Kumio Okaichi

p53 proteins accumulated by heat stress associate with heat shock proteins HSP72/HSC73 in human glioblastoma cell lines

We investigated the accumulation of p53 proteins after heat stress and their association with HSP72/HSC73 using four human glioblastoma cell lines. Human glioblastoma cell lines U-87MG and A-172 exhibited no mutation in the region between the 2nd and 11th exons of the p53 gene, whereas A-7 and T98G had mutations in exon 5 and exon 7 of the p53 gene, respectively. In U-87MG and A-172, the levels of wild-type p53 protein were slightly increased by heat stress. Levels of mutant p53 protein were apparently increased by heat stress in A-7, but not in T98G. Furthermore, wild-type p53 proteins in both U-87MG and A-172 co-immunoprecipitated with anti-HSP72/HSC73 antibody and HSP72 and HSC73 in them co-immunoprecipitated with anti-p53 antibody as did the mutant p53 proteins. These findings suggest that p53 proteins accumulated by heat stress are associated with HSP72 and HSC73.

PYRIMIDINE DIMER FORMATION AND GERMINATION OF UV‐IRRADIATED SPORES OF DICTYOSTELIUM DISCOIDEUMNC–4 ANDys–13

Abstract— Survival, UV‐photoproducts and germination of UV‐irradiated spores of Dictyostelium discoi‐deum were studied on two strains,NC–4 andys–13. The spores ofNC–4 are about 35 times more resistant to UV thanys–13 spores at 10% survival. Pyrimidine dimers were formed in UV‐irradiated spores in both strains. No photoproducts other than pyrimidine dimers were detected. The formation of pyrimidine dimers in spores was about 2% in both strains at 800 J/m2. In the germination of spores, the conversion of spores into swollen spores was not affected by UV in both strains, but the emergence of amoebae from the swollen spores was suppressed, which was more distinctive inys–13 spores than inNC–4 spores. The emerged amoebae from the UV‐irradiatedNC–4 spores were viable, while those from theys–13 spores were inviable even when they succeeded in emergence.

Mutations of a shuttle vector plasmid, pZ189, in Escherichia coli induced by boron neutron captured beam (BNCB) containing α-particles

A shuttle vector, pZ189, carrying a bacterial suppressor tRNA marker gene (supF) was dissolved in Tris-EDTA buffer containing 0.3 M 10B-enriched boric acid and then irradiated with boron neutron captured beam (BNCB) produced by the nuclear reaction 10B (n,alpha) 7Li with thermal neutrons. A DNA repair-deficient mutant, KS46 (uvrA-), of Escherichia coli was transformed with the plasmid DNA, and the transformants carrying mutations on the supF gene were selected as nalidixic acid-resistant colonies. The mutation frequency (2.4 x 10(-4)) of pZ189 at the D10 dose was about 70 times greater than the spontaneous rate (3.5 x 10(-6)). The plasmid mutations were analyzed using DNA sequencers; 88% of them were base substitutions. A few minus-one frameshifts (7%) and deletions (5%) were detected. Among these base substitutions, transversions of G:C to T:A (42%) and G:C to C:G (29%) predominated. Twenty-seven percent of the base substitutions were G:C to A:T transitions; no A:T to G:C transitions were detected.

Mutagenic specificity in DNA base sequence by irradiation of health lamp light (UV-B) in Escherichia coli

A shuttle vector, pZ189, carrying a bacterial suppressor tRNA marker gene, was irradiated with health lamp (HL) light containing UV-B. Plasmid mutations were scored by transforming an indicator strain of Escherichia coli carrying a suppressive blue amber mutation in the beta-galactosidase gene. Plasmid survival was also measured by transforming activity of the indicator strain. The majority of mutations induced by HL light were GC-AT transitions (69%) and the rest were transversions (31%). Some hot-spots in the mutations were observed by sequencing the suppressor gene. Mutagenic specificity in DNA base sequences induced by HL in E. coli agrees well with previous reports about 254-nm or 313-nm light effects on mammalian cells. This agreement may depend on the substitution of the inserted base instead of a G residue at the opposite site of a damaged C residue from conformational change of DNA structure in both bacterial and mammalian cells.

