Translocations In Lymphocytes Research Articles

Twenty-two years later: consistent dose estimation of an accidental overexposure by retrospective biological dosimetry.

The goal of this study was to retrospectively estimate the exposure dose of a victim from the Lilo radiological accident in Georgia after 22 y and compare it with the original cytogenetics-based analysis performed in our laboratory. Similar types of studies have been published, notably involving victims of the Chernobyl, Goiânia and Tammiku accidents. Nevertheless, their estimations were done after shorter periods of time post-exposure, and in some cases, the exposure might not have been exclusively of an external nature. In this study, Fluorescence In Situ Hybridization (FISH) was used to score chromosomal translocations in lymphocytes from a recent blood sample of the victim and the dose assessment was performed using our laboratory's FISH calibration curve. The resulting whole-body exposure dose of 0.35Gy [0.12, 0.72] was similar to the one obtained when reanalysing the original scoring data from 1997 (0.38Gy [0.13-0.75]), suggesting that our current calibration curve could be used for relative dose estimations long time after external exposure.

Thiol dependent NF-κB suppression and inhibition of T-cell mediated adaptive immune responses by a naturally occurring steroidal lactone Withaferin A

Withaferin A (WA), a steroidal lactone isolated from ayurvedic medicinal plant Withania somnifera, was shown to inhibit tumor growth by inducing oxidative stress and suppressing NF-κB pathway. However, its effect on T-cell mediated adaptive immune responses and the underlying mechanism has not been investigated. Since both T-cell responses and NF-κB pathway are known to be redox sensitive, the present study was undertaken to elucidate the effect of WA on adaptive immune responses in vitro and in vivo. WA inhibited mitogen induced T-cell and B-cell proliferation in vitro without inducing any cell death. It inhibited upregulation of T-cell (CD25, CD69, CD71 and CD54) and B-cell (CD80, CD86 and MHC-II) activation markers and secretion of Th1 and Th2 cytokines. WA induced oxidative stress by increasing the basal ROS levels and the immunosuppressive effects of WA were abrogated only by thiol anti-oxidants. The redox modulatory effects of WA in T-cells were attributed to its ability to directly interact with free thiols. WA inhibited NF-κB nuclear translocation in lymphocytes and prevented the direct binding of nuclear NF-κB to its consensus sequence. MALDI-TOF analysis using a synthetic NF-κB-p50 peptide containing Cys-62 residue suggested that WA can modify the cysteine residue of NF-κB. The pharmacokinetic studies for WA were also carried out and in vivo efficacy of WA was studied using mouse model of Graft-versus-host disease. In conclusion, WA is a potent inhibitor of T-cell responses and acts via a novel thiol dependent mechanism and inhibition of NF-κB pathway.

Mechanisms that Promote and Suppress Chromosomal Translocations in Lymphocytes

Recurrent chromosomal translocations are characteristic features of many types of cancers, especially lymphomas and leukemias. Several basic mechanistic factors are required for the generation of most translocations. First, DNA double-strand breaks (DSBs) must be present simultaneously at the two participating loci. Second, the two broken loci must either be in proximity or be moved into proximity to be joined. Finally, cellular DNA repair pathways must be available to join the two broken loci to complete the translocation. These mechanistic factors can vary in different normal and mutant cells and, as a result, substantially influence the frequency at which particular translocations are generated in a given cell type. Ultimately, however, appearance of recurrent oncogenic translocations in tumors is, in most cases, strongly influenced by selection for the translocated oncogene during the tumorigenesis process. In this review, we discuss in depth the factors and pathways that contribute to the generation of translocations in lymphocytes and other cell types. We also discuss recent findings regarding mechanisms that underlie the appearance of recurrent translocations in tumors.

