Frequency of BCR-ABL fusion transcripts in Serbian patients with chronic myeloid leukemia.

PURPOSE: The aim of this study was to analyze the occurrence of the most frequent BCR-ABL transcript variants (b3a2, b2a2 and e1a2) in Serbian patients with chronic myeloid leukemia (CML) and compare it with the occurrence reported in other populations. METHODS: We analyzed peripheral blood and bone marrow samples of 136 Serbian patients with CML by RT-PCR and cytogenetic methods. RESULTS: In 100 patients (73.5%) the b3a2 and in 34 (25%) the b2a2 forms of BCR-ABL were detected. One (0.75%) patient was BCR-ABL negative, but in lymphoblastic transformation he expressed the e1a2 [corrected] transcript of BCR-ABL. One (0.75%) patient displayed both b2a2 and b3a2 forms of BCR-ABL. Analysis of this group according to karyotype showed b3a2 predominance (79%) in patients with classic t(9;22); b2a2 was found in 20% and both b2a2 and b3a2 forms in 1%. In variant translocations b3a2 in 65% and b2a2 in 35% of the patients were detected. In contrast, the subgroup with normal karyotype expressed slight predominance of the b2a2 form (50%); b3a2 was found in 43% of the patients and one patient (7%) displayed e1a2. CONCLUSION: Predominance of the b3a2 form in Serbian patients with CML is in concordance with other relevant investigations, conducted mostly on Caucasian ethnic groups, but in contrast to the study performed on the Mestizo ethnic group in Ecuador. Slight predominance of the b2a2 form was also noticed among the patients with normal karyotype.

Molecular characteristics and prognostic significance of Bcl-2/IgH gene rearrangement in Serbian follicular lymphoma patients.

Follicular lymphoma (FL) is characterized by the presence of a t(14;18) chromosomal translocation that results in overexpression of bcl-2 protein. Bcl-2/IgH gene rearrangement is detected in 80-90% of follicular lymphomas in Western countries. The aim of this study was to analyze the bcl-2/IgH rearrangement in FL lymphoma patients in Serbia, by PCR technique, correlate molecular findings with clinical characteristics and outcome and assess the prognostic significance of these rearrangements. One hundred-seven patients (median age, 54 years; male/female ratio:60/47) diagnosed with FL were included in the study. DNA samples were obtained from paraffin embedded lymphoid tissue of patients. Bcl-2/IgH rearrangement was assessed for the major breakpoint region (MBR), 5' MBR and the minor cluster region (mcr) breakpoints by PCR technique. We detected a t(14;18) in 81.3% (87/107) of patients. The distribution of bcl-2-IgH rearrangement was as follows: 88,5% (77/87) in MBR breakpoint, 10,35% (9/87) in 5' MBR, whereas mcr bcl-2-IgH rearrangement was observed in one patient (1.15%). No rearrangements were detected in remaining 20 patients (18.7%). This is the first analyses of the frequency of the bcl-2/IgH gene rearrangement in Serbian FL patients, as well as in Eastern European countries. There was no correlation between presence of bcl-2/IgH gene rearrangement and clinical outcome of disease. Incidence of bcl-2/IgH gene rearrangement in Serbian FL patients is relatively high, and similar to frequency in Western countries. Presence of this rearrangement in tumor tissue is not of prognostic significance.

Localization of recognition site between transforming growth factor-beta1 (TGF-beta1) and TGF beta receptor type II: possible implications in breast cancer.

