We previously demonstrated that pre-treatment of human androgen-dependent prostate cancer cell line LNCaP with either 12-0-tetradecanoylphorbol 13-acetate (TPA, a known protein kinase C activator), or diacylglycerol-lactone (DAG-lactone) radiosensitized these cells. This effect was mediated by down-regulation of ATM protein levels, thus de-repressing the enzyme ceramide synthase (CerS), ceramide generation and apoptosis. Here we assessed whether ATM down-regulation affected DNA repair pathways. LNCaP cells were pretreated for 16 hr with 10μM DAG-lactone and irradiated with 20 or 40Gy. Pre-treatment with DAG-lactone significantly enhanced apoptosis at 48 hours from 1.1% to 13.9% (20Gy) and 19.9% (40Gy) respectively. γ-H2AX and BRCA1 foci formation at 1 and 8 hours post-radiation were scored. Immunofluorescent staining for γ-H2AX demonstrated a dose-dependent increase in γ-H2AX foci formation at 20Gy and 40Gy for both 1hr and 8 hr time points. Our data also shows that 40Gy alone produces the same amount of DNA double strand breaks (dsbs; as measured by γ-H2AX at 8 hr) as 20Gy+DAG-lactone, yet 40Gy alone does not induce apoptosis in LNCaP cells, while 20Gy+DAG-lactone does, indicating that ATM down-regulation is necessary for the apoptotic response. Similarly there was a dose-dependent response in the generation of BRCA1 foci at 20Gy and 40 Gy at 8hr post-radiation. There was no difference in the BRCA1 foci between pre-treatment with DAG-lactone either at 20Gy or at 40 Gy at both 1 hr or at 8 hr post-radiation. While apoptotic degradation is associated with γ-H2AX phosphorylation, in LNCaP cells these events would be expected at 24-48 hr. Therefore the foci observed here are unlikely to be associated with apoptotic DNA degradation and most likely result from DNA dsbs. Our recent data showed that unrepaired DNA dsbs trigger post-transcriptional activation of CerS via a mechanism that remains unknown. Accordingly MRE 11, ATM or DNA-PKcs (SCID) deficiencies sensitize mice towards CerS-mediated apoptosis in crypt stem cells clonogens.

Although these studies indicate that HR is active, further experiments need to be carried out to explore whether non-homologous end-joining (NHEJ) is or is not functional in LNCaP treated with DAG-lactone and radiation. Hence, the trigger for CerS activation still needs to be established.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1406.