Abstract

Inhibitor of differentiation (Id) proteins are members of the Helix-Loop-Helix (HLH) group of transcription factors. These proteins uniquely have no DNA-binding domain and they play vital roles in cell growth, differentiation, senescence, apoptosis, angiogenesis and neoplastic transformation. Objective: This work investigated the pro-apoptotic functions of Id proteins and their loss-or gain-of function mutants to delimit the functional domains that are essential for apoptosis in an in vitro model using human epithelial colon carcinoma cell line (HCT116). Plasmids encoding Id1, Id2, Id3 and Id4 proteins were transiently over-expressed in cultured HCT116. Initially, cell proliferation assay with MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide) was performed, followed by apoptotic and cell cycle analyses on the transfected cells. Apoptotic and cell cycle profiles were generated and statistically analyzed. Id3 protein exhibited a pro-apoptotic effect in colorectal cancer cell lines (HCT116). In addition, over-expression of Id3 resulted in growth arrest in HCT116. Id2 Asp5 showed a pro-apoptotic whilst Id2 Ala5 and Id2 HB enhanced viability of HCT116. These findings suggest that Id2 Asp5 may be loss-of-function mutant in HCT116 lines. The aspartate mutant of Id3 (Id3 Asp5) was observed to be pro-apoptotic. However Id3 Ala5 and Id3 HB showed an anti-apoptotic effect. These findings suggest that Id2 Asp5, Id3 Ala5, Id3 HB and Id3NLS may be loss-of-function mutants in HCT116 whilst Id2 Ala5, Id2 HB and Id3 Asp5 are gain-of-function mutants. These results may suggest that Id2 and Id3 may play essential roles in modulating human epithelial colon carcinoma growth and survival and could provide some hope for therapeutic opportunities in the treatment of colonic cancers.

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