Ribozyme-mediated downregulation of human metallothionein II(a) induces apoptosis in human prostate and ovarian cancer cell lines.
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.
Tekur
Seshadri
S
Division of Urology, Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Ho
Shuk-Mei
SM
D017209:Apoptosis; D001483:Base Sequence; D002455:Cell Division / Q000502:physiology; D017931:DNA Primers / Q000737:chemistry; D015536:Down-Regulation; D005260:Female; D005434:Flow Cytometry; D016259:Genes, myc / Q000502:physiology; D006801:Humans; D008297:Male; D008668:Metallothionein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D016337:RNA, Catalytic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects
D017931:DNA Primers; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D019253:Proto-Oncogene Proteins c-bcl-2; D016337:RNA, Catalytic; D012333:RNA, Messenger; D008668:Metallothionein