Sub-micrometer 20MeV protons or 45MeV lithium spot irradiation enhances yields of dicentric chromosomes due to clustering of DNA double-strand breaks.

In conventional experiments on biological effects of radiation types of diverse quality, micrometer-scale double-strand break (DSB) clustering is inherently interlinked with clustering of energy deposition events on nanometer scale relevant for DSB induction. Due to this limitation, the role of the micrometer and nanometer scales in diverse biological endpoints cannot be fully separated. To address this issue, hybrid human-hamster AL cells have been irradiated with 45MeV (60keV/µm) lithium ions or 20MeV (2.6keV/µm) protons quasi-homogeneously distributed or focused to 0.5×1µm(2) spots on regular matrix patterns (point distances up to 10.6×10.6µm), with pre-defined particle numbers per spot to provide the same mean dose of 1.7Gy. The yields of dicentrics and their distribution among cells have been scored. In parallel, track-structure based simulations of DSB induction and chromosome aberration formation with PARTRAC have been performed. The results show that the sub-micrometer beam focusing does not enhance DSB yields, but significantly affects the DSB distribution within the nucleus and increases the chance to form DSB pairs in close proximity, which may lead to increased yields of chromosome aberrations. Indeed, the experiments show that focusing 20 lithium ions or 451 protons per spot on a 10.6µm grid induces two or three times more dicentrics, respectively, than a quasi-homogenous irradiation. The simulations reproduce the data in part, but in part suggest more complex behavior such as saturation or overkill not seen in the experiments. The direct experimental demonstration that sub-micrometer clustering of DSB plays a critical role in the induction of dicentrics improves the knowledge on the mechanisms by which these lethal lesions arise, and indicates how the assumptions of the biophysical model could be improved. It also provides a better understanding of the increased biological effectiveness of high-LET radiation.

Chromosomal radiosensitivity analyzed by FISH in lymphocytes of prostate cancer patients and healthy donors.

It is known that about 5-10% of cancer patients show severe clinical side effects during and after radiotherapy due to enhanced sensitivity to ionizing radiation. Identification of those radiosensitive individuals by a reliable in vitro assay before onset of treatment would have a great impact on successful radiotherapy. We compared the radiosensitivity of the chromosomes 2, 11 and 17 in prostate cancer patients with and without severe side effects after radiotherapy and in age-matched healthy donors. Each cohort consisted of at least 10 donors. Peripheral blood lymphocytes were irradiated ex vivo with 0.5, 1 und 2 Gy ((137)Cs γ rays). We investigated the radiosensitivity of the chromosomes 2, 11 and 17 by scoring of 100 FISH painted metaphases for each dose point and donor group. Statistical analyses were performed by nonparametric tests as Mann-Whitney test and Kruskal-Wallis ANOVA, paired Wilcoxon rank test, χ(2) goodness-of-fit test and Spearman rank-order correlation at a significance level of P < 0.05. Analysis of the overall aberration yield revealed no significant differences between any donor groups. The translocation frequencies of the chromosomes 2, 11 and 17 coincided with their relative size. Thus, none of the chromosomes analyzed were more or less radiosensitive with respect to the genomic translocation frequency. Additionally, neither of the chromosomes showed enhanced or diminished radiosensitivity in one of the donor groups. Furthermore, variance analyses revealed that the distribution pattern of the aberrations per donor did not differ in each donor group even after exposure to 2 Gy. Prostate cancer patients with and without side effects cannot be distinguished from healthy donors based on aberration yield after irradiation with γ rays.

Intrachromosomal changes and genomic instability in site-specific microbeam-irradiated and bystander human-hamster hybrid cells.

