P815 Murine Mastocytoma Research Articles

Differentiation in murine mastocytoma induced by macrophage gangliosides.

In the membrane of mouse macrophages two gangliosides were detected which inhibit the division of murine mastocytoma P815 tumor cells. The two gangliosides were incorporated into the cytoplasmatic membrane of mastocytoma cells. The concentration necessary to achieve a complete inhibition of P815 tumor cell division is about 1 microM for both effective gangliosides. Macrophage ganglioside-induced inhibition of cell division is accompanied by morphological changes of the mastocytoma cells. While the cells are rounding, their diameter increases and serotonin and granules appear in the cytoplasm of the enlarged cells. Our findings suggest that macrophage gangliosides may differentiate mastocytoma cells into mast cells.

Euplectin and Coneuplectin, New Naphthopyrones from the LichenFlavoparmelia euplecta

Two new naphthopyrones, euplectin (1) and coneuplectin (2), have been isolated from the lichen Flavoparmelia euplecta and their structures elucidated using multidimensional NMR spectroscopic methods, including highfield (600 MHz) gHMBC and gNOE experiments. Cytotoxicity of the more abundant naphthopyrone (1) against the murine P-815 mastocytoma cell line (IC(50) ca. 1.67 microg/mL) has also been evaluated. These compounds are the first lichen metabolites known to contain indenone or indanone moieties in their structure. F. euplecta was also found to contain the known metabolites usnic acid, protocetraric acid, and skyrin.

Hybocarpone, a novel cytotoxic naphthazarin derivative from mycobiont cultures of the lichen Lecanora hybocarpa

Hybocarpone ( 1), a novel pentacyclic naphthazarin-derived dimer has been isolated from mycobiont cultures derived from the lichen Lecanora hybocarpa. The structure of hybocarpone ( 1) was established on the basis of 1D and 2D NMR spectroscopy. Its relative stereochemistry was predicted with the use of molecular modelling and confirmed by X-ray crystallographic analysis. Hybocarpone ( 1) was found to be a potent cytotoxin (IC 50 c 0.27 μM), active against the murine P815 mastocytoma cell line.

In VivoDifferentiation of Mast Cells from Acute Myeloid Leukemia Blasts Carrying a Novel Activating Ligand-Independent C-kit Mutation

ABSTRACT: The primary role of protooncogene c-kit in mast cell differentiation is supported by the development of mast cells from CD34+/CD117+(c-kit) myeloid precursors. Growth factor independence, neoplastic transformation and differentiation of mast cells were found in association with c-kit activating mutations in both murine and human mastocytoma and mast cell diseases. We have identified a novel c-kit mutation (D816Y) in peripheral blood mononuclear cells from a patient with AML (M2), massive presence of mast cells in bone marrow and rapid progression of the disease. The mutation, a G→T transversion at nt 2467 of the c-kit gene resulting in Asp816→Tyr substitution, corresponds to the D814Y and D817Y mutations identified and characterized in the murine P815 mastocytoma and the rat RBL-2H3 mast cell leukemia cell lines. The absence of SCF transcripts that we found by RTPCR in the patient's blasts indicates that, also in humans, this activating mutation leads to SCF independent growth. The expression of the mutant allele on Kit signaling may be further enhanced by trisomy of chromosome 4 (carrying the c-kit gene) in the patient's blasts. From these findings it is concluded that mast cells could be generated from a leukemic CD34/CD117-positive clone, that combines the antigenic expression of mast cell precursor to the growth and differentiation factor-independence which was derived by the c-kit D816Y mutation.

Photodynamic Treatment with Benzoporphyrin Derivative Monoacid Ring A Produces Protein Tyrosine Phosphorylation Events and DNA Fragmentation in Murine P815 Cells

Treatment with benozopophyrin derivative monoacid ring A (BPD-MA, verteporfin) and broad-spectrum fluorescent light rapidly produced apoptosis in murine P815 mastocytoma cells. Fragmentation of DNA, a fundamental characteristic of cells undergoing apoptosis, was evident within 3 h following the photodynamic treatment. Western immunoblot analysis using the specific antiphosphotyrosine monoclonal antibody 4G10 indicated that molecular species of > 200 kDa were phosphorylated on tyrosine residues during or immediately following the irradiation of cells loaded with BPD-MA. Increased tyrosine phosphorylation of a 15 kDa protein was evident by 15 min postirradiation. In the absence of light, BPD-MA did not affect the status of cellular protein tyrosine phosphorylation or cause DNA fragmentation. The protein kinase inhibitor staurosporine prevented tyrosine phosphorylation of the > 200 kDa species but did not affect tyrosine phosphorylation of the 15 kDa protein or the level of DNA fragmentation produced by the photodynamic treatment. The protein tyrosine phosphorylation events observed for P815 cells treated with cytotoxic levels of BPD-MA and light may not be directly related to the induction of the apoptotic cell death pathway.

