Neutron relative biological effectiveness in Hiroshima and Nagasaki atomic bomb survivors: a critical review.

The calculated risk of cancer in humans due to radiation exposure is based primarily on long-term follow-up studies, e.g. the life-span study (LSS) on atomic bomb (A-bomb) survivors in Hiroshima and Nagasaki. Since A-bomb radiation consists of a mixture of γ-rays and neutrons, it is essential that the relative biological effectiveness (RBE) of neutrons is adequately evaluated if a study is to serve as a reference for cancer risk. However, the relatively small neutron component hampered the direct estimation of RBE in LSS data. To circumvent this problem, several strategies have been attempted, including dose-independent constant RBE, dose-dependent variable RBE, and dependence on the degrees of dominance of intermingled γ-rays. By surveying the available literature, we tested the chromosomal RBE of neutrons as the biological endpoint for its equivalence to the microdosimetric quantities obtained using a tissue-equivalent proportional counter (TEPC) in various neutron fields. The radiation weighting factor, or quality factor, Qn, of neutrons as expressed in terms of the energy dependence of the maximum RBE, RBEm, was consistent with that predicted by the TEPC data, indicating that the chromosomally measured RBE was independent of the magnitude of coexisting γ-rays. The obtained neutron RBE, which varied with neutron dose, was confirmed to be the most adequate RBE system in terms of agreement with the cancer incidence in A-bomb survivors, using chromosome aberrations as surrogate markers. With this RBE system, the cancer risk in A-bomb survivors as expressed in unit dose of reference radiation is equally compatible with Hiroshima and Nagasaki cities, and may be potentially applicable in other cases of human radiation exposure.

Cancer risk at low doses of ionizing radiation: artificial neural networks inference from atomic bomb survivors.

Cancer risk at low doses of ionizing radiation remains poorly defined because of ambiguity in the quantitative link to doses below 0.2 Sv in atomic bomb survivors in Hiroshima and Nagasaki arising from limitations in the statistical power and information available on overall radiation dose. To deal with these difficulties, a novel nonparametric statistics based on the 'integrate-and-fire' algorithm of artificial neural networks was developed and tested in cancer databases established by the Radiation Effects Research Foundation. The analysis revealed unique features at low doses that could not be accounted for by nominal exposure dose, including (i) the presence of a threshold that varied with organ, gender and age at exposure, and (ii) a small but significant bumping increase in cancer risk at low doses in Nagasaki that probably reflects internal exposure to (239)Pu. The threshold was distinct from the canonical definition of zero effect in that it was manifested as negative excess relative risk, or suppression of background cancer rates. Such a unique tissue response at low doses of radiation exposure has been implicated in the context of the molecular basis of radiation-environment interplay in favor of recently emerging experimental evidence on DNA double-strand break repair pathway choice and its epigenetic memory by histone marking.

Delayed manifestation and transmission bias of de novo chromosome mutations: their relevance for radiation health effect.

The origin and transmission of de novo chromosome mutations were reviewed on the basis of our chromosome studies in retinoblastoma patients and male infertility. In a series of 264 sporadic retinoblastoma families, gross chromosome rearrangements involving the RB1 locus were identified in 23 cases (8.7%), of which 16 were non-mosaic and 7 were mosaic mutations. The newly formed chromosome mutations, whether they were non-mosaic or mosaic, had a strong bias towards paternally derived chromosome, indicating that they shared a common mechanism where a pre-mutational event or instability is carried over to zygote by sperm and manifested as gross chromosome mutation at the early stages of development. The de novo chromosome mutations are preferentially transmitted through female carriers. This transmission bias is consistent with the finding of higher frequencies of translocation carriers in infertile men (7.69% versus 0.27% in general populations) in whom meiotic progression is severely suppressed, possibly through activation of meiotic checkpoints. Such a meiotic surveillance mechanism may minimize the spreading of newly-arisen chromosome mutations in populations. A quantitative model of meiotic surveillance mechanism is proposed and successfully applied to the published data on ;humped' dose-response curves for radiation-induced spermatogonial reciprocal translocations in several mammalian species.

Development, beam characterization and chromosomal effectiveness of X-rays of RBC characteristic X-ray generator.

