NOTE: Received NCE to 12/31/2008 (from 9/30/2008) per J. Dardano/JSC (9/2008)

The results from the large scale mouse irradiation leukemogenesis study were complemented by cytogenetic studies of HZE irradiated mouse bone marrow cells. Deletions in chromosome 2 encompassing the PU.1 (Sfpi1 in the mouse) gene are characteristic in murine radiation-induced myeloid leukemias and are likely the initiating events for this type of cancer. In mouse strains that are susceptible to radiation-induced AML, bone marrow cells bearing chromosome 2 deletions can be found one day after radiation exposure and these cells and their descendants persist in the bone marrow, probably for the life of the mouse. Bone marrow cells with chromosome 2 deletions are also evident soon after irradiation of mouse strains that are resistant to radiation-induced AML, but in these strains cells with chromosome 2 deletions disappear within a month. We found that the RBE for persistent chromosome 2 deletions in 1 GeV 56Fe irradiated bone marrow cells in CBA mice was about 1. We also found that bone marrow cells with 56Fe-induced chromosome 2 deletions did not persist in, an AML resistant mouse strain, C57BL/6.

With the availability of leukemic material from AML affected mice we were able to begin the characterization of 1 GeV 56Fe and gamma-ray induced leukemias. The leukemias were assayed for chromosome 2 deletions, mutations in the non-deleted PU.1 gene, microsatellite instability, gene expression, and genomic loss or gain. The data from these assays are currently being analyzed and prepared for publication. However, a preliminary review of the data suggests that 1 GeV 56Fe and gamma-ray induced leukemias have similar molecular and cytogenetic aberrations, pointing to a similar mechanism of induction. AMLs induced by these different radiations cannot be distinguished from one-another by their gene expression profiles.

An additional, smaller scale mouse HZE irradiation study was initiated in April 2007. Twelve-week-old female BALB/cByJ mice were irradiated with 0.2 Gy of 1 GeV 56Fe ions, 0.5 Gy of 137Cs gamma-rays, or left unirradiated. There were approximately 100 mice per group. These mice were monitored to 800 days of age. All of the mice in the study have been euthanized either because they became moribund or because they reached 800 days. Tissues have been collected from the mice and are being examined by histopathology. The experiment was designed to provide preliminary data on whether the spectrum of tumors that arise in gamma-irradiated female BALB/c mice (mammary, ovarian, and lung tumors) also arise in 1 GeV 56Fe ion irradiated mice of the same sex and strain, and to determine if the high risk for HCC seen in HZE irradiated male CBA mice is also found in HZE irradiated female BALB/c mice.

In order to develop a biological model for radiation-induced AML, the cell type targeted by radiation for neoplastic transformation must be identified. Whether radiation-induced AML arises from a hematopoietic stem cell or a more restricted progenitor of myeloid lineage is currently unknown. We explored this question by using the PU.1 encompassing chromosome 2 deletion in irradiated bone marrow cells as an early marker for potential radiation-induced AML. We performed immunophenotyping combined with in situ hybridization (immunoFISH) to study the persistence of PU.1 containing chromosome 2 deletions in hematopoetic stem cells and lineage specific progenitor cells. Bone marrow cells were harvested from CBA/CaJ mice irradiated with 3 Gy of 137Cs ?-rays 1, 3 and 6 months post-irradiation and assayed by immunoFISH for PU.1 deletions and cell differentiation markers. The preliminary data demonstrated that the frequency of PU.1 deletions were similar in different cell types 1 month after irradiation but increased in the myeloid lineage and decreased in the lymphoid lineage at 3 and 6 months post-irradiation. This result indicates that radiation-induced AML is likely to originate from the more restricted progenitor of the myeloid lineage.

The original funding period for the Leukemogenesis NSCOR ended on September 30, 2008 and was followed by extensions to the end of July 2009. We took advantage of this period to undertake small, short duration experiments and to develop methodology that would be useful in a renewed of the NSCOR program. This work included a preliminary screen of Ras and Raf mutations in HCC arising in gamma-ray and 56Fe ion irradiated mice, and unirradiated controls. Codons 12, 13 and 61 of H-ras, K-ras and N-ras, and codon 624 of B-Raf were sequenced in representative tumors because these are mutation “hotspots” in chemically induced HCC. H-ras codon 61 mutations were detected in 5 of 6 tumors from unirradiated mice, 1 of 6 tumors from gamma-ray exposed mice, and 5 of 12 tumors from 56Fe irradiated mice. B-Raf codon 624 mutations were detected in one tumor each from gamma-ray and 56Fe ion irradiated mice.

