Amy Kronenberg, Group Leader And Staff Scientist
Div/biochem/molecualr Biologyuniversity Of California Berkeley
2150 Shattuck Avenue, Room 313
berkeley, Ca 947045940
Grant 5R29GM043178-05 from National Institute Of General Medical Sciences IRG: RAD
Abstract: Selected heavy ions have been shown to be potent inducers of mutations in human TK6 lymphoblasts at two distinct genetic loci thymidine kinase (tk) and hypoxanthine phosphoribosyltransferase (hgprt). The kinetics for mutation induction appear to depend on the genetic context of the particular locus in addition to LET. A systematic investigation of the dependence of mutation induction on particle charge, velocity and fluence is outlined for both the hemizygous hgprt locus and the heterozygous tk locus. Specifically, the hypothesis that considerations such as gene dosage and linkage to essential genes influence not only the kinetics of induction but also the LET vs RBE response for a given genetic locus will be tested. Secondly, we will determine whether the mutation rate may saturate more readily for a hemizygous locus with respect to particle fluence than for a heterozygous locus. This may be a result of the propensity with which accelerated heavy ions induce large scale genetic changes, including extensive deletions, such that the magnitude of those alterations resulting in viable mutants is limited by genetic linkage. This assumption will be tested by examining the spectrum of DNA structural alterations in clonally-derived populations of tk-/- or hgprt- mutants arising in cultures exposed to a single particle beam. Intragenic and multilocus change will be examined to determine the magnitude and polarity of structural alterations. The effect of particle fluence on the spectrum of DNA structural changes for each locus will be examined. The dependence of the mutation spectrum for a given locus on the absorbed dose to the nucleus will also be explored. Finally, the possibility that cryptic lethal alleles linked to the active tk or hgprt alleles of TK6 cells influence the spectrum of recoverable mutants will be examined by introducing an additional copy of chromosome 17 containing the gene for neomycin resistance into mutant TK6 subclones harboring large deletions of active tk or hgprt sequences. Neo+ revertants to tk+, or revertants to hgprt+ will be remutagenized with a heavy-ion beam. The spectrum of DNA structural alterations in neo+/tk- or newly made hgprt- lines will be compared with those obtained for tk- or hgprt- mutants of wild-type TK6 cells after exposure to the same heavy-ion beam. These studies will contribute to our understanding of the influence of genetic constraints on possible mechanisms of mutagenesis in human cell by densely ionizing radiations
Keywords: ionizing radiation, linear energy transfer, mutation, particle beam B lymphocyte, DNA, allele, chromosome, chromosome aberration, chromosome deletion, complementary DNA, cytogenetics, drug resistance, heterozygote, high energy particle, hypoxanthine phosphoribosyltransferase, linkage mapping, lymphoblast, mutant, neomycin, nucleic acid structure, particle accelerator, radiation genetics, relative biological effectiveness, thymidine kinase molecular cloning, nucleic acid probe, tissue /cell culture
Project start date: 1989-12-01
Project end date: 1995-03-31
5R29GM043178-05 (1994): $77511
Sponsored Links Excellgen http://Excellgen.com
Amy Kronenberg, Group Leader And Staff Scientist
University Of California Berkeley 2150 Shattuck Avenue, Room 313 Berkeley, Ca 947045940
Grant 5R29GM043178-04 from National Institute Of General Medical Sciences IRG: RAD
Abstract: Selected heavy ions have been shown to be potent inducers of mutations in human TK6 lymphoblasts at two distinct genetic loci thymidine kinase (tk) and hypoxanthine phosphoribosyltransferase (hgprt). The kinetics for mutation induction appear to depend on the genetic context of the particular locus in addition to LET. A systematic investigation of the dependence of mutation induction on particle charge, velocity and fluence is outlined for both the hemizygous hgprt locus and the heterozygous tk locus. Specifically, the hypothesis that considerations such as gene dosage and linkage to essential genes influence not only the kinetics of induction but also the LET vs RBE response for a given genetic locus will be tested. Secondly, we will determine whether the mutation rate may saturate more readily for a hemizygous locus with respect to particle fluence than for a heterozygous locus. This may be a result of the propensity with which accelerated heavy ions induce large scale genetic changes, including extensive deletions, such that the magnitude of those alterations resulting in viable mutants is limited by genetic linkage. This assumption will be tested by examining the spectrum of DNA structural alterations in clonally-derived populations of tk-/- or hgprt- mutants arising in cultures exposed to a single particle beam. Intragenic and multilocus change will be examined to determine the magnitude and polarity of structural alterations. The effect of particle fluence on the spectrum of DNA structural changes for each locus will be examined. The dependence of the mutation spectrum for a given locus on the absorbed dose to the nucleus will also be explored. Finally, the possibility that cryptic lethal alleles linked to the active tk or hgprt alleles of TK6 cells influence the spectrum of recoverable mutants will be examined by introducing an additional copy of chromosome 17 containing the gene for neomycin resistance into mutant TK6 subclones harboring large deletions of active tk or hgprt sequences. Neo+ revertants to tk+, or revertants to hgprt+ will be remutagenized with a heavy-ion beam. The spectrum of DNA structural alterations in neo+/tk- or newly made hgprt- lines will be compared with those obtained for tk- or hgprt- mutants of wild-type TK6 cells after exposure to the same heavy-ion beam. These studies will contribute to our understanding of the influence of genetic constraints on possible mechanisms of mutagenesis in human cell by densely ionizing radiations.
