Russell S. Taichman

Comparison of benzimidazole nucleosides and ganciclovir on the in vitro proliferation and colony formation of human bone marrow progenitor cells

Recently we have shown that certain benzimidazole ribonucleosides are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Because antiviral drugs used to treat HCMV and human immunodeficiency virus (HIV) infections can suppress marrow progenitors, we have evaluated the most promising of the new benzimidazoles for their effects on human bone marrow cells in vitro. In an initial study of the bone marrow toxicity of one of the most active compounds, 100 μm 2‐bromo‐5,6‐dichloro‐1‐(β‐d‐ribofuranosyl)‐benzimidazole (BDCRB) inhibited cell proliferation by 20% over a 10 d period compared to 52% inhibition by 100 μm ganciclovir, the drug currently most used to treat HCMV infections. The effects of these drugs and selected other benzimidazole nucleosides were evaluated more extensively in haemopoietic progenitor cell colony formation assays. Colony formation was determined at 2 weeks and scored as either burst forming units‐erythroid (BFU‐E), or colony forming units‐granulocyte/macrophage (CFU‐GM). At the highest concentration tested, 100 μm BDCRB only moderately affected BFU‐E or CFU‐GM formation (31% and 47% inhibition, respectively). This concentration is 10‐fold higher than that required to produce a 10 000‐fold reduction in virus titre. Evaluation of the 2‐chloro analog of BDCRB (TCRB) which is less potent against HCMV, its 5′‐deoxy analog (5′‐dTCRB) which is more potent, and the 2‐unsubstituted compound (DRB) gave the following order of haemopoietic toxicity: DRB > TCRB≥ 5′‐dTCRB > BDCRB. In contrast to the benzimidazoles, ganciclovir decreased colony formation by 84% for BFU‐E and 86% for CFU‐GM at 100 μm. These results establish that certain benzimidazole nucleosides are less toxic to haemopoietic progenitors than the preferred drug now being used clinically for HCMV infections. The results also establish that different structure–activity relationships exist for antiviral activity and progenitor cell toxicity, thereby suggesting that different mechanisms are involved in the two types of drug action.

Gene transfer investigations of p56-LCK function in IL-2-dependent T-cell lines: implications for mechanisms of IL-2-signal transduction.

Recently, the SRC-like non-receptor protein tyrosine kinase p56-LCK has been shown to physically associate with the interleukin-2 receptor (IL-2-R) complex and to undergo rapid elevations in its tyrosine kinase activity upon stimulation of T lymphocytes with IL-2. The functional significance of p56-LCK kinase activation for IL-2-mediated lymphocyte responses, however, has never been directly assessed. Using gene transfer approaches, we have achieved markedly elevated levels of p56-LCK kinase activity in the IL-2-dependent cytolytic T-cell line CTLL-2 and the helper line HT-2. CTLL-2 and HT-2 cells that were stably transfected with expression plasmids encoding either the normal human p56-LCK or a constitutively active version of the mouse p56-LCK kinase (LCK[Y505]) contained striking elevations in the levels of tyrosine phosphorylation on several proteins (34-36, 50-60, 62-68, 77-78, 104-110 kDa), as determined by immunoblot analysis using anti-phosphotyrosine antibodies. CTLL-2 and HT-2 LCK- and LCK(Y505F)-transfected cells remained dependent on IL-2 for their growth and survival in culture despite the findings that (i) IL-2 specifically stimulated elevations in the activity of the endogenous p56-LCK in untransfected CTLL-2 cells without affecting the activities of the other SRC-like kinases in these cells (p59-FYN, p62-YES) and that (ii) IL-2-mediated regulation of p56-LCK correlated with IL-2-driven proliferation of these T cells. Specifically, no elevation in the proliferation (DNA synthesis) or growth of these T cells was found at any of the concentrations of IL-2 examined (0.01-25 U/ml), relative to untransfected and control transfected cells. Furthermore, when cultured in the absence of IL-2, transfected T cells whose relative levels of p56-LCK activity were elevated by approximately 20-50-fold died with the same kinetics as control cells and underwent apoptosis, as defined by uptake of trypan blue dye and DNA fragmentation assays, respectively. Taken together, these data indicate that while IL-2 can up-regulate the enzymatic activity of p56-LCK, elevated levels of p56-LCK tyrosine kinase activity are insufficient to stimulate IL-2-mediated pathways required for T-cell growth and survival. These findings thus imply the existence of other signal-transducing molecules, besides p56-LCK, that physically participate in IL-2R complexes and that are necessary for initiation of the biochemical events ultimately responsible for IL-2s pleiotropic actions on lymphocytes.