Induction Of Premature Chromosome Condensation Research Articles

A faster and easier biodosimetry method based on calyculin A-induced premature chromosome condensation (PCC) by scoring excess objects

Dicentric analysis and the ring PCC assay as established biodosimetry methods both have limitations in the estimation of absorbed doses in suspected overexposure cases between 5 and 10 Gy. The proposed method based on calyculin A-induced PCC overcomes these limitations by scoring excess objects as the endpoint. This new scoring method can potentially serve as a faster and up-scalable approach that complements the existing methods with higher accuracy at different dose ranges. It can also potentially be performed by less skilled workers when no automated system is available in mass casualty emergency cases to assist with the triage of patients. Additionally, it offers the possibility to further reduce the sample size and PCC induction time. In this pilot study, a calibration curve for excess objects was constructed using the new scoring method for the first time and a blind validation test composed of three unknown doses was carried out. Almost all the dose estimates were within the 95% confidence limits of the actual test doses by scoring only 50–100 PCC spreads. This method was found to be more accurate than ring PCC for doses below 10 Gy.

An alternative approach for the induction of premature chromosome condensation in human peripheral blood lymphocytes using mitotic Akodon cells

Purpose: The premature chromosome condensation (PCC) technique is used to study exposure to external radiation through the determination of chromosome fragments observed in interphase cells. The presence of large telomeric signals in CHO cells interferes with the detection of PCC fragments and the identification of dicentric chromosomes. We present an improved method for the fusion of G0-lymphocytes with mitotic Akodon cells (few chromosomes and weakly-staining telomeric sequences) to induce PCC in combination with rapid quantification of dicentric chromosomes and centric rings as an alternative to the classical CHO cell fusion technique.Materials and methods: Whole blood from three healthy volunteers was γ-irradiated with 0, 2, or 4 Gy. Following a 24 h incubation post-exposure at 37 °C, chromosome spreads of isolated lymphocytes were prepared by standard PCC procedures using mitotic Akodon cells.Results: The percentage of scorable fusions, measured by telomere/centromere (T/C) staining, for Akodon-induced PCC was higher than that for CHO-induced PCC, irrespective of radiation exposure. Importantly, both techniques gave the same result for biodosimetry evaluation.Conclusion: The mitotic Akodon cell-induced PCC fusion assay, in combination with the scoring of dicentric chromosomes and rings by T/C staining of G0-lymphocytes is a suitable alternative for fast and reliable dose estimation after accidental radiation exposure.

Scoring rings in the cell fusion-induced premature chromosome condensation (PCC) assay for high dose radiation exposure estimation after gamma-ray exposure

Purpose: This study aimed to construct a calibration curve for high-dose exposure using cell fusion-induced premature chromosome condensation (PCC). Some of the associated practicalities and methodological details were also investigated.Materials and methods: Peripheral blood from two donors was used. PCC mediated by fusing mitotic CHO cells with interphase lymphocytes was carried out. Lymphocytes were irradiated with 60Co (0–20 Gy) and held at 37 °C for 24 h post exposure.Results: The protocol for PCC induction was effective at all doses and the number of rings increased with increasing dose. No significant difference was found between the donors (p = .896) and data were pooled. Ring aberration frequencies followed a Poisson distribution and the dose-response relationship favored a linear fitting: Y = 0.0007(±0.0004)+0.0186(±0.001)×D. Blind tests showed that the estimated doses were all within the 95% confidence limits of the delivered doses. This study has shown that it is valid to score only 100 cells per sample in a triage mode for doses above 5 Gy and that it is valid to score only hollow rings to reduce the scoring time.Conclusion: Scoring rings in cell fusion-induced PCC assay can be a feasible and fast approach for the analysis of high-dose exposures.

Ultrastructural changes associated with the induction of premature chromosome condensation in Vicia faba root meristem cells.

