sq-23377 and Mast-Cell-Sarcoma

Steel factor (SLF), the ligand for the c-Kit receptor, protects hemopoietic progenitors and mast cells from apoptosis. We show here that protection of 32D-Kit cells or mast cells from apoptosis by SLF is abrogated through concurrent inhibition of Ca2+ influx. In contrast, cell survival promoted by interleukin-3 is not affected by Ca2+ influx blockers. In the presence of blockers, increasing stimulation by SLF leads to greater levels of cell death in the population, indicating that it is the combination of activation by SLF with concurrent blockade of Ca2+ influx that results in apoptosis. The p815 mastocytoma, which expresses a mutated, constitutively active c-kit receptor, dies apoptotically in the presence of Ca2+ influx blockers alone. Ionomycin protects cells from SLF plus blocker-induced apoptosis, confirming specificity for Ca2+ ion blockade in cell death induction. Overexpression of bcl-2, which protects 32D-Kit cells from factor withdrawal, does not protect cells from apoptosis by SLF plus blocker. In contrast, caspase inhibitors YVAD-CHO, DEVD-FMK, and Boc-Asp-FMK protect cells from SLF plus blocker-induced death. These observations highlight the importance of SLF-stimulated Ca2+ influx in the protection of cells from apoptosis and demonstrate a new mechanism for inducing bcl-2 insensitive, caspase-dependent apoptosis through the combination of SLF stimulation with Ca2+ influx blockade.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calcium; Calcium Channel Blockers; Caspase 1; Cell Line; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Humans; Interleukin-3; Ion Transport; Ionomycin; Mast Cells; Mast-Cell Sarcoma; Mice; Monocytes; Oligopeptides; Poly(ADP-ribose) Polymerases; Protease Inhibitors; Proto-Oncogene Proteins c-kit; Recombinant Proteins; Stem Cell Factor; Tumor Cells, Cultured

Mast cells, mastocytoma cells and basophil leukaemia cells are well-established producers of leukotrienes when grown and stimulated appropriately. I report that the cells' ability to produce leukotrienes is dependent on the cells' proliferative status or their provision with growth factors. Proliferating MC/9 and subconfluent RBL2H3 cells respond maximally to stimulation by 1 microM ionomycin with the production of 56 and 32 pmol of cysteinyl-leukotrienes/10(6) cells respectively. In contrast, confluent RBL2H3 or growth-arrested MC/9 cells lose their ability to generate leukotrienes in response to ionomycin treatment. This rapid down-regulation of leukotriene synthesis is also observed when proliferating RBL2H3 cells are transferred to growth-factor-free medium, wherein cellular leukotriene-synthesis capacity has an apparent half-lifetime of 60 min. Transfer back into growth medium results in the regeneration of leukotriene synthesis capacity within 6 h. In growth-arrested MC/9 cells, leukotriene production ability can at least partially be restored by priming the cells with interleukin 3, but not with interleukin 4. In RBL2H3 cells, pretreatment with protein tyrosine kinase inhibitors such as genistein (5 min, 37 microM), herbimycin A (6 h, 3 microM) or tyrphostin 25 (16 h, 100 microM) completely inhibits leukotriene generation, whereas okadaic acid (15 min, 0.5 microM) has no effect. Under these conditions, both genistein and herbimycin A strongly impair ionomycin-induced protein tyrosine phosphorylation. Our study indicates that leukotriene generation in these tumour cells is tightly regulated by their proliferation status and supply with growth factors, and cell stimulation towards leukotriene synthesis appears to involve protein tyrosine kinase activity.

Topics: Animals; Benzoquinones; Catechols; Cell Division; Cell Line; Genistein; Interleukin-3; Interleukin-4; Ionomycin; Isoflavones; Kinetics; Lactams, Macrocyclic; Leukemia, Basophilic, Acute; Leukotrienes; Mast-Cell Sarcoma; Mice; Nitriles; Phosphoproteins; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Tumor Cells, Cultured; Tyrosine; Tyrphostins

We investigated the efficacy of IL-2, LPS, MDP, TRA, ionomycin and contrykal on proliferation of lymphocytes treated by tumor cell immunosuppressive factors (ISF). IL-2, LPS and/or MDP did not abolish the influence of P815 and B16 ISF on Con A or alloantigen-induced lymphocyte proliferation. TPA and in less extent ionomycin and combination of the above preparations totally abrogated the suppression of Con A-induced lymphocyte proliferation. In inverted experiments Con A abrogated ISF-mediated suppression of lymphocyte proliferation induced by TPA plus ionomycin.

