RESUMO
A new p56lck tyrosine kinase inhibitor WIN 61651 [1,4-dihydro-7-(4-methyl-1-piperizinyl)-1-(4-(4-methyl-1-piperi zinyl))phenyl- 4-oxo-3-quinolinecarboxamide) is described. WIN 61651, which is competitive with ATP, demonstrates selectivity for the lymphoid restricted tyrosine kinase p56lck over serine/threonine kinases, such as protein kinase C and protein kinase A, and over some other tyrosine kinases, including erbB2, epidermal growth factor receptor, and insulin receptor; however, it is equipotent for inhibition of p56lck and the platelet derived growth factor receptor tyrosine kinases. WIN 61651 inhibits p56lck activity in cell-free assays, tyrosine kinase activity in a T lymphocytic cell line, and T cell activation, as measured by IL-2 production by purified CD4 positive peripheral blood T lymphocytes and the mixed lymphocyte reaction. WIN 61651 constitutes a new tool for studies on the role for tyrosine kinases in lymphocyte function.
Assuntos
Inibidores Enzimáticos/farmacologia , Piperazinas/farmacologia , Quinolinas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia , Quinases da Família src/antagonistas & inibidores , Anticorpos/farmacologia , Complexo CD3/imunologia , Antígenos CD4/imunologia , Células Cultivadas , Humanos , Cinética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Fosforilação , Linfócitos T/fisiologia , Tirosina/metabolismoRESUMO
Since the poor prognosis associated with HER2 amplified breast cancers might be explained by a mechanistic association between p185HER2 overexpression and therapeutic resistance, we assessed the chemo-endocrine sensitivity of estrogen receptor (ER) containing MCF-7 breast cancer cells transfected with full-length HER2 cDNA. Of the 36 isolated MCF/HER2 subclones, 7 were found to overexpress p185HER2 surface receptor at levels 3 to 45-fold greater than parental or control transfected cells (MCF/neo). The overexpressing transfectants possessed increased inositol-1,4,5-triphosphate-3'-kinase activity comparable to enzyme activity in the endogenously HER2 amplified breast cancer cell lines SK-Br-3 and BT-474. The anti-p185HER2 monoclonal antibody and receptor-specific partial agonist, muMAb4D5 (4D5), known to inhibit growth of SK-Br-3 and BT-474 cells, produced no significant growth inhibitory effect on any of the transfectants including the 45-fold overexpressing MCF/HER2-18 cells which were studied in greater detail. MCF/HER2-18 cells contained at least partially functioning exogenous receptor since 4D5 (3 micrograms/ml) specifically stimulated phosphorylation of p185HER2 and its co-precipitating ptyr56 substrate within 5 min, and this was followed at 1 h by a transient induction of c-myc but not c-fos mRNA. ER content and the in vitro sensitivity of MCF/HER2-18 cells to 5-fluorouracil and adriamycin were identical to those of control transfectants and parental cells. However, these highly overexpressing transfectants had acquired low level (2 to 4-fold) resistance to cisplatin and were no longer sensitive to the antiestrogen tamoxifen (TAM). To compare the hormone-dependent tumorigenicity of the HER2 transfectants, MCF/HER2-18 and control cells (MCF, MCF/neo-3) were implanted into ovariectomized athymic nude mice. No tumors were produced in the absence of estradiol (E2) administration. In E2 supplemented mice, MCF/HER2-18 tumors grew most rapidly. When E2 treatment was stopped and daily TAM injections were initiated, MCF-7 and MCF/neo-3 tumor growth ceased immediately, while MCF/HER2-18 tumors continued to show an accelerated growth rate lasting weeks. This pattern of hormone-dependent, TAM-resistant growth exhibited by the MCF/HER2-18 tumors in nude mice supports the possibility that p185HER2 overexpression in human breast cancers may be linked to therapeutic resistance.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfotransferases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Tamoxifeno/farmacologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Resistência a Medicamentos , Feminino , Humanos , Camundongos , Camundongos Nus , Fosfotransferases/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Receptor ErbB-2 , Transfecção , Células Tumorais CultivadasRESUMO
