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A2780DDPfosR-8+Dex

43.0 ± 4.5

"Data are means the ± SD. IC5Q represents half the number of colonies formed in comparison to the untreated A278QS cells. Dexamethasone (Dex) was administrered to the A278QDDP cells for 24 h prior to cisplatin treatment.

"Data are means the ± SD. IC5Q represents half the number of colonies formed in comparison to the untreated A278QS cells. Dexamethasone (Dex) was administrered to the A278QDDP cells for 24 h prior to cisplatin treatment.

sequences complementary to the substrate K-ras RNA and was cloned into the plasmid pLNCX (Fig. 3). The ribozyme against activated K-ras oncogene (K-ras ribozyme) has the ability to cleave the targeted RNA in vitro. Similar results were obtained in the in vivo system; namely, the tumors were smaller in athymic mice transfected with the K-ras ribozyme than in the controls. We also evaluated the chemosensitivity of the cells to a variety of agents. The sensitivity of tumors with K-ras ribozyme was increased, as shown in Table 2. We have demonstrated the effectiveness of in vivo transfection of ribozyme for inhibiting growth as well as for increasing the sensitivity to anticancer agents. Therefore, altered K-ras genes may be an important target for strategies to enhance chemosensitivity in colon cancers.

Along this line, we have tried to identify the genes responsible for drug resistance to cisplatin in cancers and revealed that the fos and K-ras genes are implicated in cellular resistance to cisplatin. Isonishi et al. (15) also demonstrated cisplatin resistance in NIH3T3 cells transfected with a mutated H-ras gene. However, the mechanism of acquired resistance to cisplatin based on DNA damage is complex and other factors may be involved, making it important to clearly identify the effectors of cisplatin resistance.

Fig. 3. Structure of K-ras ribozyme (A) and schematic representation of retroviral vector pLNCX (B). The conserved hammerhead sequence includes a base alteration in the catalytic core. Target codon-12 is bold at cleavage site. The complementary K-ras RNA is also shown with the GUC cleavage site in mutated K-ras RNA. The position of the ribozyme and a synthetic RNA substrate used to test the ribozyme are shown with respect to exon I of K-ras. This vector contains the neo gene (NEO) and the promoter of the cytomegalovirus gene (CMV). The K-ras ribozyme as a double-stranded DNA was inserted into the HindIII site. LTR; long terminal repeat.

Fig. 3. Structure of K-ras ribozyme (A) and schematic representation of retroviral vector pLNCX (B). The conserved hammerhead sequence includes a base alteration in the catalytic core. Target codon-12 is bold at cleavage site. The complementary K-ras RNA is also shown with the GUC cleavage site in mutated K-ras RNA. The position of the ribozyme and a synthetic RNA substrate used to test the ribozyme are shown with respect to exon I of K-ras. This vector contains the neo gene (NEO) and the promoter of the cytomegalovirus gene (CMV). The K-ras ribozyme as a double-stranded DNA was inserted into the HindIII site. LTR; long terminal repeat.

The c-myc oncogene, a regulator of cell growth associated with cell division, is highly expressed in more than 70% of human colon cancers, and colon cancer is often resistant to cisplatin treatment (16). The role of c-myc protein in cellular susceptibility to anticancer drugs is controversial. In fact, overexpression of the protein has been reported to enhance tumor cell sensitiv-

Table 2

Sensitivity (IC50) of Cells Excised From Tumors by K-ras Ribozyme to Drugs In Vivo

Table 2

Sensitivity (IC50) of Cells Excised From Tumors by K-ras Ribozyme to Drugs In Vivo

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