Ece Konac

Single Nucleotide Polymorphisms in the Hypoxia-inducible Factor-1α (HIF-1α) Gene in Human Sporadic Breast Cancer

BACKGROUND DNA sequence variations in hypoxia-inducible factor-1alpha (HIF-1alpha) gene, which have been demonstrated to be correlated with tumor angiogenesis, may yield changes both in the production outcomes and in the activities of the gene. In this study, we investigated the relationship between three single nucleotide polymorphisms (SNPs) [C1772T and G1790A in exon 12 and C111A in exon 2 of the HIF-1alpha gene] in the HIF-1alpha gene coding regions and development of sporadic breast cancer in the Turkish population. These three polymorphisms result in an amino acid change from proline 582 to serine, from alanine 588 to threonine and from serine 28 to tyrosine, respectively. METHODS Genomic DNA was isolated from 102 sporadic breast cancer patients and 102 healthy female controls. All three HIF-1alpha gene regions were amplified by PCR, and genotypes were determined by RFLP and DNA sequencing. RESULTS There were no significant differences between patients and controls in terms of the distribution of C1772T and G1790A polymorphisms of HIF-1 gene (p >0.05). As for HIF-1alpha C111A polymorphism, we did not find CA and AA variants of the gene in either controls or patients. Multivariable logistic regression analysis was performed between CC and CT + TT genotypes of C1772T polymorphism. No significant differences were found between these two genotypes in terms of clinicopathological characteristics of the patients including age at enrollment, age at menarche and first delivery, number of full-term pregnancies, body mass index, use of oral contraceptives and postmenopausal hormones, family history of breast and ovarian cancers, menopausal status, histopathological features, oophorectomy, smoking habits, and alcohol consumption (p >0.05). CONCLUSIONS Our results suggest that none of the polymorphisms studied in the HIF-1alpha gene influence susceptibility to sporadic breast cancer. The present study is the first case-control study that investigates the association of HIF-1alpha polymorphisms with sporadic breast cancer in the Turkish population.

Association of Genetic Polymorphisms in Vitamin D Receptor Gene and Susceptibility to Sporadic Prostate Cancer

Genetic and environmental factors are involved in prostate cancer (PCa) etiology. Single nucleotide polymorphisms (SNPs) may contribute to the PCa pathogenesis. The goal of this study is to determine the role of vitamin D receptor (VDR) gene polymorphisms and haplotypes in the development and progression of sporadic PCa. One hundred and thirty-three PCa patients and 157 age-matched healthy controls were genotyped for the Apa I (rs7975232), Bsm I (rs1544410) and Taq I (rs731236) polymorphisms in VDR gene by using polymerase chain reaction-restriction fragment length polymorphism. An association was observed between the Apa I polymorphism and PCa predisposition (P = 0.03). When compared with AA genotype, there was a highly notable difference in the frequencies of the Aa (P = 0.02), aa (P = 0.026) and Apa I ‘‘a’’ allele carriers (Aa + aa) (P = 0.009) genotypes. Furthermore, we found a statistical difference in the allele frequencies of the Apa I polymorphism between the sporadic PCa patients and control subjects (P = 0.013). The genotype distribution for the Bsm I and Taq I polymorphisms were similar between cases and controls (P > 0.05). No clinically significant relationship was found between the three-locus haplotypes and development of sporadic PCa. The genotype frequencies for the three polymorphisms of the VDR gene within subgroups of PCa (defined by tumor stage, Gleason score, PSA levels) were also analyzed, but no statistically noteworthy difference was observed (P > 0.05). As far as we know, this is the first study which investigates the relationship between VDR genotypes and sporadic PCa in the Turkish population. Our findings suggest that the VDR ApaI (rs7975232) polymorphism may play a role in the development of sporadic PCa.

Genetic Variations in the Hypoxia-Inducible Factor-1αGene and Lung Cancer

Hypoxia-inducible factor-1 (HIF-1), an important genetic component of angiogenesis, becomes stable as a response to tumor hypoxia and facilitates tumor survival. The polymorphisms of the HIF-1αgene may cause changes in the activity of this protein, which serves as a transcription factor for many genes in tumorigenesis. In this study, we have investigated the relationship between seven HIF-1αpolymorphisms [C > T substitution in intron 8 (rs10873142), T418I (rs41508050) in exon 10, P564P (rs41492849), L580L (rs34005929), P582S (rs11549465), A588T (rs11549467) in exon 12 and dinucleotide GT repeats in intron 13 (rs10645014)] among lung cancer patients in the Turkish population. Genomic DNA was isolated from 141 lung cancer cases and 156 controls and subjected to PCR for amplification. Genotyping was carried out with RFLP and DNA sequencing methods. There was no significant difference between the lung cancer cases and controls in terms of the distribution of genotyping frequencies of seven HIF-1αpolymorphisms (P > 0.05). No significant relationship was found between the C > T substitution in intron 8 and P582S haplotypes and development of lung cancer. In addition, there were no significant associations between the genotypes and clinopathological characteristics of the cases examined. These findings show that polymorphisms in the HIF-1αgene do not confer susceptibility to lung cancer. Exp Biol Med 234:1109–1116, 2009

