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Papers by Helen Evans

Photochemistry and Photobiology, 1989

Two closely related strains of mouse lymphoma L5178Y cells, LY-R and LY-S, have been found to dif... more Two closely related strains of mouse lymphoma L5178Y cells, LY-R and LY-S, have been found to differ in their sensitivity to the cytotoxic effects of photodynamic treatment (PDT) with chloroaluminum phthalocyanine (CAPC) and red light. Strain LY-R is more sensitive to photodynamic cell killing than strain LY-S. Differences in uptake of CAPC could not account for the differences in cytotoxic effects. There was no marked difference between the two strains in the induction of single-strand breaks (which includes frank single-strand breaks and alkali-labile lesions), but substantially more DNA-protein cross-links were formed in strain LY-R by CAPC and light. Repair of single-strand breaks proceeded with similar kinetics in both strains for the first 30 min post-irradiation, suggesting that these lesions are not responsible for the differential sensitivity of the two strains to the lethal effects of photodynamic treatment. Thereafter, alkaline elution revealed the presence of increasing DNA strand breakage in strain LY-R. DNA degradation, as measured by the conversion of prelabled [14C] DNA to acid-soluble radioactivity, was more rapid and extensive in strain LY-R.

Proceedings of The National Academy of Sciences, 1987

The production and repair of radiation-induced DNA damage were measured by filter elution in stra... more The production and repair of radiation-induced DNA damage were measured by filter elution in strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. The induction of radiation-induced damage, as measured by filter elution at pH 12.1, 9.6, and 7.2, was similar in the resistant-strain LY-R and the sensitive strain LY-S. The repair of single-strand breaks and alkali-labile sites, as measured by filter elution at pH 12.1 at various times after irradiation, was somewhat slower in strain LY-S than in strain LY-R, although after a 20-min repair period the extent of

Photochemistry and Photobiology, 1997

Abstract— The mutagenicity of photodynamic therapy (PDT) using red light and either Photofrin® (p... more Abstract— The mutagenicity of photodynamic therapy (PDT) using red light and either Photofrin® (porfimer sodium) (PF) or aluminum phthalocyanine (AIPc) as the photosensitizer was determined at the thymidine kinase (TK) locus in the human lymphoblastic cell lines, TK6 and WTK1, and was compared to the mutagenicity of UVC and X-radia-tion in these cells as well as the mutagenicity of PDT in murine L5178Y lymphoblastic cell lines. Photodynamic therapy was found not to be mutagenic in TK6 cells, which possess an active p53 gene and which are relatively deficient in recombination and repair of DNA double-strand breaks. In contrast, PDT with either sensitizer was significantly mutagenic in WTK1 cells, which harbor an inactivating mutation in the p53 gene and are relatively efficient in recombination and double-strand break repair as compared to TK6 cells. The induced mutant frequency in WTK1 cells with PF as the photosensitizer was similar to that induced by UVC radiation but lower than that induced by X-radiation at equitoxic faiences/ doses. The mutant frequency induced by PDT in WTK1 cells with either photosensitizer was much lower than that induced in murine lymphoblasts at equitoxic fluences. The TK6 and WTK1 cells did not differ in their sensitivity to the cytotoxic effects of PDT, but the level of PDT-induced apoptosis was greater in TK6 than in WTK1 cells. These results indicate that the mutagenicity of PDT varies in different types of cells and may be related to the repair capabilities as well as the p53 status of the cells.

