title: Expression levels of the DNA repair enzyme HAP1 do not correlate with the radiosensitivities of human or HAP1-transfected rat cell lines.
abstract: 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.
authors.affiliation: CRC Section of Genome Damage and Repair, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Manchester, UK.
authors.forename: C J
authors.initials: CJ
authors.lastname: Herring
authors.forename: B
authors.initials: B
authors.lastname: Deans
authors.forename: R H
authors.initials: RH
authors.lastname: Elder
authors.forename: J A
authors.initials: JA
authors.lastname: Rafferty
authors.forename: J
authors.initials: J
authors.lastname: MacKinnon
authors.forename: G
authors.initials: G
authors.lastname: Barzilay
authors.forename: I D
authors.initials: ID
authors.lastname: Hickson
authors.forename: J H
authors.initials: JH
authors.lastname: Hendry
authors.forename: G P
authors.initials: GP
authors.lastname: Margison
mesh_terms: D000818:Animals; D019757:Carbon-Oxygen Lyases / Q000096:biosynthesis* / Q000235:genetics; D002470:Cell Survival / Q000528:radiation effects; D004260:DNA Repair / Q000528:radiation effects*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D015870:Gene Expression; D005910:Glioma / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D006801:Humans; D015151:Immunoblotting; D009369:Neoplasms / Q000201:enzymology* / Q000235:genetics; D011836:Radiation Tolerance; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D043223:Deoxyribonuclease IV (Phage T4-Induced); D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase