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Query: UMLS:C0004135 (ATM)
 13,001 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of ionizing radiation on the expression of two DNA-damage-inducible genes, designated gadd45 and gadd153, was examined in cultured human cells. These genes have previously been shown to be strongly and coordinately induced by UV radiation and alkylating agents in human and hamster cells. We found that the gadd45 but not the gadd153 gene is strongly induced by X rays in human cells. The level of gadd45 mRNA increased rapidly after X rays at doses as low as 2 Gy. After 20 Gy of X rays, gadd45 induction, as measured by increased amounts of mRNA, was similar to that produced by the most effective dose of the alkylating agent methyl methanesulfonate. No induction was seen after treatment of either human or hamster cells with 12-O-tetradecanoylphorbol-13-acetate, a known activator of protein kinase C (PKC). Therefore, gadd45 represents the only known mammalian X-ray-responsive gene whose induction is not mediated by PKC. However, induction was blocked by the protein kinase inhibitor H7, indicating that induction is mediated by some other kinase(s). Sequence analysis of human and hamster cDNA clones demonstrated that this gene has been highly conserved and encodes a novel 165-amino-acid polypeptide which is 96% identical in the two species. This gene was localized to the short arm of human chromosome 1 between p12 and p34. When induction in lymphoblast lines from four normal individuals was compared with that in lines from four patients with ataxia telangiectasia, induction by X rays of gadd45 mRNA was less in the cell lines from this cancer-prone radiosensitive disorder. Our results provide evidence for the existence of an X-ray stress response in human cells which is independent of PKC and which is abnormal in taxia telangiectasia.
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PMID:Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C. 199 Feb 62

Replication protein A (RPA), the trimeric single-stranded DNA-binding protein complex of eukaryotic cells, is important to DNA replication and repair. Phosphorylation of the p34 subunit of RPA is modulated by the cell cycle, occurring during S and G2 but not during G1. The function of phosphorylated p34 remains unknown. We show that RPA p34 phosphorylation is significantly induced by ionizing radiation. The phosphorylated form, p36, is similar if not identical to the phosphorylated S/G2 form. gamma-Irradiation-induced phosphorylation occurs without new protein synthesis and in cells in G1. Mutation of cdc2-type protein kinase phosphorylation sites in p34 eliminates the ionizing radiation response. The gamma-irradiation-induced phosphorylation of RPA p34 is delayed in cells from ataxia telangiectasia, a human inherited disease conferring DNA repair defects and early-onset tumorigenesis. UV-induced phosphorylation of RPA p34 occurs less rapidly than gamma-irradiation-induced phosphorylation but is kinetically similar between ataxia telangiectasia and normal cells. This is the first time that modification of a repair protein, RPA, has been linked with a DNA damage response and suggests that phosphorylation may play a role in regulating DNA repair pathways.
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PMID:The ionizing radiation-induced replication protein A phosphorylation response differs between ataxia telangiectasia and normal human cells. 824 44

Replication protein A (RPA, also called human single stranded DNA binding protein, hSSB) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair and recombination. Phosphorylation of RPA p34 subunit is observed after exposure of cells to radiation and other DNA damaging agents, which implicates the protein not only in repair but also in the regulation of replication on damaged DNA template. Here, we show that the phosphorylation observed in RPA p34 after exposure to ionizing radiation, X- or gamma-rays, is reduced and occurs later in primary fibroblasts from patients suffering from ataxia telangiectasia (AT), as compared to normal fibroblasts. We also show that in primary normal human fibroblasts, radiation-induced phosphorylation of RPA p34 is 'age'-dependent and decreases significantly as cultures senesce. Radiation-induced phosphorylation of RPA p34 is nearly absent in non-cycling cells, while the expression of p21cipl/wafl/sdil remains inducible. The results demonstrate a growth-state and culture-age dependency in radiation-induced RPA p34 phosphorylation, and suggest the operation of a signal transduction pathway that is inactivated in senescing or quiescent fibroblasts and defective in AT cells.
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PMID:Ionizing radiation-induced phosphorylation of RPA p34 is deficient in ataxia telangiectasia and reduced in aged normal fibroblasts. 873 93