Photochemistry and Photobiology, 1992

Abstract— Photosensitization mediated by Photofrin II (PFII) was found to be mutagenic at the het... more Abstract— Photosensitization mediated by Photofrin II (PFII) was found to be mutagenic at the heterozygous thymidine kinase (tk) locus in mouse L5178Y lymphoma strains LY-S1 and LY-R16 but not in strain LY-R83 which is hemizygous at the tk locus. After treatments yielding 37% survival, the mutagenicity of photosensitization with PFII in strain LY-S1 was similar to that of other mutagenic agents including x-radiation, ethyl methanesulfonate, and photosensitization with chloroaluminum phthalocyanine (AIPcCI). Although both strain LY-S1 and strain LY-R16 were mutagenized by photosensitization with PFII, only strain LY-S1 was mutagenized by photosensitization with AIPcCI. The non-mutability of strain LY-R83 following photodynamic treatment with either sensitizer may be because of the poor recovery of mutants with intergenic mutations in this TK+/0 hemizygous strain, whereas the non-mutability of strain LY-R16 subjected to photodynamic treatment with AIPcCI may be because LY-R16 cells sustaining mutagenic damage do not survive for reasons other than the loss of an essential gene.

Mutation Research-dna Repair, 1989

The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely relat... more The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely related strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. Strain LY-S is sensitive to ionizing radioation relative to strain LY-R and is deficient in the rejoining of DNA double-strand breaks induced by this agent, whereas 2 radiation-resistant variants of strain LY-S have regained the ability to rejoin these double-strand breaks. We have found that the sensitivity of these cells to m-AMSA, VP-16, and eUipticine is correlated to their sensitivity to ionizing radiation. However, this correlation did not extend to their sensitivities to novobiocin, camptothecin, hydrogen peroxide, methyl nitrosourea and UV radiation. Thus, there appears to be a unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors which stabilize the cleavable complex between the enzyme and DNA. It is possible either that (1) topoisomerase II is altered in strain LY-S and that this enzyme is involved in the repair of DNA double-strand breaks or (2) strain LY-S is deficient in a reaction which is necessary for the repair of DNA double-strand breaks induced by ionizing radiation as well as the repair of DNA damage induced by these topoisomerase II inhibitors, m-AMSA, VP-16, and ellipticine were found to be highly mutagenic at the tk locus in L5178Y strains which are heterozygous for the tk gene but not in a tk hemizygous strain, indicating that these inhibitors induce multilocus lesions in DNA, as does ionizing radiation. The differences in the sensitivity of strains LY-R and LY-S to the topoisomerase II inhibitors were paralleled by differences in the induction of protein-associated DNA double-strand breaks in the 2 strains. This correlation did not extend to the radiation-resistant variants of strain LY-S, however. The variants showed resistance to the cytotoxic effects of the inhibitors relative to strain LY-S, but exhibited DNA double-strand break induction similar to that observed in strain LY-S.

The mode of cell death of two strains of mouse lymphoma LSI78Y cells was studied following photod... more The mode of cell death of two strains of mouse lymphoma LSI78Y cells was studied following photodynamic therapy (PDT) sensitized by chloroaluminum phthalocyanine. Strains LY-R and LY-S differ in their relative sensitivities to UVC radiation, X-radiation, and PDT; both responded to PDT by undergoing apoptosis. The DNA was degraded into fragments with lengths which are multiples of approximately 180-190 base pairs (i.e., oligonucleosome size), a biochemical marker of apoptosis. The DNA fragmentation was dose and time dependent which indicates this response to be an enzymic process related to cell killing. Cycloheximide, a protein synthesis inhibitor, and actinomycin D, an RNA synthesis inhibitor, enhanced the endonucleolytic DNA fragmentation. Transmis sion electron microscopy revealed chromatin condensation around the periphery of the nucleus, which is also characteristic of apoptosis. The induction of apoptosis in L5178Y cells by PDT was rapid, with marked degradation of DNA occurring in as little as 30 min. The rapidity of the response to PDT suggests that cellular damage produced by PDT can directly activate endonucleolysis and chromatin condensation, thereby by-passing many of the early steps in the signal transduction program which are acted upon by other agents causing apoptosis.