We also developed a PCR-based assay capable of detecting rare bone marrow cells with point mutations in codon 235 of the PU.1 gene in irradiated mice. A second assay based on laser scanning cytometry is also under development. PU.1 codon 235 mutations are found in about 85% of murine radiation-induced AMLs with chromosome 2 deletion and their occurrence is thought to be a key step in leukemogenesis. Once perfected, these assays will allow us to determine the timing of the codon 235 mutation, the cells affected, and whether the mutation occurs preferentially in cells that have suffered a chromosome 2 deletion.

NOTE: Received NCE to 12/31/2008 (from 9/30/2008) per J. Dardano/JSC (9/2008)

a) In mice, 1 GeV/n 56Fe ions do not appear to be substantially more effective than gamma rays for induction of AML

b) In contrast, 56Fe irradiated mice had much higher risk for hepatocellular carcinoma than gamma ray irradiated mice

c) Chromosome 2 deletions detected in bone marrow cells one month post-irradiation may be a biomarker for AML risk

d) There are no apparent differences between 56Fe and gamma ray induced AML at the cytogenetic or molecular level

Project 1. Induction of AML in CBA/CaJ mice (project leader, Dr. Robert. L. Ullrich). This project is designed to quantitatively compare the leukemogenic effects of irradiation with gamma-rays, select HZE particles, and protons using the CBA/CaJ murine model of AML. These experiments are designed to provide sufficient quantitative data to permit the determination of their relative effectiveness by comparing slope constants obtained over specified dose ranges rather than fully defining the dose response relationships for each of these radiations.

Project 2. Quantification and characterization of specific cytogenetic changes associated initiation, progression, and development of AML (project leader, Dr. Joel S. Bedford). RBEs for a particular radiation quality can depend very much on a number of factors such as the biological effect used for comparisons, as well as the level of effect and conditions such as chemical environment. Likewise, carcinogenesis in general depends on a number of factors including not only initiating events, but also a number of tissue and organ specific conditions. Potential modifying effects associated with tissue and organ specific effects may well differ between mouse and man. On this basis, quantification and characterization of specific cytogenetic changes that are known to be important early events, and understanding effects on persistence and expansion of cells containing such alterations should be an important bridge between the mouse and human AML. Experiments in Project 2 are designed to develop such quantitative information on early events and characterize progression as a function of radiation quality.

Project 3. Molecular and cytogenetic fingerprints and biomarkers. (project leaders, Drs. Joel Bedford, Susan Bailey, Wei-Wen Cai, and Michael Story). The goals of Project 3 deal with investigations aimed at gaining a better understanding of factors at the cell and molecular level that may be involved in radiation-induced murine AML in general, and for HZE radiation exposures in particular. The overall long-term focus of the studies in Project 3 is on developing comparative information on radiation leukemogenesis in mice and humans. The studies outlined are subdivided into five sub-projects. Project 3A will investigate the possible role of the structural organization of chromatin in cytogenetic damage associated with AML. Project 3B will utilize microarray-based comparative genomic hybridization of normal vs. tumor DNA to identify regions of genomic deletions and amplifications common to the radiation-induced AML. Project 3C will investigate the possibility that HZE radiation induces chromosomal instabilities in the clonal progeny of surviving bone marrow cells after mice are exposed to HZE radiations. This sub-project also would investigate the long term stability of complex aberrations in such spleen colonies, since exposure to other high LET radiations suggest that even for low dose exposures, more than 90% of all aberrations are complex. Project 3D will investigate changes in gene expression by array technologies, in leukemogenic or pre-leukemogenic cells with a focus on changes in expression that may correlate or track with genomic changes seen in other studies in this program of studies.

Project 4. Molecular and cytogenetic targets in murine and human AML. (Project leaders, Drs. Joel Bedford, Robert Ullrich, Lalitha Nagarajan, and John Belmont). The goals of Project 4 are to identify common cytogenetic and molecular targets in mice and humans that are involved in the development of radiation-induced AML. Studies in this project are closely integrated with studies in Project 3 that focus on cell and molecular factors of importance for murine leukemogenesis. Project 4 provides the link between murine and human AML that will be necessary to extrapolate from mouse studies to estimate human risks. Project 4 contains two sub-projects. Project 4A, deals with more complete cytogenetic characterization of ã-ray and HZE induced murine AML, beyond the known chromosome 2 deletion, and aims to compare mouse and human cytogenetic changes involved in leukemogenesis. The goal of this comparison is to identify common syntenic regions or conserved linkage segments present at the breakpoints of translocations or deleted regions of the two species. Project 4B, involves the development of a human/mouse hybrid model of human leukemogenesis and the use of this model to quantify AML induction by HZE and gamma-rays. This project will also determine whether there are cytogenetic changes in irradiated human hematopoietic cells that may be predictive of leukemogenesis.