Topics: Acetylmuramyl-Alanyl-Isoglutamine; Animals; Aprotinin; Concanavalin A; Immune Tolerance; Interleukin-2; Ionomycin; Lipopolysaccharides; Lymphocyte Activation; Mast-Cell Sarcoma; Melanoma, Experimental; Mice; Sarcoma, Experimental; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Subcloning of interleukin 3 (IL-3)-dependent PB-3c mastocyte cells revealed two populations, of which only one is sensitive to oncogenic transformation by v-H-ras. The corresponding tumors produce IL-3 and grow in vitro in the absence of exogenous IL-3 [Nair, A.P.K., Diamantis, I.D., Conscience, J.F., Kindler, V., Hofer, P. & Moroni, Ch. (1989). Mol. Cell. Biol., 9, 1183-1190]. In the present investigation, IL-3 gene regulation was compared in ras transformable (rT) and ras nontransformable (rNT) lines. We report that upon expression of v-H-ras rT clones but not rNT clones express low levels of IL-3 mRNA as detected by reverse polymerase chain reaction. Treatment with ionomycin, a calcium ionophore, induced high levels of IL-3 expression only in ras-expressing rT clones. Somatic cell fusion between the rNT clone 20 and the IL-3-expressing mastocytoma line V2D1 led to down-regulation of IL-3 expression and to the requirement for exogenous IL-3 for in vitro growth and tumor suppression. In contrast, rT clone 15 lacked tumor-suppressor activity and failed to down-regulate IL-3 expression in somatic hybrids which grew in vitro without added IL-3. Our results indicate that IL-3 gene expression is a critical determinant for the generation of v-H-ras-induced mast cell tumors and show that disturbances in IL-3 gene regulation can be detected already at the premalignant level in v-H-ras transformation-sensitive cells.

Topics: Animals; Base Sequence; Cell Transformation, Neoplastic; DNA; Genes, ras; Interleukin-3; Ionomycin; Mast Cells; Mast-Cell Sarcoma; Mice; Mice, Inbred DBA; Molecular Sequence Data; RNA, Messenger

Several strategies have been used to stimulate the growth of tumor-specific T cells in place of tumor antigen. One approach is to use pharmacologic agents to activate the second messenger pathways of T-cell activation. In the present study, we examined the ability of the protein kinase C activator bryostatin 1 (B) plus the calcium ionophore ionomycin (I) to stimulate the growth of lymphocytes obtained from the axillary lymph nodes (DLN) draining a progressively growing intradermal plasmacytoma tumor. Draining lymph node cells were initially cultured with autologous tumor cells and 20 U/ml of interleukin-2 (IL-2) for 7 days. The lymphocytes were then incubated with various concentrations of bryostatin 1 plus 1 microM ionomycin and cultured for an additional 14 days in IL-2. DLN cells initially cultured with autologous tumor and then restimulated with 5 nM bryostatin 1 and 1 microM ionomycin exhibited marked in vitro proliferation and 15-fold expansion of cell numbers over 2 weeks. The cells expanded with B/I were predominantly CD8+ T cells and retained specific in vitro cytotoxicity against autologous tumor. When adoptively transferred to mice with established liver metastases, DLN cells restimulated with B/I-mediated specific tumor regression.

Topics: Animals; Bryostatins; Enzyme Activation; Immunotherapy, Adoptive; Ionomycin; Lactones; Lymph Nodes; Lymphocyte Activation; Macrolides; Mast-Cell Sarcoma; Mice; Mice, Inbred DBA; Protein Kinase C; T-Lymphocytes, Cytotoxic

Thrombin induced an increase in [Ca2+]i in mouse mastocytoma P-815 cells. This increase was markedly reduced by prior exposure to pertussis toxin (PT) but not by removal of extracellular Ca2+, suggesting that thrombin stimulates phospholipase C via a PT-sensitive GTP-binding protein. ATP also induced an increase in [Ca2+]i. This increase was insensitive to PT but completely suppressed on removal of extracellular Ca2+, suggesting that ATP stimulates Ca2+ influx in a PT-insensitive manner. Iloprost, a stable prostacyclin analogue, increased the cellular cAMP level and dose-dependently inhibited the thrombin-induced increase in [Ca2+]i, whereas the ATP-induced increase in [Ca2+]i was markedly enhanced by iloprost. Cyclic AMP analogues, dibutyryl cAMP and 8-bromo cAMP, also inhibited the increase in [Ca2+]i induced by thrombin and promoted that by ATP, indicating that the inhibitory and stimulatory effects of iloprost are mediated by cAMP. These results suggest that the prostacyclin receptor differentially regulates two distinct Ca2+ mobilizing systems via cAMP in mastocytoma cells.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Triphosphate; Aluminum; Aluminum Chloride; Aluminum Compounds; Animals; Bucladesine; Calcium; Cell Line; Chlorides; Cyclic AMP; Epoprostenol; Iloprost; Ionomycin; Kinetics; Mast-Cell Sarcoma; Mice; Pertussis Toxin; Prostaglandins; Receptors, Epoprostenol; Receptors, Prostaglandin; Sodium Fluoride; Thrombin; Virulence Factors, Bordetella