Biomedical subjects
Publications and source records attributed to J D Regan.
The rate of ultraviolet light (UV)-induced DNA excision repair was determined in embryonic cells derived from a congeneic pair of short-lived (C57BL/10.F) and long-lived (C57BL/10) mice. Excision repair was measured by both bromodeoxyuridine photolysis and arabinofuranosyl cytosine inhibition. No difference in rate of repair was observed between the two cell lines.
The tautog, Tautoga onitis, and cunner, Tautogolabrus adspersus, are marine fishes which are closely related taxonomically (family Labridae), have similar habits, the same ecological requirements and are found in the same areas. However, they differ markedly in their longevity. The tautog is longer-lived, reaching an estimated average age of 34 years. It attains sexual maturity at 3--4 years of age. The cunner, on the other hand, lives to about 5--6 years of age and is sexually mature at about one year. An examination of their respective capacities for photoreactivation using cells in tissue culture derived from these fishes revealed marked differences in the rate of monomerization of ultraviolet-induced pyrimidine dimers from their DNA. Radiochromatographic analysis of dimers in the tautog showed that half the dimers were removed within a period of 10 min of exposure to photoreactivating light whereas a 50-min exposure was required to photoreactivate dimers to this same level in the cunner. Dark repair of dimers was measured by radiochromatography and by endonuclease assay. The two methods showed similar low levels of excision repair in both species.
Excision repair of DNA in normal and xeroderma pigmentosum complementation group A fibroblasts were examined following treatment with methyl-, ethyl-, and isopropyl methanesulfonate. Studies utilizing repair synthesis methods and inhibition with arabinofuranosyl cytosine revealed two distinct phases of repair; one commencing and terminating within the first 3-5 h after the treatment, and a second much longer phase extending from 9-35 h post-treatment. Both phases of repair have a long-patch (u.v.-like) component, which establishes for the first time the existence of this mode of repair in response to alkane sulfonate damage. While xeroderma cells display somewhat fewer alkaline labile sites in their DNA following alkylation treatment than do their normal counterparts (consistent with earlier observations suggesting a deficiency in a glycosylase or apurinic endonuclease), we are unable to demonstrate a deficiency of these cells in either of the two phases of repair.
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33 chemical agents and UV- and gamma-irradiation were tested for their comparative ability to induce long-patch or short-patch repair using the 5-bromodeoxy-uridine photolysis assay. For 11 chemical agents repair was long-patch in nature as determined by calculated patch size and response of xeroderma pigmentosum cells relative to normal human cells. Typical patch sizes as measured by this assay were about 90 nucleotides for UV repair, a range of 30 to 70 nucleotides for a variety of known and suspected UV-mimetic chemicals, and 3-4 nucleotides for gamma-radiation. Alkylating agents previously shown to induce short-patch repair were shown also to induce long-patch repair.
Physical and chemical agents present in the environment can potentially damage mammalian DNA. Such damage is known in some cases to be repaired by the process of DNA excision repair. This process has been extensively studied utilizing the repair of ultraviolet irradiation damage as a model system. In this study we have used this system and the 5-bromodeoxyuridine photolysis assay to measure DNA excision repair in cells derived from 21 mammalian species. We have attempted to relate the DNA repair proficiencies and the average size of the repaired regions seen in the cell cultures with the various maximum life spans of the mammals studied. There was an approximate linear correlation between life span of the mammals and the number of DNA excision repair sites measured 20-22 hours following ultraviolet irradiation of the cell cultures. Several deviations from the linear relationships were observed which remain largely unexplained. The size of the repaired regions was shown not to be related to the maximum life spans of the mammals tested.
We have developed a technique whereby 3-h pulses of arabinofuranosyl cytosine (ara-C) and hydroxyurea (HU) are used to analyze the kinetics of repair with time after ultraviolet irradiation in human fibroblasts. We demonstrate that this technique offers a significant improvement over existing repair assays in its ability to visualize between 57 and 100% of all sites undergoing repair in a given period of time. In addition, kinetic analyses of repair are more easily made and yield more information than techniques such as repair replication or unscheduled DNA synthesis. We have also examined the nature of the inhibition event by ara-C and have determined that repair breaks accumulate in the presence of ara-C and HU only up to a certain time beyond which no further breaks appear. The time needed to reach this saturation point depends on the number of sites undergoing repair during the treatment time. This observation is discussed with respect to a possible mechanism of excision repair inhibition by ara-C and HU.
