Retroviral Vector-Mediated Transduction of K-rasAntisense RNA into Human Lung Cancer Cells Inhibits Expression of the Malignant Phenotype

Abstract

A retroviral vector system was developed to transduce a K-ras antisense construct efficiently into human cancer cells. A 2-kb fragment of K-ras gene DNA in antisense orientation was linked to a beta-actin promoter and inserted into retroviral vector LNSX in two different orientations. The constructs were transfected into amphotropic packaging cell line GP+envAm12 followed by alternating transduction between the ecotropic packaging cell line psi-2 and GP+envAm12. Titers up to 9.7 x 10(7) colony-forming units (cfu)/ml were achieved without detectable replication-competent virus. The human large cell lung carcinoma cell line H460a, which has a homozygous codon 61 K-ras mutation, was transduced with an efficiency of 95% after five to seven repeated transductions. DNA polymerase chain reaction (PCR) and genomic DNA Southern blot analysis showed that the retroviral construct was integrated into the genome of H460a cells. K-ras antisense RNA expression was detected in the cells by Northern analysis, slot blot hybridization, and reverse transcriptase-PCR. Translation of the mutated K-ras p21 protein RNA was specifically inhibited, whereas expression of other p21 species was unchanged. Proliferation of H460a cells was suppressed 10-fold following transduction by the antisense construct. Colony formation in soft agarose and tumorigenicity in an orthotopic lung cancer model in nu/nu mice were dramatically reduced in H460a cells expressing antisense K-ras. We conclude that an antisense construct for K-ras can be expressed effectively in a retroviral vector that can efficiently transduce human cancer cells.

A novel molecular staging protocol for non-small cell lung cancer

“…On a theoretical basis, if MRP-1/CD9 are reintroduced back into the appropriate lung cancer cells, there would be metastasis suppression of the tumors and aggressive surgery may be recommended. Furthermore, the antisense technology may downregulate the mutant oncogene expression; K-ras gene in antisense orientation was used to transduce human lung cancer cell lines using a retroviral vector (Zhang et al, 1993). In summary, recent remarkable progress in molecular biology has led to the elucidation of many events involved in the process of malignant transformation (Masters et al, 1996), and molecular and biological markers have been identi®ed to serve as predictors of survival.…”

Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy

“…Expression of antisense K-Ras cDNA suppressed the malignant phenotype of large cell lung carcinomas 156 and inhibited growth of H460a lung cancer cells nearly three-fold. 157 ISIS 2503 is a 20-base antisense phosphorothioate oligodeoxyribonucleotide targeting H-Ras that is currently in phase 2 clinical trials.…”

“…This suggested that T157 of p27 Kip1 is important for nuclear localization and Cdk-2 inhibition. Furthermore, T157 is located within the nuclear localization sequence of p27 Kip1 (amino acids [151][152][153][154][155][156][157][158][159][160][161][162][163][164][165][166] and is contained in a putative Akt consensus phosphorylation site (RXXRXXT 157 D). Using breast cell lines as a model, it was shown that Akt could phosphorylate p27 Kip1 on T157 both in vitro and in vivo.…”

Requisitos mínimos para o laudo de anatomia patológica em câncer de pulmão: justificativas na patogênese

“…A presença desta discreta alteração molecular tem conduzido ao desenvolvimento de novos protocolos de tratamento. Crescimento tumoral em linhagens celulares expressando mutações K-ras é marcadamente diminuído por um anti-RNA K-ras construído através da introdução de um vetor retroviral (54) . Dessa forma, melhor compreensão dessa alteração molecular no câncer de pulmão tem conduzido ao reconhecimento de um importante fator prognóstico negativo, um possível marcador de pré-malignidade e uma nova estratégia terapêutica.…”