A characteristic hot-filament type X-ray generator was constructed for irradiation of cultured cells. The source provides copper K, iron K, chromium K, molybdenum L, aluminium K and carbon K shell characteristic X-rays. When cultured mouse m5S cells were irradiated and frequencies of dicentrics were fitted to a linear-quadratic model, Y = alphaD + betaD2, the chromosomal effectiveness was not a simple function of photon energy. The alpha-terms increased with the decrease of the photon energy and then decreased with further decrease of the energy with an inflection point at around 10 keV. The beta-terms stayed constant for the photon energy down to 10 keV and then increased with further decrease of energy. Below 10 keV, the relative biological effectiveness (RBE) at low doses was proportional to the photon energy, which contrasted to that for high energy X- or gamma-rays where the RBE was inversely related with the photon energy. The reversion of the energy dependency occurred at around 1-2 Gy, where the RBE of soft X-rays was insensitive to X-ray energy. The reversion of energy-RBE relation at a moderate dose may shed light on the controversy on energy dependency of RBE of ultrasoft X-rays in cell survival experiments.

Around Semipalatinsk nuclear test site: progress of dose estimations relevant to the consequences of nuclear tests (a summary of 3rd Dosimetry Workshop on the Semipalatinsk nuclear test site area, RIRBM, Hiroshima University, Hiroshima, 9-11 of March, 2005).

The paper is an analytical overview of the main results presented at the 3rd Dosimetry Workshop in Hiroshima(9-11 of March 2005), where different aspects of the dose reconstruction around the Semipalatinsk nuclear test site(SNTS) were discussed and summarized. The results of the international intercomparison of the retrospective luminescence dosimetry(RLD) method for Dolon' village(Kazakhstan) were presented at the Workshop and good concurrence between dose estimations by different laboratories from 6 countries (Japan, Russia, USA, Germany, Finland and UK) was pointed out. The accumulated dose values in brick for a common depth of 10mm depth obtained independently by all participating laboratories were in good agreement for all four brick samples from Dolon' village, Kazakhstan, with the average value of the local gamma dose due to fallout (near the sampling locations) being about 220 mGy(background dose has been subtracted).Furthermore, using a conversion factor of about 2 to obtain the free-in-air dose, a value of local dose approximately 440 mGy is obtained, which supports the results of external dose calculations for Dolon': recently published soil contamination data, archive information and new models were used for refining dose calculations and the external dose in air for Dolon village was estimated to be about 500 mGy. The results of electron spin resonance(ESR) dosimetry with tooth enamel have demonstrated the notable progress in application of ESR dosimetry to the problems of dose reconstruction around the Semipalatinsk nuclear test site. At the present moment, dose estimates by the ESR method have become more consistent with calculated values and with retrospective luminescence dosimetry data, but differences between ESR dose estimates and RLD/calculation data were noted. For example mean ESR dose for eligible tooth samples from Dolon' village was estimated to be about 140 mGy(above background dose), which is less than dose values obtained by RLD and calculations. A possible explanation of the differences between ESR and RLD/calculations doses is the following: for interpretation of ESR data the "shielding and behaviour" factors for investigated persons should be taken into account. The "upper level" of the combination of "shielding and behaviour" factors of dose reduction for inhabitants of Dolon' village of about 0.28 was obtained by comparing the individual ESR tooth enamel dose estimates with the calculated mean dose for this settlement. The biological dosimetry data related to the settlements near SNTS were presented at the Workshop. A higher incidence of unstable chromosome aberrations, micronucleus in lymphocytes, nuclear abnormalities of thyroid follicular cells, T-cell receptor mutations in peripheral blood were found for exposed areas (Dolon', Sarjal) in comparison with unexposed ones(Kokpekty). The significant greater frequency of stable translocations (results of analyses of chromosome aberrations in lymphocytes by the FISH technique) was demonstrated for Dolon' village in comparison with Chekoman(unexposed village). The elevated level of stable translocations in Dolon' corresponds to a dose of about 180 mSv, which is close to the results of ESR dosimetry for this village. The importance of investigating specific morphological types of thyroid nodules for thyroid dosimetry studies was pointed out. In general the 3rd Dosimetry Workshop has demonstrated remarkable progress in developing an international level of common approaches for retrospective dose estimations around the SNTS and in understanding the tasks for the future joint work in this direction. In the framework of a special session the problems of developing a database and registry in order to support epidemiological studies around SNTS were discussed. The results of investigation of psychological consequences of nuclear tests, which are expressed in the form of verbal behaviour, were presented at this session as well.

