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.
© 2011 Blackwell Publishing Ltd.