Genomic cloning and characterization of the nonoccupied allele corresponding to the integration site of human papillomavirus type 16 DNA in the cervical cancer cell line SiHa.

Human papillomavirus (HPV) type 16 DNA sequences have been found integrated into the host cell genome in a large number of cervical tumors and cell lines derived therefrom. In this study, we have cloned and analyzed the nonoccupied allele corresponding to the integration site of HPV-16 in the cervical cancer cell line SiHa. Our mapping analyses revealed an approximately 7.8-kb deletion of cellular DNA upon viral integration. Computer analysis of 2.3 kb of DNA sequences from the deleted genomic region as well as 1.0 kb of sequences upstream of the viral integration site showed no significant homology to any known human sequences. DNase I mapping experiments on native chromatin demonstrated the existence of two hypersensitive sites in both the HPV-16-containing and nonoccupied alleles located approximately 1.1 and 1.7 kb upstream of the viral integration site. This suggests that viral integration occurred close to putative regulatory sequences and that recombination with host cellular DNA was not followed by a reorganization of the chromatin structure upstream of the integration site. Nuclear run-on and RT-PCR experiments showed HPV-specific transcription spanning the E2, E4, E5, and L1/L2 open reading frames (ORFs) located upstream of the HPV-16 regulatory region (URR). Taken together, our data suggest that the cellular DNA region upstream of the HPV-16 integration site in the SiHa cell line contains regulatory elements affecting transcription of HPV-16 ORFs located upstream of the HPV-16 URR.

Thyroid hormone receptors bind to the promoter of the mouse histone H10 gene and modulate its transcription.

It has been shown that the mouse histone H10 promoter contains a DNA element, composed of a direct repeat of the sequence GGTGACC separated by 7 nt, which is able to bind retinoic acid receptors and to modulate transcription of reporter genes following treatment with retinoic acid. We have now investigated whether this DNA motif is also responsive to thyroid hormone. We co-transfected CV-1 monkey kidney cells with chloramphenicol acetyltransferase (CAT) expression plasmids containing either 740 bp of the H10 wild-type promoter or five copies of the repeat element cloned in front of the thymidine kinase promoter and expression vectors for human thyroid hormone receptors (TRs) alpha or beta and retinoid X receptor alpha (RXR alpha). Treatment of transfected cells with triiodothyronine led to a dose-dependent increase in CAT activity. Transfection experiments with increasing amounts of expression vectors for either TR alpha or RXR alpha resulted in up to 6-fold enhancement of CAT transcription. Furthermore, point mutations within the half-sites of the response element of the H10 promoter, as well as deletions within the interspace region, lowered CAT activity to 60-80% of that of the wild-type control. Electrophoretic mobility shift assays showed that the repeat element was able to form retarded complexes with TR alpha homodimers, as well as with TR alpha-RXR alpha heterodimers. Our results suggest that thyroid hormone receptors are involved in the regulation of mouse histone H10 expression.

The tumour promoters dieldrin and phenobarbital increase the frequency of c-Ha-ras wild-type, but not of c-Ha-ras mutated focal liver lesions in male C3H/He mice.

The frequency and pattern of mutations at codon 61 of the c-Ha-ras protooncogene were analysed in glucose-6-phosphatase-deficient hepatic lesions of male C3H/He mice occurring either spontaneously or after continuous treatment with 10 p.p.m. dieldrin or 500 p.p.m. phenobarbital (PB) in their diet. At 52 weeks after start of promoter administration, enzyme-altered liver lesions had developed in 41% (15/37) of untreated control mice and in 67% (10/15) and 63% (10/16) of mice treated with dieldrin or PB respectively. The average numbers of focal lesions per mouse were 0.57 in the control, 1.5 in the dieldrin and 1.0 in the PB group. Lesions were punched out from frozen liver sections and used for mutation analysis by allele-specific oligonucleotide hybridization following in vitro amplification of DNA via polymerase chain reaction. In the control group, 12 out of 21 liver lesions (57%) showed c-Ha-ras mutations, while five out of 23 (22%) and four out of 16 (25%) lesions were mutated in the dieldrin and PB groups. Taking the different numbers of animals in the three experimental groups into account, our data indicate that the tumour promoters increased the frequency of c-Ha-ras wild-type but not of c-Ha-ras mutated focal liver lesions, suggesting that the mutations had occurred spontaneously and were not related to treatment. Since c-Ha-ras mutations were found to be frequent in large but infrequent in small hepatocellular lesions, these mutations may represent in livers of C3H/He mice an endogenous promoting principle that provides a selective growth advantage to the mutated progenitor cells.

