RESUMO
CEACAM1 (also known as biliary glycoprotein, C-CAM or CD66a) is a cell adhesion molecule of the immunoglobulin family behaving as a tumor inhibitory protein in colon, prostate, liver, endometrial and breast cancers. Inhibition of tumor development is dependent upon the presence of the long 71-73 amino acid cytoplasmic domain of the CEACAM1 protein (CEACAM1-L). We have recently defined a number of cis-acting motifs within the long cytoplasmic domain participating in tumor cell growth inhibition. These are Tyr488, corresponding to an Immunoreceptor Tyrosine-based Inhibition Motif, as well as the three terminal lysine residues of the protein. In this study, we provide evidence that treatment with phorbol esters leads to increased phosphorylation of in vivo (32)P-labeled CEACAM1-L in mouse CT51 carcinoma cells, in the mouse 1MEA 7R.1 liver carcinoma cells and in 293 human embryonic kidney cells transfected with the Ceacam1-L cDNA. Basal level Ser phosphorylation was abrogated by treatment with the staurosporine inhibitor, but not by the protein kinase C-specific inhibitor calphostin C or other inhibitors such as H7 or sphingosine. Specific inhibitors of protein kinase A or calmodulin kinase had only minimal effects on the levels of basal or PMA-induced Ser phosphorylation. Furthermore, PMA treatment of the CT51 cells induced cell spreading and cellular relocalization of the CEACAM1-L protein. Since Ser503 has been described as a PMA-induced phosphorylation site in other cell systems, we investigated whether Ser503 was involved in these responses in mouse intestinal cells. No differences were noticed in the basal or the PMA-induced phosphorylation levels, kinase inhibitor sensitivity or the PMA-induced relocalization of the protein between the wild-type and the Ser503Ala mutant CEACAM1-L. However, we provide evidence that Ser503 participates in CEACAM1-L-mediated tumor inhibition as its mutation to an Ala led to in vivo tumor development, contrary to the tumor inhibitory phenotype observed with the wild-type CEACAM1-L protein.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Neoplasias do Colo/patologia , AMP Cíclico/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Antígenos de Diferenciação/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Antígeno Carcinoembrionário , Testes de Carcinogenicidade , Carcinoma/tratamento farmacológico , Carcinoma/metabolismo , Carcinoma/patologia , Moléculas de Adesão Celular , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , AMP Cíclico/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Naftalenos/farmacologia , Mutação Puntual , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Serina/metabolismo , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tionucleotídeos/farmacologia , Células Tumorais CultivadasRESUMO
An abnormal stimulation of the cAMP pathway has been recognized as the primary event in various pathological situations that lead to goitrogenesis or thyroid tumors. Thyroid adenomas are monoclonal neoplasms that become independent of thyroid stimulating hormone (TSH) in their secretory function and growth. Mutated forms of the TSH receptor (TSHR) and the adenylyl cyclase-activating Gs alpha protein, which confer a constitutive activity on these proteins, have been observed in human adenomas. The FRTL-5 rat thyroid cell line is a permanent but untransformed line; the growth of which depends on the presence of TSH, and at least in part, on the stimulation of the cAMP pathway. In order to compare the oncogenic potential of the activated mutant Gs alpha protein and the constitutively activated TSHR, we have transfected FRTL-5 cells with an expression vector bearing either the cDNA of the Gs alpha gene carrying the A201S mutation or the cDNA of the TSH receptor carrying the M453T mutation recently identified in a case of congenital hyperthyroidism. The expression of these two cDNAs was driven by the bovine thyroglobulin gene promoter. We show that, although the expression of both the Gs alpha or TSHR mutant proteins leads to TSH-independent proliferation and to constitutive cAMP accumulation in FRTL-5 cells, only the mutant TSHR is able to induce neoplastic transformation, as demonstrated by growth in semi-solid medium and tumorigenesis in nude mice.
Assuntos
Transformação Celular Neoplásica , Receptores da Tireotropina/fisiologia , Animais , Bovinos , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Humanos , Camundongos , Camundongos Nus , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Ratos , Receptores da Tireotropina/biossíntese , Receptores da Tireotropina/genética , Timo/metabolismo , Timo/fisiologia , Timo/ultraestrutura , Tireotropina/fisiologia , Transcrição Gênica , TransfecçãoRESUMO
The existence of inherited aggressive forms of medullary thyroid carcinoma (MTC), and their resistance to all classical therapies, make it a prime candidate for adoptive immunotherapy. As a prelude to a vaccine for the protection of family members at risk of developing the disease, we investigated the immunological antitumour response provoked by the 6/23 rMTC cell line, compared to that of the same cell engineered to secrete interleukin-2 (rMTC-IL2), in an animal model of familial human MTC, the inbred strain of Wag/Rij rats. The rMTC cells developed a tumour that invaded the whole neck 15 days after orthotopic injection (into the thyroid), while the rMTC-IL2 cells were progressively rejected. Co-injection of rMTC-IL2 with the parental cells induced the rejection of the rMTC transplants. When injected, both tumoral cell types showed a similar positive immunoreaction with anti-MHC class I (major histocompatibility complex class I) antibodies. They both recruited natural killer cells and eosinophils at the site of injection. In addition, CD8+ T lymphocytes infiltrated the rMTC-IL2 cells, and eosinophil recruitment was amplified. Neutrophils, macrophages and CD4+ T lymphocytes were scarce. Our results suggest that the CD8+ T lymphocytes are implicated in the anti-tumour reaction elicited by the IL-2-transfected cells. As these effectors are known to induce a specific immunological response, including memory, such a protocol should be tested as a vaccine on the young population genetically at risk of developing a MTC.
Assuntos
Carcinoma Medular/imunologia , Carcinoma Medular/terapia , Imunoterapia Adotiva , Interleucina-2/metabolismo , Interleucina-2/fisiologia , Neoplasias da Glândula Tireoide/imunologia , Neoplasias da Glândula Tireoide/terapia , Animais , Linfócitos T CD8-Positivos/imunologia , Carcinoma Medular/metabolismo , Divisão Celular/fisiologia , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Injeções Subcutâneas , Interleucina-2/genética , Células Matadoras Naturais/imunologia , Transplante de Neoplasias , Ratos , Ratos Endogâmicos , Neoplasias da Glândula Tireoide/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
We have developed mouse models for tumors affecting the epithelial cellular compartment of the thyroid which has been targeted using the bovine thyroglobulin (bTg) promoter. Transgenic mice expressing the human activated c-Ha-Ras gene developed papillary thyroid carcinomas demonstrating the oncogenic potential of activated Ras gene in the thyroid gland. Transgenic mice express the mutant form of the alpha subunit of the adenylate cyclase-coupled G alpha s with mutations at codon 201 (R201H). The expression of this mutant transgene is not by itself sufficient to produce benign tumors or even hyperplasia, but the transgenic mice have inherited a predisposition to develop thyroid adenomas.