We investigated the cellular localization of the small GTPases Rab3D and Rab3A in AtT-20 cells treated with the drug Brefeldin A. Brefeldin A induces the redistribution of the Golgi complex into the ...endoplasmic reticulum and tubulation of endosomes. However, in Brefeldin A-treated wild-type AtT-20 cells, both Rab3D and Rab3A retained their distribution, indicating that they belong to a nonendosomal, post-Golgi compartment. Immunoelectron microscopy experiments indicated that both Rab3D and Rab3A localized to the ACTH-containing, large dense core granules. In contrast, in cell clones overexpressing a mutated form of Rab3D (Rab3D N135I), Rab3A did not localize to the dense core granules. Moreover, since our previous results showed that overexpression of Rab3D N135I severely impaired regulated ACTH secretion in AtT-20 cells, we sought to determine whether the impairment could depend on a redistribution of two key components of the regulated exocytosis machinery, synaptotagmin and SNAP-25. As far as synaptotagmin was concerned, in cell clones overexpressing Rab3D N135I, the protein did not localize close to the plasma membrane, in agreement with the previously reported defective docking of dense core granules to the plasma membrane. Immunofluorescence experiments showed that SNAP-25 did not change its localization in these cell clones. All in all, our findings strengthen the notion that both Rab3D and Rab3A are associated with the dense core granule compartment of AtT-20 cells, and that the impairment in the ACTH secretion caused by overexpression of a mutated Rab3D form is likely to be due to a lacking of granule docking to the plasma membrane, possibly because Rab3A fails to associate with the granules.
Disruption of the apoptotic pathways may account for resistance to chemotherapy and treatment failures in human neoplastic
disease. To further evaluate this issue, we isolated a HL-60 cell clone ...highly resistant to several drugs inducing apoptosis
and to the differentiating chemical all- trans -retinoic acid (ATRA). The resistant clone displayed an activated phosphoinositide 3-kinase (PI3K)/AKT1 pathway, with levels
of phosphatidylinositol (3,4,5) trisphosphate higher than the parental cells and increased levels of both Thr 308 and Ser
473 phosphorylated AKT1. In vitro AKT1 activity was elevated in resistant cells, whereas treatment of the resistant cell clone with two inhibitors of PI3K,
wortmannin or Ly294002, strongly reduced phosphatidylinositol (3,4,5) trisphosphate levels and AKT1 activity. The inhibitors
reversed resistance to drugs. Resistant cells overexpressing either dominant negative PI3K or dominant negative AKT1 became
sensitive to drugs and ATRA. Conversely, if parental HL-60 cells were forced to overexpress an activated AKT1, they became
resistant to apoptotic inducers and ATRA. There was a tight relationship between the activation of the PI3K/AKT1 axis and
the expression of c-IAP1 and c-IAP2 proteins. Activation of the PI3K/AKT1 axis in resistant cells was dependent on enhanced
tyrosine phosphorylation of the p85 regulatory subunit of PI3K, conceivably due to an autocrine insulin-like growth factor-I
production. Our findings suggest that an up-regulation of the PI3K/AKT1 pathway might be one of the survival mechanisms responsible
for the onset of resistance to chemotherapeutic and differentiating therapy in patients with acute leukemia.
Recent reports have highlighted that phosphoinositide-specific phospholipase Cbeta1 expression is linked to neuronal differentiation in different experimental models. We sought to determine whether ...or not this is also true for nerve growth factor (NGF)-induced neuronal differentiation of rat PC12 cells. However, we did not find differences in the expression of both the forms of phosphoinositide-specific phospholipase Cbeta1 (a and b) during sympathetic differentiation of these cells. Also, PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 were not more susceptible to the differentiating effect of NGF. Furthermore, since it is well established that phosphoinositide-specific phospholipase Cbeta1 affects cell proliferation, we investigated whether or not PC12 cell clones stably overexpressing phosphoinositide-specific phospholipase Cbeta1 showed differences in survival to serum deprivation and cell cycle, when compared to wild type cells. Nevertheless, we did not find any differences in these parameters between wild type cells and the overexpressing clones. Interestingly, in PC12 cells the overexpressed phosphoinositide-specific phospholipase Cbeta1 did not localize to the nucleus, but by immunofluorescence analysis, was detected in the cytoplasm. Therefore, our findings may represent another important clue to the fact that only when it is located within the nucleus phosphoinositide-specific phospholipase Cbeta1 is able to influence cell proliferation.
PDF
