Characterization of imatinib-resistant K562 cell line displaying resistance mechanisms.

Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the t(9; 22) and the related oncogene, BCR-ABL. Tyrosine kinase activity of fusion protein BCR-ABL is the main cause of CML. Even if imatinib is used as a tyrosine kinase inhibitor (TKI) for CML therapy, drug resistance may occur in patients and the clinical failure of imatinib treatment in resistant patients had resulted with the use of another alternative TKIs. BCR-ABL dependent and independent molecular mechanisms have crucial roles in drug resistance. To reveal the underlying molecular mechanisms which play significant roles in imatinib resistance in CML, we established K562 imatinib-resistant cell line (K562r5) which was continuously exposed to (5µM) imatinib to investigate molecular mechanisms which play significant roles in drug resistance. First of all, we analyzed T315I, M351T, F315L and F359C/L/V mutations with DNA sequencing as a BCR-ABL dependent mechanism in our cell lines. Moreover, we investigated BCR-ABL independent mechanisms such as apoptosis, autophagy, drug transport and DNA repair which affect drug resistance in these cell lines. In vitro cell viability was determined by MTT assay. DNA sequencing analysis was performed to detect BCR-ABL mutations. The apoptotic effect of imatinib on CML cell lines was tested by flow cytometric Annexin V-PE staining and caspase activation assays. Apoptotic, autophagic, drug transporter and DNA repair genes expression levels were determined by RT-PCR. The conventional cytogenetic analysis was performed on K562s and K562r cells. Our results indicate that inhibition of apoptosis, induction of autophagy, overexpression of efflux gene MDR1 and down-regulation of influx gene OCT1 play crucial roles in the progression of imatinib resistance.

Genetic Polymorphisms in PRM1, PRM2, and YBX2 Genes are Associated with Male Factor Infertility.

AIMS: The etiology of infertility is still unknown in almost half of all male infertility patients. In sperm, DNA condensation differs from somatic and female gamete cells, with the protamine (PRM) gene and its transcription factor, Y-box binding protein 2 (YBX2), playing key roles in making the structure more compact. Protamine polymorphisms have been studied in different populations, but various results have been acquired. MATERIALS AND METHODS: In our study, we examined, for the first time in a Turkish population, the association between protamine gene alleles (PRM1 c.-190C>A, PRM1 c.197G>T, and PRM2 c.248C>T), and YBX2 (c.187T>C and c.1095 + 16A>G) and male infertility. This was accomplished using polymerase chain reaction-restriction fragment length polymorphism analyses of 100 infertile and 100 fertile Turkish men. Sperm DNA fragmentation analysis was performed using the Comet technique. RESULTS: We found that the AA and CA genotypes of the PRM1 c.-190C>A polymorphism had a significant association with infertility (p < 0.001) and the AA genotype was also highly significantly associated with high sperm DNA damage (p < 0.001). CONCLUSION: This study demonstrates that the PRM1 c.-190C>A polymorphism is associated with sperm DNA fragmentation, which may impact male infertility in the Turkish population. Further research with larger groups and in various other study populations will be required to clarify the impact of protamine and YBX2 gene polymorphisms on male infertility.

Impact of follicle-stimulating hormone receptor variants in female infertility.

PURPOSE: Follicle-stimulating hormone (FSH) and its receptor play a major role in the development of follicles and regulation of steroidogenesis in the ovary and spermatogenesis in the testis. We aim to analyze the role of FSHR gene variants (single nucleotide polymorphisms (SNPs) in exon 10 (codon 307 and 680) and in the core promoter region (at position -29) and Ala189Val inactivating mutation) in Turkish infertile women. There were studies analyzing the effects of the SNPs in exon 10 (codon 307 and 680) and in the core promoter region (at position -29) of the FSHR gene on spermatogenesis, but to our knowledge, there were no studies analyzing the effects of these three SNP combinations on female fertility. METHODS: In this study, the allelic, genotype, and haplotype frequency distributions of these three SNPs in the FSHR gene were analyzed in 102 infertile women and 99 unrelated healthy control individuals. The distribution of the polymorphisms was conformed by Hardy-Weinberg equilibrium test. RESULTS: There were no statistical differences (P > 0.05) in the allele, genotype, and haplotype frequencies of the polymorphisms and FSH, luteinizing hormone (LH), estradiol (E2), and prolactin (PRL) levels between the infertile patients and the controls. However, a significant relation was found between 307 SNP GA genotype and FSH level ≥12. We did not find any homozygous or heterozygote mutations in infertile patients and healthy fertile controls. CONCLUSION: The present study was the first study analyzing gma mutation and the polymorphism of the FSHR core promoter at position -29 alone and in combination with the two common SNPs in exon 10 in Turkish infertile women population. These findings indicate the significance of Ala307Thr GA genotype may be a predictive marker for poor ovarian reserve and infertility.

