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Michael B Kastan

Publications and source records attributed to Michael B Kastan.

At least 19 recordsLinked to original sources
PubMed2006

A subtle t(3;8) results in plausible juxtaposition of MYC and BCL6 in a child with Burkitt lymphoma/leukemia and ataxia-telangiectasia.

Translocations involving 3q27 that affect the BCL6 gene are common and specific chromosomal abnormalities in B-cell precursor non-Hodgkin lymphoma (mainly diffuse large-cell and follicular lymphoma), but they have not been reported in Burkitt lymphoma. Here, we describe a case in which a BCL6 rearrangement and additional complex cytogenetic abnormalities occurred in a child with Burkitt lymphoma/leukemia and ataxia-telangiectasia. Although cytogenetic analysis of the bone marrow revealed clonal abnormalities of chromosome arms 8q and 14p and other subclonal abnormalities, the t(8;14) or its variants typically associated with Burkitt lymphoma were not observed. Fluorescence in situ hybridization with locus-specific probes and multicolor spectral karyotyping demonstrated a complex pattern of chromosomal rearrangements leading to a subtle t(3;8)(q27;q24.1) that rearranged BCL6 and placed it adjacent to MYC. We speculate that this genetic lesion occurred as a result of chromosomal instability due to the underlying disease.

Ataxia Telangiectasia↗
PubMed2006

Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks.

We show that DNA double-strand breaks (DSBs) induce complex subcompartmentalization of genome surveillance regulators. Chromatin marked by gamma-H2AX is occupied by ataxia telangiectasia-mutated (ATM) kinase, Mdc1, and 53BP1. In contrast, repair factors (Rad51, Rad52, BRCA2, and FANCD2), ATM and Rad-3-related (ATR) cascade (ATR, ATR interacting protein, and replication protein A), and the DNA clamp (Rad17 and -9) accumulate in subchromatin microcompartments delineated by single-stranded DNA (ssDNA). BRCA1 and the Mre11-Rad50-Nbs1 complex interact with both of these compartments. Importantly, some core DSB regulators do not form cytologically discernible foci. These are further subclassified to proteins that connect DSBs with the rest of the nucleus (Chk1 and -2), that assemble at unprocessed DSBs (DNA-PK/Ku70), and that exist on chromatin as preassembled complexes but become locally modified after DNA damage (Smc1/Smc3). Finally, checkpoint effectors such as p53 and Cdc25A do not accumulate at DSBs at all. We propose that subclassification of DSB regulators according to their residence sites provides a useful framework for understanding their involvement in diverse processes of genome surveillance.

Animals↗
PubMed2006

ATM-dependent suppression of stress signaling reduces vascular disease in metabolic syndrome.

Metabolic syndrome is associated with insulin resistance and atherosclerosis. Here, we show that deficiency of one or two alleles of ATM, the protein mutated in the cancer-prone disease ataxia telangiectasia, worsens features of the metabolic syndrome, increases insulin resistance, and accelerates atherosclerosis in apoE-/- mice. Transplantation with ATM-/- as compared to ATM+/+ bone marrow increased vascular disease. Jun N-terminal kinase (JNK) activity was increased in ATM-deficient cells. Treatment of ATM+/+apoE-/- mice with low-dose chloroquine, an ATM activator, decreased atherosclerosis. In an ATM-dependent manner, chloroquine decreased macrophage JNK activity, decreased macrophage lipoprotein lipase activity (a proatherogenic consequence of JNK activation), decreased blood pressure, and improved glucose tolerance. Chloroquine also improved metabolic abnormalities in ob/ob and db/db mice. These results suggest that ATM-dependent stress pathways mediate susceptibility to the metabolic syndrome and that chloroquine or related agents promoting ATM activity could modulate insulin resistance and decrease vascular disease.

Animals↗
PubMed2005

The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor.

