Enhanced phosphorylation of p53 by ATM in response to DNA damage.

The ATM protein, encoded by the gene responsible for the human genetic disorder ataxia telangiectasia (A-T), regulates several cellular responses to DNA breaks. ATM shares a phosphoinositide 3-kinase-related domain with several proteins, some of them protein kinases. A wortmannin-sensitive protein kinase activity was associated with endogenous or recombinant ATM and was abolished by structural ATM mutations. In vitro substrates included the translation repressor PHAS-I and the p53 protein. ATM phosphorylated p53 in vitro on a single residue, serine-15, which is phosphorylated in vivo in response to DNA damage. This activity was markedly enhanced within minutes after treatment of cells with a radiomimetic drug; the total amount of ATM remained unchanged. Various damage-induced responses may be activated by enhancement of the protein kinase activity of ATM.

A single ataxia telangiectasia gene with a product similar to PI-3 kinase.

A gene, ATM, that is mutated in the autosomal recessive disorder ataxia telangiectasia (AT) was identified by positional cloning on chromosome 11q22-23. AT is characterized by cerebellar degeneration, immunodeficiency, chromosomal instability, cancer predisposition, radiation sensitivity, and cell cycle abnormalities. The disease is genetically heterogeneous, with four complementation groups that have been suspected to represent different genes. ATM, which has a transcript of 12 kilobases, was found to be mutated in AT patients from all complementation groups, indicating that it is probably the sole gene responsible for this disorder. A partial ATM complementary DNA clone of 5.9 kilobases encoded a putative protein that is similar to several yeast and mammalian phosphatidylinositol-3' kinases that are involved in mitogenic signal transduction, meiotic recombination, and cell cycle control. The discovery of ATM should enhance understanding of AT and related syndromes and may allow the identification of AT heterozygotes, who are at increased risk of cancer.

ATM and ATR: networking cellular responses to DNA damage.

Maintenance of genome stability depends on the appropriate response to DNA damage. This response is based on complex networks of signaling pathways that activate numerous processes and lead ultimately to damage repair and cellular survival - or apoptosis. The protein kinases ATM and ATR are master controllers of some of these networks, acting either in concert or separately to orchestrate the responses to specific types of DNA damage or stalled replication. Understanding their mode of action is essential to our understanding of how cells cope with genotoxic stress.

Parallel induction of ATM-dependent pro- and antiapoptotic signals in response to ionizing radiation in murine lymphoid tissue.

The ATM protein kinase, functionally missing in patients with the human genetic disorder ataxia-telangiectasia, is a master regulator of the cellular network induced by DNA double-strand breaks. The ATM gene is also frequently mutated in sporadic cancers of lymphoid origin. Here, we applied a functional genomics approach that combined gene expression profiling and computational promoter analysis to obtain global dissection of the transcriptional response to ionizing radiation in murine lymphoid tissue. Cluster analysis revealed a prominent pattern characterizing dozens of genes whose response to irradiation was Atm-dependent. Computational analysis identified significant enrichment of the binding site signatures of NF-kappaB and p53 among promoters of these genes, pointing to the major role of these two transcription factors in mediating the Atm-dependent transcriptional response in the irradiated lymphoid tissue. Examination of the response showed that pro- and antiapoptotic signals were simultaneously induced, with the proapoptotic pathway mediated by p53 targets, and the prosurvival pathway by NF-kappaB targets. These findings further elucidate the molecular network induced by IR, point to novel putative NF-kappaB targets, and suggest a mechanistic model for cellular balancing between pro- and antiapoptotic signals induced by IR in lymphoid tissues, which has implications for cancer management. The emerging model suggests that restoring the p53-mediated apoptotic arm while blocking the NF-kappaB-mediated prosurvival arm could effectively increase the radiosensitivity of lymphoid tumors.

Ataxia-telangiectasia: a multifaceted genetic disorder associated with defective signal transduction.

The gene responsible for the defect in the human genetic disorder ataxia-telangiectasia, ATM, was cloned recently. The part of the gene coding for a phosphatidylinositol 3-kinase domain showed it to be related to a family of genes involved in signal transduction, cell cycle control and the response to DNA damage. The elucidation of the role of the ATM gene product will provide valuable insight into the radiosensitivity, cancer predisposition, immunodeficiency and neuropathology that characterize this syndrome.