General Results:

The CBA mouse strain has served as a valuable model for radiation-induced acute myeloid leukemia (AML) with respect to dose response relationships as well as underlying mechanisms. These mice have a very low spontaneous incidence of AML (<1%) but have been shown to be quite sensitive to induction of AML following exposure to gamma rays, x-rays and neutrons, with an RBE of 3-5 for fission spectrum neutrons. Another advantage is that few competing causes of death have been reported to occur in irradiated CBA mice. Their spontaneous frequency of other tumors is also relatively low except for liver tumors which have a frequency of approximately 12%. However, there have been no reports of an increase in liver tumor incidence following radiation exposure. This study was designed to compare the effects of Cs137 gamma rays with 1 GeV/n iron particles on the dose response for induction of AML in male CBA mice. Doses for gamma rays were based on previous studies and ranged from 1-3 Gy. For 1 GeV/n iron, doses selected were based on the assumption that the RBE for iron would be near that for neutrons, i.e., 3-5. On this basis doses ranging from 0.1 to 1 Gy were selected. As might be expected, the RBE for life shortening was estimated to be 3.7. However, unexpectedly, the RBE for AML induction by 1 GeV/n iron particles approximated 1. In addition, unlike previous studies and the present study which found very little effect of gamma-ray irradiation on the frequency of hepatocellular carcinomas, a large increase in the incidence of these tumors was found after iron irradiation where an approximate RBE of 35 was observed. These data suggest a difference in effects of HZE iron ions on the induction of leukemia compared to solid tumors suggesting potentially different mechanisms of tumorigenesis. The basis for these differences and the impact of these results on risk estimates for space travel are under investigation using cytogenetic and molecular endpoints including chromosome aberrations, expression profiling, comparative genomic hybridization (CGH), and microsatellite instability. These data are summarized below.

Bone marrow cells from CBA are more sensitive than C57BL for early PU.1 loss for both HZE Fe and g-ray exposures. PU.1 loss reasonably follows expectations from gross aberration induction. At later times after either radiation, levels of PU.1 loss return to background levels in C57BL but not CBA mice. PU.1 loss at 1 month is suggested as a biomarker for sensitivity to AML induction. Radiation-induced chromosomal instability, using delayed chromatid-type aberrations as a biomarker in a spleen colony assay, appears higher in CBA than C57BL mice. Increased levels of chromatid aberrations were seen in AMLs arising from both radiations. No instabilities resulting in chromosomal translocations were seen in spleen colonies. Evidence of microsatellite instabilities was seen in AMLs and in spleen colonies for both radiation types. Development of a system for studying changes in human bone marrow cells in humanized NOD/SCID mice appears feasible. Analysis with CGH have identified two regions with consistent changes in murine AML and alterations in gene expression in these regions confirms this observation. Studies are also directed at human AML with sample from individuals with radiation-induced AML following therapy and using a humanized mouse model. Both of which have shown promising results.

The key findings of this project to date are: 1) In mice, 1 GeV/n 56Fe ions do not appear to be substantially more effective than gamma-rays; 2) In contrast, 56Fe irradiated mice had much higher risk for hepatocellular carcinoma than gamma-ray irradiated mice; 3) Chromosome 2 deletions detected in bone marrow cells one month post-irradiation may be a biomarker for AML risk; 4)There are no apparent differences between Fe and gamma-ray induced AML at the cytogenetic or molecular level.

18th Annual NASA Space Radiation Investigators’ Workshop, Rohnert Park, California, July 13-15, 2007. p 11. , Jul-2007

18th Annual NASA Space Radiation Investigators’ Workshop, Rohnert Park, California, July 13-15, 2007. , Jul-2007

Thirteenth International Congress of Radiation Research, San Francisco, California, July 8-12 2007. , Jul-2007

Thirteenth International Congress of Radiation Research, San Francisco, California, July 8-12 2007. , Jul-2007

Thirteenth International Congress of Radiation Research, San Francisco, California, July 8-12 2007. , Jul-2007

Gravitational and Space Biology 2007 Oct;21(1):40. , Oct-2007

In project 1, this program will provide quantitative data on the relative effectiveness of protons, specific HZE particles, and gamma rays on the induction of AML in a well-defined murine model that has been shown to have substantial similarities to human AML both with respect to dose response characteristics for induction after radiation exposure and to its pathogenesis. Information on effects on latency will also be obtained. An advantage of the murine AML model is that there is already information on potential initiating lesions. Quantification of these early events, their transmissibility and on progression of initiated cells is invaluable in the extrapolation of results from murine to human AML.

