Multipotential hematopoietic progenitor cell lines have been established from nonadherent cell populations removed from continuous mouse bone marrow cultures. Clonal sublines of lines B6SUtA or B6JUt derived from single cells formed mixed colonies containing erythroid cells, neutrophil-granulocytes, and basophil/mast cells in semisolid medium containing erythropoietin and conditioned medium from pokeweed mitogen-stimulated spleen cells. Each of several subclones of cell line Ro cl formed colonies containing eosinophils, neutrophil-granulocytes, and basophil/mast cells in semisolid medium. Multipotentiality was maintained in vitro for over 2 1/2 years. In contrast, cell line 32D formed basophil/mast cell colonies with no detectable differentiation to other pathways. Multipotential cell lines did not produce detectable spleen colonies (CFUs) in vivo, nor did intravenous inoculation of up to 5 X 10(7) cells protect lethally irradiated mice from bone marrow failure. Newborn and adult mice inoculated with 5 X 10(7) cells showed no detectable leukemia or solid tumors after one year. Both multipotential and committed basophil/mast cell lines demonstrated absolute dependence upon a source of a growth factor(s) found in medium conditioned by WEHI-3 cells. These cell lines should be of value in studies of the regulation of hematopoietic stem cell differentiation in vitro.
A series of dipyridodiazepinones have been shown to be potent inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. The lead compound, BI-RG-587, had a 50% inhibitory concentration of 84 nM against HIV-1 reverse transcriptase activity. This compound reduced plaque formation of HIV-1 in HeLa cells expressing the CD4 receptor by 50% at 15 nM. BI-RG-587 at comparable concentrations inhibited the production of p24 antigen following the acute infection of CEM T-lymphoblastoid cells or primary human monocyte-derived macrophages with HIV-1. No inhibitory effects against HIV-2 or against three picornaviruses were detected. Zidovudine (3'-azido-3'-deoxythymidine [AZT])-susceptible and AZT-resistant isolates of HIV-1 were equally susceptible to BI-RG-587. AZT and BI-RG-587 exhibited synergistic inhibition of HIV-1BRU at all concentrations examined.
Transmissible retroviruses encoding human hypoxanthine phosphoribosyltransferase (HPRT) were used to infect mouse bone marrow cells in vitro, and the infected cells were transplanted into mice. Both active human HPRT-protein and chronic HPRT-virus production were detected in hematopoietic tissue of the mice, showing transfer of the gene. These results indicate the possible use of retroviruses for somatic cell therapy.
An imidazo[4,5-h]isoquinolin-7,9-dione (1) was identified as an adenosine 5'-triphosphate competitive inhibitor of lck by high throughput screening. Initial structure-activity relationship studies identified the dichlorophenyl ring and the imide NH as important pharmacophores. A binding model was constructed to understand how 1 binds to a related kinase, hck. These results suggested that removing the gem-dimethyl group and flattening the ring would enhance activity. This was realized by converting 1 to the imidazo[4,5-h]isoquinolin-9-one (20), resulting in an 18-fold improvement in potency against lck and a 50-fold increase in potency in a cellular assay.
The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did not work in vivo upon oral administration. Our current work highlights the further use of rational drug design and molecular modeling to produce a series of lck inhibitors that demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in vivo mouse model of anti-CD3-induced IL-2 production.
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