[Cytogenetic profile in patients with primary myelodysplastic syndrome].

AIM: To analyze the prevalence of chromosome aberrations presented in the revised International Prognostic Scoring System (R-IPSS) in patients with de novo myelodysplastic syndrome (MDS). Subjects and methods. Chromosome aberrations were analyzed in 197 patients aged 14 to 86 years (median age 64 years) with de novo MDS. RESULTS: Karyotype abnormalities were revealed in 129 (65.5%) patients with de novo MDS. According to the IPSS criteria, the karyotypes found 52 (26.4%) patients were assigned to an intermediate prognostic group whereas in accordance with the R-IPSS guidelines, an intermediate karyotype group included chromosome abnormalities in 32 (16.2%) patients. Out of 5 R-IPSS prognostic types, the favorable karyotype group was the largest (48.2%). The very favorable and unfavorable karyotype groups comprised few patients with MDS: 3 and 3.6%, respectively. Despite the fact that it was not mentioned in the R-IPSS, a monosomal karyotype was verified in 24 (12.2%) patients There was a correlation of the (normal and complex) karyotype with bone marrow blast counts (r=0.469; p=0.000), but not with age. CONCLUSION: A variety of cytogenetic damages cannot identify the prognostic potential of all chromosome aberrations occurring in patients with MDS even if prognostic factors increased up to 5.

[Novel splicing isoform of actin-binding protein alpha-actinin 4 in epidermoid carcinoma cells A431].

Alpha-actinin 4 (ACTN4) belongs to actin binding proteins of the spectrin superfamily. Structural organisation of actin fibres and focal contacts is considered to be its primary function in a cell. Besides that, nucleocytoplasmic shuffling of ACTN4 and its involvement in nuclear processes were demonstrated. Lately, additional isoforms of ACTN4 resulted from an alternative splicing has been described in various cell types and malignant tumours. In this study, we present investigation of a novel ACTN4 isoform of 80 kDa. The isoform was found in human epidermoid carcinoma cells A431, and it was not detected in human skin fibroblasts, normal human keratinocytes and transformed human embryonic cells HEK293T. Analysis of ACTN4 mRNA in A431 cells showed the presence of a splice variant that lacked the exons 2-8. The deleted exons code two calponin homology domains responsible for ACTN4 binding to F-actin. Intracellular distribution of the described ACTN4 isoform (ACTN4ISO) overexpressed in HEK293T cells differed from that of the full size protein. In the cytoplasm, ACTN4ISO was allocated diffusively with no colocalisation with actin cytoskeleton structures. Intranuclear distribution of ACTN4ISO also differed from that of the full size ACTN4. Nevertheless, immunochemical analysis demonstrated possibility of ACTN4ISO to form heterodimers with the full size protein. Additional investigations of novel isoform interactions with ACTN4 protein partners might clarify its functional features in A431 cells.

[Age-specific characteristics of acute myeloid leukemia karyotype].

AIM: To study distribution of some karyotype variants among patients of different age with acute myeloid leukemia (AML). MATERIAL AND METHODS: Distribution of balanced, normal, unbalanced, complex and monosomic karyotype among 244 patients with de novo AML in age groups 16-20, 21-30, 31-40, 41-50, 51-60, 61 and older was analysed. RESULTS: There is difference in frequency of balanced and complex karyotype in patients under and over 60 years. Number of AML patients with balanced aberrations including favourable variants t(8;21), t(15;17) and inv(16) falls after 60 years of age (6.7% versus 15.0% in patients aged 16-20 years; p < 0.001), while a complex karyotype occurs more frequently in AML patients at the age of 61 and older (56.8% versus 2.7% in the group 16-20 years; p < 0.001). With age, more frequently detected is the most unfavourable monosomic karyotype with aberrations similar to those in myelodysplastic syndrome (57.1% in patients aged 16-60 years and in 80.0% in the group of 61 years of age and over). CONCLUSION: Age-specific karyotype features detected may be explained by different biological mechanisms involved in leukosogenesis in young and elderly AML patients.