Keywords: ionizing radiation, linear energy transfer, mutation, particle beam, B lymphocyte, DNA, allele, chromosome, chromosome aberration, chromosome deletion, complementary DNA, cytogenetics, drug resistance, heterozygote, high energy particle, hypoxanthine phosphoribosyltransferase, linkage mapping, lymphoblast, mutant, neomycin, nucleic acid structure, particle accelerator, radiation genetics, relative biological effectiveness, thymidine kinase, molecular cloning, nucleic acid probe, tissue /cell culture
Project start date: 1989-12-01
Project end date: 1994-11-30
5R29GM043178-04 (1993): $115308
5R29GM043178-03 (1992): $118039
Grants awarded to Amy Kronenberg
RADIATION--DELAYED MUTATION & INSTABILITY IN HUMAN CELLS
Amy Kronenberg, Group Leader And Staff Scientist
Division Of Life Sciencesuniversity Of Calif-lawrenc Berkeley Lab
c/o Sponsored Projects Office
berkeley, Ca 94720
Grant 1R01CA062364-01A1 from National Cancer Institute IRG: RAD
Abstract: The goal of the proposed work is to determine whether the phenomenon known as delayed mutation occurs in human cells exposed, in vitro, to ionizing radiation. Traditional mutation assays evaluate the induction of mutations at a single snapshot in time. If radiation exposure induces a persistent instability in the genome, traditional assays may underestimate the risk of heritable alterations in somatic cells. The hypothesis to be tested is that delayed mutations arise as a result of the induction of genomic instability. Initial studies will be performed using an immortal, near-diploid human B-lymphoblastoid cell line, TK6. Subsequent studies will be performed using normal human lymphocytes. To determine whether delayed lethal mutations arise, serial sampling of the plating efficiency of irradiated cells will be performed. To determine the persistence of this phenotype, colonies arising after the initial post-irradiation plating will be subcloned in order to determine their subcultivation efficiency. We will also determine whether radiation exposure induces a persistent elevation in specific locus mutations in the progeny of irradiated cells. The candidate loci to be tested are the hypoxanthine phosphoribosyltransferase (hprt) locus and, where possible, the thymidine kinase locus (tk) and selected VNTR and microsatellite repeat loci. The spectrum of DNA structural alterations found in clonally derived hprt mutants exhibiting the delayed mutation phenotype will be determined using Southern blotting techniques or PCR-based assays to ascertain whether they resemble spontaneous mutants or whether they have a unique signature associated with the induction of instability. A separate measure of genomic instability, the ability to amplify the CAD gene, will also be performed on the cells chosen for the abovementioned studies. We will carry out somatic cell hybridization experiments to determine whether the delayed mutation phenotype in certain irradiated progeny of Chinese hamster ovary cells can be suppressed by human chromosomes. We also seek to determine whether delayed mutation is a process which is invoked in human cells only when the pathway for programmed cell death is abrogated. The overall goal of these studies is to determine whether radiation exposure induces a persistent instability in the genome of human cells, and to disclose whether a predisposition to instability is required for the delayed mutation phenotype to be evident
