Rana Dutta

Date of Graduation

2002

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Biology

Committee Chair

Donald A. Sens

Committee Co-Chair

Joginder Nath

Committee Member

John G. Killefer

Committee Member

Scott H. Garrett

Committee Member

Mary Ann Sens

Abstract

Since the expression of the brain specific metallothionein isoform, MT-3, was shown to be expressed in many, but not all prostate cancers, this gene was transfected into the commonly utilized prostate tumor cell line, PC-3, to study the consequence of MT-3 expression on growth rate and resistance to chemotherapeutic agents. PC-3 cells were stably transfected with MT-3 or MT-1E under control of the constitutive CMV promoter. The levels of MT-3 protein for the 5 overexpressing clones had an average level of 8.3 ng MT-3 per μg protein as assessed by quantitative immunodot blot analysis, and three were picked for further analysis. MT-3 expressing clones had a slower growth rate (63 hr doubling time) compared to vector-only transfected cells (35 hr doubling time). The MT-1E overexpressing clones had total MT-1/2 levels of 5.54 ± 0.85 ng/μg of protein whereas clones from vector-only transfection had 0.90 ± 0.05 ng/μg protein. The mean doubling time of the three independent clones of PC-3 cells overexpressing MT-1E was 39.2 hr compared to 36.7 hr for control PC-3 cells, and 39.9 hr for blank vector transfectants. The effect of these two isoforms of MT on cellular resistance to commonly utilized chemotherapeutic agents was determined on MT-3 and MT-1E expressing and non-expressing clones of PC-3 cells exposed to cadmium, cisplatin, vinblastine, paclitaxel, etoposide, and mitoxantrone. Both MT-3 and MT-1E expression in PC-3 cells conferred resistance to all five chemotherapeutic agents, with a 10-fold increase in resistance to paclitaxel and mitoxantrone for the MT-1E transfected clones compared MT-3 transfected clones.

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