Sefa Kizildag

Treatment of K562 cells with 1,25-dihydroxyvitamin D3 induces distinct alterations in the expression of apoptosis-related genes BCL2, BAX, BCLXL, and p21.

Apoptosis, or programmed cell death, is a very important phenomenon in cytotoxicity induced by anticancer treatment. 1α,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the active metabolite of vitamin D, inhibits the growth of multiple types of cancer cells including breast, colon, and prostate cancer cell lines. We studied alterations in the mRNA expression levels of BCL2, BAX, CYC, BCL-XL, and VDR genes in the K562 chronic myeloid leukemia cell line in response to treatment with 1,25-(OH)2D3. Morphological observation of K562 cells was evaluated by the staining with Wrights solution. Cell percentage at different phases of the cell cycle was measured, and apoptosis was measured by flow cytometry. The expression levels of the apoptosis-related genes were analyzed by real-time reverse transcription polymerase chain reaction. We found that treatment with 1,25-(OH)2D3 down-regulates BCL2 and BCL-XL mRNA expressions, as well as up-regulates expressions of BAX and p21 mRNA. The expression pattern of CYC and VDR genes were not influenced. However, K562 cells treated with 1,25-(OH)2D3 caused an arrest of cell cycle progression in G1 phase resulting in a decreased number of cells in the S phase, complemented by an accumulation of cells in the G0–G1 phases. Our data show the modulatory effects of 1,25-(OH)2D3 treatment in apoptosis-related genes in K562 cells.

Methamphetamine induces oligodendroglial cell death in vitro.

We investigated whether the psychostimulant methamphetamine (METH) has a cytotoxic effect on oligodendrocytes and which cell-death pathways are involved in the cytotoxic process. METH caused concentration- and time-dependent cytotoxicity in rat oligodendrocyte cultures. METH induced apoptotic cell death and mRNA expression of pro-apoptotic proteins (bax and DP5), but not anti-apoptotic proteins (bcl-2 and bcl-XL). These results suggest that METH induces cytotoxicity in rat oligodendrocytes via the differential regulation of the expression of genes involved in the apoptotic process.

A Novel Angiogenesis Inhibitor Bevacizumab Induces Apoptosis in the Rat Endometriosis Model

Abstract Our aim was to investigate the effects of antivascular endothelial growth factor (anti-VEGF) antibody Bevacizumab on endometrial explants and on apoptotic gene expression levels in the rat endometriosis model. Endometriotic implants were surgically formed, and rats treated with (i) 1 mg/kg single subcutaneous injection of depot leuprolide acetate; (ii) 2.5 mg/kg of single intaperitoneal injection of bevacizumab; (iii) intraperitoneal injection of saline. Histopathologic scores and adhesion scores of endometriotic foci and levels of Bcl-2-associated X protein (Bax), Cytochrome c (Cyt-c), B-cell lymphoma/ leukemia 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl) mRNA gene expressions of endometriotic foci. Bevacizumab treatment decreased the endometriotic explant size compared with control. Bevacizumab-treated rats had lower total adhesion scores when compared with the control group. Semiquantitative evaluation of the persistence of endometrial epithelial cells in the explants showed a lower score in gonadotropin-releasing hormone (GnRH) agonist-treated rats compared with control rats. In Bevacizumab increased expression of Bax 3.1-fold, Cyt-c 1.3-fold and decreased expression of Bcl-2 0.4-fold, Bcl-xl 0.8-fold compared with the control group. The GnRH agonist increased expression of Bax 3.0 fold, Cyt-c 1.3 fold and decreased expression of Bcl-2 0.4-fold, Bcl-xl 0.8-fold, compared with the control group. This study suggests that a novel angiogenesis inhibitor, anti-VEGF antibody bevacizumab is as effective as GnRH agonist in the regression of the endometriotic lesions in rat endometriosis model. One possible mechanism of this effect is the induction of apoptosis.

