PARENT SESSION
3:45 PM to 5:15 PM
Tuesday, April 23, 2002
Mini-Symposium 12
Cell Cycle and Apoptosis
Room: Nevada 4-5
, Co-Chair: Muschel, Ruth1; Haimovitz-Friedman, Adriana21University of Pennsylvania, Philadelphia, PA2Memorial Sloan-Kettering Cancer Center, New York, NY
(MS12-7) Ribozyme and antisense-mediated manipulation of poly(ADP-ribose) polmerase-1 to investigate its role in low-dose hyper-radiosensitivity.
Chalmers, Anthony*,1, Scott, Simon1, Marples, Brian1, 1 Experimental Oncology Group, Northwood, Middlesez
ABSTRACT-
The terms low-dose hyper-radiosensitivity (HRS) and increased radioresistance (IRR) describe the dual phenomena observed in many mammalian cell systems where small acute doses of radiation below ~50 cGy are more lethal per unit dose than higher doses up to ~2 Gy. There is evidence that HRS and IRR may reflect dose-dependent changes in DNA repair mechanisms. Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant, highly-conserved nuclear enzyme that rapidly binds to and is activated by DNA single and double strand breaks leading to recruitment and modulation of multiple DNA repair processes. Abrogation of the IRR response by chemical inhibitors of PARP-1 supports its role in the HRS/IRR phenomenon. To study the function of PARP-1 more specifically, we have generated a plasmid vector, pREV1, which co-expresses ribozyme (short, catalytic RNA species with site-specific cleavage activity) minigene and green fluorescent protein reporter gene sequences. A panel of six ribozyme constructs targeting different loci within the PARP-1 mRNA have been cloned in to pREV1 and are currently being assayed by Western and immunoblot techniques for their impact on cellular levels of PARP-1. Down-regulation by 50% has been achieved in transiently transfected T98G glioma cells. Stably transfected clones are being generated with a view to isolating PARP-1-depleted cell lines. Stable and transiently transfected cells will be tested in low-dose cell survival experiments to ascertain the effect of PARP-1 down-regulation on the HRS/IRR response, and compared with cells treated with chemical inhibitors of PARP-1 or transfected with the pMX18 dexamethasone-inducible PARP-1 antisense vector. Further experiments will assess the effect of PARP-1 depletion on the low dose radiation responses of (1) cell lines from our library exhibiting similar SF2 values but diverse HRS/IRR characteristics and (2) cell lines deficient in specific components of the base-excision and non-homologous end-joining pathways.
KEYWORDS: poly(adp-ribose) polymerase, low -dose hyper-radiosensitivity, ribozyme, antisense