Key message PCC induction is regulated by several signaling pathways, and all observed effects associated with PCC induction are strongly dependent on the mechanism of action of each PCC inducer used. Electron microscopic observations of cells with symptoms of premature chromosome condensation (PCC) showed that the interphase chromatin and mitotic chromosomes differed with respect to a chemical compound inducing PCC. Induction of this process under the influence of hydroxyurea and caffeine as well as hydroxyurea and sodium metavanadate led to a slight decrease in interphase chromatin condensation and the formation of chromosomes with a considerably loosened structure in comparison with the control. Incubation in the mixture of hydroxyurea and 2-aminopurine brought about clear chromatin dispersion in interphase and very strong mitotic chromosome condensation. Electron microscopic examinations also revealed the characteristic features of the structural organization of cytoplasm of Vicia faba root meristems, which seemed to be dependent on the type of the PCC inducer used. The presence of the following was observed: (i) large plastids filled with starch grains (caffeine), (ii) mitochondria and plastids of electron dense matrix with dilated invaginations of their internal membranes (2-aminopurine), and (iii) large mitochondria of electron clear matrix and plastids containing protein crystals in their interior (sodium metavanadate). Moreover, since caffeine causes either the most effective loosening of chromatin fibrils (within the prematurely condensed chromosomes) or induction of starch formation (in the plastids surrounding the nuclei), this may be a proof that demonstrates the existence of a link between physical accessibility to chromatin and the effectiveness of cellular signaling (e.g., phosphothreonine-connected).

Optimization of calyculin A-induced premature chromosome condensation assay for chromosome aberration studies

Calyculin A-induced premature chromosome condensation (PCC) assay is a simple and useful method to assess structural and numerical chromosome aberrations in cells. Our hypothesis in this study is that suboptimum calyculin A induction of PCC resulting in fuzzy compactness and/or shortened length chromosomes would decrease the detection sensitivity of numerical and structural chromosome aberrations such as small PCC rings and small excess fragments. In this study, an optimization of calyculin A exposure on chromosome morphology and PCC induction frequency was investigated using a human peripheral blood lymphocyte (PBL) ex vivo irradiation ((60) Co-γ rays; ∼0.6 Gy/min; 0-30 Gy) model. Treatment with calyculin A (50 nM) for 15 and 30 min resulted in 11.3 ± 2.7 and 9.9 ± 1.6-fold increases in the frequency of G(2) /M-PCC cells with extended length chromosomes compared with the 60-min treated group over a broad dose range (0 to 20 Gy), respectively. The G(2) /M-PCC scoring index per PCC in 15- and 30-min treated groups was increased by 1.9 ± 0.2 (P = 0.001) and 1.8 ± 0.2 (P = 0.001) compared with the 60-min treated group over 0-20 Gy, respectively. The G(2) /M-PCC efficiency of 30-min treated group was highest in the three conditions (i.e., 15-, 30-, and 60-min treatment) of calyculin A exposure. Calyculin A (50 nM) treatment for 30 min before the 48-h harvest of mitogen-stimulated human PBL is optimum for the formation of suitable chromosome morphology necessary to assess structural chromosome aberrations induced by exposure to radiation using the chemical induced-PCC assay. Published 2011 Wiley Periodicals, Inc.

Investigation of Bystander Effects in Hybrid Cells by Means of Cell Fusion and Premature Chromosome Condensation Induction

The established dogma in radiation sciences that underlies radiation protection and therapeutic applications is that radiation effects require induction of DNA damage only in cells that are directly hit by the radiation. However, extensive work during the last decade demonstrates that DNA damage responses can be detected in cells that are only bystanders. Such effects include cell killing and responses associated with DNA and chromosome damage. Here, we developed a strategy for investigating bystander effects on chromosomal integrity by premature chromosome condensation using hybrid cell formation between nontargeted human lymphocytes and targeted CHO cells or vice versa. We reasoned that signaling molecules generated in the targeted component of the hybrid will transfer to the nontargeted cell, inducing damage detectable at the chromosomal level. The results indicate that bystander cytogenetic effects between CHO and human lymphocytes cannot be detected under the experimental conditions used. This may be due either to the lack of communication of such responses between the components of the hybrid or to their abrogation by the experimental manipulations. These observations and the methodology developed should be useful in the further development of protocols for investigating bystander responses and for elucidating the underlying mechanisms.