Exposure to ionizing radiation may induce a heritable genomic instability phenotype that results in a persisting and enhanced genetic and functional change among the progeny of irradiated cells. Since radiation-induced bystander effects have been demonstrated with a variety of biological end points under both in vitro and in vivo conditions, this raises the question whether cytoplasmic irradiation or the radiation-induced bystander effect can also lead to delayed genomic instability. In the present study, we used the Radiological Research Accelerator Facility charged-particle microbeam for precise nuclear or cytoplasmic irradiation. The progeny of irradiated and the bystander human hamster hybrid (A(L)) cells were analyzed using multicolor banding (mBAND) to examine persistent chromosomal changes. Our results showed that the numbers of metaphase cells involving changes of human chromosome 11 (including rearrangement, deletion and duplication) were significantly higher than that of the control in the progeny of both nuclear and cytoplasmic targeted cells. These chromosomal changes could also be detected among the progeny of bystander cells. mBAND analyses of clonal isolates from nuclear and cytoplasm irradiations as well as the bystander cell group showed that chromosomal unstable clones were generated. Analyses of clonal stability after long-term culture indicated no significant change in the number of unstable clones for the duration of culture in each irradiated group. These results suggest that genomic instability that is manifested after ionizing radiation exposure is not dependent on direct damage to the cell nucleus.

Kinetics of CHO A L mutant expression after treatment with gamma radiation, EMS, and asbestos.

The flow cytometry mutation assay (FCMA) uses hybrid CHO A(L) cells to measure mutations of the cd59 gene located on human chromosome 11 by the absence of fluorochrome-conjugated antibody binding to the CD59 surface antigen. Mutant expression peaks between 6 and 12 days, then decreases to a stable plateau, instead of a constant mutant fraction obtained by clonogenic assays. To evaluate this variable mutant expression time, cells were treated with radiation, EMS or asbestos and cell proliferation and survival were measured at times leading up to peak mutant expression. Potential doubling time (T(pot)) values increased by at least 75% for each agent by 3 h after treatment but returned to control levels after only 3 days. Survival returned to 90% of control within a week, close to the peak expression day for all three agents. The survival of CD59(-) cells sorted on the peak day of expression was roughly half that of CD59(+) cells. Cloned EMS-treated CD59(-) cells had a doubling time of 16.7 vs. 14.1 h for CD59(+) cells. Triple mutants (CD59(-)/CD44(-)/CD90(-)) were preferentially lost from the population over time, while the proportion of CD59(-)/CD90(-) mutants increased. In conclusion, the peak day of mutant expression occurs only when cells recover from the toxic effects of the mutagen. A fraction of cells originally quantified as mutants are lost over time due to lethal deletions and slower growth.

Involvement of reactive oxygen species (ROS) in the induction of genetic instability by radiation.

Radiation generates reactive oxygen species (ROS) that interact with cellular molecules, including DNA, lipids, and proteins. To know how ROS contribute to the induction of genetic instability, we examined the effect of the anti-ROS condition, using both ascorbic acid phosphate (APM) treatment or a low oxygen condition, on the induction of delayed reproductive cell death and delayed chromosome aberrations. The primary surviving colonies of mouse m5S-derived cl. 2011-14 cells irradiated with 6 Gy of X-rays were replated and allowed to form secondary colonies. The anti-ROS treatments were applied to either preirradiation culture or postirradiation cultures for primary or secondary colony formation. Both anti-ROS conditions relieved X-ray-induced acute cell killing to a similar extent. These anti-ROS conditions also relieved genetic instability when those conditions were applied during primary colony formation. However, no effect was observed when the conditions were applied during preirradiation culture and secondary colony formation. We also demonstrated that the amounts of ROS in X-ray-irradiated cells rapidly increase and then decrease at 6 hr postirradiation, and the levels of ROS then gradually decrease to a baseline within 2 weeks. The APM treatment kept the ROS production at a lower level than an untreated control. These results suggest that the cause of genetic instability might be fixed by ROS during a 2-week postirradiation period.

Radiation-induced bystander effect and adaptive response in mammalian cells.

Two conflicting phenomena, bystander effect and adaptive response, are important in determining the biological responses at low doses of radiation and have the potential to impact the shape of the dose-response relationship. Using the Columbia University charged-particle microbeam and the highly sensitive AL cell mutagenic assay, we show here that non-irradiated cells acquire mutagenesis through direct contact with cells whose nuclei have been traversed with a single alpha particle each. Pretreatment of cells with a low dose of X-rays four hours before alpha particle irradiation significantly decreased this bystander mutagenic response. Results from the present study address some of the fundamental issues regarding both the actual target and radiation dose effect and can contribute to our current understanding in radiation risk assessment.