Radiation effects analysis in a group of interventional radiologists using biological and physical dosimetry methods

Interventional radiologists and staff members are frequently exposed to protracted and fractionated low doses of ionizing radiation, which extend during all their professional activities. These exposures can derive, due to the effects of direct and scattered radiation, in deterministic effects (radiodermitis, aged skin, cataracts, telangiectasia in nasal region, vasocellular epitelioms, hands depilation) and/or stochastic ones (cancer incidence). A methodology has been proposed for estimating the radiation risk or detriment from a group of six exposed interventional radiologists of the Hospital Universitario La Fe (Valencia, Spain), which had developed general exposition symptoms attributable to deterministic effects of ionizing radiation. Equivalent doses have been periodically registered using TLD’s and wrist dosimeters, H p (10) and H p (0.07), respectively, and estimated through the observation of translocations in lymphocytes of peripheral blood (biological methods), by extrapolating the yield of translocations to their respective dose–effect curves. The software RADRISK has been applied for estimating radiation risks in these occupational radiation exposures. This software is based on transport models from epidemiological studies of population exposed to external sources of ionizing radiation, such as Hiroshima and Nagasaki atomic bomb survivors [UNSCEAR, Sources and effects of ionizing radiation: 2006 report to the general assembly, with scientific annexes. New York: United Nations; 2006]. The minimum and maximum average excess ratio for skin cancer has been, using wrist physical doses, of [ 1.03 × 1 0 − 3 , 5.06 × 1 0 − 2 ], concluding that there is not an increased risk of skin cancer incidence. The minimum and maximum average excess ratio for leukemia has been, using TLD physical doses, of [ 7.84 × 1 0 − 2 , 3.36 × 1 0 − 1 ], and using biological doses, of [ 1.40 × 1 0 − 1 , 1.51], which is considerably higher than incidence rates, showing an excess radio-induced risk of leukemia in the group under study. Finally, the maximum radiological detriment in the group, evaluated as the total number of radio-induced cancers using physical dosimetry, has been of 2.18/1000 person-year (skin and leukemia), and using biological dosimetry of 9.20/1000 PY (leukemia). As a conclusion, this study has provided an assessment of the non-deterministic effects (rate of radio-induced cancer incidence) attributable to the group under study due to their professional activity.

T(14;18) translocations in lymphocytes of healthy dioxin-exposed individuals from Seveso, Italy

Dioxin exposure has been associated with non-Hodgkin's lymphoma (NHL) in epidemiological investigations. The NHL-related t(14;18) translocations can be detected at a low copy number in lymphocytes from healthy subjects. Exposure to NHL-associated carcinogens, such as dioxin or pesticides, may cause expansion of t(14;18)-positive clones. We investigated prevalence and frequency of circulating t(14;18)-positive lymphocytes in 144 healthy subjects from a population exposed to dioxin [plasma TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) range: <1.7-475.0 parts per trillion (p.p.t.)] after the Seveso, Italy, accident of 1976. t(14;18) translocations were measured in DNA from peripheral blood lymphocytes by high-sensitivity real-time quantitative polymerase chain reaction. We found that the frequency, but not the prevalence, of t(14;18) translocation-positive cells increased with increasing plasma TCDD. Among t(14;18)-positive subjects (n = 50;34.7%), the mean number of t(14;18) translocations/10(6) lymphocytes was 4.2 [95% confidence interval (CI), 2.9-6.2] in subjects with plasma TCDD < 10.0 p.p.t., 8.1 (95% CI, 4.9-13.3) in subjects with plasma TCDD between 10.0 and 50.0 and 12.5 (95% CI, 7.4-21.1) in subjects with plasma TCDD between 50.0 and 475.0 p.p.t. (P-trend = 0.003). As expected, t(14;18) frequency was associated with cigarette smoking and was highest in subjects who smoked for > or =16 years (mean = 12.6; 95% CI, 7.4-21.3; P = 0.01). Higher t(14;18) prevalence was found among individuals with fair hair color (P = 0.01) and light eye color (P = 0.04). No significant association between t(14;18)-and age was found. Our results show that dioxin exposure is associated with increased number of circulating t(14;18) positive cells. Whether this change in t(14;18) frequency is an indicator of elevated lymphoma risk remains speculative and needs further investigation for its potential impact on public health.

