Search PubMed⌕ Search

Biomedical subjects

M B Kastan

Publications and source records attributed to M B Kastan.

At least 19 recordsLinked to original sources
PubMed2000

ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway.

The rare diseases ataxia-telangiectasia (AT), caused by mutations in the ATM gene, and Nijmegen breakage syndrome (NBS), with mutations in the p95/nbs1 gene, share a variety of phenotypic abnormalities such as chromosomal instability, radiation sensitivity and defects in cell-cycle checkpoints in response to ionizing radiation. The ATM gene encodes a protein kinase that is activated by ionizing radiation or radiomimetic drugs, whereas p95/nbs1 is part of a protein complex that is involved in responses to DNA double-strand breaks. Here, because of the similarities between AT and NBS, we evaluated the functional interactions between ATM and p95/nbs1. Activation of the ATM kinase by ionizing radiation and induction of ATM-dependent responses in NBS cells indicated that p95/nbs1 may not be required for signalling to ATM after ionizing radiation. However, p95/nbs1 was phosphorylated on serine 343 in an ATM-dependent manner in vitro and in vivo after ionizing radiation. A p95/nbs1 construct mutated at the ATM phosphorylation site abrogated an S-phase checkpoint induced by ionizing radiation in normal cells and failed to compensate for this functional deficiency in NBS cells. These observations link ATM and p95/nbs1 in a common signalling pathway and provide an explanation for phenotypic similarities in these two diseases.

Ataxia Telangiectasia↗
PubMed2000

Ionizing radiation activates the ATM kinase throughout the cell cycle.

The ATM protein kinase is a critical intermediate in a number of cellular responses to ionizing irradiation (IR) and possibly other stresses. ATM dysfunction results in abnormal checkpoint responses in multiple phases of the cell cycle, including G1, S and G2. Though downstream targets of the ATM kinase are still being elucidated, it has been demonstrated that ATM acts upstream of p53 in a signal transduction pathway initiated by IR and can phosphorylate p53 at serine 15. The cell cycle stage-specificity of ATM activation and p53Ser15 phosphorylation was investigated in normal lymphoblastoid cell line (GM536). Ionizing radiation was found to enhance the kinase activity of ATM in all phases of the cell cycle. This enhanced activity was apparent immediately after treatment of cells with IR, but was not accompanied by a change in the abundance of the ATM protein. Since IR activates the ATM kinase in all phases of the cell cycle, DNA replication-dependent strand breaks are not required for this activation. Further, since p53 protein is not directly required for IR-induced S and G2-phase checkpoints, the ATM kinase likely has different functional targets in different phases of the cell cycle. These observations indicate that the ATM kinase is necessary primarily for the immediate response to DNA damage incurred in all phases of the cell cycle.

Ataxia Telangiectasia↗
PubMed1999

Substrate specificities and identification of putative substrates of ATM kinase family members.

Ataxia telangiectasia mutated (ATM) phosphorylates p53 protein in response to ionizing radiation, but the complex phenotype of AT cells suggests that it must have other cellular substrates as well. To identify substrates for ATM and the related kinases ATR and DNA-PK, we optimized in vitro kinase assays and developed a rapid peptide screening method to determine general phosphorylation consensus sequences. ATM and ATR require Mn(2+), but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs requires Mg(2+), DNA ends, and Ku proteins. From p53 peptide mutagenesis analysis, we found that the sequence S/TQ is a minimal essential requirement for all three kinases. In addition, hydrophobic amino acids and negatively charged amino acids immediately NH(2)-terminal to serine or threonine are positive determinants and positively charged amino acids in the region are negative determinants for substrate phosphorylation. We determined a general phosphorylation consensus sequence for ATM and identified putative in vitro targets by using glutathione S-transferase peptides as substrates. Putative ATM in vitro targets include p95/nibrin, Mre11, Brca1, Rad17, PTS, WRN, and ATM (S440) itself. Brca2, phosphatidylinositol 3-kinase, and DNA-5B peptides were phosphorylated specifically by ATR, and DNA Ligase IV is a specific in vitro substrate of DNA-PK.

Amino Acid Sequence↗
PubMed1999

Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c.

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.

