Turker Bilgen

ETS1 mediates MEK1/2-dependent overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A) in human cancer cells.

EGFR-MEK-ERK signaling pathway has an established role in promoting malignant growth and disease progression in human cancers. Therefore identification of transcriptional targets mediating the oncogenic effects of the EGFR-MEK-ERK pathway would be highly relevant. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently characterized human oncoprotein. CIP2A promotes malignant cell growth and is over expressed at high frequency (40–80%) in most of the human cancer types. However, the mechanisms inducing its expression in cancer still remain largely unexplored. Here we present systematic analysis of contribution of potential gene regulatory mechanisms for high CIP2A expression in cancer. Our data shows that evolutionary conserved CpG islands at the proximal CIP2A promoter are not methylated both in normal and cancer cells. Furthermore, sequencing of the active CIP2A promoter region from altogether seven normal and malignant cell types did not reveal any sequence alterations that would increase CIP2A expression specifically in cancer cells. However, treatment of cancer cells with various signaling pathway inhibitors revealed that CIP2A mRNA expression was sensitive to inhibition of EGFR activity as well as inhibition or activation of MEK-ERK pathway. Moreover, MEK1/2-specific siRNAs decreased CIP2A protein expression. Series of CIP2A promoter-luciferase constructs were created to identify proximal −27 to −107 promoter region responsible for MEK-dependent stimulation of CIP2A expression. Additional mutagenesis and chromatin immunoprecipitation experiments revealed ETS1 as the transcription factor mediating stimulation of CIP2A expression through EGFR-MEK pathway. Thus, ETS1 is probably mediating high CIP2A expression in human cancers with increased EGFR-MEK1/2-ERK pathway activity. These results also suggest that in addition to its established role in invasion and angiogenesis, ETS1 may support malignant cellular growth via regulation of CIP2A expression and protein phosphatase 2A inhibition.

Chk1 targeting reactivates PP2A tumor suppressor activity in cancer cells

Checkpoint kinase Chk1 is constitutively active in many cancer cell types and new generation Chk1 inhibitors show marked antitumor activity as single agents. Here we present a hitherto unrecognized mechanism that contributes to the response of cancer cells to Chk1-targeted therapy. Inhibiting chronic Chk1 activity in cancer cells induced the tumor suppressor activity of protein phosphatase protein phosphatase 2A (PP2A), which by dephosphorylating MYC serine 62, inhibited MYC activity and impaired cancer cell survival. Mechanistic investigations revealed that Chk1 inhibition activated PP2A by decreasing the transcription of cancerous inhibitor of PP2A (CIP2A), a chief inhibitor of PP2A activity. Inhibition of cancer cell clonogenicity by Chk1 inhibition could be rescued in vitro either by exogenous expression of CIP2A or by blocking the CIP2A-regulated PP2A complex. Chk1-mediated CIP2A regulation was extended in tumor models dependent on either Chk1 or CIP2A. The clinical relevance of CIP2A as a Chk1 effector protein was validated in several human cancer types, including neuroblastoma, where CIP2A was identified as an NMYC-independent prognostic factor. Because the Chk1-CIP2A-PP2A pathway is driven by DNA-PK activity, functioning regardless of p53 or ATM/ATR status, our results offer explanative power for understanding how Chk1 inhibitors mediate single-agent anticancer efficacy. Furthermore, they define CIP2A-PP2A status in cancer cells as a pharmacodynamic marker for their response to Chk1-targeted therapy.

Effects of hormone replacement therapy on bone mineral density in Turkish patients with or without COL1A1 Sp1 binding site polymorphism

Aim:  To evaluate the effects of hormone replacement therapy (HRT) on bone mineral density (BMD) in patients with or without COL1A1 Sp1 binding site polymorphism.

