Modulation of platelet-derived growth factor-beta mRNA expression and cell growth in a human mesothelioma cell line by a hammerhead ribozyme.
Malignant mesothelioma in humans is a rare disease, but it has recently received much public attention and concern because of its strong relationship to exposure to asbestos. We have found overexpression of the gene for platelet-derived growth factor (PDGF)-beta in several mesothelioma xenografts in nude mice. Because some mesothelioma cell lines such as VAMT-1 overexpress PDGF-beta and PDGF-beta receptors, it was considered that an autocrine loop involving PDGF-beta and its receptor may contribute to the malignant phenotype of these cells. To investigate this possibility we have developed a hammerhead ribozyme against PDGF-beta mRNA. This c-sis ribozyme was able to cleave an artificial PDGF-beta RNA substrate in a cell-free system. Transduction of this ribozyme, with the aid of a constitutive expression vector, in the VAMT-1 cell line led to a decrease in the PDGF-beta mRNA level. The ribozyme expressed in these cells was functional in cleaving the artificial RNA substrate in vitro. Ribonuclease protection assays using the ribozyme and whole PDGF-beta mRNA showed that this ribozyme was capable of cleaving the whole mRNA in vivo. Transfectant clones containing the wild-type ribozyme showed decreased cell growth, in parallel with the decreases in PDGF-beta expression. The disabled ribozyme was inactive in the cleavage reaction in vitro and in decreasing the cell growth rate in vivo. Our data indicate that in some mesothelioma cells the PDGF-beta autocrine loop may be functional and transduction of the PDGF-beta ribozyme leads to a significant reduction of cell growth. The c-sis ribozyme may be applicable in the treatment of patients with malignant mesothelioma.
Dorai
T
T
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029.
Kobayashi
H
H
Holland
J F
JF
Ohnuma
T
T
D000818:Animals; D001194:Asbestos / Q000009:adverse effects; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D015870:Gene Expression / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008654:Mesothelioma / Q000139:chemically induced / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis*; D017479:Receptors, Platelet-Derived Growth Factor / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D014407:Tumor Cells, Cultured
D010982:Platelet-Derived Growth Factor; D016337:RNA, Catalytic; D012333:RNA, Messenger; D001194:Asbestos; D017479:Receptors, Platelet-Derived Growth Factor; D012260:Ribonucleases