Jun-ichiro Komura

A PHOSPHATIDYLINOSITOL 3-KINASE INHIBITOR WORTMANNIN INDUCES RADIORESISTANT DNA SYNTHESIS AND SENSITIZES CELLS TO BLEOMYCIN AND IONIZING RADIATION

ATM and DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) have been shown to have sequences homologous to the catalytic domains of mammalian phosphatidylinositol 3‐kinase (PI3‐kinase). In order to determine the contribution of ATM and DNA‐PKcs to the increased sensitivity of cells to DNA‐damaging agents observed in the presence of PI3‐kinase inhibitors, we examined the effects of a PI3‐kinase inhibitor, wortmannin, on cellular sensitivity to bleomycin (BLM), mitomycin C (MMC), X‐irradiation and ultraviolet (UV)‐irradiation using 2 human tumor cell lines (T98G and A172), a human fibroblast cell line (LM217), an ataxia telangiectasia (AT) cell line (AT3BISV), a scid murine cell line (SCF) and a control murine cell line (CBF). Wortmannin sensitized all of the cells, including AT3BISV and SCF, to BLM and X‐irradiation, but not to MMC or UV‐irradiation. Hypersensitivity to BLM and X‐irradiation and normal sensitivity to MMC and UV‐irradiation are characteristic phenotypes of both AT and scid mice. DNA‐dependent protein kinase (DNA‐PK) activity was suppressed by wortmannin to 45–65% of the control values in all of the cells except SCF, in which DNA‐PK activity was not detected. Wortmannin also induced radioresistant DNA synthesis, which is a cellular phenotype of AT, in T98G and SCF cells, but did not change the DNA synthesis rates after X‐irradiation in AT3BISV. Our data suggest that wortmannin decreases the activities of both the ATM protein and DNA‐PK, indicating that it might be of use as a sensitizing agent for radiotherapy and chemotherapy. Int. J. Cancer 78:642–647, 1998.

Chromatin structure analysis by ligation-mediated and terminal transferase-mediated polymerase chain reaction

Publisher Summary This chapter discusses chromatin structure analysis by ligation-mediated and terminal transferase-mediated polymerase chain reaction (PCR). The chapter describes several protocols for chromatin analysis, each of which depends on the ligation of a linker (or an adapter) oligonucleotide to DNA prior to PCR. These ligation-mediated PCR assays provide single-nucleotide resolution and sensitivity adequate for the study of mammalian cells. The chapter discusses ligation-mediated PCR (LM-PCR) and transferase-dependent PCR (TD-PCR). Both methods can be used to obtain information on minimally perturbed in vivo chromatin structure, and each requires only a microgram or less of total genomic DNA per lane of a sequencing gel. Good quality sequence-type ladders are obtained that contains information on DNA structure and DNA-bound proteins. The chapter describes a short procedure that allows LM-PCR data to be obtained in two days or less rather than the four or five days required for the standard procedure.

Age-dependent alterations in mRNA level and promoter methylation of collagen α1(I) gene in human periodontal ligament

In an attempt to understand the molecular mechanisms of age-dependent degenerative alteration in human periodontal tissues, we examined mRNA level and DNA methylation of collagen alpha1(I) gene. Using healthy periodontal ligament tissues from humans aged 9-76 years, we found that the collagen alpha1(I) mRNA level decreased almost linearly with age. It was observed in both Northern blot and dot blot hybridization. Examination of DNA methylation in the collagen alpha1(I) gene promoter region by its susceptibility to methylation-sensitive restriction enzyme followed by Southern blot analysis showed age-dependent increase of DNA methylation at -1705 and -80 positions located upstream of the gene. The data suggest the possible importance of alterations in collagen alpha1(I) gene expression and its DNA methylation in promoter region in age-dependent degeneration of periodontal ligament.

Repression of transient expression by DNA methylation in transcribed regions of reporter genes introduced into cultured human cells.

