Abstract
BackgroundPlatinum‐based chemotherapy and radiotherapy are standard treatments for non‐small cell lung cancer, which is the commonest, most lethal cancer worldwide. As a marker of treatment‐induced cancer cell death, we have developed a radiodiagnostic imaging antibody, which binds to La/SSB. La/SSB is an essential, ubiquitous ribonuclear protein, which is over expressed in cancer and plays a role in resistance to cancer therapies.AimIn this study, we examined radiation‐induced DNA double strand breaks (DSB) in lung cancer cell lines and examined whether La/SSB associated with these DSB.MethodThree lung cancer lines (A549, H460 and LL2) were irradiated with different X‐ray doses or X‐radiated with a 5 Gy dose and examined at different time‐points post‐irradiation for DNA DSB in the form of γ‐H2AX and Rad51 foci. Using fluorescence microscopy, we examined whether La/SSB and γ‐H2AX co‐localise and performed proximity ligation assay (PLA) and co‐immunoprecipitation to confirm the interaction of these proteins.ResultsWe found that the radio‐resistant A549 cell line compared to the radio‐sensitive H460 cell line showed faster resolution of radiation‐induced γ‐H2AX foci over time. Conversely, we found more co‐localised γ‐H2AX and La/SSB foci by PLA in irradiated A549 cells.ConclusionThe co‐localisation of La/SSB with radiation‐induced DNA breaks suggests a role of La/SSB in DNA repair, however further experimentation is required to validate this.
Highlights
Lung cancer and its major form, non-small cell lung cancer (NSCLC), is globally the commonest and most lethal cancer
Given that DNA damage may happen as rapidly as electron-transfer, the transcripts involved in the DNA damage response (DDR) are expressed before the DNA repair process begins, and dynamic and intricate regulation of transcript stability allows cells to react promptly to the damage and maintain genomic integrity
The DDR involves at least hundreds of RNA molecules and proteins including mRNA, non-coding RNA molecules and RNA-binding proteins (RBP)
Summary
Lung cancer and its major form, non-small cell lung cancer (NSCLC), is globally the commonest and most lethal cancer. During apoptotic tumour cell death in vitro, the La/SSB protein translocates from nucleus to cytoplasm, and as necrosis develops with loss of cell membrane integrity, the La/SSB protein becomes available for antigenspecific antibody binding in the dead tumour cells.[12,13,19,20,21,22] after DNA-damaging anti-cancer treatments such as some cytotoxic chemotherapy drugs or ionising radiation, the binding of specific antibodies to La/SSB in dead tumour cells is even greater because of two major effects. We made an initial series of experimental observations to address the gap in our understanding of the conditions and context for binding of the chDAB4 mAb to tumour cells dying after DNAdamaging treatment and to explore the potential involvement of La/SSB in the DNA repair response to DNA-damaging treatment in lung cancer cells. We observed that, in response to DNA-damaging stimuli such as ionising radiation, the levels of La/SSB expression in tumour cells increased before plasma cell membrane integrity was lost.[12,21] immunocytological observations of La/SSB protein interactions in the current study were made after fixation and permeabilisation of the cancer cells
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