; thus, the distribution changes with growth rate. The frequency of cells with different numbers of bud scars (i.e., different genealogical ages) was determined for all growth rates, and the observed distribution changed with the growth rate in the manner predicted. In this haploid strain new buds formed adjacent to the previous buds in a regular pattern, but at slower growth rates the pattern was more irregular. The median volume of the cells and the volume at start in the cell cycle both increased at faster growth rates. The implications of these findings for the control of the cell cycle are discussed.

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The mean size and percentage of budded cells of a wild-type haploid strain of Saccharomyces cerevisiae grown in batch culture over a wide range of doubling times (tau) have been measured using microscopic measurements and a particle size analyzer. Mean size increased over a 2.5-fold range with increasing growth rate (from tau = 450 min to tau = 75 min). Mean size is principally a function of growth rate and not of a particular carbon source. The duration of the budded phase increased at slow growth rates according to the empirical equation, budded phase = 0.5 tau + 27 (all in minutes). Using a recent model of the cell cycle in which division is thought to be asymmetric, equations have been derived for mean cell age and mean cell volume. The data are consistent with the notion that initiation of the cell cycle occurs at "start" after attainment of a critical cell size, and this size is dependent on growth rate, being, at slow growth rates, 63% of the volume of fast growth rates. Previous reports are reanalyzed in the light of the unequal division model and associated population equations.

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