Retroviral vector-mediated transduction of K-ras antisense RNA into human lung cancer cells inhibits expression of the malignant phenotype.
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.
Department of Thoracic Surgery, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Y
Y
Zhang
T
T
Mukhopadhyay
L A
LA
Donehower
R N
RN
Georges
J A
JA
Roth
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D011905:Genes, ras; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D015689:Oncogene Protein p21(ras) / Q000096:biosynthesis / Q000235:genetics; D010641:Phenotype; D016372:RNA, Antisense / Q000235:genetics / Q000494:pharmacology*; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D016372:RNA, Antisense; D015689:Oncogene Protein p21(ras)