Regulation of apoptotic protease activating factor-1 oligomerization and apoptosis by the WD-40 repeat region

Apoptotic protease activating factor-1 (Apaf-1) has been identified as a proximal activator of caspase-9 in cell death pathways that trigger mitochondrial damage and cytochrome c release. The mechanism of Apaf-1 action is unclear but has been proposed to involve the clustering of caspase-9 molecules, thereby facilitating autoprocessing of adjacent zymogens. Here we show that Apaf-1 can dimerize via the CED-4 homologous and linker domains of the molecule providing a means by which Apaf-1 can promote the clustering of caspase-9 and facilitate its activation. Apaf-1 dimerization was repressed by the C-terminal half of the molecule, which contains multiple WD-40 repeats, but this repression was overcome in the presence of cytochrome c and dATP. Removal of the WD-40 repeat region resulted in a constitutively active Apaf-1 that exhibited greater cytotoxicity in transient transfection assays when compared with full-length Apaf-1. These data suggest a mechanism for Apaf-1 function and reveal an important regulatory role for the WD-40 repeat region.

HPV16 activates the AIM2 inflammasome in keratinocytes

Human papillomaviruses (HPV) are double-stranded DNA viruses, which selectively infect keratinocytes in stratified epithelia. After an initial infection, many patients clear HPV. In some patients, however, HPV persist, and dysfunctional innate immune responses to HPV infection could be involved in the ineffective clearing of these viruses. In this study, the mechanisms of HPV-induced immune responses in keratinocytes were investigated. Binding of viral DNA leads to AIM2 inflammasome activation and IL-1β release, while IFI16 activation results in IFN-β release. Using immunohistochemistry, AIM2 and IFI16-two recently identified sensors for cytosolic DNA-were also detected in HPV positive skin lesions. CISH stainings further confirmed the presence of cytosolic HPV16 DNA in biopsy samples. Moreover, active IL-1β and cleaved caspase-1 were detected in HPV infected skin, suggesting inflammasome activation by viral DNA. In subsequent functional studies, HPV16 DNA triggered IL-1β and IL-18 release via the AIM2 inflammasome in normal human keratinocytes. Although HPV DNA did not induce IFN-β in keratinocytes, IFN-β secretion was observed when AIM2 was blocked. Meanwhile, blocking of IFI16 increased HPV16 DNA-induced IL-1β, but not IL-18, secretion. These findings suggest crosstalk between IFI16 and AIM2 in the immune response to HPV DNA. In sum, novel aspects concerning HPV-induced innate immune responses were identified. Eventually, understanding the mechanisms of HPV-induced inflammasome activation could lead to the development of novel strategies for the prevention and treatment of HPV infections.

VDR and MEK-ERK dependent induction of the antimicrobial peptide cathelicidin in keratinocytes by lithocholic acid

Cathelicidin is an antimicrobial peptide (AMP) and signaling molecule in innate immunity and a direct target of 1,25-dihydroxyvitamin D3 (1,25D3) in primary human keratinocytes (NHEK). The expression of cathelicidin is dysregulated in various skin diseases and its regulation differs depending on the epithelial cell type. The secondary bile acid lithocholic acid (LCA) is a ligand of the vitamin D receptor (VDR) and can carry out in vivo functions of vitamin D3. Therefore we analyzed cathelicidin mRNA- and peptide expression levels in NHEK and colonic epithelial cells (Caco-2) after stimulation with LCA. We found increased expression of cathelicidin mRNA and peptide in NHEK, in Caco-2 colon cells no effect was observed after LCA stimulation. The VDR as well as MEK-ERK signaled the upregulation of cathelicidin in NHEK induced by LCA. Collectively, our data indicate that cathelicidin induction upon LCA treatment differs in keratinocytes and colonic epithelial cells. Based on these observations LCA-like molecules targeting cathelicidin could be designed for the treatment of cutaneous diseases that are characterized by disturbed cathelicidin expression.

Manganese Superoxide Dismutase Is a Promising Target for Enhancing Chemosensitivity of Basal-Like Breast Carcinoma

AIMS: Although earlier reports highlighted a tumor suppressor role for manganese superoxide dismutase (MnSOD), recent evidence indicates increased expression in a variety of human cancers including aggressive breast carcinoma. In the present article, we hypothesized that MnSOD expression is significantly amplified in the aggressive breast carcinoma basal subtype, and targeting MnSOD could be an attractive strategy for enhancing chemosensitivity of this highly aggressive breast cancer subtype.