Project 2 focuses on RBE estimates for induction of initial lesions involving chromosome 2 and examine the impact of different radiation qualities on the persistence, clonal expansion and progression of these initiated cells.

Projects 3 and 4 of this program will provide the fundamental knowledge needed to facilitate the application of the data derived from projects 1 and 2 for human risk estimates.

Project 3 is designed to identify, quantify, and characterize cytogenetic and molecular targets and early biomarkers in murine AML. This project will also determine the impact of factors such as radiation quality, time after exposure, tissue and cell lineage specific effects, and individual susceptibility on these fingerprints and biomarkers.

Project 4 serves as the primary link between murine and human AML. The aim of project 4 is two-fold. First, to develop comparative molecular and molecular cytogenetic information on potential common targets involved in the pathogenesis of murine and human AML. Second, to develop model systems to facilitate translation of translate the results from mice to human AML. The program outlined is a dynamic scientific program focused on achieving long-term goals. As such the specific projects outlined in this proposal are meant as a starting point for a comprehensive analysis of radiation-induced AML. Based on progress and results from the studies described in this proposal, new directions will arise, projects may be completed, and new projects initiated. Changes are coordinated through consultations with our advisory groups and with appropriate NASA officials who may be involved in scientific and programmatic oversight. This year all of the investigators in the program met at CSU along with the members of the internal and external advisory committees for a comprehensive program review and to develop strategic plans for the next year.

In addition to these research projects, this program contains two cores. Core A coordinates the distribution of cells, tissues and other biological materials needed for all the projects and coordinate irradiation of animals, tissues and cells. Core C provides support to the research projects and cores within this program, to coordinate interactions among investigators and institutions, and to provide scientific and operational management and fiscal oversight.

The projects are briefly described below. Details of progress for each project during this reporting period follow this description.

Project 1. Induction of AML in CBA/CaJ mice (project leader, Dr. Robert. L. Ullrich).

This project is designed to quantitatively compare the leukemogenic effects of irradiation with gamma rays, select HZE particles, and protons using the CBA/CaJ murine model of AML. These experiments are designed to provide sufficient quantitative data to permit the determination of their relative effectiveness by comparing slope constants obtained over specified dose ranges rather than fully defining the dose response relationships for each of these radiations. Over this project a large number of animals have died or were sacrificed at 800 days of age and analysis of lifeshortening and AML frequencies are underway. We also finished dosimetry for the low dose rate (LDR) irradiation groups and are beginning irradiations. This is the time at which we should expect to begin to see AML and indeed, we are beginning to find mice with AML in these groups. Likewise for the acute gamma ray group, we are beginning to see the development of AML. Besides AML we have also observed a high frequency of liver tumors and some hemangiomas and hemangiosarcomas. The relationship of radiation dose and quality on the frequency as well as the malignancy and metastatic potential for these tumors is also being examined.

Project 2: Quantification and characterization of specific cytogenetic changes associated with initiation, progression, and development of AML (Project leader, J.S. Bedford).

The aim of Project 2 is to measure the relative biological effectiveness (RBE) of select HZE radiations relative to gamma-radiation over specified dose ranges with respect to the production of chromosomal abnormalities that have been shown in previous studies (for x-or gamma-ray exposures) to be associated with radiation induced AML in CBA/CaJ mice. For x- or gamma-radiations, virtually all (at least 95%) of the cells of leukemias that do develop in the irradiated CBA mice contain a deletion of the PU.1 gene located on chromosome 2. During the past 2.5 years we have been measuring the loss of a small (200kb) segment of mouse chromosome 2 where this gene resides, in bone marrow cells sampled periodically after irradiation in the two mouse strains for the different doses and different radiations. There is some question as to whether the dose response is linear, though it cannot be ruled out. The slope of the dose response relationship in the lower dose region was perhaps somewhat less than that seen after 1 day, but the dose response appeared to plateau for higher doses. After 1 year, we have scored only a few mice from the CBA group, but it is immediately apparent that the percent of bone marrow cells with PU.1 loss was greatly increased relative to mice sampled at both the 1 day, and especially 1 month after irradiation.

We cannot, with any certainty, explain the differences in the dose responses for cells sampled from mice at different times after irradiation. One possibility is that cells experiencing nuclear traversals by the HZE iron ions are much more likely to be eliminated from the population after several divisions, as would be the case for the samples taken 1 month after irradiation. The samples taken after 1 year, on the other hand, may result from a small growth advantage of cells that did survive. We are continuing our investigations into these possibilities.

Project 3: Molecular and cytogenetic fingerprints and biomarkers. (project leaders, Drs. Joel Bedford, Susan Bailey, Wei-Wen Cai, and Michael Story).