Role of mutations at codon 61 of the c-Ha-ras gene during diethylnitrosamine-induced hepatocarcinogenesis in C3H/He mice.

Liver tumors of certain strains of mice frequently contain mutations at codon 61 of the c-Ha-ras gene. In our study, we investigated the significance of these mutations in the carcinogenic process. Male C3H/He mice received a single injection of diethylnitrosamine (DEN) on day 15 after birth, and groups of animals were killed at various time intervals between 11 and 52 wk after treatment. At the earlier time points (11-29 wk), we analyzed microdissected tissue from precancerous glucose-6-phosphatase-deficient liver lesions larger than approximately 200 microns in diameter, for the presence and pattern of c-Ha-ras codon 61 mutations. In parallel, the growth characteristics of these liver lesions were studied by pulse labeling with [3H]thymidine and by determining the size distribution of the lesions. At the later time points (42-52 wk after DEN treatment), liver tumors were dissected and also analyzed for the presence of c-Ha-ras mutations. We found mutations to be already present in some of the enzyme-altered liver lesions at weeks 11-29, suggesting that the mutations occurred early in the carcinogenic process. Whereas about 10% of the precancerous focal liver lesions showed mutations in the c-Ha-ras gene, the mutation frequency was increased to about 50% in the later-appearing hepatocellular adenomas and carcinomas, suggesting that c-Ha-ras codon 61 mutations may provide a selective advantage to the mutated cell clones.

p53 mutations are absent from carcinogen-induced mouse liver tumors but occur in cell lines established from these tumors.

Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas. We have examined, by single-strand conformation polymorphism analysis of polymerase chain reaction products, a total of 93 carcinogen-induced liver tumors from mice of three different strains (C3H/He, C57BL/6J, and B6C3F1) for the presence of p53 aberrations. Hepatoma lines, established from 12 liver tumors, were also included in the analysis. While structural aberrations of the p53 gene were not detected in any of the primary mouse liver tumors analyzed, single-base substitutions occurred at different locations within the p53 gene in three of the cell lines during in vitro propagation. One hepatoma line carried two point mutations on separate alleles. All four mutations were either G:C----C:G or C:G----G:C transversions. Mutations at codon 61 of the c-Ha-ras gene, which were frequent in primary liver tumors from C3H/He and B6C3F1 mice, were not detected in the hepatoma lines. Our data indicate (i) that c-Ha-ras but not p53 mutations play an important role during the early stages of mouse hepatocarcinogenesis and (ii) that p53 mutations confer a selective growth advantage to the mutated hepatoma cells in vitro.

Mutational activation of the c-Ha-ras gene in liver tumors of different rodent strains: correlation with susceptibility to hepatocarcinogenesis.

The frequency and pattern of mutations at codon 61 of the c-Ha-ras gene have been analyzed in 195 liver tumors and 132 precancerous liver lesions from various rodent strains with differing susceptibility to hepatocarcinogenesis. By using the polymerase chain reaction and allele-specific oligonucleotide hybridization, C----A transversions at the first base and A----T transversions or A----G transitions at the second base of c-Ha-ras codon 61 were detected in 20-60% of spontaneous or carcinogen-induced liver tumors of the C3H/He, CBA, CF1, and B6C3F1 mouse strains, which are highly susceptible to hepatocarcinogenesis. No such mutations, however, could be found in any of the 31 liver tumors of the insensitive C57BL/6J and BALB/c mouse strains or in any of the 35 liver tumors of the comparatively resistant Wistar rat. Further analyses of c-Ha-ras codon 12 mutations in liver tumors from the three insensitive rodent strains also failed to give any positive results. In early precancerous liver lesions, c-Ha-ras codon 61 mutations were found in 13-14% of lesions of the sensitive C3H/He and B6C3F1 mouse strains but not in any of the 34 lesions of the insensitive C57BL/6J mouse. Taken together, our results indicate a close correlation between the mutational activation of the c-Ha-ras gene in liver tumors of the different rodent strains and their susceptibility to hepatocarcinogenesis, whereby the mutations appear to provide a selective growth advantage, leading to a clonal expansion of the mutated liver cell population, only in livers of sensitive but not of insensitive strains.