Fragments of ATM which have dominant-negative or complementing activity.

The ATM protein has been implicated in pathways controlling cell cycle checkpoints, radiosensitivity, genetic instability, and aging. Expression of ATM fragments containing a leucine zipper motif in a human tumor cell line abrogated the S-phase checkpoint after ionizing irradiation and enhanced radiosensitivity and chromosomal breakage. These fragments did not abrogate irradiation-induced G1 or G2 checkpoints, suggesting that cell cycle checkpoint defects alone cannot account for chromosomal instability in ataxia telangiectasia (AT) cells. Expression of the carboxy-terminal portion of ATM, which contains the PI-3 kinase domain, complemented radiosensitivity and the S-phase checkpoint and reduced chromosomal breakage after irradiation in AT cells. These observations suggest that ATM function is dependent on interactions with itself or other proteins through the leucine zipper region and that the PI-3 kinase domain contains much of the significant activity of ATM.

Jun NH2-terminal kinase phosphorylation of p53 on Thr-81 is important for p53 stabilization and transcriptional activities in response to stress.

The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH2-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.

Kinetics of O6-methylguanine repair in human normal and ataxia telangiectasia cell lines and correlation of repair capacity with cellular sensitivity to methylating agents.

Human lymphoblastoid cell lines from normal individuals and from patients with ataxia telangiectasia were either proficient or deficient in their ability to repair the mutagenic DNA adduct O6-methylguanine that is induced by methylating carcinogens. There was no relationship between the capacity to repair O6-methylguanine and the ataxia telangiectasia phenotype. Time-course studies done following a short pulse (2 min) of alkylation with 0.5 microgram of N-[3H]methyl-N'-nitro-N-nitrosguanidine per ml revealed that the repair of O6-methylguanine in human lymphoblastoid lines proficient in this ability is a rapid process, which proceeds with a half-life of 10 to 15 min. Lymphoblastoid lines with deficient capacity to repair this DNA adduct were hypersensitive to the cytotoxic effect of the methylating carcinogens N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, and methyl methanesulfonate, and this hypersensitivity was correlated with the relative amount of O6-methylguanine induced by each of the three chemicals. This was taken as an indication of the lethality of unrepaired O6-methylgluanine. The extent of DNA repair synthesis induced by the three carcinogens was the same in cell lines proficient and deficient in O6-methylguanine repair, indicating no major deficiency in an excision repair pathway in the hypersensitive cell lines.

Cellular hypersensitivity to neocarzinostatin in ataxia-telangiectasia skin fibroblasts.

Cellular sensitivity of human skin fibroblast strains from three healthy donors, eight ataxia-telangiectasia (A-T) patients belonging to six sibships, and two A-T heterozygotes to the lethal action of the antitumor antibiotic neocarzinostatin was tested, using colony-forming ability as the criterion for survival. All the A-T strains were significantly more sensitive to killing by neocarzinostatin than were the control strains. The average D0 for the A-T strains following neocarzinostatin treatment was 14.6 ng/ml, as compared to 37.9 ng/ml for the normal strains. The two A-T heterozygous strains showed intermediate sensitivity with an average D0 of 26.9 ng/ml. Neocarzinostatin sensitivity of A-T cells could therefore serve as a convenient aid for the laboratory diagnosis of A-T. Since A-T cells are also known to be hypersensitive to ionizing radiation and bleomycin, it would appear that they are primarily hypersensitive to DNA-breaking agents.

Importance of phenotypic and molecular characterization for identification of a neuroepithelioma tumor cell line, NUB-20.

A neuroblastic-like cell line (NUB-20) was derived from a case of histopathologically diagnosed metastatic neuroblastoma. The metastatic tumor and nude mouse heterotransplant resembled neuroblastoma by histological criteria, in contrast to the primary tumor, which was differentially classified as Ewing's sarcoma. However, the cell line demonstrated a unique phenotype in culture with respect to morphology, immunohistochemical markers, and sensitivity to a battery of differentiation modulators. These characteristics, together with the presence of a chromosomal translocation (11;22),(q24;q12) and amplification with enhanced expression of the c-myc protooncogene rather than N-myc, established this tumor as neuroepithelioma. Neuroepithelioma is a tumor type distinct from, but related to, neuroblastoma in its development from the neural crest lineage. These results emphasize the growing importance of cytogenetic and molecular markers in the classification and characterization of human tumors.