Photochemistry and Photobiology, 1988

Abstract— Chloroaluminum phthalocyanine (CAPC) is an efficient photosensitizer for the inactivati... more Abstract— Chloroaluminum phthalocyanine (CAPC) is an efficient photosensitizer for the inactivation of Chinese hamster V79 cells. In order to investigate possible molecular mechanisms in the photo-dynamic action of CAPC and visible light, the induction and repair rate of two classes of DNA lesions have been determined, i.e. DNA single-strand breaks and DNA-protein cross-links. In cells pretreated with 1 μ.M CAPC, a fluence of 12 kJ/m2 of red light (>600 nm) kills approximately 50% of the cells and induces 3 to 3.5 Gy-equivalents of single-strand breaks. The repair of these breaks was slower than the repair of single-strand breaks induced by m̀-irradiation. The photodynamic action of CAPC also induces a large number of DNA-protein cross-links which, in contrast to m̀-radiation-induced DNA-protein cross-links, do not appear to be repaired during 4 h of post-treatment incubation in fresh medium. These studies suggest that DNA may be an important target for the cytotoxicity of CAPC + red light.

Photochemistry and Photobiology, 1989

Abstract The cytotoxic and mutagenic effects of chloroaluminum phthalocyanine (CAPC) plus red lig... more Abstract The cytotoxic and mutagenic effects of chloroaluminum phthalocyanine (CAPC) plus red light have been measured in strains of L5178Y mouse lymphoma cells which differ in their DNA repair capacities. Strain LY-R, deficient in the excision repair of UV-induced dimers, was found to be relatively more sensitive to the cytotoxic effects of CAPC plus light, whereas strain LY-S, deficienl in the repair of DNA double-strand breaks, was more sensitive than strain LY-R to the mutagenic effects of the treatment. Mutation frequencies were measured in LY-S and LY-R sub-strains which were heterozygous or hemizygous at the thymidine kinase (tk) locus. The mutation frequency at the tk locus induced in the heterozygous strain LY-SI by CAPC plus light was lower than that induced by an equitoxic dose of ionizing radiation but similar to that induced by an equitoxic dose of UVC radiation: The mutation frequency at the F., dose of CAPC plus light was approximately 1100 per 106 surviving cells. The induced frequency in strain LY-S1 was much higher than in either tk+l-heterozygous or ik+10 hemizygous strains of LY-R. The rate and extent of incorporation of CAPC by the LY-R strains was somewhat greater than for strain LY-S1 at early times after CAPC addition, but by the time the cells were irradiated (18 h after CAPC addition) the difference was not great enough to account for the difference in cytotoxicity. It is possible that the cytotoxic and mutagenic lesions differ and that either the quantities of the respective lesions induced or the efficiencies of repair of the respective lesions differ inversely in the two strains.light have been measured in strains of L5178Y mouse lymphoma cells which differ in their DNA repair capacities. Strain LY-R, deficient in the excision repair of UV-induced dimers, was found to be relatively more sensitive to the cytotoxic effects of CAPC plus light, whereas strain LY-S, deficienl in the repair of DNA double-strand breaks, was more sensitive than strain LY-R to the mutagenic effects of the treatment. Mutation frequencies were measured in LY-S and LY-R sub-strains which were heterozygous or hemizygous at the thymidine kinase (tk) locus. The mutation frequency at the tk locus induced in the heterozygous strain LY-SI by CAPC plus light was lower than that induced by an equitoxic dose of ionizing radiation but similar to that induced by an equitoxic dose of UVC radiation: The mutation frequency at the F., dose of CAPC plus light was approximately 1100 per 106 surviving cells. The induced frequency in strain LY-S1 was much higher than in either tk+l-heterozygous or ik+10 hemizygous strains of LY-R. The rate and extent of incorporation of CAPC by the LY-R strains was somewhat greater than for strain LY-S1 at early times after CAPC addition, but by the time the cells were irradiated (18 h after CAPC addition) the difference was not great enough to account for the difference in cytotoxicity. It is possible that the cytotoxic and mutagenic lesions differ and that either the quantities of the respective lesions induced or the efficiencies of repair of the respective lesions differ inversely in the two strains.