Mutations in the Ha-ras proto-oncogene in spontaneous and chemically induced liver tumours of the CF1 mouse.

Mutations in codon 61 of the Ha-ras proto-oncogene have been shown to occur with a high frequency in both spontaneous and carcinogen-induced liver tumours of the B6C3F1 mouse. In the present study we analysed the frequency and pattern of Ha-ras mutations in liver tumours in a different strain of mice, namely the CF1 mouse. These liver tumours occurred spontaneously or after administration of either aflatoxin B1, phenobarbital or dieldrin. Mutation analysis was performed by in vitro amplification of DNA using the polymerase chain reaction combined with selective oligonucleotide hybridization. Our results demonstrate the presence of mutations in the Ha-ras gene in liver tumours of the CF1 strain. In total, 6 out of 35 liver tumours of male CF1 mice, two of them occurring after treatment with the tumour promoter phenobarbital solely, contained mutations at either the first or second base of codon 61 of the Ha-ras gene. The types of mutations found in liver tumours of the CF1 mouse were very similar to those described in the B6C3F1 mouse, indicating that mutations in the Ha-ras proto-oncogene may represent a critical event in mouse hepatocarcinogenesis.

Detection of genomic alterations in carcinogen-induced mouse liver tumors by DNA fingerprint analysis.

DNA fingerprint analysis was used to study structural abnormalities in the genome of mouse liver tumor cells. Liver tumors were induced in three strains of mice, namely C57BL/6J, C3H/He and B6C3F1, by a single injection of 20 micrograms/g body wt. diethylnitrosamine on day 15 after birth. DNA from liver tumors was digested with Hinfl restriction enzyme and hybridized on Southern blots with wild-type bacteriophage M13 DNA as probe. The resulting fingerprints of tumor DNA were compared with those of DNA from normal liver tissue. Genomic aberrations were detected in two out of 68 tumors analyzed, one stemming from a C57BL/6J and the other from a C3H/He mouse.

Mutations at codon 61 of the Ha-ras proto-oncogene in precancerous liver lesions of the B6C3F1 mouse.

Liver tumors of the B6C3F1 mouse frequently contain mutations at specific sites of codon 61 of the Ha-ras proto-oncogene. To address whether these mutations occur early or late during carcinogenesis, we analyzed mutations in the Ha-ras gene in small precancerous liver lesions of the B6C3F1 mouse. For this purpose, 10-microns frozen liver sections were prepared and stained for glucose-6-phosphatase activity. Using punching cannuli, we then took small tissue samples of approximately 5-30 micrograms from enzyme-deficient liver lesions and from normal parts of the liver. These tissue samples were analyzed for mutations in the Ha-ras gene by in vitro amplification of DNA via the polymerase chain reaction combined with selective oligonucleotide hybridization. By this approach we were able to analyze mutations in the Ha-ras gene within lesions with diameters of less than 0.5 mm. Our results demonstrate that approximately 15% of the glucose-6-phosphatase-negative lesions that occurred 24-28 wk after a single injection of diethylnitrosamine contain either C----A transversions at the first base or A----G transitions and A----T transversions at the second base of codon 61 of the Ha-ras gene. The same types of mutations, although with a somewhat higher frequency (33%), were found in liver tumors taken 68 wk after diethylnitrosamine treatment. These findings demonstrate that Ha-ras mutations can be detected even in very small precancerous liver lesions, suggesting that these mutations may be an early, perhaps even the first, critical event during murine hepatocarcinogenesis.