Microsatellite instability in early-onset breast cancer

Breast cancer in a young person is considered a rare and very aggressive disease. The theories addressing the underlying genetic mechanisms of this disease are controversial. Therefore, additional genetic concepts playing a possible role in its pathogenesis and prognosis must be investigated. Microsatellite instability (MSI) characterized by a mutational process of insertions or deletions in microsatellite repeats might constitute a sensitive indicator for genomic instability in cancer. MSI has been described in a wide variety of tumors, particularly in hereditary non-polyposis colorectal cancer. The reports regarding its occurrence and prognostic significance in breast cancer are in conflict with each other. The purpose of this study was to investigate MSI in early-onset breast cancer and to correlate its occurrence with clinicopathological prognisticators. In this study, 16 female patients with primary breast cancer under 35 years of age (range 29-34) were investigated for the incidence of MSI in five microsatellite loci (D2S123, D3S1611, D17S807, D17S796 and Xq11-12) by comparing paired normal and tumor tissue DNA after PCR amplification from paraffin-embedded tissues. No instability was found in any of these five microsatellite loci. Although care must be taken not to overstate the importance of this result due to the inadequate number of microsatellite markers and DNA samples studied, this preliminary report indicates that MSI phenotype is uncommon in human early-onset breast cancer. Therefore, it does not appear to be related to the prognosis of disease.

Consistent Loss of Heterozygosity at 14q32 in Lymphoid Blast Crisis of Chronic Myeloid Leukemia

Little is understood about the basic biological mechanisms that underlie the reasons for acute transformation in chronic myeloid leukemia (CML). Progression of disease may include inactivation of one or more tumor suppressor genes (TSGs). A widely used methodology for indirectly detecting somatic inactivation of TSGs is searching loss of heterozygosity (LOH) for polymorphic loci located in or near the gene(s) of interest. We aimed to analyze DNA of chronic phase and blastic phase archive material of 15 CML patients for LOH using D1S430, D2S123, D3S1611, D11S29, D14S65, D17S520, BAT 40 markers, the dinucleotide repeat located in the ABL gene and the trinucleotide repeat located in the BCR gene (amplification of the trinucleotide in the BCR gene could not be succeeded). LOH was identified by a %50 lost of one of the alleles intensity. LOH was detected with the ABL dinucletide repeat and D2S123 marker in two patients and with the D14S65 marker in three patients. The three patients exhibiting LOH at the D14S6S locus, all proceeded through lymphoid blast crisis. The D14S65 marker is located at the 14q32 locus which contains the immunglobulin heavy chain gene and the TCL1 oncogene. 14q32 abnormalities at the molecular level, may be predictive for lymphoid blast crisis, whether or not they are detectable cytogenetically

ACVR1 gene mutations in four Turkish patients diagnosed as fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized with congenital malformations of the great toes and progressive heterotopic ossifications in the skeletal muscles and soft tissue. FOP has been associated with a specific point mutation on the ACVR1 (Activin A receptor type I) gene. Four sporadic cases clinically diagnosed as FOP have been included in this study for mutational analysis. In three patients, heterozygote c.617G>A; p.R206H mutation was detected by both DNA sequence analyses and by HphI restrictive enzyme digestion. In the fourth patient, a heterozygote c.774G>T; p.R258S mutation in exon 5 was detected by DNA sequence analysis.

Analysis of cytotoxic T lymphocyte antigen-4 (CTLA-4) exon 1 polymorphism in patients with type 1 diabetes mellitus in a Turkish population.

Recent studies have described linkage and association between cytotoxic T-lymphocyte antigen-4 (CTLA-4) gene polymorphism and type 1 diabetes mellitus (DM1) in some ethnic populations, but not others. This finding suggests that CTLA-4 gene association with DM1 may be influenced by the racial composition of the population. Thus, it is important to study the polymorphism of the CTLA-4 gene in different ethnic groups. In this case-control association study, the CTLA-4 gene exon 1 A/G polymorphism was analyzed in 48 children with DM1 and 80 healthy controls using polymerase chain reaction-restriction fragment length polymorphism analysis. The possible interaction of the CTLA-4 gene polymorphism with the presence of established genetic markers (HLA-DR genotyping) was also evaluated in 29 patients. The results of the present study do not suggest an association of the known polymorphism in exon 1 of the CTLA-4 gene with DM1 in this Turkish population, and G-allele containing CTLA-4 genotypes were not preferentially associated with age at clinical presentation or with presence of other genetic (HLA-DR3 or -DR4) markers of DM1.

Role of transforming growth factor-beta 1 gene polymorphisms in childhood idiopathic thrombocytopenic purpura.