Proceedings of The National Academy of Sciences, 1986

Mouse L5178Y strain LY-S and its parental strain LY-R differ in their comparative sensitivities t... more Mouse L5178Y strain LY-S and its parental strain LY-R differ in their comparative sensitivities to the cytotoxic effects of various mutagenic agents--i.e., strain LY-S has been found to be more sensitive, less sensitive, or similarly sensitive to individual agents in comparison to strain LY-R. Nevertheless, strain LY-S has been found to be uniformly less mutable than strain LY-R at the hypoxanthine (guanine) phosphoribosyltransferase (Hprt) locus following treatment with x-radiation, UV radiation, or alkylating agents. In the present work we have isolated subclones of strains LY-R and LY-S that are heterozygous at the thymidine kinase (Tk) locus (chromosome 11). We have found that a heterozygous LY-S Tk+/Tk- strain shows as high or higher mutability at the Tk locus than do heterozygous LY-R strains following treatment with x-radiation, UV radiation, or ethyl methanesulfonate. Mutability of all heterozygous strains at the Tk locus is much higher than at the Hprt locus following treatment with these mutagenic agents, with the exception of one heterozygous LY-R strain that possesses only one chromosome 11 and that is poorly mutable at the Tk locus by x-radiation. On the basis of these results, we have suggested that because of a repair deficiency, multilocus lesions are formed in the DNA of LY-S strains following treatment with radiation or alkylating agents; multilocus lesions lead to poor recovery of viable mutants when the target locus is closely linked to essential genes and situated on a hemizygous chromosomal region (e.g., the Hprt locus on the X chromosome or the Tk locus in strains monosomic for chromosome 11); and x-radiation is a relatively poor mutagen at loci situated on hemizygous chromosomal regions, in repair-efficient and repair-deficient cells, because of its tendency to form multilocus lesions.

Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal ... more Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal disease, contains two loci, rgpA and rgpB, encoding the extracellular Arg-X specific proteases (RGPs, Arg-gingipains), and kgp, which encodes a Lys-X specific protease (KGP, Lys-gingipain). The rgpA and kgp genes encode polyproteins comprising pro-peptide and catalytic domain with large N-and C-terminal extensions which require proteolytic processing at several Arg and Lys residues to generate mature enzymes. The product of rgpB contains only a pro-peptide and the catalytic domain which requires processing at an Arg residue to generate active enzyme. An rgpA rgpB double mutant (E8) of P. gingivalis was constructed to study the role of RGPs in the processing of KGP. A kgp mutant (K1A) was also studied to investigate the role of KGP in the generation of RGPs. E8 was stable in the absence of the antibiotics tetracycline and clindamycin (selection markers for rgpA and rgpB, respectively) and exhibited the same pigmentation, colony morphology and identical growth rates to the parent W50 strain in the absence of antibiotics, in both complex and chemically defined media. The KGP activity of E8, grown in the absence of tetracycline, in whole cultures and in culture supernatants (up to 6 d) was identical to levels in W50. However, in the presence of tetracycline in the growth medium, the level of KGP was reduced to 50 % of levels present in whole cultures of W50. Since tetracycline had no effect on RGP or KGP activity when incorporated into assay buffer, this effect is most likely to be on the synthesis of Kgp polypeptide. K1A was also stable in the absence of antibiotics but was unable to pigment, and remained straw-coloured throughout growth. RGP activity in whole cultures of K1A was identical to levels in W50, but RGP activity in 6 d culture supernatants was reduced to 50 % of levels present in W50. Thus, although KGP is not required for generation of RGP activity from RgpA and RgpB polypeptides, its absence affects the release/transport of RGP into culture supernatant.