Synthesis, Characterization and Antitumour Activity of 5-Chlorouracil and 5-Chlorouracil-Histidine Complexes with Some Metal(III) Ions

Complexes of the type [AIL(OH)CI], [ML(OH)2] and [M′LL′(H2O)CI], where HL = 5-chlorouracil; HL′ = histidine; M = Cr(III) or Fe(III) and M′ = Al(III), Cr(III) or Fe(III), were synthesized and characterized. The complexes are polymeric in nature showing high decomposition points and are insoiuble in water and common organic solvents. 5-Chlorouracil is coordinated to the metal ion through the O atom of C(4)˭O and the N atoni of N(1) while histidine through the O atom of -COO− and the N atom of -NH2 group. The μeff values, electronic spectral bands and ESR spectra suggest a polymeric 6-coordinate spin-free octahedral stereochemistry for the Cr(III) and Fe(III) complexes. In vivo and in vitro antitumour activity results indicate that the Cr(III) complexes have significant activity against P815 murine mastocytoma but the Al(III) complexes show poor activity.

Treatment of the P815 murine mastocytoma with cisplatin or etoposide up‐regulates cell‐surface Fas (CD95) expression and increases sensitivity to anti‐Fas antibody‐mediated cytotoxicity and to lysis by anti‐CD3‐activated killer‐T cells

Treatment of the P815 murine mastocytoma with cisplatin or etoposide up‐regulates cell‐surface Fas (CD95) expression and increases sensitivity to anti‐Fas antibody‐mediated cytotoxicity and to lysis by anti‐CD3‐activated killer‐T cells

Treatment of the P815 murine mastocytoma with cisplatin or etoposide up-regulates cell-surface Fas (CD95) expression and increases sensitivity to anti-Fas antibody-mediated cytotoxicity and to lysis by anti-CD3-activated killer-T cells.

We have investigated the effect of pre-treatment with the anti-cancer drugs cisplatin and etoposide on the susceptibility of P815 murine mastocytoma cells to lysis by murine spleen-derived anti-CD3-activated killer-T (AK-T) cells. A 20 hr pre-treatment with cisplatin (0.2-2 microg/ml) or etoposide (0.01-1 microg/ml) rendered P815 cells significantly more sensitive to AK-T cell-mediated lysis in a 4 hr 51Cr-release assay than untreated control tumor cells. At lower concentrations, pre-treatment with cisplatin or etoposide had no direct cytotoxic effects on P815 tumor cells, as measured by the MTT assay. AK-T cell-mediated killing of P815 tumor cells pre-treated with 2 microg/ml cisplatin or 1 microg/ml etoposide was only partially inhibitable by the Ca2+ chelator EGTA, suggesting that the Ca2+-independent Fas (CD95)/Fas ligand cytolytic pathway of AK-T cells contributes to cytotoxicity. In comparison to untreated control P815 cells, 2 microg/ml cisplatin- or 1 microg/ml etoposide-treated P815 cells exhibited increased expression of Fas mRNA and cell-surface Fas, which correlated with increased sensitivity to lysis by AK-T cells. In addition, pre-treatment with cisplatin or etoposide caused P815 tumor cells to become sensitive to the cytotoxic effects of anti-Fas antibody in a 4 hr 51Cr-release assay. Taken together, our results demonstrate that short-term exposure to concentrations of cisplatin and etoposide in the low cytotoxic range and below up-regulates Fas expression by P815 tumor cells, thereby facilitating cytotoxicity mediated through the Fas/Fas ligand cytolytic pathway.

Targeting an anti-viral CD8+ T cell response to a growing tumor facilitates its rejection.

