AJMB  Vol.2 No.4 , October 2012
GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing (siRNA) in senescent human diploid fibroblasts
Abstract: Human diploid fibroblasts (HDFs) undergo a limited number of cell divisions in culture. After certain population doublings, they reach a state of irreversible growth arrest known as replicative senescence. Senescent HDFs showed several molecular and cytological changes such as large flat morphology, expression of senescence-associated β-galactosidase (SA β-gal) activity and altered gene expression. Small interfering RNA (siRNA) has been demonstrated to be a potential research tool to analyse gene function and pathway. Expression of an appropriate housekeeping or reference gene can be used as a measurement of transfection efficiency in siRNA. Therefore this study was designed to determine the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model. GAPDH knockdown with an appropriate transfection reagent was measured by quantitative real time RT-PCR while cellular senescence was characterized based on morphological changes, expression of SA β-gal and p16INK4a expression levels. Our findings showed that GAPDH knockdown represents silencing efficiency and down regulation of p16INK4a in senescent transfected HDFs caused morphological alterations which results in the formation of spindle shaped fibroblasts. This study demonstrated the suitability of GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing in HDFs aging model.
Cite this paper: Makpol, S. , Zainuddin, A. and Chua, K. (2012) GAPDH expression as a measurement of transfection efficiency for p16INK4a gene silencing (siRNA) in senescent human diploid fibroblasts. American Journal of Molecular Biology, 2, 390-397. doi: 10.4236/ajmb.2012.24041.

[1]   Hayflick, L. and Moorhead, P.S. (1961) The serial cultivation of human diploid cell strains. Experimental Cell Research, 25, 585-621. doi:10.1016/0014-4827(61)90192-6

[2]   Zheng, W., Wang, H., Xue, L., Zhang, Z. and Tong, T. (2004) Regulation of Cellular Senescence and p16INK4a Expression by Id1 and E47 Proteins in Human Diploid Fibroblast. The Journal of Biological Chemistry, 279, 31524-31532. doi:10.1074/jbc.M400365200

[3]   Campisi, J. (2000) Cancer, aging and cellular senescence. In vitro, 14, 183-188.

[4]   Park, W.Y., Park, J.S., Cho, K.A., Kim, D.I., Ko, Y.G., Seo, J.S. and Park, S.C. (2000) Upregulation of caveolin attenuates epidermal growth factor signaling in senescent cells. Journal of Biological Chemistry, 275, 20847-20852. doi:10.1074/jbc.M908162199

[5]   Dimri, G.P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens, M., Rubelj, I., Pereira-Smith, O., Peacocke, M. and Campisi, J. (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proceedings of the National Academy of Sciences of the United States of America, 92, 9363-9367. doi:10.1073/pnas.92.20.9363

[6]   Wagner, M., Hampel, B., Bernhard, D., Hala, M., Zwerschke, W. and Jansen-Durr, P. (2001) Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploidization and senescence-associated apoptosis. Experimental Gerontology, 36, 1327-1347. doi:10.1016/S0531-5565(01)00105-X

[7]   Cho, K.A., Ryu, S.J., Oh, Y.S., Park, J.H., Lee, J.W., Hwang-Phill, K., Kim, K.T., Jang, I.S. and Park, S.C. (2004) Morphological Adjustment of Senescent Cells by Modulating Caveolin-1 Status. The Journal of Biological Chemistry, 279, 42270-42278. doi:10.1074/jbc.M402352200

[8]   Cristofalo, V.J. (1988) Cellular biomarkers of aging. Experimental Gerontology, 23, 297-305. doi:10.1016/0531-5565(88)90032-0

[9]   Alcorta, D.A., Xiong, Y., Phelps, D., Hannon, G., Beach, D. and Barrett, J.C. (1996) Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts. Proceedings of the National Academy of Sciences of the United States of America, 93, 13742-13747. doi:10.1073/pnas.93.24.13742

[10]   Hara, E., Smith, R., Parry, D., Tahara, H., Stone, S. and Peters, G. (1996) Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence. Molecular Cell Biology, 16, 859-867.