The effect on DNA repair in ultraviolet-irradiated human skin fibroblasts by hydroxyurea has been examined in this study using three independent methods for measuring DNA repair:the 5-bromodeoxyuridine photolysis assay which measures DNA repair replication, chromatographic measurement of thymine-containing dimers, and measurement of specific ultraviolet-endonuclease-sensitive sites in irradiated DNA. Little effect of hydroxyurea was observed at the concentration of 2 mM, which is often used to inhibit semiconservative DNA synthesis; however, 10 mM hydroxyurea resulted in marked inhibition (65--70%) of excision repair. This inhibition was accompanied by a possible doubling in the size of the repaired region. The accumulation of large numbers of single-strand breaks following ultraviolet irradiation and hydroxyurea incubation seen by other investigators was not observed with the normal skin fibroblasts used in this study. A comparison of hydroxyurea effects on the different DNA repair assays indicates inhibition of one step in DNA repair also results in varying degrees of inhibition of other steps as well.
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The repair of human DNA after damage by known and potential metabolites of benzo(a)pyrene has been examined utilizing the bromodeoxyuridine photolysis assay. Repair was characterized as either ultraviolet ("long") or ionizing radiation type ("short") repair utilizing normal cells and cells deficient in ultraviolet-type repair endonuclease from a patient with xeroderma pigmentosum (XP). We have found that only (+/-)-7beta,8alpha-dihydroxy-9beta,-10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide 1) and its disastereomer, (+/-)-7beta,8alpha,-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide 2) elicit damage to DNA which is recognizable by the ultraviolet excision repair system in normal human cells. Benzo(a)pyrene 4,5-, 9,10-, 11,12-oxides do not elicit damage which is repairable by this repair system. The 1,2-diol-3,4-epoxides from naphthalene have no measurable activity in our assay. These results indicate that both the benzo(a)pyrene ring structure and the diol epoxide groups are important in causing the damage to DNA which is repairable by the ultraviolet excision repair system. These results parallel the reported high mutagenic activity of these compounds and support the concept that benzo(a)pyrene 7,8-diol-9,10-epoxides may be the ultimate, metabolically activated forms of benzo(a)pyrene.
We have examined the ability of normal fibroblasts and of excision-deficient xeroderma pigmentosum (XP) and XP variant fibroblasts to perform postreplication DNA repair after increasing doses of either ultraviolet (UV) irradiation or mutagenic benzo(a)pyrene derivatives. XP cells defective in the excision of both UV-induced pyrimidine dimers and guanine adducts induced by treatment with the 7,8-diol-9,10-epoxides of benzo(a)pyrene were partially defective in their ability to synthesize high molecular weight DNA after the induction of both classes of DNA lesions. This defect was more marked in XP variant cells, despite their ability to remove by excision repair both pyrimidine dimers and the diol epoxide-induced lesions to the same degree as observed in normal cells. The benzo(a)pyrene 9,10-oxide had no effect in any of the 3 cell lines. The response of the excision and postreplication DNA repair mechanisms operating in human fibroblasts treated with benzo(a)pyrene 7,8-diol-9,10-epoxides, therefore, appears to resemble closely that seen after the induction of pyrimidine dimers by UV irradiation.
We compared three methods for estimating the induction and removal of UV-induced pyrimidine dimers from the DNA of human fibroblasts. Results indicate that after UV doses of 5-20 J/m2, 50% of the dimers are removed by 24 hours. Almost complete excision can be observed if the cells are incubated for periods not less than 72 hours after a dose of 5 J/m2. After higher doses, it probably takes even longer for such complete removal to doses, it probably takes even longer for such complete removal to be seen.
Neoplastic cell lines exhibit RNA synthesis and process patterns which are related to phenotypic attributes more complex than merely the rate of proliferation. Mouse neuroblastoma cells of the same genotype but different differentiated states have different ribosomal RNA precursor processing patterns, while plasmacytoma cells of different genotypes but the same differentiated state have the same pre-ribosomal RNA processing pattern. In addition, our observations indicate that chromatin-associated RNA is involved in cytodifferentiation and is closely related to phenotypic variability. When neuroblastoma cells are induced to differentiate, there is a 2- to 3-fold increase in the labeling of chromatin-associated RNA. Both of the differentiated cell lines, human myeloma and mouse neuroblastoma, have slow-labeling, stable chromatin-associated RNA while this same fraction from HeLa cells is labeled rapidly and is unstable.
Normal human skin cells were treated with six insecticide esters of N-methylcarbamic acid or their N-nitroso derivatives. The DNA of the cells was sedimented in alkaline sucrose gradients at various times after treatment. The insecticides used were aldicarb, baygon, BUX-TEN, carbofuran, landrin, and methomyl. Numerous single-strand breaks were apparent in the DNA of all the nitroso derivative-treated cells but not in the DNA of those treated with the parent insecticides. Since the effect of the nitroso derivatives on the DNA could be observed for at least 20 h after removal of the chemical from the cultures, the DNA repairing events normally occurring in human cells after damage initiated by these chemical agents was not repaired as UV-type DNA damage or ionizing-type DNA damage in human cells. These observations suggest that the human cellular DNA in vivo is irreversibly altered by nitrosated N-methyl carbamate insecticides resulting in numerous alkali-sensitive bonds.