Flowchart: Preparation: hTert


Text Box: siRNA





Text Box: hTertg 

Cervical cancer

Life span                                         

Text Box: Trail


Text Box: p53



Text Box: p16(INK4a)


Text Box: RB



Int J Oncol. 2007 Aug;31(2):361-8. Links

Lentiviral vector mediated siRNA knock-down of hTERT results in diminished capacity in invasiveness and in vivo growth of human glioma cells in a telomere length-independent manner.

Zhao P, Wang C, Fu Z, You Y, Cheng Y, Lu X, Lu A, Liu N, Pu P, Kang C, Salford LG, Fan X.

Department of Neurosurgery, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, P.R. China.

Glioma cells are characterized by their invasiveness and resistance against conventional therapeutics. Telomerase activity has been suggested to be an important target for glioma treatment. Here we assessed the anticancer effects and its potential mechanisms of lentiviral vector mediated siRNA knock-down of the human telomerase reverse transcriptase (hTERT) in U87MG human glioblastoma cells. Stable expression of anti-hTERT siRNA reduced the hTERT expression and TRAP assay telomerase activity to barely detectable levels. Injection of lentiviral vectors encoding anti-hTERT siRNA significantly inhibited the growth of pre-established macroscopic xenograft tumors, which was in contrast to the finding that no obvious effects on cell growth, cell cycle progression and telomere length were observed in anti-hTERT siRNA expressing U87MG cells during short-term in vitro cultures. The in vivo glioma growth inhibition effect was already evident in the period coincided with no detectable telomere length changes, suggesting that hTERT inhibition may hinder glioma cell growth in a telomere length-independent manner. Importantly, transwell migration assay showed profound inhibitory effect on the invasive capacity of U87MG cells following short-term anti-hTERT siRNA expression. Thus, efficient knock-down of hTERT can inhibit glioma cell proliferation and migration prior to its effect on telomere length.

PMID: 17611693 [PubMed - in process]

Mol Biol Cell. 2005 Mar;16(3):1491-9. Epub 2005 Jan 12.Click here to read Click here to readLinks

Immortalization of human fetal cells: the life span of umbilical cord blood-derived cells can be prolonged without manipulating p16INK4a/RB braking pathway.

Terai M, Uyama T, Sugiki T, Li XK, Umezawa A, Kiyono T.

Department of Reproductive Biology and Pathology, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan.

Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are expected to serve as an excellent alternative to bone marrow-derived human mesenchymal stem cells. However, it is difficult to study them because of their limited life span. To overcome this problem, we attempted to produce a strain of UCBMSCs with a long life span and to investigate whether the strain could maintain phenotypes in vitro. UCBMSCs were infected with retrovirus carrying the human telomerase reverse transcriptase (hTERT) to prolong their life span. The UCBMSCs underwent 30 population doublings (PDs) and stopped dividing at PD 37. The UCBMSCs newly established with hTERT (UCBTERTs) proliferated for >120 PDs. The p16INK4a/RB braking pathway leading to senescence can be inhibited by introduction of Bmi-1, a polycomb-group gene, and human papillomavirus type 16 E7, but the extension of the life span of the UCBMSCs with hTERT did not require inhibition of the p16INK4a/RB pathway. The characteristics of the UCBTERTs remained unchanged during the prolongation of life span. UCBTERTs provide a powerful model for further study of cellular senescence and for future application to cell-based therapy by using umbilical cord blood cells.

PMID: 15647378 [PubMed - indexed for MEDLINE]

Mol Cell Biol. 2002 Jul;22(14):5157-72.Click here to read Click here to readLinks

A two-stage, p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status.

Rheinwald JG, Hahn WC, Ramsey MR, Wu JY, Guo Z, Tsao H, De Luca M, Catricalà C, O'Toole KM.

Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. JRheinwald@rics.bwh.harvard.edu

With increasing frequency during serial passage in culture, primary human keratinocytes express p16(INK4A) (p16) and undergo senescence arrest. Keratinocytes engineered to express hTERT maintain long telomeres but typically are not immortalized unless, by mutation or other heritable event, they avoid or greatly reduce p16 expression. We have confirmed that keratinocytes undergo p16-related senescence during growth in culture, whether in the fibroblast feeder cell system or in the specialized K-sfm medium formulation, and that this mechanism can act as a barrier to immortalization following hTERT expression. We have characterized the p16-related arrest mechanism more precisely by interfering specifically with several regulators of cell cycle control. Epidermal, oral mucosal, corneal limbal, and conjunctival keratinocytes were transduced to express a p16-insensitive mutant cdk4 (cdk4(R24C)), to abolish p16 control, and/or a dominant negative mutant p53 (p53DD), to abolish p53 function. Expression of either cdk4(R24C) or p53DD alone had little effect on life span, but expression of both permitted cells to divide 25 to 43 population doublings (PD) beyond their normal limit. Keratinocytes from a p16(+/-) individual transduced to express p53DD alone displayed a 31-PD life span extension associated with selective growth of variants that had lost the wild-type p16 allele. Cells in which both p53 and p16 were nonfunctional divided rapidly during their extended life span but experienced telomere erosion and ultimately ceased growth with very short telomeres. Expression of hTERT in these cells immortalized them. Keratinocytes engineered to express cdk4(R24C) and hTERT but not p53DD did not exhibit an extended life span. Rare immortal variants exhibiting p53 pathway defects arose from them, however, indicating that the p53-dependent component of keratinocyte senescence is telomere independent. Mutational loss of p16 and p53 has been found to be a frequent early event in the development of squamous cell carcinoma. Our results suggest that such mutations endow keratinocytes with extended replicative potential which may serve to increase the probability of neoplastic progression.

PMID: 12077343 [PubMed - indexed for MEDLINE

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2007 Jul;23(7):638-40. Links

[hTERT promoter regulated tumor targeting TRALL in cervical cancer cell.]

[Article in Chinese]

Zhang M, Xin XY, Li HM, Song H.

Department of Obstetrics and Gynecology, Xijing Hospital, Xi'an 710032, China.

AIM: To investigate the effect of hTERT promoter which regulated tumor targeting TRALL on the cervical cancer cell line HeLa. METHODS: The mRNA expression of TRAIL on HeLa was examined by RT-PCR. The proliferation, apoptosis, motion of the transfected cervical cancer cell were detected by MTT, flow cytometry, and cell invasion assay respectively. The ultrastructure was observed by electron microscope. RESULTS: Compared with the control group, the expression of TRAILmRNA was significantly upregulated in hTERT-TRAIL (P<0.05). The apotosis rate and the inhibitory rate of cell growth of hTERT-TRAIL was significantly high (P<0.05). Electron microscope results indicated that hTERT promoter regulated tumor targeting TRALL facilitated the apoptosis of HeLa. CONCLUSION: The hTERT-TRAIL significantly inhibits the malignant proliferation and invasion ability of the cervical cancer cell line, and facilitates the apoptosisof HeLa, which lays a foundation for the treatment of patients with cervical cancer.

PMID: 17618588 [PubMed - in process]