Photochemistry and Photobiology, 1989

Two closely related strains of mouse lymphoma L5178Y cells, LY-R and LY-S, have been found to dif... more Two closely related strains of mouse lymphoma L5178Y cells, LY-R and LY-S, have been found to differ in their sensitivity to the cytotoxic effects of photodynamic treatment (PDT) with chloroaluminum phthalocyanine (CAPC) and red light. Strain LY-R is more sensitive to photodynamic cell killing than strain LY-S. Differences in uptake of CAPC could not account for the differences in cytotoxic effects. There was no marked difference between the two strains in the induction of single-strand breaks (which includes frank single-strand breaks and alkali-labile lesions), but substantially more DNA-protein cross-links were formed in strain LY-R by CAPC and light. Repair of single-strand breaks proceeded with similar kinetics in both strains for the first 30 min post-irradiation, suggesting that these lesions are not responsible for the differential sensitivity of the two strains to the lethal effects of photodynamic treatment. Thereafter, alkaline elution revealed the presence of increasing DNA strand breakage in strain LY-R. DNA degradation, as measured by the conversion of prelabled [14C] DNA to acid-soluble radioactivity, was more rapid and extensive in strain LY-R.

Proceedings of The National Academy of Sciences, 1987

The production and repair of radiation-induced DNA damage were measured by filter elution in stra... more The production and repair of radiation-induced DNA damage were measured by filter elution in strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. The induction of radiation-induced damage, as measured by filter elution at pH 12.1, 9.6, and 7.2, was similar in the resistant-strain LY-R and the sensitive strain LY-S. The repair of single-strand breaks and alkali-labile sites, as measured by filter elution at pH 12.1 at various times after irradiation, was somewhat slower in strain LY-S than in strain LY-R, although after a 20-min repair period the extent of

Photochemistry and Photobiology, 1997

Abstract— The mutagenicity of photodynamic therapy (PDT) using red light and either Photofrin® (p... more Abstract— The mutagenicity of photodynamic therapy (PDT) using red light and either Photofrin® (porfimer sodium) (PF) or aluminum phthalocyanine (AIPc) as the photosensitizer was determined at the thymidine kinase (TK) locus in the human lymphoblastic cell lines, TK6 and WTK1, and was compared to the mutagenicity of UVC and X-radia-tion in these cells as well as the mutagenicity of PDT in murine L5178Y lymphoblastic cell lines. Photodynamic therapy was found not to be mutagenic in TK6 cells, which possess an active p53 gene and which are relatively deficient in recombination and repair of DNA double-strand breaks. In contrast, PDT with either sensitizer was significantly mutagenic in WTK1 cells, which harbor an inactivating mutation in the p53 gene and are relatively efficient in recombination and double-strand break repair as compared to TK6 cells. The induced mutant frequency in WTK1 cells with PF as the photosensitizer was similar to that induced by UVC radiation but lower than that induced by X-radiation at equitoxic faiences/ doses. The mutant frequency induced by PDT in WTK1 cells with either photosensitizer was much lower than that induced in murine lymphoblasts at equitoxic fluences. The TK6 and WTK1 cells did not differ in their sensitivity to the cytotoxic effects of PDT, but the level of PDT-induced apoptosis was greater in TK6 than in WTK1 cells. These results indicate that the mutagenicity of PDT varies in different types of cells and may be related to the repair capabilities as well as the p53 status of the cells.