Genetic and cytologic data from Saccharomyces cerevisiae and mammals implicate the Mre11 complex, consisting of Mre11, Rad50, and Nbs1, as a sensor of DNA damage, and indicate that the complex influences the activity of ataxia-telangiectasia mutated (ATM) in the DNA damage response. Rad50(S/S) mice exhibit precipitous apoptotic attrition of hematopoietic cells. We generated ATM- and Chk2-deficient Rad50(S/S) mice and found that Rad50(S/S) cellular attrition was strongly ATM and Chk2 dependent. The hypomorphic Mre11(ATLD1) and Nbs1(Delta)(B) alleles conferred similar rescue of Rad50(S/S)-dependent hematopoietic failure. These data indicate that the Mre11 complex activates an ATM-Chk2-dependent apoptotic pathway. We find that apoptosis and cell cycle checkpoint activation are parallel outcomes of the Mre11 complex-ATM pathway. Conversely, the Rad50(S) mutation mitigated several phenotypic features of ATM deficiency. We propose that the Rad50(S) allele is hypermorphic for DNA damage signaling, and that the resulting constitutive low-level activation of the DNA damage response accounts for the partial suppression of ATM deficiency in Rad50(S/S) Atm(-/-) mice.

ATP-Binding Cassette Transporters↗
PubMed2005

Regulation of p53 translation and induction after DNA damage by ribosomal protein L26 and nucleolin.

Increases in p53 protein levels after DNA damage have largely been attributed to an increase in the half-life of p53 protein. Here we demonstrate that increased translation of p53 mRNA is also a critical step in the induction of p53 protein in irradiated cells. Ribosomal protein L26 (RPL26) and nucleolin were found to bind to the 5' untranslated region (UTR) of p53 mRNA and to control p53 translation and induction after DNA damage. RPL26 preferentially binds to the 5'UTR after DNA damage, and its overexpression enhances association of p53 mRNA with heavier polysomes, increases the rate of p53 translation, induces G1 cell-cycle arrest, and augments irradiation-induced apoptosis. Opposite effects were seen when RPL26 expression was inhibited. In contrast, nucleolin overexpression suppresses p53 translation and induction after DNA damage, whereas nucleolin downregulation promotes p53 expression. These findings demonstrate the importance of increased translation of p53 in DNA-damage responses and suggest critical roles for RPL26 and nucleolin in affecting p53 induction.

5' Untranslated Regions↗
PubMed2005

ATM activation in normal human tissues and testicular cancer.

The ATM kinase is a tumor suppressor and key regulator of biological responses to DNA damage. Cultured cells respond to genotoxic insults that induce DNA double-strand breaks by prompt activation of ATM through its autophosphorylation on serine 1981. However, whether ATM-S1981 becomes phosphorylated in vivo, for example during physiological processes that generate DSBs, is unknown. Here we produced phospho-specific monoclonal antibodies against S1981-phosphorylated ATM (pS-ATM), and applied them to immunohistochemical analyses of a wide range of normal human tissues and testicular tumors. Our data show that regardless of proliferation and differentiation, most human tissues contain only the S1981-nonphosphorylated, inactive form of ATM. In contrast, nuclear staining for pS-ATM was detected in subsets of bone-marrow lymphocytes and primary spermatocytes in the adult testes, cell types in which DSBs are generated during physiological V(D)J recombination and meiotic recombination, respectively. Among testicular germ-cell tumors, an aberrant constitutive pS-ATM was observed especially in embryonal carcinomas, less in seminomas, and only modestly in teratomas and the pre-invasive carcinoma-in-situ stage. Compared with pS-ATM, phosphorylated histone H2AX (gammaH2AX), another DNA damage marker and ATM substrate, was detected in a higher proportion of cancer cells, and also in normal fetal gonocytes, and a wider range of adult spermatocyte differentiation stages. Collectively, our results strongly support the physiological relevance of the recently proposed model of ATM autoactivation, and provide further evidence for constitutive activation of the DNA damage machinery during cancer development. The new tools characterized here should facilitate monitoring of ATM activation in clinical specimens, and help develop future treatment strategies.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2004

Cell-cycle checkpoints and cancer.

