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P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)
- Event: WCLC 2013
- Type: Poster Session
- Track: Biology
- Presentations: 1
- Coordinates: 10/28/2013, 09:30 - 16:30, Exhibit Hall, Ground Level
P1.05-024 - PARP inhibition increases sensitivity of NSCLC cells to cisplatin (ID 3300)
09:30 - 16:30 | Author(s): J. Thomale
Primary and acquired resistance to platinum agents is a serious clinical problem in lung cancer. Its mechanisms are probably multifactorial and remain poorly understood. Enhanced DNA repair can lead to increased cell viability in the face of DNA damage and has been proposed to be important in mediating platinum resistance. PARPs (poly(ADP-ribose) polymerases) are a family of nuclear enzymes that regulate the repair of DNA single-strand breaks (SSBs). Cisplatin sensitivity and DNA repair mechanisms following treatment with the PARP inhibitor, PJ34, was investigated in this study.
A panel of isogenic cisplatin resistant (CisR) NSCLC cells lines (MOR, SKMES-1, H1299) previously generated in our laboratory were used. The cisplatin resistant phenotype was initially assessed by treating CisR and parental (PT) cells with increasing doses of cisplatin (0-80uM) for 72h, after which time, cell proliferation was measured (BrdU). The effects of PJ34 on cell survival were also examined in a similar dose-response study. IC~25~ concentrations were calculated for each cell line using GraphPad statistical software. Cells were treated with PJ34 (IC~25~) alone, or in combination with cisplatin and cell survival/proliferation measured after 72h. Under similar experimental conditions, RNA was isolated from cells from which cDNA was reverse transcribed. All cell lines were screened for PARP1, PARP2, BRCA1, BRCA2 and ERCC1 mRNA at basal levels, and in response to treatment (RT-PCR). To investigate DNA double strand break (DSB) repair capacity in our panel of cell lines in response to PARP inhibition and cisplatin, phosphorylated γH2AX foci was examined by High Content Analysis (HCA) following treatment of cell lines for 24h. Cisplatin-DNA adduct formation (Pt-GpG) was studied following treatment of cells for 24h. Cells (1x10/ml) were spotted on Superfrost® Gold glass slides. Immunofluorescence staining of specific DNA platination products, and quantification of adducts, was performed using an antibody that specifically recognises cisplatin-GpG DNA adducts.
MOR and H1299 CisR cells were significantly more resistant to cisplatin (10µM and 20µM) compared to PT cells. SKMES-1 CisR cells were also significantly more resistant at 10µM, 20µM and 40µM cisplatin. While PJ34 had no effect on NSCLC cells when treated as a single agent, cell proliferation was significantly inhibited in MOR and H1299 cells when used in combination with cisplatin. No effect however was observed in our panel of CisR cell lines. While baseline expression levels of PARP1/2, BRCA1/2 and ERCC1 mRNA levels were similar in PT and CisR cell lines, BRAC1/2 mRNA expression was increased in cells treated with cisplatin alone, and in combination with PJ34 in PT cells but not in CisR cells. The formation of γH2AX foci and measurement of cisplatin-GpG DNA adducts in response to PARP inhibition and cisplatin are currently being investigated.
Data from this study show that inhibition of NSCLC cells with the PARP inhibitor, PJ34, sensitises lung cancer cells to the cytotoxic effects of the platinum drug, cisplatin. Further studies are warranted to investigate the role of PARP inhibitors in cisplatin resistant NSCLC cells.