Inhibition of human telomerase by a retrovirus expressing telomeric antisense RNA.

Human telomerase, the RNA-dependent DNA polymerase that adds TTAGGG repeats to chromosome ends, is selectively expressed in immortalised cells and most tumours, suggesting a potential role for telomerase inhibitors in cancer therapy. Replication-deficient retroviruses were used to determine whether mRNA containing UUAGGG, the complementary sequence to the template region of the hTR telomerase RNA, is sufficient to inhibit telomerase activity. Telomerase activities measured by the telomeric repeat amplification protocol (TRAP) assay in extracts prepared from immortalised mouse fibroblasts, human HeLa cells and human kidney carcinoma cells were inhibited by 75% or greater in 26 of 56 cell clones expressing UUAGGG. Telomerase activity was not inhibited by expression of mRNA containing a transposed sequence, GGGAUU. Telomerase activities in vivo were inferred from changes in cellular morphology, proliferation capacity, growth rate and measurement of the content of telomere DNA. Giant senescent-like cells emerged shortly after cloning mouse PA317 and human HeLa cells expressing UUAGGG. The fraction of giant cells varied from 100% at the fifth population doubling (PD) in one culture to 2-6% at 50 PD in several other cultures. Giant cells were absent in all parental cells and clones expressing GGGAUU. The average cellular content of telomere DNA was independent of telomerase activity over 50 PD. The results indicate that expression of RNA complementary to the template region of hTR is sufficient to inhibit telomerase in vitro and in vivo, but that the effect of inhibition on individual cells is highly variable.

Vitalethine modulates erythropoiesis and neoplasia.

Novel compounds based upon the thiol N-(carboxy)-beta-alanyl-cysteamine (vitaletheine) have strikingly potent and seemingly diverse biological activities. Concentrations of vitaletheine modulators from 1 femtograms/ml to 100 picograms/ml medium regulate RBC production from progenitors initially deprived of erythropoietin. Similarly, as little as attograms/ml concentrations of the disulfide vitalethine stimulate immunological responses of murine splenocytes toward sheep RBC in a hemolytic plaque assay. Because dosages of vitalethine as low as femtograms/kg substantially diminish tumor size and incidence and increase survival to 80% in mice inoculated with a uniformly fatal melanoma (Cloudman S-91), activities of these compounds have in vivo significance. A preliminary probe of the benzyl derivative of vitalethine in a myeloma model (NS-1) suggests efficacy (100% survival) as well. The high potencies of the vitaletheine modulators, both in cell culture and in vivo, indicate that these or similar regulatory components, if constitutively present, probably occur endogenously at vanishingly small concentrations and may be prone to deficiency resulting from metabolic imbalances, irradiation, aging, diet, pathogenic or parasitic infections, or exposure to environmental pollutants. Pathways for the biosynthesis of vitaletheine are proposed and chemical syntheses of the vitaletheine modulators are described. Possible molecular mechanisms of action, including interactions with peptidyl hormones, other endogenous effectors, and xenobiotic and pharmaceutical compounds, are explored. Indications for the treatment of other diseases are identified.

Isoprenoid pathway activity is required for IgE receptor-mediated, tyrosine kinase-coupled transmembrane signaling in permeabilized RBL-2H3 rat basophilic leukemia cells.

Previously, we reported that the isoprenoid pathway inhibitor, lovastatin, blocks the activation by IgE receptor cross-linking of 45Ca2+ influx, 1,4,5-inositol trisphosphate production, secretion, and membrane changes (ruffling, spreading) in intact RBL-2H3 rat basophilic leukemia cells. These results indicated that an isoprenoid pathway intermediate, very likely an isoprenylated protein, is importantly involved in the control of IgE receptor-mediated signal transduction. Here, we show that 20 h of pretreatment with lovastatin also inhibits antigen-induced secretion and membrane responses in streptolysin O-(SLO)-permeabilized cells. However, lovastatin does not inhibit secretion stimulated by the nonhydrolyzable GTP analog, GTP gamma S. Furthermore, the membrane responses to GTP gamma S persist, although in an attenuated form, in lovastatin-treated permeabilized cells. The relative insensitivity of GTP gamma S-induced responses to lovastatin was one of several indications that antigen and GTP gamma S may activate separate pathways leading to transmembrane responses in permeabilized cells. Further experiments showed that the beta-thio derivative of GDP, GDPBAS, inhibits the secretory and membrane responses to GTP gamma S, as expected for a GTP-binding protein-dependent signaling pathway, while having little effect on antigen-induced responses. Conversely, genistein blocks the secretory and membrane responses to antigen, as expected for a tyrosine kinase-dependent pathway, without altering the GTP gamma S-induced responses. From these results, and from additional data from cells treated with tyrphostins and sodium orthovanadate, we propose that IgE receptor-mediated secretion from permeabilized RBL-2H3 cells occurs by a tyrosine kinase-dependent pathway that requires isoprenoid pathway activity for function. We propose further that RBL-2H3 cells contain a separate GTP-binding protein-mediated signaling pathway whose direct activation by GTP gamma S is either independent of isoprenoid pathway activity or depends on the activity of an isoprenylated protein that is not significantly depleted after 20 h of lovastatin treatment.