Alexander Bloch

Differential expression of proteins regulating cell cycle progression in growth vs. differentiation

The level of various G1 cyclins and cyclin-dependent kinases (cdks) present in the nuclei of synchronized ML-1 human myeloblastic leukemia cells was determined as a function of time after initiation of cell growth with insulin-like growth factor-1 (IGF-1) and transferrin (Tf), and following induction of differentiation with transforming growth factor-beta1 (TGF-beta1). Cyclin E and cdk2 were expressed at relatively high levels in the nuclei of proliferation-stimulated cells, whereas cyclin D1 and cdk5 were expressed at comparably high levels in the nuclei of differentiation-induced cells. In the nuclear extracts from proliferation-stimulated cells, cyclin E complexed specifically with cdk2, whereas in nuclear extracts from differentiation-induced cells, cyclin D1 bound specifically to cdk5. Increased cyclin E/cdk2 expression was accompanied by increased DNA synthesis, whereas increased cyclin D1/cdk5 levels correlated with decreased DNA synthesis. In both growth- and differentiation-induced cells, cyclin D2 expression preceded the expression of cyclin D3, and a significantly larger amount of these cyclins was present in differentiation- as compared to proliferation-induced cells. In contrast, cdk4 and cdk6 were present at similar levels in the nuclear extracts from both growth- and differentiation-induced cells. These data show that, in ML-1 cells, the proliferation-associated progression from G1 to S, as well as the differentiation-associated transit from G1 to maturation is accompanied by the expression of specific cyclin/cdk pairs, comprising cdk2/cyclin E in growth and cdk5/cyclin D1 in differentiation.

Differential ability of mitogen-stimulated human leukocyte-conditioned media to induce Fc receptors in human leukemia cells

The ability of mitogen-stimulated human leukocyte-conditioned media (M-CM) to induce the in vitro differentiation of various human leukemic cell lines was evaluated by measuring the appearance of Fc receptors (FcR) through their ability to form EA rosettes. Only cells of myeloid lineage were induced by M-CM to express FcR; T-, and B-, and non-T/non-B cells failed to respond. As determined with ML-1, a line of human myeloblastic leukemia cells, pokeweed mitogen-conditioned medium, at concentrations of 1-10%, stimulated the expression of FcR in 70-98% of the cells within 1 day after treatment. Phytohemagglutinin-, concanavalin A-, and lipopolysaccharide-conditioned media were less active. The FcR-inducing activity was partially separated from M-CM by chromatography on Sephadex G-75. It was stable between pH 4 and 10, and lost activity at temperatures above 40 degrees C.

Differentiation-associated changes in human non-T, non-B leukemia cell lines after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA)

The ability of TPA to induce stable phenotypic changes that normally serve as markers of differentiation was examined in the four human non-T, non-B cell lines, NALL-1, NALM-16, REH and KM-3. In all four lines, noncytotoxic concentrations of the phorbol ester caused an extensive reduction in the number of cells expressing cALL surface antigen and terminal deoxynucleotidyl transferase. The disappearance of these markers correlated with the loss of cell proliferation. In one of the cell lines, NALL-1, TPA treatment gave rise to a significant increase in Ia-like antigen and antigen T-101, markers which represent more advanced stages of cell maturation. However, surface or cytoplasmic immunoglobins, indicators of mature B cells, were not detectable. Antigen 3A1, specific for myeloid and for T cells, antigen Leu-4, specific for T cells and antigen CM1, specific for monocytes, were also absent. In all cell lines, exposure to TPA resulted in an approximately two-fold increase in acid phosphatase and beta-glucuronidase activity. The emergence of these phenotype changes was not altered upon repeated washing of the TPA-treated cells. These results demonstrate that while TPA is capable of inducing various non-T, non-B cell lines to differentiate to a limited degree, differences exist between the lines in the extent to which they can mature towards the B-cell stage.

A Practical Synthesis of The Antibiotic Toyocamycin

Abstract The nucleoside antibiotic toyocamycin was synthesized by condensation of the silylated 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose, followed by debromination and deblocking.

Synthesis of 5′-Substituted Derivatives of the Pyrrolo[2,3-d]-Pyrimidine Nucleoside Sangivamycin and Their Effect on Protein Kinase A and C Activity

Abstract Under cell-free conditions, where the antibiotic sangivamycin is not phosphorylated, it is an effective inhibitor of PKC and to a lesser extent of PKA activity. In intact cells, the antibiotic is phosphorylated, thereby, extending its range of activity to other targets including DNA and RNA. To preserve selective inhibitory activity for the protein kinases, analogs potentially resistant to phosphorylation were prepared by replacing the 5′-hydroxy group with O-nitro, O-sulfamoyl, O-methane-sulfonyl or azido groups. These compounds were more potent inhibitors of PKA and PKC activity than was the parent nucleoside.

