Document Detail


Ribozyme-mediated downregulation of human metallothionein II(a) induces apoptosis in human prostate and ovarian cancer cell lines.
MedLine Citation:
PMID:  11807957     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Human metallothioneins (MTs) are low-molecular-weight, cysteine-rich, metal ion-binding proteins that constitute the majority of intracellular protein thiols. They are overexpressed in prostate and ovarian cancers and are believed to confer resistance to radiation and cytotoxic anticancer drugs. The aim of this study was to investigate the roles of MTs in prostate and ovarian cancer cells and their possible relationship with other cancer development and progression factors. The main problem in investigating the role of MT, however, is the absence of any known specific inhibitor. To this end, in a previous study, we had developed sequence-specific ribozymes (Rzs) targeting MT and had shown their in cellulo efficacy. Here we show that transient transfection of a vector carrying a hammerhead Rz (Rz4-9), designed to cleave class II MT, in the human prostate cancer cell line PC-3 and the ovarian cancer cell line SKOV-3 resulted in a dose-dependent attenuation of MT-II(a) transcripts and dramatic cell loss. Transient transfection with 2 microg of Rz4-9 vector DNA completely abolished MT-II(a) mRNA levels and induced a 94% and a 67% reduction in cell number in PC-3 cells and SKOV-3 cells, respectively. Fluorescence-activated cell sorting (FACS) showed that the Rz-induced cell loss probably was due to apoptosis, because it was associated with marked increases in the hypodiploid cell population, reaching maximums of 52% and 64% in cultures of PC-3 and SKOV-3, respectively. Additionally, annexin V-propidium iodide double-staining, followed by FACS, confirmed that Rz4-9-induced cell death was due to apoptosis and showed a vector DNA-dependent increase in late apoptotic cell numbers that reached maximums of 80% and 42%, respectively, in PC-3 and SKOV-3 cell cultures transfected with the highest concentration of vector DNA. In parallel experiments, transfection with a vector containing the enzymatically inactive mutant Rz-3-3 or the empty vector was not effective in inducing similar responses. The Rz-induced loss of MT-II(a) mRNA-associated cell death in these cancer cell lines was attended by dose-dependent downregulation of the proto-oncogene c-myc and the apoptosis inhibitory mediator bcl-2, suggesting that these signaling pathways are involved in the process. In conclusion, our data indicate that MT-II(a) is an important cell-survival or anti-apoptotic factor for prostate and ovarian cancer cells and that downregulation of its expression via transgene expression of a sequence-specific Rz is a feasible target for cancer therapy.
Authors:
Seshadri Tekur; Shuk-Mei Ho
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Molecular carcinogenesis     Volume:  33     ISSN:  0899-1987     ISO Abbreviation:  Mol. Carcinog.     Publication Date:  2002 Jan 
Date Detail:
Created Date:  2002-01-24     Completed Date:  2002-02-07     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  8811105     Medline TA:  Mol Carcinog     Country:  United States    
Other Details:
Languages:  eng     Pagination:  44-55     Citation Subset:  IM    
Copyright Information:
Copyright 2002 Wiley-Liss, Inc.
Affiliation:
Division of Urology, Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
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MeSH Terms
Descriptor/Qualifier:
Apoptosis*
Base Sequence
Cell Division / physiology
DNA Primers / chemistry
Down-Regulation
Female
Flow Cytometry
Genes, myc / physiology
Humans
Male
Metallothionein / antagonists & inhibitors,  genetics,  metabolism*
Ovarian Neoplasms / metabolism*
Prostatic Neoplasms / metabolism*
Proto-Oncogene Proteins c-bcl-2 / metabolism
RNA, Catalytic / pharmacology*
RNA, Messenger / metabolism
Reverse Transcriptase Polymerase Chain Reaction
Transfection
Tumor Cells, Cultured / drug effects
Grant Support
ID/Acronym/Agency:
CA15776/CA/NCI NIH HHS; CA62269/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/DNA Primers; 0/Proto-Oncogene Proteins c-bcl-2; 0/RNA, Catalytic; 0/RNA, Messenger; 9038-94-2/Metallothionein

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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