MDR1 gene polymorphisms may be associated with Behçet's disease and its colchicum treatment response.

Behçet's disease (BD) is a chronic multisystem disorder. Infectious agents, immune system mechanisms, and genetic factors are implicated in the etiopathogenesis of BD, which remains to be explained. The human MDR1 (ABCB1) gene encoder P-glycoprotein (P-gp) plays a key role in drug disposition, serves as a protective mechanism against xenobiotics, and provides additional protection for the brain, testis, and fetus. We investigated the genotype and haplotype distributions of three MDR1 gene polymorphisms (C1236T, G2677T/A, and C3435T) in 104 BD patients and 130 control subjects. The genotyping analysis was performed by using PCR-RFLP methods. No statistically significant differences were found for the genotypic and allelic distributions of three individual single nucleotide polymorphisms (SNPs) in the MDR1 gene between BD patients and control subjects in this study (p>0.05). However, combined genotype and haplotype frequencies have found statistically significant differences between BD and control subjects for some combinations (p<0.05). The CC-GG binary genotype for C1236T-G2677T/A loci couple in particular may have a high degree of predisposition to BD (p=0.009; OR, 3.03; 95% CI, 1.41-6.54). Furthermore, significant differences between colchicine-responsive and -nonresponsive groups were found. Genotypic and allelic distributions of C3435T and G2677T/A loci, as well as their genotype and haplotype combinations, were found to have statistically significant differences (p<0.05). The TT genotype for the C3435T locus (p=0.001; OR, 6.59; 95% CI, 1.86-23.30) and T allele (p=0.009; OR, 2.09; 95% CI, 1.18-3.70) plays a substantial role in the colchicine response. Our study showed that MDR1 genes and their polymorphisms may affect a patient's BD susceptibility and colchicine response.

Stem cell mediated cardiovascular repair.

Recent increase in the interest in stem and progenitor cells may be attributed to their behavioural characteristics. A consensus has been reached that embryonic or adult stem cells have therapeutic potential. As cardiovascular health issues are still the major culprits in many developed countries, stem and progenitor cell driven approaches may give the clinicians a new arsenal to tackle many significant health issues. However, stem and progenitor cell mediated cardiovascular regeneration can be achieved via complex and dynamic molecular mechanisms involving a variety of cells, growth factors, cytokines, and genes. Functional contributions of transplanted cells on target organs and their survival are still critical problems waiting to be resolved. Moreover, the regeneration of contracting myocardial tissue has controversial results in human trials. Thus, moderately favourable clinical results should be interpreted carefully. Determining the behavioural programs, genetic and transcriptional control of stem cells, mechanisms that determine cell fate, and functional characteristics are the primary targets. In addition, ensuring the long-term follow-up of cells with efficient imaging techniques in human clinical studies may provide a resurgence of the initial enthusiasm, which has faded over time. Here, we provide a brief historical perspective on stem cell driven cardiac regeneration and discuss cardiac and vascular repair in the context of translational science.

Haplotype-based analysis of MDR1/ABCB1 gene polymorphisms in a Turkish population.

The three functional single-nucleotide polymorphisms (SNPs) of the MDR1 gene, C1236T, G2677T/A, and C3435T, exhibit an interpopulation difference. In this study, we analyzed the haplotype frequencies of these three SNPs in 107 unrelated healthy Turkish subjects and compared them with those of other reported populations. We found that C1236T, G2677T/A, and C3435T SNPs are expected to be structured in 10 different haplotypes, with 4 prominent haplotypes T-T-T (33.7%), C-G-C (25.0%), T-G-C (10.9%), and C-T-T (8.7%). There was a statistically significant linkage disequilibrium between all C1236T, G2677T/A, and C3435T SNPs (p < 0.0001); however, our results indicated that only loci 2677 and 3435 show relatively strong linkage disequilibrium (Lewontin's coefficient [D'] = 0.74, Pearson's correlation [r(2)] = 0.47). The haplotype frequency distribution of our study group was found to be significantly different from that in Han Chinese, Uygur Chinese, Kazakh Chinese, Indian, Malay, Japanese, Caucasian, and Ashkenazi Jewish populations (p < 0.0001). The results of this study may contribute to population-specific haplotype data on the MDR1 gene and may serve as a basis for studies on response to P-glycoprotein substrate drugs as well as for future association studies of certain diseases in Turkish population.