Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination In vivo.

Ku is a complex of two proteins, Ku70 and Ku80, and functions as a heterodimer to bind DNA double-strand breaks (DSB) and activate DNA-dependent protein kinase. The role of the Ku70 subunit in DNA DSB repair, hypersensitivity to ionizing radiation, and V(D)J recombination was examined in mice that lack Ku70 (Ku70(-/-)). Like Ku80(-/-) mice, Ku70(-/-) mice showed a profound deficiency in DNA DSB repair and were proportional dwarfs. Surprisingly, in contrast to Ku80(-/-) mice in which both T and B lymphocyte development were arrested at an early stage, lack of Ku70 was compatible with T cell receptor gene recombination and the development of mature CD4+CD8- and CD4-CD8+ T cells. Our data shows, for the first time, that Ku70 plays an essential role in DNA DSB repair, but is not required for TCR V(D)J recombination. These results suggest that distinct but overlapping repair pathways may mediate DNA DSB repair and V(D)J recombination.

Strand-specific postreplicative processing of mammalian telomeres.

Telomeres are specialized nucleoprotein structures that stabilize the ends of linear eukaryotic chromosomes. In mammalian cells, abrogation of telomeric repeat binding factor TRF2 or DNA-dependent protein kinase (DNA-PK) activity causes end-to-end chromosomal fusion, thus establishing an essential role for these proteins in telomere function. Here we show that TRF2-mediated end-capping occurs after telomere replication. The postreplicative requirement for TRF2 and DNA-PKcs, the catalytic subunit of DNA-PK, is confined to only half of the telomeres, namely, those that were produced by leading-strand DNA synthesis. These results demonstrate a crucial difference in postreplicative processing of telomeres that is linked to their mode of replication.

Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs.

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.

New syndrome with the Sakoda complex, bilateral anophthalmia, and cortical dysgenesis.

An 8-year-old Japanese boy had Sakoda complex (basal encephalomeningocele, agenesis of the corpus callosum, and cleft lip and/or palate) associated with bilateral anophthalmia, dysgenesis of the cerebral cortex, severe mental retardation, and intractable epilepsy as core symptoms and hemiparesis, microcephalus, short stature, and hemivertebra. Tada and Nakamura described the first case of the Sakoda complex associated with bilateral anophthalmia, cortical dysgenesis, neonatal-onset seizures, and severe mental retardation. Fourteen patients with the Sakoda complex with or without ocular dysplasia were reviewed. It is proposed that these cases belong to a clinical entity that is distinguishable from the remaining 12 patients because of bilateral anophthalmia, cortical dysgenesis, and its resulting severe mental retardation and intractable epilepsy. There is a possibility that these two cases are one severe end of certain spectrum disorders in which certain common gene(s) might be implicated.

Proteus syndrome: report of the first Japanese case with special reference to differentiation from Klippel-Trenaunay-Weber syndrome.

This is the first report of a Japanese girl with Proteus syndrome. She presented with growth acceleration and precocious development of the left breast as well as macrodactyly, hemihypertrophy, a subcutaneous preaxillary mass, portwine stains, connective tissue nevi, and a depigmented macule. All these abnormalities were confined to the left side of her body. Although most of the manifestations fit those of Proteus syndrome, the presence of the portwine stains and hemihypertrophy also suggested Klippel-Trenaunay-Weber syndrome. The findings in our patient suggest that the most important characteristic distinguishing Proteus syndrome from Klippel-Trenaunay-Weber syndrome is the presence of functional abnormalities such as a growth spurt and precocious breast development. Proteus syndrome may be genetically different from the Klippel-Trenaunay-Weber syndrome.

Effect of rectal administration of rebamipide on dextran sulfate sodium-induced colitis: role of hepatocyte growth factor.

OBJECTIVE AND DESIGN: Since rebamipide is effective for the treatment of ulcerative colitis (UC), we examined the involvement of hepatocyte growth factor (HGF) in the action of rebamipide. MATERIALS: Fifty-five and forty female Balb/c mice, respectively, were used in Exp. 1 and 2. TREATMENT: 50 mg/kg/day rebamipide (Exp. 1) and 1 x 10(7) pfu pAxCAHGF (the CAG promoter-driving HGF gene in adenovirus vector) (Exp. 2) were intrarectally introduced after induction of colitis by 4 % dextran sulfate sodium (DSS). METHODS: Therapeutic effects were assessed by cell proliferation and apoptosis. RESULTS: Rebamipide caused proliferation of epithelial cells at 10 days after treatment, and decreased apoptosis at 10, 14 and 21 days, compared with controls. Expression of HGF was greatly increased in rebamipide-treated mice. pAxCAHGF caused cell proliferation and apoptosis, which showed the same pattern as with rebamipide treatment. CONCLUSIONS: Rectal administration of rebamipide is effective for DSS-induced colitis in association with induction of HGF.