Differential radio-sensitivities of human chromosomes 1 and 2 in one donor in interphase- and metaphase-spreads after 60Co γ-irradiation

BackgroundRadiation-induced chromosome aberrations lead to a plethora of detrimental effects at cellular level. Chromosome aberrations provide broad spectrum of information ranging from probability of malignant transformation to assessment of absorbed dose. Studies mapping differences in radiation sensitivities between human chromosomes are seldom undertaken. Consequently, health risk assessment based on radio-sensitivities of individual chromosomes may be erroneous. Our efforts in this article, attempt to demonstrate differences in radio-sensitivities of human chromosome-1 and/or -2, both in interphase and metaphase spreads.MethodsUpon blood collection, dosimetry and irradiation were performed. Lymphocytes were isolated after whole-blood irradiation with 60Co γ-rays in the dose range of 0–5 Gy for both interphase, and metaphase aberration studies. Induction of premature chromosome condensation in interphase cells was accomplished using a phosphatase inhibitor, calyculin-A. Metaphase spreads were harvested from short-term peripheral blood lymphocyte cultures following colcemid arrest and using an automated metaphase harvester and spreader. Aberration analysis in both interphase and metaphase spreads were done using FISH.ResultsIn interphase, aberrant cell and aberration frequency involving chromosome 1 and/or 2 increased linearly with radiation dose. In metaphase, aberrations increased in a linear-quadratic manner with dose. Our studies ascertain that chromosome-2 is more radio-sensitive than chromosome-1 in both interphase and metaphase stages, albeit the DNA content of chromosome-2 is lesser than chromosome-1 by almost 10 million base pairs.ConclusionDifferences in radio-sensitivities of chromosomes have implications in genetic damage, chromosome organization, and chromosome function. Designing research experiments based on our vital findings may bring benefit to radiation-induced risk assessment, therapeutics and development of chromosome specific biomarkers.

Transient inhibition of Calyculin A induced premature chromosome condensation by hyperthermia

The analysis of chromosomal aberrations by premature chromosome condensation (PCC) induced by Calyculin A (Cal) is feasible in tumor biopsies from patients and has the potential to predict sensitivity to radiotherapy. As hyperthermia (HT) improves radiotherapy outcome in certain tumor sites, it was investigated whether PCC induction is still possible after temperatures reached in the clinic. Human cervical carcinoma (CaSki) and lung carcinoma (SW-1573) cells were incubated with Cal to induce PCC immediately after 1 h treatment at temperatures ranging from 41°C to 43°C and after recovery for up to 24 h after treatment with 43°C. Levels of phosphorylated Cdc2 (at the Tyr15 residue), histone H3 (at the Ser10 residue) and Cyclin B1 were investigated by immunoblotting. The amount of cells positive for phosphorylated histone H3 was determined by flow cytometry. Temperatures ≥42.5°C inhibited the induction of PCC by Cal, while recovery of PCC-induction was observed at >20 h after treatment in both cell lines. The phosphorylation status of Cdc2 as well as of histone H3 in cells treated with Cal directly after HT at 43°C was similar to that of cells treated with Cal alone or treated with Cal 24 h after HT at 43°C. HT alone did not affect the levels of phosphorylated Cdc2, while phosphorylation levels of histone H3 were increased as compared with control status of these two proteins. Phosphorylated and total Cyclin B1 levels were not influenced by any of the treatments. Flow cytometric analysis confirmed that HT at 43°C did not interfere with phosphorylation of histone H3. Our data indicate that HT transiently inhibits PCC induction by Cal in a temperature-dependent manner. Therefore, an interval of at least 24 h after HT should be applied before taking tumor biopsies for karyogram analysis of patients treated with temperatures above 42.5°C.