All life on earth must cope with constant exposure to DNA-damaging agents such as the Sun's radiation. Highly conserved DNA-repair and cell-cycle checkpoint pathways allow cells to deal with both endogenous and exogenous sources of DNA damage. How much an individual is exposed to these agents and how their cells respond to DNA damage are critical determinants of whether that individual will develop cancer. These cellular responses are also important for determining toxicities and responses to current cancer therapies, most of which target the DNA.

Animals↗
PubMed2004

The telomeric protein TRF2 binds the ATM kinase and can inhibit the ATM-dependent DNA damage response.

The telomeric protein TRF2 is required to prevent mammalian telomeres from activating DNA damage checkpoints. Here we show that overexpression of TRF2 affects the response of the ATM kinase to DNA damage. Overexpression of TRF2 abrogated the cell cycle arrest after ionizing radiation and diminished several other readouts of the DNA damage response, including phosphorylation of Nbs1, induction of p53, and upregulation of p53 targets. TRF2 inhibited autophosphorylation of ATM on S1981, an early step in the activation of this kinase. A region of ATM containing S1981 was found to directly interact with TRF2 in vitro, and ATM immunoprecipitates contained TRF2. We propose that TRF2 has the ability to inhibit ATM activation at telomeres. Because TRF2 is abundant at chromosome ends but not elsewhere in the nucleus, this mechanism of checkpoint control could specifically block a DNA damage response at telomeres without affecting the surveillance of chromosome internal damage.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2004

Initiating cellular stress responses.

The phosphoinositide 3-kinase related kinases (PIKKs) mediate responses to diverse stresses, including DNA double-strand breaks (DSBs), abnormal replication fork progression, the recognition of premature termination codons in mRNAs, and inadequate nutrient availability. Rigorous control of these kinases limits cellular damage and promotes cell viability in the presence of stress. Control mechanisms include the localization of PIKKs into multiprotein complexes at the sites of damage and mediation of protein-protein contacts such that substrates are allowed access to the PIKK catalytic domains.

Animals↗
PubMed2004

Phosphorylation of SMC1 is a critical downstream event in the ATM-NBS1-BRCA1 pathway.

The ATM protein kinase is activated by intermolecular autophosphorylation in response to DNA damage and initiates cellular signaling pathways that facilitate cell survival and reduce chromosomal breakage. Here, we show that NBS1 and BRCA1 are required for the recruitment of previously activated ATM to the sites of DNA breaks after ionizing irradiation, and that this recruitment is required for the phosphorylation of SMC1 by ATM. To explore the functional importance of SMC1 phosphorylation, murine cells were generated, in which the two damage-induced phosphorylation sites in SMC1 are mutated. Although these cells demonstrate normal phosphorylation and focus formation of ATM, NBS1, and BRCA1 proteins after IR, they exhibit a defective S-phase checkpoint, decreased survival, and increased chromosomal aberrations after DNA damage. These observations suggest that many of the abnormal stress responses seen in cells lacking ATM, NBS1, or BRCA1 result from a failure of ATM migration to sites of DNA breaks and a resultant lack of SMC1 phosphorylation.

Animals↗
PubMed2004

Disappearance of the telomere dysfunction-induced stress response in fully senescent cells.

Replicative senescence is a natural barrier to cellular proliferation that is triggered by telomere erosion and dysfunction. Here, we demonstrate that ATM activation and H2AX-gamma nuclear focus formation are sensitive markers of telomere dysfunction in primary human fibroblasts. Whereas the activated form of ATM and H2AX-gamma foci were rarely observed in early-passage cells, they were readily detected in late-passage cells. The ectopic expression of telomerase in late-passage cells abrogated ATM activation and H2AX-gamma focus formation, suggesting that these stress responses were the consequence of telomere dysfunction. ATM activation was induced in quiescent fibroblasts by inhibition of TRF2 binding to telomeres, indicating that telomere uncapping is sufficient to initiate the telomere signaling response; breakage of chromosomes with telomeric associations is not required for this activation. Although ATM activation and H2AX-gamma foci were readily observed in late-passage cells, they disappeared once cells became fully senescent, indicating that constitutive signaling from dysfunctional telomeres is not required for the maintenance of senescence.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2004

Distinct functional domains of Nbs1 modulate the timing and magnitude of ATM activation after low doses of ionizing radiation.