A partially agonistic monoclonal antibody, 4D5, known to bind to the extracellular domain of p185HER2 and shown to inhibit long term growth of p185HER2-overexpressing breast cancer cells, was used to study signal transduction and phosphotyrosyl protein substrates associated with this receptor. Normal breast epithelial cells and breast carcinoma cells expressing low levels of p185HER2 were not affected by 4D5. HER2/neu-overexpressing breast cancer cells (BT-474 and SK-Br-3) exposed to 4D5 exhibited rapid phosphorylation of both p185HER2 and an associated 56-kDa phosphotyrosyl protein (ptyr56). Paralleling the 4D5- stimulated phosphorylation of p185HER2 and ptyr56 was a 5-10-fold induction of c-fos mRNA and phosphatidylinositol 4-kinase activity and a 2-fold induction of inositol 1,4,5-trisphosphate 3'-kinase activity. The increased phosphatidylinositol 4-kinase activity immunoprecipitated with p185HER2 and also co-eluted with ptyr56 from an antiphosphotyrosine immunoaffinity column. These results indicate that short term (less than 6 h) 4D5 activation of p185HER2 in overexpressing breast cancer cells produces agonistic-like signaling typical of homologous tyrosine kinase growth factor receptors such as epidermal growth factor receptor. The data also suggest that ptyr56 represents a novel phosphorylated substrate associated with 4D5-stimulated p185HER2.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Western Blotting , Neoplasias da Mama/patologia , Eletroforese em Gel de Poliacrilamida , Humanos , Fosfatidilinositóis/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-fos , Receptor ErbB-2 , Tensoativos , Células Tumorais CultivadasRESUMO
The HER2 protooncogene encodes a growth factor receptor-like transmembrane protein tyrosine kinase (p185HER2) whose ligand remains to be fully characterized. The overexpression of p185HER2 is implicated in aggressive forms of breast and ovarian cancers. The role of p185HER2 in aggressive malignancy, as well as its cell surface localization, makes it an attractive target for therapeutic monoclonal antibodies. In this report we have studied the modulation of p185HER2 function with 2 monoclonal antibodies, termed 4D5 and 6E9, which bind the extracellular domain of p185HER2. 4D5 inhibited proliferation of p185HER2 overexpressing SK-BR-3 human breast carcinoma cells (ED50 of approximately 1 nM) but did not inhibit proliferation of cultured human breast carcinoma MCF7 cells, low expressors of p185HER2. Monoclonal antibody 6E9 does not inhibit the growth of either cell line. Antibody binding studies revealed 2 populations of p185HER2 molecules on SK-BR-3 cells: one of high abundance (approximately 2 x 10(6) sites/cell) recognized by 4D5 (Kd approximately 6 nM) and the other of low abundance (2 x 10(4) sites/cell) recognized by 6E9 (Kd approximately 0.1 nM). 4D5, in an agonistic manner, downregulated SK-BR-3 cell surface p185HER2, was internalized, and stimulated p185HER2 phosphorylation in intact cells. Phosphoamino acid analysis of p185HER2 derived from SK-BR-3 cells incubated with the 4D5 monoclonal antibody demonstrated increased tyrosine, serine and threonine phosphorylation. 4D5, on short term (5 min) exposure to SK-BR-3 cells, stimulated inositol lipid hydrolysis as evidenced by increased intracellular levels of inositol polyphosphates (InsP) and sn-1,2-diacylglycerol (sn-1,2-DAG). On longer (24 h) exposure to the cells, the antibody appeared to downregulate this signalling pathway since the intracellular levels of InsP and sn-1,2-DAG decreased by 30 to 40%. 6E9 did not inhibit SK-BR-3 cell proliferation, downregulate surface p185HER2, stimulate receptor phosphorylation, or stimulate the second messenger pathway. Despite these agonistic properties, 4D5 was an inhibitor of SK-BR-3 cell proliferation at all concentrations tested (0.7 to 70 pM). The data suggest that 4D5 is a partial or weak agonist and thus may inhibit cell proliferation by mimicking ligand-like receptor downregulation.
