PMID: 16677834;Abstract:
Intracellular levels of the heat stress protein Hsp70 are elevated following exposure to elevated temperature. The cochaperone HspBP1 is an intracellular protein that is known to bind to and regulate Hsp70 activity. The purpose of this study was to determine if HspBP1 levels changed when Hsp70 levels were altered. Heat stress resulted in an increase in Hsp70 levels but no change in HspBP1 levels. Treatment of cells with the apoptosis inducing drug camptothecin lowered Hsp70 levels but again had no effect on HspBP1 levels. Cells treated with camptothecin plus heat stress did not exhibit an increase in Hsp70 levels. Over-expression in cells stably transfected with HspBP1 cDNA resulted in a 290% increase in HspBP1 levels without a similar change in Hsp70 levels. These results demonstrate that Hsp70 and HspBP1 are not coordinately regulated but provide evidence that an increase in the ratio of HspBP1 to Hsp70 correlates with apoptosis, in a similar way to reducing the amount of Hsp70. © 2006 International Federation for Cell Biology.
We have developed a cell model to investigate steroid control of differentiation using a subline of HT1080 cells (HT-AR1) that have been engineered to express the human androgen receptor. Dihydrotestosterone (DHT) treatment of HT-AR1 cells induced growth arrest and cytoskeletal reorganization that was associated with the expression of fibronectin and the neuroendocrine markers chromogranin A and neuron-specific enolase. Expression profiling analysis identified the human FERM domain-encoding gene EHM2 as uniquely induced in HT-AR1 cells as compared to 16 other FERM domain containing genes. Since FERM domain proteins control cytoskeletal functions in differentiating cells, and the human EHM2 gene has not been characterized, we investigated EHM2 steroid-regulation, genomic organization, and sequence conservation. We found that DHT, but not dexamethasone, induced the expression of a 3.8 kb transcript in HT-AR1 cells encoding a 504 amino acid protein, and moreover, that human brain tissue contains a 5.8 kb transcript encoding a 913 amino acid isoform. Construction of an unrooted phylogenetic tree using 98 FERM domain proteins revealed that the human EHM2 gene is a member of a distinct subfamily consisting of nine members, all of which contain a highly conserved 325 amino acid FERM domain.
PMID: 11801526;Abstract:
To better understand androgen action in normal prostate cells, we characterized the androgen growth response of an immortalized nontumorigenic rat prostate cell line called CA25 that had been stably transfected with androgen receptor (AR) cDNA. Surprisingly, we found that AR(+) CA25 cells grew slower in the presence of dihydrotestosterone (DHT), whereas the growth of AR(-) CA25 cells was not affected by the hormone. DHT-mediated growth inhibition of CA25 cells was not attributable to an increase in apoptosis but rather to a morphological conversion consistent with terminal differentiation. Specifically, we found that DHT treatment of CA25 cells resulted in a striking change in cell architecture, localization of desmoplakin to cell-cell boundaries, and an increase in α6p integrin levels, a newly described marker of cell differentiation. Although no androgen-dependent changes were observed in the transcript levels of the mitochondrial aspartate aminotransferase or c-Myc genes by Northern blot analysis, RNA expression profiling of DHT-treated CA25 cells identified 282 genes of 1,018 that were continually expressed over a 48-h period. It was found that 63 of these genes were up-regulated >5-fold within the first 4 h of treatment and encoded functions involved in transport, signal transduction, and metabolism. These expression profile data are consistent with the striking morphological changes we observed in DHT-treated CA25 cells and provide a starting point for molecular analysis of in vitro prostate cell differentiation.
A variety of stimuli have been identified which initiate transcription-dependent programmed cell death (apoptosis) in specific target cells. Since the withdrawal of androgens induces regression and apoptosis in rat ventral prostate (RVP) epithelial cells, and it is known that the androgen receptor is a transcriptional regulator, we used subtraction cDNA cloning to isolate differentially expressed transcripts from the RVP of androgen ablated rats. In addition to sulfated glycoprotein-2 and glutathione S-transferase (GST), which had been previously described, several other transcripts were found to be elevated 3- to 8-fold in the regressing RVP. DNA sequencing revealed that two of these cDNA clones encode matrix carboxyglutamic acid and gamma-actin, respectively. A third cDNA contained novel sequence information and was named RVP.1. The RVP.1 transcript is expressed at very low levels in the RVP and epididymis of normal adult rats (less than 0.01% of the total mRNA) and is undetectable in other tissues, such as kidney, liver, and muscle. RVP.1 encodes a putative 280-amino acid protein, which shares no significant homology with previously described protein functional domains. We examined the expression of these transcripts in serum-starved NIH 3T3 cells to determine whether any of them are elevated in cells that are growth arrested. It was found that only GST mRNA levels are increased under these conditions. These data may suggest that induction of some genes, such as RVP.1, could be associated with apoptosis, whereas other transcripts, such as GST, may be up-regulated in response to altered rates of cellular metabolism.
