Overexpression of Id3 induces apoptosis of A549 human lung adenocarcinoma cells

Objectives:  Inhibitor of differentiation 3 (Id3) protein has been implicated in the control of multiple cell death signalling pathways and in aetiology of numerous diseases. The aims of this study were to construct a recombinant eukaryotic expression vector (pEGFP/Id3), containing human Id3 (hId3) fused with enhanced green fluorescent protein (EGFP), and to determine effects of ectopic Id3 overexpression, on human lung adenocarcinoma cell (A549) proliferation. Materials and methods:  Human Id3 cDNA was inserted into pEGFP‐N1 vector to yield the recombinant eukaryotic expression vector pEGFP/Id3. Cells were transfected with pEGFP or pEGFP/Id3, and proliferation of EGFP‐expressing cells was monitored by flow cytometry (FCM) and confocal fluorescence microscopy. RT‐PCR, immunoblotting and immunocytochemistry were used to assess Id3 mRNA transcription and protein expression. Apoptosis was evaluated by Annexin V/7‐AAD staining and FCM, while nuclear morphology of apoptotic cells was examined using Hoechst 33258 staining. Results:  Over 4 days transfection with pEGFP, the proportion of EGFP‐positive A549 cells peaked at approximately 60% by 48 h and remained stable over the next 48 h. In contrast, the proportion of EGFP‐positive cells in cultures transfected with pEGFP/Id3 decreased from a peak of 60% at 48 h to <5% at 96 h, suggesting that Id3 expression inhibited cell proliferation or survival. Annexin V/7‐AAD and Hoechst 33258 staining revealed significantly higher rates of apoptosis in pEGFP/Id3‐transfected cells. Conclusion:  Overexpression of Id3 triggered apoptosis in A549 human lung adenocarcinoma cells, implicating Id3 in negative control of tumour growth. These Id3‐induced pro‐apoptotic signalling pathways require further study, but this preliminary investigation suggests that Id3 regulation could be exploited in anti‐tumour therapies.