Assignment of Chinese hamster p53 gene (TP53) to chromosome band 2p31, a region not involved in the karyotypic changes of a tumorigenic cell line.

The Chinese hamster tumor-derived cell line 835T2 exhibits specific karyotypic changes, including the loss and/or translocation of genetic material. To investigate whether the p53 tumor suppressor gene was involved in the exchanges, cDNA from primary Chinese hamster cells was isolated by using sense and antisense primers of the human p53 gene. The cDNA was sequenced, and the sequence was compared with the Syrian and human p53 cDNA reported sequences. The sequence homology was very elevated, demonstrating that the cloned fragment contained part of the Chinese hamster p53 gene. The corresponding genomic fragment was also cloned and used as a biotin-labeled probe for in situ hybridization on Chinese hamster chromosome spreads. Hybridization was visualized by avidin-FITC, and the assignment was done comparing the banding obtained with BamHI restriction enzyme and the location of the fluorescent signals pattern of the same metaphase. The signals revealed that the p53 gene (TP53) is localized on Chinese hamster chromosome band 2p31, which is not involved in the karyotypic changes specific to the 835T2 cell line.

Absence of UV-induced non-homologous recombination in repair-deficient CHO cell lines transfected with ERCC genes.

The nucleotide excision repair pathway removes a broad spectrum of DNA lesions, including UV-induced damage. To ascertain whether the repair of the latter has a causative role in the enhancement of non-homologous recombination, Chinese hamster CHO cell lines proficient and deficient in the ability to repair UV-induced damage were transfected with a plasmid containing the bacterial neoR gene. Following UV-treatment an enhancement of non-homologous recombination above the spontaneous level was observed in repair-proficient cells, whereas no increase was observed in repair-deficient cell lines. Hence, the latter were transfected with the corresponding excision repair cross complementing human genes and the resulting repair-proficient transfectants were tested for UV-induced non-homologous recombination. In both untreated and UV-treated transfectants, the frequencies of the event were not significantly different. Cumulatively, the results suggest that non-homologous recombination induced by UV-irradiation is not restored by the correction of the excision repair defect.

dCTP misincorporation in a Chinese hamster mutator phenotype: the role of GGA genetic context.

Clone CSA7 is a CHEF18 hamster cell line that shows an increased intracellular accumulation of dCTP. To localize the mutations that accumulate spontaneously in a functional gene of such a mutator phenotype, independent CSA7 mutants of the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene were isolated and screened by a polymerase chain reaction-single strand conformation polymorphism technique. Sixty-two percent of mutants produced detectable changes of the strand migration profile and the mutations were preferentially localized in the exons 3 (31%) and 6 (62%). The sequencing of such exons revealed that the rate of C base incorporation was the major mutation pathway and that the A base of a GGA sequence was the preferential site of misincorporation.

Prevalence of hepatitis C virus genotypes in Italy.

Hepatitis C viruses (HCV) present in 110 Italian patients were characterized by genotype-specific PCRs. Among the 65 cases of community-acquired hepatitis, HCV genotype II was dominant (60%), followed by genotypes IV (15%), III (11%), and I (3%). Among the 45 hemophilia-associated cases, the distribution of the four HCV genotypes was markedly different: genotype I was the most prevalent (61%), followed by genotypes II (25%), III (4%), and IV (2%). Double infections were observed in eight patients. Two HCV remained unclassified. For the 45 community-acquired cases from which a liver biopsy was available, genotype II was associated with more severe liver damage than the other types.

Gene amplification and gene deletion are induced at different rates in V79/AP4 Chinese hamster cells.

The effect of mitomycin C (MMC) treatment on gene amplification and gene deletion induction in a V79/AP4 Chinese hamster cell line was investigated. Spontaneous and induced cellular variants resistant to N-phosphonacetyl-L-aspartate (PALA), which selects for carbamyl-P-synthetase, aspartate transcarbamylase and dihydroorotase (CAD) gene amplification events, and to intermediate concentrations of 2,6-diaminopurine (DAP), which selects for adenine phosphoribosyl transferase (aprt) gene deletion events, were isolated. The molecular analysis of spontaneous and induced PALA- and DAP-resistant clones showed that while in the former the CAD gene was amplified, in the latter both mutations and deletions occurred at the aprt gene. These results indicate that MMC favored the rise of gene amplification rather than gene deletion events, and suggest that aberrant DNA replication processes were responsible for the observed gene amplification.

Analysis of electroporation-induced genetic damages in V79/AP4 Chinese hamster cells.