Detection of ataxia telangiectasia heterozygous cell lines by postirradiation cumulative labeling index: measurements with coded samples.

Coded skin fibroblast cell strains from ataxia telangiectasia (AT) families and apparently normal individuals were obtained from two different sources. AT homozygous strains were clearly identified on the basis of marked hypersensitivity to cell killing by X-irradiation. AT heterozygotes were intermediate in their cytotoxic response between AT homozygotes and five normal reference cell strains. When density-inhibited cultures were X-irradiated and immediately subcultured to low density, a large fraction of AT heterozygous cells were irreversibly blocked in G1 as determined by cumulative labeling indices following incubation with [3H]thymidine. No such block occurred in four reference normal or AT homozygous strains. Three coded cell strains from apparently normal individuals resembled AT heterozygotes in their response; two of these strains were heterozygous for lysosomal storage disease. Thus, although the phenotype associated with the cellular response of AT heterozygous cells to X-irradiation is not specific to this disorder, the cumulative labeling indices assay may be a useful method for the detection of AT heterozygotes in kindreds with known AT.

Increased oxidative stress in ataxia telangiectasia evidenced by alterations in redox state of brains from Atm-deficient mice.

Ataxia-telangiectasia (A-T) is a genetic disorder caused by mutational inactivation of the ATM gene. A-T patients display a pleiotropic phenotype and suffer primarily from progressive ataxia caused by degeneration of cerebellar Purkinje and granule neurons. Disruption of the mouse Atm locus creates a murine model of A-T that exhibits most of the clinical features of the human disease. We previously hypothesized that some aspects of A-T, such as the preferential loss of certain neurons, could result from a continuous state of increased oxidative stress (G. Rotman and Y. Shiloh, Cancer Surv., 29: 285-304, 1997; G. Rotman and Y. Shiloh, BioEssays, 19: 911-917, 1997). The present work tests this hypothesis by analyzing markers of redox state in brains of Atm-deficient mice. We found alterations in the levels of thiol-containing compounds in Atm (-/-) brains, as well as significant changes in the activities of thioredoxin, catalase, and manganese superoxide dismutase in Atm (-/-) cerebella. These changes are indicative of increased levels of reactive oxygen species, which are seen primarily in the cerebellum of Atm-deficient mice. Our findings support the hypothesis that the absence of functional ATM results in oxidative stress, which may be an important cause of the degeneration of cerebellar neurons in A-T.

Cellular and molecular characteristics of an immortalized ataxia-telangiectasia (group AB) cell line.

Ataxia-telangiectasia (A-T) is a multisystem hereditary disease featuring neurodegeneration, immunodeficiency, extreme cancer proneness, chromosomal instability, and radiosensitivity. A-T is found in many ethnic groups, and is genetically heterogeneous: four complementation groups have been identified in A-T so far. Attempts to isolate the A-T gene are based in part on gene transfer experiments, using permanent A-T fibroblast lines, obtained by transformation with SV40. "Immortalization" of A-T primary diploid fibroblasts using SV40 is difficult, possibly because of the chromosomal instability of these cells. The number of currently available permanent A-T fibroblast lines is small, and not all of them have been assigned to specific complementation groups. Using the assay of X-ray induced inhibition of DNA synthesis, we have assigned the A-T strain AT22IJE to complementation group AB. Origin-defective SV40 was used to transfect these cells, and one transformant (AT22IJE-T), which survived crisis, was found to have the typical characteristics of permanent cell lines obtained in this way. "In-gel renaturation" analysis did not show any DNA amplification of high degree in AT22IJE-T. Cytogenetic analysis showed considerable chromosomal instability in the new cell line, and medium conditioned by these cells contained the clastogenic activity which is characteristic of the parental strain as well. Other parameters of the "cellular A-T phenotype" have also been retained in the immortalized cells: hypersensitivity to the lethal effects of X-rays and neocarzinostatin, as well as "radioresistant" DNA synthesis. However, the sensitivity of AT22IJE-T to both DNA-damaging agents is less pronounced than that of the parental cells. The capacity of the cells for uptake of foreign DNA was tested by introducing into them the plasmid pRSVneo, using three different transfection methods. Satisfactory frequency of G418-resistant transfectants (0.66%) was achieved using a protocol recently published by Chen and Okayama (Mol. Cell Biol., 7: 2745-2752, 1987), which was found to be superior to the traditional calcium phosphate transfection method and to the polybrene-based method.