Relationship Between the Structure of Sangivamycin-Derived Nucleosides and Their Effect on Leukemic Cell Growth and on Protein Kinase A and C Activity

Abstract The nucleoside antibiotic sangivamycin (4-amino-7-(β-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-5-carboxamide, (1) is an effective inhibitor of protein kinase A (PKA) and protein kinase C (PKC) but, upon its phosphorylation in intact cells, it gains the ability to affect other targets as well. To retain its selectivity for the protein kinases, a series of nonphosphorylatable sangivamycin derivatives was prepared by replacing the 5′-hydroxyl group with other functions including N3, F, SO2NH2, NO2, and NH2, These derivatives were more potent inhibitors of PKA and PKC than were the phosphorylatable compounds, although the latter were more potent inhibitors of leukemic cell growth.

A regulatory role for tumor necrosis factor (TNF) in ML-1 human myeloblastic leukemia cell maturation

ML-1 human myeloblastic leukemia cells are induced to differentiate to monocytes by conditioned medium (CM) derived from tetradecanoylphorbol acetate (TPA)-treated ML-1 cells. Antibodies to tumor necrosis factor (TNF) inhibited the differentiation-stimulating activity of CM, indicating that this activity is due to the presence of TNF in CM. TNF added to non-conditioned medium was as effective as CM in stimulating ML-1 cell differentiation. In the presence of a low (0.12 ng/ml) concentration of TPA, TNF-induced maturation was synergistically increased and Type I macrophages were formed. With higher (1-10 ng/ml) TPA concentrations, Type II macrophages were also obtained. As the TNF/TPA concentration increased, ML-1 cell differentiation was increasingly inhibited. Mature cells derived from ML-1 cells were found to secrete TNF at concentrations ranging from less than 2 U/ml to greater than 180 U/ml, the amount depending upon the number of cells and the stage of cell maturation. These results indicate that TNF participates in the regulation of precursor cell maturation. Low concentrations of TNF produced by small numbers of mature cells stimulate differentiation, whereas high concentrations of TNF generated by elevated numbers of macrophages inhibit the maturation process, possibly in combination with other cytokines. Because TNF serves as a competence factor for ML-1 cells, (Guan X.-P., Takuma T., Hromchak R. & Bloch A. (1990) Competence and progression in cell differentiation. Proc. Am. Assoc. Cancer Res. 31, 28.), the TNF-induced stimulation of differentiation depends additionally on the action of serum-contained differentiation-specific progression factors.

Differential effect of growth- and differentiation-inducing factors on the release of eicosanoids and phospholipids from ML-1 human myeloblastic leukemia cells

In the absence of serum, growth of ML-1 human myeloblastic leukemia cells is induced by the insulin-like growth factor-1 (IGF1) together with transferrin (Tf), whereas monocytic differentiation is initiated by the transforming growth factor-beta (TGF-beta) in combination with Tf. Initiation of growth was followed by the rapid release of arachidonic acid (AA), hydroxyeicosatetraenoic acids (HETEs) and phospholipids into the culture medium. In contrast, induction of differentiation occurred without the release of these lipids beyond the level present in control. Inhibitors of enzymes involved in the formation of AA and of HETEs, including phospholipase A2 and lipoxygenases, caused interference with growth but not with differentiation, and an inhibitor of the cyclooxygenase path affected neither growth nor differentiation. These results indicate that the initiation of ML-1 cell growth but not of cell differentiation is dependent upon the increased formation of AA and its derivatives formed primarily via the lipoxygenase path.

Induction of protein kinase C translocation and cell differentiation in ML-1 human myeloblastic leukemic cells by tumor necrosis factor-alpha, transforming growth factor-beta, or tetradecanoylphorbol acetate.

Tumor necrosis factor-alpha and transforming growth factor-beta, like 12-O-tetradecanoylphorbol 13-acetate, induce differentiation of ML-1 human myeloblastic leukemia cells along the monocyte path. As measured at 5 min following exposure of the cells to either of these agents, extensive translocation of protein kinase C from the cytosolic to the membrane fraction occurred. A correlation was observed to exist between protein kinase C translocation, cell differentiation, and cessation of cell growth induced by transforming growth factor-beta and tumor necrosis factor-alpha.

Synthesis and Biological Activity of 6-Hydroxyguanidino-and 6-Hydroxyureidopurine And Their Ribonucleosides

Abstract N 6 −(1-hydroxyguanidino)purine IIa, and its 9-β-D-ribonucleoside derivative IIb were prepared by reacting at room temperature 6-hydroxyadenine Ia and 6-hydroxyadenosine Ib, with 1-guanyl-3,5-dimethylpyrazole nitrate in DMF. Refluxing IIa and IIb in 95% ethanol gave N6−(1-hydroxyureido)purine and its ribonucleoside derivative respectively; the latter compound was also obtained by refluxing Ib with 1-guanyl-3,5-dimethylpyrazole nitrate in ethanol. The two base analogs were inactive against L1210 cells in vitro, but the nucleoside derivatives inhibited the growth of these cells by 50% at 5 × 10 -6 and 6 × 10−7 M respectively. Compound IIb, at 200 mg/kg/day × 5, increased the life span of L1210-bearing DBA/2N mice by 57%. Cytofluorometric determinations showed that IIb inhibited cell growth in the G2 phase of the cell cycle. also found to inhibit adenosine deaminase activity with a Ki = 3.47 μM.