Abstract
Mutation of c-K-ras oncogene is an important step in progression of colon cancer. We used a hammerhead ribozyme (KrasRz) against mutated K-ras gene transcripts (codon 12, GTT) to inactivate mutant K-ras function in the colon cancer cell line SW480, harbouring a mutant K-ras gene. The β-actin promoter-driven KrasRz sequence (pHβ/KrasRz) was introduced into these cells (SW480/KrasRz), and we evaluated its effects on growth of the colon cancer. The gene expression of angiogenesis-related molecules (vascular endothelial growth factor and thrombospondin) was also estimated in SW480/KrasRz. KrasRz specifically and efficiently cleaved the mutant K-ras mRNA but not wild-type mRNA in vitro. SW480/KrasRz showed decreased growth rate under tissue culture conditions (P< 0.01, Dunnett’s test). The xenotransplantability of SW480/KrasRz (XeSW480/KrasRz) was significantly decreased in nude mice (P< 0.05, Fisher’s exact test). Tumour volume of the xenografts XeSW480/KrasRz was significantly smaller than that of XeSW480/DisKrasRz (P< 0.01, Dunnett’s test). Gene expression of VEGF was suppressed in SW480/KrasRz, while TSP1 gene expression was enhanced. The SW480/KrasRz cells showed apoptosis-related features including nuclear condensation and DNA fragmentation. These results suggested that the hammerhead ribozyme-mediated inactivation of the mutated K-ras mRNA induced growth suppression, apoptosis and alteration of angiogenic factor expression. © 2000 Cancer Research Campaign
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References
Andreyev HJ, Norman AR, Cunningham D, Oates JR and Clarke PA (1998) Kirsten ras mutations in patients with colorectal cancer: the multicenter ‘RASCAL’ study. J Natl Cancer Inst 90: 675–684
Bouffard DY, Ohkawa T, Kijima H, Irie A, Suzuki T, Curcio LD, Holm PS, Sassani A and Scanlon KJ (1996) Oligonucleotide modulation of multidrug resistance. Eur J Cancer 32A: 1010–1018
Cech TR, Zaug AJ and Grabowski PJ (1981) In vitrosplicing of the ribosomal RNA precursor of tetrahymena: Involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell 27: 487–496
Chi S, Kitanaka C, Noguchi K, Mochizuki T, Nagashima Y, Shirouzu M, Fujita H, Yoshida M, Chen W, Asai A, Himeno M, Yokoyama S and Kuchino Y (1999) Oncogenic Ras triggers cell suicide through the activation of a caspase-independent cell death program in human cancer cells. Oncogene 18: 2281–2290
Cho KR and Vogelstein B (1992) Genetic alterations in the adenoma carcinoma sequence. Cancer 70: 1727–1731
Feng M, Cabrera G, Deshane J, Scanlon KJ and Curiel DT (1995) Neoplastic reversion accomplished by high efficiency adenoviral-mediated delivery of an anti-ras ribozyme. Cancer Res 55: 2024–2028
Fukushima Y, Oshika Y, Tsuchida T, Tokunaga T, Hatanaka H, Kijima H, Yamazaki H, Ueyama Y, Tamaoki N and Nakamura M (1998) Brain-specific angiogenesis inhibitor 1 expression is inversely correlated with vascularity and distant metastasis of colorectal cancer. Int J Oncol 13: 967–970
Grugel S, Finkenzeller G, Weindel K, Barleon B and Marme D (1995) Both v-Ha-Ras and v-Raf stimulate expression of the vascular endothelial growth factor in NIH 3T3 cells. J Biol Chem 270: 25915–25919
Hsu SC, Volpert OV, Steck PA, Mikkelsen T, Poverini PJ, Rao S, Chou P and Bouck N (1996) Inhibition of angiogenesis in human glioblastomas by chromosome 10 induction of thrombospondin-1. Cancer Res 56: 5684–5691
Kieser A, Weich HA, Brandner G, Marme D and Kolch W (1994) Mutant p53 potentiates protein kinase C induction of vascular endothelial growth factor expression. Oncogene 9: 964–969
Kijima H, Ishida H, Ohkawa T, Kashani-Sabet M and Scanlon KJ (1995) Therapeutic applications of ribozymes. Pharmacol Ther 68: 247–267
Kijima H, Bouffard DY and Scanlon KJ (1998) Ribozymes as a novel approach for the treatment of human pancreatic carcinoma. Methods Mol Med 11: 193–208
Jednak MA and Nostrant TT (1998) Screening for colorectal cancer. Prim Care 25: 293–308
Kohl NE, Mosser SD, Desolmes SJ, Giuliani EA, Pompliano DL, Graham SL, Smith RL, Scolnick EM, Oliff A and Gibbs JB (1993) Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor. Science 260: 1934–1942
Matsuguchi T and Kraft AS (1998) Regulation of myeloid cell growth by distinct effectors of Ras. Oncogene 17: 2701–2709
Mukhopadhyay D, Tsiokas L and Sukhatme PV (1995) Wild-type p53 and v-Src exert opposing influences on human vascular endothelial growth factor gene expression. Cancer Res 55: 6161–6165
Ohta Y, Kijima H, Kashani-Sabet M and Scanlon KJ (1996) Tissue-specific expression of an anti-ras ribozyme inhibits proliferation of human malignant melanoma cells. Nucleic Acids Res 24: 938–942