We demonstrate in a murine model that targeting an anti-viral T cell response to a growing tumor facilitates priming of a tumor-associated antigen (TAA)-specific, rejecting T cell response. Murine P815 mastocytoma cells grow aggressively in a syngeneic host. Transfected P815/S cells (expressing the hepatitis B surface antigen, HBsAg) also grow as subcutaneous tumors, but occasional 'spontaneous' rejections after transient growth are observed. Growth of P815/S tumors (but not of P815 tumors) is efficiently suppressed by a CD8+ cytotoxic T lymphocyte (CTL)-dependent immune mechanism in mice primed to HBsAg by DNA-immunization. In hosts immunized against HBsAg by DNA vaccination, HBsAg-specific CTL are generated. This specific CTL reactivity was targeted to s.c.-growing P815 tumors by intra tumor injections of either HBsAg-encoding plasmid DNA or viable P815/S cells; this treatment led to tumor rejection in 70-80% of the tumor-bearing animals. All rejecting animals showed a CD8+ CTL-dependent resistance to subsequent challenges by native, non-transfected P815 tumors. Targeting an established anti-viral ('strong') CTL response to a growing tumor hence is an efficient strategy to facilitate priming of a rejecting CTL response against ('weak') TAA in this system.

Antitumor activity studies of newly synthesized N-salicyloyl- N′-( p-hydroxybenzthioyl)hydrazine and its Copper(II) complex both in vivo and in vitro

A new ligand N-salicyloyl- N′-( p-hydroxybenzthioyl) hydrazine (H 2STPH) and its Cu II complex [Cu(SPTH)] were prepared and characterized by analytical and physicochemical studies. In vivo antitumor activity of [Cu(STPH)] has been tested against breast tumor in C 3H/J strain mice and in vitro on P-815 (murine mastocytoma) and K-562 (human erythroleukemia) cells. LD 50 values were estimated at a dose level of 100 mg/kg body weight and a light microscopic study also carried out.

Synthesis, characterization and antitumour activity of uracil and uracil–histidine complexes with metal(III) ions

Complexes of the[Al(L-H)(OH)Cl],[M(L-H)(H2O)2- (OH)Cl] and [M′(L-H)(L′-H)(H2O)Cl] type, where L = uracil (URL); L′ = histidine (HISD); M = CrIII or FeIII and M′ = AlIII, CrIII or FeIII were synthesized and characterized. The complexes are polymers, with high decomposition points and are insoluble in water and common organic solvents. Uracil is coordinated to the metal ion through the O atom of C(4)-O and the N atom of N(1), while histidine coordinates through the O atom of -CO2− and the N atom of the -NH2 groups. The μeff values, electronic spectral bands and e.s.r. spectra suggest a polymeric six coordinate spin-free octahedral stereochemistry for the CrIII and FeIII complexes. The in vivo and in vitro antitumour activity results indicate that CrIII and FeIII complexes have significant activity against P815 murine mastocytoma but AlIII complexes show poor activity.

5-Fluorouracil and 5-fluorouracil-histidine complexes with Al III, Cr III and Fe III ions and their antitumour activity

Complexes of the type [M(L-H)(OH)Cl], [Cr(L-H)(H 2O) 2(OH)Cl] and [M′(L-H)(L′-H)(H 2O)Cl], where L = 5-fluorouracil; L′ = histidine (HISD); M = Al III or Fe III and M′ = Al III, Cr III or Fe III were synthesized and characterized. The complexes are insoluble in water and common organic solvents. 5-Fluorouracil is coordinated to the metal ion through the O atom of C 4 = O and the N atom of N 1 while histidine coordinates through the O atom of −COO − and the N atom of −NH 2 groups. The μ eff values, electronic spectral bands and ESR spectra suggest a polymeric 6-coordinate spin free octahedral stereochemistry for Cr III and Fe III complexes. The in vivo antitumour effect of 5-fluorouracil and its complexes was examined on C 3H/He mice against P815 murine mastocytoma. As evident from their T/C values Cr III and Fe III complexes display significant and higher antitumour activity compared to 5-fluorouracil while the Al III complexes show lower activity. The in vitro results of the complexes on the same cells indicate that Cr III and Fe III complexes show higher inhibition on 3H-thymidine and 3H-uridine incorporation in DNA and RNA replication, respectively.

Synthesis and characterization of binary and heterobimetallic complexes of dithiofuroate with 3d-metal ions

New complexes [M(dtf)2] (M=MnII or CuII), Cr(dtf)3 and V(dtf)4 (dtf=dithiofuroate) and heterobimetallic complexes M[Cd(dtf)4] (M=VO2+, MnII, FeII, CoII, NiII or ZnII) have been prepared in H2O−EtOH and characterized by elemental analyses, magnetic susceptibility measurements and spectroscopic studies. The known complexes Fe(dtf)3, Co(dtf)3, Ni(dtf)2, and Zn(dtf)2 have been screened towards the bacteria Pseudomonas spergillus and Bacillus spergillus. In vitro results of the binary complexes on P-815 (murine mastocytoma) indicate that these complexes show significant inhibition of [3H]thymidine and [3H]uridine incorporation in DNA and RNA in the tumour cells.