[11]   Noda, A., Ning, Y., Venable, S.F., Pereira-Smith, O.M. and Smith, J.R. (1994) Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Experimental Cell Research, 211, 90-98. doi:10.1006/excr.1994.1063

[12]   Stein, G.H., Drullinger, L.F., Soulard, A. and Dulic, V. (1999) Differential roles for cyclin-dependent kinase inhibitors p21 and p16 in the mechanisms of senescence and differentiation in human fibroblasts. Molecular and Cellular Biology, 19, 2109-2117.

[13]   Wong, H., & Riabowol, K. (1996) Differential CDK inhibitor gene expression in aging human diploid fibroblasts. Experimental Gerontology, 31, 311-325. doi:10.1016/0531-5565(95)00025-9

[14]   Berthet, C., Klarmann, K.D., Hilton, M.B., Suh, H.C., Keller, J.R., Kiyokawa, H., Kaldis, P. (2006) Combined loss of Cdk2 and Cdk4 results in embryonic lethality and Rb hypophosphorylation. Developmental Cell, 10, 563- 573. doi:10.1016/j.devcel.2006.03.004

[15]   Malumbres, M. and Barbacid, M. (2007) Cell cycle kinases in cancer. Current Opinion in Genetic Development, 17, 60-65. doi:10.1016/j.gde.2006.12.008

[16]   Gil, J. and Peters, G. (2006) Regulation of the INK4b- ARF-INK4a tumour suppressor locus: All for one or one for all. National Review of Molecular and Cellular Biology, 7, 667-677. doi:10.1038/nrm1987

[17]   Campisi, J. (2001) Cellular senescence as a tumor-suppressor mechanism. Trends in Cell Biology, 11, S27-S31.

[18]   Ohtani, N., Yamakoshi, K., Takahashi, A. and Hara, E. (2004) The p16INK4a-RB pathway: Molecular link between cellular senescence and tumor suppression. The Journal of Medical Investigation, 51, 146-153. doi:10.2152/jmi.51.146

[19]   Campisi, J., Dimiri, G. and Hara, E. (1996) Control of replicative senescence. In: Schneider, E.L., Rowe, J.W., Johnson, T.G., Holbrook, N.J. and Morrison, J.H., Eds., Handbook of the Biology of Aging, Academic Press, New York.

[20]   Brenner, A.J., Stampfer, M.R. and Aldaz, C.M. (1998) Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation. Oncogene, 17, 199-205. doi:10.1038/sj.onc.1201919

[21]   Jarrad, D.F., Sarkar, S., Shi, Y., Yeager, T.R., Magrane, G., Kinoshita, H., Nassif, N., Meisner, L., Newton, M.A., Waldman, F.M. and Reznikoff, C.A. (1999) p16/pRb pathway alterations are required for bypassing senescence in human prostate epithelial cells. Cancer Research, 59, 2957-2964.

[22]   You, Y.O., Lee, G. and Min, B.M. (2000) Retinoic acid extends the in vitro life span of normal human oral keratinocytes by decreasing p16 (INK4A) expression and maintaining telomerase activity. Biochemical and Biophysical Research Communications, 268, 268-274. doi:10.1006/bbrc.2000.2101

[23]   Sandhu, C., Peehl, D.M. and Slingerland, J. (2000) p16INK4A mediates cyclin dependent kinase 4 and 6 inhibition in senescent prostatic epithelial cells. Cancer Research, 60, 2616-2622.

[24]   Zindy, F., Quelle, D.E., Roussel, M.F. and Sherr, C.J. (1997) Expression of the p16INK4a tumor suppressor versus other INK4 family members during mouse development and aging. Oncogene, 15, 203-211. doi:10.1038/sj.onc.1201178

[25]   Gao, C.Y. and Zelenka, P.S. (1997). Cyclins, cyclindependent kinases and differentiation. BioEssays, 19, 307. doi:10.1002/bies.950190408

[26]   Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E. and Mello, C.C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenor-habditis elegans. Nature, 391, 806-811. doi:10.1038/35888

