Kras

Pancreatic cancer provides the most genetically clear-cut indication for antisense diagnosis. Ninety percent of the tumors carry a mutation in the twelfth codon of the K-RAS oncogene (28). K-RAS, H-RAS, and N-RAS are among the most frequently mutated oncogenes detected in human tumors (18). The mutational spectrums of the K-RAS oncogenes detected in colon, lung, and pancreatic tumors have been characterized. For example, the G-to-T and G-to-A mutations in the second base of codon 12 account for 65% of the K-RAS mutations observed in pancreatic tumors. Mutant K-RAS genes may be detected in circulating cells, bile, and pancreatic juice, as well as in biopsied tissue of patients with advanced disease (29).

The human K-RAS protooncogene codes for an evolutionarily conserved G-protein, K-Ras p21, which binds guanine nucleotides with high affinity and is associated with the inner surface of the plasma membrane. A broad range of eukaryotes carries the RAS gene family, whose members code for immunologically related proteins of approx 21 kDa with 188 to 189 amino acid residues. Many varieties of Ras proteins have been found, mostly differing at their carboxy termini. The Ras:guanosine 5'-diphosphate (GDP) complex transduces signals from growth factors binding to cell-surface receptors (30), whereupon the GDP is exchanged for guanosine 5'-triphosphate (GTP) to convert the inactive Ras:GDP complex to the active Ras:GTP complex (31). The Ras:GTP complex transmits the proliferative signal downstream through a cascade of kinases to activate nuclear transcription factors for proliferative genes. The active GTP complex with Ras is restored to the inactive GDP complex by hydrolysis of GTP to GDP. The Ras protein itself possesses intrinsic guanosine 5'-triphosphatase (GTPase) activity. In vivo, however, this intrinsic activity is very slow unless enhanced by GAP (GTPase-activating protein). After the discovery of GAP, it was shown that the main biochemical difference between wild-type p21 and oncogenic Ras proteins with mutations in codon 12, 13, or 61 is the ability of GAP to induce GTP hydrolysis in the active Ras:GTP complex. GAP-induced hydrolysis can be as much as 1000 times faster with wild-type Ras than with these mutant forms of Ras (32). These mutant forms thus remain in the active GTP form much longer than the wild type. The continuous transmission of a growth signal by the mutant forms is responsible for the oncogenic properties.

Inhibition of K-Ras protein appears to be at least a part of the antiprolifera-tive mechanism of paclitaxel, a natural product that binds to microtubules, along which K-Ras may traverse on its way from the endoplasmic reticulum to the inner leaflet of the cell membrane (33), which H-Ras does not. Additionally, prevention of K-Ras post-translational farnesylation by farnesyltrans-ferase inhibitors has been shown to inhibit the growth of RAS-dependent tumors in immunocompromised mice (34). Unfortunately, paclitaxel displays strong dose-limiting toxicity, which limits its efficacy (35).

The high rate of K-RAS mutations makes it a reasonable target for antisense single photon emission computerized tomography (SPECT), positron emission tomography (PET), or magnetic resonance imaging (MRI) detection of early stage pancreatic cancer. Regarding therapy with antisense K-RAS, although the presence of the twelfth codon mutation does not necessarily imply causation, reducing the level of K-RAS gene expression might inhibit proliferation or reverse transformation in malignant cells transformed by mutated K-RAS. These results were observed in several cell lines and nude mouse models with antisense RNA (36,37) or ribozyme expression from vectors (38), and by antisense DNA treatment (19,39-42).

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