The association between intragenic SNP haplotypes and mutations of the beta globin gene in a Turkish population

Identification of the beta globin gene mutation-related haplotypes is of interest for the delineation of the clinical heterogeneity as well as understanding of the origin and spreading of the beta globin gene mutations. We screened the whole beta globin gene in 197 Turkish patients by direct sequencing and performed Haploview analyses for beta globin gene haplotyping using five common intragenic SNPs; rs713040, rs10768683, rs7480526, rs7946748, and rs1609812. We found 25 different beta globin gene point mutations by sequencing. A Turkish type of inv/del mutation by MLPA and Gap-PCR was also detected with additional studies. The seven most common mutations with higher frequency of 5% were IVS-I-110 (G>A) (35.6%), Hb S(10.6%), IVS-I-6 (T>C) (7.4%), IVS-I-1 (G>A) (6.9%), IVS-II-1 (G>A) (6.9%), Cod8(-AA) (6%), IVS-II-745 (C>G) (5.1%) and accounted for 78.7% of all mutations. We identified seven different haplotypes (Haplotype I-VII) using five intragenic single nucleotide polymorphisms (SNPs) genotyped by sequencing of the beta globin gene. The association between the mutations and the haplotypes was defined for 16 different mutations. We suggest that haplotyping by these five intragenic SNPs will provide useful information about the origin of the mutations and gene flow among as well as the explanation of the clinical heterogeneity.

Frequencies of four genetic polymorphisms in the CYP1A2 gene in Turkish population

Cytochrome P450 (CYP) 1A2 gene is involved in the metabolic activation of several carcinogens and altered metabolization of some clinically used drugs. We aimed to investigate the distributions of genetic polymorphisms-3860 (G/A)(CYP1A2*1C) and-2467 (T/del)(CYP1A2*1D) in the 5′-flanking region and-739 (T/G)(CYP1A2*1E) and-163(C/A)(CYP1A2*1F) in the first intron of the CYP1A2 gene in 110 unrelated healthy Turkish volunteers by PCR-RFLP technique. The frequencies of each polymorphism in Turkish population were found as 0.04, 0.92, 0.01, 0.27 for CYP1A2*1C, CYP1A2*1D, CYP1A2*1E, CYP1A2*1F, respectively. Compared with other populations, CYP1A2*1Dhas been found to be significantly increased in Turkish population. On the other hand, in general, the frequencies of the other polymorphisms were concordant with those in the Egyptian and Caucasian populations, and were different from those in the Japanese, Chinese and Ethiopian populations. Our results suggest that due to increased frequency of CYP1A2*1D in Turkish population, unctional significance of CYP1A2*1D should be evaluated. It might be screened to determine the relationship between CYP1A2*1D and CYP1A2 related drug metabolisms in associated groups.

The effect of HBB: c.*+96T>C (3'UTR +1570 T>C) on the mild b-thalassemia intermedia phenotype.

Hemoglobin beta (HBB): c.*+96T>C substitution is very rare among β-globin gene mutations and its clinical significance remains to be clarified. The present study aimed to investigate the role of HBB: c.*+96T>C in the β-thalassemia intermedia phenotype in a Turkish family. The proband and parents were screened for β-globin gene mutations via direct sequencing. Hematological and physical examination results were recorded, and correlated according to genotype. The proband was compound heterozygous for Cod 8 (-AA) and HBB: c.*+96T>C, whereas his mother and father were heterozygous for Cod 8 (-AA) and HBB: c.*+96T>C, respectively. The father had almost normal hematological findings, whereas the mother had the typical β-thalassemia trait phenotype. The proband was diagnosed as mild β-thalassemia intermedia based on hepatosplenomegaly and hematological findings. To the best of our knowledge this is the first report of HBB: c.*+96T>C mutation in a Turkish family. HBB: c.* 96T>C substitution is a very rare, but clinically relevant β-globin gene mutation. Additionally, we think that if 1 spouse is a carrier for β-globin gene mutation the other should be screened for silent mutations, such as HBB: c.*+96T>C mutation of the β-globin gene, even if she/he does not have any clinical or hematological signs of the β-thalassemia trait phenotype.

First Observation of Hemoglobin G-Waimanalo and Hemoglobin Fontainebleau Cases in the Turkish Population.

