Growth inhibition of human pancreatic cancer cell lines by anti-sense oligonucleotides specific to mutated K-ras genes

Kita, Keiichiro; Saito, Seiji; Morioka, Cintia Yoko; Watanabe, Akïharu

doi:10.1002/(sici)1097-0215(19990209)80:4<553::aid-ijc12>3.0.co;2-6

Cited by 73 publications

(17 citation statements)

References 16 publications

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“…How do we rationalize this apparent incongruence? The three RAS genes are expressed in all tissues, albeit to different levels (32,51). It is, however, not known if the different isoforms are involved in separate signal pathways or if each protein functions similarly and together constitute a functional redundancy within the same RAS pathway.…”

Section: Discussionmentioning

confidence: 99%

“…In this way the levels of KRAS and GAPDH transcripts refer to the same amount of DNA competitor and can be directly compared. Figure 5 shows the results obtained with BxPC3 cells, homozygotic for KRAS, and Panc-1 cells that are heterozygotic for KRAS but with the mutant allele prevailing over the wild-type allele (32). It can be seen that in Panc-1 cells, 13 h following PNA treatment, P14 strongly suppresses the transcription of KRAS but not that of GAPDH, at all three concentrations used.…”

Section: Measurements Of the Level Of Kras Mrna By Competitive Rt-pcrmentioning

confidence: 96%

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Transcription Inhibition of Oncogenic KRAS by a Mutation-Selective Peptide Nucleic Acid Conjugated to the PKKKRKV Nuclear Localization Signal Peptide

et al. 2005

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Mutations in the Kirsten ras (KRAS) gene are present in almost all pancreatic adenocarcinomas, and one common mutation is at codon 12: GGT (Gly) is transformed into GAT (Asp). In this work we have targeted the KRAS coding sequence embracing the GAT mutation with a sense PNA molecule (P14), with the aim of downregulating the expression of the mutant allele. P14 was designed with a 15-base sequence complementary to the antisense strand of KRAS at the GAT (Asp) mutation and conjugated to the nuclear localization signal peptide PKKKRKV. CD spectra as a function of temperature show that P14 (2 microM) binds to the antisense strand of the GAT target in the mutated allele with a T(M) of 78 degrees C and to the antisense strand of the GGT target in the wild-type allele with a T(M) of 69 degrees C, in 50 mM Tris-HCl, pH 7.4, and 1 M NaCl. Moreover, P14 showed a high capacity to enter and accumulate in the nuclei of pancreatic cells (Panc-1 and BxPC3), whereas the nonconjugated analogue did not. Quantitative RT-PCR showed that 1 microM P14 was able to specifically suppress KRAS transcription in Panc-1 cells, which harbor mutant KRAS, but not in BxPC3 cells, which contain only wild-type KRAS. However, P14 inhibited KRAS transcription also in BxPC3 cells when used at concentrations of 5 and 10 microM. Following a single PNA treatment, changes in protein level were evident only in Panc-1 cells. As we found that all three genes of the ras family are expressed in the pancreatic cells, we designed PNA-NLS conjugates (P16 and P17) to target also HRAS and NRAS. The binding of each PNA conjugate to the ras genes was assayed by electrophoresis, and their capacity to inhibit transcription was measured by RT-PCR. All of the data obtained, both in vivo and in vitro, are discussed in terms of sequence specificity in the binding between PNA-NLS molecules and genomic DNA.

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Section: Discussionmentioning

confidence: 99%

Section: Measurements Of the Level Of Kras Mrna By Competitive Rt-pcrmentioning

confidence: 96%

Transcription Inhibition of Oncogenic KRAS by a Mutation-Selective Peptide Nucleic Acid Conjugated to the PKKKRKV Nuclear Localization Signal Peptide

et al. 2005

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“…One is to control the RAS expression level. For instance, both siRNAs [54] and antisense nucleotides [55,56] have been used to reduce RAS expression in tumor cells and some are under clinical studies [57]. Another strategy is to intervene the posttranslational modifications of the RAS Cterminus, including farnesylation, endoproteolysis, and methylation [7,37].…”