Keywords: gene expression, gene mutation, ionizing radiation, radiation genetics chemical stability, enzyme inhibitor, genome, lymphocyte, nucleic acid structure, phenotype, programmed cell death, protein tyrosine kinase CHO cell, human tissue, immortalized cell, nucleic acid hybridization, polymerase chain reaction, southern blotting
Project start date: 1994-07-01
Project end date: 1997-06-30
1R01CA062364-01A1 (1994): $164284
Radiation-Induced Mutagenesis And Apoptotic Regulation
Amy Kronenberg, Group Leader And Staff Scientist
University Of Calif-lawrenc Berkeley Lab C/o Sponsored Projects Office Berkeley, Ca 94720
Grant 5R01CA092277-04 from National Cancer Institute IRG: RAD
Abstract: Programmed cell death (PCD) is a fundamental process that eliminates extraneous or potentially dangerous cells. Many human tumors have found ways to evade PCD. We discovered a novel role for PCD in limiting mutagenesis in human cells. Over-expression of BCL-2 or BCL-X L increased x-ray-induced mutation at the autosomal TK1 locus in TK6 cells. We also saw an increased frequency of loss of heterozygosity (LOH) mutations with duplications of the inactive allele - suggesting that more cells were mutated via homologous recombinational repair (HRR). Our goal is to understand the basis for increased autosomal mutageness mediated by BCL-2 family members. Four aims are proposed 1) We will test the hypothesis that the elevated level of LOH mutations at the TK1 locus in TK6 cells that over-express BCL-2 or BCL-X L is associated with an increase in homology directed repair (HDR) of DNA double-strand breaks (DSBs). We will use an integrated DR-GFP reporter to measure gene conversion following a single, site-specific DSB. We recently showed that high BCL-X L expression promotes HDR in TK6 cells. We plan to generalize this finding to other BCL-2 family members and to FL5.12 cells in which PCD regulation by BCL-2 family members has been well studied. 2) We will test the hypothesis that the elevated frequencies of TK1 mutations in TK6-bclXL cells result from a novel activity of BCL-X L distinct from its anti-apoptotic function. We will develop isogenic TK6 cells that express mutated BCL-X L that can t block PCD to assess if x-ray-induced TK1 mutagenesis is modulated. 3) We will test the hypothesis that BCL-X L promotes x-ray-induced TK1 mutations by maintaining higher levels of HsRAD51 in TK6 cells post-IR. We will use isogenic TK6 cells that express a mutant HsRAD51 (HsRAD51D-A) insensitive to caspase cleavage that promotes HRR, wildtype HsRAD51, or a cleavage fragment of HsRAD51 that can t promote HRR. If HsRAD51D-A elevates x-ray- induced TK1 mutagenesis, we will determine if it acts in the same pathway as BCL-X L by co-expressing these proteins in TK6 cells. New strategies are included in the event that an elevated level of HsRAD51 is toxic. 4) We will test the hypothesis that PCD suppression per se can enhance x-ray-induced TK1 mutagenesis. We will use TK6 cells expressing mutant procaspase-9 (cys287ala) that acts as a dominant negative to suppress PCD. Elucidating the mechanisms through which BCL-2 and BCL-X L mediate mutagenesis in vitro should illuminate mechanisms of mutation in B-cell follicular lymphomas that express high levels of BCL-2. It is the secondary mutations that lead to advance, aggressive disease in people.
