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Many genes of the human genome display pleiotropic activity, playing an important role in two or more unrelated pathways. Surprisingly, some of these functions can even be antagonistic, often letting to divergent functional outcomes depending on microenviromental cues and tissue/cell type-dependent parameters. Lately, the Bruton’s tyrosine kinase (BTK) has emerged as one of such pleiotropic genes, with opposing effects in cancer pathways. While it has long been considered oncogenic in the context of B cell malignancies, recent data shows that BTK can also act as a tumour suppressor in other cells, as an essential member of the p53 and p73 responses to damage. Since BTK inhibitors are already being used clinically, it is important to carefully review these new findings in order to fully understand the consequences of blocking BTK activity in all the cells of the organism.

Bruton’s tyrosine kinase (BTK) is a key component of B cell receptor signalling. Because of this, BTK plays an important role in cell proliferation and survival in various B cell malignancies. However, in certain contexts, BTK can also have tumour suppressor functions. We have previously shown that BTK activates the p53 transcriptional activity by binding to and phosphorylating p53, as well as acting on MDM2 to reduce its inhibitory effects. This results in increased p53 functions, including enhanced cell death. Here, we report that BTK can also induce cell death and increase responses to DNA damage independently of p53. This is concomitant to the induction of p21, PUMA and MDM2, which are classic target genes of the p53 family of proteins. Our results show that these p53-independent effects of BTK are mediated through p73. Similar to what we observed in the p53 pathway, BTK can upregulate p73 after DNA damage and induce expression of its target genes, suggesting that BTK is a modulator of p73 functions and in the absence of p53. This effect allows BTK to have pro-apoptotic functions independently of its effects on the p53 pathway and thus play an important role in the DNA damage-related induction of apoptosis in the absence of p53. This provides a novel role of BTK in tumour suppression and contributes to the understanding of its complex pleiotropic functions

CUDC‐907, a dual PI3K/HDAC inhibitor, has been proposed to have therapeutic potential in hematopoietic malignancies. However, the molecular mechanisms of its effects in chronic lymphocytic leukaemia (CLL) remain elusive. We show that CLL cells are sensitive to CUDC‐907, even under conditions similar to the protective microenvironment of proliferation centres. CUDC‐907 inhibited PI3K/AKT and HDAC activity, as expected, but also suppressed RAF/MEK/ERK and STAT3 signalling and reduced the expression of anti‐apoptotic BCL‐2 family proteins BCL‐2, BCL‐xL, and MCL‐1. Moreover, CUDC‐907 downregulated cytokines BAFF and APRIL and their receptors BAFFR, TACI, and BCMA, thus blocking BAFF‐induced NF‐κB signalling. T cell chemokines CCL3/4/17/22 and phosphorylation of CXCR4 were also reduced by CUDC‐907. These data indicated that CUDC‐907 abrogates different protective signals and suggested that it might sensitize CLL cells to other drugs. Indeed, combinations of low concentrations of CUDC‐907 with inhibitors of BCL2, BTK, or the NF‐κB pathway showed a potent synergistic effect. Our data indicate that, apart from its known functions, CUDC‐907 blocks multiple pro‐survival pathways to overcome microenvironment protection in CLL cells. This provides a rationale to evaluate the clinical relevance of CUDC‐907 in combination therapies with other targeted inhibitors.