Hemagglutinating virus of Japan-artificial viral envelope liposome-mediated cotransfer of bag-1 and bcl-2 genes protects hepatic cells against ischemic injury through BAG-1-assisted preferential enhancement of bcl-2 protein expression.

Hepatic injury subsequent to ischemia-reperfusion (I/R) was demonstrated in our previous study to be prevented by hemagglutinating virus of Japan (HVJ)-artificial viral envelope (AVE) liposome-mediated gene transfer of the antiapoptotic gene, human bcl-2 (h-bcl-2). In the present study, we introduced simultaneously both mouse Bcl-2-associated athanogene 1 (m-bag-1) and the h-bcl-2 gene by the same HVJ-AVE liposome transfection method, and found that I/R-induced hepatic injuries such as release of hepatic marker enzymes into blood, cell morphological degeneration, and cellular DNA strand cleavage were suppressed more effectively than by transfection with either gene singly. In addition, the h-Bcl-2 expression level in the ischemic state, but not in the nonischemic state, was markedly higher in h-bcl-2/m-bag-1-cotransfected liver than in h-bcl-2-transfected liver. In contrast, the m-BAG-1 expression level in the ischemic state, but not in the nonischemic state, was only slightly higher in h-bcl-2/m-bag-1-cotransfected liver than in m-bag-1-transfected liver. Thus, with dual gene cotransfer, coexistent Bcl-2 protein exerts no activity to assist a marked enhancement of BAG-1 protein, whereas the function of overexpressed BAG-1 as a Bcl-2-binding protein may lead to the enhancement of efficient expression of h-Bcl-2 in I/R-treated liver as compared with nonischemic liver, which results in repression of diverse I/R-induced cell death symptoms, presumably through the formation of functional complexes of BAG-1 and Bcl-2.

Genetic reversion in an acute myelogenous leukemia cell line from a Fanconi anemia patient with biallelic mutations in BRCA2.

A 2-year old boy was diagnosed with Fanconi anemia (FA) and acute myeloid leukemia (AML). A cell line (termed FA-AML1) was established from blast cells obtained after a second relapse after a successful bone marrow transplant. Histochemical and surface marker analysis confirmed that the cells were derived from the myeloid lineage. Cytogenetic analysis revealed multiple chromosomal aberrations, including a ring 7. Stable proliferation of the cultured cells was absolutely dependent on the presence of granulocyte macrophage colony-stimulating factor or interleukin 3. This is the first AML cell line successfully established from a FA patient. Remarkably, FA-AML1 cells appeared to lack the characteristic cellular FA phenotype, i.e., a hypersensitivity to growth inhibition and chromosomal breakage by the cross-linking agent mitomycin C. Genomic DNA from the patient showed biallelic mutations [8415G>T (K2729N)and 8732C>A (S2835STOP)] in the breast cancer susceptibility gene FANCD1/BRCA2 [N. Howlett et al., Science (Wash. DC), 297: 606-609, 2002]. In the AML cells, however, the 8732C>A nonsense mutation was changed into a missense mutation by a secondary alteration, 8731T>G, resulting in 2835E, which restored the open-reading frame of the gene and could explain the reverted phenotype of these cells. Loss of the FA phenotype by genetic correction of a FA gene mutation during AML progression may be a common late event in the pathogenesis of AML in FA patients, which may be treatment related. This finding suggests a novel mechanistic principle of tumor progression based on the genetic correction of an early caretaker gene defect.

Life span shortening of normal fibroblasts by overexpression of BCL-2: a result of potent increase in cell death.