Although overexpression of TGF-beta1 protein has been demonstrated in advanced breast cancer (BC) patients, as well as in other solid tumours, the molecular mechanism of this process remains obscure. This paper proposes that a genetic/epigenetic alteration might occur in the TGF-beta1 gene, within the region coding for the recognition site with TGFbeta receptor type II, leading to a disruption of the ligand-receptor interaction and triggering the TGF-beta1 cascade-related BC progression. To establish the operational framework for this hypothesis, in the present study, this recognition site was identified by the Informational Spectrum Method (ISM) to comprise two TGF-beta1 peptides (positions 47-66 aa and 83-112 aa) and one receptor peptide at positions 112-151 aa of the extracellular domain of the receptor (TbetaRIIM). The TbetaRIIM locus was further evaluated by ISM-derived deletion analysis of the TbetaRII sequences. To provide experimental support for the proposed model, a pilot study of plasma TGF-beta1 analysis was performed in advanced BC patients (n = 8). Two commercial ELISA assays, one with specific alphaTGF-beta1 MAb (MAb) and other with TbetaRIIM as the immobilized phase, revealed pronounced differences in the pattern of plasma TGF-beta1 elevation. In MAb-profile, the TGF-beta1 increase was detected in 7 of 8 patients, whereas analogous TbetaRIIM-profile revealed the elevation in 3 of 8 patients, taking a 50% of maximal elevation as the cut-off value. These findings are consistent with the proposed aberration of TGF-beta1 ligand within the TbetaRII recognition site. Summarizing, this model system is a good starting point for further genetic studies, particularly on genetic/epigenetic alterations of sequences involved in TGF-beta1 and TbetaRIIM interaction, with putative prognostic value for breast cancer.

Busulfan-induced loss of Ph chromosome in chronic myeloid leukemia.

We report a 44-yr-old female with Philadelphia chromosome positive chronic myeloid leukemia who was initially treated with busulfan, and clinical remission has been achieved. After 4 yr, low-dose busulfan therapy was started again and induced bone marrow aplasia. The patient spontaneously has recovered from aplasia, and complete cytogenetic remission with loss of Ph+ chromosome in bone marrow has been achieved. However, reverse-transcription-polymerase chain reaction analysis showed presence of the bcr-abl transcript in bone marrow.

Analysis of 5' non-coding region in hepatitis C virus by single-strand conformation polymorphism and low-stringency single specific primer PCR.

Single-strand conformation polymorphism (SSCP) and low-stringency single specific primer (LSSP)-PCR in hepatitis C virus (HCV) genotyping were examined for informativeness and reliability. The analysis of HCV isolates included seven type 1 isolates, two type 2 isolates, and two type 3 isolates. We also analyzed five isolates that presented as mixed infections determined by type-specific PCR. Among mixed isolates, one isolate was 1a/1b and four isolates were 1b/3a. SSCP and LSSP-PCR were applied to the analysis of 5' non-coding region of HCV (-289 to -5) that contains genotype-specific sequences. Direct cycle sequencing of this region determined sequence divergences that define genotype and sequence alterations within the same genotype. Optimized conditions for the SSCP analysis clearly distinguished between genotypes 1, 2 and 3. In addition, the SSCP analysis detected sequence variants within the same genotype. However, the SSCP analysis and DNA sequencing did not confirm the presence of mixed infections. LSSP analysis, not previously employed in HCV genotyping, enabled clear distinction between genotypes 1, 2 and 3, however, this method did not differentiate between sequence variants within a genotype. Importantly, the LSSP profile demonstrated distinction between mixed infection isolates.

Distribution of HCV genotypes among risk groups in Serbia.

Blood samples from 190 patients that were anti-hepatitis C virus (HCV) positive were genotyped and 165 were found to contain HCV-RNA. Genotyping was performed by PCR based on type-specific primers (117 isolates) and LiPA test (48 isolates) and verifying by sequencing. In Serbia, the most frequent genotype was 1b (49.1%), followed by genotype 3 (21.2%) and genotype 1a (8.5%). The frequency of genotypes 2 and 4 was below 5% and mixed infections were encountered in 9.1% of cases. Distribution of genotypes was monitored in different risk groups: intravenous drug abusers, patients under blood transfusion, patients with previous history of surgery, patients undergoing hemodialysis and those with unknown risk factors. Genotype distribution is essentially the same in all the groups, except for the patients undergoing hemodialysis and those with previous history of surgery where significant difference exists compared with the group with unknown route of transmission (p < 0.001 and p < 0.05, respectively). There exists significant age-dependent genotype 3 distribution in Serbian population (p < 0.01).

