Download FACULTY SPONSOR`S NAME AND DEGREE:

Summer Student Research Program
Project Description
FACULTY SPONSOR’S NAME AND DEGREE: Harvey L. Ozer, M.D. and Satnam S. Banga, Ph.D.
PHONE: (973) 972 - 8953
DEPARTMENT AND INTERNAL MAILING ADDRESS: Department of Microbiology and Molecular
Genetics
E-MAIL:[email protected] / [email protected]
PROJECT TITLE (200 Characters max):
Analysis of Candidate Genes Involved in SV40-transformed Human Cells
HYPOTHESIS:
Inactivation of the candidate gene (SEN6) in the 6q27 region leads to immortalization of SV40-transformed
human cells
PROJECT DESCRIPTION (Include design, methodology, data collection, techniques, data analysis to
be employed and evaluation and interpretation methodology)
The finite division potential of normal human fibroblasts (HF) in culture provides an in vitro model
system for studying the mechanism of cellular aging, senescence, and immortalization.
Normal mammalian cells can be propagated in cell culture for only a limited time, eventually ceasing
to proliferate ("senescence"). This phenomenon is a model for the cellular basis of human aging. On the
other hand, cancer cells grow continuously in culture (and in the animal) and have overcome senescence;
that is, they are "immortal". Hence replicative senescence is a mechanism of protection against cancer. We
have been studying human diploid fibroblasts (HF) to understand the mechanism of multi-step
carcinogenesis ("transformation") of such cells in culture and its effect on cellular aging. We have found that
introduction of genes from the DNA tumor virus SV40 allows us to identify two key steps in this process.
The SV40-encoded T antigens induce several changes in growth properties in HF but additional changes in
cellular gene(s) are required for immortalization. We have isolated a matched series of clonally derived
SV40-transformed HF with pre-immortal and immortal growth phenotypes, permitting the direct test of
hypotheses concerning biochemical and genetic bases for immortalization of human cells. We have also
mapped a specific chromosome rearrangement on chromosome 6 in different cell lines which is directly
associated with immortalization. We are currently examining candidate genes (designated SEN6) based on
their known location at 6q27. To identify the candidate SEN6 gene, we will be using RNAi (RNA
interference) approach to inactivate each of the candidate genes in pre-immortal cells. Inactivation of the
candidate gene(s) by RNAi may increase the frequency of immortal clones as compared to untreated preimmortal cells. We will also monitor the expression of candidate genes in RNAi treated and untreated
control cultures using RT-PCR or Western blotting. We will also be determining the growth suppressive
activities of candidate genes in our immortal cell lines. To do that, we will express each of the candidate
genes under the control of regulated promoter such as Tetracycline in SV40-transformed immortal cells.
Induction of candidate gene(s) by Tetracycline may result in the suppression of growth and thus may lead to
reduced colony formation as compared to immortal cells stably transfected with an empty vector. Overexpression of candidate genes will be monitored by RT-PCR or western blotting. Both RNAi and growth
suppression studies may identify SEN6 gene whose inactivation leads to immortalization SV 40-transformed
human fibroblasts.
SPONSOR’S MOST RECENT PUBLICATIONS RELEVANT TO THIS RESEARCH:
1. Harvey, B.P., Banga, S.S. and H.L. Ozer (2004) Regulation of Multifunctional Ca2+/Calmodulin-dependent Protein
Kinase II by the PP2C Phosphatase PPM1F in Fibroblasts. J. Biological Chemistry, 279:24889-24898.
2. Benanti, J.A., Williams D., Robinson, K.L., Ozer, H.L. and D.A. Galloway (2002) Induction of extracellular matrixremodeling genes by the senescence associated protein APA-1. Mol. Cell. Biol. 22:7385-7397.
Summer Student Research Program
Project Description
3. Macera- Bloch, L., Houghton, J., Lenahan, M., Jha, K.K. and H.L. Ozer. (2001) Termination of lifespan of SV40transformed human fibroblasts in crisis is due to apoptosis. J. Cellular Physiology 190:332-344.
4. Jha, K.K., Banga, S., Palejwala, V. and Ozer, H.L. (1998) SV40-mediated immortalization. Exp. Cell Res. 245:1-7.
5. Banga, S.S., Kim, S., Hubbard, K., Dasgupta, T., Jha, K.K., Patsalis, P., Hauptschein, R., Gamberi, B., DallaFavera, R., Kraemer, P. and H.L. Ozer (1997) SEN6, a locus for SV40-mediated immortalization of human cells,
maps to 6q26-27. Oncogene, 14:313-321.
IS THIS PROJECT SUPPORTED BY EXTRAMURAL FUNDS?
Yes
or
No
(IF YES, PLEASE SUPPLY THE GRANTING AGENCY’S NAME)
AG04821
THIS PROJECT IS:
Clinical
Laboratory
Behavioral
Other
THIS PROJECT IS CANCER-RELATED
THIS PROJECT IS HEART, LUNG & BLOOD- RELATED
THIS PROJECT EMPLOYS RADIOISOTOPES
THIS PROJECT INVOLVES THE USE OF ANIMALS
PENDING
APPROVED
IACUC PROTOCOL #
THIS PROJECT INVOLVES THE USE OF HUMAN SUBJECTS
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IRB PROTOCOL # M
THIS PROJECT IS SUITABLE FOR:
UNDERGRADUATE STUDENTS
ENTERING FRESHMAN
SOPHMORES
ALL STUDENTS
THIS PROJECT IS WORK-STUDY: Yes
or
No
WHAT WILL THE STUDENT LEARN FROM THIS EXPERIENCE?
Tissue culture, molecular biology (RNA isolation, RT-PCR, protein isolation, Western blotting,
immunofluorescence)