Kanji Ishizaki

DNA damage response pathway in radioadaptive response

Radioadaptive response is a biological defense mechanism in which low-dose ionizing irradiation elicits cellular resistance to the genotoxic effects of subsequent irradiation. However, its molecular mechanism remains largely unknown. We previously demonstrated that the dose recognition and adaptive response could be mediated by a feedback signaling pathway involving protein kinase C (PKC), p38 mitogen activated protein kinase (p38MAPK) and phospholipase C (PLC). Further, to elucidate the downstream effector pathway, we studied the X-ray-induced adaptive response in cultured mouse and human cells with different genetic background relevant to the DNA damage response pathway, such as deficiencies in TP53, DNA-PKcs, ATM and FANCA genes. The results showed that p53 protein played a key role in the adaptive response while DNA-PKcs, ATM and FANCA were not responsible. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), mimicked the priming irradiation in that the inhibitor alone rendered the cells resistant against the induction of chromosome aberrations and apoptosis by the subsequent X-ray irradiation. The adaptive response, whether it was afforded by low-dose X-rays or wortmannin, occurred in parallel with the reduction of apoptotic cell death by challenging doses. The inhibitor of p38MAPK which blocks the adaptive response did not suppress apoptosis. These observations indicate that the adaptive response and apoptotic cell death constitute a complementary defense system via life-or-death decisions. The p53 has a pivotal role in channeling the radiation-induced DNA double-strand breaks (DSBs) into an adaptive legitimate repair pathway, where the signals are integrated into p53 by a circuitous PKC-p38MAPK-PLC damage sensing pathway, and hence turning off the signals to an alternative pathway to illegitimate repair and apoptosis. A possible molecular mechanism of adaptive response to low-dose ionizing irradiation has been discussed in relation to the repair of DSBs and implicated to the current controversial observations on the expression of adaptive response.

Activation of Ataxia Telangiectasia-mutated DNA Damage Checkpoint Signal Transduction Elicited by Herpes Simplex Virus Infection

Eukaryotic cells are equipped with machinery to monitor and repair damaged DNA. Herpes simplex virus (HSV) DNA replication occurs at discrete sites in nuclei, the replication compartment, where viral replication proteins cluster and synthesize a large amount of viral DNA. In the present study, HSV infection was found to elicit a cellular DNA damage response, with activation of the ataxia-telangiectasia-mutated (ATM) signal transduction pathway, as observed by autophosphorylation of ATM and phosphorylation of multiple downstream targets including Nbs1, Chk2, and p53, while infection with a UV-inactivated virus or with a replication-defective virus did not. Activated ATM and the DNA damage sensor MRN complex composed of Mre11, Rad50, and Nbs1 were recruited and retained at sites of viral DNA replication, probably recognizing newly synthesized viral DNAs as abnormal DNA structures. These events were not observed in ATM-deficient cells, indicating ATM dependence. In Nbs1-deficient cells, HSV infection induced an ATM DNA damage response that was delayed, suggesting a functional MRN complex requirement for efficient ATM activation. However, ATM silencing had no effect on viral replication in 293T cells. Our data open up an interesting question of how the virus is able to complete its replication, although host cells activate ATM checkpoint signaling in response to the HSV infection.

Perturbed gap-filling synthesis in nucleotide excision repair causes histone H2AX phosphorylation in human quiescent cells

Human histone H2AX is rapidly phosphorylated on serine 139 in response to DNA double-strand breaks and plays a crucial role in tethering the factors involved in DNA repair and damage signaling. Replication stress caused by hydroxyurea or UV also initiates H2AX phosphorylation in S-phase cells, although UV-induced H2AX phosphorylation in non-cycling cells has recently been observed. Here we study the UV-induced H2AX phosphorylation in human primary fibroblasts under growth-arrested conditions. This reaction absolutely depends on nucleotide excision repair (NER) and is mechanistically distinct from the replication stress-induced phosphorylation. The treatment of cytosine-β-D-arabinofuranoside strikingly enhances the NER-dependent H2AX phosphorylation and induces the accumulation of replication protein A (RPA) and ATR-interacting protein (ATRIP) at locally UV-damaged subnuclear regions. Consistently, the phosphorylation appears to be mainly mediated by ataxia-telangiectasia mutated and Rad3-related (ATR), although Chk1 (Ser345) is not phosphorylated by the activated ATR. The cellular levels of DNA polymerases δ and ϵ and proliferating cell nuclear antigen are markedly reduced in quiescent cells. We propose a model that perturbed gap-filling synthesis following dual incision in NER generates single-strand DNA gaps and hence initiates H2AX phosphorylation by ATR with the aid of RPA and ATRIP.

