Effect of Peptide AEDG on Telomere Length and Mitotic Index of PHA-Stimulated Human Blood Lymphocytes.

We studied the effect of peptide AEDG on telomere length and mitotic index of PHA-stimulated blood lymphocytes from young (18-22 years, N=5) and middle-aged (49-54 years, N=6) men. In the younger age group, no significant changes in the mitotic index were detected, while in the middle-aged group, a decrease in this parameter was found in one case. The relative length of telomeric regions of metaphase chromosomes was evaluated by in situ fluorescence hybridization with DNA probes specific to telomeres. After incubation with peptide AEDG, significant changes in the relative telomere length were found in 7 of 11 individuals (3 cases in the younger age group and 4 cases in the middle age group). Significant increase in telomere length after exposure to peptide AEDG was revealed in 5 cases, including two individuals of the younger age group (by 41 and 55%) and three individuals of the middle age group (by 156, 18, and 76%). In one individual of the younger age group and in one of the middle-age group, a significant decrease in telomere length (by 37 and 15%, respectively) was found. A tendency to normalization of telomere lengths was noted: this parameter increased in individuals with initially lower telomere length relative to the group mean value and decreased in individuals with initially longer telomeres compared to the mean length in the group.

Differential DNA Hydroxymethylation in Human Uterine Leiomyoma Cells Depending on the Phase of Menstrual Cycle and Presence of MED12 Gene Mutations.

Using immunofluorescence with specific antibodies, we analyzed DNA hydroxymethylation in uncultured cells from 25 human uterine leiomyomas considering the menstrual cycle phase during surgery and the presence of MED12 gene mutations. It was found that each tumor node had specific DNA hydroxymethylation level that did not depend on the presence of mutations in MED12 gene, but depended on the phase of menstrual cycle. The degree of DNA hydroxymethylation was significantly lower in cells of leiomyomas excised during the luteal phase compared to the follicular phase (p=0.0431). Hormonal status changing at various phases of menstrual cycle is a factor affecting DNA hydroxymethylation in leiomyoma cells.

DNA methylation patterns of metaphase chromosomes in human preimplantation embryos.

We performed a stage-by-stage study of DNA methylation patterns in metaphase chromosomes from blastomeres of triploid and abnormal diploid human embryos. QFH-banded homologous parental chromosomes differ in their DNA methylation patterns at the metaphase of the 1st cleavage division. Chromosomes of both parental genomes are gradually demethylated at subsequent cleavages, undergoing hemimethylation in 2-cell embryos. At the 4-cell stage hypomethylated chromosomes initially appear and are further registered until the blastocyst stage. The proportion of hemimethylated and hypomethylated chromosomes varies between the blastomeres since the 4-cell stage with no preference for certain chromosomes to be hemi- or hypomethylated demonstrates random segregation of hypomethylated, undermethylated and methylated chromatids during cell cleavage. By the blastocyst stage the chromosomes acquire band- and, thus, chromosome-specific methylation patterns, with 5-methylcytosine-rich DNA preferentially accumulated in R- and T-bands and in the short arms of acrocentric chromosomes. Thus, demethylaton and remethylation of parental genomes of human embryos proceeds in the same manner from the 1st metaphase stage up to the blastocyst. These processes involve all chromosomes and all bands from each chromosome and lead to establishment of chromosome-specific DNA methylation patterns by the blastocyst stage with no differences between homologous chromosomes.

Alterations of cytological and karyological profile of human mesenchymal stem cells during in vitro culturing.

Transplantation of human bone marrow mesenchymal stem cells is considered as a promising therapeutic approach to the therapy of many diseases. However, the problem of possible alterations of the properties of mesenchymal stem cells during their expansion in in vitro cultures before transplantation is not solved. In our study, one of two hundred examined cultures of mesenchymal stem cell cultures derived from donors without bone marrow pathologies and developed under standard culturing conditions demonstrated spontaneous disturbances in morphology, proliferation, and karyotype at early passages. The cells of this abnormal culture retained immunophenotype characteristic of normal mesenchymal stem cells, but some of them (15-25%) had numerous numerical and structural chromosome aberrations.

[Peculiarities of metaphase chromosome methylation pattern in preimplantation human embryos].

Methylation pattern peculiarities revealed by immunocytochemical analysis of metaphase chromosomes from preimplanted human embryos with monoclonal antibodies against 5-methylcytosine are described. Chromosomes of 2-8-cell triploid human embryos are undermethylated, if compared to those from PHA-stimulated fetal cord blood lymphocytes. Hemimethylation (asymmetric labeling of sister chromatids) is typical for the most of embryonic chromosomes at 2-cell--blastocyst stages due most probably to a passive loss of methylation during initial cleavages. Diffuse labeling and sister chromatid exchanges are two other cytogenetic peculiarities revealed by immunofluorescent staining of early human embryos. Hypomethylation of pericentromeric heterochromatin of chromosomes 1, 9, 16 and different methylation status of some homologous chromosomes may distinguish them from metaphase chromosomes of lymphocytes. M-banding pattern typical for chromosomes from adult and cord blood lymphocytes initially appears in embryonic metaphase chromosomes as early as at a 8-cell stage to be established for most part of chromosomes of the karyotype at the morula-blastocyst stage only.

