Design, synthesis, and antiviral activity of certain 2,5,6-trihalo-1-(beta-D-ribofuranosyl)benzimidazoles.

A new series of 2-substituted 5,6-dichlorobenzimidazole ribonucleosides has been synthesized and tested for activity against two human herpes viruses and for cytotoxicity. 2,5,6-Trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) was prepared by ribosylation of the heterocycle 2,5,6-trichlorobenzimidazole followed by a removal of the protecting groups. The 2-bromo derivative (BDCRB) was made in a similar fashion from 2-bromo-5,6-dichlorobenzimidazole.

Multiple myeloma: high incidence of chromosomal aneuploidy as detected by interphase fluorescence in situ hybridization.

Because metaphase cytogenetic studies in multiple myeloma (MM) are hampered by a low proliferative activity of myeloma cells in vitro, interphase cytogenetics by means of fluorescence in situ hybridization (FISH) should improve the detection of chromosomal abnormalities in MM. We therefore investigated chromosomal aneuploidy in 36 patients with MM using interphase FISH and alpha-satellite DNA probes for chromosomes 1, 3, 7, 8, 11, 12, 16, 17, 18, and X. By FISH, myeloma cells from 32 patients (88.

Glucocorticoid-mediated immunomodulation: hydrocortisone enhances immunosuppressive endogenous retroviral protein (p15E) expression in mouse immune cells.

To define glucocorticoid (GC)-regulated genes contributing to the anti-inflammatory and immunosuppressive effects of GC, previous work from our laboratory revealed up-regulation of transcripts from endogenous type B mouse mammary tumour virus (Mtv) and type C murine leukaemia virus (Emv) loci by high dose GC treatment of P388D1 macrophage-like cells. This study demonstrates enhancement of expression from Mtv and Emv loci in P388D1 cells by more physiological hydrocortisone concentrations (1 microM), and shows direct transcriptional mode of regulation by blocking GC-mediated signal transduction at different levels. Furthermore, we found up-regulation of Emv mRNA steady-state levels in murine lymphoid lineage cells (T-like EL4 and BW5147 cells; B-like X63 cells) upon GC treatment.

Low incidence of MDR1 expression in acute promyelocytic leukaemia.

The MDR1 gene product, P-glycoprotein, functions as a transmembrane efflux pump for certain cytotoxic agents including anthracyclines. Based upon the clinical observation that patients with acute promyelocytic leukaemia (APL) respond favourably to anthracyclines, we hypothesized that APL cells may have low levels of MDR1 expression. We therefore investigated MDR1 expression in 10 patients with APL and compared results with those obtained in 18 patients with other subtypes of acute myelogenous leukaemia (AML).

Expression of MDR1 by normal bone marrow cells and its implication for leukemic hematopoiesis.

Expression of MDR1 is a well-characterized mechanism leading to resistance of tumor cells to drugs like vinca-alkaloids, anthracyclines, and epipodophyllotoxins. In hematopoiesis, recent data indicate that not only leukemic cells, but also some populations of normal hematopoietic cells, particularly CD34+ progenitor cells as well as peripheral blood lymphocytes, express a functional multidrug-resistant phenotype. Among CD34+ cells, we found evidence that myeloid committed precursor cells (CD34+/CD33+) have lower levels of MDR1 expression than earlier CD34+ cell populations, but there was no difference in MDR1 expression between CD34+/HLA-DR- and CD34+/HLA-DR+ subpopulations.

Growth-factor stimulation reveals two mechanisms of retinoblastoma gene inactivation in human myelogenous leukemia cells.

Mutation or deletion of the retinoblastoma tumor suppressor gene (Rb) or abnormal Rb protein expression is found in many types of human solid tumors. Low or absent levels of Rb protein are usually found in the leukemic cells of patients with acute myelogenous leukemia (AML) who have an extremely poor prognosis. The absence of Rb protein in these AML cells could result from defects in the Rb gene or from abnormal cell cycle regulation that affects Rb expression.

Inhibition of human cytomegalovirus in culture by alkenyl guanine analogs of the thiazolo[4,5-d]pyrimidine ring system.

A series of alkyl and alkenyl guanine analogs containing a thiazolo[4,5-d]pyrimidine ring system were prepared by reaction of the appropriate alkyl halide with the sodium salt of the heterocycle. In preliminary antiviral efficacy evaluations against laboratory strains of both human cytomegalovirus (HCMV) and herpes simplex virus types 1 and 2, it was determined that two of the compounds (T70072 and T01132) were more active and less toxic in stationary-phase cell monolayers than were the other derivatives tested. T01132 and T70072, which have 2-pentenyl and 3-methyl-2-butenyl moieties attached to position 3 of the 5-aminothiazolo[4,5-d]pyrimidine-2,7-dione, respectively, were then more extensively evaluated for anti-HCMV activity.

Granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, PIXY-321, stem cell factor, interleukin-3, and interleukin-7: receptor binding and effects on clonogenic proliferation in acute lymphoblastic leukemia.