O6-alkylguanine-DNA alkyltransferase activity of human malignant glioma and its clinical implications

SummaryActivity of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) is an important determinant of responsiveness of tumor cells to chloroethylnitrosoureas (CENUs), representative chemotherapeutic agents for primary malignant gliomas. In order to assess the real states of this repair protein in human malignant gliomas, we assayed AGT activity in surgically extirpated 42 malignant glioma samples and studied the distribution of the activity under certain clinical conditions. There were wide variations in AGT activity between individuals. No significant difference in AGT activity on average was seen either between glioblastoma and anaplastic astrocytoma, nor between primary and recurrent tumors. Among 42 malignant gliomas, 7 samples (16.7%) had low AGT activity less than 0.1 pmoles/mg protein. In the case of glioblastoma, tumors possessing higher AGT activity tended to be less responsive to post-operation remission-induction therapy including CENUs. The result of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) chemosensitivity assay by using the corresponding surgical specimens suggested a close relationship between cellular resistance to CENUs and AGT activity. It was found to be unlikely that a short term administration of CENUs had a significant effect on AGT activity of brain tumors in human body. We could detect a bit of definite evidences of the relevance of AGT to resistance to CENUs and need to conduct further investigations for other resistance factors.

Chromosomal assignments of 17 structural genes and 11 related DNA fragments in rats (Rattus norvegicus) by Southern blot analysis of rat × mouse somatic cell hybrid clones

DNA from 18 rat x mouse somatic cell hybrid clones, which segregated individual rat chromosomes, was analyzed by Southern blot for chromosomal gene assignments. Through the use of 17 DNA probes cloned from 7 rat genes, A2M, ATP1A1, ATP1A2, ATP1A3, B2M, GSTP, and SMST; 5 mouse genes, Ncam, Ngfg, Pim-1, Tcp-1, and Trp53; and 5 human genes, MBP, MYB, NEFM, SCN2A, and TCRGC1, 17 structural genes including 15 newly assigned genes and 11 related DNA fragments were assigned to particular rat chromosomes. Syntenic conservation of the genes among rats, mice, and humans is discussed.

Frequent somatic mutations of mitochondrial DNA in esophageal squamous cell carcinoma

Recent studies of various cancers, such as those of the breast, head and neck, bladder and lung, reported that 46–64% of somatic mutations in the D‐loop region of mitochondrial DNA (mtDNA) are observed. However, in esophageal cancer, only a low rate (5%) of somatic mutations has so far been reported in one article (Hibi, K. et al., Int J Cancer 2001;92:319–321). Thus, to confirm this we analyzed the somatic mutations for hypervariable regions (HVR‐I and HVR‐II) in the D‐loop of mtDNA to reevaluate the possibility of mitochondrial genetic instability in this cancer. We amplified both HVRs by PCR and DNA samples obtained from 38 esophageal tumors and matched normal tissues, and then sequenced them. Comparing the sequences of tumors to those of normal tissues, we found 14 somatic mutations in 13 patients (34.2%). Eleven mutations were at the C consecutive stretch from position 303 to 309 of MITOMAP in the mitochondria databank (http://www.mitomap.org/), 1 at position 215 in HVR‐II and 2 at positions 16,304 and 16,324 in HVR‐I. There were 41 types of germ line variations in HVR‐I including 2 not so far recorded in the mtDNA databank and 17 in HVR‐II including 1 not yet recorded. We also determined nuclear genome instability of these 38 specimens by analyzing 3 independent microsatellite sequences. While 4 specimens showed a single microsatellite change, which is tumor specific, we did not find any co‐relation between a somatic mtDNA mutation and microsatellite instability of nuclear genome DNA. These results suggest that mtDNA mutations might show a genetic instability in esophageal cancer independently from a nuclear genome instability.

Infrequent alterations of the p16INK4A gene in liver cancer

We examined the genomic status of the p16INK4A (inhibitor of cyclin‐dependent kinase 4 A) and cyclin‐dependent kinase 4 (CDK4) genes in 62 human hepatocellular carcinomas (HCCs), 5 cholangiocellular carcinomas and 6 cell lines derived from human liver cancers. Although no samples showed the homozygous deletion of the p16INK4A gene, we detected intragenic mutations of the p16INK4A gene in 3 HCCs and one HCC cell line, which led to an amino‐acid substitution or a frameshift. In 2 HCC samples with mis‐sense mutations of the p16INK4A gene, loss of heterozygosity on 9p22 was also detected, suggesting that the loss of function of p16 was induced during hepatocarcinogenesis. On the other hand, amplification or rearrangement of the CDK4 gene was not detected in any samples examined in this study. These results indicated that the mutations or deletions of the p16INK4A gene are not frequent, but may play a role in a sub‐set of human HCC.

