PTEN mutation, expression and LOH at its locus in ovarian carcinomas. Relation to TP53, K-RAS and BRCA1 mutations.

OBJECTIVE: We aimed to evaluate frequency of PTEN mutation, LOH and expression in ovarian tumors. In search for a molecular pathway, we confronted PTEN gene mutations with TP53, K-RAS and BRCA1 gene status in the same tumors. We also evaluated clinical significance of PTEN expression in a subgroup of patients uniformly treated with platinum-based regimens. METHODS: Molecular analysis was performed on 105 ovarian tumors (100 carcinomas) with the use of the SSCP and sequencing. Seventy-six tumors were analyzed for LOH at 10q23 locus with the use of six polymorphic markers. Immunohistochemical PTEN expression was done on paraffin-embedded material. Multivariate and univariate analysis was performed with the STATA program. RESULTS: PTEN mutations occurred in 5/100 (5%) of all carcinomas and in 3/15 (20%) of endometrioid carcinomas (EC). Low-grade EC that developed in borderline tumors had PTEN and/or K-RAS mutation (4/5, 80%), while high-grade EC had TP53 mutations only. There was a reverse association between PTEN and TP53 mutations (P = 0.005). LOH at PTEN locus was found in 60% of endometrioid and in 28% of serous and clear cell carcinomas. PTEN expression did not associate with PTEN mutations or LOH. Strong PTEN expression diminished risk of death in a TP53 positive group only (HR = 0.35, P = 0.029). CONCLUSION: Our results suggest that PTEN mutations may play a role in a development of low-grade endometrioid tumors. PTEN haploinsufficiency caused by LOH or epigenetic events may possibly contribute to development of other histological types and may be an adverse prognostic factor.

Mg2+ does not induce isomerization of the open transcription complex of Escherichia coli RNA polymerase at the model Pa promoter bearing consensus -10 and -35 hexamers.

The kinetics and thermodynamics of the formation of the transcriptional open complex (RPo) by Escherichia coli RNA polymerase at the synthetic Pa promoter bearing consensus -10 and -35 recognition hexamers were studied in vitro. Previously, this promoter was used as a control one in studies on the effect of DNA bending by An x Tn sequences on transcription initiation and shown to be fully functional in E. coli (Lozinski et al., 1991, Nucleic Acids Res. 19, 2947; Lozinski & Wierzchowski, 1996, Acta Biochim. Polon. 43, 265). The data now obtained demonstrate that the mechanism of Pa-RPo formation and dissociation conforms to the three-step reaction model: bind-nucleate-melt, commonly accepted for natural promoters. Measurements of the dissociation rate constant of Pa-RPo as a function of MgCl2 concentration allowed us to determine the number of Mg2+ ions, nMg approximately/= 4, being bound to the RPo in the course of renaturation of the melted DNA region. This number was found constant in the temperature range of 25-37 degrees C, which indicates that under these conditions the complex remaines fully open. This observation, taken together with the recent evidence from KMnO4 footprinting studies that the length of the melted region in Pa-RPo at 37 degrees C is independent of the presence of Mg2+ ions (Lozinski & Wierzchowski, 2001, Acta Biochim. Polon. 48, 495), testifies that binding of Mg2+ to RPo does not induce its further isomerization, which has been postulated for the lambdaP(R)-RPo complex (Suh et al., 1992, Biochemistry 31, 7815; 1993, Science 259, 358).

Chloroacetaldehyde-induced mutagenesis in Escherichia coli: specificity of mutations and modulation by induction of the adaptive response to alkylating agents.

The mutational specificity of chloroacetaldehyde (CAA), one of the metabolites of the human carcinogen vinyl chloride (VC), has been determined through the examination of Arg+ revertants in Escherichia coli AB2497 (Arg-) and identification of their tRNA suppressors. The predominant mutations were GC-->AT transitions (65%) followed by AT-->TA transversions (12.5%). The observed mutational specificity of CAA is very similar to the reported specificity of the other VC metabolite, chloroethylene oxide. The induction of the adaptive response to alkylating agents significantly decreased the frequency of CAA-induced Rifr and Arg+ mutants in E. coli AB2497 and increased the cell survival. Likewise, the adaptation of bacterial cells decreased the frequency of GC-->AT transitions in CAA-treated M13glyU phage transformed to E. coli JC15419 and increased the phage survival. Experiments with strain MS23, which is an alkA mutant deficient in 3-methyladenine-DNA glycosylase II, and with MS23 harboring the pYN1000 plasmid carrying the alkA+ gene, have shown that induction of this repair enzyme is responsible for reduction of the level of CAA-induced mutations. The role of N2,3-ethenoguanine, among the other etheno-adducts, in CAA-induced mutagenesis and as a target for repair in 3-methyladenine-DNA glycosylase II proficient bacterial cells is discussed.