Replication protein A (RPA) is a trimeric single-stranded DNA-binding protein complex involved in DNA replication, repair, and recombination. DNA damage induces phosphorylation of the RPA p34 subunit, and it has been speculated that this phosphorylation could contribute to the regulation of the DNA damage-induced S-phase checkpoint. To further examine this potential relationship, human cell lines expressing ataxia telangiectasia (AT)-mutated dominant-negative fragments, which fail to arrest in S phase in response to ionizing radiation (IR), and AT cells expressing AT-mutated-complementing fragments, which regain the ability to arrest replicative DNA synthesis in response to IR, were analyzed for radiation-induced RPA phosphorylation. Results from these studies demonstrate that IR-induced RPA phosphorylation can be uncoupled from the S-phase checkpoint, suggesting that RPA phosphorylation in response to IR is neither necessary nor sufficient for an S-phase arrest.
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PMID:Dissociation of radiation-induced phosphorylation of replication protein A from the S-phase checkpoint. 927 1

Ataxia telangiectasia-mutated gene (ATM) is a 350-kDa protein whose function is defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM protein varied from undetectable in most AT cell lines to highly expressed in HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that ATM is predominantly a nuclear protein associated with the chromatin and nuclear matrix. ATM protein levels remained constant throughout the cell cycle and did not change in response to serum stimulation. Ionizing radiation had no significant effect on either the expression or distribution of ATM. ATM immunoprecipitates from HeLa cells and the human DNA-dependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA-dependent manner. Phosphorylation of p34 RPA occurred on threonine and serine residues. Phosphopeptide analysis demonstrates that the ATM-associated protein kinase phosphorylates p34 RPA on similar residues observed in vivo. The DNA-dependent protein kinase activity observed for ATM immunocomplexes, along with the association of ATM with chromatin, suggests that DNA damage can induce ATM or a stably associated protein kinase to phosphorylate proteins in the DNA damage response pathway.
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PMID:Characterization of ATM expression, localization, and associated DNA-dependent protein kinase activity. 972 99

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4-8 h) to UV radiation (10-30 J/m(2)). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.
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PMID:UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein. 1135 16

The heterotrimeric DNA-binding protein, replication protein A (RPA), consists of 70-, 34-, and 14-kDa subunits and is involved in maintaining genomic stability by playing key roles in DNA replication, repair, and recombination. RPA participates in these processes through its interaction with other proteins and its strong affinity for single-stranded DNA (ssDNA). RPA-p34 is phosphorylated in a cell-cycle-dependent fashion primarily at Ser-29 and Ser-23, which are consensus sites for Cdc2 cyclin-dependent kinase. By systematically examining RPA-p34 phosphorylation throughout the cell cycle, we have found there are distinct phosphorylated forms of RPA-p34 in different cell-cycle stages. We have isolated and purified a unique phosphorylated form of RPA that is specifically associated with the mitotic phase of the cell cycle. The mitotic form of RPA (m-hRPA) shows no difference in ssDNA binding activity as compared with recombinant RPA (r-hRPA), yet binds less efficiently to double-stranded DNA (dsDNA). These data suggest that mitotic phosphorylation of RPA-p34 inhibits the destabilization of dsDNA by RPA complex, thereby decreasing the binding affinity for dsDNA. The m-hRPA also exhibits altered interactions with certain DNA replication and repair proteins. Using highly purified proteins, m-hRPA exhibited decreased binding to ATM, DNA pol alpha, and DNA-PK as compared to unphosphorylated recombinant RPA (r-hRPA). Dephosphorylation of m-hRPA was able to restore the interaction with each of these proteins. Interestingly, the interaction of RPA with XPA was not altered by RPA phosphorylation. These data suggest that phosphorylation of RPA-p34 plays an important role in regulating RPA functions in DNA metabolism by altering specific protein-protein interactions.
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PMID:RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions. 1264 57

