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 β-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 ψ-2 and GP + envAm12. Titers up to 9.7 × 107 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. Direct alteration of oncogene expression in human cancer cells can decrease cell proliferation and tumorigenicity. Successful gene therapy of human cancers will necessitate efficient delivery of gene constructs that can effectively alter oncogene expression. Zhang et al. describe a retroviral antisense K-ras expression vector that eliminates expression of the mutant p21 protein. They show through a repetitive infection strategy that transduction of human lung cancer cells is efficient and that transduction of the antisense construct inhibits proliferation, colony formation, and tumorigenicity.