Dual Phospholipase C/Diacylglycerol Requirement for Protein Kinase D1 Activation in Lymphocytes

The serine/threonine kinase protein kinase D1 (PKD1) is a protein kinase C (PKC) substrate that mediates antigen receptor signal transduction in lymphocytes. PKC phosphorylates serines 744/748 within the PKD1 catalytic domain, and this is proposed to be necessary and sufficient for enzyme activation. Hence, a PKD1 mutant with alanine substituted at positions 744 and 748 (PKD-S744A/S748A) is catalytically inactive. Conversely, a PKD1 mutant with glutamic residues substituted at positions 744 and 748 as phospho-mimics (PKD-S744E/S748E) is constitutively active when expressed in Cos7 or HeLa cells. The present study reveals that Ser-744/Ser-748 phosphorylation is required for PKD1 activation in lymphocytes. However, PKD-S744E/S748E is not constitutively active but, like the wild type enzyme, requires antigen receptor triggering or phorbol ester stimulation. Antigen receptor activation of wild type PKD is dependent on phospholipase C (PLC)/diacylglycerol (DAG) and PKC, whereas PKD-S744E/S748E is only dependent on PLC/DAG but no longer requires PKC. Hence, substitution of serines 744 and 748 with glutamic residues as phospho-mimics bypasses the PKC requirement for PKD1 activation but does not bypass the need for antigen receptors, PLC, or DAG. In lymphocytes, PKD1 is, thus, not regulated by PLC and PKC in a linear pathway; rather, PKD1 activation has more stringent requirements for integration of dual PLC signals, one mediated by PKCs and one that is PKC-independent.

Translocation analysis by the FISH-painting method for retrospective dose reconstruction in individuals exposed to ionizing radiation 10 years after exposure

Fluorescence in situ hybridization (FISH) is a powerful method largely used for detecting chromosomal rearrangements, translocations in particular, which are important biomarkers for dose assessment in case of human exposure to ionizing radiation. To test the possibility of using the translocation analysis by FISH-painting method in retrospective dose assessment, we carried out in vitro experiments in irradiated human lymphocytes, in parallel with the analysis of translocations in lymphocytes from 10 individuals, who were exposed to 137cesium in the Goiânia (Brazil) accident (samples collected 10 years after exposure). The in vitro dose–response curve for the genomic translocation frequencies (FGs) fits a linear quadratic model, according to the equation: Y=0.0243X2+0.0556X. The FG values were also calculated for the individuals exposed to 137cesium, ranging from 0.58 to 5.91 per 100 cells, and the doses were estimated and compared with the results obtained by dicentric analysis soon after the accident, taking the opportunity to test the validity of translocation analysis in retrospective biodosimetry. A tentative of retrospective dosimetry was performed, indicating that the method is feasible only for low level exposure (below 0.5Gy), while for higher doses there is a need to apply appropriate correction factors, which take into consideration mainly the persistence of chromosomal translocations along with time, and the influence of endogenous and exogenous factors determining the inter-individual variability in the cellular responses to radiation.

Three somatic genetic biomarkers and covariates in radiation-exposed Russian cleanup workers of the chernobyl nuclear reactor 6-13 years after exposure.

Three somatic mutation assays were evaluated in men exposed to low-dose, whole-body, ionizing radiation. Blood samples were obtained between 1992 and 1999 from 625 Russian Chernobyl cleanup workers and 182 Russian controls. The assays were chromosome translocations in lymphocytes detected by FISH, hypoxanthine phosphoribosyltransferase (HPRT) mutant frequency in lymphocytes by cloning, and flow cytometic assay for glycophorin A (GPA) variant frequency of both deletion (N/Ø) and recombination (N/N) events detected in erythrocytes. Over 30 exposure and lifestyle covariates were available from questionnaires. Among the covariates evaluated, some increased (e.g. age, smoking) and others decreased (e.g. date of sample) biomarker responses at a magnitude comparable to Chernobyl exposure. When adjusted for covariates, exposure at Chernobyl was a statistically significant factor for translocation frequency (increase of 30%, 95% CI of 10%-53%, P = 0.002) and HPRT mutant frequency (increase of 41%, 95% CI of 19%-66%, P < 0.001), but not for either GPA assay. The estimated average dose for the cleanup workers based on the average increase in translocations was 9.5 cGy. Translocation analysis is the preferred biomarker for low-dose radiation dosimetry given its sensitivity, relatively few covariates, and dose-response data. Based on this estimated dose, the risk of exposure-related cancer is expected to be low.