Duran Canatan1,2, Turker Bilgen3, Vildan Ciftci1, Gulsum Yazici1, Serpil Delibas2, Ibrahim Keser4 1Antalya Genetic Diagnostic Center, Antalya, Turkey 2Hemoglobinopathy Diagnostic Center of Mediterranean Blood Diseases Foundation, Antalya, Turkey 3Namik Kemal University Research and Application Center for Scientific and Technological Investigations, Tekirdag, Turkey 4Akdeniz University Faculty of Medicine, Department of Biology and Genetics, Antalya, Turkey

First Observation of Hemoglobin Kansas [β102(G4)Asn→Thr, AAC>ACC] in the Turkish Population.

1. Aydogdu I, Erkurt MA, Ozhan O, Kaya E, Kuku I, Yitmen E, Aydin NE. Reversible bone marrow necrosis in a patient due to overdosage of diclofenac sodium. Am J Hematol 2006;81:298. 2. Bhasin TS, Sharma S, Chandey M, Bhatia PK, Mannan R. A case of bone marrow necrosis of an idiopathic aetiology: the report of a rare entity with review of the literature. J Clin Diagn Res 2012;7:525-528. 3. Rosenbaum H. Hemorrhagic aspects of Gaucher disease. Rambam Maimonides Med J 2014;5:e0039.

FRI0339 Prevelance of MEFV gene mutations in chidren with juvenil idiopathic arthritis

Background Mutations of the MEFV gene, which encodes pyrin protein, a negative regulator of inflammation, leads to Familial Mediterranean Fever (FMF). Recent studies with adults suggest that MEFV gene mutations may have a risk factor for other rheumatic diseases. Objectives In this study, we aimed to study the frequency of MEFV gene mutations in children with Juvenile Idiopathic Arthritis (JIA). Methods Children with JIA who had no typical symptoms of familial Mediterranean fever (FMF) were screened for the mutations in exon 2 and exon 10 of the MEFV gene. Each sample was screened for the mutations located in exon 2 and exon 10 of the MEFV gene by direct sequencing. Results A total of 52 children, 29 girls (55.8%), with a mean age of 9.6±4.4 years (2-16.6 years) were included. Patients were classified according to JIA subgroups as oligoarthritisin 26 (50%), polyarthritisin 13 (24.8%), systemic arthritis in11 (21.2%) patients and arthritis related with enthesitis and with inflammatory bowel disease one each. Eleven patients (21.2%) were heterozygous, two (3.8%) were homozygous and another two were compound heterozygous for MEFV gene. The allele frequency of MEFV mutations was found to be 18.2%, which is higher than the general population. No significant difference was found between the subgroups of JIA. Conclusions These finding suggest that mutations of the MEFV gene may present with varied distinct clinical presentations other than FMF. The MEFV gene may be responsible for diseases other than FMF. Disclosure of Interest None Declared

Expressional Analyses of NM23-H1, KAI1 and MKK4 Metastasis-Related Genes in Metastatic Ovarian Carcinomas

Objective: Cytoreductive surgery as a basis of therapy in epithelial ovarian carcinomas (EOC) provides the primary tumor and metastatic tumor samples from a same patient. This gives an excellent opportunity for evaluation of metastatic factors by excluding inter-individual differences. Therefore, we aimed to define changes at mRNA levels of NM23-H1, KAI1 and MKK4 metastasisrelated genes in the paired normal tissue, primary tumor and omental metastatic tumor samples obtained from a same patient. Material and Methods: mRNA levels were quantified by quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) following total RNA extraction in normal tissues, primary malign tissues of EOC, and its metastatic lesions on omentum for 41 patients with stage III-C (FIGO) EOC. Results: We found that mRNA level of NM23-H1 was significantly higher in metastatic samples compared to primary tumors (p=0.009). On the other hand, MKK4 was found to be significantly lower in primary tumor samples compared to normal tissues (p=0.024). There was no significant change at mRNA level of KAI1 among normal tissues, primary tumors and omental metastatic tumor samples. Conclusion: We suppose that in detailed functional studies, approaches that suppress NM23-H1 gene and restore MKK4 gene would make these genes important molecular targets for treatment of metastatic EOC in the future.