RESUMEN
We have previously found that the mRNA and protein levels of the folate receptor alpha (FRalpha) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FRalpha has not fully been determined. We investigated the effect of FRalpha over-expression in the mouse gonadotroph alphaT3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FRalpha were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FRalpha promotes cell proliferation. These effects were abrogated in the same alphaT3-1 cells when transfected with a mutant FRalpha cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FRalpha over-expressing cells. In summary, our data suggests that FRalpha regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.
Asunto(s)
Proteínas Portadoras/metabolismo , Ácido Fólico/metabolismo , Gonadotrofos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Notch/metabolismo , Adenoma/metabolismo , Adenoma/patología , Animales , Proteínas Portadoras/genética , Línea Celular , Proliferación Celular , Modelos Animales de Enfermedad , Receptores de Folato Anclados a GPI , Humanos , Ratones , Neoplasias Hipofisarias/metabolismo , Neoplasias Hipofisarias/patología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Receptor Notch3 , Receptores de Superficie Celular/genética , Transfección , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Clinically nonfunctional pituitary adenomas cause hypopituitarism or compression of regional structures. Unlike functional tumors, there is no available medical treatment or specific imaging technique for these tumors. We have recently discovered that both folate receptor (FR)alpha mRNA and protein are uniquely overexpressed in nonfunctional pituitary tumors, but not in functional adenomas. We hypothesized that FRalpha may hold significant promise for medical treatment by enabling novel molecular imaging and targeted therapy. Here, we used murine pituitary tumor cell line alphaT3-1 as a model to investigate the biological significance of FRalpha and its mutant FR67. We demonstrate that overexpression of FR facilitated tumor cell growth and anchorage-independent growth in soft agar. More colonies were observed in FR overexpressing cells than in mutant FR67 clones in soft agar. Cell proliferation rate was increased, the percentage of cells in S-phase was increased, and high PCNA staining was detected in cells overexpressing the receptor. In alphaT3-1 cells transfected with mutant FR67, cell proliferation rate was reduced, the percentage of cells residing in S-phase was slightly decreased, and low PCNA staining was observed. By real-time quantitative PCR, the genes involved in NOTCH3 pathway including NOTCH3, HES-1, and TLE2 were altered; the mRNA expression of FGFR1 was upregulated, and ERbeta mRNA was downregulated in FR overexpressing cells. Our findings suggest that FRalpha plays a role in pituitary tumor formation, and this effect may in part be due to its regulation of the NOTCH3 pathway.
Asunto(s)
Adenoma/metabolismo , Neoplasias Hipofisarias/metabolismo , Receptores de Superficie Celular/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular , Proliferación Celular , Femenino , Receptor 1 de Folato , Ácido Fólico/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Persona de Mediana Edad , Mutación , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor Notch3 , Receptores de Superficie Celular/genética , Receptores Notch/metabolismoRESUMEN
Myeloperoxidase (MPO), an enzyme active against bacterial and fungal infections, is expressed specifically in myeloblasts and promyelocytes and minimal in other cell types. We recently identified and partially characterized an upstream enhancer located between -4100 and -3844 bp of the MPO gene. We showed that an AML1 site contributes to enhancer activity and specificity. We now demonstrate three additional footprints within the MPO enhancer and provide evidence that C/EBP and c-Myb sites contribute to its functional, tissue-specific activity. This distal enhancer appears to play an important role in the control of MPO transcription during differentiation of myeloid cells.