Immortalization of human cells

Figure 26.1 Immortalization of human cells Cells enter replicative senescence at mortality stage 1 (Ml Hayflick limit) after about 60 population doublings (PD). The protein p 16 accumulates in senescent cells. The simian virus 40 (SV40) large T antigen as well as the human papilloma virus (HPV) type 16-E6 and E7 proteins sequester the retinoblastoma protein (Rb) and/or p53 constitutively releases the transcription factor E2F. E2F induces expression proteins required for progression through Gl/S transition, thus the cells escape cell cycle arrest. At mortality stage 2 (M2), transformed cells must overcome senescence and crisis before they are immortalized. This is likely to involve the activation of telomerase either by the introduction of hTERT cDNA or by a genetic change that activates telomerase.

Bryan, T. M., and R. R. Reddel. 1994. SV40-induced immortalization of human cells. CritRev Oncog 5(4) 331—57. 

X, Gandia K, Vaughan MB, Wright WE, Gazdar AF, Shay JW, Minna JD (2004) Immortalization of human bronchial epithelial cells in the absence of viral oncoproteins. Cancer Res 64(24) 9027-9034. 

Bednarz J, Teifel M, Friedl P, Engelmann K. Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells. Acta Ophthalmol Scand 78 130-136 (2000). 

Takahashi, K., Y. Sawasaki, J. Hata, K. Mukai, and T. Goto. 1990. Spontaneous transformation and immortalization of human endothelial cells. In Vitro Cell Dev Biol 26(3 Pt 1) 265-74. 

Rice, R. H., K. E. Steinmann, L. A. deGraffenried, Q. Qin, N. Taylor, and R. Schlegel. 1993. Elevation of cell cycle control proteins during spontaneous immortalization of human keratinocytes. Mol Biol Cell 4(2) 185-94. 

Wynford-Thomas, D., J. A. Bond, F. S. Wyllie, J. S. Burns, E. D. Williams, T. Jones, D. Sheer, and N. R. Lemoine. 1990. Conditional immortalization of human thyroid epithelial cells A tool for analysis of oncogene action. Mol Cell Biol 10(10) 5365-77. 

Peterson, S. R., D. M. Gadbois, E. M. Bradbury, and P. M. Kraemer. 1995. Immortalization of human fibroblasts by SV40 large T antigen results in the reduction of cyclin D1 expression and subunit association with proliferating cell nuclear antigen and Wafl. Cancer Res 55(20) 4651-7. 

Immortalization of human hepatocytes would help to overcome the hmited availability of human cells, thereby avoiding the use of mahgnant-derived cell hnes however, care will still be required with these cells. A better approach would be to develop methods for the culture of primary human hepatocytes using hormonally defined media containing growth factors in which cells are stimulated to undergo division. 

PER.C6 cells have been used extensively for the production of gene therapy vectors. This cell line was derived from the immortalization of human embryonic retina cells through the use of adenovirus El gene (Fallaux et al., 1998). This cell line has been well characterized since its establishment, and no retroviruses or adventitious viruses have been detected in it. This cell line is easily adapted to different growth conditions and stably produces high levels of recombinant proteins. 

Primary cultures of cerebromicrovascular endothelial cells (CEC) derived from rat, bovine, porcine, mouse and human can rapidly lose key phenotypic markers of the blood-brain barrier (BBB) and undergo cellular senescence after a limited number of divisions in vitro. Furthermore, expression of BBB markers varies considerably among BCEC obtained from different species. These issues, compounded with the problems associated with very limited availability of human brain biopsies, small initial yield of cells and short proliferative life span of human cells, greatly restrict the utility of primary human BCEC as a reliable in vitro BBB model. Therefore, Muruganandam et al. (1997) developed an immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier. 

KowolikCM, Liang S, Yu Y, and Yee JK. Cre-mediated reversible immortalization of human renal proximal tubular epithelial cells. 

Immortal or Extended Life Span of Human Cells. More than 30 years ago, it was demonstrated for the hrst time that SV40 [17], a DNA tumor virus of the papova... 

Meinl E, Hohlfeld R, Wekerle H, Fleckenstein B (1995) Immortalization of human T cells by herpesvirus saimiri. Immunol Today 16 55-58... 

Few immortalized cell lines derived from human renal epithelial cells are available, particularly from normal kidney. Probably the best-known and most commonly used cell line derived from normal human kidney is the HK-2 cell, which was developed in 1994 (Ryan et al., 1994). In discussing this model elsewhere (Lash et al., 2014b), we noted that despite validation of several PT functions, immortalization of the cells by viral transduction undoubtedly will cause changes in cellular function, particularly related to processes such as cell proliferation and stress response. Additionally, a recent study (Huang et al., 2015) compared the sensitivity... 

Kemp, S. J. Thorley, A. J. Gorelik, J. Seckl, M. J. O Hare, M. J. Arcaro, A. Korchev, Y. Goldstraw, R Tetley, T. D., Immortalization of human alveolar epithelial cells to investigate nanoparticle uptake. American Journal of Respiratory Cell and Molecular Biology 2008, 39, 591-597. 

Shammas M.A., Simmons C.G., Corey D.R., Reis R.J.S. Telomerase inhibition by peptide nucleic acids reverses immortality of transformed human cells. Oncogene 1999 18 6191-6200... 

Herbert B. S., Pitts A. E., Baker S. I., Hamilton S. E., Wright W. E., Shay J.W., Corey D.R. Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death. Proc. Natl Acad. Sci. USA 1999 96 14276-14281. 

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