Photochemistry and Photobiology, 1992

Abstract— Photosensitization mediated by Photofrin II (PFII) was found to be mutagenic at the het... more Abstract— Photosensitization mediated by Photofrin II (PFII) was found to be mutagenic at the heterozygous thymidine kinase (tk) locus in mouse L5178Y lymphoma strains LY-S1 and LY-R16 but not in strain LY-R83 which is hemizygous at the tk locus. After treatments yielding 37% survival, the mutagenicity of photosensitization with PFII in strain LY-S1 was similar to that of other mutagenic agents including x-radiation, ethyl methanesulfonate, and photosensitization with chloroaluminum phthalocyanine (AIPcCI). Although both strain LY-S1 and strain LY-R16 were mutagenized by photosensitization with PFII, only strain LY-S1 was mutagenized by photosensitization with AIPcCI. The non-mutability of strain LY-R83 following photodynamic treatment with either sensitizer may be because of the poor recovery of mutants with intergenic mutations in this TK+/0 hemizygous strain, whereas the non-mutability of strain LY-R16 subjected to photodynamic treatment with AIPcCI may be because LY-R16 cells sustaining mutagenic damage do not survive for reasons other than the loss of an essential gene.

Mutation Research-dna Repair, 1989

The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely relat... more The cytotoxic and mutagenic effects of topoisomerase II inhibitors were measured in closely related strains of mouse lymphoma L5178Y cells differing in their sensitivity to ionizing radiation. Strain LY-S is sensitive to ionizing radioation relative to strain LY-R and is deficient in the rejoining of DNA double-strand breaks induced by this agent, whereas 2 radiation-resistant variants of strain LY-S have regained the ability to rejoin these double-strand breaks. We have found that the sensitivity of these cells to m-AMSA, VP-16, and eUipticine is correlated to their sensitivity to ionizing radiation. However, this correlation did not extend to their sensitivities to novobiocin, camptothecin, hydrogen peroxide, methyl nitrosourea and UV radiation. Thus, there appears to be a unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors which stabilize the cleavable complex between the enzyme and DNA. It is possible either that (1) topoisomerase II is altered in strain LY-S and that this enzyme is involved in the repair of DNA double-strand breaks or (2) strain LY-S is deficient in a reaction which is necessary for the repair of DNA double-strand breaks induced by ionizing radiation as well as the repair of DNA damage induced by these topoisomerase II inhibitors, m-AMSA, VP-16, and ellipticine were found to be highly mutagenic at the tk locus in L5178Y strains which are heterozygous for the tk gene but not in a tk hemizygous strain, indicating that these inhibitors induce multilocus lesions in DNA, as does ionizing radiation. The differences in the sensitivity of strains LY-R and LY-S to the topoisomerase II inhibitors were paralleled by differences in the induction of protein-associated DNA double-strand breaks in the 2 strains. This correlation did not extend to the radiation-resistant variants of strain LY-S, however. The variants showed resistance to the cytotoxic effects of the inhibitors relative to strain LY-S, but exhibited DNA double-strand break induction similar to that observed in strain LY-S.

The mode of cell death of two strains of mouse lymphoma LSI78Y cells was studied following photod... more The mode of cell death of two strains of mouse lymphoma LSI78Y cells was studied following photodynamic therapy (PDT) sensitized by chloroaluminum phthalocyanine. Strains LY-R and LY-S differ in their relative sensitivities to UVC radiation, X-radiation, and PDT; both responded to PDT by undergoing apoptosis. The DNA was degraded into fragments with lengths which are multiples of approximately 180-190 base pairs (i.e., oligonucleosome size), a biochemical marker of apoptosis. The DNA fragmentation was dose and time dependent which indicates this response to be an enzymic process related to cell killing. Cycloheximide, a protein synthesis inhibitor, and actinomycin D, an RNA synthesis inhibitor, enhanced the endonucleolytic DNA fragmentation. Transmis sion electron microscopy revealed chromatin condensation around the periphery of the nucleus, which is also characteristic of apoptosis. The induction of apoptosis in L5178Y cells by PDT was rapid, with marked degradation of DNA occurring in as little as 30 min. The rapidity of the response to PDT suggests that cellular damage produced by PDT can directly activate endonucleolysis and chromatin condensation, thereby by-passing many of the early steps in the signal transduction program which are acted upon by other agents causing apoptosis.