Assuntos
Anticorpos Monoclonais/química , Proteínas Oncogênicas Virais/imunologia , Receptores de Superfície Celular/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos dos fármacos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Sítios de Ligação de Anticorpos , Neoplasias da Mama/tratamento farmacológico , Diglicerídeos/biossíntese , Feminino , Humanos , Proteínas Oncogênicas Virais/biossíntese , Neoplasias Ovarianas/tratamento farmacológico , Fosfatidilinositóis/biossíntese , Proteínas Tirosina Quinases/química , Receptor ErbB-2 , Transdução de Sinais , Células Tumorais Cultivadas/metabolismoRESUMO
The HER2 protooncogene encodes a 185-kDa transmembrane protein (p185HER2) with extensive homology to the epidermal growth factor (EGF) receptor. Clinical and experimental evidence supports a role for overexpression of the HER2 protooncogene in the progression of human breast, ovarian, and non-small cell lung carcinoma. These data also support the hypothesis that p185HER2 present on the surface of overexpressing tumor cells may be a good target for receptor-targeted therapeutics. The anti-p185HER2 murine monoclonal antibody (muMAb) 4D5 is one of over 100 monoclonals that was derived following immunization of mice with cells overexpressing p185HER2. The monoclonal antibody is directed at the extracellular (ligand binding) domain of this receptor tyrosine kinase and presumably has its effect as a result of modulating receptor function. In vitro assays have shown that muMAb 4D5 can specifically inhibit the growth of tumor cells only when they overexpress the HER2 protooncogene. MuMAb 4D5 has also been shown to enhance the TNF-alpha sensitivity of breast tumor cells that overexpress this protooncogene. Relevant to its clinical application, muMAb 4D5 may enhance the sensitivity of p185HER2-overexpressing tumor cells to cisplatin, a chemotherapeutic drug often used in the treatment of ovarian cancer. In vivo assays with a nude mouse model have shown that the monoclonal antibody can localize at the tumor site and can inhibit the growth of human tumor xenografts which overexpress p185HER2. Modulation of p185HER2 activity by muMAb 4D5 can therefore reverse many of the properties associated with tumor progression mediated by this putative growth factor receptor. Together with the demonstrated activity of muMAb 4D5 in nude mouse models, these results support the clinical application of muMAb 4D5 for therapy of human cancers characterized by the overexpression of p185HER2.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias/terapia , Proto-Oncogenes , Animais , Expressão Gênica , Humanos , Imunoterapia , Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/terapia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Receptor ErbB-2 , Células Tumorais Cultivadas/imunologiaRESUMO
The proto-oncogene HER2/neu encodes a protein tyrosine kinase (p185HER2) that is homologous to the human epidermal growth factor receptor. Amplification and/or overexpression of HER2/neu occurs in multiple human malignancies and appears to be integrally involved in progression of some breast and ovarian cancers. Because of this fact, HER2/neu is an intriguing target for specific cancer therapeutic strategies. One such strategy is active specific immunotherapy, in which the immune system is targeted at specific antigens expressed by tumor cells. We have employed a transfected cell line that secretes the extracellular domain of p185HER2 as a source of HER2-derived immunogen in a guinea pig model. The immunized animals developed a cellular immune response, as monitored by delayed-type hypersensitivity, and antisera derived from immunized animals specifically inhibited the in vitro growth of human breast tumor cells overexpressing p185HER2. These data provide support for an immunotherapeutic approach to cancers characterized by overexpression of the HER2/neu proto-oncogene.