Mutagenic effect of low energy neutrons on human chromosome 11.

PURPOSE: The shape of the dose-effect curve for neutrons, i.e. the question as to whether the curve is linear or supralinear in the low-dose region, is still not clear. Therefore, the mutagenic effect of very low doses of low-energy neutrons was determined. MATERIALS AND METHODS: Human-hamster hybrid A(L) cells contain human chromosome 11, which expresses the membrane protein CD59. This membrane protein can be detected immunologically and quantified by flow cytometry. The A(L) cells were irradiated with neutrons of 0.565, 2.5 or 14.8 MeV and the results were compared with those after 200 kVp X-rays. Before irradiation, cells spontaneously mutated in the CD59 gene were removed by magnetic cell sorting (MACS). RESULTS: The relative biological effectiveness (RBE) for CD59 mutation induction was 19.8 (+/-2.7) for 0.565 MeV, 10.2 (+/-1.9) for 2.5 MeV, and 10.2 (+/-1.6) for 14.8 MeV neutrons. Linear mutation responses were obtained with all radiations except for 14.8 MeV neutrons where a supralinear curve may be a better fit. The deletion spectrum of mutated cell clones showed 29 Mbp deletions on average after irradiation with 0.069 Gy of 0.565 MeV neutrons. This scale of deletions is similar to that after 3 Gy 100 kV X-rays (=34 Mbp). For 50% cell survival, the RBE of the neutrons was 11 compared with 200 kV X-rays. CONCLUSIONS: Neutrons of low energies (0.565 or 2.5 MeV) produce a linear dose-response for mutation in the tested dose range of 0.015-0.15 Gy. The neutron curve of 14.8 MeV can be approximated by a curvilinear or linear function.

Induction of a bystander chromosomal damage of He-ion microbeams in mammalian cells.

We report here a bystander effect in chromosomal damage using He-ion microbeam. Human-hamster hybrid cells were irradiated with a precision He-ion microbeam generated by the Columbia microbeam system. When 20% of the cells were exposed to single He ion, the incidence of cells with chromatid-type breaks detected with the PCC technique was covered wide range from 0 to 6 breaks per cell. In contrast, the distribution showed a mixed two-peak pattern, such as non-exposed and all-cell exposed patterns, under the condition of assuming no bystander effect by treating with an effective inhibitor of cell-cell communication. These findings provide clear evidence that single He-ion irradiated cells can induce bystander chromosomal alterations in neighboring cells not directly hit by He ion.

Interaction between radiation-induced adaptive response and bystander mutagenesis in mammalian cells.

Two conflicting phenomena, the bystander effect and the adaptive response, are important in determining biological responses at low doses of radiation and have the potential to have an impact on the shape of the dose-response relationship. Using the Columbia University charged-particle microbeam and the highly sensitive AL cell mutagenic assay, we reported previously that nonirradiated cells acquired mutagenesis through direct contact with cells whose nuclei had previously been traversed with either a single or 20 alpha particles each. Here we show that pretreatment of cells with a low dose of X rays 4 h before alpha-particle irradiation significantly decreased this bystander mutagenic response. Furthermore, bystander cells showed an increase in sensitivity after a subsequent challenging dose of X rays. Results from the present study address some of the pressing issues regarding both the actual target size and the radiation dose response and can improve on our current understanding of radiation risk assessment.

Secondary acute myelogenous leukemia with MLL gene rearrangement following radioimmunotherapy (RAIT) for non-Hodgkin's lymphoma.

Targeted therapy with conjugated and unconjugated monoclonal antibodies for non-Hodgkin's lymphoma has revolutionized the approach to this disease. The efficacy and low toxicity of these agents have allowed introduction of this strategy in the early stages of therapy. Longer follow-up is needed before validating the safety of these agents. Since monoclonal antibodies are being given as front-line therapy, it is important to identify all potential adverse events. We report a case of secondary acute myelogenous leukemia (AML) with 11q23 cytogenetic abnormality and mixed lymphoid leukemia (MLL) gene expression in a patient treated with Y90 labeled anti-CD20 antibody (Zevalin). The patient was not exposed to topoisomerase II inhibitors. Our observations suggest a relationship between 11q23 leukemia and radioimmunotherapy (RAIT) and further studies are needed.

