Association of p53 polymorphisms with breast cancer: a case-control study in Slovak population.

Protein p53 is the tumor suppressor involved in cell cycle control and apoptosis. As a transcription factor p53 controls many cell processes and helps in prevention of cancer development. The p53 gene is polymorphic. Polymorphisms can affect the important regions involved in protein tumor suppressor activity. The well-known polymorphisms are the polymorphisms BstUI in exon 4 and MspI in intron 6. Both are supposed to be associated with cancer development. The purpose of this study was to investigate the genotype frequencies and associations of these polymorphisms with breast cancer in Slovak population. We observed the prevalence of BstUIPro (27.47%) and MspIA1 (17.58%) alleles and BstUIPro/Pro (8.79%) and MspIA1/A1 (5.49%) genotypes in breast cancer patients in comparison with controls 23.40%, 14.10%, 5.77%, 1.92% respectively. However the differences were not significant. After division of the cases and controls according to the age the prevalence of the risk alleles and genotypes in women at the age 50 years or less was higher as compared to women older than 50 years. In the younger women group, the p53 BstUI polymorphism genotype frequencies were 6.2% for BstUIPro/Pro, 31.0% for BstUIArg/Pro and 62.8% for BstUIArg/Arg in controls and 11.11 %, 40.74% and 48.15% in cases respectively. The risk of disease for BstUIPro/Pro genotype was more than two-fold higher in comparison with the BstUIArg/Arg (OR=2.34, 95% CI=0.53-10.24). In p53 MspI the genotype frequencies were 1.77% for MspIA1/A1, 24.78% for MspIA1/A2 and 73.45% for MspIA2/A2 in controls and 11.11%, 18.52% and 70.37% in cases respectively. The risk of disease for MspIA1/A1 genotype was more than six-fold higher in comparison with the MspIA2/A2 (OR=6.55, 95% CI=1.02-41.98). When we evaluated the association of both polymorphisms together with the breast cancer risk we observed that the highest risk was connected with the genotype BstUIPro/Pro / MspIA1/A1 (OR=2.99, 95% CI=0.69-13.06). Our results indicate that both BstUI and MspI p53 polymormphisms might play the role in the breast cancer development especially in women younger than 50 years.

A new extremely large allele at the D1S80 (MCT118) locus.

A new extremely large allele at locus D1S80, segregating in a three-generation family is described. The length of PCR-generated allele is approximately 1000 bp. Restriction analysis indicates that this increase is due to an increased number of basic core sequence. The assessed number of tandem repeats is in range 52-55, corresponding to 979-1027 bp exact length of the PCR-generated fragment.

PKU in Slovakia: mutation screening and haplotype analysis.

The restriction fragment length polymorphism haplotypes and seven common mutations in the phenylalanine hydroxylase gene were analysed in 49 unrelated Slovak phenylketonuria (PKU) families of Caucasian origin. The predominant mutation in this population sample is R408W, with a frequency of 45.9%. In addition, four other mutations have been identified at relatively high frequencies: IVS12nt1, 10.2%; R158Q, 7.1%; R261Q, 7.1%; R252W, 2.0%. The mutation-haplotype associations correspond to those described in other European populations. The high proportion of mutations (72.4%) amenable to simple rapid detection based on the polymerase chain reaction provides a good basis for direct DNA-diagnosis of PKU in the Slovak population.

Distribution of ApoBII, MCT118 (D1S80), YNZ22 (D17S30), and COL2A1 Amp-FLPs (amplified fragment length polymorphisms) in Caucasoid population of Slovakia.

Amp-FLPs are simple and rapid tools for genetic characterization of both individuals and populations. This paper presents allele frequencies of four Amp-FLPs (ApoBII, MCT118, YNZ22, and COL2A1) based on the analysis of more than 100 unrelated Caucasoid Slovaks. The proportion of heterozygotes observed and expected, and the probability that two individuals taken at random from the population would be identical in a given polymorphism (PI), was determined for each Amp-FLP.

An evaluation of three pesticides: piritione, supercypermethrin and metolachlor in transformation bioassays of BHK21 and hamster embryo cells.

In a study of potential carcinogenicity of pesticides, Piritione, metolachlor (in the form of Dual and VUCHT 524) and Supercypermethrin (in the form of Supercypermethrin EC and Supercypermethrin TP) were assayed for induction of anchorage independent growth of BHK21 cells and morphological transformation of Syrian hamster embryo cells. The activity of these substances in both transformation assays was compared to the activity of the direct-acting ultimate carcinogen N-methyl-N-nitrosourea. In comparison to the very strong transforming activity of N-methyl-N-nitrosourea all pesticides tested with or without S9 fraction manifested a very weak, weak, medium or strong effect. The ability to induce anchorage independent growth was graded as follows: Dual < Supercypermethrin EC < Supercypermethrin TP < or = Piritione < VUCHT 524. Results of Syrian hamster embryo cell transformation assay were very similar to the BKH21 transformation assay. VUCHT 524 strongly induced transformation whereas Dual was inactive. Piritione and Supecypermethrin EC and Supercypermethrin TP elicited a slight but significant positive response.

