PTEN mutations as predictive marker for the high-grade endometrial cancer development in slovak women.

Endometrial carcinoma (ECa) is one of the most common neoplasia of the female genital tract. The phosphatase and tensin (PTEN) homolog is the most frequently mutated tumor suppressor gene in endometrial carcinoma. PTEN encodes a phosphatase, a key regulatory enzyme involved in a signal transduction pathway that regulates cell growth, migration and apoptosis. The study evaluates an association between the morphological appearance of endometrial hyperplasia and ECa, and the presence of PTEN variations, PTEN protein´s level and intracellular localization. A total of 67 archived formalin-fixed and paraffin-embedded human biopsy tissue specimens with normal proliferative and secretory endometrium, endometrial hyperplasia without atypia and endometrial atypical hyperplasia, endometrioid the grade G1 and G3 and serous subtype of ECa were evaluated by sequencing for the presence of mutations in coding regions of PTEN gene of endometrial epithelial cells. The PTEN gene expression and intercellular localization of PTEN protein were evaluated immunohistochemically by immunoreactive score (IRS). PTEN mutation spectrum in endometrial carcinoma was identified for Slovak population. 28 non-silent mutations were identified in PTEN, twelve of them were novel, not annotated in Catalogue of Somatic Mutations in Cancer. Higher frequency of PTEN mutations was observed in serous carcinoma compared to global average. No correlation was observed between samples´ IRS, PTEN cellular localization and identified mutations. PTEN sequencing can be beneficial for patients considering prognosis of disease and sensitivity to treatment.

Distribution of the most common polymorphisms in TYMS gene in Slavic population of central Europe.

Thymidylate synthetase (TS) plays a critical role in the de novo synthesis of dTMP inside the cell. Therefore, TS is a suitable target for cytotoxic drugs such as fluoropyrimidines. Drug efficacy and toxicity depend on the intracellular level of TS, which is significantly influenced by the polymorphisms in the 5'UTR (TSER - rs45445694, TSER*3G>C - rs2853542) and 3'UTR (1494del TTAAAG - rs151264360) of TYMS gene. Polymorphic variants of TYMS gene affect TS activity via gene expression and transcript stability. Patients who undergo fluoropyrimidine therapy may benefit from genetic testing prior to the administration of chemotherapy. At the 5' terminus of TYMS, there is a polymorphic region represented by a variable number of 28bp long tandem repeats (2-9 tandems) with the G or C nucleotide variant (SNP G>C). The 3'end of TYMS gene may decrease the stability of mRNA in the case of 6 base deletion (1494del6, D). In our study, we have focused on testing of TYMS gene polymorphisms, determination of TYMS variant frequencies in Western Slavic population and comparison of Slovak population with other populations.We performed identification of 5'UTR (rs45445694 - TSER*2 or TSER*3; rs2853542 - TSER*3G>C; TSER*3+ins6) and 3'UTR (rs151264360/1494del6/D) polymorphic regions of TYMS gene among 96 volunteers by PCR-RFLP and fragment analysis. Slovak frequencies of selected polymorphisms were established as follows: the frequency of TSER*2, TSER*3, TSER*3G>C, 1494del6/D and I to be 41%, 59%, 34%, 37.5% and 62.5% respectively. The high resolution of the capillary electrophoresis technique allowed among TSER*3 group identification of a subgroup of four individuals with rare 6bp insertion in 3R allele, id est 2.1% TSER*3+ins6 allele frequency. In our study, we have revealed individuals with rare G>C substitution in the first 28bp tandem repeat of TSER*2 promoter enhancer region (rs183205964) as well, the overall frequency of this polymorphic allele in Slovak population was 2.1%. Our results proved that Slovak population is in Hardy-Weinberg equilibrium and proportion of TYMS polymorphisms is in accordance with other published data.

Treatment of human tumor cells by combine gene therapy harnessing plasmids expressing human tumor necrosis factor alpha and bacterial cytosine deaminase suicide gene.

We have assessed the effect of combine cancer gene therapy with exogenous human tumor necrosis factor alpha (hTNFalpha) and Escherichia coli cytosine deaminase (CD) suicide gene on two human breast adenocarcinoma cell lines MDA-MB-361 and SK-BR-3. Transfection of a plasmid containing hTNFalpha under the control of a hybrid promoter resulted in expression of hTNFalpha gene in vitro. Transduction of retroviral plasmid containing bacterial cytosine deaminase led to the expression of cytosine deaminase in adenocarcinoma cell lines as well. The significant increase in apoptotic cells and decrease of cell proliferation in both tumor cell lines was observed using combination treatment with hTNFalpha expression plus CD/5-FC suicide system. Corresponding data were generated by MTT cell proliferation assay and by flow cytometric analysis. The presence of both genes after transduction of retroviral vector containing CD and transfection of hTNFalpha plasmid was confirmed by PCR and RT-PCR. The additive cell killing effect due to the presence of bacterial CD and hTNFalpha gene after activation of non-toxic prodrug was observed. Whether the bicistronic vector containing both therapeutic genes improve the therapy need to be assessed in the future.

Combine cancer gene therapy harnessing plasmids expressing human tumor necrosis factor alpha and Herpes simplex thymidine kinase suicide gene.

We have assessed the effect of combine cancer gene therapy with exogenous human tumor necrosis factor alpha (hTNFalpha) and suicide gene therapy on three human cancer cell lines MCF-7 (breast adenocarcinoma), U-118MGand 42-MG-BA (human gliomas). Transfection of a plasmid containing hTNFalpha under the control of a hybrid promoter resulted in expression of hTNFalpha gene in vitro. Transduction of retroviral plasmid containing Herpes simplex thymidine kinase (HSVtk) led to the expression of thymidine kinase in all three cell lines. MTT cell proliferation assay and flow cytometric analysis showed a significant increase in apoptotic and necrotic cells and decrease of proliferation in all cell lines after combine therapy with hTNFalpha expression plus thymidine kinase/GCV suicide system. The presence of these two genes after transduction of retroviral vector containing thymidine kinase and hTNFalpha was confirmed by PCR. The expression of HSVtk gene was proved by Western blot analysis, and the expression of both genes was confirmed by RT-PCR. Additive cell killing effect due to presence of HSVtk and hTNFalpha therapeutic genes after activation of non-toxic prodrug was observed. Whether the bicistronic plasmid containing both genes would improve the therapeutic effect need to be assessed in the future.

Tumor targeted gene therapy with plasmid expressing human tumor necrosis factor alpha in vitro and in vivo.

We have assessed the effect of exogenous human tumor necrosis factor alpha (hTNFalpha) in three human cancer cell lines; MDA-MB-361 (breast adenocarcinoma), HCT 116 (colon carcinoma) and 8-MG-BA (glioma). In vitro transfection of a plasmid containing hTNFalpha under the control of a hybrid promoter resulted in expression of hTNFalpha gene in all three cell lines and secretion into the culture medium was seen with MDA-MB-361 cells. Flow cytometric analysis showed a significant increase in apoptotic and necrotic cells in MDA-MB-361 and to a lesser extent in HCT 116 cells. Increased apoptosis was confirmed by an increase in pro-caspase 3 activation. No effects of hTNFalpha expression were seen in 8-MG-BA cells. Intratumoral delivery of the hTNFalpha expression plasmid into MDA-MB-361 tumor xenografts grown in nude mice caused hemorrhagic tumor necrosis. This strategy may be a simple and promising gene therapy approach to the treatment of some human tumors.