It is well known that BCL-2 protects against cell death by both apoptosis and necrosis. The culture of bcl-2-transfected normal fibroblasts showed a shorter life span by about 12 population doubling levels compared to that of vector transfectants (64 vs 76 population doubling levels, respectively). An MTT assay revealed that BCL-2-overexpressing cells (HCA2/bcl-2) showed more severe growth suppression due to hydrogen peroxide or doxorubicin treatment than vector control cells (HCA2/vector). We observed a significant number of dead cells in the HCA2/bcl-2 culture, but not in the HCA2/vector culture. Other BCL-2 family proteins with both antiapoptotic and proapoptotic activity and other apoptosis-related factors were maintained at similar levels, indicating that overexpression of BCL-2 is the major reason that normal fibroblasts are sensitized to cell death. A broad caspase inhibitor (z-Val-Ala-Asp-fmk) and inhibitors of specific caspases (acetyl-Asp-Glu-Val-Asp-CHO, acetyl-Ile-Glu-Thr-Asp-CHO, and acetyl-Leu-Glu-His-Asp-CHO) suppressed cell death of HCA2/bcl-2 effectively, suggesting involvement of caspase 3-, 8-, and 9-dependent pathways in cell death and that the form of death is apoptosis. Unexpectedly, involvement of active MEK in cell death was shown by the use of its inhibitor, suggesting that crosstalk between BCL-2 and the MAP kinase cascade regulates death as well as life span.

Bcl-2 in cancer and normal tissue cells as a prediction marker of response to 5-fluorouracil.

Bcl-2 in cancer cells was shown to be a potent indicator of 5-FU efficacy, but the protein in normal tissue cells appeared not to be a marker of 5-FU toxicity probably due to the functional alteration of Bcl-2 associated with cell senescence. Transfection analysis of Bcl-2-S and Bcl-2-AS into A549 lung cancer cells revealed that Bcl-2 suppressed cell death induced by 5-FU, and the gene expression level of Bcl-2 was closely correlated with the IC50 for 5-FU in 21 fresh human gastric tumor specimens. Such correlation could not be observed in a neonatal human foreskin fibroblast strain, MJ90 (HCA2), and 21 human normal tissues adjacent to tumors. Transfection analysis of Bcl-2-S and Bcl-2-AS into MJ90 cells showed that Bcl-2 correlated with the resistance to 5-FU in the transfectants at PDL60 as in A549 cells, but increased Bcl-2 in the PDL72 senescent transfectant did not cause an increase of the resistance to 5-FU. Cell aging was observed in MJ90 cells and Bcl-2 in the cells was found to decrease with the cell senescence. The senescent cells, however, were more resistant to 5-FU than the younger PDL60 cells having proliferation activity.

Electrophoretic variant of a lactate dehydrogenase isoenzyme and selective promoter methylation of the LDHA gene in a human retinoblastoma cell line.

BACKGROUND: Lactate dehydrogenase (LD), a tetrameric product of the genes LDHA and LDHB, may be increased in sera of cancer patients. A variant isoenzyme with electrophoretic mobility between LD2 and LD3 (LD2ex) has been described in patients, but its molecular nature is largely unknown. METHODS: A newly established retinoblastoma cell line, NCC-RbC-51 (R51), showed an isoenzyme pattern with only two bands, LD1 and LD2ex. We investigated the isoenzymes by Northern blot, Western blot, and methylation analysis and PCR. RESULTS: Northern blot analysis revealed that R51 cells expressed no wild-type/somatic LDHA mRNA, but did express a small amount of LDHA-related mRNA with a slightly higher molecular mass. Western blot analysis confirmed the anti-LDHA-reactive protein with a 3-kDa higher molecular mass. Treatment of R51 cells with the demethylating agent 5-aza-2'-deoxycytidine restored the expression of the LD2, -3, -4, and -5 isoenzymes. PCR analysis of sodium bisulfite-treated genomic DNA revealed that the CpG island in the promoter region around exon a of the LDHA gene was completely methylated. Reverse transcription-PCR analysis and direct sequencing revealed that R51 cells expressed a RNA with the sequence of the human homolog of a murine testis-specific variant that has exon 0 as the 5' noncoding sequence. LDHB was expressed normally in R51 cells. CONCLUSIONS: The somatic LDHA in R51 cells is transcriptionally silenced by promoter hypermethylation around exon a, leaving only LDHB to be expressed normally and a testis-specific variant transcript of LDHA containing exon 0. LD2ex possibly results from tetramerization of three wild-type LDHB molecules and one variant LDHA product.

Characteristics of the end-joining of DNA double-strand breaks by the ataxia-telangiectasia nuclear extract.

