Hiroshi Hayakawa

The basic and clinical implications of ABC transporters, Y‐box‐binding protein‐1 (YB‐1) and angiogenesis‐related factors in human malignancies

In our laboratories, we have been studying molecular targets which might be advantageous for novel cancer therapeutics. In this review, we focus on how ATP‐binding cassette (ABC) transporter superfamily genes, Y‐box‐binding protein‐1 (YB‐1), and tumor angiogenesis‐associated factors could contribute to the development of novel strategies for molecular cancer therapeutics. ABC transporters such as P‐glycoprotein/MDR1 and several MRP family proteins function to protect cells from xenobiotics, drugs and poisons, suggesting that ABC transporters are a double‐edged sword. In this regard, P‐glycoprotein/MDR1 is a representative ABC transporter which plays a critical role in the efflux of a wide range of drugs. We have reported that gene amplification, gene rearrangements, transcription factor YB‐1 and CpG methylation on the promoter are involved in MDR1 gene overexpression in cultured cancer cells. Among them, two mechanisms appear to be relevant to the up‐regulation of MDR1 gene in human malignancies. We first reported that MDR1 gene promoter is activated in response to environmental stimuli, and is modulated by methylation/demethylation of CpG sites on the MDR1 promoter. We also demonstrated that YB‐1 modulates not only transcription of various genes associated with cell growth, drug resistance and DNA synthesis, but also translation, mRNA stabilization and DNA repair/self‐defense processes. Angiogene‐sis is also involved in tumor growth, invasion and metastasis of various malignancies, and so angiogenesis‐related molecules also offer novel molecular targets for anticancer therapeutics. (Cancer Sci 2003; 94: 9–14)

Expression and cloning of complementary DNA for a human enzyme that repairs O6-methylguanine in DNA.

A cell line with an increased resistance to alkylating agents and an extremely high level of O6-methylguanine-DNA methyltransferase activity was isolated after transfection of methyltransferase-deficient Mer- cells with a cDNA library, prepared from methyltransferase-proficient human Mer+ (Raji) cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis revealed that a protein, with a molecular weight of approximately 25,000, accepted 3H label from DNA that had been treated with [3H]methylnitrosourea. Since the cDNA for methyltransferase was integrated into the chromosomal DNA, it was recovered by using the polymerase chain reaction. When the cDNA placed in an expression vector p500 was introduced into Mer- cells, the cells acquired an increased resistance to alkylating agents and exhibited a high level of O6-methylguanine-DNA methyltransferase activity. From the transformants the cDNA could be recovered as a part of the autonomously replicating plasmid. The nucleotide sequence of the cDNA was determined, and an open reading frame comprising 207 amino acid residues was found. The molecular weight of methyltransferase, calculated from the predicted amino acid sequence, was 21,700. The predicted amino acid sequence of the human methyltransferase exhibits an intensive homology with those of the bacterial counterparts, Ada and Ogt proteins of Escherichia coli and Dat protein of Bacillus subtilis, especially around possible methyl acceptor sites.

Mouse MTH2 protein which prevents mutations caused by 8-oxoguanine nucleotides

MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis, in the nucleotide pool, thereby preventing DNA replication errors. During a search of GenBank EST database, we found a new member of MutT-related protein, MTH2, which possesses the 23-amino acid MutT module. The cloned mouse MTH2 (mMTH2) cDNA was expressed in Escherichia coli mutT(-) cells and the protein was purified. mMTH2 protein hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with Km of 32 microM. Expression of cDNA for mMTH2 reduced significantly the elevated level of spontaneous mutation frequency of E. coli mutT(-) cells. Thus, MTH2 has a potential to protect the genetic material from the untoward effects of endogenous oxygen radicals. MTH2 could act as an MTH1 redundancy factor.

A novel mechanism for preventing mutations caused by oxidation of guanine nucleotides

MutT‐related proteins, including the Escherichia coli MutT and human MutT homologue 1 (MTH1) proteins, degrade 8‐oxo‐ 7,8‐dihydrodeoxyguanosine triphosphate (8‐oxo‐dGTP) to a monophosphate, thereby preventing mutations caused by the misincorporation of 8‐oxoguanine into DNA. Here, we report that human cells have another mechanism for cleaning up the nucleotide pool to ensure accurate DNA replication. The human Nudix type 5 (NUDT5) protein hydrolyses 8‐oxo‐dGDP to monophosphate with a Km of 0.77 µM, a value considerably lower than that for ADP sugars, which were originally identified as being substrates of NUDT5. NUDT5 hydrolyses 8‐oxo‐dGTP only at very low levels, but is able to substitute for MutT when it is defective. When NUDT5 is expressed in E. coli mutT− cells, the increased frequency of spontaneous mutations is decreased to normal levels. Considering the enzymatic parameters of MTH1 and NUDT5 for oxidized guanine nucleotides, NUDT5 might have a much greater role than MTH1 in preventing the occurrence of mutations that are caused by the misincorporation of 8‐oxoguanine in human cells.