Comparison of X-ray dose-response curves obtained by chromosome painting using conventional and PAINT nomenclatures.

PURPOSE: The compare the suitability of PAINT and conventional nomenclature systems for the construction of chromosome aberration dose-effect curves for X-rays using FISH techniques, and to compare these curves with those based on solid-stained dicentrics analysed in first division metaphases by the FPG technique. MATERIALS AND METHODS: Blood samples were irradiated at 0.1, 0.25, 0.50, 0.75, 1, 1.5, 2, 3, 4 and 5 Gy 180 kV X-rays. FISH painting was performed using probes for chromosomes 1, 4 and 11 in combination with a pan-centromeric probe. RESULTS: Translocations showed a higher background frequency than dicentrics. This influences the ratio of translocations:dicentrics at the lower doses and the uncertainties of dose-effect curves for translocations. The dose-effect curves for dicentrics obtained by FISH and solid stain were in close agreement. CONCLUSION: For short-term biological dosimetry purposes by FISH, the use of dic(BA) (PAINT nomenclature) or total dicentrics (conventional nomenclature) should give similar dose estimates. For dose reconstruction, the use of total or complete translocations result in similar uncertainties.

Analysis of mutant quantity and quality in human-hamster hybrid AL and AL-179 cells exposed to 137Cs-gamma or HZE-Fe ions.

We measured the number of mutants and the kinds of mutations induced by 137Cs-gamma and by HZE-Fe (56Fe [600 MeV/amu, LET = 190 KeV/micrometer) in standard AL human hamster hybrid cells and in a new variant hybrid, AL-179. We found that HZE-Fe was more mutagenic than 137Cs-gamma per unit dose (about 1.6 fold), but was slightly less mutagenic per mean lethal dose, DO, at both the S1 and hprt- loci of AL cells. On the other hand, HZE-Fe induced about nine fold more complex S1- mutants than 137Cs-gamma rays, 28% vs 3%. 137Cs-gamma rays induced about twice as many S1- mutants and hprt-mutants in AL-179 as in AL cells, and about nine times more of the former were complex, and potentially unstable kinds of mutations.

A panel of transferable fragments of human chromosome 11q.

Cytogenetic and molecular studies have implicated one or more tumor suppressor genes on the long arm of human chromosome 11 in the malignant progression of several human solid tumors, including malignant melanoma and carcinomas of the breast, cervix, ovary, and lung. Microcell-mediated chromosome transfer of an intact copy of chromosome 11 into tumor cell lines has provided additional evidence of tumor suppressor gene function in melanoma, breast cancer, and cervical cancer. However, sublocalization of the region(s) conferring the tumor suppressive effect has been difficult. To facilitate mapping of tumor suppressor gene(s) on chromosome 11, we have generated a panel of 25 mouse donor cell lines containing neo-tagged fragments of human chromosome 11q which can be transferred into cell lines to test for tumor suppressor activity. The chromosome fragments in these cell lines have been characterized by fluorescence in situ hybridization with probes to human DNA and to the centromere of chromosome 11, and also by analysis of microsatellite markers spanning chromosome 11. Finally, to demonstrate the usefulness of these cell lines as donors for microcell-mediated chromosome transfer, two fragments were transferred into the human melanoma cell line UACC 903. This panel of selectable subchromosomal fragments, derived from the long arm of human chromosome 11, will be useful for the regional localization of tumor suppressors and other genes by means of functional assays.

A low, adaptive dose of gamma-rays reduced the number and altered the spectrum of S1- mutants in human-hamster hybrid AL cells.