A double-strand break was introduced in plasmid pZErO-2 at a specific site within the ccdB gene that is lethal to Escherichia coli cells and treated with nuclear extracts from human cells. The efficiency of rejoining was monitored by Southern blot analysis and the fidelity of rejoining was measured by expressing the ccdB gene after bacterial transformation. The efficiency of rejoining in the nuclear extract from an ataxia-telangiectasia (A-T) cell line was comparable to that from a control cell line. However, the accuracy of rejoining was much lower for the A-T cell extract than for the control cell extract. All mutations were deletions, most of which contained short direct repeats at the breakpoint junctions. The deletion spectrum caused by the A-T nuclear extract was distinct from that of the control extract. These results indicate that the ccdB gene is useful for analysis of mis-rejoining and that A-T cells have certain deficiencies in end-joining of double-strand breaks in DNA.

Effect of BCL-2 down-regulation on cellular life span.

Reactive oxygen species (ROS) are toxic for cells. BCL-2 is known as the anti-death protein and acts as an antioxidant. When the BCL-2 level of normal fibroblasts was suppressed by antisense bcl-2 oligodeoxynucleotide or antisense bcl-2 RNA expression, the life span of the culture was shortened by about 11 population doublings (approx. 15% of the total life span) in comparison to the control culture. Since about twice as many cell deaths were observed in the antisense culture than in the vector culture, the life span shortening was probably caused by ROS-induced death. Acceleration of telomere shortening was not evident in the antisense culture. Other BCL-2 family proteins showed no significant change in expression. Cell death was suppressed by N-acetyl-L-cysteine, an antioxidant, suggesting that ROS were the major cause of cell death. In conclusion, reduction of BCL-2 makes cells more sensitive to death induced by ROS and leads to shortening of the culture's life span.

DNA damage response pathway in radioadaptive response.

Radioadaptive response is a biological defense mechanism in which low-dose ionizing irradiation elicits cellular resistance to the genotoxic effects of subsequent irradiation. However, its molecular mechanism remains largely unknown. We previously demonstrated that the dose recognition and adaptive response could be mediated by a feedback signaling pathway involving protein kinase C (PKC), p38 mitogen activated protein kinase (p38MAPK) and phospholipase C (PLC). Further, to elucidate the downstream effector pathway, we studied the X-ray-induced adaptive response in cultured mouse and human cells with different genetic background relevant to the DNA damage response pathway, such as deficiencies in TP53, DNA-PKcs, ATM and FANCA genes. The results showed that p53 protein played a key role in the adaptive response while DNA-PKcs, ATM and FANCA were not responsible. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), mimicked the priming irradiation in that the inhibitor alone rendered the cells resistant against the induction of chromosome aberrations and apoptosis by the subsequent X-ray irradiation. The adaptive response, whether it was afforded by low-dose X-rays or wortmannin, occurred in parallel with the reduction of apoptotic cell death by challenging doses. The inhibitor of p38MAPK which blocks the adaptive response did not suppress apoptosis. These observations indicate that the adaptive response and apoptotic cell death constitute a complementary defense system via life-or-death decisions. The p53 has a pivotal role in channeling the radiation-induced DNA double-strand breaks (DSBs) into an adaptive legitimate repair pathway, where the signals are integrated into p53 by a circuitous PKC-p38MAPK-PLC damage sensing pathway, and hence turning off the signals to an alternative pathway to illegitimate repair and apoptosis. A possible molecular mechanism of adaptive response to low-dose ionizing irradiation has been discussed in relation to the repair of DSBs and implicated to the current controversial observations on the expression of adaptive response.

Cellular sensitivity determinants to docetaxel in human gastrointestinal cancers.

beta-tubulin (beta-TUB), Bcl-XL, and additionally glutathione S-transferase pi (GSTpi) were found to participate in sensitivity to docetaxel (TXT) in 7 human gastrointestinal cancer cell lines. The gene expression level of beta-TUB, Bcl-XL, and GSTpi was closely correlated with the IC50 for TXT. beta-TUB amount related to TXT resistance, and GST activity was correlated with IC50 for TXT in the 30-min treatment setting. Bcl-XL transfection increased TXT resistance of COLO201 cells, whereas GST inhibition by ethacrynic acid enhanced TXT cytotoxicity. Continuous TXT treatment increased beta-TUB and GSTpi expression, but the increased GSTpi mRNA was observed in TXT-resistant HCC-48 cells alone.