Apoptosis resistance of senescent human fibroblasts is correlated with the absence of nuclear IGFBP-3.

Signaling through the insulin/IGF axis plays a major role in determining the rate of aging in many species. IGF-binding proteins (IGFBPs) modulate the IGF pathway in higher organisms. IGFBP-3 accumulates in conditioned medium of senescent human fibroblasts, suggesting that it may contribute to the senescent phenotype. IGFBP-3 can enhance apoptotic cell death in tumor cells due to its ability to target intracellular regulators of apoptosis, including nuclear transcription factors. Senescent fibroblasts are highly resistant to apoptosis, suggesting that IGFBP-3 fails to induce apoptosis in this cell type; however, mechanisms of apoptosis resistance in senescent cells are poorly understood. To address this question, we studied the production and intracellular localization of IGFBP-3 in senescent fibroblasts. Whereas IGFBP-3 is highly overexpressed by senescent fibroblasts, IGFBP-3 was not detectable in the nucleus of senescent fibroblasts. In tumor cells, IGFBP-3 can be internalized by endocytosis, which is considered as a prerequisite for the intracellular functions of IGFBP-3 and probably also for its transport to the nucleus; we show here that endocytotic uptake of IGFBP-3 does not occur in senescent human fibroblasts. This is correlated with a generally decreased endocytotic activity of these cells, as shown with the model substrate transferrin. The data are consistent with a model where IGFBP-3 accumulation in conditioned medium of senescent fibroblasts contributes to growth arrest of these cells, whereas the failure to endocytose IGFBP-3 and the absence of nuclear IGFBP-3 may contribute to the well-established apoptosis resistance of senescent human fibroblasts.

APC inactivation associates with abnormal mitosis completion and concomitant BUB1B/MAD2L1 up-regulation.

BACKGROUND & AIMS: Chromosomal instability, a hallmark of most colorectal cancers, has been related to altered chromosome segregation and the consequent deficit in genetic integrity. A role for the tumor suppressor gene APC has been proposed in colorectal cancer that leads to compromised chromosome segregation even though the molecular mechanism is not yet understood. Here, we tackled the genetic basis for the contribution of APC to chromosomal instability in familial adenomatous polyposis and sporadic colorectal cancer. METHODS: We have used video-microscopy of primary cultures and molecular genetic methods to address these issues in human samples and in genetically defined mouse models that either recapitulate the familial adenomatous polyposis syndrome (Apc(1638N)), or develop tumors in the absence of APC mutations (pvillin-KRASV12G). RESULTS: Mutations in APC were associated with an increased incidence in cell cycle defects during the completion of cytokinesis. Transcriptome analysis performed on mouse models indicated a significant up-regulation of genes that regulate accurate mitosis. Notably, we identified up-regulated expression of BUB1B and MAD2L1, 2 genes that are involved in the mitotic checkpoint, but have so far not been implicated in chromosomal instability induced by APC loss of function. In vitro modulation of APC expression suggested a causal association for this upregulation, which was consistently found in sporadic and familial adenomatous polyposis lesions, as an early event in colorectal tumorigenesis. CONCLUSIONS: In addition to the known function of APC during correct spindle assembly and positioning, we propose a concomitant involvement of APC in the surveillance mechanism of accurate mitosis.

Microarray analysis supports a role for ccaat/enhancer-binding protein-beta in brain injury.

CCAAT/enhancer-binding protein-beta (C/EBPbeta) is a transcription factor that plays an important role in regulating cell growth and differentiation. This protein plays a central role in lymphocyte and adipocyte differentiation and hepatic regeneration and in the control of inflammation and immunity in the liver and in cells of the myelomonocytic lineage. Our previous studies suggested that this protein could also have important functions in the brain. Therefore, we were interested in the identification of downstream targets of this transcription factor in cells of neural origin. We performed cDNA microarray analysis and found that a total of 48 genes were up-regulated in C/EBPbeta-overexpressing neuronal cells. Of the genes that displayed significant changes in expression, several were involved in inflammatory processes and brain injury. Northern blot analysis confirmed the up-regulation of ornithine decarboxylase, 24p3/LCN2, GRO1/KC, spermidine/spermine N(1)-acetyltransferase, xanthine dehydrogenase, histidine decarboxylase, decorin, and TM4SF1/L6. Using promoter-luciferase reporter transfection assays, we showed the ornithine decarboxylase and 24p3 genes to be biological downstream targets of C/EBPbeta in neuroblastoma cells. Moreover, the levels of C/EBPbeta protein were significantly induced after neuronal injury, which was accompanied by increased levels of cyclooxygenase-2 enzyme. This strongly supports the concept that C/EBPbeta may play an important role in brain injury.