Gordana Nikcevic

Improved transfection efficiency of cultured human cells

We simultaneously tested the transfection efficiency of NT2/D1 and HeLa cells with Lipofectamine™ (Life Technologies) and Effectene™ (Qiagen) transfection reagents using the pCH110 eukaryotic assay vector, which contains the lacZ reporter gene. Under our culture conditions for NT2/D1 and HeLa cells, Effectene™ transfection efficiency could be augmented by simply increasing the amount of plasmid DNA 1.5–3 times above the recommended concentration without any visible cytotoxicity. With the Lipofectamine™ reagent, optimal transfection efficiency was obtained for both cell lines within the recommended concentrations, but at the top of the range. The results indicate that optimization of the transfection process should include plasmid DNA concentrations above the levels suggested by the manufacturers, in order to accomplish the highest transfection efficiency.

Up‐regulation of the SOX3 gene expression by retinoic acid: characterization of the novel promoter‐response element and the retinoid receptors involved

Sox3/SOX3 gene is considered to be one of the earliest neural markers in vertebrates and it is implicated in the genetic cascades that direct brain formation. We have previously shown that early phases of differentiation and neural induction of NT2/D1 embryonal carcinoma cells by retinoic acid (RA) involve up‐regulation of the SOX3 gene expression. Here, we present identification of a novel positive regulatory promoter element involved in RA‐dependent activation of the SOX3 gene expression in NT2/D1 cells. This element represents a direct repeat 3‐like motif that directly interacts with retinoid X receptor (RXR) α in a sequence‐specific manner. It is capable of independently mediating the RA effect in a heterologous promoter context and its disruption caused significant reduction of RA/RXR transactivation of the SOX3 promoter. Furthermore, by using synthetic antagonists of retinoid receptors, we have shown for the first time, that RA‐induced SOX3 gene expression could be significantly down‐regulated by the synthetic antagonist of RXR. Also, this data showed that RXRs, but not RA receptors, are mediators of RA effect on the SOX3 gene up‐regulation in NT2/D1 cells. Presented data will be valuable for future investigation of SOX3 gene expression, not only in NT2/D1 model system, but also in diverse developmental, physiological and pathological settings.

Mechanical activity in heart regulates translation of α-myosin heavy chain mRNA but not its localization

Mechanical inactivity depresses protein expression in cardiac muscle tissue and results in atrophy. We explore the mechanical transduction mechanism in spontaneously beating neonatal rat cardiomyocytes expressing the α-myosin heavy chain (α-MyHC) isoform by interfering with cross-bridge function [2,3-butanedione monoxime (BDM), 7.5 mM] without affecting cell calcium. The polysome content and α-MyHC mRNA levels in fractions from a sucrose gradient were analyzed. BDM treatment blocked translation at initiation (162 ± 12% in the nonpolysomal RNA fraction and 43 ± 6% in the polysomal fraction, relative to control as 100%; P < 0.05). There was an increase in α-MyHC mRNA from the nonpolysomal fraction (120.5 ± 7.7%; P < 0.05 compared with control) with no significant change in the heavy polysomes. In situ hybridization of α-MyHC mRNA was used to estimate message abundance as a function of the distance from the nucleus. The mRNA was dispersed through the cytoplasm in spontaneously beating cells as well as in BDM-treated cells (no significant difference). We conclude that direct inhibition of contractile machinery, but not calcium, regulates initiation of α-MyHC mRNA translation. However, calcium, not pure mechanical signals, appears to be important for message localization.

6-mercaptopurine influences TPMT gene transcription in a TPMT gene promoter variable number of tandem repeats-dependent manner

AIM TPMT activity is characterized by a trimodal distribution, namely low, intermediate and high methylator. TPMT gene promoter contains a variable number of GC-rich tandem repeats (VNTRs), namely A, B and C, ranging from three to nine repeats in length in an A(n)B(m)C architecture. We have previously shown that the VNTR architecture in the TPMT gene promoter affects TPMT gene transcription. MATERIALS, METHODS & RESULTS: Here we demonstrate, using reporter assays, that 6-mercaptopurine (6-MP) treatment results in a VNTR architecture-dependent decrease of TPMT gene transcription, mediated by the binding of newly recruited protein complexes to the TPMT gene promoter, upon 6-MP treatment. We also show that acute lymphoblastic leukemia patients undergoing 6-MP treatment display a VNTR architecture-dependent response to 6-MP. CONCLUSION These data suggest that the TPMT gene promoter VNTR architecture can be potentially used as a pharmacogenomic marker to predict toxicity due to 6-MP treatment in acute lymphoblastic leukemia patients.