RESULTS: Using MDA-MB-231 and BT549 as a model of basal breast cancer cell lines, we show that knockdown of MnSOD decreased the colony-forming ability and sensitized the cells to drug-induced cell death, while drug resistance was associated with increased MnSOD expression. In an attempt to develop a clinically relevant approach to down-regulate MnSOD expression in patients with basal breast carcinoma, we employed activation of the peroxisome proliferator-activated receptor gamma (PPARγ) to repress MnSOD expression; PPARγ activation significantly reduced MnSOD expression, increased chemosensitivity, and inhibited tumor growth. Moreover, as a proof of concept for the clinical use of PPARγ agonists to decrease MnSOD expression, biopsies derived from breast cancer patients who had received synthetic PPARγ ligands as anti-diabetic therapy had significantly reduced MnSOD expression. Finally, we provide evidence to implicate peroxynitrite as the mechanism involved in the increased sensitivity to chemotherapy induced by MnSOD repression.

INNOVATION AND CONCLUSION: These data provide evidence to link increased MnSOD expression with the aggressive basal breast cancer, and underscore the judicious use of PPARγ ligands for specifically down-regulating MnSOD to increase the chemosensitivity of this subtype of breast carcinoma.

Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1

The breast cancer susceptibility gene BRCA1 encodes a protein implicated in the cellular response to DNA damage, with postulated roles in homologous recombination as well as transcriptional regulation. To identify downstream target genes, we established cell lines with tightly regulated inducible expression of BRCA1. High-density oligonucleotide arrays were used to analyze gene expression profiles at various times following BRCA1 induction. A major BRCA1 target is the DNA damage-responsive gene GADD45. Induction of BRCA1 triggers apoptosis through activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), a signaling pathway potentially linked to GADD45 gene family members. The p53-independent induction of GADD45 by BRCA1 and its activation of JNK/SAPK suggest a pathway for BRCA1-induced apoptosis.

Recognition of O6MeG lesions by MGMT and mismatch repair proficiency may be a prerequisite for low-dose radiation hypersensitivity

Low-dose hyper-radiosensitivity (HRS) is the phenomenon whereby cells exposed to radiation doses of less than approximately 0.5 Gy exhibit increased cell killing relative to that predicted from back-extrapolating high-dose survival data using a linear-quadratic model. While the exact mechanism remains to be elucidated, the involvement of several molecular repair pathways has been documented. These processes in turn are also associated with the response of cells to O6-methylguanine (O6MeG) lesions. We propose a model in which the level of low-dose cell killing is determined by the efficiency of both pre-replicative repair by the DNA repair enzyme O6-methylguanine methyltransferase (MGMT) and post-replicative repair by the DNA mismatch repair (MMR) system. We therefore hypothesized that the response of cells to low doses of radiation is dependent on the expression status of MGMT and MMR proteins. MMR (MSH2, MSH6, MLH1, PMS1, PMS2) and MGMT protein expression signatures were determined in a panel of normal (PWR1E, RWPE1) and malignant (22RV1, DU145, PC3) prostate cell lines and correlated with clonogenic survival and cell cycle analysis. PC3 and RWPE1 cells (HRS positive) were associated with MGMT and MMR proficiency, whereas HRS negative cell lines lacked expression of at least one (MGMT or MMR) protein. MGMT inactivation had no significant effect on cell survival. These results indicate a possible role for MMR-dependent processing of damage produced by low doses of radiation.

Dual targeting of tumour cells and host endothelial cells by novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines

PURPOSE: Some members of a novel series of pyrrolo-1,5-benzoxazepines (PBOXs) are microtubule-targeting agents capable of inducing apoptosis in a variety of human cancerous cells, hence, they are currently being developed as potential anti-cancer agents. The purpose of this study was to first characterise the activities of a novel PBOX analogue, PBOX-16 and then investigate the anti-angiogenic potential of both PBOX-16 and its prototype PBOX-6.

METHODS: The effects of PBOX-6 and -16 on cancerous cells (chronic myeloid leukaemia K562 cells and ovarian carcinoma A2780 cells) and primary cultured human umbilical vein endothelial cells (HUVECs) were examined by assessing cell proliferation, microtubular organisation, DNA analysis of cell cycle progression and caspase-3/7 activity. Their anti-angiogenic properties were then investigated by examining their ability to interfere with HUVEC differentiation into capillary-like structures and vascular endothelial growth factor (VEGF)-stimulated HUVEC migration.

