Transcriptional regulation of prostate kallikrein-like genes by androgen.

Using gene-specific synthetic oligonucleotides the expression and regulation of kallikrein-like genes in the human prostatic cancer cell line LNCaP were studied. Prostate-specific antigen (PSA) and human glandular kallikrein (hGK-1) together constitute a subfamily of serine proteases exclusively produced in the human prostate. RNA analysis revealed that both genes are expressed in LNCaP cells with PSA basal levels being 2-fold higher than hGK-1 levels. Both mRNAs are induced over a period of 24 h in the presence of 3.3 nM of the synthetic androgen mibolerone. Stimulation of PSA RNA is about 5- fold,whereas hGK-1 stimulation is less pronounced. Nuclear run-on analysis revealed that androgen induction of kallikrein-like genes in LNCaP cells is a rapid event (c3 h) occurring at the level of transcription initiation. Treatment of cells with cycloheximide demonstrates that, while PSA/hGK-1 basal transcription strictly depends on continuous protein synthesis, transcriptional induction by androgen does not. This suggests the direct involvement of the androgen receptor in the induction process independent of additional labile protein factors necessary for kallikrein basal transcription. A binding motif is present in the PSA and hGK-1 promoters, closely resembling the consensus sequence for steroidresponsive elements. The androgen antagonist cyproterone acetate was also able to stimulate transcription of kallikrein-like genes in LNCaP cells. In contrast, androgen-dependent transcriptional suppression of the protooncogene c-myc was strongly counteracted by cyproterone acetate. Thus, antiandrogens act differentially on androgen-regulated prostate-specific (PSA, hGK-1) and growthrelated (c-myc) gene expression in LNCaP cells

Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen.

Autoregulation is a control mechanism common to several proteins of the steroid/thyroid hormone receptor superfamily. In this work the effect of androgens and antiandrogens on the expression of the human androgen receptor (hAR) in prostate and breast cancer cell lines was studied. Northern blot analysis revealed a decrease in hAR steady state RNA levels in LNCaP cells by 3.3 nht of the synthetic androgen mibolerone. Maximal down-regulation of hAR RNA to 30% of control levels occurred 48 h after hormone addition. T47D breast cancer cells showed a similar effect with mibolerone, while hAR expression in normal skin fibroblasts did not respond to androgen treatment. As shown by nuclease Sl analysis, hAR transcripts initiate at three principal start sites, all of which are equally sensitive to androgen. Steroidal as well as nonsteroidal antiandrogens were capable of partially antagonizing androgen-mediated hAR RNA down-regulation in LNCaP and T47D cells, while not exerting a significant effect when administered alone. While hAR RNA stability was increased by hormone, nuclear run-on analysis revealed a 4-fold reduction of hAR gene transcrip tion 98 h after androgen treatment. Although decreased hAR RNA levels did not coincide with a parallel decrease in AR protein levels, analysis of androgen-inducible reporter constructs demonstrated that prolonged androgen administration to ceils results in a progressively impaired sensitivity of the intracellular androgen response mechanism. These results show that prolonged androgen exposure leads, besides its effect on hAR RNA levels, to functional inactivation of the AR. Thus, in viva, posttranslational control of AR activity appears to be a novel mechanism of negative autoregulation of androgen effects on gene expression

Thermal analysis of high-bandwidth and energy-efficient 980 nm VCSELs with optimized quantum well gain peak-to-cavity resonance wavelength offset

This article may be downloaded for personal use only. Any other use requires prior permission of the author and AIP Publishing. This article appeared in Appl. Phys. Lett. 111, 243508 (2017) and may be found at https://doi.org/10.1063/1.5003288.The static and dynamic performance of vertical-cavity surface-emitting lasers (VCSELs) used as light-sources for optical interconnects is highly influenced by temperature. We study the effect of temperature on the performance of high-speed energy-efficient 980 nm VCSELs with a peak wavelength of the quantum well offset to the wavelength of the fundamental longitudinal device cavity mode so that they are aligned at around 60 °C. A simple method to obtain the thermal resistance of the VCSELs as a function of ambient temperature is described, allowing us to extract the active region temperature and the temperature dependence of the dynamic and static parameters. At low bias currents, we can see an increase of the −3 dB modulation bandwidth f−3dB with increasing active region temperature, which is different from the classically known situation. From the detailed analysis of f−3dB versus the active region temperature, we obtain a better understanding of the thermal limitations of VCSELs, giving a basis for next generation device designs with improved temperature stability

The F-box protein SKP2 mediates androgen control of p27 stability in LNCaP human prostate cancer cells

BACKGROUND: The cyclin-dependent kinase inhibitor p27 is a putative tumor suppressor that is downregulated in the majority of human prostate cancers. The mechanism of p27 down-regulation in prostate cancers in unknown, but presumably involves increased proteolysis mediated by the SCF(SKP2) ubiquitin ligase complex. Here we used the human prostate cancer cell line LNCaP, which undergoes G1 cell cycle arrest in response to androgen, to examine the role of the SKP2 F-box protein in p27 regulation in prostate cancer. RESULTS: We show that androgen-induced G1 cell cycle arrest of LNCaP cells coincides with inhibition of cyclin-dependent kinase 2 activity and p27 accumulation caused by reduced p27 ubiquitylation activity. At the same time, androgen decreased expression of SKP2, but did not affect other components of SCF(SKP2). Adenovirus-mediated overexpression of SKP2 led to ectopic down-regulation of p27 in asynchronous cells. Furthermore, SKP2 overexpression was sufficient to overcome p27 accumulation in androgen arrested cells by stimulating cellular p27 ubiquitylation activity. This resulted in transient activation of CDK2 activity, but was insufficient to override the androgen-induced G1 block. CONCLUSIONS: Our studies suggest that SKP2 is a major determinant of p27 levels in human prostate cancer cells. Based on our in vitro studies, we suggest that overexpression of SKP2 may be one of the mechanisms that allow prostate cancer cells to escape growth control mediated by p27. Consequently, the SKP2 pathway may be a suitable target for novel prostate cancer therapies