Photochemistry and Photobiology, 1988

Abstract— Chloroaluminum phthalocyanine (CAPC) is an efficient photosensitizer for the inactivati... more Abstract— Chloroaluminum phthalocyanine (CAPC) is an efficient photosensitizer for the inactivation of Chinese hamster V79 cells. In order to investigate possible molecular mechanisms in the photo-dynamic action of CAPC and visible light, the induction and repair rate of two classes of DNA lesions have been determined, i.e. DNA single-strand breaks and DNA-protein cross-links. In cells pretreated with 1 μ.M CAPC, a fluence of 12 kJ/m2 of red light (>600 nm) kills approximately 50% of the cells and induces 3 to 3.5 Gy-equivalents of single-strand breaks. The repair of these breaks was slower than the repair of single-strand breaks induced by m̀-irradiation. The photodynamic action of CAPC also induces a large number of DNA-protein cross-links which, in contrast to m̀-radiation-induced DNA-protein cross-links, do not appear to be repaired during 4 h of post-treatment incubation in fresh medium. These studies suggest that DNA may be an important target for the cytotoxicity of CAPC + red light.

Photochemistry and Photobiology, 1989

Abstract The cytotoxic and mutagenic effects of chloroaluminum phthalocyanine (CAPC) plus red lig... more Abstract The cytotoxic and mutagenic effects of chloroaluminum phthalocyanine (CAPC) plus red light have been measured in strains of L5178Y mouse lymphoma cells which differ in their DNA repair capacities. Strain LY-R, deficient in the excision repair of UV-induced dimers, was found to be relatively more sensitive to the cytotoxic effects of CAPC plus light, whereas strain LY-S, deficienl in the repair of DNA double-strand breaks, was more sensitive than strain LY-R to the mutagenic effects of the treatment. Mutation frequencies were measured in LY-S and LY-R sub-strains which were heterozygous or hemizygous at the thymidine kinase (tk) locus. The mutation frequency at the tk locus induced in the heterozygous strain LY-SI by CAPC plus light was lower than that induced by an equitoxic dose of ionizing radiation but similar to that induced by an equitoxic dose of UVC radiation: The mutation frequency at the F., dose of CAPC plus light was approximately 1100 per 106 surviving cells. The induced frequency in strain LY-S1 was much higher than in either tk+l-heterozygous or ik+10 hemizygous strains of LY-R. The rate and extent of incorporation of CAPC by the LY-R strains was somewhat greater than for strain LY-S1 at early times after CAPC addition, but by the time the cells were irradiated (18 h after CAPC addition) the difference was not great enough to account for the difference in cytotoxicity. It is possible that the cytotoxic and mutagenic lesions differ and that either the quantities of the respective lesions induced or the efficiencies of repair of the respective lesions differ inversely in the two strains.light have been measured in strains of L5178Y mouse lymphoma cells which differ in their DNA repair capacities. Strain LY-R, deficient in the excision repair of UV-induced dimers, was found to be relatively more sensitive to the cytotoxic effects of CAPC plus light, whereas strain LY-S, deficienl in the repair of DNA double-strand breaks, was more sensitive than strain LY-R to the mutagenic effects of the treatment. Mutation frequencies were measured in LY-S and LY-R sub-strains which were heterozygous or hemizygous at the thymidine kinase (tk) locus. The mutation frequency at the tk locus induced in the heterozygous strain LY-SI by CAPC plus light was lower than that induced by an equitoxic dose of ionizing radiation but similar to that induced by an equitoxic dose of UVC radiation: The mutation frequency at the F., dose of CAPC plus light was approximately 1100 per 106 surviving cells. The induced frequency in strain LY-S1 was much higher than in either tk+l-heterozygous or ik+10 hemizygous strains of LY-R. The rate and extent of incorporation of CAPC by the LY-R strains was somewhat greater than for strain LY-S1 at early times after CAPC addition, but by the time the cells were irradiated (18 h after CAPC addition) the difference was not great enough to account for the difference in cytotoxicity. It is possible that the cytotoxic and mutagenic lesions differ and that either the quantities of the respective lesions induced or the efficiencies of repair of the respective lesions differ inversely in the two strains.