Restoration of the cholesterol metabolism in 3T3 cell lines derived from the sphingomyelinosis mouse (spm/spm) by transfer of a human chromosome 18.

We searched for a human chromosome that would restore the cholesterol metabolism in 3T3 cell lines (SPM-3T3) derived from homozygous sphingomyelinosis mice (spm/spm). Mouse A9 cells containing a single copy of pSV2neo-tagged chromosomes 9, 11, or 18 derived from normal human fibroblasts served as donor cells for transfer of human chromosomes. Purified A9 microcells were fused with SPM-3T3 cells, and the microcell hybrids were selected in medium containing G418 antibiotics. The microcell hybrids that contained human chromosomes 9, 11, or 18 in a majority of cells were examined. The accumulation of intracellular cholesterol in the microcell hybrids containing a chromosome 18 decreased markedly, whereas in the microcell hybrids containing either chromosomes 9 or 11 it was similar to that in SPM-3T3 cells. The SPM-3T3 cells with an intact chromosome 18 were further passaged and subcloned. Clones which again accumulated intracellular cholesterol had concurrently lost the introduced chromosome 18. The abnormal accumulation was associated with a decrement in the esterification of exogenous cholesterol. These findings suggest that the gene responsible for the abnormal cholesterol metabolism in the spm/spm mice can be restored by a human chromosome 18. The gene was tentatively mapped on 18pter-->18p11.3 or 18q21.3-->qter that was lost during subcloning, thereby resulting in reaccumulation of the intracellular cholesterol.

ATM phosphorylates histone H2AX in response to DNA double-strand breaks.

A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ATM and Rad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs-/- and Atm-/- cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs-/- cells and severely compromised in Atm-/- cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm-/- fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm-/- fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks.

A human gene that restores the DNA-repair defect in SCID mice is located on 8p11.1-->q11.1.

In order to map the gene that is responsible for the DNA-repair defect in severe combined immune deficient (SCID) mice, a mixture of microcells independently isolated from mouse A9 cells containing pSV2neo-tagged human chromosomes 5, 7, 8, 9, 11, 15, 18 or 20 were fused with SCID fibroblast cell lines SCVA2 and SCVA4, which were originally established from lung tissue of the C.B.17-scid/scid mouse by SV40 virus transfection. After irradiation with 60Co gamma-rays and selection with antibiotic G418, 12 independent clones were obtained, of which 4 contained an intact chromosome 8, 3 clones contained a deleted chromosome 8 [del(8)q22-->qter or del(8)q23--> qter] and remaining 5 had no detectable or specific human chromosome. We further independently transferred a single human chromosome 8 or 11 into the SCVA cells via microcell fusion, and examined the radiation sensitivity of the microcell hybrids. Complementation of the radiation sensitivity was correlated with the presence of human chromosome 8 in microcell hybrids, whereas no correlation was observed in clones following the transfer of human chromosome 11. Thus, the results indicate that human chromosome 8 restored high sensitivity to ionizing radiation. A number of subclones that were radiation resistant or sensitive were isolated from the microcell hybrids. The concordance of the radiation sensitivity with the presence or absence of specific DNA fragments on chromosome 8 indicates that the human gene is located on the centromeric region of chromosome 8, i.e., 8p11.1--> q11.1.

Genetic evidence of a role for ATM in functional interaction between human T-cell leukemia virus type 1 Tax and p53.

Recent evidence from several investigators suggest that the human T-cell leukemia virus type 1 Tax oncoprotein represses the transcriptional activity of the tumor suppressor protein, p53. An examination of published findings reveals serious controversy as to the mechanism(s) utilized by Tax to inhibit p53 activity and whether the same mechanism is used by Tax in adherent and suspension cells. Here, we have investigated Tax-p53 interaction simultaneously in adherent epithelial (HeLa and Saos) and suspension T-lymphocyte (Jurkat) cells. Our results indicate that Tax activity through the CREB/CREB-binding protein (CBP), but not NF-kappaB, pathway is needed to repress the transcriptional activity of p53 in all tested cell lines. However, we did find that while CBP binding by Tax is necessary, it is not sufficient for inhibiting p53 function. Based on knockout cell studies, we correlated a strong genetic requirement for the ATM, but not protein kinase-dependent DNA, protein in conferring a Tax-p53-repressive phenotype.

Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells.

The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an approximately 350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway.

Cosmids and transcribed sequences from chromosome 11q23.

To obtain cosmid markers and transcribed sequences from a specific chromosome region, a series of radiation-reduced hybrids (RHs) containing various regions of human chromosome 11 was prepared from microcell hybrid A9 (neo11) cells containing a normal human chromosome 11 tagged with pSV2neo at 11p11.2. Among 15 radiation hybrid clones isolated, RH(11)-9 which contains a q23 fragment in addition to the neo integration site, was used for the construction of a cosmid library. Cosmid clones having human DNA sequences were screened, and localized by Southern hybridization with the radiation hybrid panel. Fifty-nine cosmids were assigned to 11q23 and 6 cosmids to 11p11.2. Exon amplification proceeded with 23 of the 59 cosmids and 16 putative exons were cloned. Three of them were identical to those constituting a known gene which locates on q23 (ATDC), and the others were unknown. Thus, the RHs containing various subchromosomal fragments of chromosome 11 were useful for constructing region-specific DNA markers. The RH(11)-9 cells and putative exons also facilitate the positional cloning of genes in the 11q23 region.

DNA-PKcs is critical for telomere capping.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is critical for DNA repair via the nonhomologous end joining pathway. Previously, it was reported that bone marrow cells and spontaneously transformed fibroblasts from SCID (severe combined immunodeficiency) mice have defects in telomere maintenance. The genetically defective SCID mouse arose spontaneously from its parental strain CB17. One known genomic alteration in SCID mice is a truncation of the extreme carboxyl terminus of DNA-PKcs, but other as yet unidentified alterations may also exist. We have used a defined system, the DNA-PKcs knockout mouse, to investigate specifically the role DNA-PKcs specifically plays in telomere maintenance. We report that primary mouse embryonic fibroblasts (MEFs) and primary cultured kidney cells from 6-8 month-old DNA-PKcs-deficient mice accumulate a large number of telomere fusions, yet still retain wild-type telomere length. Thus, the phenotype of this defect separates the two-telomere related phenotypes, capping, and length maintenance. DNA-PKcs-deficient MEFs also exhibit elevated levels of chromosome fragments and breaks, which correlate with increased telomere fusions. Based on the high levels of telomere fusions observed in DNA-PKcs deficient cells, we conclude that DNA-PKcs plays an important capping role at the mammalian telomere.

Signal joint formation is also impaired in DNA-dependent protein kinase catalytic subunit knockout cells.

The effort to elucidate the mechanism of V(D)J recombination has given rise to a dispute as to whether DNA-dependent protein kinase catalytic subunit (DNA-PKcs) contributes to signal joint formation (sjf). Observations reported to date are confusing. Analyses using DNA-PKcs-deficient cells could not conclude the requirement of DNA-PKcs for sjf, because sjf can be formed by end-joining activities which are diverse among cells other than those participating in V(D)J recombination. Here, we observed V(D)J recombination in DNA-PKcs knockout cells and showed that both signal and coding joint formation were clearly impaired in the cells. Subsequently, to directly demonstrate the requirement of DNA-PKcs for sjf, we introduced full-length cDNA of DNA-PKcs into the knockout cells. Furthermore, several mutant DNA-PKcs cDNA constructs designed from mutant cell lines (irs-20, V3, murine scid, and SX9) were also introduced into the cells to obtain further evidence indicating the involvement of DNA-PKcs in sjf. We found as a result that the full-length cDNA complemented the aberrant sjf and that the mutant cDNAs constructs also partially complemented it. Lastly, we looked at whether the kinase activity of DNA-PKcs is necessary for sjf and, as a result, demonstrated a close relationship between them. Our observations clearly indicate that the DNA-PKcs controls not only coding joint formation but also the sjf in V(D)J recombination through its kinase activity.

Construction of 110 cosmid markers and a 4.5-Mb YAC contig on human chromosome 8p12-q11.

Microcell hybrids containing various regions of human chromosome 8 were formed by microcell-mediated transfer of neo-tagged chromosome 8 into the cells derived from severe combined immunodeficiency (SCID) mouse. Thus, 110 cosmid markers were isolated from SV40-transformed SCID fibroblast cell line (SCVA) containing a p12-q11.1 region of human chromosome 8 and were assigned to eight regions in 8p12-q11.1, using a microcell-hybrid panel. For positional cloning of a human gene that restores the DNA-repair defect in a mouse with SCID on 8p11.1-q11.1 (SCID region), we constructed a yeast artificial chromosome (YAC) contig of about 4.5 Mb. Overlapping YACs were further aligned by restriction mapping, using rare-cutting restriction endonucleases. The cosmids and YAC contig should facilitate isolation of the SCID gene and other genes, such as the Werner syndrome-responsible gene in or near this region.