High frequency of spontaneous translocations revealed by FISH in cells from patients with the cancer-prone syndromes ataxia telangiectasia and Nijmegen breakage syndrome

The application of fluorescence in situ hybridization (FISH) using whole-chromosome paints (WCPs) is proving to be a very powerful technique for revealing chromosomal instability that, for the most part, has gone undetected by conventional cytogenetic analysis. We have analyzed the frequency of translocations in lymphocytes and lymphoblastoid cell lines from ataxia telangiectasia (AT) and Nijmegen breakage syndrome (NBS) homozygotes and heterozygotes using a three-color chromosome-painting technique (WCP 1, 2, 4). With this assay we were able to detect an increased frequency of spontaneous translocations in AT homozygotes (median, 18.47 ± 10.82 translocations per 1,000 metaphase cells; 10 patients) and AT heterozygotes (median, 7.87 ± 3.15 translocations per 1,000 cells; 7 patients), in comparison to controls (median, 2.26 ± 1.75 translocations per 1,000 cells; 10 controls). Analysis of NBS homozygotes (median, 19.05 ± 11.27 translocations per 1,000 cells; 5 patients) and NBS heterozygotes (median, 6.93 ± 3.04 translocations per 1,000 cells; 6 patients) also showed an increased frequency of translocations in these patients compared to controls. The presence of such hitherto undetected chromosomal aberrations corroborate previous findings of spontaneous chromosomal instability in AT and NBS patients, as manifested by an increased rate of open breaks and rearrangements involving chromosomes 7 and 14. Moreover, we show that the degree of genomic instability in AT and NBS patients is even higher than previously established and that some AT and NBS heterozygotes evidence spontaneous chromosomal instability as well. These increased levels of nonspecific translocations could be an important risk factor for the development of malignancies in homozygotes and heterozygotes for ATM or NBS1 gene mutations.

Biologically-based risk estimation for radiation-induced chronic myeloid leukemia.

Radiation cancer risks are typically determined by the use of simple statistical descriptions of epidemiological data. It is important in risk assessment in general, however, to attempt to incorporate as much biological information into the risk models as possible. We illustrate this by presenting a biologically-based linear-quadratic-exponential (LQE) incidence rate model for radiation-induced chronic myeloid leukemia (CML). The model consists of a linear-quadratic dose-response for the induction of BCR-ABL, a waiting time distribution between BCR-ABL formation and detection of CML, and an exponential cell-killing term that multiplies both the background and induced incidence rates. Using data exclusive of the A-bomb survivor cohort, Bayesian priors are defined for each of the nine parameters in this LQE model. The priors are based on chromosomal translocations in lymphocytes, hematopoietic stem cell survival experiments, CML waiting times in women irradiated for benign disease, the background CML incidence rate in the U.S. population, and genomic DNA target sizes of BCR and ABL. Fixing three of the LQE model parameters to the means of their priors, maximum likelihood estimates of the remaining six parameters were obtained using A-bomb survivor incidence data for Hiroshima males. The likelihood estimates and the corresponding six prior distributions, both approximated as multivariate normal, were then used to form Bayesian posteriors for the six parameters not fixed. With these posteriors the LQE model yields Qgamma*=0.0042 Gy(-1) where Qgamma* is the upper 95% confidence bound of the lifetime CML risk per person-gray in the limit of low doses of gamma-rays. This value is slightly less than Qgamma*=0.0049 Gy(-1) obtained from likelihood estimates of the LQE parameters, and substantially less than Qgamma*=0.0158 Gy(-1) obtained for a simple statistical model linear in dose for kermas less than 4 Gy.

