The aberrant asynchronous replication - characterizing lymphocytes of cancer patients - is erased following stem cell transplantation.

BACKGROUND: Aberrations of allelic replication timing are epigenetic markers observed in peripheral blood cells of cancer patients. The aberrant markers are non-cancer-type-specific and are accompanied by increased levels of sporadic aneuploidy. The study aimed at following the epigenetic markers and aneuploidy levels in cells of patients with haematological malignancies from diagnosis to full remission, as achieved by allogeneic stem cell transplantation (alloSCT). METHODS: TP53 (a tumor suppressor gene assigned to chromosome 17), AML1 (a gene assigned to chromosome 21 and involved in the leukaemia-abundant 8;21 translocation) and the pericentomeric satellite sequence of chromosome 17 (CEN17) were used for replication timing assessments. Aneuploidy was monitored by enumerating the copy numbers of chromosomes 17 and 21. Replication timing and aneuploidy were detected cytogenetically using fluorescence in situ hybridization (FISH) technology applied to phytohemagglutinin (PHA)-stimulated lymphocytes. RESULTS: We show that aberrant epigenetic markers are detected in patients with hematological malignancies from the time of diagnosis through to when they are scheduled to undergo alloSCT. These aberrations are unaffected by the clinical status of the disease and are displayed both during accelerated stages as well as in remission. Yet, these markers are eradicated completely following stem cell transplantation. In contrast, the increased levels of aneuploidy (irreversible genetic alterations) displayed in blood lymphocytes at various stages of disease are not eliminated following transplantation. However, they do not elevate and remain unchanged (stable state). A demethylating anti-cancer drug, 5-azacytidine, applied in vitro to lymphocytes of patients prior to transplantation mimics the effect of transplantation: the epigenetic aberrations disappear while aneuploidy stays unchanged. CONCLUSIONS: The reversible nature of the replication aberrations may serve as potential epigenetic blood markers for evaluating the success of transplant or other treatments and for long-term follow up of the patients who have overcome a hematological malignancy.

Terahertz radiation increases genomic instability in human lymphocytes.

Terahertz radiation is increasingly being applied in new and evolving technologies applied in areas such as homeland security and medical imaging. Thus a timely assessment of the potential hazards and health effects of occupational and general population exposure to THz radiation is required. We applied continuous-wave (CW) 0.1 THz radiation (0.031 mW/ cm(2)) to dividing lymphocytes for 1, 2 and 24 h and examined the changes in chromosome number of chromosomes 1, 10, 11 and 17 and changes in the replication timing of their centromeres using interphase fluorescence in situ hybridization (FISH). Chromosomes 11 and 17 were most vulnerable (about 30% increase in aneuploidy after 2 and 24 h of exposure), while chromosomes 1 and 10 were not affected. We observed changes in the asynchronous mode of replication of centromeres 11, 17 and 1 (by 40%) after 2 h of exposure and of all four centromeres after 24 h of exposure (by 50%). It is speculated that these effects are caused by radiation-induced low-frequency collective vibrational modes of proteins and DNA. Our results demonstrate that exposure of lymphocytes in vitro to a low power density of 0.1 THz radiation induces genomic instability. These findings, if verified, may suggest that such exposure may result in an increased risk of cancer.

Increased levels of numerical chromosome aberrations after in vitro exposure of human peripheral blood lymphocytes to radiofrequency electromagnetic fields for 72 hours.

Mazor, R., Korenstein-Ilan, A., Barbul, A., Eshet, Y., Shahadi, A., Jerby, E. and Korenstein, R. Increased Levels of Numerical Chromosome Aberrations after In Vitro Exposure of Human Peripheral Blood Lymphocytes to Radiofrequency Electromagnetic Fields for 72 Hours. Radiat. Res. 169, 28-37 (2008). We investigated the effects of 72 h in vitro exposure of 10 human lymphocyte samples to radiofrequency electromagnetic fields (800 MHz, continuous wave) on genomic instability. The lymphyocytes were exposed in a specially designed waveguide resonator at specific absorption rates (SARs) of 2.9 and 4.1 W/kg in a temperature range of 36-37 degrees C. The induced aneuploidy of chromosomes 1, 10, 11 and 17 was determined by interphase FISH using semi-automated image analysis. We observed increased levels of aneuploidy depending on the chromosome studied as well as on the level of exposure. In chromosomes 1 and 10, there was increased aneuploidy at the higher SAR, while for chromosomes 11 and 17, the increases were observed only for the lower SAR. Multisomy (chromosomal gains) appeared to be the primary contributor to the increased aneuploidy. The effect of temperature on the level of aneuploidy was examined over the range of 33.5-40 degrees C for 72 h with no statistically significant difference in the level of aneuploidy compared to 37 degrees C. These findings suggest the possible existence of an athermal effect of RF radiation that causes increased levels of aneuploidy. These results contribute to the assessment of potential health risks after continuous chronic exposure to RF radiation at SARs close to the current levels set by ICNIRP guidelines.