Assuntos
Neoplasias da Mama/terapia , Imunoterapia Ativa , Proteínas Proto-Oncogênicas/imunologia , Vacinas Sintéticas , Animais , Especificidade de Anticorpos/genética , Divisão Celular/imunologia , Separação Celular , Proteínas do Sistema Complemento/imunologia , Citotoxicidade Imunológica/imunologia , Feminino , Citometria de Fluxo , Cobaias , Humanos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Receptor ErbB-2RESUMO
The erbB2 oncogene encodes a 185-kilodalton transmembrane protein whose sequence is similar to the epidermal growth factor receptor (EGFR). A 30-kilodalton factor (gp30) secreted from MDA-MB-231 human breast cancer cells was shown to be a ligand for p185erbB2. An antibody to EGFR abolished the tyrosine phosphorylation induced by EGF and transforming growth factor-alpha (TGF-alpha) but only partially blocked that produced by gp30 in SK-BR-3 breast cancer cells. In two cell lines that overexpress erbB2 but do not expresss EGFR (MDA-MB-453 breast cancer cells and a Chinese hamster ovary cell line that had been transfected with erbB2), phosphorylation of p185erbB2 was induced only by gp30. The gp30 specifically inhibited the growth of cells that overexpressed p185erbB2. An antibody to EGFR had no effect on the inhibition of SK-BR-3 cell colony formation obtained with gp30. Thus, it appeared that gp30 interacted directly with the EGFR and erbB2. Direct binding of gp30 to p185erbB2 was confirmed by binding competition experiments, where gp30 was found to displace the p185erbB2 binding of a specific antibody to p185erbB2. The evidence described here suggests that gp30 is a ligand for p185erbB2.
Assuntos
Receptores ErbB/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Anticorpos Monoclonais , Ligação Competitiva , Neoplasias da Mama , Linhagem Celular , Cromatografia de Afinidade , Receptores ErbB/isolamento & purificação , Feminino , Humanos , Cinética , Ligantes , Peso Molecular , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proto-Oncogenes , TransfecçãoRESUMO
The kinetics and regulation of nucleoside phosphorylation by highly purified human deoxycytidine kinase from leukemic lymphoblasts were studied. The phosphorylation of purine nucleosides by this enzyme showed sensitivity to the endogenous inhibitors dCTP and UDP three times greater than the phosphorylation of dCyd. Examination of nucleotide pools in human T and B lymphoblasts disclosed that the levels of dCTP and UDP in these cells were sufficient to regulate kinase activity. The enhanced sensitivity of the kinase to dCTP and UDP was related to its reduced ability to interact with purine nucleosides. Comparison of the phosphorylation kinetics for pyrimidine and purine dideoxynucleosides used in antiviral therapy showed that the purine nucleosides were at least 50-fold less efficient as enzyme substrates. These results suggest that the phosphorylation of pharmacologically active purine nucleosides by deoxycytidine kinase is regulated by cellular nucleotide pools.