Endometrial mRNA expression of matrix metalloproteinases, their tissue inhibitors and cell adhesion molecules in unexplained infertility and implantation failure patients

The aim of this study was to analyse whether some cases of unexplained infertility and implantation failure after IVF could be explained by different expression levels of the matrix metalloproteinases (MMP-2, 9), their tissue inhibitors (TIMP-2, 3) and intercellular (ICAM-1) and vascular (VCAM-1) adhesion molecules in endothelial cells. Total RNA was extracted from the endometrial tissues of 41 women (unexplained infertile, group 1, n = 15; fertile volunteers, group 2, n = 15 and patients with implantation failure after IVF, group 3, n = 11). MMP-2, MMP-9, TIMP-2, TIMP-3, ICAM-1 and VCAM-1 mRNA expression levels were measured quantitatively using real-time polymerase chain reaction. In the endometrium from women with unexplained infertility and implantation failure after IVF, MMP-2 and TIMP-3 expression were significantly decreased when compared with the fertile group (P < 0.05 and P </= 0.001 respectively). In addition, a marked decrease was observed in the expression of VCAM-1 in women with unexplained infertility. These results suggest that the expression of gelatinase A (MMP-2), TIMP-3 and VCAM-1, at least at the transcriptional level, might be regulated by common factors and signalling pathways. The present study adds new and important data in this field and highlights the complex preparation of the endometrium for implantation at the molecular level.

Effects of AG490 and S3I-201 on regulation of the JAK/STAT3 signaling pathway in relation to angiogenesis in TRAIL-resistant prostate cancer cells in vitro

The aim of the present study was to analyze the molecular mechanisms involved in blocking the signaling pathway and the effects of this on the progression of prostate cancer (CaP) cells in vitro. LNCaP human CaP cell line was stimulated with interleukin-6 (IL-6) in the presence/absence of Janus kinase (JAK) 2 (AG490), signal transducer and activator of transcription 3 [(STAT3) S3I-201] inhibitors and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Cytotoxic activity, the activation of phosphorylated (p)-STAT3 protein, caspase (CASP) 3 activity at protein level, vascular endothelial growth factor (VEGF) A, VEGFC, vascular endothelial growth factor receptor 2, STAT3, matrix metalloproteinase-2, myeloid cell leukemia sequence 1 (MCL-1), CASP8 and CASP9 messenger RNA (mRNA) levels were determined. Morphology and apoptosis were confirmed by DAPI staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. IL-6 rapidly induced the phosphorylation of STAT3 in a dose- and time-dependent manner with a peak expression at 3 h at a concentration of 25 ng/ml. In addition, AG490 (50 μM) and S3I-201 (300 μM) inhibited STAT3 activation. Western blotting results revealed that p-STAT3 protein expression decreased significantly with AG490 and S3I-201 treatment in LNCaP cells. AG490 and S3I-201 induced the downregulation of VEGFA, MCL-1 and STAT3 and the upregulation of CASP8 and CASP9 mRNA transcription levels. In addition, the inhibitors increased the level of CASP3 protein. Combinations of AG490- and S3I-201-TRAIL did not result in an increase in this effect. Parallel results were found by DAPI staining and TUNEL assay. To the best of our knowledge, this is the first study to investigate the possible clinical use of AG490 or S3I-201, together with the reduced use of chemotherapeutic agents with high cytotoxicity, for their ability to exert an apoptotic effect, targeting the JAK/STAT3 pathway.

The realm of microRNAs in cancers

MicroRNAs (miRNAs) are members of the non-protein coding RNA family. miRNAs, which can regulate genes on transcriptomic level through either degrading the target messenger RNA (mRNA) or suppressing the protein synthesis, also take part in a number of biological functions that involve development, differentiation, proliferation and apoptosis. The mutations and polymorphisms in the expression levels of miRNA genes or alterations in their epigenetic mechanisms may play their roles in the formation of malignancies. Increasing evidence shows that aberrant miRNA expression profiles are present in a variety of cancers. Therefore, it has been suggested that these profiles could be useful for diagnosis and classification of different tumor types and that these small RNAs might provide significant opportunities for the development of future miRNA-based therapies. In this review, we aimed to look into the realm of miRNAs, which is a recent area of research, appraise their biological activities on molecular level and their probable benefits on clinical practice.