We examined the effects of a low, adaptive dose of 137Cs-gamma-irradiation (0.04 Gy) on the number and kinds of mutants induced in AL human-hamster hybrid cells by a later challenge dose of 4 Gy. The yield of S1- mutants was significantly less (by 53%) after exposure to both the adaptive and challenge doses compared to the challenge dose alone. The yield of hprt- mutants was similarly decreased. Incubation with cycloheximide (CX) or 3-aminobenzamide largely negated the decrease in mutant yield. The adaptive dose did not perturb the cell cycle, was not cytotoxic, and did not of itself increase the mutant yield above background. The adaptive dose did, however, alter the spectrum of S1- mutants from populations exposed only to the adaptive dose, as well as affecting the spectrum of S1- mutants generated by the challenge dose. The major change in both cases was a significant increase in the proportion of complex mutations compared to small mutations and simple deletions.

Human chromosome-specific changes in a human-hamster hybrid cell line (AL) assessed by fluorescent in situ hybridization (FISH).

PURPOSE: To quantitatively assess all gamma-ray induced chromosomal changes confined to one human chromosome using fluorescence microscopy and in situ hybridization with a fluorescently labeled human chromosome specific nucleic acid probe. METHODS AND MATERIALS: Synchronized human-hamster hybrid cells containing human chromosome 11 were obtained by a modified mitotic shake-off procedure. G1 phase cells (> 95%) were irradiated with 137Cs gamma rays (0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, and 10.0 Gy) at a dose rate of 1.1 Gy/min and mitotic cells collected 16-20 h later; chromosomal spreads were prepared, denatured, and hybridized with a fluorescein-tagged nucleic acid probe against total human DNA. Chromosomes were examined by fluorescence microscopy and all categories of change involving the human chromosome 11 as target, recorded. RESULTS: Overall, of the 3104 human-hamster hybrid cells examined, 82.1% were euploid, of which 88.6% contained one copy of human chromosome 11, 6.2% contained two copies, and 5.2% contained 0 copies. This is compatible with mitotic nondisjunction in a small fraction of cells. Of the remaining 17.9% of cells, 85.2% were tetraploid cells with two copies of human chromosome 11. For all aberrations involving human chromosome 11 there was a linear relationship between yield and absorbed dose of 0.1 aberrations per chromosome per Gy. The yield of dicentrics, translocations, and terminal deletions that involve one lesion on the human chromosome was linear, while the yield of interstitial deletions that arise from two interacting lesions on the human chromosome was curvilinear. The frequencies of dicentrics and translocations were about equal, while there was a high (40-60%) incidence of incomplete exchanges between human and hamster chromosomes. CONCLUSIONS: Fluorescent in situ hybridization (FISH) procedures allow for the efficient detection of a broad range of induced changes in target chromosomes. Symmetrical exchanges induced in G1 (translocations) were readily scored and found to equate with the complementary asymmetrical exchanges (dicentrics). That is, nonlethal stable changes, which might be of concern in carcinogenic processes, complement lethal, unstable changes. Interstitial deletions that may contribute to the loss of antioncogenes as well as to lethality are also readily detected with enhanced levels detected at higher doses. The high level of induced terminal deletions and of incomplete dicentrics and translocations indicate a partial failure of interaction between lesions induced in human and hamster DNA, and suggest that such interspecies interactions lack the fidelity of intraspecies DNA lesion interactions. This suggests caution in the use of such model systems as indicators of human cell responsiveness.

Molecular analysis of mutagenesis by high LET radiation.

Mutation induction by high linear energy transfer [LET] alpha particles and gamma-rays was scored in the human hamster hybrid [AL] cells. Southern blotting technique was used to analyse the molecular changes in the DNA from both the HGPRT- and S1- mutants. Dose dependent mutagenesis in the AL cells irradiated with the charged particles was higher by almost 20 fold at the S1 than the corresponding HGPRT locus. Southern analysis of the mutants induced by the high LET particles showed mostly multilocus deletion at both the HGPRT and S1 genes.

In vitro radiation-induced neoplastic progression of low-grade uroepithelial tumors.