Cytokines are frequently used after chemotherapy of leukemias and solid tumors to augment recovery of normal hematopoiesis. While the regulation of normal and leukemic myelopoiesis is well investigated, little is known about effects of cytokines on growth and differentiation of lymphoblastic leukemia. In this study, we investigated the expression of receptors for G-CSF, GM-CSF, SCF, IL-3, and IL-7 on acute lymphoblastic leukemia (ALL) blasts and the effects of these growth factors (GF) on ALL blast colony formation.

Karyotype and prognosis in non-Hodgkin lymphoma.

In malignant non-Hodgkin lymphomas (NHL), cytogenetic analysis may provide prognostic information including prediction of histologic evolution and responsiveness to therapy. In this study, we correlate clinical data and chromosomal aberrations in 70 adult patients with newly diagnosed NHL followed for a median of 20 months. Clonal aberrations were detected in 68/70 patients (97%).

Benzimidazole ribonucleosides: design, synthesis, and antiviral activity of certain 2-(alkylthio)- and 2-(benzylthio)-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazoles.

Several 2-alkylthio- and 2-benzylthio derivatives of 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB) have been designed and synthesized from 5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole-2-thione. All compounds were evaluated for activity against human cytomegalovirus (HCMV) and/or herpes simplex virus type-1 (HSV-1). Three different cytotoxicity assays were used to determine if the compounds were toxic to uninfected cells.

Relationship between cytotoxicity and conversion of thiosangivamycin analogs to toyocamycin analogs in cell culture medium.

Non-nucleoside analogs of the pyrrolopyrimidine nucleosides toyocamycin, sangivamycin and thiosangivamycin have been synthesized and their cytotoxicity in mammalian cells determined. While studying the effects of 5-thioamide-substituted analogs on cell growth, we observed an interesting phenomenon in which cells recovered spontaneously from growth inhibition during extended incubations. HPLC studies demonstrated that the 5-thioamide moiety of several structurally dissimilar 7-substituted 4-aminopyrrolo[2,3-d]pyrimidines, including thiosangivamycin, is unstable in cell culture medium and is converted to the corresponding 5-nitrile with a half-life of approximately 48 h.

Large-scale preparation of highly purified, frozen/thawed CD34+, HLA-DR- hematopoietic progenitor cells by sequential immunoadsorption (CEPRATE SC) and fluorescence-activated cell sorting: implications for gene transduction and/or transplantation.

The purification of early hematopoietic progenitor cells for autologous transplantation is based on two rationales: (1) elimination of clonogenic tumor cells, and/or (2) gene transfer into indefinitely self-replicating hematopoietic stem cells. Primitive CD34+ stem cells can be separated from more mature stem cells, or probably from clonogenic tumor cells, by differences in HLA-DR surface antigen expression. The objective of this study was to establish a large-scale technique for purification of CD34+, DR- progenitor cells from a large volume marrow harvest.

Retinoic acid-induced expression of CD38 antigen in myeloid cells is mediated through retinoic acid receptor-alpha.

CD38 is a leukocyte differentiation antigen that has been thought to be a phenotypic marker of different subpopulations of T- and B-lymphocytes. In myeloid cells, CD38 is expressed during early stages of differentiation. Virtually no information is available on regulation and functions of CD38.

Growth inhibition of follicular small-cleaved-cell lymphoma cells in short-term culture by interleukin-3.

Background: The role of interleukin-3 (IL-3) in stimulating the growth of early myeloid progenitor cells is very well established. Therefore, IL-3 has been incorporated into many post-bone-marrow transplantation and intensive chemotherapy programs for the treatment of solid tumors and hematologic malignancies, including lymphomas. However, the effect of IL-3 on normal and malignant lymphocytes has not been well studied.

Synthesis, antiproliferative, and antiviral activity of 4-amino-1-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyridazin-7(6H)-one and related derivatives.

The synthesis of 4-amino-1-beta-D-ribofuranosylpyrrolo[2,3-d]pyridazin-7 (6H)-one (3) from the reaction of ethyl 3-cyano-1-beta-D-ribofuranosylpyrrole-2-carboxylate (10) and hydrazine is described. The 5:6 pyrrolo[2,3-d]pyridazin-7(6H)-one structure of 3 was established via a three-step conversion of 3 into 1-beta-D-ribofuranosylpyrrolo[2,3-d]pyridazin-4,7(5H,6H)- dio ne (14). 4-Amino-3-chloro-1-beta-D-ribofuranosylpyrrolo[2,3-d]pyridazin+ ++-7(6H)-one (16) 4-amino-3-bromo-1-beta-D-ribofuranosylpyrrolo[2,3-d]pyridazin++ +-7(6H)-one (18) were prepared via N-chlorosuccinimide or N-bromosuccinimide treatment of 4-amino-1-(2,3,5-tri-O-benzyl-beta- D-ribofuranosyl)pyrrolo[2,3-d]pyridazin-7(6H)-one (7) followed by a removal of the benzyl groups with boron trichloride.

Rapid induction of CD38 antigen on myeloid leukemia cells by all trans-retinoic acid.