Keywords: apoptosis, gene mutation, radiation carcinogenesis, radiation genetics, radiation related neoplasm /cancer, BCL2 gene /protein, DNA damage, cysteine endopeptidase, loss of heterozygosity, cell line, enzyme linked immunosorbent assay, flow cytometry, western blotting
Project start date: 2003-07-01
Project end date: 2008-06-30
5R01CA092277-04 (2006): $328068
5R01CA092277-03 (2005): $335963
5R01CA092277-02 (2004): $335963
1R01CA092277-01A2 (2003): $335963
RADIATION AND GENOMIC INSTABILITY IN FINITE LIFESPAN HUMAN MAMMARY EPITHELIUM
Amy Kronenberg
University Of Calif-lawrenc Berkeley Lab, Ofc Of Sponsored Projects & Industry Partnerships, Berkeley, Ca 94720-8202
Grant 5R21ES016889-02 from National Institute Of Environmental Health Sciences
Abstract: Ionizing radiation was the first environmental mutagen identified by H.J. Muller in 1928, and radiation is a known human carcinogen. Epidemiological evidence from the Japanese atomic bomb survivors indicates that the female breast is exquisitely sensitive to radiogenic cancer. Women treated for breast cancer are at risk for a new treatment associated breast cancer as are women treated with radiation to the chest for Hodgkin´s disease. Recent data indicate that younger women (
Keywords: ARF; Accidents; Address; Age; Background Radiation; Benign; Biopsy; Biopsy Sample; Biopsy Specimen; Breast; Breast Tissue; CDK4I; CDKN2; CDKN2A; CDKN2A gene; CMM2; Cancer Causing Agents; Cancer of Breast; Cancer, Radiation-Induced; Cancers; Carcinogens; Carcinoma, Intraductal; Causality; Cells; Chest; Chromosomal Instability; Chromosome Instability; Cyclin-Dependent Kinase Inhibitor 2A Gene; DCIS; Data; Development; Diagnostic; Disease; Disorder; Dose; Ductal Breast Carcinoma In Situ; Ductal Carcinoma In Situ; Electromagnetic Radiation, Ionizing; Environmental Carcinogens; Environmental genotoxicant; Epidemiology; Epithelial Cells; Etiology; Exhibits; Exposure to; Female breast; Follow-Up Studies; Followup Studies; Frequencies (time pattern); Frequency; Gamma Rays; Genes, CDKN2; Genes, p16; Genes, p16INK4A; Genome; Genome Instability; Genomic Instability; Histologic; Histologically; Hodgkin Disease; Hodgkin Disorder; Hodgkin lymphoma; Hodgkin`s; Hodgkin`s Lymphoma; Hodgkin`s disease; Hodgkins lymphoma; Human; Human Mammary Epithelium; Human, General; Hyperplasia; Hyperplastic; INK4; INK4A; Individual; Intraductal Carcinoma of the Breast; Ionizing radiation; Japanese; Japanese Population; Length of Life; Longevity; Low-Level Radiation; Lymphogranuloma, Malignant; MLM; MTS1; MTS1 Genes; Malignant; Malignant - descriptor; Malignant Neoplasms; Malignant Tumor; Malignant Tumor of the Breast; Malignant neoplasm of breast; Mammary Gland Parenchyma; Mammary Gland Tissue; Mammary Glands, Human; Mammary gland; Man (Taxonomy); Man, Modern; Medical; Natural Radiation; Neoplasms; Non-Infiltrating Ductal Breast Adenocarcinoma; Non-Infiltrating Ductal Carcinoma of the Breast; Non-Infiltrating Intraductal Adenocarcinoma; Non-Infiltrating Intraductal Breast Adenocarcinoma; Non-Infiltrating Intraductal Carcinoma; Non-Invasive Ductal Breast Adenocarcinoma; Non-Invasive Ductal Carcinoma of the Breast; Non-Invasive Intraductal Breast Adenocarcinoma; Noninfiltrating Intraductal Carcinoma; Oncogens; Pathology; Persons; Phenotype; Population; Predisposition; Radiation; Radiation, Gamma; Radiation-Induced Cancer; Radiation-Induced Malignant Neoplasm; Radiation-Ionizing Total; Radiation-Related Malignant Neoplasm; Risk; Role; Sampling; Staging; Survey Instrument; Surveys; Survivors; Susceptibility; TP16; TSG9A; Testing; Therapeutic; Thorace; Thoracic; Thorax; Time; Tumors; Woman; Work; base; cell type; disease causation; disease etiology; disease/disorder; disease/disorder etiology; disorder etiology; environmental mutagenesis; environmental mutagens; experiment; experimental research; experimental study; high risk; human female; irradiation; life span; lifespan; lymphogranulomatosis; lymphogranulomatosis (malignant); malignancy; malignant breast neoplasm; mammary; neoplasia; neoplasm/cancer; neoplastic growth; p14ARF; p16INK4 Genes; p16INK4a; public health relevance; radiation related cancer; radiation related neoplasm/cancer; ray (radiation); research study; senescence; social role