Sporadic amplification of the HER2/neu protooncogene in adenocarcinomas of various tissues.

The HER2/neu protooncogene was found to be amplified in 6 of 109 primary adenocarcinoma tumors. No HER2/neu amplification was found in 29 other primary nonadenocarcinomatous tumors. In two colon tumors, in addition to the amplification, DNA rearrangement of HER2/neu gene was also observed. The rearrangement was explored in detail in one tumor and it was shown to be confined to the 3' region of the gene. Moreover, this tumor expressed an aberrant HER2/neu polypeptide with a molecular weight of 190,000, which is larger by approximately 5,000 than the molecular weight of the normal HER2/neu protein. The aberrant HER2/neu protein was immunoprecipitated with site-specific antibodies against a synthetic peptide from the COOH-terminal end of the normal HER2/neu protein; it also displayed intrinsic protein tyrosine kinase activity leading to self-phosphorylation.

Amplification and rearrangement of DNA sequences from the chromosomal region 2p24 in human neuroblastomas.

Seven DNA fragments which map to or very near human chromosome band 2p24 are shown to be differentially amplified in DNA from specific subsets of an enlarged series of human neuroblastoma cell lines and primary neuroblastomas. Of these DNA fragments, the probe NB-19-21 for the oncogene N-myc is the most frequently amplified, with a second expressed sequence (pG21) amplified in 9 of those 11 cell lines and 16 of those 25 tumors exhibiting amplification of N-myc. The remaining probes are in turn each amplified in progressively smaller, nested subsets of the cell lines and tumors in which both N-myc and pG21 are amplified. These data permit construction of models for the organization of a "neuroblastoma amplicon," i.e., an originally amplified DNA domain, with N-myc positioned most central and the other DNA fragments increasingly peripheral; comparable models result for the cell lines and the tumors. Five of the seven probes examined detect novel DNA fragments in these specimens, reinforcing previous observations that extensive DNA rearrangement can occur during DNA amplification in neuroblastoma cell lines and in primary neuroblastomas. Such rearrangements could contribute significantly to the evolution of the neuroblastoma amplicon in different specimens to progressively smaller units, preserving, in the limit, amplification of N-myc.

Definition and refinement of chromosome 11 regions of loss of heterozygosity in breast cancer: identification of a new region at 11q23.3.

Chromosome 11 is frequently altered in several types of human neoplasms. In breast cancer, loss of heterozygosity has been described in two regions of this chromosome, 11p15 and 11q22-23. In this report we have dissected the two regions using high-density polymorphic markers, and have found that there are at least two independent areas of loss of heterozygosity in each region, suggesting that multiple genes on chromosome 11 may be targets of genetic alteration during tumor establishment or progression. The regions defined are: at 11p15, between loci D11S576 and D11S1318 and between D11S988 and D11S1318; at 11q23, between D11S2000 and D11S897 and between D11S528 and D11S990. The narrowing of these regions of loss should facilitate the cloning of the regions in yeast artificial chromosomes to identify the critical tumor suppressor genes.

Atm knock-in mice harboring an in-frame deletion corresponding to the human ATM 7636del9 common mutation exhibit a variant phenotype.

ATM, the gene mutated in the human immunodeficiency disorder ataxia-telangiectasia (A-T), plays a central role in recognizing ionizing radiation damage in DNA and in controlling several cell cycle checkpoints. We describe here a murine model in which a nine-nucleotide in-frame deletion has been introduced into the Atm gene by homologous recombination followed by removal of the selectable marker cassette by Cre-loxP site-specific, recombination-mediated excision. This mouse, Atm-DeltaSRI, was designed as a model of one of the most common deletion mutations (7636del9) found in A-T patients. The murine Atm deletion results in the loss of three amino acid residues (SRI; 2556-2558) but produces near full-length detectable Atm protein that lacks protein kinase activity. Radiosensitivity was observed in Atm-DeltaSRI mice, whereas the immunological profile of these mice showed greater heterogeneity of T-cell subsets than observed in Atm(-/-) mice. The life span of Atm-DeltaSRI mice was significantly longer than that of Atm(-/-) mice when maintained under nonspecific pathogen-free conditions. This can be accounted for by a lower incidence of thymic lymphomas in Atm-DeltaSRI mice up to 40 weeks, after which time the animals died of other causes. The thymic lymphomas in Atm-DeltaSRI mice were characterized by extensive apoptosis, which appears to be attributable to an increased number of cells expressing Fas ligand. A variety of other tumors including B-cell lymphomas, sarcomas, and carcinomas not seen in Atm(-/-) mice were observed in older Atm-DeltaSRI animals. Thus, expression of mutant protein in Atm-DeltaSRI knock-in mice gives rise to a discernibly different phenotype to Atm(-/-) mice, which may account for the heterogeneity seen in A-T patients with different mutations.