PURPOSE To investigate whether transforming growth factor-beta 1 (TGF-beta 1) gene polymorphisms have a role in the development, clinical progress, and treatment response in children with idiopathic thrombocytopenic purpura (ITP). PATIENTS AND METHODS Thirty-five children with acute ITP, 40 children with chronic ITP, and 97 healthy children were enrolled. After genomic DNA was extracted, TGF-beta 1 gene 509 (C-->T), codon 25 (Arg-->Pro), and codon 10 (Leu-->Pro) polymorphisms were studied using a coupled polymerase chain reaction-restriction enzyme digestion method. RESULTS The genotype and allele frequencies of TGF-beta 1 polymorphisms between acute ITP, chronic ITP, and control group did not differ significantly. No significant association was found between TGF-beta 1 polymorphisms and therapy response. CONCLUSIONS These results demonstrate that the frequency of TGF-beta1 gene 509 (C-->T), codon 25 (Arg-->Pro), and codon 10 (Leu-->Pro) polymorphisms and alleles do not play a role as a genetic risk factor in the development and clinical progress of ITP. Different results may be obtained with further studies involving larger patient populations and other TGF-beta 1 gene polymorphisms.

Molecular diagnosis of maturity-onset diabetes of the young (MODY) in Turkish children by using targeted next-generation sequencing.

Abstract Aim: To perform molecular analysis of pediatric maturity onset diabetes of the young (MODY) patients by next-generation sequencing, which enables simultaneous analysis of multiple genes in a single test, to determine the genetic etiology of a group of Turkish children clinically diagnosed as MODY, and to assess genotype-phenotype relationship. Methods: Forty-two children diagnosed with MODY and their parents were enrolled in the study. Clinical and laboratory characteristics of the patients at the time of diagnosis were obtained from hospital records. Molecular analyses of GCK, HNF1A, HNF4A, HNF1B, PDX1, NEUROD1, KLF11, CEL, PAX4, INS, and BLK genes were performed on genomic DNA by using next-generation sequencing. Pathogenicity for novel mutations was assessed by bioinformatics prediction software programs and segregation analyses. Results: A mutation in MODY genes was identified in 12 (29%) of the cases. GCK mutations were detected in eight cases, and HNF1B, HNF1A, PDX1, and BLK mutations in the others. We identified five novel missense mutations – three in GCK (p.Val338Met, p.Cys252Ser, and p.Val86Ala), one in HNF1A (p.Cys241Ter), and one in PDX1 (p.Gly55Asp), which we believe to be pathogenic. Conclusion: The results of this study showed that mutations in the GCK gene are the leading cause of MODY in our population. Moreover, genetic diagnosis could be made in 29% of Turkish patients, and five novel mutations were identified.

Long-term effects of GnRH agonist, GnRH antagonist, and estrogen plus progesterone treatment on apoptosis related genes in rat ovary.

OBJECTIVE To define the long-term effects of GnRH antagonist, GnRH agonist, and estrogen plus progesterone treatments on apoptosis and apoptosis-related gene expressions, including bcl2, bax, and cyt c in rat ovary. DESIGN Prospective placebo-controlled experimental study. SETTING Obstetrics and Gynecology and Medical Biology and Genetics university departments. ANIMAL(S) Forty female wistar rats that were 3 to 4 months of age. INTERVENTION(S) Forty rats were randomly divided into 4 groups of 10 each. In group 1 (control) each rat received normal saline as placebo by gastric lavage. In group 2 (GnRH agonist) 1 mg/kg leuprolide acetate in depot form was given for 30 days. In group 3 (GnRH antagonist) each animal received 0.1 mg/kg cetrorelix every 2 days. In group 4 (estrogen plus progesterone) 0.5 mg/kg estradiol valerate and norethisterone enantate in depot form was given every 30 days. After 60 days, the animals were killed. MAIN OUTCOME MEASURE(S) Assessment of morphology, histology of ovaries, determination of the number of apoptotic cells, and analysis of apoptosis-related gene expression of bcl2, bax, and cyt c in the rat ovaries. RESULT(S) Long-term GnRH antagonist treatment significantly increased bax gene expression, but the ratio of bcl2:bax gene expression was constant compared with control group. The GnRH agonist treatment significantly increased cyt c gene expression, and estrogen plus progesterone treatment significantly decreased bcl 2 and significantly increased cyt c expressions. In the estrogen plus progesterone group, ovaries were cystic and larger than in the other groups. There was no significant morphologic change between the other groups. CONCLUSION(S) Long-term administration of GnRH agonist, GnRH antagonist, and estrogen plus progesterone can modulate the apoptosis-related genes in rat ovary. Although GnRH antagonist treatment does not influence apoptosis, GnRH antagonist and estrogen plus progesterone treatments seem to influence apoptosis in rat the ovary. Further clinical studies focusing on the effect of these agents on apoptosis-related genes could be performed.