The goals of Project 3 deal with investigations aimed at gaining a better understanding of factors at the cell and molecular level that may be involved in radiation induced murine AML in general, and for HZE radiation exposures in particular. The overall long-term focus of the studies in project 3 is on developing comparative information on radiation leukemogenesis in mice and humans. The studies outlined are subdivided into five sub-projects.

Project 3A. Work this past year has involved laying the groundwork to investigate the possible role of the structural organization of chromatin in cytogenetic damage associated with AML. We have isolated and tested the hybridization of several BACs and other markers along mouse chromosome 2 that will be used to measure the proximity of the two breakpoint cluster regions surrounding the PU.1 gene. Using Metamorph software with a deconvolution package to simulate confocal microscopy, we can begin to measure interphase breakpoint cluster distances as previously outlined.

Project 3B. This project involves utilization of microarray-based comparative genomic hybridization of normal vs. tumor DNA to identify regions of genomic deletions and amplifications common to the radiation-induced AML. The aim is to use mouse genome tiling BAC to characterize the genomic abnormalities in the mouse leukemia or other tumor samples collected in this project. While most of the mice subjected to heavy ion radiation are still in the process of developing leukemia or other type of tumors we are currently analyzing samples from Simon Boufler’s lab in the UK. On the new tilling arrays about 60 % of the clones have an independent overlapping clone. The redundant clones are expected significantly increase the confidence level of abnormalities detected by single clones. Most significant progress has been made with regard to the automation of scanning and image quantitation. This improvement has significantly increased our array analysis throughput. Other aspects of our technology such as blocking and hybridization of arrays have also been improved over the last year benefiting from the support of this project. We expect in the coming year we will be focusing on routine analysis of the leukemia samples. The first set of histologically confirmed AML cases have recently been received and we should have some preliminary results from this sub-project soon.

Project 3C involves a study to investigate the possibility that HZE radiation induces chromosomal instabilities in the clonal progeny of surviving bone marrow cells after mice are exposed to HZE radiations. This sub-project also would investigate the long term stability of complex aberrations in such spleen colonies, since exposure to other high LET radiations suggest that even for low dose exposures, more than 90% of all aberrations are complex. We will also use another aliquot of the spleen samples for assessment of microsatellite instability. Project 3-D. Project 3-D has received 4 control samples and 10 samples from leukemic mice, 3 of which were lost because of degraded RNA. This left us with 4 normal and 7 leukemic samples. We used these samples to examine expression on our array platform (Illumina mouse whole genome). First we examined technical replicates. Within experimental samples, the biological replicates were most similar. Normal spleen samples were all found under one branch. However, leukemic samples showed the greatest diversity as a group. We will also examine signal transduction pathways when we have more samples.

Project 4. Molecular and cytogenetic targets in murine and human AML. (Project leaders, Drs. Ullrich, Bedford, Nagarajan, and Belmont).

The goals of project 4 are to identify common cytogenetic and molecular targets in mice and humans that are involved in the development of radiation-induced AML. Studies in this project are closely integrated with studies in projects 2 and 3 which focused on focus on cell and molecular factors of importance for murine leukemogenesis. Project 4 also was designed to provide a link between murine and human AML that will be necessary to extrapolate from mouse studies to estimate human risks. Project 4 contains two sub-projects.

Project 4A. This project aims to compare the mouse vs. human changes involved in leukemogenesis. Tumors have now appeared in appreciable numbers of animals from HZE irradiated and some of the gamma irradiated mice, so we are now beginning to have material for analysis of PU.1 loss along with the pathology and histology necessary for diagnosis of AML as opposed to other tumors. All the animals (CBA mice) irradiated with gamma-rays that have been diagnosed with AML have a very high percentage of PU.1 loss in cells harvested from the enlarged spleens. In very striking contrast to the results for gamma-irradiations, cells harvested from the enlarged spleens of animals irradiated with 1GeV/n iron ions that were later diagnosed with AML did not have an appreciable proportion with PU.1 loss. The interesting finding here, of course is that the AMLs resulting from the HZE radiations may arise through a different pathway. The results so far suggest an even greater need to carry out the more detailed cytogenetic analysis of chromosomal changes, possible induction of instabilities (chromosomal and microsatellite) as judged by the spleen colony assays, and the changes that may be seen in the expression arrays as well as the amplification and deletion analysis from the array CGH analyses for AMLs induced by gamma vs HZE irradiations in these mice.