Recent interest has focused on the identification of molecular genetic mechanisms in multistep neoplastic transformation. In vitro exposure of simian virus 40 (SV40)-immortalized human uroepithelial cells (SV-HUC) that are environmentally relevant to bladder carcinogens has been shown to produce tumorigenic transformation, as assessed by the ability of cells exposed to a carcinogen to form xenograph tumors with heterogeneous cancer phenotypes ranging from very aggressive, invasive high-grade carcinomas to superficial low-grade indolent tumors. In addition, exposure of a low-grade indolent tumor generated in the SV-HUC system, MC-T11, to the same carcinogens results in neoplastic progression as assessed by the production of high-grade aggressive cancers. In the present study, we show neoplastic progression of MC-T11 after in vitro exposure to a single dose of 6 Gy X rays. In addition, we show that the chromosome deletions, including losses of 4q, 11p, 13q and 18, observed in these radiation-induced tumors are similar to those observed in carcinogen-induced tumors, thus supporting the hypothesis that the experimental cell system, not the transforming agent, dictates the genetic losses required for tumorigenic transformation and progression.

Radiation-induced damage, repair and exchange formation in different chromosomes of human fibroblasts determined by fluorescence in situ hybridization.

We have used fluorescence in situ hybridization with whole-chromosome probes for human chromosomes 1, 4, 8 and 13 to investigate the extent to which the induction of damage and its repair after exposure to ionizing radiation is distributed randomly among these chromosomes. All the studies were performed with AG1522 human fibroblasts irradiated with 6 Gy and maintained in a nondividing state for at least 6 h after irradiation except for the measurements of initial damage. The extent of initial damage was determined by fusion of the cells immediately after irradiation with metaphase HeLa cells to obtain premature chromosome condensation (PCC). Breaks and exchanges were also scored by PCC 24 h after irradiation and in metaphase spreads at the first division after irradiation. The data obtained were consistent with random breakage and repair in these chromosomes. Comparing PCC 24 h after irradiation with first metaphase, there was a deficit in aberrations at metaphase, particularly in unrejoined breaks, implying loss or slowing of cells containing aberrations prior to the first division. An analysis of dicentrics and translocations in chromosome 4 at first and in subsequent divisions showed that there was an equal number of dicentrics and translocations at first metaphase with loss of dicentrics, but no loss of translocations in subsequent divisions. These data are supportive of the hypothesis tht the total number of chromosome aberrations in cells can be estimated from a single chromosome pair.

A novel general strategy for cloning tumor suppressor genes using radiation-reduced chromosomal superfragments.

To identify the tumor suppressor gene on human chromosome 11p15, we generated mouse microcell hybrids containing small transferable chromosome 11p15 fragments, which we have termed "DNA superfragments". These hybrids will be used to identify which fragments contain a tumor suppressor gene by direct transfer of the fragments to tumor cells via microcell fusion.

A radiation hybrid map of the proximal long arm of human chromosome 11 containing the multiple endocrine neoplasia type 1 (MEN-1) and bcl-1 disease loci.

We describe a high-resolution radiation hybrid map of the proximal long arm of human chromosome 11 containing the bcl-1 and multiple endocrine neoplasia type 1 (MEN-1) disease gene loci. We used X-ray irradiation and cell fusion to generate a panel of 102 hamster-human somatic cell hybrids containing fragments of human chromosome 11. Sixteen human loci in the 11q12-13 region were mapped by statistical analysis of the cosegregation of markers in these radiation hybrids. The most likely order for these loci is C1NH-OSBP-(CD5/CD20)-PGA-FTH1-COX8-PYGM -SEA-KRN1-(MTC/P11EH/HSTF1/INT2)-GST3- PPP1A. Our localization of the human protooncogene SEA between PYGM and INT2, two markers that flank MEN-1, suggests SEA as a potential candidate for the MEN-1 locus. We map two mitogenic fibroblast growth factor genes, HSTF1 and INT2, close to bcl-1, a mapping that is consistent with previously published data. Our map places the human leukocyte antigen genes CD5 and CD20 far from the bcl-1 locus, indicating that CD5 and CD20 expression is unlikely to be altered by bcl-1 rearrangements. PPP1A, which has been postulated as a MEN-1 candidate tumor suppressor gene, and GST3, a gene transcriptionally active in many human cancers, both map distal to the bcl-1 translocation cluster and the region containing MEN-1, and therefore are unlikely to be directly involved in bcl-1 or MEN-1.