Animals↗
PubMed1999

Phenylbutyrate-induced G1 arrest and apoptosis in myeloid leukemia cells: structure-function analysis.

The aromatic fatty acid phenylbutyrate (PB) induces cytostasis, differentiation, and apoptosis in primary myeloid leukemic cells at clinically achievable concentrations. In the present study, we have investigated the structural and cellular basis for PB-induced cytostasis, using the ML-1 human myeloid leukemia cell line as a model system. PB induced a dose-dependent increase in cells in G1 with a corresponding decrease in cells in S-phase of the cell cycle. At comparable doses, PB induced expression of CD11b, indicating myeloid differentiation. At higher doses, the drug induced apoptosis. The antitumor activity was independent of the aromatic ring, as butyric acid (BA) was of equal or greater potency at producing these biological changes. In contrast, shortening of the fatty acid carbon chain length, as demonstrated with phenylacetate (PA), significantly diminished drug potency. Consistent with their effects on cell cycle, PB and BA, but not PA, induced the cyclin-dependent kinase inhibitor, p21(WAF1/CIP1), and led to the appearance of hypophosphorylated Rb, suggesting a role for p21(WAF1/CIP1) in PB-induced cytostasis. Therefore, it appears that the fatty acid moiety of PB, rather than its aromatic ring, is critical for its activity in myeloid leukemic cells. These data provide a potential mechanistic basis for the increased potency of PB over PA previously demonstrated in primary leukemic samples, and support the further clinical development of PB in the treatment of hematologic malignancies.

Antineoplastic Agents↗
PubMed1998

Loss of atm radiosensitizes multiple p53 null tissues.

An unusual clinical finding in ataxia-telangiectasia, a human disorder caused by mutations in atm, is exquisite sensitivity to gamma irradiation. By contrast, homozygous deletion of p53 is marked by radiation resistance in certain tissue compartments. Previous studies (A. J. Levine, Cell, 88: 323-331, 1997) have shown that, in vitro, p53-deficient bone marrow cells are resistant to gamma irradiation. Furthermore, the gastrointestinal radiosensitization engendered by the loss of atm has recently been shown (C. H. Westphal et al., Nat. Genet., 16: 397-401, 1997) to be independent of p53. Expanding on previous work, we have looked at in vivo bone marrow resistance in p53-deficient mice. Our results indicate that inbred FVB strain p53 null mice survive lethal irradiation doses because of bone marrow resistance. Moreover, the deletion of atm radiosensitizes even p53 null bone marrow and mouse embryonic fibroblast cells. The results presented here argue that the loss of atm radiosensitizes multiple tissues in a p53-independent manner. Hence, functional inhibition of atm in p53 null and p53 wild-type human tumors may be a useful adjunct to gamma irradiation-based antitumor therapy.

Animals↗
PubMed1998

Defective potassium currents in ataxia telangiectasia fibroblasts.

Similarities exist between the progressive cerebellar ataxia in ataxia telangiectasia (AT) patients and a number of neurodegenerative diseases in both mouse and man involving specific mutations in ion channels and/or ion channel activity. These relationships led us to investigate the possibility of defective ion channel activity in AT cells. We examined changes in the membrane potential of AT fibroblasts in response to extracellular cation addition and found that the ability of AT fibroblasts to depolarize in response to increasing concentrations of extracellular K+ is significantly reduced when compared with control fibroblasts. Electrophysiological measurements performed with a number of AT cell lines, as well as two matched sets of primary AT fibroblast cultures, reveal that outward rectifier K+ currents are largely absent in AT fibroblasts in comparison with control cells. These K+ current defects can be corrected in AT fibroblasts transfected with the full-length ATM cDNA. These data implicate, for the first time, a role for ATM in the regulation of K+ channel activity and membrane potential.

Ataxia Telangiectasia↗
PubMed1998

Activation of the ATM kinase by ionizing radiation and phosphorylation of p53.