The CD38 or T10 molecule is one of the least understood differentiation antigens. Virtually no information is available on the regulation and functions of CD38 antigen in hematopoietic cells. Using human promyelocytic leukemia cells, we demonstrate that all trans-retinoic acid is a potent and specific inducer of CD38 expression in myeloid lineage.

Immunoglobulin gene rearrangement in plasma cell dyscrasias: detection of small clonal cell populations in peripheral blood and bone marrow.

The bone marrow (BM) and peripheral blood (PB) samples of 71 patients with plasma cell dyscrasias were analysed by the Southern blot technique for the presence of clonal immunoglobulin (Ig) gene rearrangements. 53% of BM samples examined were archival material such as air dried BM slides or frozen trephine biopsies. The results were related to bone marrow plasmacytosis as determined by cytology and flow cytometry, and other clinical parameters.

Fluorescent in situ hybridization and cytogenetic studies of trisomy 12 in chronic lymphocytic leukemia.

Cytogenetic studies (CG) of 475 chronic lymphocytic leukemia (CLL) cases showed trisomy 12 in 6.1% or 26% of patients with abnormal karyotypes. Fluorescence in situ hybridization (FISH) detected trisomy 12 in 35% of 117 CLL patients.

Induction of differentiation in myeloid leukemia cell lines and acute promyelocytic leukemia cells by liposomal all-trans-retinoic acid.

All-trans-retinoic acid (ATRA) induces complete remissions in the majority of patients with acute promyelocytic leukemia. Despite continuous p.o.

Flow cytometric detection of cytoplasmic antigens in acute leukemias: implications for lineage assignment.

This study aimed at optimizing the conditions for flow cytometric detection of myeloperoxidase (MPO), cytoplasmic CD3 (cCD3), and cytoplasmic CD22 (cCD22), which seem to be more reliable lineage-associated markers in acute leukemia than surface antigens. Fixation methods employing saponin as detergent resulted in accurate detection of MPO and cCD3, whereas cCD22 was detectable only after buffered-formaldehyde-acetone fixation. MPO was detected in 16/17 AML, but only in 1/6 ALL, the MPO positive ALL being also CD13 positive.

Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype.

The multidrug-resistance gene, MDR1 is expressed in many normal tissues, but little is known about its expression in normal hematopoietic cells. Using the monoclonal antibody C219 and flow cytometric analysis, P-glycoprotein (P-gp) was found to be expressed in all peripheral blood (PB) subpopulations (CD4, CD8, CD14, CD19, CD56) except granulocytes. To specifically determine MDR1 gene expression, these PB subpopulations were isolated by fluorescence-activated cell sorting (FACS) and analyzed for MDR1 mRNA by polymerase chain reaction (PCR).

Detection of activity of P-glycoprotein in human tumour samples using rhodamine 123.

Based on the fluorescent properties of the dye rhodamine 123 (Rh123), which is transported by the membrane efflux pump P-glycoprotein (P-gp), we developed a functional flow cytometric assay for the detection of multidrug-resistant (MDR) cells. Using drug sensitive cell lines (KB-3-1) and MDR mutants (KB-8-5, KB-C1) experimental conditions were established that enabled demonstration of significant differences in Rh123 efflux and accumulation. Subsequently we investigated the applicability of this functional assay for the prediction of MDR in human peripheral blood and bone marrow samples.

Improved synthesis and biological evaluation of an acyclic thiosangivamycin active against human cytomegalovirus.

We previously described the synthesis and in vitro antiviral activity of an acyclic thiosangivamycin analog (Gupta et al., 1989a). In order to extend these initial studies, a new, multi-gram synthesis of 4-amino-7-[(2-hydroxy- ethoxy)methyl]pyrrolo]2,3-d]pyrimidine-5-thiocarboxamide (compound 229) was achieved in 5 steps from the known 5-amino-2-bromo-3,4-dicyanopyrrole in good overall yield.

Proliferation of hematopoietic cells is accompanied by suppressed expression of heat shock protein 70.

In this study, we have examined the synthesis of heat shock protein (HSP70) in leukemia cells from acute myelogenous leukemia (AML) patients and in mononuclear cells from normal individuals, before and after growth factor stimulation. We have shown that the HSP70 protein was expressed in these cells in the absence of temperature elevation. Stimulation of proliferation of AML cells by the growth factors interleukin-3 and granulocyte-macrophage colony-stimulating factor and stimulation of normal lymphocytes by phytohemagglutinin (PHA) resulted in decreased synthesis of HSP70, suggesting that high levels of HSP70 are associated with cellular differentiation.

Regulation of the expression of ICAM-1 on human monocytes and monocytic tumor cell lines.

In this study we investigated the mechanisms regulating expression of ICAM-1 that plays an important role for Ag presentation, on peripheral blood monocytes, and three myelomonocytic cell lines representing different stages of monocyte maturation. ICAM-1 expression on freshly isolated monocytes was low in all donors tested. After 16- to 20-h incubation, about a 20-fold increase was observed, whereas ICAM-1 expression on lymphocytes did not change.