Published:  Jan 20, 2017DOI: 10.7324/JABB.2017.50102
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.
Kyei F, Asante D, Sarpong E, Edekor J.A.M, Konja D, Gavor E. Over-expression of Id2 and Id3 Proteins Regulates Growth and Survival of Human Colon Carcinoma (HCT116) Cells. J App Biol Biotech. 2017; 5 (01): 010-017. DOI: 10.7324/JABB.2017.50102
1. Sikder HS, Meghann KD, Shariff D, Byungwoo R, Alani RM. Id proteins in cell growth and tumourigenesis. Cancer cell. 2003; 3:525-530.
2. Askew DS, Ashmun RA, Simmons BC, Clevaland JL. Constitutive c-myc expression in an IL-3-dependent myeloid cell line suppresses cell cycle arrest and accelerates apoptosis. Oncogene. 1991; 6:1915-1922.
3. Wagner AJ, Kokontis MJ, Hay N. Myc-mediated apoptosis requires wild-type p53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21 (WAF-1/CIP-1). Genes and Development. 1994; 8:2817-2830.
4. Chen X, Ko JK, Jayaraman L, Prives C. P53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells. Genes and Development. 1996; 10:2438-2451.
5. Florio M, Hernandez MC, Yang H, Shu HK, Cleveland JK, Israel MA. Id2 promotes apoptosis by a novel mechanism independent of dimerisation to basic helix-loop-helix factors. Molecular and Cell Biology. 1998; 18:2371-2381.
6. Wilson JW, Deed RW, Inoue T, et al. Expression of Id helix-loop-helix proteins in colorectal adenocarcinoma correlates with p53 expression and mitotic index. Cancer Research. 2001; 61:8803-8810.
7. Nishiyama K, Takaji K, Kataoka K, et al. Id1 gene transfer confers angiogenic property on fully differentiated endothelial cells and contributes to therapeutic angiogenesis. Circulation. 2005; 112:2840-2850.
8. Parrinello S, Lin CQ, Murata K, et al. Id-1, ITF-2, and Id-2 comprise a network of helix-loop-helix proteins that regulate mammary epithelial cell proliferation, differentiation, and apoptosis. Journal of Biological Chemistry. 2001; 276:39213-39219.
9. Andres-Barquin PJ, Hernandez MC, Israel MA. Id genes in nervous system development. Histology and Histopathology. 2000; 15:603-618.
10. Tanaka K, Pracyk JB, Takeda K, et al. Expression of Id1 results in apoptosis of cardiac myocytes through a redox-dependent mechanism. Journal of Biological Chemistry. 1998; 273:25922-25928.
11. Norton JD. Id helix-loop-helix proteins in cell growth, differentiation and tumourigenesis. Journal of Cell Science. 2000; 113:3897-3905.
12. Hara E, Hall M, Peters G. CDK2-dependent phosphorylation of Id2 modulates activity of E2A-related transcription factors. European Molecular Biology Organization Journal. 1997; 16:101-110.
13. Kyei F. Knock-down of Id1 and Id3 proteins induces apoptosis in human colon carcinoma (HCT116) cells. Journal of Biology and Life Science. 2015; 6:194-208.
14. Kee BL, Rivera RR, Murre C. Id3 inhibits B lymphocyte progenitor growth and survival in response to TGF b. Nature Immunology. 2001; 2:242-247.
15. Sakurai D, Tsuchiya N, Yamaguchi A, et al. Crucial Role of Inhibitor of DNA binding/differentiation in the vascular endothelial growth factor-induced activation and angiogenic processes of human endothelial cells. Journal of Immunology. 2004; 173:5801-5809.
16. Hara E, Zebedee Z. Id proteins in cell cycle control and cellular senescence. Oncogene. 2001; 20:8317-8325.
17. Ling MT, Wang X, Ouyang XS, Xu K, Tsao SW, Wong YC. Id-1 expression promotes cell survival through activation of NF-B signalling pathway in prostate cancer cells. Oncogene. 2003; 22:4498-4508.
18. Barndt RJ, Zhuang Y. Controlling lymphopoiesis with a combinatorial E-protein code, Cold Spring Harbor Symposia on Quantitative Biology, New York, Cold Spring Harbor Laboratory Press; 1999, p 45-50.
19. Norton JD, Atherton GT. Coupling of cell growth control and apoptosis functions of Id proteins. Molecular Cell Biology. 1998: 18;2371-2381.
20. Tu X, Baffa R, Luke S, Prisco M, Baserga R. Intracellular redistribution of nuclear and nucleolar proteins during differentiation of 32D murine hemopoietic cells. Experimental Cell Research. 2003; 288;119-130.
21. Lasorella A, Iavarone A. The protein ENH is a cytoplasmic sequestration factor for Id2 in normal and tumor cells from the nervous system. Proceedings of the National Academy of Sciences USA. 2006; 103;4976-4981.
22. Matsumura ME, Lobe DR, McNamara CA. Contribution of the helix-loop- helix factor Id2 to regulation of vascular smooth muscle cell proliferation. Journal of Biological Chemistry. 2002: 277:7293-7297.
23. Li X, Luo Y, Starremans PG, McNamara CA, Pei Y, Zhou J. Polycystin-1 and polycystin-2 regulate the cell cycle through the helix-loop-helix inhibitor Id2. Nature Cell Biology. 2005; 7:1102-1112.
24. Xu L, Massague J. Nucleocytoplasmic shuttling of signal transducers. Nature Reviews Molecular Cell Biology. 2004; 5:209-219.
25. Deed RW, Hara E, Atherton GT, Peters G, Norton J. D. Regulation of Id3 cell cycle function by Cdk2-dependent phosphorylation. Molecular Cell Biology. 1997; 17:6815-6821.
26. Butler DC, Haramizu S, Williamson D. L, Always S. E. Phospho-ablated Id2 is growth suppressive and pro-apoptotic in proliferating myoblasts. PloS One. 2009; 4: e6302.
27. Deed WR, Jasiok M, Norton JD. Lymphoid-specific expression of the Id3 gene in hematopoietic cells: selective antagonism of E2A basic helix-loop-helix protein associated with Id3-induced differentiation of erythroleukaemia cells. Journal of Biological Chemistry. 1998; 273:8278-8286.
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