Loss of heterozygosity on chromosome 17 and mutation of the p53 gene in retinoblastoma

Loss of heterozygosity (LOH) on chromosome 17 and mutations of the p53 gene were examined in 25 retinoblastomas (RB), consisting of three familial tumors, nine hereditary tumors without family history, 11 non-hereditary tumors, one recurrent tumor and one lung-metastatic tumor. LOH on chromosome 17 was detected in only one of the 23 primary RB. No mutations of the p53 gene were detected in the primary tumors. A recurrent tumor showed LOH on the short arm region of chromosome 17. LOH on chromosome 17 and a point mutation of the p53 gene were also detected in a metastatic tumor. These results suggest that LOH on chromosome 17 and mutation of the p53 gene may not be associated with the development of primary RB, but may play a role in the progression of RB.

Multiple types of aberrations in the p16 (INK4a) and the p15(INK4b) genes in 30 esophageal squamous-cell-carcinoma cell lines.

To determine the role and mode of inactivation of the p16 and p15 genes in human esophageal tumors, we examined alterations and expression of the α and β forms of the p16 gene, 5′ CpG island methylation of p16 exon 1α, and alterations of the p15 gene in 30 esophageal squamous‐cell‐carcinoma cell lines. Of 30 such cell lines examined, 28 (93%) showed aberrations of the α form of the p16 gene: 18 homozygous deletions, 6 point mutations and 4 hypermethylation. Methylation was exclusively observed in cell lines with the wild‐type α form. Of the 6 point mutations, one was observed in exon 1 α, one in the splice acceptor site of intron 1 and the remaining 4 were in exon 2. In the β form, 18 homozygous deletions and 3 point mutations in exon 2 were detected, but no point mutation was found in exon 1 β. All mutations in exon 2 gave rise to premature termination codons in the reading frame of the α transcript, while no non‐sense mutations were observed in the reading frame of the β transcript. Among 12 cell lines without homozygous deletions of the α and β forms of the p16 gene, the expected wild‐type β transcript was observed in 8 cell lines, whereas only one cell line expressed the expected wild‐type α transcript. Homozygous deletions of the p15 gene were observed in 16 cell lines (53%), and no point mutations were detected. Twelve cell lines had alterations only in the α form of the p16 gene, while none showed aberrations exclusively in the p15 gene. Taken together, these results indicate that inactivation of the β form of the p16 gene and the p15 gene are not so frequent as that of the α form of the p16 gene in ESC cell lines, suggesting that aberration of the α form of p16 gene is the primary target of 9p loss in ESC. Int. J. Cancer, 70:437–442, 1997.

Genetic alterations in patients with esophageal cancer with short- and long-term survival rates after curative esophagectomy.

OBJECTIVE The objective of this study was to ascertain the exact relation between specific oncogenes and long- and short-term survival rates in patients with esophageal cancer. SUMMARY BACKGROUND DATA Recent developments in molecular biology have shown that several oncogenes and suppressor genes are involved in the development of esophageal cancer. However, the role of these genes still is unknown. METHODS The clinical outcome of 84 cases of R0-resected esophageal carcinomas (from 1986-1993) and the molecular and biologic characteristics of these tumors were studied. The patients studied were divided into three groups, which were designated as follows: shortest term survivors (up to 6 months), short-term survivors (7-12 months), and long-term survivors (>5 years). These groups included 23, 17, and 44 subjects, respectively. For the genomic analysis, CyclinD1, int-2, murine double minute 2 (MDM2), retinoblastoma, p53, adenomatous polyposis coli (APC), deleted in colorectal carcinoma (DCC), and human papillomavirus were studied in these patients. The regrowth capability of primary cultures and the clinicopathologic characteristics of these patients also were analyzed. RESULTS The CyclinD1 and int-2 genes, which are located in the 11q13 chromosome, and the MDM2 gene were related to short survival. However, the p53 mutation and human papillomavirus infection were not related to short-term survival. The average ratio of genomic abnormalities to genes examined was higher in the shortest and short-term survival groups than in the long-term survival group. Regrowth capability in primary cultures also was related to short-term survival. Among the long-term survival patients, 7 (16%) of 44 cases suffered further cancer after esophagectomy. CONCLUSIONS These results suggest that the 11q13 amplicon and MDM2 may play an important role in the progression of esophageal cancer, and an accumulation of genomic abnormalities may result in poor prognosis. Careful follow-up testing for double cancer is needed in long-term survivors of esophageal cancer.