Mammalian enzymes for preventing transcriptional errors caused by oxidative damage

8-Oxo-7,8-dihydroguanine (8-oxoGua) is produced in cells by reactive oxygen species normally formed during cellular metabolic processes. This oxidized base can pair with both adenine and cytosine, and thus the existence of this base in messenger RNA would cause translational errors. The MutT protein of Escherichia coli degrades 8-oxoGua-containing ribonucleoside di- and triphosphates to the monophosphate, thereby preventing the misincorporation of 8-oxoGua into RNA. Here, we show that for human the MutT-related proteins, NUDT5 and MTH1 have the ability to prevent translational errors caused by oxidative damage. The increase in the production of erroneous proteins by oxidative damage is 28-fold over the wild-type cells in E.coli mutT deficient cells. By the expression of NUDT5 or MTH1 in the cells, it is reduced to 1.4- or 1.2-fold, respectively. NUDT5 and MTH1 hydrolyze 8-oxoGDP to 8-oxoGMP with Vmax/Km values of 1.3 × 10−3 and 1.7 × 10−3, respectively, values which are considerably higher than those for its normal counterpart, GDP (0.1–0.5 × 10−3). MTH1, but not NUDT5, possesses an additional activity to degrade 8-oxoGTP to the monophosphate. These results indicate that the elimination of 8-oxoGua-containing ribonucleotides from the precursor pool is important to ensure accurate protein synthesis and that both NUDT5 and MTH1 are involved in this process in human cells.

Nucleotide excision repair DNA synthesis by excess DNA polymerase β: a potential source of genetic instability in cancer cells

The nucleotide excision repair pathway contributes to genetic stability by removing a wide range of DNA damage through an error‐free reaction. When the lesion is located, the altered strand is incised on both sides of the lesion and a damaged oligonucleotide excised. A repair patch is then synthesized and the repaired strand is ligated. It is assumed that only DNA polymerases δ and/or ε participate to the repair DNA synthesis step. Using UV and cisplatin‐modified DNA templates, we measured in vitro that extracts from cells overexpressing the error‐prone DNA polymerase β exhibited a five‐to sixfold increase of the ultimate DNA synthesis activity compared with control extracts and demonstrated the specific involvement of Pol β in this step. By using a 28 nt gapped, double‐stranded DNA substrate mimicking the product of the incision step, we showed that Pol β is able to catalyze strand displacement downstream of the gap. We discuss these data within the scope of a hypothesis previously presented proposing that excess error‐prone Pol β in cancer cells could perturb the well‐defined specific functions of DNA polymerases during error‐free DNA transactions.—Canitrot, Y., Hoffmann, J.‐S., Calsou, P., Hayakawa, H., Salles, B., Cazaux, C. Nucleotide excision repair DNA synthesis by excess DNA polymerase β: a potential source of genetic instability in cancer cells. FASEB J. 14, 1765–1774 (2000)

Repair of ultraviolet radiation damage in xeroderma pigmentosum cells belonging to complementation group F

DNA-repair characteristics of xeroderma pigmentosum belonging to complementation group F were investigated. The cells exhibited an intermediate level of repair as measured in terms of (1) disappearance of T4 endonuclease-V-susceptible sites from DNA, (2) formation of ultraviolet-induced strand breaks in DNA, and (3) ultraviolet-induced unscheduled DNA synthesis during post-irradiation incubation. The impaired ability of XP3YO to perform unscheduled DNA synthesis was restored, to half the normal level, by the concomitant treatment with T4 endonuclease V and ultraviolet-inactivated Sendai virus. It is suggested that xeroderma pigmentosum cells of group F may be defective, at least in part, in the incision step of excision repair.

Organization and expression of the human gene for O6-methylguanine-DNA methyltransferase

O6-Methylguanine-DNA methyltransferase plays an important role in cellular defence against mutagens and carcinogens with alkylating activity. Certain tumor-derived cell lines, termed Mer-, are defective in the enzyme activity and have an increased sensitivity to alkylating agents. We cloned the genomic sequence coding for the human O6-methylguanine-DNA methyltransferase and elucidated the structure. The gene consisted of 5 exons and spanned more than 170 kb, while mRNA for the enzyme was 950 nucleotides long. No or only little mRNA for the enzyme was formed in Mer- cells, though there was no gross difference in the coding and promoter regions of the gene between Mer+ and Mer- cells. The putative promoter region, derived from Mer+ cells, was placed upstream of the chloramphenicol acetyltransferase reporter gene and the constructs were introduced into Mer+ and Mer- cells. In Mer- cells, a lowered level of transient expression of the gene was observed as compared with Mer+ cells, but this difference alone does not account for the in vivo difference of expression of the gene in the two types of cells; there might be difference in cis-acting elements. The DNA sequence in the 5 upstream region of the gene was extremely GC-rich and there were no consensus sequences, such as the TATA and CAAT boxes. There were lower levels of methylation in the putative promoter of various Mer- cells, as compared with findings in Mer+ cells. Methylation in this region may be involved in regulating expression of the gene.

A human enzyme that liberates uracil from DNA.

Abstract An enzyme activity which acts specifically on uracil-containing DNA was found in human placenta and cultured fibroblasts. The enzyme liberates uracil from DNA in the presence of EDTA at pH 7.5. Almost equal levels of the activity were found in normal and xeroderma pigmentosum cell lines (complementation group A).

Intracellular localization and function of DNA repair methyltransferase in human cells

An antibody preparation specific for human O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) was obtained by immunoaffinity purification on two types of affinity columns with the purified human and mouse methyltransferase proteins as ligands. The antibodies were used in Western blotting analysis of fractionated cell extracts. More than 90% of the methyltransferase protein was recovered in the cytoplasmic fractions with both human HeLa S3 cells and MR-M cells, the latter overproducing the enzyme 36 times as much as the former. Cytoplasmic localization of the methyltransferase in HeLa S3 cells was further confirmed by in situ immunostaining. By Western blotting analysis of fractionated cell extracts from HeLa S3 cells treated with alkylating agents, we found that amounts of the enzyme decreased more rapidly in the nuclear fraction than in the cytoplasmic fraction, and recovery of the enzyme level in the cytoplasmic fraction was slower than that in the other. These results suggest that the methyltransferase protein is degraded in the nucleus after it commits the repair reaction and that the cytoplasmic enzyme is transported into the nucleus as the nuclear methyltransferase is used up in this manner.