Relevance: NARRATIVE Ionizing radiation is a human carcinogen, and results from the Japanese atomic bomb survivors and medically exposed populations have demonstrated that the young female breast is very sensitive to radiation-induced cancer. Others have shown that genomic instability first appears in human breast biopsy materials at the earliest malignant stage - ductal carcinoma in situ - suggesting that instability is an important early feature in the development of breast cancer. The experiments proposed address whether radiation exposure elicits persistent genomic instability in the progeny of apparently normal finite lifespan human mammary epithelial cells from donors below age 40, whether this is a common phenotype amongst individuals, and whether transition past the first senescence barrier is required for finite lifespan human mammary epithelial cells to exhibit radiation induced genomic instability
Project start date: 2009-06-15
Project end date: 2011-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
PFA/PA: PA-06-181
5R21ES016889-02 (2010): $269034
1R21ES016889-01A1 (2009): $223691
HIGH LET RADIATION And GENOMIC INSTABILITY IN HUMAN CELLS
Amy Kronenberg, Group Leader And Staff Scientist
University Of Calif-lawrenc Berkeley Lab C/o Sponsored Projects Office Berkeley, Ca 94720
Grant 5R01CA073966-05 from National Cancer Institute IRG: ZCA1
Abstract: Applicant s Description) Human exposure to densely ionizing radiation occurs environmentally on the earth and in space. Occupational exposures to neutrons also occur in the nuclear power industry and near medical accelerators. While most of these exposures involve irradiation with a very low fluence of high LET radiations, the carcinogenic risks of such exposures remain poorly understood. The goals of the proposed investigation are focused to test the hypothesis that densely ionizing radiations, such as accelerated iron ions, are more potent inducers of a persistent state of genomic instability in human lymphoid cells, as compared with sparsely ionizing radiations such as energetic protons. Karyotypic heterogeneity will serve as an initial indicator of genomic instability. More comprehensive experiments are outlined to evaluate other features of chromosomal-scale instability, including increased rates of mutation at defined sequences within the human genome. will also delineate the molecular mechanisms involved in maintenance of persistent and progressive radiation-induced instability. In particular, she will test the hypothesis that low fluence exposures to high LET radiations induce instability through increased rates of aberrant recombination leading to loss of heterozygosity, as assayed along a segment of chromosome 17q. She presents an experimental plan to test the hypothesis that programmed cell death masks expression of persistent genomic instability following low fluence exposure to densely ionizing iron ions or comparable doses of protons. Specific experimental approaches are outlined to determine when programmed cell death is of critical importance in the selective removal of heavily damaged cells at early times post-exposure, at later times to weed out late-arising clones with inappropriately rearranged genomes, or a combination thereof. The proposed investigations will provide quantitative and mechanistic information regarding iron ion- and proton-induced genomic instability. The approaches outlined focus on the genesis of the types of heritable alterations that have often been associated with human carcinogenesis. The results obtained will be of specific importance in the assessment of radiation risks to astronaut health. The results obtained with low fluence iron-ion exposures will serve as a model for terrestrial exposures to radon and neutrons.
Keywords: high energy particle, mutagen, neoplasm /cancer, radiation carcinogen, radiation carcinogenesis, apoptosis, gene deletion mutation, genetic recombination, ion, iron, linear energy transfer, loss of heterozygosity, radiation genetics, thymidine kinase, carcinogen testing, human genetic material tag, mutagen testing, tissue /cell culture
Project start date: 1997-09-30
Project end date: 2002-09-30
5R01CA073966-05 (2001): $144441
5R01CA073966-04 (2000): $281841
5R01CA073966-03 (1999): $273968
Sponsored Links Excellgen http://Excellgen.com
5R01CA073966-02 (1998): $267974
1R01CA073966-01 (1997): $131397
RADIATION--DELAYED MUTATION & INSTABILITY IN HUMAN CELLS
Amy Kronenberg, Group Leader And Staff Scientist
University Of Calif-lawrenc Berkeley Lab C/o Sponsored Projects Office Berkeley, Ca 94720
Grant 5R01CA062364-03 from National Cancer Institute IRG: RAD
Project start date: 1994-07-01
Project end date: 1998-06-30
5R01CA062364-03 (1996): $233910
3R01CA062364-03S1 (1996): $11999