[Immunocytochemical analysis of human metaphase chromosome methylation status].

The present paper describes a distribution of 5-methylcytosine-rich DNA in human metaphase chromosomes from PHA-stimulated lymphocytes. Immunocytochemical detection of 5-methylcytosine was carried out with monoclonal antibodies. Fluorescent signals were preferentially localized in certain chromosomal regions, corresponding to R-, some T-bands, pricentromeric heterochromatin, and short arms of acrocentric chromosomes. Specificity of fluorescent signals distribution along chromosomes allowed to describe a new type of human metaphase chromosomes banding pattern, which we call M-banding. Specific M-markers of landmarks were identified for each chromosome pair. The analysis of M-bands methylation status was carried out taking into account data available in literature on their nucleotide structure features, namely GC-rich H3 isochore content and CpG-islands concentration. According to our results, a high level of methylation is typical for the majority of GC-rich regions. However, certain bands of 6, 9, 10, 13, 15 chromosomes (6q15, 6q21, 6q23, 9p13, 9p22, 9p32, 10q24, 13q22, 15q15, 15q24) were shown to be hypomethylated, suggesting their special functional status in lymphocytes.

[Metaphase chromosomes of human multiple myeloma cells of line U-266: karyotype and morpho-functional characteristics of nucleolar organizer regions]. / Metafaznye khromosomy kletok linii mnozhestvennoi mielomy cheloveka U-266: kariotip i morfofunktsional'naia kharakteristika iadryshkoobrazuiushchikh raionov.

Human multiple myeloma (MM) cell lines are widely used to investigate chromosome rearrangements typical for this disease. However, during cell cultivation both numeral and structural chromosome rearrangements usually take place in addition to changes of structural and functional status of particular chromosome regions. We investigated karyotype and morpho-functional status of nucleolar organizer regions (NORs) in human cell line MM U-266. Cytogenetic analysis (G-banding) showed karyotypic stability and balanced chromosome set in hypodiploid (n = 44) U-266 cells. We found the presence of rearranged chromosomes, typical for U-266, which retained throughout many year cell cultivation. At the same time, further chromosome rearrangements were shown, along with a tendency of cells to polyploidization. Using FISH (rDNA-probe) and AgNOR-staining techniques, we found that only 4 of 8 NORS were Ag positive (AgNOR), whose dimensions varied from 1 to 3 units of arbitrary scale (u.a.s.). The average summarized AgNOR size was 7.19 +/- 0.03 u.a.s. Peculiarities of the NOR morpho-functional status in U-266 cells are discussed.

[The methylation peculiarities of pericentromeric heterochromatin of chromosomes 1,9 and 16 in human embryo]. / Osobennosti metilirovaniia pritsentromernykh geterokhromatinovykh raionov khromosom 1,9 i 16 u émbrionov cheloveka.

By means of in situ nick-translation technique, methylation patterns of pericentric heterochromatin of chromosomes 1, 9 and 16 in extraembryonic (chorion) and embryonic cells of 5-8 week old human fetuses with normal karyotype (5), and in one specimen with trisomy for chromosome 16 were studied. Fixed metaphase chromosomes from direct chromosome preparations were digested with either endonuclease Msp I or its isoshizomer Hpa II recognizing and restricting the same sDNA sequence C decreases CGC with Hpa II, but not Msp I sensitive to methylation state of internal cytosin. According to our results, heterochromatin of extraembryonic, but not embryonic cells is hypomethylated. An obvious difference was registered in signal strength between homologous regions in iq12 of both parental chromosomes 1 in early (5-6 week old), but not in more advanced fetuses. Methylation pattern difference was detected in pericentric chromatin of triple copies of chromosome 16 in extraembryonic tissues of the 47,XY, + 16 fetus. These results are in line with a hypothesis of intraheterochromatin location of "early" genes governing initial stages of embryonic development in humans.

[Polymorphism of nucleolar organizer regions of chromosomes in human embryos]. / Polimorfizm iadryshkoobrazuiushchikh raionov khromosom u émbrionov cheloveka.

NOR activity in metaphase chromosomes from extraembryonic and embryonic tissues of 9-12 week human fetuses was studied after standard silver staining. Significant interidividual variations in the average cummulative NOR activity was assessed by means of one-factor dispersion analysis. No significant intertissue fluctuation of NOR activity was found. Total number of NOR+ chromosomes demonstrated no correlation with the embryonic age. Steady growth of an average cummulative NOR activity respective of progressive embryonic age was proven by correlation analysis method. Unequal participation of NOR-bearing chromosomes of D- and G-groups during early embryonic development in human was shown.