Abstract

The purpose of this study was to learn more about the role of the retinoblastoma tumor suppressor protein (pRb) in cell proliferation, differentiation and senescence. To do so, a mammalian expression vector containing the cDNA of a temperature-sensitive (ts) mutant of pRb was prepared and transfected together with the puromycin resistance gene into mouse 3T3 pRb (-/-) fibroblasts. After several weeks of selection in puromycin-containing cell culture medium, eighteen independent 3T3 clones were isolated and expanded into the cell lines 3T3 ts pRb 1-18. These cell lines were then analyzed for expression of the ts pRb protein using immunofluorescence and Western Blot techniques. Cells were grown on coverslips and subsequently fixed, permeabilized and subjected to immunofluorescence staining for pRb with a monoclonal anti-pRb antibody and a rhodamine-conjugated secondary antibody. Three of the 3T3 cell lines were determined to be positive for ts pRb expression in this assay. In order to confirm this result, whole-cell protein extracts of all cell lines were prepared. Proteins in these extracts were separated by SDS-PAGE and the presence of pRb was determined by Western Blotting.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Unknown (K16)
Project #
5K16DE000275-11
Application #
6474599
Study Section
Project Start
2001-07-01
Project End
2002-06-30
Budget Start
Budget End
Support Year
11
Fiscal Year
2001
Total Cost
$208,185
Indirect Cost

  Institution

Name
Harvard University
Department
Type
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115

  Publications

McDermott, Nancy E; Chuang, Sung-Kiang; Woo, Valerie V et al. (2006) Maxillary sinus augmentation as a risk factor for implant failure. Int J Oral Maxillofac Implants 21:366-74
Chuang, S K; Cai, T; Douglass, C W et al. (2005) Frailty approach for the analysis of clustered failure time observations in dental research. J Dent Res 84:54-8
Chuang, S-K; Hatch, J P; Rugh, J et al. (2005) Multi-center randomized clinical trials in oral and maxillofacial surgery: modeling of fixed and random effects. Int J Oral Maxillofac Surg 34:341-4
Treister, Nathaniel S; Woo, Sook-Bin; O'Holleran, Eileen W et al. (2005) Oral chronic graft-versus-host disease in pediatric patients after hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 11:721-31
Woo, Valerie V; Chuang, Sung-Kiang; Daher, Shadi et al. (2004) Dentoalveolar reconstructive procedures as a risk factor for implant failure. J Oral Maxillofac Surg 62:773-80
Basile, John R; Eichten, Alexandra; Zacny, Valerie et al. (2003) NF-kappaB-mediated induction of p21(Cip1/Waf1) by tumor necrosis factor alpha induces growth arrest and cytoprotection in normal human keratinocytes. Mol Cancer Res 1:262-70
McDermott, Nancy E; Chuang, Sung-Kiang; Woo, Valerie V et al. (2003) Complications of dental implants: identification, frequency, and associated risk factors. Int J Oral Maxillofac Implants 18:848-55
Halpern, Leslie R; Carter, Jeffrey B; Chuang, Sung-Kiang et al. (2003) A comparison of 2 consultation and treatment strategies to manage impacted third molars. J Oral Maxillofac Surg 61:779-84
Chuang, S K; Tian, L; Wei, L J et al. (2002) Predicting dental implant survival by use of the marginal approach of the semi-parametric survival methods for clustered observations. J Dent Res 81:851-5
Chuang, S K; Wei, L J; Douglass, C W et al. (2002) Risk factors for dental implant failure: a strategy for the analysis of clustered failure-time observations. J Dent Res 81:572-7

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