We developed a convenient method to methylate all CpG dinucleotides in both strands in a selected region of a plasmid, and investigated the effect of DNA methylation in the transcribed regions of reporter genes on the transient expression in HeLa cells. In a construct containing the chloramphenicol acetyltransferase (CAT) gene linked to the SV40 early promoter, methylation in the CAT structural gene repressed CAT activity. Methylation in the transcribed region of the Escherichia coli lacZ gene driven by the human cytomegalovirus immediate early promoter also inhibited expression of beta-galactosidase activity. These results suggest that methylation in the transcribed region as well as promoter methylation may affect transcription.

Molecular nature of mutations induced by a high dose of X-rays in spleen, liver, and brain of the lacZ-transgenic mouse

DNA sequences of 103 spontaneous mutants and 102 X‐ray‐induced mutants of the lacZ transgene from spleen, liver, and brain of the Muta™Mouse were examined and compared to elucidate characteristics of radiation‐induced mutations in vivo. The radiation‐induced mutants were isolated from genomic DNA of each tissue collected at 3.5 days after 200 Gy of whole body irradiation. Base substitution was predominant (80% or more) in nonirradiated tissues, while deletion was prevalent (about 55%) in irradiated tissues. The other types of mutation appeared at similar frequencies in both control and irradiated tissues. The size of the deletions was smaller than 438 nucleotides, with a predominance of one basepair deletions in both control and irradiated tissues. A close look at the nucleotides at the deletion endpoints revealed that many of the radiation‐induced deletions did not have repeated sequences at the break point termini, whereas all deletions found in unirradiated tissues showed one or more bases of repeated sequences at the termini. Further, eight complex‐type deletion mutations were found only in irradiated tissues. Comparison among the three types irradiated tissue did not reveal any tissue‐specificity. The data indicate that the molecular nature of mutations induced in tissues with ionizing radiation is different from that of spontaneous mutations. Environ. Mol. Mutagen. 34:97–105, 1999

Disappearance of Nucleosome Positioning in Mitotic Chromatin in Vivo

During mitosis, transcription is silenced and most transcription factors are displaced from their recognition sequences. By in vivo footprinting analysis, we have confirmed and extended previous studies showing loss of transcription factors from an RNA polymerase II promoter (c-FOS) and, for the first time, an RNA polymerase III promoter (U6) in HeLa cells. Because little was known about nucleosomal organization in mitotic chromosomes, we performed footprinting analysis for nucleosomes on these promoters in interphase and mitotic cells. During interphase, each of the promoters had a positioned nucleosome in the region intervening between proximal promoter elements and distal enhancer elements, but the strong nucleosome positioning disappeared during mitosis. Thus, the nucleosomal organization that appears to facilitate transcription in interphase cells may be lost in mitotic cells, and nucleosome positioning during mitosis does not seem to be a major component of the epigenetic mechanisms to mark genes for rapid reactivation after this phase.

Chromatin fine structure of the c-MYC insulator element/DNase I-hypersensitive site I is not preserved during mitosis

During mitosis in higher eukaryotic cells, transcription is silenced and transcription complexes are absent from promoters in the condensed chromosomes; however, epigenetic information concerning the pattern of expressed and silent genes must be preserved. Recently, it has been reported that CTCF, a major protein in vertebrate insulator elements, remains associated with mitotic chromatin. If the structure of insulators is preserved during mitosis, then it is possible that insulators can function as components or elements of the mechanism involved in the transfer of epigenetic information through the mitotic phase and can help guide the reconstitution of domain structure and nuclear organization after the completion of this phase. We have studied the chromatin structure of the insulator upstream of the c-MYC gene in mitotic HeLa cells. The region of the insulator corresponds to the DNase I hypersensitive site I, but Southern blot analysis revealed that hypersensitivity was lost during mitosis. High resolution in vivo footprinting analysis using dimethyl sulfate, UV light, psoralen, and DNase I also demonstrated the disappearance of the sequence-specific direct binding of CTCF and the absence of detectable structures during mitosis. Thus, it appears that the nucleoprotein complex involving this insulator element must be reassembled de novo with each new cell generation.