RESULTS: PBOX-6 and -16 inhibited proliferation of K562, A2780 and HUVEC cells in a concentration-dependent manner. PBOX-16, confirmed as a novel depolymerising agent, was approximately tenfold more potent than PBOX-6. Inhibition of cell proliferation was mediated by G(2)/M arrest followed by varying degrees of apoptosis depending on the cell type; endothelial cells underwent less apoptosis than either of the cancer cell lines. In addition to the antitumourigenic properties, we also describe a novel antiangiogenic function for PBOXs: treatment with PBOXs inhibited the spontaneous differentiation of HUVECs into capillary-like structures when grown on a basement membrane matrix preparation (Matrigel™) and also significantly reduced VEGF-stimulated HUVEC migration.

CONCLUSION: Dual targeting of both the tumour cells and the host endothelial cells by PBOX compounds might enhance the anti-cancer efficacy of these drugs.

Nuclear translocation and functions of growth factor receptors

Cellular signal transduction in response to environmental signals involves a relay of precisely regulated signal amplifying and damping events. A prototypical signaling relay involves ligands binding to cell surface receptors and triggering the activation of downstream enzymes to ultimately affect the subcellular distribution and activity of DNA-binding proteins that regulate gene expression. These so-called signal transduction cascades have dominated our view of signaling for decades. More recently evidence has accumulated that components of these cascades can be multifunctional, in effect playing a conventional role for example as a cell surface receptor for a ligand whilst also having alternative functions for example as transcriptional regulators in the nucleus. This raises new challenges for researchers. What are the cues/triggers that determine which role such proteins play? What are the trafficking pathways which regulate the spatial distribution of such proteins so that they can perform nuclear functions and under what circumstances are these alternative functions most relevant?

Tumor necrosis factor receptor expression and signaling in renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC), a tubular epithelial cell (TEC) malignancy, frequently secretes tumor necrosis factor (TNF). TNF signals via two distinct receptors (TNFRs). TNFR1, expressed in normal kidney primarily on endothelial cells, activates apoptotic signaling kinase 1 and nuclear factor-kappaB (NF-kappaB) and induces cell death, whereas TNFR2, inducibly expressed on endothelial cells and on TECs by injury, activates endothelial/epithelial tyrosine kinase (Etk), which trans-activates vascular endothelial growth factor receptor 2 (VEGFR2) to promote cell proliferation. We investigated TNFR expression in clinical samples and function in short-term organ cultures of ccRCC tissue treated with wild-type TNF or specific muteins selective for TNFR1 (R1-TNF) or TNFR2 (R2-TNF). There is a significant increase in TNFR2 but not TNFR1 expression on malignant TECs that correlates with increasing malignant grade. In ccRCC organ cultures, R1-TNF increases TNFR1, activates apoptotic signaling kinase and NF-kappaB, and promotes apoptosis in malignant TECs. R2-TNF increases TNFR2, activates NF-kappaB, Etk, and VEGFR2 and increases entry into the cell cycle. Wild-type TNF induces both sets of responses. R2-TNF actions are blocked by pretreatment with a VEGFR2 kinase inhibitor. We conclude that TNF, acting through TNFR2, is an autocrine growth factor for ccRCC acting via Etk-VEGFR2 cross-talk, insights that may provide a more effective therapeutic approach to this disease.

Inhibition of endosome fusion by wortmannin persists in the presence of activated Rab5

Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3-kinase inhibitor, wortmannin. It has been proposed that phosphoinositide 3-kinase activity may be required for activation of rab5 by influencing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to wortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5(Q79L) or of a xanthosine triphosphate-binding mutant, rab5(D136N), in the presence of the nonhydrolysable analogue XTPgammaS. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the endosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despite prior incubation with the nonhydrolysable nucleotide analogue GTPgammaS. Consistent with these results, direct measurement of clathrin-coated vesicle-stimulated nucleotide dissociation from exogenous rab5 was insensitive to the presence of wortmannin. A large excess of rab5(Q79L), beyond levels required for maximal stimulation of the fusion assay, afforded protection against wortmannin inhibition, and partial protection was also observed with an excess of wild-type rab5 independent of GTPgammaS.

Dilated cardiomyopathy and impaired cardiac hypertrophic response to angiotensin II in mice lacking FGF-2.

