Dorai T, Kobayashi H, Holland J F, Ohnuma T
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029.
Mol Pharmacol. 1994 Sep;46(3):437-44.
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.
恶性间皮瘤在人类中是一种罕见疾病，但由于其与接触石棉的密切关系，最近受到了公众的广泛关注。我们发现，血小板衍生生长因子（PDGF）-β基因在裸鼠的几种间皮瘤异种移植瘤中过表达。由于一些间皮瘤细胞系（如VAMT-1）过表达PDGF-β及其受体，因此认为涉及PDGF-β及其受体的自分泌环可能促成了这些细胞的恶性表型。为了研究这种可能性，我们开发了一种针对PDGF-β mRNA的锤头状核酶。这种c-sis核酶能够在无细胞系统中切割人工合成的PDGF-β RNA底物。借助组成型表达载体，将这种核酶转导至VAMT-1细胞系中，导致PDGF-β mRNA水平降低。在这些细胞中表达的核酶在体外能够切割人工RNA底物。使用该核酶和完整的PDGF-β mRNA进行的核糖核酸酶保护分析表明这种核酶能够在体内切割完整的mRNA。含有野生型核酶的转染克隆显示细胞生长减慢，这与PDGF-β表达的降低相一致。失活的核酶在体外切割反应中无活性，在体内也不能降低细胞生长速率。我们的数据表明，在一些间皮瘤细胞中，PDGF-β自分泌环可能发挥作用，转导PDGF-β核酶可导致细胞生长显著降低。c-sis核酶可能适用于恶性间皮瘤患者的治疗。