Endometrial cancer (EC) is now the most prevalent gynaecological malignancy in the Western world. Black or African American women (BoAA) have double the mortality of Caucasian women, and their tumours tend to be of higher grade. Despite these disparities, little is known regarding the mutational landscape of EC between races. Hence, we wished to investigate the molecular features of ECs within The Cancer Genome Atlas (TCGA) dataset by racial groupings. In total 374 Caucasian, 109 BoAA and 20 Asian patients were included in the analysis. Asian women were diagnosed at younger age, 54.2 years versus 64.5 years for Caucasian and 64.9 years for BoAA women (OR 3.432; p=0.011); BoAA women were more likely to have serous type tumors (OR 2.061; p=0.008). No difference in overall survival was evident. The most frequently mutated gene in Caucasian and Asian tumours was PTEN (63% and 85%), unlike BoAA cases where it was TP53 (49%). Mutation and somatic copy number alteration (SCNA) analysis revealed an enrichment of TP53 mutations in BoAAs; whereas POLE and RPL22 mutations were more frequent in Caucasians. Major recurrent SCNA racial differences were observed at chromosomes 3p, 8, 10, and 16, which clustered BoAA tumors into 4 distinct groups and Caucasian tumors into 5 groups. There was a significantly higher frequency of somatic mutations in DNA mismatch repair genes in Asian tumours, in particular PMS2 (p=0.0036). In conclusion, inherent racial disparities appear to be present in the molecular profile of EC, which could have potential implications on clinical management.

p53 is a tumour suppressor that is activated in response to various types of stress. It is regulated by a complex pattern of over 50 different post-translational modifications, including ubiquitination by the E3 ligase MDM2, which leads to its proteasomal degradation. We have previously reported that expression of Bruton's Tyrosine Kinase (BTK) induces phosphorylation of p53 at the N-terminus, including Serine 15, and increases its protein levels and activity. The mechanisms involved in this process are not completely understood. Here, we show that BTK also increases MDM2 and is necessary for MDM2 upregulation after DNA damage, consistent with what we have shown for other p53 target genes. Moreover, we found that BTK binds to MDM2 on its PH domain and induces its phosphorylation. This suggested a negative regulation of MDM2 functions by BTK, supported by the fact BTK expression rescued the inhibitory effects of MDM2 on p53 transcriptional activity. Indeed, we observed that BTK mediated the loss of the ubiquitination activity of MDM2, a process that was dependent on the phosphorylation functions of BTK. Our data together shows that the kinase activity of BTK plays an important role in disrupting the MDM2-p53 negative feedback loop by acting at different levels, including binding to and inactivation of MDM2. This study provides a potential mechanism to explain how BTK modulates p53 functions.

To the editor: The inhibitor of Bruton’s tyrosine kinase (BTK) ibrutinib has transformed the treatment of chronic lymphocytic leukemia (CLL); many patients with previously untreatable disease may now enter durable remissions.[1][1],[2][2] Nevertheless, the kinome of ibrutinib is broad, resulting

ISSN 2045-1393 Q What exactly is meant by the term ‘precision medicine’ Although the empirical chemotherapy of the last century allowed some spectacular advances, the outlook for many patients with cancer remains unaltered. Moreover, the toxicities associated with chemotherapy are often severe, particularly in the elderly. The aim of the multibillion dollar cancer industry is to develop medicines that will allow targeted therapy of malignancy, in much the same manner as we use antibiotics to treat bacterial infections. ‘Precision medicines’ are the means by which we will be able to deliver effective ‘personalized cancer therapy’ without significant systemic toxicities. Precision medicines come in various shapes and sizes. Monoclonal antibodies, owing to their specificity for cell surface epitopes, have been used for many years. Novel antibody drug conjugates with enhanced efficacy and antibodies that are able to redirect the immune response are two notable recent advances. However, advances in structural biology, with the resolution of the structure of many key kinases and apoptotic inhibitors have allowed the development of a plethora of highly specific

The Nobel Prize in Physiology or Medicine for 1906 was shared by two scientists that set the basis for understanding how the brain works: Camillo Golgi and Santiago Ramón y Cajal were awarded the honour “in recognition of their work on the structure of the nervous system”. Yet, contrary to what usually happens in these situations, one of them was wrong and tried to sabotage the theories of the other one, refusing to admit his mistakes even when he gave his acceptance speech. How did Santiago Ramón y Cajal, a humble Spanish doctor, manage to upstage the legendary Italian pathologist and change forever the way we see the brain?