Detection of p53 protein in induced sputum after occupational exposure to crystalline silica.

OBJECTIVE: To examine the possibility of detecting p53 protein in the supernatant of induced sputum (IS) of workers exposed to crystalline silica. METHODS: Personal interviews were used to obtain demographic data, occupational and exposure histories, and health habits of the study participants. Sputum samples were collected from all subjects. RESULTS: The all-male study cohort included 35 workers (mean age 43.8 years) exposed to silica and 7 unexposed workers (34.7 years, P < 0.05). The mean duration of exposure was 13.4 years, and the range of exposure levels to silica was 0.02 to 0.33 ppm. The mean level of p53 protein was higher in the exposed group compared with in the unexposed group (76.47 pg/mL and 62.43 pg/mL, respectively). CONCLUSIONS: p53 may serve as a biomarker to identify workers at high risk for developing pulmonary malignancies. IS can detect p53 protein in sputum.

Granulocyte colony-stimulating factor generates epigenetic and genetic alterations in lymphocytes of normal volunteer donors of stem cells.

OBJECTIVE: Because the effect of granulocyte colony-stimulating factor (G-CSF), which is widely used for allogeneic stem cell transplantation, on DNA function and stability has not yet been unequivocally elucidated, the aim of this study was to determine whether G-CSF leads to epigenetic and/or genetic modifications. MATERIALS AND METHODS: Molecular cytogenetic techniques based on fluorescence in situ hybridization technology were used. RESULTS: Lymphocytes of G-CSF mobilized donors displayed epigenetic (altered replication timing of alleles) and genetic (aneuploidy) alterations similar to those observed in lymphocytes of cancer patients. Specifically, in the donors' lymphocytes, biallelically expressed genes (TP53 and AML1) and a repetitive noncoding DNA sequence associated with chromosome segregation (CEN17) showed loss of synchrony in allelic replication timing (allele-specific replication). Each displayed a highly asynchronous pattern of allelic replication similar to that characterizing monoallelic expressed genes. This non-locus-specific epigenetic phenomenon, which also affects DNA sequences associated with chromosome segregation, was accompanied by aneuploidy. Although the loss of replication synchrony in the lymphocytes of G-CSF mobilized donors was a transient epigenetic modification, aneuploidy remained unchanged. The G-CSF effect also was observed after G-CSF administration in vitro. 5-Azacytidine, a DNA methylation blocking agent, inhibited G-CSF in vitro induction of allele-specific replication. CONCLUSION: G-CSF, probably via changes in DNA methylation capacity, leads to cancer-characteristic DNA modifications in lymphocytes of normal mobilized donors.

Allele-specific replication associated with aneuploidy in blood cells of patients with hematologic malignancies.

We hypothesize that coordination between the two DNA parental sets in somatic cells is essential for the stability of the diploid genome, and that its disruption is associated with the many alterations observed in the various cancerous phenotypes. As coordination between two allelic counterparts is well exemplified by synchrony in replication timing, we examined, in blood cells of patients suffering from various hematologic malignancies, replication patterns of five loci. These loci were three cancer-implicated genes (TP53, AML1, and RB1) and two nontranscribed sequences engaged in chromosome segregation. All five loci normally display synchrony in allelic replication timing. In addition, in order to exemplify an asynchronous mode of allelic replication, we followed the replication of allelic counterparts of an imprinted gene (SNRPN), which is distinguished by its asynchronous mode of allelic replication (allele-specific replication). Allelic replication patterns were studied by fluorescence in situ hybridization (FISH), which has been shown to distinguish between nonreplicated and replicated regions of the genome in interphase cells, based on the structure of the specific hybridization signals that are being detected. Using the FISH replication assay we observed, for all loci which normally exhibit synchrony in allelic replication, loss of synchrony when present in blood cells of patients with hematologic malignancies. The loss of synchrony in allelic replication in patients' cells was accompanied by aneuploidy (chromosome losses and gains), the hallmark of cancer. We were able to reinstate the normal pattern of replication in the patients' cells by introducing an inhibitor of DNA methylation. It thus appears loss of allelic coordination is an epigenetic alteration characterizing cancer, which is easily identified by simple cytogenetic means and has a potential use in both cancer investigation and detection.