FGF-2 has been implicated in the cardiac response to hypertrophic stimuli. Angiotensin II (Ang II) contributes to maintain elevated blood pressure in hypertensive individuals and exerts direct trophic effects on cardiac cells. However, the role of FGF-2 in Ang II-induced cardiac hypertrophy has not been established. Therefore, mice deficient in FGF-2 expression were studied using a model of Ang II-dependent hypertension and cardiac hypertrophy. Echocardiographic measurements show the presence of dilated cardiomyopathy in normotensive mice lacking FGF-2. Moreover, hypertensive mice without FGF-2 developed no compensatory cardiac hypertrophy. In wild-type mice, hypertrophy was associated with a stimulation of the c-Jun N-terminal kinase, the extracellular signal regulated kinase, and the p38 kinase pathways. In contrast, mitogen-activated protein kinase (MAPK) activation was markedly attenuated in FGF-2-deficient mice. In vitro, FGF-2 of fibroblast origin was demonstrated to be essential in the paracrine stimulation of MAPK activation in cardiomyocytes. Indeed, fibroblasts lacking FGF-2 expression have a defective capacity for releasing growth factors to induce hypertrophic responses in cardiomyocytes. Therefore, these results identify the cardiac fibroblast population as a primary integrator of hypertrophic stimuli in the heart, and suggest that FGF-2 is a crucial mediator of cardiac hypertrophy via autocrine/paracrine actions on cardiac cells.

Remyelination in vitro following protein kinase C activator-induced demyelination.

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.

Evidence of a Ca(2+)-(center dot)NO-cGMP signaling pathway controlling zoospore biogenesis in the aquatic fungus Blastocladiella emersonii

The sporulation stage of the aquatic fungus Blastocladiella emersonii culminates with the formation and release to the medium of a number of zoospores, which are motile cells responsible for the dispersal of the fungus. The presence in the sporulation solution of 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a potent and selective inhibitor of nitric oxide-sensitive guanylyl cyclases, completely prevented biogenesis of the zoospores. In addition, this compound was able to significantly reduce cGMP levels, which increase drastically during late sporulation, suggesting the existence of a nitric oxide-dependent mechanism for cGMP synthesis. Furthermore, increased levels of nitric oxide-derived products were detected during sporulation by fluorescence assays using DAF-2 DA, whose signal was drastically reduced in the presence of the nitric oxide synthase inhibitor N omega-Nitro-L-arginine methyl ester (L-NAME). These results were confirmed by quantitative chemiluminescent determination of the intracellular levels of nitric oxide-derived products. A putative nitric oxide synthase (NOS) activity was detected throughout sporulation, and this enzyme activity decreased significantly when L-NAME and 1-[2-(Trifluoromethyl)phenyl]imidazole (TRIM) were added to the assays. NOS assays carried out in the presence of EGTA showed decreased enzyme activity, suggesting the involvement of calcium ions in enzyme activation. Additionally, expressed sequence tags (ESTs) encoding putative guanylyl cyclases and a cGMP-phosphodiesterase were found in B. emersonii EST database (http://blasto.iq.usp.br), and the mRNA levels of the corresponding genes were observed to increase during sporulation. Altogether, data presented here revealed the presence and expression of guanylyl cyclase and cGMP phosphodiesterase genes in B. emersonii and provided evidence of a Ca(2+)-(center dot)NO-cGMP signaling pathway playing a role in zoospore biogenesis. (C) 2009 Elsevier Inc. All rights reserved.

Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 gag-pol

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) target HIV-1 reverse transcriptase (RT) by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation.

Regulation of glycogen synthase and phosphorylase during recovery from high-intensity exercise in the rat

The aim of this study was to determine the role of the phosphorylation state of glycogen synthase and glycogen phosphorylase in the regulation of muscle glycogen repletion in fasted animals recovering from high-intensity exercise. Groups of rats were swum to exhaustion and allowed to recover for up to 120 min without access to food. Swimming to exhaustion caused substantial glycogen breakdown and lactate accumulation in the red, white and mixed gastrocnemius muscles, whereas the glycogen content in the soleus muscle remained stable. During the first 40 min of recovery, significant repletion of glycogen occurred in all muscles examined except the soleus muscle. At the onset of recovery, the activity ratios and fractional velocities of glycogen synthase in the red, white and mixed gastrocnemius muscles were higher than basal, but returned to pre-exercise levels within 20 min after exercise. In contrast, after exercise the activity ratios of glycogen phosphorylase in the same muscles were lower than basal, and increased to pre-exercise levels within 20 min. This pattern of changes in glycogen synthase and phosphorylase activities, never reported before, suggests that the integrated regulation of the phosphorylation state of both glycogen synthase and phosphorylase might be involved in the control of glycogen deposition after high-intensity exercise.