Figure S1. Western blots showing that the reduction in RAD51 levels after cisplatin treatment in melanoma cells is not seen in other cell types. Blots support the RAD51 quantification shown in Fig. 2 of the main text. Figure S2. Images showing that RAD51 repair foci are not induced by short term cisplatin treatment of melanoma cells. Figure S3. The cisplatin-induced reduction in RAD51 levels in melanoma cells is not affected by proteasome or lysosome inhibitor treatment. Figure S4. Melanoma cells show only a weak cisplatin-induced G2 arrest. Figure S5. Specificity of antibodies to translesion synthesis DNA Polymerases. Figure S6. Western blots showing increased levels of DNA Polymerase zeta in cisplatin-treated melanoma cells. Blots support the DNA Pol ζ and Pol η expression data shown in Fig. 7 of the main text. (PDF 2393 kb

Ann-Marie Ritchie (4692964)

Charlie Gourley (438864)

David Melton (4692970)

Ewan McNeil (4692967)

Katy Astell (517935)

Liang Song (40318)

BMC Cancer

Abstract Background The global incidence of melanoma has been increasing faster than any other form of cancer. New therapies offer exciting prospects for improved survival, but the development of resistance is a major problem and there remains a need for additional effective melanoma therapy. Platinum compounds, such as cisplatin, are the most effective chemotherapeutics for a number of major cancers, but are ineffective on metastatic melanoma. They cause monofunctional adducts and intrastrand crosslinks that are repaired by nucleotide excision repair, as well as the more toxic interstrand crosslinks that are repaired by a combination of nuclease activity and homologous recombination. Methods We investigated the mechanism of melanoma resistance to cisplatin using a panel of melanoma and control cell lines. Cisplatin-induced changes in levels of the key homologous recombination protein RAD51 and compensatory changes in translesion synthesis DNA polymerases were identified by western blotting and qRT-PCR. Flow cytometry, immunofluorescence and western blotting were used to compare the cell cycle and DNA damage response and the induction of apoptosis in cisplatin-treated melanoma and control cells. Ectopic expression of a tagged form of RAD51 and siRNA knockdown of translesion synthesis DNA polymerase zeta were used to investigate the mechanism that allowed cisplatin-treated melanoma cells to continue to replicate. Results We have identified and characterised a novel DNA damage response mechanism in melanoma. Instead of increasing levels of RAD51 on encountering cisplatin-induced interstrand crosslinks during replication, melanoma cells shut down RAD51 synthesis and instead boost levels of translesion synthesis DNA polymerase zeta to allow replication to proceed. This response also resulted in synthetic lethality to the PARP inhibitor olaparib. Conclusions This unusual DNA damage response may be a more appropriate strategy for an aggressive and rapidly growing tumour like melanoma that enables it to better survive chemotherapy, but also results in increased sensitivity of cultured melanoma cells to the PARP inhibitor olaparib

Liang Song

Ewan M. McNeil

Ann-Marie Ritchie

Katy R. Astell

Charlie Gourley

David W. Melton

Directory of Open Access Journals

Melanoma cells replicate through chemotherapy by reducing levels of key homologous recombination protein RAD51 and increasing expression of translesion synthesis DNA polymerase ζ

FigShare

Additional file 1: of Melanoma cells replicate through chemotherapy by reducing levels of key homologous recombination protein RAD51 and increasing expression of translesion synthesis DNA polymerase ζ

Background: The global incidence of melanoma has been increasing faster than any other form of cancer. New therapies offer exciting prospects for improved survival, but the development of resistance is a major problem and there remains a need for additional effective melanoma therapy. Platinum compounds, such as cisplatin, are the most effective chemotherapeutics for a number of major cancers, but are ineffective on metastatic melanoma. They cause monofunctional adducts and intrastrand crosslinks that are repaired by nucleotide excision repair, as well as the more toxic interstrand crosslinks that are repaired by a combination of nuclease activity and homologous recombination.Methods: We investigated the mechanism of melanoma resistance to cisplatin using a panel of melanoma and control cell lines. Cisplatin-induced changes in levels of the key homologous recombination protein RAD51 and compensatory changes in translesion synthesis DNA polymerases were identified by western blotting and qRTPCR. Flow cytometry, immunofluorescence and western blotting were used to compare the cell cycle and DNA damage response and the induction of apoptosis in cisplatin-treated melanoma and control cells. Ectopicexpression of a tagged form of RAD51 and siRNA knockdown of translesion synthesis DNA polymerase zeta were used to investigate the mechanism that allowed cisplatin-treated melanoma cells to continue to replicate.Results: We have identified and characterised a novel DNA damage response mechanism in melanoma. Instead of increasing levels of RAD51 on encountering cisplatin-induced interstrand crosslinks during replication, melanoma cells shut down RAD51 synthesis and instead boost levels of translesion synthesis DNApolymerase zeta to allow replication to proceed. This response also resulted in synthetic lethality to the PARP inhibitor olaparib.Conclusions: This unusual DNA damage response may be a more appropriate strategy for an aggressive andrapidly growing tumour like melanoma that enables it to better survive chemotherapy, but also results inincreased sensitivity of cultured melanoma cells to the PARP inhibitor olaparib

Song, Liang

McNeil, Ewan M.

Ritchie, Ann-Marie

Astell, Katy R

Gourley, Charlie

Melton, David W.

Edinburgh Research Explorer

Melanoma cells replicate through chemotherapy by reducing levels of key homologous recombination protein RAD51 and increasing expression of translesion synthesis DNA Polymerase ζ

Crossref

Sekkingstad, Dorthea

Syse, Ingrid

NORA - Norwegian Open Research Archives