The ATM kinase is a tumour suppressor and a key activator of genome integrity checkpoints in mammalian cells exposed to ionizing radiation (IR) and other insults that elicit DNA double-strand breaks (DSBs). In response to IR, autophosphorylation on serine 1981 causes dissociation of ATM dimers and initiates cellular ATM kinase activity. Here, we show that the kinetics and magnitude of ATM Ser1981 phosphorylation after exposure of human fibroblasts to low doses (2 Gy) of IR are altered in cells deficient in Nbs1, a substrate of ATM and a component of the MRN (Mre11-Rad50-Nbs1) complex involved in processing/repair of DSBs and ATM-dependent cell cycle checkpoints. Timely phosphorylation of both ATM Ser1981 and the ATM substrate Smc1 after IR were rescued via retrovirally mediated reconstitution of Nbs1-deficient cells by wild-type Nbs1 or mutants of Nbs1 defective in the FHA domain or nonphosphorylatable by ATM, but not by Nbs1 lacking the Mre11-interaction domain. Our data indicate that apart from its role downstream of ATM in the DNA damage checkpoint network, the MRN complex serves also as a modulator/amplifier of ATM activity. Although not absolutely required for ATM activation, the MRN nuclease complex may help reach the threshold activity of ATM necessary for optimal genome maintenance and prevention of cancer.

Ataxia Telangiectasia↗
PubMed2004

Phosphatases join kinases in DNA-damage response pathways.

An inappropriate imbalance of kinase and phosphatase activities could be deleterious to cellular processes such as proliferation. Cellular responses to DNA damage use signal-transduction pathways involving phosphorylation events, and such modifications must be reversible to make these responses transient, rather than permanent, events. Three recent articles describe roles for two phosphatases in signaling pathways that are activated after DNA damage.

DNA Damage↗
PubMed2004

BRCA1 is required for common-fragile-site stability via its G2/M checkpoint function.

Common fragile sites are loci that form chromosome gaps or breaks when DNA synthesis is partially inhibited. Fragile sites are prone to deletions, translocations, and other rearrangements that can cause the inactivation of associated tumor suppressor genes in cancer cells. It was previously shown that ATR is critical to fragile-site stability and that ATR-deficient cells have greatly elevated fragile-site expression (A. M. Casper, P. Nghiem, M. F. Arlt, and T. W. Glover, Cell 111:779-789, 2002). Here we demonstrate that mouse and human cells deficient for BRCA1, due to mutation or knockdown by RNA interference, also have elevated fragile-site expression. We further show that BRCA1 functions in the induction of the G(2)/M checkpoint after aphidicolin-induced replication stalling and that this checkpoint function is involved in fragile-site stability. These data indicate that BRCA1 is important in fragile-site stability and that fragile sites are recognized by the G(2)/M checkpoint pathway, in which BRCA1 plays a key role. Furthermore, they suggest that mutations in BRCA1 or interacting proteins could lead to rearrangements at fragile sites in cancer cells.

Animals↗
PubMed2004

NAD+ modulates p53 DNA binding specificity and function.

DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD(+) binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B(3)) increases the rate of intracellular NAD(+) synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD(+) on p53, as a molecule structurally related to part of NAD(+), TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B(1)), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53.

Adenosine Diphosphate↗
PubMed2004

Analyzing cell cycle checkpoints after ionizing radiation.

Several methods to measure cell cycle perturbation after exposure to ionizing radiation are presented in this chapter. These methods include the G1 checkpoint assay by 5' bromode-oxyuridine (BrdUrd) labeling followed by flow cytometric analysis, the S-phase checkpoint assay by measuring DNA synthesis in response to ionizing radiation, and the G2/M checkpoint assay by quantitating histone H3 phosphorylation after ionizing radiation.

Bromodeoxyuridine↗