Experimental and Clinical Therapeutics

Monday, October 17, 2005 3:00 PM-5:00 PM Exhibit Hall

(PP116) Together, active EGFR and EGFR trans-activated by irradiation over-activate PARP depleting NAD+ and increasing cell-killing.

Yacoub, Adly1, Dent, Paul1, Hagan, Michael *,1, 1 Radiation Oncology, Richmond, VA, USA

ABSTRACT- Purpose/Objective: Although EGF and ionizing radiation (IR) independently activate EGFR to up-regulate proteins involved in DNA repair, ligand activation of EGFR at irradiation enhances cell-killing. Because one of the up-regulated proteins, XRCC1, binds Poly(ADP-ribose) polymerase (PARP) reducing auto-modification, we examined PARP activation following ligand activation, radiation trans-activation and the combination. Materials/Methods: Using human prostate cancer cell lines that express high (DU145) or low (LNCaP) levels of EGFR, we measured NAD+ stores by the enzyme cycling assay, PARP activation via 32P-NAD and 10H MaB, and cell survival following irradiation in the presence or absence of EGF (1-5ng/ml) or the PARP inhibitor, PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide). We examined the impact of NAD+ synthesis by RNAi knockdown of NMNAT and EGFR-ERK signal interruption by the tyrphostin AG1478 and the MEK inhibitor PD98059. Results: EGF addition immediately before or after irradiation led to increased cell-killing for both cell lines. Cell survival was markedly reduced for LNCaP cells. Western analysis showed that both ionizing radiation and EGF addition led to prompt synthesis of polyADP-ribose (PAR), which in each case was accompanied by reduction in cellular NAD+. EGF addition, however, did not increase ionizing radiation-induced DNA damage, assayed using the comet assay. The combination of EGF and irradiation, however, produced stronger PARP activation and NAD depletion, 25% for DU145 cells and 41% for LNCaP cells. Addition of the PARP inhibitor PJ34 prior to EGF addition or irradiation increased the NAD+ levels and survival of LNCaP cells. Each EGF effect is eliminated by the tyrphostin AG1478, while MEK1/2 inhibition actually exacerbates each response. Conclusions: EGFR activation produces prompt activation of PARP, which is markedly increased by simultaneous irradiation. This increased PARP activation is apparently not due to increased DNA damage; is increased by blocking EGFR-ERK signaling; and is eliminated by blocking EGFR auto-phosphorylation. Thus, PARP activation appears to be a downstream effect of EGFR signaling, not mediated by ERK1/2. EGF/IR Over-activation of PARP in LNCaP cells, which decreases both NAD+ and cell survival, can be blocked at the level of PARP as well as at the receptor level.

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2005 RRS