Mituo Ikenaga

Genetic complementation groups in cockayne syndrome

Skin fibroblasts from patients with Cockayne syndrome (CS cells) exhibited marked ultraviolet (UV) sensitivity as measured by colony-forming ability. Further, recovery of semiconservative DNA synthesis following UV irradiation was absent in CS cells, as it is in xeroderma pigmentosum cells. We found that the rate of semiconservative DNA synthesis measured at 12 h after 12 J/m2 of UV irradiation had recovered to nearly normal levels in binuclear cells obtained by the fusion of CS strains CS3BE (GM1856) and CS7SE (GM1428) and of CS3BE (GM1856) and CS1BE (GM1629), but not of CS7SE (GM1428) and CS1BE (GM1629). These results indicate that there are at least two genetic complementation groups in CS.

The major cause of inactivation and mutation by 4-Nitroquinoline 1-Oxide in Escherichia coli: Excisable 4NQO-purine adducts

Abstract The potent carcinogen, 4-nitroquinoline 1-oxide, is known to mimic the biological effects of ultraviolet light on various living organisms. We conclude that the 4NQO † effects on Escherichia coli are mostly due to covalent binding of 4NQO to DNA producing 4NQO-guanine and 4NQO-adenine adducts in a ratio of about 4:1 without repair and about 7:1 after repair. This is based on the following experimental results. From E. coli cells treated with [ 3 H]4NQO, DNA was extracted and subjected to radiochromatography. We detected two peaks of 4NQO-guanine adduct, one peak of 4NQO-adenine adduct and a peak due to 4-aminoquinoline 1-oxide released from a labile fraction of 4NQO-guanine adducts during acid hydrolysis of DNA before streaking it on paper for chromatography. These four kinds of 4NQO-purine adducts disappeared from DNA of the normal strain at almost the same rate, about 85% in 60 minutes by post-incubating in nutrient broth, but these adducts did not disappear for the uvrA − derivative lacking the excision-repair ability for ultraviolet-induced pyrimidine dimers, except for slight disappearance of 4AQO-releasing adduct. The number of DNA lesions per genome of the uvrA − strain at 37% survival was found to be nearly equal between the 4NQO-purine adducts (~200 lesions) and pyrimidine dimers (~100 lesions). These findings at the molecular level quantitatively parallel the previous findings at the cellular level that the uvrA − strain is about 25 to 30 times as sensitive as its parental strain to killing and mutation by either 4NQO or ultraviolet light. The unique characteristics of 4NQO-purine products are discussed in relation to the mutational specificity of 4NQO and the more-than-supposed complexity of excision repair for DNA.

Excision repair of DNA base damage in human cells treated with the chemical carcinogen 4-nitroquinoline 1-oxide.

Abstract Excision repair of DNA damage produced by 4-nitroquinoline 1-oxide (4NQO), a potent chemical carcinogen, was compared in a normal human amnion FL cell line and a xeroderma pigmentosum (XP) cell line unable to repair ultraviolet-induced pyramidine dimers. The main objective of this study was to investigate, by a direct assay of the loss of damage from DNA, whether DNA damage induced by 4NQO in human cells is repaired by the excision-repair system as in Escherichia coli cells. DNA was extracted from FL and XP cells treated with [ 3 H]4NQO, hydrolyzed and subjected to radiochromatographic analysis in order to quantitate the initial formation of 4NQO damage and subsequent disappearance during post-incubation. Two peaks of stable 4NQO-quanine adducts appeared on the chromatogram, together with one peak of stable 4NQO-adenine adduct and a peak due to 4-aminoquinoline 1-oxide (4AQO) released from a labile fraction of 4NQO-guanine adduct during hydrolysis. The three kinds of stable 4NQO-purine adduct disappeared from DNA of the FL cells at almost the same rate of about 60% during 24-h post-incubation in culture medium, and 4AQO disappeared somewhat faster. In the XP cells, however, the stable adducts did not disappear from DNA, whereas about 40% of the 4AQO-releasing adduct disappeared from DNA. These findings at the molecular level quantitatively parallel the previous findings at the cellular level that the XP cells are several times as sensitive as normal cells to killing by 4NQO. These results lead to the conclusion that in human cells 4NQO-induced lethality is mainly due to the four kinds of 4NQO-purine adduct as it is in E. coli , and that the adducts are excisable by the same excision-repair mechanism that works on pyramidine dimers.

Prenatal diagnosis of Cockayne syndrome using assay of colony-forming ability in ultraviolet light irradiated cells.