Enhanced phosphorylation of p53 serine 18 following DNA damage in DNA-dependent protein kinase catalytic subunit-deficient cells.

DNA-dependent protein kinase (DNA-PK) controls signal transduction following DNA damage. However, the molecular mechanism of the signal transduction has been elusive. A number of candidates for substrates of DNA-PK have been reported on the basis of the in vitro assay system. In particular, the Ser-15 amino acid residue in p53 was one of the first such in vitro substrates to be described, and it has drawn considerable attention due to its biological significance. Moreover, p53 Ser-15 is a site that has been shown to be phosphorylated in response to DNA damage. In addition, crucial evidence indicating that DNA-PK controls the transactivation of p53 following DNA damage was reported quite recently. To clarify these important issues, we conducted the experiments with dna-pkcs null mutant cells, including gene knockout cells. As a result, we detected enhanced phosphorylation of p53 Ser-18, which corresponds to Ser-15 of human p53, and significant expression of p21 and mdm2 following ionizing radiation. Furthermore, we identified a missense point mutation in the p53 DNA-binding motif region in SCGR11 cells, which were established from severe combined immunodeficient (SCID) mice and used for previous study on the role of DNA-PK in p53 transactivation. Our observation clearly indicates that DNA-PK catalytic subunit does not phosphorylate p53 Ser-18 in vivo or control the transactivation of p53 in response to DNA damage, and these results further emphasize the different pathways in which ataxia telangiectasia-mutated (ATM) and DNA-PK operate following radiation damage.

ATM: a mediator of multiple responses to genotoxic stress.

The ATM protein kinase is the product of the gene responsible for the pleiotropic recessive disorder ataxia-telangiectasia. ATM-deficient cells show enhanced sensitivity and greatly reduced responses to genotoxic agents that generate DNA double strand breaks (DSBs), such as ionizing radiation and radiomimetic chemicals, but exhibit normal responses to DNA adducts and base modifications induced by other agents. Therefore, DSBs are most likely the predominant signal for the activation of ATM-mediated pathways. Identification of the ATM gene triggered extensive research aimed at elucidating the numerous functions of its large multifaceted protein product. While ATM has both nuclear and cytoplasmic functions, this review will focus on its roles in the nucleus where it plays a central role in the very early stages of damage detection and serves as a master controller of cellular responses to DSBs. By activating key regulators of multiple signal transduction pathways, ATM mediates the efficient induction of a signaling network responsible for repair of the damage, and for cellular recovery and survival.

Downregulation of the type 1 insulin-like growth factor receptor in mouse melanoma cells is associated with enhanced radiosensitivity and impaired activation of Atm kinase.

The type 1 insulin-like growth factor receptor (IGF1R) is required for growth, tumorigenicity and protection from apoptosis. IGF1R overexpression is associated with radioresistance in breast cancer. We used antisense (AS) RNA to downregulate IGF1R expression in mouse melanoma cells. Cells expressing AS-IGF1R transcripts were more radiosensitive in vitro and in vivo than controls. Also they showed reduced radiation-induced p53 accumulation and p53 serine 18 phosphorylation, and radioresistant DNA synthesis. These changes were reminiscent of the cellular phenotype of the human genetic disorder ataxia-telangiectasia (A-T), caused by mutations in the ATM gene. Cellular Atm protein levels were lower in AS-IGF1R-transfected cells than in control cells, although there was no difference in Atm expression at the transcriptional level. AS-IGF1R cells had detectable basal Atm kinase activity, but failed to induce kinase activity after irradiation. This suggests that IGF1R signalling can modulate the function of Atm, and supports the concept of targeted IGF1R downregulation as a potential treatment for malignant melanoma and other radioresistant tumours.