The p53 tumor suppressor protein is activated and phosphorylated on serine-15 in response to various DNA damaging agents. The gene product mutated in ataxia telangiectasia, ATM, acts upstream of p53 in a signal transduction pathway initiated by ionizing radiation. Immunoprecipitated ATM had intrinsic protein kinase activity and phosphorylated p53 on serine-15 in a manganese-dependent manner. Ionizing radiation, but not ultraviolet radiation, rapidly enhanced this p53-directed kinase activity of endogenous ATM. These observations, along with the fact that phosphorylation of p53 on serine-15 in response to ionizing radiation is reduced in ataxia telangiectasia cells, suggest that ATM is a protein kinase that phosphorylates p53 in vivo.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1998

ATM binds to beta-adaptin in cytoplasmic vesicles.

Inherited mutations in the ATM gene lead to a complex clinical phenotype characterized by neuronal degeneration, oculocutaneous telangiectasias, immune dysfunction, and cancer predisposition. Using the yeast two-hybrid system, we demonstrate that ataxia telangiectasia mutated (ATM) binds to beta-adaptin, one of the components of the AP-2 adaptor complex, which is involved in clathrin-mediated endocytosis of receptors. The interaction between ATM and beta-adaptin was confirmed in vitro, and coimmunoprecipitation and colocalization studies show that the proteins also associate in vivo. ATM also interacts in vitro with beta-NAP, a neuronal-specific beta-adaptin homolog that was identified as an autoantigen in a patient with cerebellar degeneration. Our data describing the association of ATM with beta-adaptin in vesicles indicate that ATM may play a role in intracellular vesicle and/or protein transport mechanisms.

Adaptor Protein Complex 3↗
PubMed1998

Human smooth muscle alpha-actin gene is a transcriptional target of the p53 tumor suppressor protein.

Smooth muscle (sm) alpha-actin is expressed in vascular smooth muscle cells and fibroblast cells. Its expression is regulated by cell proliferation and repressed during oncogenic transformation. In this study, we demonstrate that p53 activation is associated with a dramatic increase in organized microfilament bundles and an increase in sm alpha-actin mRNA level. Wild-type p53, but not mutant p53, strongly stimulated human sm alpha-actin promoter activity in p53 null cell lines. The sequences homologous to the p53 consensus sequence and to the p53 binding sequence from the muscle creatine kinase, were found within a specific region of the sm alpha-actin promoter. This sequence was sufficient to confer p53-dependent activation to a heterologous promoter and p53 was capable of binding to this sequence as assessed by gel shift analysis. Ionizing irradiation of colorectal tumor cells caused an increase in alpha-actin mRNA level in a p53-dependent manner. Taken together, these results demonstrate that human sm alpha-actin gene is a transcriptional target for p53 tumor suppressor protein and represents the first example of a cytoskeletal gene with a functionally defined p53 response element.

Actins↗
PubMed1998

Small contribution of G1 checkpoint control manipulation to modulation of p53-mediated apoptosis.

Deregulation of the S-phase promoting E2F-1 transcription factor has been shown to cooperate with p53 to induce apoptosis. BaF3 cells undergo rapid, p53-dependent apoptosis when irradiated in the absence of IL-3. Rapid apoptosis induced by ionizing radiation (IR) coincides with attenuated p21(WAF1/Cip1) induction. Failure to adequately induce p21 could result in inappropriate release of E2F from Rb which may then cooperate with p53 to induce apoptosis in cells deprived of growth factor. We engineered BaF3 cells to express exogenous p21 and tested whether overexpressing p21 in cells irradiated in the absence of IL-3 protects from IR-induced apoptosis. Enforced p21 expression resulted in a consistent, but partial, protection of cells from undergoing IR-induced apoptosis. However, deregulating E2F activity through expression of HPV E7 failed to sensitize cells to IR-induced apoptosis in the presence of IL-3. Together, these data strongly suggest that the IL-3-responsive factors which modulate p53-mediated apoptosis in BaF3 cells are largely independent of G1 cell cycle checkpoint control mediated by p21.

Animals↗
PubMed1998

Modulation of cell death by Bcl-XL through caspase interaction.