Proceedings of The National Academy of Sciences, 1986

Mouse L5178Y strain LY-S and its parental strain LY-R differ in their comparative sensitivities t... more Mouse L5178Y strain LY-S and its parental strain LY-R differ in their comparative sensitivities to the cytotoxic effects of various mutagenic agents--i.e., strain LY-S has been found to be more sensitive, less sensitive, or similarly sensitive to individual agents in comparison to strain LY-R. Nevertheless, strain LY-S has been found to be uniformly less mutable than strain LY-R at the hypoxanthine (guanine) phosphoribosyltransferase (Hprt) locus following treatment with x-radiation, UV radiation, or alkylating agents. In the present work we have isolated subclones of strains LY-R and LY-S that are heterozygous at the thymidine kinase (Tk) locus (chromosome 11). We have found that a heterozygous LY-S Tk+/Tk- strain shows as high or higher mutability at the Tk locus than do heterozygous LY-R strains following treatment with x-radiation, UV radiation, or ethyl methanesulfonate. Mutability of all heterozygous strains at the Tk locus is much higher than at the Hprt locus following treatment with these mutagenic agents, with the exception of one heterozygous LY-R strain that possesses only one chromosome 11 and that is poorly mutable at the Tk locus by x-radiation. On the basis of these results, we have suggested that because of a repair deficiency, multilocus lesions are formed in the DNA of LY-S strains following treatment with radiation or alkylating agents; multilocus lesions lead to poor recovery of viable mutants when the target locus is closely linked to essential genes and situated on a hemizygous chromosomal region (e.g., the Hprt locus on the X chromosome or the Tk locus in strains monosomic for chromosome 11); and x-radiation is a relatively poor mutagen at loci situated on hemizygous chromosomal regions, in repair-efficient and repair-deficient cells, because of its tendency to form multilocus lesions.

Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal ... more Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal disease, contains two loci, rgpA and rgpB, encoding the extracellular Arg-X specific proteases (RGPs, Arg-gingipains), and kgp, which encodes a Lys-X specific protease (KGP, Lys-gingipain). The rgpA and kgp genes encode polyproteins comprising pro-peptide and catalytic domain with large N-and C-terminal extensions which require proteolytic processing at several Arg and Lys residues to generate mature enzymes. The product of rgpB contains only a pro-peptide and the catalytic domain which requires processing at an Arg residue to generate active enzyme. An rgpA rgpB double mutant (E8) of P. gingivalis was constructed to study the role of RGPs in the processing of KGP. A kgp mutant (K1A) was also studied to investigate the role of KGP in the generation of RGPs. E8 was stable in the absence of the antibiotics tetracycline and clindamycin (selection markers for rgpA and rgpB, respectively) and exhibited the same pigmentation, colony morphology and identical growth rates to the parent W50 strain in the absence of antibiotics, in both complex and chemically defined media. The KGP activity of E8, grown in the absence of tetracycline, in whole cultures and in culture supernatants (up to 6 d) was identical to levels in W50. However, in the presence of tetracycline in the growth medium, the level of KGP was reduced to 50 % of levels present in whole cultures of W50. Since tetracycline had no effect on RGP or KGP activity when incorporated into assay buffer, this effect is most likely to be on the synthesis of Kgp polypeptide. K1A was also stable in the absence of antibiotics but was unable to pigment, and remained straw-coloured throughout growth. RGP activity in whole cultures of K1A was identical to levels in W50, but RGP activity in 6 d culture supernatants was reduced to 50 % of levels present in W50. Thus, although KGP is not required for generation of RGP activity from RgpA and RgpB polypeptides, its absence affects the release/transport of RGP into culture supernatant.