UNIQUE DNA REPAIR PROPERTY OF AN ULTRAVIOLET-SENSITIVE (radC MUTANT OF Dictyostelium discoideum

Abstract— Dictyostelium discoideum is an organism that shows higher UV resistance than other organisms, such as Escherichia coli and human cultured cells. We examined the removal of cyclobutane pyrimidine dimers (CPD) and 6–4 photoproducts from DNA in the radC mutant and the wild‐type strain using an enzyme‐linked immunosorbent assay with monoclonal antibodies. Wild‐type cells excised more than 90% of both CPD and 6–4 photoproducts within 4 h. Dictyostelium discoideum appeared to have a special repair system, because 6–4 photoproducts were repaired faster than CPD in E. coli and human cultured cells. In radC mutant cells, although only 50% of CPD were excised from DNA within 8 h, effective removal of 6–4 photoproducts (80% in 8 h) was observed. Excision repair‐deficient mutants generally cannot remove both CPD and 6–4 photoproducts. Though the radC mutant shows deficient excision repair, it can remove 6–4 photoproducts to a moderate degree. These results suggest that D. discoideum has two kinds of repair systems, one mainly for CPD and the other for 6–4 photoproducts, and that the radC mutant has a defect mainly in the repair enzyme for CPD.

REMOVAL OF PYRIMIDINE DIMERS IN UV-IRRADIATED SPORES OF Dictyostelium discoideum DURING GERMINATION

The spores of Dictyostelium discoideum TW‐8 (radC) are about twice as sensitive to UV than the parental strain NC‐4 spores at a 10% survival level. Ultraviolet irradiation apparently suppressed the emergence of amoebae from swollen TW‐8 spores as compared with NC‐4 spores, though the conversion of spores into swollen spores was not affected by UV irradiation in either strain. About 85% removal of pyrimidine dimers was detected in UV‐irradiated NC‐4 spores at 200 J/m2 during spore germination for 9 h, but no removal of pyrimidine dimers was detected in TW‐8 spores under the same conditions. The removal of pyrimidine dimers from the NC‐4 spores began at around 2 h germination when the spores have become swollen. The number of enzyme‐sensitive sites (ESS) detected by Micrococcus luteus endonuclease in the DNA of UV‐irradiated NC‐4 spores also began to decrease at about 2 h into germination. The decrease in ESS, however, was hardly detectable in UV‐irradiated TW‐8 spores at any step during germination.

Ki-ras oncogene activation in transplantable rat thyroid carcinoma induced by N-bis(2-hydroxypropyl)nitrosamine.

We have established 17 transplantable rat thyroid carcinoma cell lines from primary thyroid tumors of rats induced by N-bis(2-hydroxypropyl)nitrosamine (DHPN) (Cancer Res. (1993) 53, 4408-4412). The present study was designed to evaluate point mutations in the murine c-Ki-ras gene of these carcinoma cell lines. Using PCR amplification and direct sequencing, we found that the activated form of the Ki-ras oncogene was present in 4 (23%) of a total of 17 cell lines, all the Ki-ras gene mutations being GC-->AT transitions. In three of the cell lines, the mutations occurred in codon 12 (GTP-binding domain), and in the remaining one the first nucleotide of codon 63 was affected. Histologically, three of the carcinomas with Ki-ras mutation were diagnosed as well-differentiated carcinomas, and the other as poorly differentiated carcinoma. Mutations of the ras gene are relatively uncommon in tumors of these histological types. From these experimental results, we suggest that the mutation induced by DHPN is due to damage to guanine in cellular DNA. In addition, Ki-ras activation may play an important role in the initiation of thyroid carcinogenesis.

PHOTOREACTIVATION OF UV‐KILLING IN Vibrio parahaemolyticus WP28*

Abstract— Vibrio parahaemolyticus WP28 cells possess efficient photoreactivation (PR) from U V‐killing. The action spectrum of PR in these cells is quite analogous to that in Escherichia coli cells, which have a broad efficient range from 350 to 425 nm with a maximum at 375 nm. PR at 315 nm is also significantly present.