Novel transcriptional regulatory element in the phenylalanine hydroxylase gene intron 8

We present the first transcriptional regulatory element found in a PAH gene intron. The element is located in the PAH gene intron 8, acts as an enhancer specifically in the hepatoma cell line, and binds GATA-1 transcription factor. Herein the presented data could unlock a new area for the analysis of PAH gene expression and could contribute to refining genotype-phenotype correlation.

The influence of novel transcriptional regulatory element in intron 14 on the expression of Janus kinase 2 gene in myeloproliferative neoplasms

The expression of Janus kinase 2 (JAK2) gene is altered in myeloproliferative neoplasms (MPN) and the regulation of transcription could be a mechanism that modulates JAK2 gene expression. We analyzed the transcriptional potential of single-nucleotide polymorphism (SNP) rs12343867 T > C in JAK2 intron 14, tagging 46/1 haplotype, and its influence on JAK2 gene expression. Functional analysis of JAK2 intron 14 was performed using the pBLCAT5 reporter system in K562 cells. Identification of the proteins binding to the intron 14 regulatory element was accomplished by electrophoretic mobility shift assay (EMSA) and supershift assays. Quantification of the expression of JAK2 gene in a cohort of 51 MPN patients and 12 healthy controls was performed by real-time quantitative polymerase chain reaction (RQ-PCR). Functional analysis revealed that the intronic DNA element harboring SNP rs12343867 T > C acts as a transcriptional repressor in vitro. The repressor activity was significantly attenuated by the presence of nucleotide C. Supershift analysis showed the enrolment of transcriptional factor Meis1 in this process. RQ-PCR experiments showed increased JAK2 expression in patients with the JAK2V617F mutation, with a significant difference between essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) patients. SNP rs12343867 showed no statistically significant influence on the expression of JAK2 gene in MNP patients.

Translation is regulated via the 3' untranslated region of alpha-myosin heavy chain mRNA by calcium but not by its localization.

Posttranscriptional regulation plays an important role in α-myosin heavy chain (α-MyHC) protein synthesis in cardiac muscle cells. In the present study, we test the effects of calcium and mRNA mislocalization on α-MyHC translation in order to determine the mechanism(s) contributing to translational block via the 3′ untranslated region (3′UTR). Neonatal rat cardiac myocytes were treated for 6 h with L-isoproterenol (10 μM) to enhance beating, with 10 μM verapamil to block beating and mislocalize mRNA, or with 3 μM colchicine to enhance beating but mislocalize mRNA by depolymerization of the microtubules. In order to determine whether translation is regulated by the 3′UTR, either a control (SV40 3′UTR) or the experimental (α-MyHC 3′UTR) was placed after a luciferase reporter gene and transfected into the myocytes. The amount of luciferase protein only decreased significantly in verapamil arrested cells transfected with the α-MyHC 3′UTR construct (P < 0.01). To control for the possibility that pharmacological treatments might affect transcription or message stability, we analyzed neomycin and luciferase mRNA levels transcribed from the same transfected plasmid. No significant changes were found with an RNase protection assay. These results suggest that calcium but not mRNA localization regulates protein synthesis and further, this is mediated by the 3′ untranslated region of α-MyHC.

HLA genotyping in pediatric celiac disease patients

Celiac disease (CD) is a chronic inflammatory disease in the small intestine triggered by gluten uptake that occurs in genetically susceptible individuals. HLA-DQ2 protein encoded by HLA-DQA1*05 and DQB1*02 alleles is found in 90-95% of CD patients. All of the remaining patients carry HLA-DQ8 protein encoded by HLA-DQA1*03 and DQB1*03:02 alleles. Specific HLA-DQ genotypes define different risk for CD incidence. Presence of susceptible HLA-DQ genotypes does not predict certain disease development, but their absence makes CD very unlikely, close to 100%. Here we presented for the first time the distribution of HLA-DQ genotypes in the group of pediatric celiac patients from the University Childrens Hospital, Belgrade, Serbia and estimated risk for CD development that these genotypes confer. Seventy three celiac disease patients and 62 healthy individuals underwent genotyping for DQA1, DQB alleles and DRB1 allele. 94.5% of patients carried alleles that encode DQ2 protein variant and 2.7% carried alleles that encode DQ8 protein variant. Two patients carried single DQB1*02 allele. No patients were negative for all the alleles predisposing to CD. The highest HLA-DQ genotype risk for CD development was found in group of patients homozygous for DQ2.5 haplotype, followed by the group of heterozygous carriers of DQ2.5 haplotype in combination with DQB1*02 allele within the other haplotype. The lowest risk was observed in carriers of a single copy of DQB1*02 or DQA1*05 allele or other non-predisposing alleles. HLA genotyping, more informative than serological testing commonly used, proved to be a useful diagnostic tool for excluding CD development.