Okada F, Rak J, Croix B, Lieubeau B, Kaya M, Rncari L, Shirasawa S, Sasazuki T and Kerbel R (1998) Impact of oncogenes in tumor angiogenesis: Mutant K-ras up-regulation of vascular endothelial growth factor/vascular permeability factor is necessary, but not sufficient for tumorigenicity of human colorectal carcinoma cells. Proc Natl Acad Sci USA 95: 3609–3614
Oshika Y, Masuda K, Tokunaga T, Hatanaka H, Kamiya T, Abe Y, Ozeki Y, Kijima H, Yamazaki H, Tamaoki N, Ueyama Y and Nakamura M (1998) Thrombospondin2 gene expression is correlated with decreased vascularity in non-small cell lung cancer. Clin Cancer Res 4: 1785–1788
Rak J, Mitsuhashi Y, Bayko L, Filmus J, Shirasawa S, Sasazuki T and Kerbel RS (1995) Mutant ras oncogenes upregulate VEGF/VPF expression: implications for induction and inhibition of tumor angiogenesis. Cancer Res 55: 4575–4580
Saraga E, Bautista D, Dorta G, Chaubert P, Martin P, Sordat B, Protiva P, Blum A, Bosman F and Benhattar J (1997) Genetic heterogeneity in sporadic colorectal adenomas. J Pathol 181: 281–286
Scanlon KJ, Jiao L, Funato T, Wang W, Tone T, Rossi JJ and Kashani-Sabet M (1991) Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein. Proc Natl Acad Sci USA 88: 10591–10595
Scanlon KJ, Ohta Y, Ishida H, Kijima H, Ohkawa T, Kaminski A, Tsai J, Horng G and Kashani-Sabet M (1995) Oligonucleotide-mediated modulation of mammalian gene expression. FASEB J 6: 1288–1296
Siemeister G, Weindel K, Mohrs K, Barleon B, Martiny G and Marme D (1996) Reversion of deregulated expression of vascular endothelial growth factor in human renal carcinoma cells by von Hippel-Lindau tumor suppressor protein. Cancer Res 56: 2299–2301
Shirasawa S, Furuse M, Yokoyama N and Sasazuki T (1993) Altered growth of human colon cancer cell lines disrupted at activated Ki-ras. Science 260: 85–88
Tokunaga T, Oshika Y, Abe Y, Ozeki Y, Sadahiro S, Kijima H, Tsuchida T, Yamazaki H, Ueyama Y, Tamaoki N and Nakamura M 1998a) Vascular endothelial growth factor (VEGF) mRNA isoform expression pattern is correlated with liver metastasis and poor prognosis in colon cancer. Br J Cancer 77: 998–1002
Tokunaga T, Nakamura M, Oshika Y, Tsuchida T, Kazuno M, Fukushima Y, Kawai K, Abe Y, Kijima H, Yamazaki H, Tamaoki N and Ueyama Y 1998b) Alterations in tumour suppressor gene p53 correlate with inhibition of thrombospondin-1 gene expression in colon cancer cells. Virchows Arch 433: 415–418
Tokunaga T, Nakamura M, Oshika Y, Abe Y, Ozeki Y, Fukushima Y, Sadahiro S, Kijima H, Tsuchida T, Yamazaki H, Tamaoki N and Ueyama Y (1999) Thrombospondin-2 expression is correlated with inhibition of angiogenesis and metastasis of colon cancer. Br J Cancer 79: 354–359
Tomisawa M, Tokunaga T, Oshika Y, Tsuchida T, Fukushima Y, Sato H, Kijima H, Yamazaki H, Ueyama Y, Tamaoki N and Nakamura M (1999) Expression of vascular endothelial growth factor isoform is closely correlated with tumor stage and vascularisation in renal cell carcinoma. Br J Cancer 35: 133–137
Tsuchida T, Kijima H, Oshika Y, Tokunaga T, Abe Y, Yamazaki H, Tamaoki N, Ueyama Y, Scanlon KJ and Nakamura M (1998) Hammerhead ribozyme specifically inhibits mutant K-ras mRNA of human pancreatic cancer cells. Biochem Biophys Res Common 253: 368–378
Tsuchida T, Kijima H, Tokunaga T, Oshika Y, Hatanaka H, Fukushima Y, Abe Y, Kawai K, Yoshida Y, Miura S, Yamazaki H, Tamaoki N, Ueyama Y and Nakamura M (1999) Expression of the thrombospondin 1 receptor CD36 is correlated with decreased stromal vascularisation in colon cancer. Int J Oncol 14: 47–51
Tsuchida T, Kijima H, Hori S, Oshika Y, Tokunaga T, Kawai K, Yamazaki H, Ueyama Y, Scanlon KJ, Tamaoki N and Nakamura M (2000) Adenovirus-mediated anti-K-ras ribozyme induces apoptosis and growth suppression of human pancreatic carcinoma. Cancer Gene Ther,
Volpert OV, Dameron KM and Bouck N (1997) Sequential development of an angiogenic phenotype by human fibroblasts progressing to tumorigenity. Oncogene 14: 1495–1502
Ward RL, Todd AV, Santiago F, O’Connor T and Hawkins NJ (1997) Activation of the K-ras oncogene in colorectal neoplasms is associated with decreased apoptosis. Cancer 79: 1106–1113
Yamazaki H, Kijima H, Ohnishi Y, Abe Y Oshika Y Tsuchida T Tokunaga T Tsugu A Ueyama Y Tamaoki N Nakamura M (1998) Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expression. J Natl Cancer Inst 90: 581–587
Zabrenetsky V, Harris CC, Steeg PS and Roberts DD (1994) Expression of the extracellular matrix molecule thrombospondin inversely correlates with malignant progression in melanoma, lung, and breast carcinoma cell lines. Int J Cancer 59: 191–195
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Tokunaga, T., Tsuchida, T., Kijima, H. et al. Ribozyme-mediated inactivation of mutant K-ras oncogene in a colon cancer cell line. Br J Cancer 83, 833–839 (2000). https://doi.org/10.1054/bjoc.2000.1363
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DOI: https://doi.org/10.1054/bjoc.2000.1363
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