Treatment with Proteasome Inhibitor MG132 during Cloning Improves Survival and Pronuclear Number of Reconstructed Rat Embryos

In several mammalian species including rats, successfully cloned animals have been generated using somatic cell nuclear transfer (SCNT). However, in the case of rats, additional treatment with MG132, a proteasome inhibitor, before enucleation of oocytes seems to be required for successful cloning because ovulated rat oocytes are spontaneously activated, and hence, their suppression is the key to successful cloning. A previous study on rats demonstrated that matured oocytes potentially possess lower cytostatic factor (CSF) activity compared to mouse oocytes, resulting in a low incidence of premature chromosome condensation in the reconstructed embryos after SCNT. It is known that mice having more than two pronuclei are generally observed in nuclear-transferred oocytes after induction of premature chromosome condensation, which implies successful reprogramming. This leads us to the hypothesis that MG132 treatment affects not only the inhibition of spontaneous activation but also the reprogramming and developmental ability of reconstructed rat embryos. If so, prolonged MG132 treatment during and/or after SCNT may further improve the survivability. However, the effect of MG132 treatment on reconstructed embryos after SCNT has been very limited in rats and other species. We show here that prolonged MG132 treatment during and after SCNT improves survival and the number of pronuclei in reconstructed rat embryos after activation. These reconstructed embryos treated before, during, and after SCNT showed significantly higher p34(cdc2) kinase activity involving CSF activity compared to that of the control embryos. On the other hand, p34(cdc2) kinase activity was not recovered in nuclear-transferred oocytes without MG132, which suggested that the enucleation had detrimental effects on the development of reconstructed oocytes. Taken together, MG132 treatment during SCNT increases survival and pronuclear numbers in reconstructed rat embryos via maintenance of high CSF activity. The data suggest that MG132 treatment is indispensable for at least rat SCNT.

Calyculin-A improves chromosome condensation for cytogenetic analysis of blastomeres from bovine and murine eight-cell stage embryos

This study evaluated the serine/threonine phosphatase inhibitor calyculin-A for rapid, efficient induction of premature chromosome condensation (PCC) in blastomeres obtained from Day 3 bovine and Day 2 murine eight-cell stage embryos, and its potential for use in cytogenetic analysis. Experiment 1 tested calyculin-A duration (0, 60, 120, and 180min) to induce PCC in bovine blastomeres. More blastomeres that underwent PCC had chromosomes suitable for cytogenetic analysis if treated for 120 or 180min (P<0.005). Experiment 2 compared doses of calyculin-A (0, 10, 50, and 100nM) on bovine blastomeres; calyculin-A (50nM, 120min) induced PCC suitable for cytogenetic analysis in the greatest number of blastomeres when compared to other doses (52.5%; P<0.005). Effects of calyculin-A (50nM) on murine blastomeres at durations of 0, 60, 90, and 120min to induce PCC were tested in Experiment 3, with 90min inducing the highest frequency of condensed chromosomes suitable for cytogenetic analysis (34%; P<0.05). Finally, Experiment 4 evaluated calyculin-A treated bovine embryos under optimal conditions (50nM, 120min) for use in gender and cytogenetic analysis. Whole chromosome paint probes were successfully hybridized to chromosomes along with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI) counterstaining, allowing detection of embryo gender (54% F:46% M) and ploidy of individual blastomeres within embryos (64% diploid:36% mixoploid embryos). In conclusion, we inferred that calyculin-A was useful for rapid induction of PCC, producing chromosome spreads suitable for cytogenetic analysis of blastomeres in G1 or G2/M phase of the cell cycle.