Distinct spectrum of N-ras mutations in aml and mds patients in yugoslavia.

Mutations that activate ras genes were demonstrated to be associated with certain types of malignancies. Multiple point mutations were predominantly found in the N-ras and occasionally in the K-ras genes. The analysis of 4 MDS, 23 AML and 11 CML patients from Yugoslavia revealed the prevalence of the N-ras mutation (83%) over K-ras mutations (17%). Although the frequencies of the N- and K-ras mutations in these patients were similar to the ones reported for patients from USA and Japan, the N-ras mutational spectra considerably differed. The prevailing type of mutation in patients from Yugoslavia was G-to-T transversion at the first position in the codon 12 of the N-ras gene. This study supports a hypothesis that different geographical and environmental factors may cause the accumulation of different type of point mutations in the same target gene.

Prevalence of G-to-T transversions among K-ras oncogene mutations in human colorectal tumors in Yugoslavia.

Human colorectal carcinoma tissue sampled from 37 patients, routinely graded into Dukes' stages A, B and C and histologically examined for the level of differentiation, were analyzed for the presence of point mutations in the K-ras oncogene. Seventeen cases out of the 37 analyzed were found to have a mutation in either the 12th or the 13th codon of the K-ras gene, giving an overall frequency of mutation of 46%. The incidence of mutations in Dukes' stages A, B and C was 33, 46 and 58% respectively. Although the frequency of mutation appears to be similar to that reported for the USA population, the spectrum of point mutations in codons 12 and 13 of the K-ras gene in the Yugoslav population appears to differ significantly. G-to-T transversions make up 77% of all mutations present, with the distribution as follows: 18% at the first base and 59% at the second base of codons 12 and 13. G-to-A transitions at the second base is the only other mutation identified, occurring mainly in codon 13 in colorectal tumors of all 3 stages.

Soman intoxication-induced increase in the levels of mRNAs coding for acute phase reactants.

The effect of lethal and repetitive sublethal soman intoxication on the composition of rat liver mRNA was examined by cell-free translation and hybridization. It was found that lethal as well as sustained sublethal soman poisoning of rats elicited a typical acute phase response as judged by a several-fold increase in levels of mRNAs coding for the major acute phase proteins and the vast number of systemic and metabolic changes creating the acute phase response should be taken into account when the metabolic events during the recovery from organophosphate intoxication are under consideration.

Comparative studies of rat liver and sea urchin embryo nuclear matrices: partial fractionation and protein kinase activity distribution.

Rat liver and sea urchin embryo nuclear matrices were found to differ in composition and in the strength of the association of their structural elements. Apart from the qualitative differences in composition, the embryonic matrices retained greater amounts of nuclear proteins and DNA, and were less susceptible to ultrasonic treatment than those of rat liver. They were essentially resistant to mild sonication, by which the rat liver matrix structure was resolved into two distinct fractions, referred to by Berezney (1980) as matricin and ribonucleoprotein (RNP). Both sub-fractions exhibited a protein kinase activity; the phosphorylating capacity of the RNP-associated protein kinases was found to be higher than that of the matricin-bound enzyme. The preferred substrate was among the secondary matrix proteins. In sea urchin embryos, sonication introduced no change in the type and lesion of the matrix proteins phosphorylated by the associated enzyme.

Embryonic stage-related properties of sea urchin embryo chromatin.

Two chromatin components, obtained by buoyant density centrifugation of the unsheared blastula and gastrula chromatin of a sucrose/glucose gradient, have been comparatively characterized. When compared to the heavy fraction the light fraction (i) represents a far smaller part of chromatin, (ii) contains a higher RNA/DNA mass ratio and a higher proportion of newly synthesized nonhistone proteins and (iii) possesses greater template activity for RNA synthesis. Gastrulation of the embryos was found to render the dense chromatin fraction less compact and both chromatin subpopulations more transcriptable and enriched with newly synthesized non-histone proteins.