Frameshifts, base substitutions and minute deletions constitute X-ray-induced mutations in the endogenous tonB gene of Escherichia coli K12

We have analyzed the DNA sequence changes in a total of 127 X-ray-induced mutations in the endogenous tonB gene of Escherichia coli cells. Frameshifts accounted for 61 mutations among which 51 were a - 1 frameshift. The second most commonly found mutations were base substitutions (20 transversions and 8 transitions). Twelve of the 16 deletion mutations were the minute-size deletion of 3-25 base pairs, three were the medium-size deletion of 294-643 base pairs and the remaining one was the deletion of 8375 base pairs. Half of the frameshifts and deletions had a run of several identical bases or short direct repeats at the sites of mutation. The spectrum was not in good agreement with the spectrum of spontaneous endogenous tonB mutation nor with the spectra obtained from a mutated gene on a plasmid which had been irradiated in vitro and used to transfect cells for the assay. We discuss the possibility that an X-ray-induced DNA strand break produces local alteration of DNA structure which increases aberrant DNA replication leading to frameshift and minute-size deletion mutations.

Radiation-induced mutations in the spleen and brain of lacZ transgenic mice.

PURPOSE To study the dose-response and molecular nature of radiation-induced mutations in the spleen and brain of lacZ transgenic mice. MATERIALS AND METHODS Line 60 transgenic mice containing the bacterial lacZ gene in a plasmid background were used. Mutants were selected using phenyl-beta-D-galactoside. The nature of mutants was determined by sequencing DNAs of mutant lacZ genes found in control and irradiated tissues. RESULTS X-ray irradiation at 50 and 100 Gy showed linear dose-responses for mutation induction in both tissues. The slope, however, was about twice as steep in spleen than in brain. DNA sequence analyses showed that the predominant type of mutation induced by radiation in both tissues were large deletions. CONCLUSIONS Radiation induces mutations in spleen and brain at different efficiencies but the molecular nature of the induced mutations are similar in the two issues.Purpose : To study the dose-response and molecular nature of radiation-induced mutations in the spleen and brain of lacZ transgenic mice. Materials and methods : Line 60 transgenic mice containing the bacterial lacZ gene in a plasmid background were used. Mutants were selected using phenyl-beta-D-galactoside. The nature of mutants was determined by sequencing DNAs of mutant lacZ genes found in control and irradiated tissues. Results : X-ray irradiation at 50 and 100 Gy showed linear dose-responses for mutation induction in both tissues. The slope, however, was about twice as steep in spleen than in brain. DNA sequence analyses showed that the predominant type of mutation induced by radiation in both tissues were large deletions. Conclusions : Radiation induces mutations in spleen and brain at different efficiencies but the molecular nature of the induced mutations are similar in the two issues.

XPC is involved in genome maintenance through multiple pathways in different tissues.

In an attempt to evaluate the role of the Xpc gene in maintaining genomic stability in vivo under normal conditions, the age-dependent accumulation of spontaneous mutations in different tissues was analyzed in Xpc-deficient lacZ-transgenic mice. Brain, testis, and small intestine revealed no effects from the Xpc-deficiency, whereas liver, spleen, heart, and lung showed an enhanced age-related accumulation of mutations in Xpc-deficient mice. In the spleen, the effect was not obvious at 2 and 12 months of age, but became apparent at 23 months. The magnitude of the observed effect at an advanced age was similar in the liver, spleen and heart, but was comparatively smaller in the lung. Haploinsufficiency was observed in liver and spleen but not in heart and lung. Analysis of DNA sequences in the mutants revealed that the frequency of G:C to T:A changes were elevated in the liver and heart of Xpc-deficient aged mice, supporting the possible involvement of XPC in base excision repair of oxidized guanine. The occurrence of two or more mutations within a single lacZ gene was termed a multiple mutation and was also elevated in old Xpc-deficient mice. Among the clones examined, two mutant clones showed as many as four mutations within a short stretch of DNA. This is the first demonstration to support suggestions for the existence of a role for XPC in the suppression of multiple mutations. These multiple mutations could conceivably be generated by error-prone trans-lesional DNA synthesis. Overall, these results indicate that there may be diverse roles or mechanisms through which XPC participates in genome maintenance in different tissues.