41 NUCLEAR REMODELING IS AFFECTED BY TREATMENT OF BOVINE OOCYTES WITH A PROTEASOME INHIBITOR BEFORE NUCLEAR TRANSFER

Complete reprogramming of somatic cell nuclei after nuclear transfer (NT) depends on extensive remodeling of chromatin by factors present in the recipient cytoplast. M-Phase Promoting Factor (MPF) activity, responsible for nuclear remodeling in metaphase II recipients, may be lowered by oocyte enucleation and handling prior to NT. Then, a partial nuclear envelope breakdown or incomplete premature chromosome condensation (PCC) may be, in turn, associated with an inefficient reprogramming. The aim of the present study was to maintain the bovine recipient cytoplast at a high level of MPF activity during the fusion procedure by using a proteasome inhibitor, MG132, and to assess the consequences on nuclear remodeling and developmental potential. Bovine COCs were in vitro-matured for 23 h. Matured oocytes were denuded, and then incubated in TCM-199 for 45 min and enucleated in the presence (treated group) or absence (control group) of 5 µm MG132. Embryos were reconstructed by fusion with adult fibroblasts and activated in 10 µg mL–1 cycloheximide and 5 µg mL–1 cytochalasin B. In Experiment 1, MPF activity was analyzed immediately after fusion/activation by measuring the phosphorylation of exogenous histone H1, and Cyclin B expression was assessed by Western blotting. In Experiment 2, microtubules revealed by immunofluorescense with anti-tubulin antibody and chromatin stained with 10 µg mL–1 propidium iodide were analyzed by confocal microscopy 1 h after fusion/activation. In Experiment 3, NT embryos activated for 5 h were cultured in vitro for 7 days. Rate of development and cell counts in both groups were then recorded at the blastocyst stage. Remarkably, in Experiment 1, a high MPF activity was found in only 50% of the control oocytes, but MG132 treatment did not enhance this rate. On the other hand, cyclin B persisted for 2 h after activation in treated oocytes whereas it had dropped in controls. Experiment 2 revealed a higher rate of PCC in the treated embryos (n = 51) than in control embryos (n = 54): 96.0% v. 24.0% (chi-square, P &lt; 0.001). Moreover, microtubules reorganized in a metaphasic spindle in embryos undergoing PCC, whereas cytoplasmic microtubules were observed in the others. In Experiment 3, cleavage and blastocyst rates were not significantly different between the treated (n = 92) and the control groups (n = 105): 83.7% and 53.3% v. 78.1% and 50.5%, respectively. However, the mean cell number in treated embryos (n = 27) was significantly higher than in controls (n = 20): 134 � 25 v. 109 � 43 (P &lt; 0.05). This study suggests that MG132 treatment improved the maintenance of oocyte factors responsible for PCC in bovine NT embryos, although it did not modify MPF activity, thus questioning the role of MPF in the induction of PCC. Accordingly, PCC may be important for blastocyst quality and nuclear reprogramming in NT embryos. Full-term development of MG132-derived embryos is under investigation.

Sequential phosphorylation of Ser‐10 on histone H3 and ser‐139 on histone H2AX and ATM activation during premature chromosome condensation: Relationship to cell‐cycle phase and apoptosis

Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser-10 and Ser-139, respectively, as well as ATM activation through phosphorylation on Ser-1981, during PCC, and relate these events to cell-cycle phase and to initiation of apoptosis. To induce PCC, A549 and HL-60 cells were exposed to the phosphatase inhibitor calyculin A (Cal A). Phosphorylation of histone H3 and H2AX as well as ATM activation were detected immunocytochemically concurrent with analysis of cellular DNA content and activation of caspase-3, a marker of apoptosis. The intensity of cellular fluorescence was measured by flow- or laser scanning cytometry. Induction of PCC led to rapid histone H3 phosphorylation, followed by activation of ATM and then H2AX phosphorylation in both, HL-60 and A549 cells. All these events occurred sequentially, prior to caspase-3 activation, and affected cells in all phases of the cell cycle. ATM activation and H2AX phosphorylation was seen during mitosis of A549 but not HL-60 cells. Because the Cal A-induced phosphorylation of histone H3 and H2AX, and of ATM, precede caspase-3 activation these modifications are pertinent to PCC and not to apoptosis-associated chromatin condensation. The sequence of histone H3 and H2AX phosphorylation and ATM activation during PCC is compatible with a role of ATM in mediating phosphorylation of H2AX but not H3. Mitosis in some cell types may proceed without ATM activation and H2AX phosphorylation.