Electroporation is a recent technique used to introduce exogenous DNA into eukaryotic cells. It is important to establish that the gene of interest is transferred into a functional, non-mutated recipient cell. V79/AP4 Chinese hamster cells were exposed to high-voltage pulsed electric fields and some biological and genetic effects were measured. The results showed that cytotoxicity was related in a dose-dependent manner to the number of applied pulses. Thioguanine-resistant colony-forming cells as well as chromosomal aberrations were also induced whereas ouabain resistants and sister-chromatid exchanges were not or slightly induced. Spontaneous and electroporation-induced clones that were phenotypically TGR/HATS were used to investigate the hprt locus. Molecular screening of the locus showed that the number of deleted exons was significantly higher in induced than in spontaneous TG-resistant clones, suggesting that the genetic damages induced by electroporation concern the loss of regions well over the size of the hprt locus.

Accumulation of anchorage independent cells showing amplified genes (CAD) during the in vitro propagation of CHEF18 Chinese hamster cells.

Anchorage independence and gene amplification have frequently been associated with a transformed or tumorigenic phenotype in cultured mammalian cells. However, it is unknown whether these two traits occur as related events during transformation, or are independent features of the transformed phenotype. To clarify this point, immortalized, untransformed CHEF18 Chinese hamster cells were propagated in culture until they became transformed and tumorigenic. The frequencies with which CHEF18 cells formed colonies either in soft agar, in medium containing N-phosphonacetyl-L-aspartate or in the two selective media simultaneously, were determined. The results indicate that anchorage independence and CAD gene amplification spontaneously arose during the propagation of the cells and that their concurrent emergence was not the consequence of independent events. However, the kinetics of their appearance suggests that anchorage independence is the early event whereas gene amplification might represent one of the numerous events which can be dynamically selected in anchorage-independent cells.

Specific chromosomal aberrations correlated to transformation in Chinese hamster cells.

Cytogenetic changes were investigated during the spontaneous progression of CHEF18 Chinese hamster cells towards tumorigenicity. We further report the chromosomal characterization of a series of spontaneous anchorage-independent clones, as well as of a series of tumor-derived cell lines resulting from injection of late passage cells in nude mice. The high karyotypic homogeneity (presence of four marker chromosomes strictly associated in all the metaphases analyzed) in all clones and tumor-derived cell lines prompted us to alter the specific pattern of chromosomal aberrations in order to identify which if any of the aberrations were more strictly related to transformation. For this purpose we treated a tumor-derived cell line with Colcemid and analyzed the reversion of anchorage-independent phenotype in the subclones showing an altered association of the four marker chromosomes. We conclude that two of four marker chromosomes contribute to anchorage independence.

Responsiveness of tumorigenic and non-tumorigenic CHEF18 Chinese hamster cells to 1-beta-D-arabinofuranosylcytosine treatment.

In cultured mammalian cells, sister chromatid exchanges are easily induced by agents that perturb the scheduled timing of DNA replication. In this work a blockage of DNA synthesis induced by 1-beta-D-arabinofuranosylcytosine was applied to non-tumorigenic and tumorigenic CHEF18 Chinese hamster cells, and their responsiveness was compared. The data show that both the induction of sister chromatid exchanges and the reduction of the colony-forming ability were less extensive in non-tumorigenic than in tumorigenic CHEF18 cells. The results suggest that a tight control of the scheduled timing of DNA replication is present in non-tumorigenic CHEF18 cells and perhaps this feature avoids the generation of those chromosomal structures that are responsible for the abnormal induction of sister chromatid exchanges and for the elevated cytotoxicity seen in tumorigenic cells.

Reduction of proliferative heterogeneity of CHEF18 Chinese hamster cell line during the progression toward tumorigenicity.

Doubling time and generation time represent two parameters by which the proliferation of cultured mammalian cells can be monitored. In this study we report the characterization of CHEF18 Chinese hamster cell line during the progression toward tumorigenicity by analysis of doubling time and generation time. The two parameters reveal that the proliferation was initially different, indicating the presence of a proliferative heterogeneity among the cycling cells. The progressive reduction up to the disappearance of this discrepancy suggests that a modification of the length of some phases of the cell cycle may have occurred during the progression toward tumorigenicity. However, the hypothesis that the shortening of doubling time might be due to a continuous coming out of cells from the cell cycle rather than to a shortening of the cell cycle is presented.

Folate-sensitive fragile sites in Chinese hamster cell lines.

The expression of fragile sites in three different Chinese hamster cell lines was studied. Results showed that folate-sensitive fragile sites were expressed in the pericentromeric regions of chromosomes 1, 3, 4, 6, and 7 and in band 1q22. A comparison of the breakpoints involved in formation of chromosome rearrangements in some established Chinese hamster cell lines was also made. Results showed that while the specific type of rearrangement was random, the breakpoints were not. Three of the chromosomal sites most frequently involved in breaks were regions in which fragile sites were expressed.