Chromosomal translocations in lymphocytes before and after radiotherapy for testicular seminoma

Chromosomal translocations in lymphocytes before and after radiotherapy for testicular seminoma

Chromosome Translocations in Lymphocytes from Individuals Exposed to 137Cs 7.5 Years After the Accident in Goi nia (Brazil)

A cytogenetic study was carried out for 12 individuals, 7.5 y after exposure to 137Cs in Goiânia (Brazil). The frequencies of translocations were determined by the fluorescence in situ hybridisation (FISH) method, using two cocktails of chromosome probes: #1,6,11 and #3,4,8, and ranges of 0.5 to 9.7 and zero to 4.2 translocations per 100 cells were found, respectively. The mean genomic translocation frequencies were 5.15 ± 1.11, 8.84 ± 1.63 and 22.58 translocations/100 cells for different levels of absorbed doses: <1.0, 1.5-2.0 and 4.6 Gy, respectively, indicating a 10-45 fold increase in relation to the normal frequency. For seven probands, the frequencies of dicentrics were 10 times higher than the control value. The present results are also relevant to a discussion of the reliability and limitations of the analysis of translocations (for a limited number of chromosomes) as a parameter for retrospective biological dosimetry, in cases of human exposure to ionising radiation.

Differences in the Yields of Dicentrics and Reciprocal Translocations Observed in the Chromosomes of Irradiated Human Skin Fibroblasts and Blood Lymphocytes from the Same Healthy Individuals

Plateau-phase human dermal fibroblasts and whole blood G0 lymphocytes obtained from the same three healthy donors were irradiated with different doses of 200 kV X rays. The genomic yields of dicentrics were evaluated in the first postirradiation mitosis. Fluorescence in situ hybridization was used to "paint" the chosen whole chromosomes. The yields of reciprocal translocations were scored for all donors for chromosome 4, and also for the third donor for chromosomes 7 and 9. The amounts of reciprocal translocations were scored in the first as well as in later (4th or 5th) postirradiation cell divisions. The yields of dicentrics involving the painted chromosomes were scored for one donor as well. The yields of dicentrics observed in the lymphocytes and in the skin fibroblasts from the same individual were significantly different. Yields of dicentrics were much lower in fibroblasts than in lymphocytes. This was observed for all doses up to 4 Gy for all three donors studied. Fibroblasts from two individuals were also irradiated with 6 Gy. The interindividual variability was similar in both cell types. The yields of reciprocal translocations in lymphocytes and in fibroblasts were similar. The yields of reciprocal translocations were practically constant when comparing the first mitotic divisions and later divisions. The observed ratio of translocations:dicentrics was higher in fibroblasts than in lymphocytes, where it was about 1 for one donor and about 2 for the other two donors. Significant differences between the yields of dicentrics and translocations as well as between the shapes of their dose responses were observed in lymphocytes and fibroblasts from the same individual. Our experimental data suggest that repair processes with different efficiencies or different repair processes might be active in cells of different types of tissue.

FISH analysis of translocations in lymphocytes of children exposed to the Chernobyl fallout: preferential involvement of chromosome 10.

Using whole-chromosome painting probes, we have analyzed the frequency of translocations of chromosomes 1, 3, and 10 in peripheral lymphocytes of 20 Gomel (Belarus) children, including both thyroid tumor affected and healthy individuals. Gomel was one of the most heavily radiocontaminated areas due to fallout from the Chernobyl nuclear power plant disaster. As controls, 14 healthy children from Pisa (Italy) were investigated simultaneously. Translocation rates were significantly higher in the tumor affected (1.71 +/- 0.68) and healthy Gomel children (2.69 +/- 0.50) than in the Italian controls (0.79 +/- 0.24). We also observed, in healthy Gomel children, an approximately three-fold higher frequency of chromosome 10 translocations compared to translocations affecting chromosomes 1 or 3 (P = 0.0096), a difference that was made even larger (about four fold) after correcting for chromosome size (P = 0.0009). This finding suggests a preferential involvement of chromosome 10 in translocations induced in vivo by low levels of ionizing radiation.