Mutagenic and cytotoxic action of heated pork meat extracts in human diploid fibroblasts.

Extracts from lean pork heated at 200 degrees C have a strong mutagenic activity in the Ames Salmonella assay (strain TA98 +S9). The formation of mutagenicity is highly temperature dependent, thus an extract heated at 100 degrees C is not mutagenic in this system. This paper shows that the 200 degrees C extract also causes mutations at the hprt locus in normal human fibroblasts, as demonstrated by a dose-dependent increase of 6-thioguanine resistant mutants. The mutation frequencies were increased 6 and 13 times, respectively, for extract concentrations corresponding to 100 and 200 mg meat/ml medium. Heterocyclic amines, previously shown to be present in the 200 degrees C extract are conceivably responsible for at least part of the observed mutagenicity. The extracts prepared at 100 degrees C had no significant effect on the mutant frequency in human fibroblasts. A pronounced, dose-dependent, decrease in cell survival was observed with both the 100 and the 200 degrees C extracts. The nature of the cytotoxic components is not clear and might be different in the two extracts.

Induction of 6-thioguanine-resistant mutants in human diploid fibroblasts in vitro with ethylene oxide.

Human diploid fibroblasts (strain VH-10) were exposed to the direct-acting alkylating agent, ethylene oxide (EtO), in vitro, and the frequency of HPRT mutants was evaluated by selection in medium containing 6-thioguanine. A dose-dependent increase of the mutant frequency was found in the dose range of 2.5-10 mMh of EtO. The EtO-induced mutant frequency increased 5-19 times the background frequency at low or moderately toxic doses, which indicates that EtO is a strong mutagen in human fibroblasts in vitro. The mutagenic potency was 9.8 x 10(-6) per mMh.

Decemtione (Imidan)-induced single-strand breaks to human DNA, mutations at the hgprt locus of V79 cells, and morphological transformations of embryo cells.

To study the genotoxic activity of Decemtione (Imidan), this substance was subjected to a series of tests. After preliminary cytotoxicity testing, the capacity of Decemtione to damage human DNA was determined by alkaline elution of DNA and DNA unwinding. Both tests gave positive results, suggesting that Decemtione was able to induce single-strand breaks in DNA. This capacity was higher in the absence and lower in the presence of the S9 fraction. The potential mutagenicity of Decemtione was followed on the basis of its ability to induce resistance to 6-thioguanine in V79 hamster cells. Unlike the induction of single-strand breaks, Decemtione showed, in the absence of the metabolic activation system, a very weak mutagenic effect, which was, however, significantly higher in the presence of the S9 fraction. The ability of the substance to transform diploid cells under in vitro conditions was followed on the basis of morphological transformation of Syrian hamster embryo cells. The results showed that Decemtione, like positive carcinogenes, induced a significant elevation in morphologically transformed colonies of embryo cells. The results suggest a carcinogenic potential of this organophosphate insecticide.

Study of the biological effects of theophylline on V79 cells: viability, membrane permeability, and metabolic cooperation.

The effect of theophylline, a specific inhibitor of phosphodiesterase, on gap junction-mediated intercellular communication between Chinese hamster V79 cells was examined. It was found that addition of theophylline to coculture of 6-thioguanine-resistant (TGr) and 6-thioguanine-sensitive (TGs) V79 cells significantly increased the recovery of TGr cells. This finding indicates an inhibition of metabolic cooperation of V79 cells by theophylline. Theophylline was tested at concentrations less than 0.3 mg/ml, which were neither cytotoxic (after short or continuous exposure) nor inhibited the synthesis of DNA, RNA, and proteins. At the tested concentrations, no change was found in the membrane permeability of cells. Theophylline did not increase the incorporation of glucose into the cells.

Inhibitory effect of theophylline on repair of potentially lethal MMS-induced damages to DNA in V79 cells.

In a study of Chinese hamster V79 cells growing in the presence of sublethal concentrations of theophylline, we followed both the nature of DNA replication and the cells' response to toxic and DNA-damaging effects of methyl methanesulfonate (MMS). We found that cells cultured at low concentrations of theophylline (less than or equal to 0.3 mg/ml medium) showed deviations in the rate of DNA replication which, however, did not depress either the growth activity of the cells or their colony-forming ability. Considerable differences as against the controls appear in theophylline-cultured cells after treatment with MMS. Not only are they more sensitive to the toxic effects of this alkylating agent, but also their DNA synthesis is strikingly inhibited. More unrepaired lesions remain in parental DNA, and short fragments of daughter DNA, synthesized following cell treatment with MMS, are not elongated during a 2-h post-MMS treatment. Theophylline obviously belongs among agents inhibiting repair of potentially lethal MMS-induced DNA damages in Chinese hamster V79 cells.