bryostatin-1 and Cell-Transformation--Neoplastic

Although the role of serine/threonine protein kinase C (PKC) in malignant transformation is known from decades, an anti-PKC based approach in cancer therapy was hampered for the difficulties in developing pharmacological compounds able to selectively inhibit specific PKC isoforms. In this review, the role of PKC-epsilon and PKC-delta in promoting and counteracting tumor progression in different types of cancer, respectively, will be discussed in relationship with promising therapeutic perspectives based either on small molecule inhibitors or on natural compounds. Among a myriad of molecules able to modulate PKC activity, we will focus on the role of the enzastaurin and briostatin-1, which already entered clinical trials for several human cancers.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bryostatins; Cell Cycle; Cell Transformation, Neoplastic; Clinical Trials as Topic; Humans; Indoles; Neoplasm Metastasis; Neovascularization, Pathologic; Plant Extracts; Protein Kinase C-delta; Protein Kinase C-epsilon; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Topics: 3T3 Cells; Animals; Bryostatins; Carcinogens; Cell Transformation, Neoplastic; Enzyme Activation; Humans; Isoenzymes; Lactones; Macrolides; Mice; Mice, Inbred C3H; Phorbol Esters; Protein Binding; Protein Kinase C; Receptors, Cell Surface; Signal Transduction; Skin Neoplasms; Structure-Activity Relationship

It is well known that the differentiation of acute promyelocytic leukaemia (APL) cells by all-trans-retinoic acid (ATRA) may be enhanced by myeloid growth factors, but the requirement for growth factors in this process is unclear. Our previous studies in multiple myeloma and non-APL acute myeloid leukaemia demonstrated that lineage-specific growth factors are required for the maximal activity of many pharmacologic differentiating agents in vitro. Thus, we studied whether the differentiation of APL is similarly dependent on growth factors. We found that the myeloid growth factors granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor markedly increased the differentiation of NB4 cells or APL blasts from clinical samples treated with ATRA, arsenic trioxide (ATO), or bryostatin-1 as evidenced by the enhanced expression of myeloid surface antigens and the inhibition of clonogenic growth. Furthermore, myeloid growth factors were necessary for the differentiation of APL cells since the activity of each pharmacologic agent could be blocked by specific growth factor-neutralizing antibodies. Each differentiating agent was active only at concentrations that inhibited cell cycling, suggesting that this property is also required for differentiation. These data demonstrate that both pharmacologic differentiating agents and myeloid growth factors are required, but neither sufficient, for the differentiation of APL cells. The combined use of pharmacologic differentiating agents and growth factors may improve the clinical efficacy of differentiation therapy in APL.

Topics: Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Bryostatins; Cell Cycle; Cell Line, Tumor; Cell Transformation, Neoplastic; Flow Cytometry; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Inhibitors; Humans; Leukemia, Promyelocytic, Acute; Macrolides; Myelopoiesis; Oxides; Tretinoin

Tumor-promoting phorbol esters activate, but then deplete cells of, protein kinase C (PKC) with prolonged treatment. It is not known whether phorbol ester-induced tumor promotion is due to activation or depletion of PKC. In rat fibroblasts overexpressing the c-Src proto-oncogene, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent growth and other transformation-related phenotypes. The appearance of transformed phenotypes induced by TPA in these cells correlated not with activation but rather with depletion of expressed PKC isoforms. Consistent with this observation, PKC inhibitors also induced transformed phenotypes in c-Src-overexpressing cells. Bryostatin 1, which inhibited the TPA-induced down-regulation of the PKCdelta isoform specifically, blocked the tumor-promoting effects of TPA, implicating PKCdelta as the target of the tumor-promoting phorbol esters. Consistent with this hypothesis, expression of a dominant negative PKCdelta mutant in cells expressing c-Src caused transformation of these cells, and rottlerin, a protein kinase inhibitor with specificity for PKCdelta, like TPA, caused transformation of c-Src-overexpressing cells. These data suggest that the tumor-promoting effect of phorbol esters is due to depletion of PKCdelta, which has an apparent tumor suppressor function.