Synthesis, characterization and antitumour studies on N-salicyl-N?-thiobenzohydrazide and its copper(II) complex

The new potential tetradentate ligand N-salicyl-N′-thiobenzohydrazide (H2SBTH) and its Cu complex, [Cu(SBTH)], have been prepared and characterized by physico-chemical studies. In vivo antitumour activity of [Cu(SBTH)] has been tested against breast tumour in C3H/J strain mice; LD50 values were also calculated. The cytotoxicity and antitumour effect of [Cu(SBTH)] is a maximum at 100 mg kg-1 body weight injected intraperitoneally in mice carrying breast tumour. In vitro results of the ligand and the complex on P-815 (murine mastocytoma) and K-562 (human erythroleukemia cells) indicate that these compounds show significant inhibition on 3H-thymidine and 3H-uridine incorporation in DNA and RNA in these tumour cells. Light microscopic study of the treated tumour mass demonstrated that certain cellular degradation, such as disappearance of mitotic figures, loss in cellular compactness, distortion of nucleus and disruption of cytoplasmic boundaries, take place in complex-treated mice with tumours.

A retroviral peptide encoded by mutated env p15E gene is recognized by specific CD8 + T lymphocytes on drug-treated murine mastocytoma P815

Highly immunogenic (“xenogenized”) tumour variants appear after treatment of murine mastocytoma P815 with the triazene derivative DTIC, a phenomenon associated with the appearance of structurally abnormal p15E env proteins in the variant cells. In the present study, we have isolated and sequenced several p15E cDNA gene fragments amplified by means of polymerase chain reaction (PCR) from parental (P815) and xenogenized (P815/DTIC) tumour cells. Compared to known p15E sequences in parental cells, one p15E sequence from xenogenized cells presented three distinct nucleotide changes, one of which was apparently unique to P815/DTIC DNA and cDNA upon single-nucleotide primer extension assay. One major histocompatibility complex (MHC) class I-binding peptide, corresponding to a putative mutation in the p15E sequence, was tested in parallel with the parental peptide for recognition by P815/DTIC-specific cytotoxic T cells in vitro. The results suggested that the amino acid substitution at the relevant position of the p15E protein may produce an antigenic T cell epitope. By skin test assay of mice primed with either the synthetic peptide or P815/DTIC cells, evidence was obtained that the mutated peptide is immunogenic in vivo, and that the neoepitope is expressed by P815/DTIC cells. In accordance with previous data in the L5178Y/DTIC tumour model system, these findings reinforce the notion that xenogenization of tumour cells may result in the expression of class I-binding mutated peptides of retroviral origin.

Anti‐estrogens enhance the therapeutic effect of lymphokine‐activated killer cells on the P815 murine mastocytoma

Anti‐estrogens enhance the therapeutic effect of lymphokine‐activated killer cells on the P815 murine mastocytoma

Anti-estrogens enhance the therapeutic effect of lymphokine-activated killer cells on the P815 murine mastocytoma.

Tamoxifen (TX) and toremifene (TO) enhanced the lysis of P815 mastocytoma cells in vitro by syngeneic DBA2 spleen cells that have been activated by human recombinant interleukin-2 (IL-2) for 6 days (lymphokine-activated killer [LAK] cells). Similarly, enhanced tumor suppression occurred when TX- or TO-treated P815 cells were mixed with LAK cells and injected s.c. into normal DBA2 recipients. Tumor suppression could be increased further by treating such recipients orally with TX or TO and by the repeated injections of LAK cells into the tumor site. The treatment of animals bearing tumors (5 mm in diameter) orally with TX or TO or with LAK cells i.p. resulted in tumor suppression. When the drug treatment was combined with LAK cells, tumor suppression was more pronounced, and complete tumor regression was induced in a significant number of the animals so treated. Our results indicate that the immunotherapeutic effect of LAK cells can be significantly amplified by combined treatment with the anti-estrogens TX or TO.

CD28-mediated cytotoxicity by the human leukemic NK cell line YT involves tyrosine phosphorylation, activation of phosphatidylinositol 3-kinase, and protein kinase C.