The catalytic subunit of DNA-dependent protein kinase selectively regulates p53-dependent apoptosis but not cell-cycle arrest.

DNA damage induced by ionizing radiation (IR) activates p53, leading to the regulation of downstream pathways that control cell-cycle progression and apoptosis. However, the mechanisms for the IR-induced p53 activation and the differential activation of pathways downstream of p53 are unclear. Here we provide evidence that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) serves as an upstream effector for p53 activation in response to IR, linking DNA damage to apoptosis. DNA-PKcs knockout (DNA-PKcs-/-) mice were exposed to whole-body IR, and the cell-cycle and apoptotic responses were examined in their thymuses. Our data show that IR induction of apoptosis and Bax expression, both mediated via p53, was significantly suppressed in the thymocytes of DNA-PKcs-/- mice. In contrast, IR-induced cell-cycle arrest and p21 expression were normal. Thus, DNA-PKcs deficiency selectively disrupts p53-dependent apoptosis but not cell-cycle arrest. We also confirmed previous findings that p21 induction was attenuated and cell-cycle arrest was defective in the thymoctyes of whole body-irradiated Atm-/- mice, but the apoptotic response was unperturbed. Taken together, our results support a model in which the upstream effectors DNA-PKcs and Atm selectively activate p53 to differentially regulate cell-cycle and apoptotic responses. Whereas Atm selects for cell-cycle arrest but not apoptosis, DNA-PKcs selects for apoptosis but not cell-cycle arrest.

A 3-Mb physical map of the chromosome region 8p21.3-p22, including a 600-kb region commonly deleted in human hepatocellular carcinoma, colorectal cancer, and non-small cell lung cancer.

To isolate a putative tumor suppressor gene(s), we have constructed a physical map and a detailed deletion map of chromosome region 8p21.3-p22, where loss of heterozygosity (LOH) has been frequently seen in human hepatocellular carcinomas (HCC), colorectal cancers (CRC), and non-small cell lung cancers (NSCLC). The smallest commonly deleted region at 8p21.3-p22 in HCC and CRC was between the loci defined by C18-245 and C18-2644; in NSCLC, a region between C18-1051 and C18-2644 was commonly deleted. A contiguous physical map of 12 cosmid markers in the 8p21.3-p22 region was constructed by means of multi-color fluorescence in situ hybridization (FISH) and pulsed-field gel electrophoresis (PFGE). On the basis of this physical map, which spans roughly 3.1 Mb, the estimated sizes of the commonly deleted regions were at most 1.2 Mb in HCC and CRC and 0.6 Mb in NSCLC. As four of the 12 physically ordered markers are located within the 0.6 Mb region commonly deleted in all three tumor types, nearly one fourth to one fifth of the target region has already been covered with cosmid inserts.

DNA-dependent protein kinase-independent activation of p53 in response to DNA damage.

Phosphorylation at serine 15 of the human p53 tumor suppressor protein is induced by DNA damage and correlates with accumulation of p53 and its activation as a transcription factor. The DNA-dependent protein kinase (DNA-PK) can phosphorylate serine 15 of human p53 and the homologous serine 18 of murine p53 in vitro. Contradictory reports exist about the requirement for DNA-PK in vivo for p53 activation and cell cycle arrest in response to ionizing radiation. While primary SCID (severe combined immunodeficiency) cells, that have defective DNA-PK, show normal p53 activation and cell cycle arrest, a transcriptionally inert form of p53 is induced in the SCID cell line SCGR11. In order to unambiguously define the role of the DNA-PK catalytic subunit (DNA-PKcs) in p53 activation, we examined p53 phosphorylation in mouse embryonic fibroblasts (MEFs) from DNA-PKcs-null mice. We found a similar pattern of serine 18 phosphorylation and accumulation of p53 in response to irradiation in both control and DNA-PKcs-null MEFs. The induced p53 was capable of sequence-specific DNA binding even in the absence of DNA-PKcs. Transactivation of the cyclin-dependent-kinase inhibitor p21, a downstream target of p53, and the G1 cell cycle checkpoint were also found to be normal in the DNA-PKcs -/- MEFs. Our results demonstrate that DNA-PKcs, unlike the related ATM protein, is not essential for the activation of p53 and G1 cell cycle arrest in response to ionizing radiation.