Assuntos
Desoxicitidina Quinase/antagonistas & inibidores , Nucleosídeos/fisiologia , Nucleotídeos/fisiologia , Fosfotransferases/antagonistas & inibidores , Nucleosídeos de Purina/metabolismo , Linfócitos T/metabolismo , Linfócitos B/metabolismo , Linhagem Celular , Desoxicitidina Quinase/fisiologia , Humanos , FosforilaçãoRESUMO
Specific progesterone binding by cultured human breast carcinoma T47D, MCF-7, and ZR75-1 cells was decreased 25-40% by epidermal growth factor (EGF), with a 50% effective dose of 0.1 nM EGF. Studies with the soluble and particulate fractions prepared after homogenization of T47D cells grown in glass roller bottles revealed equivalent EGF-induced decreases in progesterone binding to receptors in both fractions. Equilibrium progesterone binding studies with these soluble and particulate fractions revealed that EGF decreased the receptor number, but had no effect on affinity. With cells grown adherent to plastic dishes, EGF treatment induced a greater decrease in binding to receptors recovered in the particulate fraction, than to receptors recovered in the soluble fraction. The decrease in progesterone binding induced by 20 nM EGF was maximal after 2 min of cellular EGF treatment for receptors recovered in the soluble fraction, but was only half-maximal after 15 min for receptors recovered in the particulate fraction. Decreased progesterone binding persisted for at least 8 days in cells cultured with 1 nM EGF. Either insulin or EGF stimulated T47D cell proliferation by two- to threefold with a 50% effective dose of 100 nM for insulin and 0.1 nM for EGF. The progestin, R5020, decreased T47D cell growth by 30% with a 50% effective dose of 1 nM. Either EGF or insulin antagonized the inhibitory effect of R5020 on cell reproduction, but progestins did not antagonize the growth stimulatory response of cells to EGF. Progestins increased the number of EGF receptors within 12 h of their addition to T47D cells, but this response was lost after 6 days. These data show that EGF or progesterone can regulate the receptor number of the other, but for cell reproduction, the effect of EGF is dominant over that of progestins.
Assuntos
Neoplasias da Mama/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Progesterona/metabolismo , Animais , Linhagem Celular , Feminino , Humanos , Insulina/farmacologia , Masculino , Camundongos , Promegestona/metabolismo , Receptores de Progesterona/metabolismo , Células Tumorais CultivadasRESUMO
Each of four independent experimental approaches showed that human breast carcinoma T47D cells contain both high and low affinity progesterone receptors. (i) Equilibrium-specific [3H]progesterone binding to adherent cultured cells revealed dissociation constants (Kd) of 1.5 and 60 nM and 0.33 and 2.4 X 10(6) sites/cell, respectively. Both the high and low affinity receptors were specific for progestins as demonstrated by steroid binding competition studies conducted at 5 and 50 nM [3H]progesterone. (ii) Equilibrium [3H]progesterone binding to the resolved soluble and particulate fractions from a cell homogenate sedimented at 40,000 X g.min revealed Kd = 1.4 nM high affinity binding sites exclusively in the supernatant fraction and Kd = 24 nM low affinity sites exclusively in the particulate fraction. Extraction of the particulate fraction with a high ionic strength buffer solubilized the low affinity receptors stoichiometrically; but once solubilized, they displayed Kd = 2.4 nM high affinity progesterone binding. Characterizations of 3H-ligand bound specifically to progesterone receptors in intact cells or resolved subcellular fractions revealed no [3H]progesterone metabolites that could account for the low affinity binding. (iii) Calculations based on the rate constants of [3H] progesterone association with or dissociation from adherent cells revealed the same dissociation constants for both high and low affinity binding as those determined by equilibrium measurements. (iv) Nonionic detergent extraction of cells incubated with a wide range of [3H]progesterone concentrations revealed high affinity progesterone binding to receptors in the detergent-soluble fraction and low affinity binding associated primarily with the particulate residue, consistent with the data on equilibrium progesterone binding to resolved cell homogenate fractions. The rate of extraction of the high affinity receptor-progesterone complex with nonionic detergent (t1/2 = 1 min at 0 degrees C) equaled the rate of extraction of a representative lysosomal enzyme, beta-acetylglucosaminidase.