The analysis of colony‐forming ability after ultraviolet light exposure is described with amniotic fluid cells from a pregnancy at risk for Cockayne syndrome. The amniotic fluid cells were considerably more sensitive to ultraviolet light than normal amniotic fluid cells and skin fibroblasts from normal donors. It took about 5 weeks from amniocentesis to identify the affected fetus by this colony assay. The diagnosis was confirmed in fibroblast cultures from both skin and lung of the aborted fetus.

DNA REPAIR AND CLINICAL CHARACTERISTICS OF 96 XERODERMA PIGMENTOSUM PATIENTS IN JAPAN

ABSTRACT Ninety six xeroderma pigmentosum patients in Japan were examined for DNA repair capacities of their cells and clinical characteristics. A half of the patients were children under 10 years old, most of them having very low repair capacities. There were two older patients, 45 and 64, who showed very mild symptom but whose cells showed low amount of unscheduled DNA synthesis. One of them was identified to belong to a new complementation group, F. There were many group A patients and no group B, C., or E patients. Apparently the development of clinical symptoms were related to the repair capacities of the cells with possible exception of above two patients, although the host-cell reactivation experiment on the group F cells revealed considerably higher repair capacity.

ACTION SPECTRUM FOR PHOTOREACTIVATION OF ULTRAVIOLET‐INDUCED MORPHOLOGICAL ABNORMALITY IN SEA URCHIN EGGS

Abstract An action spectrum was obtained for photoreactivation (PR) of morphological abnormality arising from ultraviolet (UV)‐irradiation of sea urchin sperm. The wavelength dependence of PR was measured by the restoration of the formation of normal pluteus larvae after the exposure of fertilized eggs to various fluences of monochromatic PR light (313 to 500 nm). The PR action spectrum showed a maximum around 365 nm and a secondary peak somewhere above 400 nm. High PR activity beyond 400 nm wavelengths may reflect an advantageous or adaptational ability to cope with harmful effects of solar UV radiation.

DNA PHOTOREACTIVATING ENZYME FROM SILKWORM

An ammonium‐sulfate‐precipitable (33–70%) fraction in extracts from eggs of silkworm Bombyx mori contains photoreactivating enzyme that reactivates the transforming activity of UV inactivated Hemophilus influenzae DNA. The action spectrum for in vitro photoreactivation with the enzyme has a broad peak around 365–385 nm, with a shoulder extending to 460 nm. This relatively higher photoreactivation efficiency at wavelengths longer than 450 nm seems to be a unique feature of DNA photoreactivating enzyme of silkworm. Using gel filtration, a mol wt of 42,000 was estimated for the enzyme. Optimum and isoionic pH of the enzyme were 7.2 and 5.4, respectively. These properties of silkworm enzyme are within the range of variations in reported biochemical characteristics of photoreactivating enzymes from different species.

The sensitivities to radiations and radiomimetic chemicals of cells from patients with ataxia telangiectasia

SummaryThe lethal action of physical and chemical agents on a fibroblast strain derived from a Japanese patient with ataxia telangiectasia (AT) was measured by the cellular colony forming ability, in comparison with a British AT cell strain. Both of the AT strains showed a significantly increased sensitivity to X-rays and an antitumor agent bleomycin, as compared with normal fibroblasts. Also, the two AT strains were slightly more sensitive to methyl methanesulfonate than normal cells. The British AT strain (AT4BI) exhibited a marked sensitivity to 4-nitroquinoline 1-oxide and to mitomycin C compared with normal cells, whereas the Japanese AT strain (AT1OS) showed normal response to 4-nitroquinoline 1-oxide and a reduced sensitivity to mitomycin C. Sensitivity of AT1OS cells to N-methyl-N′-nitro-N-nitrosoguanidine was also normal under our experimental conditions.

Rapid procedures for prenatal diagnosis of Cockayne syndrome

SummaryChanges in the rate of semi-conservative DNA synthesis with time after ultraviolet light irradiation were measured with amniotic fluid cells at 6 to 10th subcultures, which were originally derived from the pregnancy with the fetus affected with Cockayne syndrome. The rate of DNA synthesis at 12 hr after ultraviolet exposure relative to that of unirradiated control was much less in the Cockayne amniotic fluid cells than those observed with normal amniotic fluid cells or with fibroblasts from normal donors. These results suggest that by measuring the rate of DNA synthesis prenatal diagnosis of Cockayne syndrome may be accomplished within 3 to 4 weeks, in contrast to the actual length of time, 5 weeks, required for the prenatal diagnosis of this syndrome by measuring colony forming ability of ultraviolet irradiated cells.