Project 4B. Deletions of chromosomes 5q, 7q, 11q,17p and 20q are recurrent anomalies in human acute myelogenous leukemia (AML), secondary to radiation and or alkylating agent therapy. As part of the NSCOR, our focus in the past twelve months has been on two goals:

(I) To characterize the expression of candidate leukemia suppressor genes within the minimally deleted interval in 7q. Quantitative PCR suggests decreased expression for HIC/MDFIC, TES and CAV in AML cell lines. Interestingly these genes span the FRA7G fragile site. Among these, the transcription factor HIC/MDFIC is of particular interest because of its potential role in enhancing GMCSF sensitivity in myeloid progenitors. We will be interested in evaluating HIC, TES and CAV expression in the murine heavy ion induced AML models developed in other parts of the consortium.

(II) To characterize genetic pathway in AML patients secondary to radiotherapy for primary malignancy. This subset is distinguished from secondary AML studied by others in that these patients did not receive alkylating agents. We have examined 145 patients for recurrent, non-random anomalies. A number of interesting conclusions can be drawn from this analysis: (i) Deletions of chromosome 5 and 7 co-segregate in these patients (ii) A significant fraction of these cases (>30%) harbored no gross cytogenetic anomalies and (iii) Recurrent anomalies of chromosome 11q, found in a small subset raise a potential leukemia suppressor role for genes encoded in this interval.

COSPAR Colloquium on Space Radiation Biology, Xi’an China, July 22, 2006. , Jul-2006

Fourth International Workshop on Space Radiation Research, and 17th Annual NASA Space Radiation Health Investigators’ Workshop, Moscow–St Petersburg, June 5–9, 2006. , Jun-2006

Fourth International Workshop on Space Radiation Research, and 17th Annual NASA Space Radiation Health Investigators’ Workshop, Moscow–St Petersburg, June 5–9, 2006. , Jun-2006

Fourth International Workshop on Space Radiation Research, and 17th Annual NASA Space Radiation Health Investigators’ Workshop, Moscow–St Petersburg, June 5–9, 2006. , Jan-2008

Over this project period irradiations of mice for the acute HZE and gamma ray irradiations were completed. We are finishing dosimetry for the low dose rate (LDR) irradiation groups and will begin irradiations in the fall. In all almost 1100 mice (extra mice were added to allow for loss during travel post irradiation) have been entered into the project 1 study with HZE particles and approximately 200 are over 400 days post irradiation. This is the time at which we should expect to begin to see AML. Indeed, we are beginning to find mice with AML in these groups. Likewise for the acute gamma ray group, all 800 mice have been irradiated and we are beginning to see the development of AML.

Project 2: Quantification and characterization of specific cytogenetic changes associated with initiation, progression, and development of AML (Project leader, J.S. Bedford).

Determination of the relative biological effectiveness (RBE) of select HZE radiations relative to gamma-radiation over specified dose ranges for induction of AML in CBA/CaJ mice is well underway as outlined under project 1 above. In parallel, groups of mice exposed to the same radiation doses are being studied to follow the relative effectiveness of these radiations for the production of chromosomal abnormalities that are known to be associated with radiation induced AML in these mice, as well as the relative effectiveness for production of the same chromosome abnormalities in another strain of mice, C57/BL6, shown in previous studies to be more resistant to radiation induction of AML. Virtually all (at least 95%) of the cells of leukemias that do develop in the irradiated CBA mice contain a deletion of the PU.1 gene located on chromosome 2. The experimental plan we have been carrying out during the past year and a half involves measurement of the loss of a small segment of mouse chromosome 2 where this gene resides, in bone marrow cells sampled 24 hours, 1 month, and 1 year after irradiation in the two mouse strains for the different doses and different radiations. To this end, we have isolated and fluorescently labeled BAC clones containing the PU.1 gene; in particular we chose BAC RP23-263H8, which is197 kb in size. It is located on chromosome 2 in region 2E1, mapping at 49.3 cM or (by physical mapping) between 90,840 - 91,037 kb from the centromere. We verified that the correct probe (BAC) was isolated utilizing a PCR method with unique primers for this BAC, and the labeled BAC was then used as a FISH probe on metaphase cells from mouse bone marrow. The result from PCR verification confirms our BAC was indeed located on chromosome 2 and in the expected location.