The caspases are cysteine proteases that have been implicated in the execution of programmed cell death in organisms ranging from nematodes to humans. Many members of the Bcl-2 family, including Bcl-XL, are potent inhibitors of programmed cell death and inhibit activation of caspases in cells. Here, we report a direct interaction between caspases and Bcl-XL. The loop domain of Bcl-XL is cleaved by caspases in vitro and in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3 withdrawal. Mutation of the caspase cleavage site in Bcl-XL in conjunction with a mutation in the BH1 homology domain impairs the death-inhibitory activity of Bcl-XL, suggesting that interaction of Bcl-XL with caspases may be an important mechanism of inhibiting cell death. However, once Bcl-XL is cleaved, the C-terminal fragment of Bcl-XL potently induces apoptosis. Taken together, these findings indicate that the recognition/cleavage site of Bcl-XL may facilitate protection against cell death by acting at the level of caspase activation and that cleavage of Bcl-XL during the execution phase of cell death converts Bcl-XL from a protective to a lethal protein.

Apoptosis↗
PubMed1998

Frequent detection of tumor cells in hematopoietic grafts in neuroblastoma and Ewing's sarcoma.

Many poor-risk neuroblastomas and tumours of the Ewing's sarcoma family (ET) recur despite autologous transplants. Recurrence may be due to tumor cells contained in the BM harvests or PBSC harvests. The objectives of this prospective study were to: (1) determine the incidence and degree of tumor cell contamination in paired BM and PBSC harvests; and (2) determine the efficacy of tumor cell purging by immunomagnetic CD34+ cell selection. 198 samples from 11 consecutive patients with neuroblastoma or Ewing's sarcoma were analyzed. We assayed tumor contamination by RT-PCR assay for PGP 9.5, plus immunohistochemistry for neuroblastoma-specific antigens (the latter in neuroblastoma only). None of these patients had tumor cells detected in their BM by clinical histology immediately before BM or PBSC harvests. However, 82% of PBSC and 89% of backup BM harvests were contaminated with tumor by RT-PCR and/or immunocytochemistry assays. Unselected PBSC and BM harvests contained similar quantities of tumor cells (median, approximately 200000 cells). Cyclophosphamide plus G-CSF mobilization did not affect the incidence or level of contamination in PBSC harvests, as compared to blood obtained before mobilization. Immunomagnetic CD34+ cell selection depleted tumor cells by a median of 3.0 logs for PBSC, and 2.6 logs for BM harvests.

Adolescent↗
PubMed1998

Tumor-suppressor p53: implications for tumor development and prognosis.

The p53 protein plays a central role in modulating cellular responses to cytotoxic stresses by contributing to both cell-cycle arrest and programmed cell death. Loss of p53 function during tumorigenesis can lead to inappropriate cell growth, increased cell survival, and genetic instability. p53 gene mutations occur in approximately half of all malignancies from a wide range of human tumors. In some tumor types, these p53 mutations are associated with poor prognosis and treatment failure. Based on these insights, new approaches are being developed to prevent, diagnose, and treat cancer.

Apoptosis↗
PubMed1997

DNA damage induces phosphorylation of the amino terminus of p53.

Data are presented demonstrating that DNA damage leads to specific post-translational modifications of p53 protein. Using two-dimensional peptide mapping of in vivo radiolabeled p53 tryptic phosphopeptides, recombinant truncated p53 protein, and synthetic p53 tryptic peptides, a unique p53 phosphopeptide was identified after exposure of ML-1 cells to ionizing irradiation. This peptide represents the first 24 amino acids of p53 and contains three phosphorylated serine residues. A specific p53 phosphopeptide antibody identified serine-15 as one of the two serines in p53 that becomes phosphorylated following DNA damage induced by either ionizing irradiation (IR) or ultraviolet (UV) irradiation in multiple cell types. IR-induced phosphorylation of p53 does not affect the kinetics of p53 binding to or dissociating from DNA as assessed by electrophoretic mobility-shift assays. However, p53 phosphorylation induced by DNA damage correlates with enhanced transcription of downstream p53 target genes. Low levels of phosphoserine-15 p53 are detectable within 6 hr after IR in AT cells, whereas lymphoblasts from normal individuals exhibit this modification within 1 hr. In contrast, phosphorylation of p53 on serine-15 is similar in normal and AT cells after UV irradiation. Our results indicate that p53 is phosphorylated in response to DNA damage, that this de novo phosphorylation may be involved in the subsequent induction and activation of p53, and that although ATM affects the kinetics of p53 phosphorylation after IR, it is not absolutely required for phosphorylation of p53 on serine-15.

Ataxia Telangiectasia↗