Abstract #521: Poly(ADP-ribose) polymerase-1 polymorphisms, expression and activity in a panel of human tumor cell lines

Background: (PARP-1) is a DNA-binding enzyme activated by DNA breaks and involved in DNA repair, genomic stability, transcription control, cell death and proliferation. PARP activity is reported to be higher in various cancers than adjacent normal tissue. Contrastingly, the prevalence of some cancers is reported to be greater in individuals with a single nucleotide polymorphism (SNP) V762A (T2444C) in the catalytic domain that causes a reduction inPARP-1 activity. We aimed to resolve these opposing observations by determining PARP-1 genotype, PARP-1 protein levels, and PARP-1 activity in a panel of 20 human cancer cell lines.Methods: We studied 5 neuroblastoma cell lines (TR14, NB1691, NGP, LS, SK-N-BE2C), 3 breast cancer cell lines (T47D, MCF-7, MDA-MB-231), 7 leukaemia cell lines (Nalm 6, PreB 697, HL-60, K562, CCRF-CEM, Jurkat, Molt-4), Burkitt\#8217;s lymphoma cell line (Raji), thyroid carcinoma cell line (ML-1), colorectal carcinoma cell line (LoVo), hereditary spherocytosis cell line (TK-6) and bone marrow stromal cell line (HS-5). The T2444C SNPwas detected by pyrosequencing and the (CA)n microsatellite in the promoter was analyzed by capillary-electrophoresis. PARP-1 activity and expression were determined using GCLP validated immunoblot andWestern blot, respectively. Results: Analysis of the active site (T2444C) SNP revealed that most of the cell lines had the wild genotype (T/T), PreB and LoVo cells were heterozygous (T/C) while LS and Jurkat cells were homozygous variant (C/C). A long (CA)15-20 polymorphism in the promoter region may result in increased PARP-1 expression but most of the cells had short microsatellites 56% CA11/CA11, 28% CA11/CA15, 6% CA11/CA12, 6% CA13/CA14, and only 6% CA15/CA15.There was a wide variation in PARP activity in the cell line panel with a 35-fold difference between the lowest (2460 pmol PAR/106 HS5 cells) and the highest activity (85750 pmol PAR/106 NGP cells). There was less variation in PARP-1 protein expression with a 3.5-fold difference between the lowest (2.0 ng/\#956;g for HS5 cells) and the highest (7.1 ng/\#956;g for ML-1 cells). The mean activity in the cancer cells was 45 fold higher than mean activity in normal human lymphocytes (Proc Annu Meet Am Assoc Cancer Res 2008 #540) and the PARP-1 protein levels were 23-fold higher. Conclusion: Overall our data showed significant upregulation of PARP-1 expression and activity in cancer cell lines compared to human lymphocytes. However, we did not find any significant correlation between PARP-1 expression and length of the (CA)n-microsatellite in the promoter or activity with either PARP-1 protein levels or the T2444C SNP between the cell lines. This suggests that post-translational mechanisms contribute significantly to the increase in activity. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 521.