Project 3A: Proximity of Radiation Induced Deletion Breakpoints in Mouse Chromosome 2 in Relation to Myeloid Tissue-Specific Chromatin Structure. (Joel Bedford, Yuanlin Peng, and David Maragnon)

Nikiforova and co-workers reported in 2000 that two regions on human chromosome 10 are associated with the breakpoints producing an inversion that involving fusion of part of the RET gene with the H4 gene located some 30 Mb away. This inversion occurs in an appreciable number of patients with thyroid cancer in patients who had been exposed to radiation as a result to the Chernobyl accident. Using BAC probes containing these regions, their report showed that the breakpoint regions on chromosome 10 were much closer together in normal thyroid cells than predicted, and closer than actually measured in breast epithelial cells, i.e., another tissue. Since the data of Bouffler and Cox and co-workers at Harwell in the U.K. had demonstrated both a proximal and a distal cluster of breakpoints around the PU.1 gene involved in the radiation induced leukemias of CBA mice, we hypothesized that a similar proximity of these breakpoints may be involved in the bone marrow cell precursors of the leukemias.

To test this hypothesis, we isolated additional BAC probes reported in the breakpoint regions. We hasten to add that this situation is not the exact equivalent of a precise inversion but involves a cluster, so we had planned to look at several regions in the cluster. The first step was to isolate and label probes and confirm they were indeed located on chromosome 2 in about the expected location. The first preliminary result shows a metaphase in which the PU.1 probe, already established to be located on chromosome 2, is located between two green fluorescent signals on the same chromosome. One of the green (FITC) labeled probes was from BAC RP23-1011 located at 36.8cM on chromosome 2 and proximal (nearer the centromere) to the PU.1-containing probe. The other green labeled probe was from BAC RP23-34E24 located at 56.8 cM on chromosome 2 and distal to the PU.1-containing probe. There was some indication of a more diffuse labeling and we attribute this to slightly suboptimal hybridization of these two breakpoint cluster probes at this time. The probe size after labeling by nick-translation was sub-optimal. Nevertheless, results demonstrated that the probes we have isolated are on the expected chromosome at the expected position.

Project 3B: CGH Signatures of Spontaneous and Radiation-Induced AMLs (Project Leader, W-W Cai). Our project involves using whole genome BAC arrays to analyze mouse leukemia samples generated in the project. We are ready to accept samples for analysis and have performed some analysis for AML samples from Simon Boufler’s lab in UK. In the meantime we continue to improve the quality and resolution of our mouse whole genome BAC arrays. We have recently selected a new set of tiling path clones with unique sequences at both ends. This will remove any potential chimeric clones and the map information for clone set will be reliable and relatively stable. The new set contains close to 22,000 BAC clones covering the genome in contigs with less than 300 gaps, most of which are located in the unclonable regions. The Y chromosome is also covered in this clones set. We have finished DNA preparation for the new clone set and high quality new arrays will be printed in the next two months.

Another significant development is our success in development of a new procedure for making Cot I blocking DNA. The homemade Cot I DNA works as well as the commercial Cot I DNA but the cost is significantly reduced. We are currently evaluating an automatic image quantitation software package. We expect that implementation of this package will greatly increase our sample throughput.

Project 3C: HZE Induced Chromosomal Instabilities and Complex Aberration Biomarker Stability in the Clonal Progeny of Mouse Myeloid Cells Irradiated and Assayed in vivo. (Project Leaders: S.M. Bailey and J.S. Bedford). We have carried out some preliminary studies to perfect the tail vein injections and determine the optimum dose to use for bone marrow ablation, but have not isolated HZE surviving bone marrow clonal cell populations for cytogenetics analysis.

Project 3D: Gene Expression in AML (Project Leader, M.D. Story) . At this time we await samples for processing. We have worked out the logistics associated with shipping samples and tested RNA quality as a result of shipping small tumor samples in RNAzol B. RNA quality and quantity are more than sufficient for array analysis. All components for array hybridization and data analysis are in place.

Project 4. Molecular and cytogenetic targets in murine and human AML. (Project leaders Drs. Joel Bedford and Robert Ullrich, Lalitha Nagarajan, and John Belmont).

4a. Common molecular and cytogenetic targets in murine and human AML (Project leaders Drs. Joel Bedford and Lalitha Nagarajan) Deletions of chromosomes 5q, 7q, 11q, 17p and 20q are recurrent anomalies in human acute myelogenous leukemia, secondary to radiation and or alkylating agent therapy. As part of the NSCOR, our focus in the past eighteen months has been to characterize deletion 7q. Based on the completed genomic sequence of chromosome 7, we have generated a transcript map of the critical region of loss (between the D7S525 and D7S2502 loci). Notably, a recently characterized tumor suppressor gene DOCK4 and an evolutionarily conserved zinc finger gene ZNF277 localize to this interval, head to head, within <0.5 kb of each other. A paper on this on recently been accepted for publication (Hong Liang, Patricia D. Castro, Jin Ma, and Lalitha Nagarajan. Finer Delineation and Transcript Map of the 7q31 Locus Deleted in Myeloid Neoplasms. Cancer Genet. Cytogenetics, in press). The reagents generated in this study will be valuable in elucidating the role of loss 7q31 loci in the pathogenesis AML. We will be interested in evaluating these genes in the murine heavy ion induced AML models that are being developed in other parts of the consortium.

