Non-stimulated Peripheral Blood Lymphocytes Research Articles

Characteristics of mitochondrial DNA in the peripheral blood cells of residents of Kazakhstan around Semipalatinsk Nuclear Test Site

The study was undertaken to address the question whether there are differences in the molecular features of mitochondrial DNA (mtDNA) extracted from peripheral blood lymphocytes between the individuals possibly exposed to radiation around Semipalatinsk Nuclear Testing Site and those who lived in noncontaminated areas in Kazakhstan. Subjects were residents from three villages considered to be highly contaminated with radioactive fallout. Control individuals were from one settlement in Kazakhstan and a Japanese noncontaminated area. Two parameters, the relative mtDNA content and abundance of large-scale deletions in mtDNA were estimated by real-time PCR and fluorescence-mediated PCR, respectively. The number of large-scale deletions generally decreased with the increase of mtDNA content both in Kazakhstan and in the control Kazakhstan and Japanese individuals. The character of relationship between the number of large-scale deletions and the mtDNA content was similar in any group tested irrespective of the radiation history of a blood donor, and this tendency was preserved between different age groups. We conclude that examination of large-scale mtDNA deletions in nonstimulated peripheral blood lymphocytes is unlikely to be informative as a bioindicator of radiation exposure.

DNA repair capacity after γ-irradiation and expression profiles of DNA repair genes in resting and proliferating human peripheral blood lymphocytes

DNA repair plays an important role in maintaining genomic integrity, and deficiencies in repair function are known to promote cancer development. Several studies have used the individual capacity to repair DNA damage in peripheral blood lymphocytes (PBLs) as a cancer risk marker. As the cell’s ability to remove DNA damage may be correlated with proliferative activity, it is an important question whether quiescent or dividing cells should be used in such studies. The aim of our study was to compare DNA repair capacity and expression profiles of 70 known DNA repair genes, both in resting and phytohemagglutinin (PHA) stimulated human PBLs. Using the comet assay, γ-radiation-induced DNA damage and repair in lymphocytes was analyzed. No difference, neither in the rate of radiation-induced DNA damage nor in DNA repair capacity between PHA-stimulated and non-stimulated PBLs was observed. Stimulated cells, however, showed significantly elevated values for background damage. Transcriptional profiles of repair genes were analyzed using cDNA arrays. Hybridization experiments were performed with mRNA isolated from both unstimulated and PHA-stimulated PBLs. More than 70% of all evaluated genes had constant expression levels. Twelve genes responded with a more than two-fold increase of transcripts to the mitogenic stimulus. Most of the up-regulated repair enzymes are also known to play a role in DNA replication. In conclusion, the data presented here suggest that all repair proteins needed for the repair of γ-irradiation induced DNA-damage, that can be detected by the alkaline comet assay, are already present in G 0 cells at sufficient amounts and do not need to be induced once lymphocytes are stimulated to start cycling. Our results thus do not support a general increase in DNA repair activity of PBLs by PHA stimulation, and the use of stimulated PBLs in molecular epidemiological studies on DNA repair of γ-irradiation induced DNA damage seems not to be mandatory.

Differential transcriptional regulation of silencer of death domains in cord blood and peripheral blood lymphocytes.

We have shown that neonatal lymphocytes are deficient in TNF-induced apoptosis. Recently, a 60 kDa protein termed silencer of death domains (SODD) has been shown to prevent spontaneous apoptosis. The binding of SODD to DD of the TNFR-I maintains DD in monomeric form and prevents their aggregation which is required for the apoptotic signal. We examined the expression of SODD mRNA during TNF-induced apoptosis. Although in non-stimulated cord blood lymphocytes the constitutive expression of SODD mRNA was lower than that of non-stimulated peripheral blood lymphocytes, following anti-CD3 stimulation SODD mRNA was significantly increased in cord blood lymphocytes at 12 and 24 h, returning to the baseline at 48 h. In contrast, a modest decrease in SODD transcript was observed in peripheral blood lymphocytes. Furthermore, TNF downregulated SODD mRNA expression in peripheral blood lymphocytes, whereas it had no significant effect on SODD transcripts in cord blood lymphocytes. Therefore, differential regulation of SODD may be one of the mechanisms responsible for decreased TNF-induced apoptosis in neonatal lymphocytes.