[FLT3 and NPM1 gene mutations in patients with acute myeloid leukemias and the impact of FLT3-ITD mutations on the survival of patients with a normal karyotype].

AIM: To estimate the extent of FLT3 and NPM1 gene mutations and the impact of mutations of FLT3-ITD on the survival of patients with acute myeloid leukemias (AML). MATERIALS AND METHODS: The nucleus-containing cells of bone marrow and blood were studied in 43 patients with AML. Polymerase chain reaction analysis of total genomic DNA was applied. RESULTS: Mutations of FLT3-ITD, FLT3-TDK, and the NPM1 gene were found in 16 (37.2%) patients. A total of 19 mutations were revealed. There were 8 mutations of FLT3-ITD, 5 of FLT3-TKD, and 6 in the NPM1 gene. Single damages to genes were detected in 13 patients: FLT3-ITD in 6 (13.9%), FLT3-TKD in 4 (9.3%), and NPM1 in 3 (7%). Three (7%) patients exhibited 2 mutations simultaneously: in the NPM1 and FLT3-ITD in 2 (4.7%) and in the NPM1 gene and FLT3-TKD in 1 (2.3%). In AML patients with a normal karyotype and the FLT3-ITD-/NPM1 and FLT3-ITD+/ NPM-T genotypes, median overall survival was 17.3 versus 8 months (p = 0.069); and event-free survival (EFS) was 11 versus 5 months (p = 0.026). Univariate analysis established the negative impact of FLT3-1TD mutation on EFS. CONCLUSION: The findings allow AML patients with a normal karyotype and the FLT3-ITD-/NPM-genotypes to be identified as a poor prognosis group.

[Investigation of FLT3-ITD and NPM1 mutations in patients with myelodysplastic syndrome and mixed myeloid diseases].

Two FLT3-ITD mutations, one FLT3-TKD) and five NPM1 mutations were detected in 7 patients with de novo myelodysplastic syndrome (MDS) out of 44 cases of MDS and MDS/mixed myeloid diseases. Expression of one of the three investigated mutations was identified: 4 in gene NPM1 (9.1%) and 2--FLT3-ITD (4.5%); simultaneous FLT3-ITD and NPM1 mutation--1 (2.3%); no progression in NPM1 within 9-20 months--3, although with chromosome 7 damage--2. It was suggested that NPM1 mutation without complex karyotype may serve as marker of relatively favorable course.

[Analysis of nuclear protein complexes comprising alpha-actinin-4 by 2D-electrophoresis and mass-spectrometry].

Actin-binding protein alpha-actinin-4 is a member of spectrin super family. It is located in the cytoplasm and in the nucleus. However, nuclear functions of alpha-actinin-4 are still not clear. In this study, we analyzed composition of nuclear protein complexes associated with alpha-actinin-4 in A431 cells. Using 2D electrophoresis, we have determined that about 50 different proteins may be associated with nuclear alpha-actinin-4. Using mass-spectrometry, we analyzed major proteins of these complexes. beta-Actin, alpha- and beta-tubulins, ribonucleoprotein A2/B1, which regulates splicing and is associated with beta-actin, peroxiredoxin-1, which is involved in oxidative stress, and glycolytic enzyme D-3-phosphoglycerate dehydrogenase were identified by MALDI-TOF. Detection of these proteins in nuclear complexes with alpha-actinin-4 may suggest that alpha-actinin-4 is involved in transcription and splicing. Presence of beta-actin in the investigated complexes was confirmed by tandem mass-spectrometry (MALDI-TOF-TOF). Immunoprecipitation of nuclear proteins with antibodies against alpha-tubulin confirmed association of alpha-actinin-4 with alpha-tubulin in the protein complex. Nuclear alpha-actinin-4 constitutes of 105 KDa fullsize isoform and two truncated isoforms of 65 and 75 kDa, whereas only the truncated isoform have been found in nuclear complexes with alpha-tubulin. These data suggest that alpha-actinin-4 is associated with a number of different nuclear protein complexes which may carry out different functions in the cell nucleus.