Project 4b. Development of a mouse/human hybrid model of human leukemogenesis. (Project leader, Dr. John W. Belmont). These studies were designed to determine whether hematopoietic cells transplanted from the bone marrow of CBA strain mice to irradiated NOD/LtSz/Rag1null mice remain susceptible to radiation-induced AML and determine if transplantation affects the latency. CBA/CaJ and NOD/LtSzRag1null mice were obtained from Jackson laboratory (Bar Harbor, Maine), and colonies were established and expanded in the Baylor College of Medicine Center for Comparative Medicine Barrier facility. Currently, we are maintaining 20 breeding cages (one male and two female per cage) for the NOD/Rag strain and 12 breeding cages for the CBA/CaJ strain to have the sufficient number of age-appropriate animals to perform the experiments. The NOD/Rag strain showed particularly slow breeding habits.

Serial experiments have been performed to estimate the minimum number of CBA-CaJ Sca1+cKit+Lin- cells to be transplanted in NOD/Rag mice in order to obtain engraftment. Usually, but for different strains of mice, that number ranges from 1000 to 100,000 Sca1+cKit+ Lin-cells. Previous experience indicated that 30-50 million cells could be harvested from a murine BM (four bones collection) and that Sca1+cKit+Lin- constitute about 0.5% (about 100,000-200,000 cells/mouse). The Sca1+cKit+Lin- sorting has been optimized. Briefly, CBA/CaJ bone marrow mononuclear cells are stained with the BD Pharmigen mouse lineage panel, which contains biotinylated monoclonal antibodies to mouse CD3e, CD11b (Mac-1), CD45-B220, Ly-6G (Gr-1), and Ly-6c, and TER-119/Erythoroid cells (BD Pharmigen, Cat. No 559971). In a second step, cells are incubated with Avidin-FITC (BD Pharmigen , Cat. No. 554057), and with PE-Anti-mouse Ly-6A/E (Sca-1; Caltag Code No. MSCA04-3), and PE-Rat-Anti-CD117 (cKit; Caltag RM6204), and eventually sorted. We are recovering an average of 0.1% Sca-1+cKit+Lin- cells/mouse.

Five groups of two to ten NOD/Rag mice have received 25,000 to 50,000 CBA/CaJ Sca-1+cKit+Lin- cells by intra-tail vein injections. The recipient mice were irradiated immediately before the transplants with 4 Gy (1 Gy/min) in a 137Cs small animal irradiator. Engraftment of CBA cells in the host mice has been monitored from the third week post-transplantation by flow cytometric analysis for the marker H2-K specific of the CBA strain (NOD/LtSz/Rag1null carry the marker H2-D) from the murine peripheral blood. The technique has been optimized to reduce the level of background due to the monoclonal antibody capturing by the Fc receptor on the host cells. Briefly, NOD/Rag-transplanted-CBA/CaJ peripheral blood cells are preincubated with purified Rat anti-mouse CD16/CD32 (Fcg III/II Receptor; BD Pharmigen Cat. No. 553141) prior of the H2-K staining (BD Pharmigen Cat. No. 553592; FITC-labeled). We have observed an engraftment ranging from 2% to 16% in five NOD/Rag mice which received 50,000 CBA/CaJ cells. No engraftment was found in the mice which received 25,000 donor cells. CBA/CaJ positive-NOD/Rag mice were irradiated with 3Gy as established by the experimental procedure.

Presentations: Invited presentation at 3rd International Workshop on Space Radiation and 15th Annual NASA Space Radiation Health Investigator’s Workshop, May 16-20, 2004: Modulation of genetic effects by RNA interference of NHEJ. S.M. Bailey, J.S. Bedford and H.L. Liber

Invited presentation at NASA Bioastronautics Investigators’ Workshop, Galveston TX, January 10-12, 2005: DNA-PK knockdown by siRNA has differential quantitative effects on telomere dysfunction and mutagenesis in human lymphoblasts treated with gamma-rays or HZE particles. Qinming Zhang, Eli S. Williams, Joel S. Bedford, Howard L. Liber and Susan M. Bailey

Invited presentation at 16th Annual Space Radiation Health Investigator’s Workshop, Port Jefferson, NY, May 15-18, 2005: Modulation of Genetic Effects by RNA interference of NHEJ. Susan M. Bailey, Joel S. Bedford and Howard L. Liber

Twelfth International Congress of Radiation Research. Brisbane, Australia, 17 – 22 August 2003. , Aug-2003