[27]   Lee, S.H. and Sinko, P.J. (2006) SiRNA-Getting the message out. European Journal of Pharmaceutical Sciences, 27, 401-410. doi:10.1016/j.ejps.2005.12.002

[28]   Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494-498. doi:10.1038/35078107

[29]   Rozema, D.B. and Lewis, D.L. (2003) SiRNA delivery technologies for mammalian systems. Targets, 2, 253-260 doi:10.1016/S1477-3627(03)02381-X

[30]   Touchberry, C.D., Wacker, M.J., Richmond, S.R., Whitman, S.A. and Godard, M.P. (2006) Age-Related Changes in Relative Expression of Real-Time PCR Housekeeping Genes in Human Skeletal Muscle. Journal of Biomolecular Techniques, 17, 157-162.

[31]   Corbin, I.R., Gong, Y., Zhang, M. and Minuk, G.Y. (2002) Proliferative and nutritional dependent regulation of glycer-aldehyde-3-phosphate dehydrogenase expression in the rat liver. Cell Proliferation, 35, 173-182. doi:10.1046/j.1365-2184.2002.00236.x

[32]   Chua, K.H., Aminuddin, B.S., Fuzina, N.H. and Ruszymah, B.H.I. (2005) Insulin-transferrin-selenium prevent human chondrocyte dedifferentiation and promote the formation of high quality tissue engineered human hyaline cartilage. European Cells and Materials, 9, 58-67.

[33]   Zainuddin, A., Chua, K.H., Abdul Rahim, N. and Makpol, S. (2010) Effects of experimental treatment on GAPDH mRNA expression as a housekeeping gene in human diploid fibroblasts. BMC Molecular Biology, 11, 59. doi:10.1186/1471-2199-11-59

[34]   Carthagena, L., Bergamaschi, A., Luna, J.M., David, A., Uchil, P.D., Margottin-Goguet, F., Mothes, W., Hazan, U., Transy, C., Pancino, G. and Nisole, S. (2009) Human TRIM gene expression in response to interferons. PloS One, 4, e4894. doi:10.1371/journal.pone.0004894

[35]   Thellin, O., Zorzi, W., Lakaye, B., De Borman, B., Coumans, B., Hennen, G., Grisar, T., Igout, A. and Heinen, E. (1999) Housekeeping genes as internal standards: Use and limits. Journal of Biotechnology, 75, 291-295. doi:10.1016/S0168-1656(99)00163-7

[36]   Chad, D.T., Michael, J.W., Scott, R.R., Samantha, A.W. and Michael, P.G. (2006) Age-related changes in relative expression of real time RT PCR housekeeping genes in human skeletal muscle. Journal of Biomolecular Techniques, 17, 157-162.

[37]   Sharpless, N.E. (2003) The persistence of senescence. Science of Aging Knowledge Environment, PE24. doi:10.1126/sageke.2003.33.pe24

[38]   Chen, Q.M., Tu, V.C., Catania, J., Burton, M., Toussaint, O. and Dilley, T. (2000) Involvement of Rb family proteins, focal adhesion proteins and protein synthesis in senescent morphogenesis induced by hydrogen peroxide. Journal of Cell Science, 113, 4087-4097.

[39]   Sellers, W.R., Novitch, B.G., Miyake, S., Heith, A., Ot- terson, G.A., Kaye, F.J., Lassar, A.B. and Kaelin, W.G. (1998) Stable binding to E2F is not required for the retinoblastoma protein to activate transcription, promote differentiation and suppress tumor cell growth. Genes & Development, 12, 95-106. doi:10.1101/gad.12.1.95

[40]   Makpol, S., Zainuddin, A., Chua, K.H., Yusof, Y.A., Wan, N.W.Z. (2012) Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts. Clinics, 67, 135-143. doi:10.6061/clinics/2012(02)08

[41]   Kato, D., Miyazawa, K., Starborg, M., Wada, I., Oka, T., Sakai, T., Peters, G. and Hara, E. (1998) Features of replicative senescence induced by direct addition of antennapedia-p16INK4A fusion protein to human diploid fibroblasts. FEBS Letters, 427, 203-208. doi:10.1016/S0014-5793(98)00426-8