The impact of disruption of the PI3K (phosphatidylinositol 3-kinase) pathway on the response of human leukemia cells to pharmacological cyclin-dependent kinase (CDK) inhibitors has been examined. Exposure of U937 monocytic leukemia cells to minimally toxic concentrations of flavopiridol (FP), roscovitine, or CGP74514A for 3 h in conjunction with the PI3K inhibitor LY294002 (abbreviated LY in the article) resulted in a marked decrease in Akt phosphorylation. Coexposure of cells to LY and CDK inhibitors also resulted in an early (i.e., within 3 h) and striking increase in mitochondrial damage [e.g., cytochrome c, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis (IAP)-binding protein with low isoelectric point (Smac/DIABLO), and apoptosis-initiating factor (AIF) release], caspase activation, and apoptosis. Similar interactions were observed in a variety of other leukemia cell types (e.g., HL-60, Jurkat, Raji, and NB4). Apoptosis, induced by FP/LY, was substantially blocked by ectopic expression of Bcl-2, but to a considerably lesser extent by dominant-negative caspase-8. FP-induced apoptosis was not enhanced by agents that inhibited protein kinase (PK) A (H89), PKC (GFX), mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK1/2; U0126), p38 MAP kinase (MAPK; SB202190), m-target of rapamycin (TOR; rapamycin), or ataxia-telangiectasia mutation (ATM; caffeine), whereas the PI3K inhibitor wortmannin exerted effects similar to those of LY. The dramatic potentiation of CDK inhibitor-induced apoptosis by LY was accompanied by diminished Bad phosphorylation, induction of Bcl-2 cleavage, and down-regulation of X-linked IAP (XIAP) and Mcl-1. Cells exposed to CDK inhibitors + LY also exhibited reduced phosphorylation of glycogen synthase kinase (GSK)-3, forkhead transcription factor (FKHR), p70(S6K), and ERK, but increased activation of p34(cdc2) and p38 MAPK. LY/CDK inhibitor-treated cells also displayed diminished pRb dephosphorylation on CDK2- and CDK4-specific sites, retinoblastoma protein cleavage, and down-regulation of cyclin D(1). Inducible expression of constitutively active (myristolated) Akt significantly, albeit partially, attenuated apoptosis in Jurkat leukemia cells treated with either FP alone or the combination of FP and LY. Finally, cotreatment with LY and FP resulted in a dramatic increase in apoptosis in primary leukemic blasts obtained from a patient with acute myeloblastic leukemia. Together, these findings suggest that the PI3K/Akt pathway plays a major role in regulating the apoptotic response of human leukemia cells to pharmacological CDK inhibitors and raise the possibility that combined interruption of CDK- and PI3K-related pathways may represent a novel therapeutic strategy in hematological malignancies.
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PMID:The lethal effects of pharmacological cyclin-dependent kinase inhibitors in human leukemia cells proceed through a phosphatidylinositol 3-kinase/Akt-dependent process. 1270 69

Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, gamma-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and gamma-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and gamma-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and gamma-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and gamma-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with gamma-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and gamma-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with gamma-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells.
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PMID:Replication protein A and gamma-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks. 1547 97

The presence of DNA damage activates a specific response cascade culminating in DNA repair activity and cell cycle checkpoints. Although the type of lesion dictates what proteins are involved in the response, replication protein A (RPA) and the Mre11/ Rad50/Nbs1 complex (MRN) respond to most types of lesions. To examine the relationship of RPA and the MRN complex in DNA damage responses, we used siRNA-mediated protein depletion of RPA-p70 and Mre11. Depletion of RPA-p70 decreased the ability of cells to form phospho-Nbs1 foci and increased levels of DNA double-strand breaks (DSBs) following treatment with etoposide (ETOP). In contrast, depletion of Mre11 led to increased levels of RPA-p34 foci formation, but abrogated phospho-RPA-p34 foci formation. These data support a role for RPA as an initial signal/sensor for DNA damage that facilitates recruitment of MRN and ATM/ATR to sites of damage, where they then work together to fully activate the DNA damage response.
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PMID:Replication protein A is required for etoposide-induced assembly of MRE11/RAD50/NBS1 complex repair foci. 1770 70


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