Abstract
BackgroundInhibitor of differentiation 4 (Id4), a member of the Id gene family is also a dominant negative regulator of basic helix loop helix (bHLH) transcription factors. Some of the functions of Id4 appear to be unique as compared to its other family members Id1, Id2 and Id3. Loss of Id4 gene expression in many cancers in association with promoter hypermethylation has led to the proposal that Id4 may act as a tumor suppressor. In this study we provide functional evidence that Id4 indeed acts as a tumor suppressor and is part of a cancer associated epigenetic re-programming.MethodsData mining was used to demonstrate Id4 expression in prostate cancer. Methylation specific polymerase chain reaction (MSP) analysis was performed to understand molecular mechanisms associated with Id4 expression in prostate cancer cell lines. The effect of ectopic Id4 expression in DU145 cells was determined by cell cycle analysis (3H thymidine incorporation and FACS), expression of androgen receptor, p53 and cyclin dependent kinase inhibitors p27 and p21 by a combination of RT-PCR, real time-PCR, western blot and immuno-cytochemical analysis.ResultsId4 expression was down-regulated in prostate cancer. Id4 expression was also down-regulated in prostate cancer line DU145 due to promoter hyper-methylation. Ectopic Id4 expression in DU145 prostate cancer cell line led to increased apoptosis and decreased cell proliferation due in part by an S-phase arrest. In addition to S-phase arrest, ectopic Id4 expression in PC3 cells also resulted in prolonged G2/M phase. At the molecular level these changes were associated with increased androgen receptor (AR), p21, p27 and p53 expression in DU145 cells.ConclusionThe results suggest that Id4 acts directly as a tumor suppressor by influencing a hierarchy of cellular processes at multiple levels that leads to a decreased cell proliferation and change in morphology that is possibly mediated through induction of previously silenced tumor suppressors.
Highlights
Inhibitor of differentiation 4 (Id4), a member of the inhibitors of differentiation (Id) gene family is a dominant negative regulator of basic helix loop helix transcription factors
Based on our observations that ectopic expression of Id4 can decrease proliferation, increase apoptosis and induce a transition towards an epithelial phenotype, we considered the possibility that Id4
The tumor suppressor role of Id4 appears to be unique as compared to other members of the Id gene family (Id1, Id2 and Id3) that may act as oncogenes or co-operating oncogenes in many cancers [24,27,30]
Summary
Inhibitor of differentiation 4 (Id4), a member of the Id gene family is a dominant negative regulator of basic helix loop helix (bHLH) transcription factors. The Id proteins lack the DNA binding basic domain but have intact HLH domain [2,3] This domain configuration allows the Id family to dimerize with bHLH transcription factors, but the lack of the basic domain renders the Id-bHLH dimer transcriptionally inactive, as it fails to bind and regulate promoter activity of genes dependent on E-box (CANNTG) response element [4]. The four different isoforms of Ids (Id1, Id2, Id3 and Id4) have a highly conserved HLH domain but divergent Nand C-terminal domains This sequence divergence may account for protein-specific interactions possibly resulting in differential functions of Id proteins [5,6,7]. Consistent with gene specific interactions, the Id proteins exhibit isoform specific functions such as modulation of breast cancer 1, early onset (BRCA1) promoter activity by Id4 [15,16], localization of Id1 to the centrosomes [17] leading to accumulation of cells with abnormal centrosome number and induction of apoptosis by Id2 in myeloid precursors, osteosarcoma [18] and neuronal cells [19] by an HLH independent mechanism
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