Section: Strategies For Targeting Ras Signaling For Cancer Therapymentioning

confidence: 99%

Targeting Prenylated RAS Modifying Enzymes in Cancer Cells

Chen¹

2006

CST

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RAS oncogenes have been identified in about 30% of all human cancers, particularly in 90% of human pancreatic cancers, 50% of colorectal tumors, and 30% of lung cancers. RAS is a central switch for many signal transduction pathways. The RAS proteins undergo three major posttranslational modification steps to become fully functional: prenylation (farnesylation or geranylgeranylation) of the cysteine residue of the CAAX of the RAS C-terminus (C, cysteine, A, aliphatic amino acid; X, Ser, Met, Glu, and Leu), endoproteolysis to remove the AAX amino acid sequence, and methylation of the newly formed prenylated cysteine C-terminus. It is hypothesized that any of these three steps could be an interference point for targeting RAS signaling to block the growth of the mutant RAS-dominant cancer cells. In the last decade, intensive efforts have been directed to target the prenylation of RAS, resulting in many RAS farnesylation inhibitors, which are in the clinical trials with mixed results. On the other hand, both recent chemical genetic and traditional genetic studies demonstrate that targeting two prenylation-dependent modification enzymes, RAS endoprotease and methyltransferase, might be two additional targets in killing mutant RAS-dependent cancer cells. This mini-review discusses the implications of both RAS endoprotease and methyltransferase as anticancer targets and their respective inhibitors as anticancer agents in cancer therapy. A. INTRODUCTIONThe RAS oncogenes were the first human transforming genes identified in human cancer cells ([1-3]; reviewed in ref.[4]). Since the RAS oncogenetic products were shown to potential anticancer targets [5,6], targeting the RAS signaling pathway for cancer therapy has been pursued for more than a decade [7][8][9][10][11]. Many cell-active inhibitors of Ras C-terminal modifying enzymes have been made over the last decade to target the RAS post-modification processes in order to selectively block the RAS-mediated tumor growth, including RAS farnesyltransferase inhibitors [12][13][14][15], RAS methyltransferase inhibitors [16][17][18][19], and RAS endoprotease inhibitors [20][21][22][23]. Several farnesyltransferase inhibitors (FTIs) have been in different stages of clinical trials with mixed results (reviewed in refs. [24-26]). Interestingly, the receptor tyrosine kinase (RTK) inhibitors such as imatinib and gefitinib, which were developed during the similar period as RAS C-terminal processing enzyme inhibitors, instead showed great clinical results and became the first group of small molecule signal transduction inhibitors as clinical anticancer drugs approved by the Food and Drug Administration in the United States of America [27]. In case of kinase inhibitors, more than 30 kinase inhibitors are now under clinical trials [28,29]. The mixed results from the FTI clinical trials and recent interesting results from chemical biology [19,22,23,30] and genetic studies [31-34] of prenylated RAS modifying enzymes prompt the scientists to re-examine the potential of targeting prenylated ...

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“…Complimentary 'antisense' RNA is introduced into a tumour cell in order to bind to mutant K-ras mRNA, thus inactivating it and preventing the production of mutant K-ras protein (84). The antisense RNA must be tailored to the speci c mutation identi ed in each tumour.…”

Section: Gene Therapymentioning

confidence: 99%

New Techniques and Agents in the Adjuvant Therapy of Pancreatic Cancer

et al. 2002

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Pancreatic ductal adenocarcinoma represents a major oncological challenge. Despite improvements in surgical techniques, long-term survival after resection is poor, with few patients surviving after 5 years. Until recently, there have been no large randomized trials of adjuvant therapy in pancreatic ductal adenocarcinoma. However, major trials such as the European Study Group for Pancreatic Cancer (ESPAC-1) and ESPAC-3 trials have set new standards for patient recruitment and development in this field. Adjuvant therapy has the potential to improve both patient survival and quality of life after curative resection. Currently, the best treatment is with 5-fluorouracil with folinic acid, but in the light of ongoing clinical trials, this may be supplanted by gemcitabine as the treatment of choice. Chemoradiotherapy does not appear to be beneficial in the adjuvant setting, but trials of a wide variety of other techniques and agents in the treatment of advanced disease are being undertaken and some of these will almost certainly be extended into the adjuvant setting in time. Great progress has been made in the adjuvant treatment of pancreatic cancer in the past 10 years and similar advances are likely over the next decade.

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Growth inhibition of human pancreatic cancer cell lines by anti-sense oligonucleotides specific to mutated K-ras genes

Cited by 73 publications

References 16 publications

Transcription Inhibition of Oncogenic KRAS by a Mutation-Selective Peptide Nucleic Acid Conjugated to the PKKKRKV Nuclear Localization Signal Peptide

Transcription Inhibition of Oncogenic KRAS by a Mutation-Selective Peptide Nucleic Acid Conjugated to the PKKKRKV Nuclear Localization Signal Peptide

Targeting Prenylated RAS Modifying Enzymes in Cancer Cells

New Techniques and Agents in the Adjuvant Therapy of Pancreatic Cancer

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