Herring, C J; Deans, B; Elder, Rhoderick H; Rafferty, Joseph A; MacKinnon, J; Barzilay, G; Hickson, I D; Hendry, Jolyon H; Margison, Geoffrey P (1999-06)
Apurinic/apyrimidinic (AP) sites in DNA are potentially lethal and mutagenic. They can arise spontaneously or following DNA damage from reactive oxygen species or alkylating agents, and they constitute a significant product of DNA damage following cellular exposure to ionizing radiation. The major AP endonuclease responsible for initiating the repair of these and other DNA lesions in human cells is HAP1, which also possesses a redox function. We have determined the cellular levels of this enzyme in 11 human tumour and fibroblast cell lines in relation to clonogenic survival following ionizing radiation. Cellular HAP1 levels and surviving fraction at 2 Gy (SF2) varied five- and tenfold respectively. However, no correlation was found between these two parameters following exposure to gamma-irradiation at low (1.1 cGy per min) or high (108 cGy per min) dose rates. To examine this further, wild-type and mutant versions of HAP1 were overexpressed, using an inducible HAP1 cDNA expression vector system, in the rat C6 glioma cell line which has low endogenous AP endonuclease activity. Induction of wild-type HAP1 expression caused a > fivefold increase in the capacity of cellular extracts to cleave an oligonucleotide substrate containing a single abasic site, but increased expression did not confer increased resistance to gamma-irradiation at high- or low-dose rates, or to the methylating agent methyl methanesulphonate (MMS). Expression in C6 cell lines of mutant forms of HAP1 deleted for either the redox activator or DNA repair functions displayed no apparent titrational or dominant negative effects. These studies suggest that the levels of endogenous AP endonuclease activities in the various cell lines examined are not limiting for efficient repair in cells following exposure to ionizing radiation or MMS. This contrasts with the correlation we have found between HAP1 levels and radiosensitivity in cervix carcinomas (Herring et al (1998) Br J Cancer 78: 1128-1133), indicating that HAP1 levels in this case assume a critical survival role and hence that established cell lines might not be a suitable model for such studies.
Brent, T P; Von Wronski, M A; Edwards, C C; Bromley, Michael; Margison, Geoffrey P; Rafferty, Joseph A; Pegram, C N; Bigner, D D (1993)
Cellular levels of O6-methylguanine-DNA methyltransferase (MGMT) correlate strongly with cellular resistance to carcinogenic and chemotherapeutic agents that produce adducts at the O6-position of guanine in DNA. Although biochemical and molecular assays can indicate the average MGMT content of tissues or tumors, they cannot distinguish mixed populations of cells, such as those that exist in tumor biopsy samples. We have determined MGMT at the cellular level in a panel of pediatric rhabdomyosarcoma xenografts by in situ immunostaining with a human MGMT-specific antibody employing a very sensitive procedure that involves biotin-avidin coupled horseradish peroxidase with silver-enhanced diaminobenzidine-nickel staining. Two xenograft tumor lines known to be MGMT-deficient were not stained, whereas the nuclei in three MGMT-expressing lines were clearly stained. This is the first demonstration of an in situ procedure that discriminates drug-sensitive MGMT-deficient tumors from drug-resistant MGMT expressing tumors. This procedure should prove useful, therefore, for predicting the susceptibility of tissues and tumors to O6-guanine alkylating agents.
Jelínek, Jaroslav; Fairbairn, Leslie J; Dexter, T Michael; Rafferty, Joseph A; Stocking, C; Ostertag, W; Margison, Geoffrey P (1996-03-01)
A human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA-containing retrovirus was used to infect murine long-term primary bone marrow cultures. High levels of ATase expression were obtained, and colony-forming cells of the granulocyte-macrophage lineage from the cultures transduced with the human ATase retrovirus were three times more resistant to the alkylating agent, N-methyl-N-nitrosourea (MNU), than control cultures. Furthermore, expression of the human ATase protected long-term hematopoiesis, measured as the output of progenitor cells to the nonadherent fraction of the culture, against the cytotoxic effects of repeated exposures to MNU. These results clearly show that a human ATase cDNA-containing retrovirus can be used to infect long-term primary bone marrow cultures and that this attenuates their sensitivity to nitrosoureas.
McElhinney, R S; Donnelly, Dorothy J; McCormick, J E; Kelly, Jane; Watson, Amanda J; Rafferty, Joseph A; Elder, Rhoderick H; Middleton, Mark R; Willington, Mark; McMurry, T Brian H; et al. (1998-12-17)
A number of novel guanine derivatives containing heterocyclic moieties at the O6-position have been synthesized using a purine quaternary salt which reacts with alkoxides under mild conditions. Initially O6-substituents were investigated in which the benzene ring of the known agent, O6-benzylguanine, was replaced by unsubstituted heterocyclic rings. The ability of these agents to inactivate the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase), both as pure recombinant protein and in the human lymphoblastoid cell line Raji, has been compared with that of O6-benzylguanine. The present paper focuses on O6-substituents with basic rings, and under standard conditions several of them proved more effective than benzyl for inactivation of both recombinant and Raji ATase. Among the pyridine derivatives, the 2-picolyl compound 7 is not very active in contrast to the 3- and 4-picolyl compounds, and this influenced our choice of isomers of other basic ring systems for study. Since halogen substitution in the thiophene ring considerably increased the activity (17 versus 6), similar modifications in the pyridine series were examined. The more polar O6-substituents in this study are on the whole compatible with the stereochemical requirements of the ATase protein, and their pharmacological properties may be valuable in subsequent in vivo investigations, particularly the thenyl (6), 5-thiazolylmethyl (12), 5-bromothenyl (17), and 2-chloro-4-picolyl (21) derivatives.
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