Tumor inhibitory activity of anti-ras ribozymes delivered by retroviral gene transfer.

This study reports the successful introduction into tumor cells of a ribozyme directed against an oncogene using a retroviral gene delivery system. A hammerhead ribozyme that selectively targets and cleaves the activated Ha-ras (V12Ras) oncogene was delivered using a retroviral vector and expressed under the control of a transfer RNA promoter in V12Ras-transformed 3T3 murine fibroblast and rat colon epithelial cells. The expression of V12Ras messenger RNA and V12Ras P21 protein was reduced by up to 100% and 75%, respectively, in cells transduced with functional ribozyme. Reductions in V12Ras expression correlated with the stable expression of the functional ribozyme in vivo and decreased tumorigenicity in nude mice. Ribozyme constructs containing identical antisense flanking regions, but a mutant catalytic center, did not attenuate V12Ras expression or decrease the tumorigenicity of transduced tumor cells. These data support the potential therapeutic role of antioncogene ribozymes.

Early clonality and high-frequency proviral integration into the c-myc locus in AKR leukemias.

Blot hybridization of thymocyte DNA from AKR/J mice was used to detect new proviral junction fragments as markers of clonality at different stages of viral leukemogenesis and to detect DNA rearrangements at the c-myc locus due to proviral insertion. Clonal populations of thymocytes were observed in mink cell focus-forming virus-injected mice as early as 35 days postinjection, at a stage distinguishable from frank leukemia by flow cytometric analysis and transplantation bioassay. Specific proviral integrations in the c-myc locus were detected in 15% of these early clones and in up to 65% of late-developing thymomas and frank leukemias. Thus, in this system c-myc activation appears to be a common mechanism in T-cell leukemogenesis.

Cloning and characterization of a vertebrate cellular myosin regulatory light chain complementary DNA.

We have isolated two series of complementary DNAs (cDNAs) from a chicken gizzard cDNA library encoding two isoforms of phosphorylatable myosin regulatory light chain (RLC). One of the cDNAs encodes a previously isolated smooth muscle myosin RLC (also referred to as LC20-A); the other encodes a protein that shares 92% homology with the LC20-A isoform. The phosphorylatable threonine and serine residues at positions 18 and 19 of the two myosin RLC sequences are conserved. The two cDNAs are 81% homologous at the nucleotide level over the coding region; the 5' and 3' untranslated regions are divergent. Most of the DNA nonhomology in the coding region does not affect the protein sequence, indicating strong evolutionary conservation pressure to maintain the myosin RLC structure. Northern blot analysis using 3' untranslated region probes reveals restrictive tissue specific expression of one myosin RLC isoform (LC20-A) in smooth muscle tissue and not in other tissues examined. In contrast, the novel myosin RLC isoform messenger RNA (mRNA) is uniformly expressed in all smooth and nonmuscle tissues examined and is designated as cellular myosin RLC for this reason. Our results indicate that cellular and smooth muscle myosin RLC isoforms are distinct and are encoded by separate genes. This report describes the cloning of a novel vertebrate cellular myosin RLC mRNA that differs from previously characterized smooth muscle RLC isoform mRNAs in both primary sequence and expression pattern.