Mutational Status and Gene Repertoire of IGHV-IGHD-IGHJ Rearrangements in Serbian Patients With Chronic Lymphocytic Leukemia

UNLABELLED The mutational status and configuration of immunoglobulin heavy variable (IGHV) gene rearrangements was analyzed in 85 Serbian patients with chronic lymphocytic leukemia (CLL). We found that 55.3% of cases belonged to mutated and 44.7% to unmutated CLL, progressive disease predominating in the unmutated subset. IGHV gene use resembled that obtained for Mediterranean countries, except for underrepresentation of the IGHV4 subgroup in our cohort. BACKGROUND Chronic lymphocytic leukemia (CLL) results from the clonal expansion of mature B lymphocytes and is characterized by extreme clinical heterogeneity. One of the most reliable prognostic markers in chronic lymphocytic leukemia (CLL) is the mutational status of immunoglobulin heavy variable (IGHV) genes, which defines 2 subsets, mutated CLL (M-CLL) and unmutated CLL (U-CLL), with different clinical courses. Biased IGHV gene use between M-CLL and U-CLL clones, as well as population differences in the IGHV gene repertoire have been reported. PATIENTS AND METHODS In this study, mutational status and configuration of IGHV-IGHD-IGHJ rearrangements in 85 Serbian patients were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing methodology. RESULTS We found that 55.3% of cases belonged to M-CLL and 44.7% belonged to U-CLL, with progressive disease predominating in the unmutated subset. Most frequently expressed was the IGHV3 subgroup (55.7%), followed by IGHV1 (27.3%), IGHV4 (12.5%), IGHV5 (2.3%), IGHV2 (1.1%), and IGHV6 (1.1%). The distribution of IGHD subgroups was as follows: IGHD3, 39.1%; IGHD2, 21.8%; IGHD6, 12.6%; IGHD1, 10.3%; IGHD4, 8%; IGHD5, 6.9%; and IGHD7, 1.1%. The most frequent IGHJ gene was IGHJ4 (48.9%), followed by IGHJ6 (28.4%), IGHJ3 (11.4%), and IGHJ5 (11.4%). In 15.3% of cases, heavy complementarity-determining region 3 (VH CDR3) amino acid sequences could be assigned to previously defined stereotyped clusters. CONCLUSIONS Our study showed a strong correlation between IGHV gene mutational status and clinical course of CLL. IGHV gene use was comparable to that obtained for Mediterranean countries, with the exception of the IGHV4 subgroup, which was underrepresented in our cohort.

Prognostic Impact of NPM1 Mutations in Serbian Adult Patients with Acute Myeloid Leukemia

Based on current findings, the presence of NPM1 mutations in acute myeloid leukemia (AML) patients is associated with an increased probability of complete remission (CR) and better overall survival (OS). We determined the incidence and prognostic relevance of NPM1 mutations, their association with FLT3 and IDH mutations, and other clinical characteristics in Serbian adult AML patients. Samples from 111 adult de novo AML patients, including 73 AML cases with a normal karyotype (NK-AML), were studied. NPM1, FLT3, and IDH mutations were detected by PCR and direct sequencing. NPM1 mutations were detected in 22.5% of patients. The presence of NPM1 mutations predicted a low CR rate and shorter OS. NPM1 mutations showed an association with both FLT3 and IDH mutations. Survival analysis based on NPM1/FLT3 mutational status revealed a lower OS for NPM1+/FLT3– compared to the NPM1–/FLT3– group in NK-AML patients. The lack of impact or unfavorable prognostic effect of NPM1 mutations found in this study can be assigned to a small cohort of analyzed AML patients, as can the presence of FLT3 and IDH mutations or other genetic lesions that cooperate with NPM1 mutations influencing prognosis.