RESUMO
P-selectin glycoprotein ligand-1 (PSGL-1) is an adhesive molecule that is known to be a ligand for P-selectin. An anti-adhesive property of PSGL-1 has not been previously reported. In this study, we show that PSGL-1 expression is anti-adhesive for adherent cells and we have elucidated the underlying mechanism. Overexpression of PSGL-1 induced cell rounding and floating in HEK293T cells. Similar phenomena were demonstrated in other adherent cell lines with overexpression of PSGL-1. PSGL-1 overexpression inhibits access of antibodies to cell surface molecules such as integrins, HLA and CD25. Cells transfected with PSGL-1 deletion mutants that lack a large part of the extracellular domain and chimeric construct expressing extracellular CD86 and intracellular PSGL-1 only showed rounded morphology, but there are no floating cells. These results indicated that PSGL-1 causes steric hindrance due to the extended structure of its extracellular domain that is highly O-glycosylated, but intracellular domain also has some effect on cell rounding. This study implies that PSGL-1 has Janus-faced functions, being both adhesive and anti-adhesive.
Assuntos
Adesão Celular , Glicoproteínas de Membrana/fisiologia , Animais , Linhagem Celular Tumoral , Forma Celular , Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Integrinas/metabolismo , Glicoproteínas de Membrana/química , Camundongos , Estrutura Terciária de ProteínaRESUMO
Five novel, canine lymphoma cell lines (Ema, CLC, CLK, Nody-1 and UL-1) were established from dogs suffering from lymphoma and characterized in vitro and in vivo. All cell lines, except CLC, were characterized with T-cell phenotypes, by flow cytometric analysis and polymerase chain reaction for antigen receptor rearrangement. Cell proliferation rates and transcriptional levels of MYC, PTEN, KIT and FLT3 varied between each cell line. Intraperitoneal xenotransplantation of Ema, CLC, Nody-1 and UL-1 lymphoma cell lines into NOD/SCID mice induced ascites, intraperitoneal tumors and severe infiltration of lymphoma cells into the pancreas and mesentery. Establishment of novel canine lymphoma cell lines with different characteristics is critical for elucidating the pathophysiology of canine lymphoma and improving current therapies.
Assuntos
Linhagem Celular Tumoral , Doenças do Cão/patologia , Linfoma/veterinária , Animais , Proliferação de Células , Doenças do Cão/genética , Cães , Feminino , Imunofenotipagem/veterinária , Linfoma/genética , Linfoma/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Neoplásico/química , RNA Neoplásico/genética , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Receptores de Antígenos de Linfócitos T/genética , Organismos Livres de Patógenos Específicos , Transplante Heterólogo/veterináriaRESUMO
Thirteen different monoclonal antibodies against canine P-selectin glycoprotein ligand-1 (cPSGL-1) were obtained by immunization of rats with cells of a canine lymphoma cell line (Ema). O-sialoglycoprotein endopeptidase treatment of Ema cells showed that all of these antibodies recognized O-glycosylated peptides of canine PSGL-1. Experiments using deletion or point mutants of cPSGL-1 indicated that these antibodies could be categorized into several groups based on their cPSGL-1 recognition characteristics. These anti-cPSGL-1 monoclonal antibodies will be useful for analysis of the canine P-selectin and PSGL-1 system.