Biological Dosimetry Using Human Interphase Peripheral Blood Lymphocytes

Conventional metaphase-spread chromosome-aberration-based biodosimetry techniques for radiation dose assessment, although robust, are laborious and time consuming. The molecular cytogenetic laboratory of the Armed Forces Radiobiology Research Institute is developing simple and rapid interphase-based cytological assays that will be applicable to a broad range of radiation exposure scenarios. These assays include analysis of chromosome aberrations (premature chromosome condensation-fluorescence in situ hybridization assay) and mitochondrial DNA mutations (mtDNA4977 deletion assay) using resting human peripheral blood lymphocytes. The dose-effect relationship for radiation-induced aberrations involving chromosome 1 after 24 hours of repair at 37 degrees C in resting human peripheral blood lymphocytes was studied using fluorescence in situ hybridization after chemical induction of premature chromosome condensation as previously explained. In the present study, we examined whether gamma irradiation in the range of 0 to 7.5 Gy induces a dose-dependent increase in aberrations manifested as "excess spots." The number of excess spots per cell, reflecting aberrations involving chromosome 1, increased from 0.035 at 0.5 Gy to 0.236 at 7.5 Gy. This observed dose-effect relationship was fit with a nonlinear power model. This technique may be extended to the study of radiation-induced translocations in interphase cells for retrospective dose reconstruction. With a recently developed in situ polymerase chain reaction method to detect and quantify mtDNA deletion in interphase cells after radiation exposure in cultured human peripheral blood lymphocytes, 90% to 95% of cells are analyzable. We discuss the potential use of the mtDNA deletion assay in biological dosimetry applications. Interphase-based cytological assays may eliminate some inherent problems associated with metaphase-spread-based assays. These problems involve (1) the limited number of analyzable cells containing chromosome aberrations, which is due to various factors including radiation-induced cell death and delay in cell cycle progression into mitosis, and (2) the requirements for radiation cytogenetics expertise and tedious labor to manually score chromosome aberrations.

Induction of premature chromosome condensation by a phosphatase inhibitor and a protein kinase in unstimulated human peripheral blood lymphocytes: a simple and rapid technique to study chromosome aberrations using specific whole-chromosome DNA hybridization probes for biological dosimetry

We developed a simple and rapid method to study chromosome aberrations involving specific chromosomes using unstimulated human peripheral blood lymphocytes (HPBL). Premature chromosome condensation (PCC) was induced by incubating unstimulated HPBL in the presence of okadaic acid (OA, a phosphatase inhibitor), adenosine triphosphate (ATP), and p34 cdc2/cyclin B kinase [an essential component of mitosis-promoting factor (MPF)], which eliminated the need for fusion with mitotic cells. OA concentration and duration of incubation for PCC induction was optimized using mitogen-stimulated HPBL; a final concentration of 0.75 μM incubated for 3 h was optimum, resulting in approximately 20% PCC yield. In unstimulated HPBL, PCC was induced by the addition of p34 cdc2/cyclin B kinase at concentrations as low as 5 units/ml to a cell culture medium containing OA. Increases in the concentration of p34 cdc2/cyclin B kinase from 5 to 50 units/ml resulted in a concentration-dependent increase in PCC yield (30% to 42%). We demonstrate that this technique of inducing PCC in unstimulated HPBL is suitable for studying radiation-induced aberrations involving a specific chromosome (chromosome 1) after 24 h repair using a whole-chromosome in situ hybridization probe and chromosome painting. Cells with aberrant chromosome number 1 are characterized with more than two chromosome spots. The frequency of cells with aberrant chromosome 1 increased with 60Co gamma-radiation doses in the region 0–7.5 Gy. The observed dose–effect relationship for the percentage of cells with aberrant chromosome 1 ( Y) was explained by using both a linear [ Y=(2.77±0.230) D+0.90±0.431, r 2=0.966] and a nonlinear power [ Y=(5.70±0.46) D (0.61±0.05), r 2=0.9901) model. This technique can be applied to biological dosimetry of radiation exposures involving uniform whole-body low linear energy transfer (LET) exposures.

Increased G2 chromosomal radiosensitivity in cancer patients: the role of cdk1/cyclin-B activity level in the mechanisms involved

