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Título: The regulation of parathyroid hormone-related protein (PTHRP) gene expression in hypercalcemia of malignancy /
Autores: Aklilu, Fasika.
Fecha: 1999
Publicador: McGill University - MCGILL
Fuente:
Tipo: Electronic Thesis or Dissertation
Tema: Biology, Molecular.
Biology, Genetics.
Health Sciences, Oncology.
Descripción: The studies included in this thesis were aimed at identifying the mechanisms that lead to aberrant expression of the PTHRP gene in cancer.
We have used the hepatocyte growth factor receptor oncogene, Tpr-Met, as a model and examined the effect of this oncogene on PTHRP expression. When transfected into Fisher rat 313 (Fr3T3) fibroblasts, Tpr-Met increased the transcription of PTHRP mRNA and secretion of the protein. To identify the signaling pathways involved we analyzed a mutant of Tpr-Met, Tyr489, that was impaired in activating a number of downstream effectors, including Phosphatidylinositol-3 kinase, Grb2 and Shc. The ability of Tpr-Met/Tyr 489 mutant to induce PTHRP expression was significantly reduced. Furthermore, inhibiting Ras using lovastatin, in wild-type Tpr-Met transfected cells, Completely Suppressed PTHRP levels, suggesting that the mechanism was Ras-dependent.
We next directly investigated the effect of Ras on PTHRP expression in vitro, and on hypercalcemia of malignancy in vivo. When transfected PTHRP cells the activated mutant of Ras (RasV12) potently increased PTHRP mRNA and protein levels. When RasV12 expressing cells were subcutaneously injected into BALB/c/nu/ nu mice, the tumors developed rapidly, and signs of hypercalcemia were detected within 2 weeks. Inhibiting Ras using a specific inhibitor, B-1096, completely blocked expression of PTHRP, in vitro, and suppressed the sips of hypercalcemia in vivo. These results show that inhibiting Ras was sufficient to block tumor expression of PTHRP and development of hypercalcemia.
Using rat Leydig tumor H-500 cells, we next investigated effector pathways downstream of Ras that mediate serum stimulated PTHRP expression. PTHRP mRNA was decreased by a dominant negative mutant of Raf (Raf C4B) and by a MEK inhibitor (PD 098059), implicating the involvement of Ras-Raf-MEK pathway in the serum response. In addition, stimulation with UV light or expression of an activated form of Rac (Rac V12) was sufficient to increase PTHRP mRNA. Furthermore, a dominant negative mutant Of Rac (Rac N17) also blocked serum induced expression of mRNA. This suggests that the stress-activated pathways may provide alternative mechanisms that can regulate the PTHRP gene. These pathways also appear to be important in the serum induced response. Collectively, the results from these studies contribute to our limited knowledge of the mechanisms governing PTHRP expression in cancer. The findings also provide novel targets to explore for improved therapy of hypercalcemia.
Idioma: en