Apoptosis and nitric oxide release induced by thalidomide, gossypol and dexamethasone in cultured human chronic myelogenous leukemic K‐562 cells

The effects of different dosages of thalidomide, gossypol and dexamethasone on the levels of apoptosis and nitric oxide (NO) production were studied in a human chronic myelogenous leukemic cell line, K‐562. Increases in the levels of apoptosis were induced by both 15 and 30 μM of thalidomide but only 15 μM significantly increased NO production. All dosages of gossypol used increased the production of NO and all dosages except 200 μM raised the level of apoptosis. After dexamethasone treatment the level of NO either decreased or stayed constant. Thus, some dosages of thalidomide and gossypol concomitantly raise the levels of apoptosis and NO production, but dexamethasone, though it induced apoptosis, had no significant effect on NO production.

Expression of the imprinted IGF2 and H19 genes in the endometrium of cases with unexplained infertility

OBJECTIVE As genomic imprinting plays a critical role in the development of the placenta, the aim of this study was to detect whether the expression levels of the imprinted genes IGF2 and H19 in the endometrium differ between infertile and fertile women. STUDY DESIGN Total RNA was extracted from 30 (15 unexplained infertile and 15 fertile) womens endometrial tissue. cDNA was synthesized from total RNAs of each sample. IGF2 and H19 mRNA expression levels were measured quantitatively using the Real Time PCR method. In order to determine the allelic expression of IGF2 and H19, genomic DNA was extracted from endometrial tissues. RESULTS When compared with the control group, increased mRNA expression of IGF2 was detected (1.5-fold change, P=0.015) in the unexplained infertility group. In contrast, H19 expression was lower in the infertility group as compared to the control group (4-fold change, P<0.0001). Restriction analysis of cDNA-derived PCR product showed that all patients and controls indicated monoallelic expression of IGF2 and H19. CONCLUSION Our results showed that altered expression of these imprinted genes might affect implantation and that their timely and appropriate activation is important for proper functioning. To understand the molecular epigenetic basis of implantation and placental development, genomic imprinted genes should be further investigated.

Is cytogenetic diagnosis of 46,XX karyotype spontaneous abortion specimens erroneous? Fluorescence in situ hybridization as a confirmatory technique.

Aim: Performing the standard cytogenetic technique on spontaneous abortion material is still a valuable tool, but finding a normal 46,XX karyotype can confuse investigators and lead to a problem in diagnosis. This is mainly because it is possible for the female or male conceptus to retain contaminating maternal cells. To address this possibility, we used fluorescence in situ hybridization technique (FISH). X (DXZ1: p11.1‐q11.1 region) and Y (DYZ3: p11.1‐q11.1 region) chromosome α‐satellite probes were employed to confirm the karyotypes previously diagnosed as 46,XX by our cytogenetic laboratory, or to verify the occurrence of ‘Y chromosome component’.

DNA methyltransferase inhibitor-mediated apoptosis in the Wnt/β-catenin signal pathway in a renal cell carcinoma cell line.

The Wnt signaling pathway is activated in most cancer types when Wnt antagonist genes are inactivated. Glycogen synthase kinase 3 (GSK3β) is an important regulator of the Wnt/β-catenin signaling pathway. The mechanisms underlying GSK3β regulation of neoplastic transformation and tumor development are unclear. Studies have raised the possibility that the Wnt signaling pathway may be implicated in renal cell carcinoma (RCC). Therefore, in the present study, we hypothesize that the expression and methylation status of the secreted frizzled-related protein 2 (sFRP2) gene, one of the secreted antagonists that bind Wnt protein, and re-expression of this gene with the demethylation agent (5-aza-2′-deoxycytidine; DAC) may induce apoptosis in RCC cells. To test this hypothesis, we investigated the relationship among epigenetic inactivation of sFRP2 and p-GSK3β (Ser9) and other Wnt antagonists (sFRP1, DKK3, WIF-1) and apoptotic factors (Bax and Caspase3) as well as the anti-apoptotic factor BCL2. Our results indicate that DAC-mediated inhibition of DNA methylation led to a re-activation of sFRP2 expression and increased expression levels of the Wnt antagonists and apoptotic factors. In contrast, the level of β-catenin (CTNNB1) expression decreased. The p-GSK3β (Ser9) protein level in Caki-2 cells was significantly down-regulated, while the DNA fragmentation rate increased after treatment with 5 μM DAC at 96 h. Our data show that sFRP2 functions as a tumor suppressor gene in RCC and that its restoration may offer a new therapeutic approach for the treatment of RCC. Moreover, our study draws attention to the regulatory features of epigenetic molecules and analyses their underlying molecular mechanisms of action and their potential use in clinical practice.