Purpose : To test the hypothesis that deficient DNA repair as measured by increased G2 chromosomal radiosensitivity results from up-regulation of cdk1/cyclinB and cell cycle control mechanisms during the G2 to M transition. Materials and methods : A total of 185 cancer patients and 25 normal individuals were tested for G2 chromosomal radiosensitivity. The chromatid breaks were analysed in metaphase using the G2 assay or directly in G0 and G2 phase using premature chromosome condensation (PCC). The activity of cdk1/cyclinB, a key regulator of the G2 to M-phase transition, was measured by histone H1 kinase activity and correlated with the development of chromatid breaks after irradiation of cell lines in vitro. Results : Based on the G2 assay, cancer patients on average showed increased chromosomal radiosensitivity above controls. When the analysis was carried out directly in G0 or G2 lymphocytes using PCC, no differences in the induction of chromosomal damage and its repair were observed between G2 assay-sensitive and G2-normal donors. Using the G2 assay to test G2 radiosensitivity in various cell lines, it was found that the higher the cdk1/cyclinB activity level of the cell line tested, the higher the yield of chromatid breaks scored. Furthermore, when mitotic cells from these cell lines were used for PCC induction in irradiated G2 lymphocytes it was observed that the higher the cdk1/cyclinB activity level of mitotic cells used, the higher was the induced yield of chromatid breaks. Conclusion : The cdk1/cyclin-B activity levels during the G2 to M transition impair DNA repair processes and play a major role in the yield of chromatid breaks induced after G2-irradiation. Regulation of cdk1/cyclinB complex activity rather than deficient repair enzymes of DNA damage may underlie the mechanisms of G2 radiosensitivity.

Chemically induced premature chromosome condensation in human fibroblast cell lines: fundamental study for applications to the biodosimetry of local exposure.

The premature chromosome condensation (PCC) of human peripheral lymphocytes treated with inhibitors of protein phosphatase has been demonstrated to be an excellent tool for the estimation of high-dose whole-body exposure. To develop a new biodosimetry for local exposure, the cytogenetical reaction of human fibroblast lines to PCC inducers was examined and compared with that of lymphocytes. The efficiency of the induction by calyculin A was greater than that by okadaic acid in both cell types. Calyculin A induced PCC in 5-Gy-irradiated and unirradiated samples at almost the same frequency in the lymphocytes, whereas the efficacy was considerably lower in irradiated fibroblasts than in unirradiated ones. Calcium ionophore enhanced the induction of PCC in irradiated fibroblasts, although PCC frequencies were still much lower than those in the lymphocytes. The frequency of ring chromosomes observed in 2- and 5-Gy-irradiated fibroblasts was too low to be used as a marker for cytogenetic dosimetry, and that of excess fragments, scored as the observed chromosome number minus 46, might be substituted. The frequency of excess fragments for 2-, 5-, and 10-Gy-irradiated fibroblasts was less than 0.75, about 1 and a few per cell, respectively, although these values changed with the culture period. The prospects and limitations of the application of PCC techniques to fibroblasts are discussed.

Detection of radiation-induced chromosome aberrations using fluorescence in situ hybridization in drug-induced premature chromosome condensations of tumour cell lines with different radiosensitvities

A potential assay for radiosensitivity of human tumours is that of radiation-induced chromosome damage determined on metaphase spreads of human solid tumours. It is often difficult, however, to obtain enough metaphases for cytogenetic analysis after radiation. A possible solution would be to use the technique of premature chromosome condensation (PCC), enabling the study of interphase cells. The induction of PCCs using mitotic inducer cells is technically difficult, however, and the frequency of induction relatively low. We have attempted to use another approach to induce PCCs using the phosphatase inhibitors okadaic acid and calyculin A. Both inhibitors were found to induce PCCs in several human tumour cell lines, with calyculin A producing the higher incidence. Determination of radiation-induced chromosome aberrations using fluorescence in situ hybridization on these chemically induced PCCs showed a clear difference between a radiosensitive (SCC61) and a radioresistant (A549) tumour cell line, with more aberrations in the sensitive line. Owing to incomplete condensation compared with that in standard metaphases, accurate classification of aberration types was not possible. Despite this limitation, the present data indicate that this relatively quick and simple method may be useful for determining chromosome aberrations in interphase cells and potentially in human solid tumours for predictive assay purposes.

Hyperthermic induction of premature chromosome condensation in human lymphocytes

Hyperthermic induction of chromosomal aberrations was examined in human lymphocytes. For this purpose, whole blood cultures were exposed to an elevated temperature (43°C) at the 46th h of culture initiation, followed by 24 h of recovery. Detailed chromosomal analysis showed significantly higher levels of aberrations as compared to control cultures of the same individuals. These aberrations include breaks, gaps of both chromosome and chromatid type, aneuploidy and high levels of polyploidy. The interesting observation was the occurrence of S-phase premature chromosome condensation (PCC). The S-phase PCC observed here could be due to asynchronous nuclei induced by hyperthermia. A possible interpretation for the occurrence of asynchronous nuclei leading to the induction of S-phase PCC is discussed.