Tumor Necrosis Factor-α Contributes to Ischemia- and Reperfusion-Induced Endothelial Activation in Isolated Hearts

During myocardial reperfusion, polymorphonuclear neutrophil (PMN) adhesion involving the intercellular adhesion molecule-1 (ICAM-1) may lead to aggravation and prolongation of reperfusion injury. We studied the role of early tumor necrosis factor-α (TNF-α) cleavage and nuclear factor-κB (NF-κB) activation on ICAM-1 expression and venular adhesion of PMN in isolated hearts after ischemia (15 minutes) and reperfusion (30 to 480 minutes). NF-κB activation (electromobility shift assay) was found after 30 minutes of reperfusion and up to 240 minutes. ICAM-1 mRNA, assessed by Northern blot, increased during the same interval. Functional effect of newly synthesized adhesion molecules was found by quantification (in situ fluorescence microscopy) of PMN, given as bolus after ischemia, which became adherent to small coronary venules (10 to 50 mm in diameter). After 480 minutes of reperfusion, ICAM-1–dependent PMN adhesion increased 2.5-fold compared with PMN adhesion obtained during acute reperfusion. To study the influence of NF-κB on PMN adhesion, we inhibited NF-κB activation by transfection of NF-κB decoy oligonucleotides into isolated hearts using HJV-liposomes. Decoy NF-κB but not control oligonucleotides blocked ICAM-1 upregulation and inhibited the subacute increase in PMN adhesion. Similar effects were obtained using BB 1101 (10 μg), an inhibitor of TNF-α cleavage enzyme. These data suggest that ischemia and reperfusion in isolated hearts cause liberation of TNF-α, activation of NF-κB, and upregulation of ICAM-1, an adhesion molecule involved in inflammatory response after ischemia and reperfusion

Conservation of the COP9/signalosome in budding yeast

BACKGROUND: The COP9/signalosome (CSN), a multiprotein complex consisting of eight subunits, is implicated in a wide variety of regulatory processes including cell cycle control, signal transduction, transcriptional activation, and plant photomorphogenesis. Some of these functions have been linked to CSN-associated enzymes, including kinases and an activity that removes the ubiquitin-like protein NEDD8/Rub1p from the cullin subunit of E3 ligases. CSN is highly conserved across species from fission yeast to humans, but sequence comparison has failed to identify the complex in budding yeast, except for a putative CSN5 subunit called Rri1p. RESULTS: We show that disruption of four budding yeast genes, PCI8 and three previously uncharacterized ORFs, which encode proteins interacting with Rrr1p/Csn5p, each results in the accumulation of the cullin Cdc53p exclusively in the Rub1p-modified state. This phenotype, which resembles that of fission yeast csn mutants, is due to a biochemical defect in deneddylation that is complemented by wild-type cell lysate and by purified human CSN in vitro. Although three of the four genes encode proteins with PCI domains conserved in metazoan CSN proteins, their disruption does not confer the DNA damage sensitivity described in some fission yeast csn mutants. CONCLUSIONS: Our studies present unexpected evidence for the conservation of a functional homologue of the metazoan CSN, which mediates control of cullin neddylation in budding yeast

Direct effects of CO2 concentration on growth and isotopic composition of marine plankton.

The assessment of direct effects of anthropogenic CO2 increase on the marine biota has received relatively little attention compared to the intense research on CO2-related responses of the terrestrial biosphere. Yet, due to the rapid air–sea gas exchange, the observed past and predicted future rise in atmospheric CO2 causes a corresponding increase in seawater CO2 concentrations, [CO2], in upper ocean waters. Increasing [CO2] leads to considerable changes in the surface ocean carbonate system, resulting in decreases in pH and the carbonate concentration, [CO2−3]. These changes can be shown to have strong impacts on the marine biota. Here we will distinguish between CO2-related responses of the marine biota which (a) potentially affect the ocean's biological carbon pumps and (b) are relevant to the interpretation of diagnostic tools (proxies) used to assess climate change on geological times scales. With regard to the former, three direct effects of increasing [CO2] on marine plankton have been recognized: enhanced phytoplankton growth rate, changing elemental composition of primary produced organic matter, and reduced biogenic calcification. Although quantitative estimates of their impacts on the oceanic carbon cycle are not yet feasible, all three effects increase the ocean's capacity to take up and store atmospheric CO2 and hence, can serve as negative feedbacks to anthropogenic CO2 increase. With respect to proxies used in palaeo-reconstructions, CO2-sensitivity is found in carbon isotope fractionation by phytoplankton and foraminifera. While CO2- dependent isotope fractionation by phytoplankton may be of potential use in reconstructing surface ocean pCO2 at ancient times, CO2-related effects on the isotopic composition of foraminiferal shells confounds the use of the difference in isotopic signals between planktonic and benthic shells as a measure for the strength of marine primary production. The latter effect also offers an alternative explanation for the large negative swings in δ13C of foraminiferal calcite between glacial and interglacial periods. Changes in [CO2−3] affect the δ18O in foraminiferal shells. Taking this into account brings sea surface temperature estimates for the glacial tropics closer to those obtained from other geochemical proxies