Assuntos
Neoplasias da Mama/metabolismo , Receptores de Progesterona/metabolismo , Ligação Competitiva , Detergentes , Humanos , Cinética , Concentração Osmolar , Cloreto de Potássio , Progesterona/metabolismo , Promegestona/metabolismo , Receptores de Progesterona/isolamento & purificação , Frações Subcelulares/metabolismo , Células Tumorais CultivadasRESUMO
Cell extracts from human leukemic T lymphoblasts and myeloblasts were chromatographed on DEAE-cellulose columns to separate purine deoxyribonucleoside, deoxyadenosine (dAdo) and deoxyguanosine (dGuo), phosphorylating activities. Three distinct purine deoxyribonucleoside kinases, a deoxycytidine (dCyd) kinase, an adenosine (Ado) kinase, and a deoxyguanosine (dGuo) kinase (the latter appears to be localized in mitochondria), were resolved. dCyd kinase contained the major phosphorylating activity for dAdo, dGuo, and 9-beta-D-arabinofuranosyladenine (ara-A). Ado kinase represented a second kinase for dAdo and ara-A while a third kinase for dAdo was found in mitochondria. dCyd kinase was purified about 2000-fold with ion-exchange, affinity, and hydrophobic chromatographies. On gel electrophoresis, both dCyd and dAdo phosphorylating activities comigrated, indicating that the activities are associated with the same protein. The enzyme showed a broad pH optimum ranging from pH 6.5 to pH 9.5. Divalent cations Mg2+, Mn2+, and Ca2+ stimulated dCyd kinase activity; Mg2+ produced the maximal activity. dCyd kinase from either lymphoid or myeloid cells showed broad substrate specificity. The enzyme used several nucleoside triphosphates, but ATP, GTP, and dTTP were the best phosphate donors. dCyd was the best nucleoside substrate, since dCyd kinase had an apparent Km of 0.3, 85, 90, and 1400 microM for dCyd, dAdo, dGuo, and ara-A, respectively. The enzyme exhibited substrate activation with both pyrimidine and purine deoxyribonucleosides, suggesting that there is more than one substrate binding site on the kinase. These studies show that, in lymphoblasts and myeloblasts, purine deoxyribonucleosides and their analogues are phosphorylated by dCyd kinase, Ado kinase, and dGuo kinase.
Assuntos
Leucemia Linfoide/enzimologia , Leucemia Mieloide/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool) , Fosfotransferases/metabolismo , Nucleosídeos de Purina/metabolismo , Nucleosídeos de Pirimidina/metabolismo , Adenosina Quinase/metabolismo , Linhagem Celular , Criança , Ativação Enzimática , Humanos , Cinética , Mitocôndrias/enzimologia , Fosforilação , Fosfotransferases/isolamento & purificação , Especificidade por SubstratoRESUMO
The biochemical basis of cellular resistance to 9-beta-D-arabinofuranosyladenine (ara-A) and its natural purine derivative, deoxyadenosine, was investigated with two mutants of cultured human T-lymphoblastoid CCRF-CEM cells. One mutant that lacked deoxycytidine kinase activity, designated CEM/ara-C, retained about 10% of wild-type deoxyadenosine kinase and deoxyguanosine kinase activity each but maintained normal adenosine kinase or thymidine kinase activity. This suggested that in these human T-lymphoblastoid cells, as in other previously studied mammalian cells, deoxycytidine and purine deoxyribonucleosides are phosphorylated by the same enzyme. Despite this extensive reduction of purine nucleoside kinase activities, the cytotoxicity of ara-A or deoxyadenosine was not appreciably affected, decreasing by only 2.5- and 6-fold, respectively. A second mutant, isolated by selecting CEM/ara-C mutants that were resistant to ara-A, showed a 100-fold increase in resistance to ara-A cytotoxicity. This ara-A-resistant subline was deficient in the activities of two enzymes, deoxycytidine kinase and adenosine kinase, and showed a high degree of resistance to deoxyadenosine, adenosine, and pyrazofurin but not to pyrimidine analogs, such as 5-fluorodeoxyuridine or 5-fluorouridine. Further studies of ara-A and deoxyadenosine phosphorylation in wild-type and resistant cell lines disclosed that, although deoxycytidine kinase is the principal enzyme for their phosphorylation in vitro, their intracellular conversion to cytotoxic nucleotides depends on the joint action of deoxycytidine kinase and adenosine kinase rather than purine-specific deoxynucleoside kinase, as previously thought.