Topics: Animals; Antineoplastic Agents; Bryostatins; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Down-Regulation; Fibroblasts; Isoenzymes; Lactones; Macrolides; Phenotype; Protein Kinase C; Protein Kinase C-alpha; Protein Kinase C-delta; Protein Kinase C-epsilon; Proto-Oncogene Proteins pp60(c-src); Rats; Tetradecanoylphorbol Acetate; Transfection

It has been previously reported that Bryostatin 1 (Bryo1) induces differentiation of the human acute lymphoblastic leukemia (ALL) cell line, Reh, to a monocytoid B-cell stage. In this study we demonstrate that a novel protein, ubiquitin COOH-terminal hydrolase (UCH-L1), is associated with this differentiation. Reh cells were treated with 200 nmol/l of Bryo1 for 72 h and analyzed for changes in morphology, surface immunophenotype, acid phosphatase and terminal deoxynucleotidyl transferase. Protein patterns of the parent and differentiated cells, by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), were studied. Bryo1-treated cells expressed morphologic, phenotypic and enzymatic features of the monocytoid B-cell stage. The UCH-L1 enzyme (MW-pl 34-5.3) was detected by 2 D PAGE in the differentiated, but not in parent cells. The presence of UCH-L1 in the Bryo1-treated cells was further confirmed by immunoblotting of 2 D PAGE using UCH-L1 polyclonal antibody. Ubiquitin expression was studied in parent and Bryo1-treated cells and was compared with 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells. Both agents, TPA and Bryo1, increased the level of ubiquitin expression as detected by flow cytometry. Sodium borohydride, an inhibitor of UCH-L1, inhibited the Bryo1-induced differentiating effect on Reh cells. To date, the mechanism by which Bryo1, exerts its B-cell differentiating effect is not fully understood. This study shows that UCH-L1 expression may play a major role in Bryo1-induced differentiation in pre-B-ALL.

Topics: Adolescent; Animals; Bryostatins; Cell Division; Cell Transformation, Neoplastic; Electrophoresis, Gel, Two-Dimensional; Enzyme Induction; Female; Gene Expression Regulation, Neoplastic; Humans; Immune Sera; Immunoblotting; Lactones; Leukemia, Lymphoid; Macrolides; Mitogens; Rabbits; Thiolester Hydrolases; Tumor Cells, Cultured; Ubiquitin Thiolesterase

Previously it was reported that transformation of NIH 3T3 fibroblast by the Ha-ras, v-src, v-fms, and A-raf oncogenes decreased the stimulatory effects of phorbol 12-myristate 13-acetate (PMA; 'TPA'), an activator of protein kinase C (PKC), on the phosphorylation of an endogenous 80 kDa substrate and on 86Rb uptake [Wolfman, Wingrove, Blackshear & Macara (1987) J. Biol. Chem. 262, 16546-16552], as well as on sphingomyelin synthesis [Kiss, Rapp & Anderson (1988) FEBS Lett. 240, 221-226]. Here, we investigated how transformation affects the PMA-stimulated hydrolysis of phosphatidylethanolamine (PtdEtn), a recently characterized mechanism which may contribute to the generation of the second messengers phosphatidic acid and 1,2-diacylglycerol. The effects of PMA were compared with those of bryostatin, a non-tumour-promoter activator of PKC. Transformation of NIH 3T3 cells with Ha-ras, v-raf, or A-raf enhanced the stimulatory effect of PMA on the phospholipase D-mediated hydrolysis of PtdEtn. On the other hand, the effects of bryostatin on PtdEtn hydrolysis were only slightly increased, if at all, in cells transformed with these oncogenes. In crude membrane preparations isolated from these transformed cells, PMA, but not bryostatin, enhanced the combined stimulatory effects of ATP and the GTP analogue guanosine 5'-[gamma-thio]triphosphate on phospholipase D-mediated PtdEtn hydrolysis. The PKC inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine inhibited the stimulatory effect of PMA only in intact cells. These results indicate that transformation of cells by certain oncogenes differentially affects phospholipase D-mediated hydrolysis of PtdEtn induced by PMA and bryostatin, suggesting that the action of PMA might involve two different mechanisms.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Bryostatins; Cell Line; Cell Transformation, Neoplastic; Genes, ras; Guanosine 5'-O-(3-Thiotriphosphate); Hydrolysis; Kinetics; Lactones; Macrolides; Mice; Mice, Inbred Strains; Oncogene Proteins v-raf; Oncogenes; Phosphatidylethanolamines; Phospholipase D; Protein Kinase C; Protein-Tyrosine Kinases; Retroviridae Proteins, Oncogenic; Rubidium; Tetradecanoylphorbol Acetate