The human leukemic cell line YT displays spontaneous cytotoxicity against CD80+ and/or CD86+ and ICAM-1+ target cells. In this work, we report that CD28-mediated cytotoxicity of YT involves tyrosine phosphorylation and activation of phosphatidylinositol (PI) 3-kinase, the Tec kinase Itk/Emt, and protein kinase C (PKC). YT mediates lysis of CD80+/CD86+ B lymphoblastoid cell lines and the murine mastocytoma p815 transfected with CD80 or CD86. The lysis was inhibited by two different Pi 3-kinase inhibitors, wortmannin and LY294002. The PKC inhibitors calphostin C and bisindolylmaleimide GF109203X also abolished YT-mediated cytotoxicity. Furthermore, exocytosis of cytolytic effector molecules was also inhibited by PI 3-kinase inhibitors and PKC inhibitors. PMA together with Ionomycin did not induce granule exocytosis or cytotoxicity by YT cells. Treatment of YT cells with PMA for up to 20 h, which depleted PMA-responsive PKC isoforms, had no effect on the CD28-mediated cytotoxicity. This cytotoxicity displayed by PMA-treated YT cells, however, could still be inhibited by Pi 3-kinase inhibitors and PKC inhibitors. Taken together, these results are consistent with a model in which activation of CD28 and LFA-1 induces tyrosine phosphorylation of the CD28 cytoplasmic domain, recruitment and activation of PI 3-kinase, as well as the Tec kinase Itk/Emt, and the activation of PMA-nonresponsive PKC isoenzymes. Activation of PI 3-kinase and PMA-nonresponsive PKC isoenzymes is shown to be involved directly in cytolytic granule release by YT cells.

Modulation of lymphokine‐activated killer cell‐mediated cytotoxicity by estradiol and tamoxifen

The effect of tamoxifen (TX) and estradiol (E2) on interleukin-2 (IL-2)-activated killer (LAK) cell-mediated cytotoxicity was examined using spleen cells of Fisher 344 rats as the source of effectors and P815 murine mastocytoma cells as targets. Treatment of target cells with either TX or E2 for 4 or 18 hr rendered them highly sensitive to LAK cell-mediated lysis. When TX and E2 were applied jointly, cytotoxicity remained at the level of TX treatment alone. The cytotoxic potential of IL-2-primed LAK cells was not modified consistently by TX and E2. When TX-treated target and effector cells were combined, high cytotoxicity characteristic of sensitized target cells was observed. In similar experiments with E2-treated cells, both enhancement and inhibition of cytotoxicity by treated effector cells was seen in some designs. Target cells could be sensitized for LAK cell-mediated destruction by physiological concentrations (I nM) of E2 and equimolar concentrations of TX. Sensitization led to the accelerated release of the nuclear label 3H-thymidine from target cells after the cytotoxic insult and could be prevented by treatment with the metabolic inhibitors cycloheximide and actinomycin D. Enhanced 3H-thymidine release from TX-treated targets was also demonstrated after induction of Ca2+ influx by exposure to the ionophore A23187. Neither E2 nor TX exerted a direct cytotoxic effect on P815 cells. P815 cells had no classical receptors for E2 or progesterone. © 1996 Wiley-Liss, Inc.

Modulation of lymphokine-activated killer cell-mediated cytotoxicity by estradiol and tamoxifen.

The effect of tamoxifen (TX) and estradiol (E2) on interleukin-2 (IL-2 )-activated killer (LAK) cell-mediated cytotoxicity was examined using spleen cells of Fischer 344 rats as the source of effectors and P815 murine mastocytoma cells as targets. Treatment of target cells with either TX or E2 for 4 or 18 hr rendered them highly sensitive to LAK cell-mediated lysis. When TX and E2 were applied jointly, cytotoxicity remained at the level of TX alone. The cytotoxic potential of IL-2-primed LAK cells was not modified consistently by TX and E2. When TX-treated target and effector cells were combined, high cytotoxicity characteristic of sensitized target cells was observed. In similar experiments with E2-treated cells, both enhancement and inhibition of cytotoxicity by treated effector cells was seen in some designs. Target cells could be sensitized for LAK cell-mediated destruction by physiological concentrations (1 nM) of E2 and equimolar concentration of TX. Sensitization led to the accelerated release of the nuclear label 3H-thymidine from target cells after cytotoxic insult and could be prevented by treatment with the metabolic inhibitors cycloheximide and actinomycin D. Enhanced 3H-thymidine release from TX-treated targets was also demonstrated after induction of Ca2+ influx by exposure to the ionophore A23187. Neither E2 nor TX exerted a direct cytotoxic effect on P815 cells. P815 cells had no classical receptors for E2 or progesterone.