Hypertonic Treatment Does Not Affect the Radiation Yield of Interphase Chromosome Breaks in DNA Double-Strand Break Repair-Deficient xrs-5 Cells

A DNA double-strand break (DSB) repair-deficient CHO cell line, xrs-5, was used to evaluate the effect of hypertonic treatment on the radiation yield of interphase chromosome breaks, as visualized by means of premature chromosome condensation (PCC). For this purpose, interphase chromosome breaks were measured in plateau-phase G1 cells after exposure to 10 Gy X rays using either the standard protocol for PCC induction or a revised protocol that includes a brief (20 min) treatment in hypertonic medium (500 mM NaCl) during chromosome condensation. Experiments at the chromosome level were complemented by experiments at the DNA and the cellular level performed under similar postirradiation conditions. The results obtained were compared to those reported previously for CHO cells. Radiation yields of interphase chromosome breaks, as measured using standard protocols for PCC induction, were higher in xrs-5 than in CHO cells. However, the yields became similar between the two cell lines when a 20-min incubation in hypertonic medium was introduced immediately after fusion with mitotic cells. This equalization was due to the fact that incubation in hypertonic medium did not affect the radiation yield of interphase chromosome breaks in xrs-5 cells but increased it in CHO cells to levels similar to those measured in xrs-5 cells. This result suggests that xrs-5 cells constitutively express as interphase chromosome breaks a subset of DSBs which in repair-proficient CHO cells requires incubation in hypertonic medium for transformation to interphase chromosome breaks. Rejoining of DSBs, when measured in isotonic medium, was incomplete in xrs-5 cells compared to CHO cells. Rejoining of DSBs normally repaired in xrs-5 cells was completely inhibited during treatment in hypertonic medium. Postirradiation treatment in hypertonic medium affected the survival of xrs-5 cells only modestly, suggesting that the sector of PLD (beta-PLD) which required treatment in hypertonic medium for fixation in repair-proficient CHO cells is fixed constitutively in xrs-5 cells. These results are in agreement with a model developed to interpret results obtained in similar experiments with CHO cells (Iliakis et al., Radiat. Res 135, 160-170, 1993). The model postulates that potentiation in CHO cell killing induced by hypertonic treatment is caused by the transformation of a subset of fast-repairing DSBs to irreparable interphase chromosome breaks and assumes that this transformation is equivalent to fixation of beta-PLD.(ABSTRACT TRUNCATED AT 400 WORDS)

Determination of the radiation sensitivity of the stromal cells in the murine long-term bone marrow culture by measuring the induction and rejoining of interphase chromosome breaks

Purpose : To determine the radiosensitivity of bone marrow stromal cells, the rate of interphase chromosome breakage and rejoining of stromal cells in the murine long term bone marrow culture and of human skin fibroblasts were compared. Methods and Materials : The cells were irradiated with doses up to 6 Gy and repair times up to 6 hr were investigated. After induction of premature chromosome condensation by fusing the cells with mitotic HeLa cells, the number of interphase chromosome fragments was counted. Results : The number of radiation induced breaks was found to be not significantly different for both cell types with 6.16 ± 0.26 breaks per Gray for the fibroblasts and 5.96 ± 0.20 breaks per Gray for the stromal cells. A significant difference was observed in the repair rate. The fibroblasts rejoined 39.6% of the breaks induced initially during the first hour after irradiation and 5.6 ± 1.84 breaks remained unrejoined after 6 hr, while the stromal cells were able to rejoin 63.2% in 1 hr and had 2.05 ± 0.07 breaks unrejoined after 6 hr. Conclusion : If the well substantiated assumption is made, that the capacity to repair DNA double strand breaks or interphase chromosome breaks is correlated with the cellular radiosensitivity, this findings indicate, that murine bone marrow stromal cells are more radioresistant than human skin fibroblasts.