Senescence is a cellular process that is thought to have prognostic and therapeutic relevance in conditions such as cancer, aging, and fibrosis. However, current protocols for identifying senescent cells in vitro and in vivo have several drawbacks. Most markers used lack sufficient specificity and false positives and negatives in common. In addition, classical staining techniques often require lengthy protocols and do not offer objective quantification. Recently, several novel markers of senescence associated with the plasma membrane have been identified. Here, we propose to take advantage of these markers to define a customizable FACS-based protocol to detect senescent cells using antibodies tagged with fluorescence dyes. This method has the advantage of being fast and allowing quantitation. Furthermore, its specificity is increased using several markers simultaneously.

Senescence is a cellular process that is thought to have prognostic and therapeutic relevance in conditions such as cancer, aging, and fibrosis. However, current protocols for identifying senescent cells in vitro and in vivo have several drawbacks. Most markers used lack sufficient specificity and false positives and negatives in common. In addition, classical staining techniques often require lengthy protocols and do not offer objective quantification. Recently, several novel markers of senescence associated with the plasma membrane have been identified. Here, we propose to take advantage of these markers to define a customizable FACS-based protocol to detect senescent cells using antibodies tagged with fluorescence dyes. This method has the advantage of being fast and allowing quantitation. Furthermore, its specificity is increased using several markers simultaneously.

Chronic lymphocytic leukemia (CLL) cells multiply and become more resistant to immunochemotherapy in “proliferation centers” within tissues, whereas apoptosis occurs in the periphery. Various models recapitulate these microenvironments in vitro, such as stimulation with CD154 and IL4. Using this system, we observed a 30- to 40-fold induction of wild-type p53 protein in 50 distinct human CLL specimens tested, without the induction of either cell-cycle arrest or apoptosis. In contrast, the mRNA levels for p53 did not increase, indicating that its elevation occurred posttranscriptionally. Mechanistic investigations revealed that under the conditions studied, p53 was phosphorylated on residues associated with p53 activation and increased half-life. However, p53 protein induced in this manner could transcriptionally activate only a subset of target genes. The addition of a DNA-damaging agent further upregulated p53 protein levels, which led to apoptosis. p53 induction relied on the increase in intracellular reactive oxygen species observed after CD154 and IL4 stimulation. We propose that chronic oxidative stress is a characteristic of the microenvironment in B-cell “proliferation centers” in CLL that are capable of elevating the basal expression of p53, but to levels below the threshold needed to induce arrest or apoptosis. Our findings suggest that reactivation of the full transcriptional activities of p53 in proliferating CLL cells may offer a possible therapeutic strategy. Cancer Res; 76(21); 6311–9. ©2016 AACR.

p53 is a tumor suppressor that prevents the emergence of transformed cells by inducing apoptosis or senescence, among other responses. Its functions are regulated tightly by posttranslational modifications. Here we show that Bruton's tyrosine kinase (BTK) is a novel modulator of p53. We found that BTK is induced in response to DNA damage and p53 activation. BTK induction leads to p53 phosphorylation, which constitutes a positive feedback loop that increases p53 protein levels and enhances the transactivation of its target genes in response to stress. Inhibiting BTK reduced both p53-dependent senescence and apoptosis. Further, BTK expression also upregulated DNA damage signals and apoptosis. We conclude that despite being involved in oncogenic signals in blood malignancies, BTK has antineoplastic properties in other contexts, such as the enhancement of p53's tumor suppressor responses. Along with evidence that BTK expression correlates with good prognosis in some epithelial tumors, our findings may encourage a reevaluation of the clinical uses of BTK inhibitors in cancer therapy. Cancer Res; 76(18); 5405–14. ©2016 AACR.

p53 is a critical tumor suppressor in humans. It functions mostly as a transcriptional factor and its activity is regulated by numerous post-translational modifications. Among different covalent modifications found on p53 the most controversial one is lysine methylation. We found that human G9a (hG9a) unlike its mouse orthologue (mG9a) potently stimulated p53 transcriptional activity. Both ectopic and endogenous hG9a augmented p53-dependent transcription of pro-apoptotic genes, including Bax and Puma, resulting in enhanced apoptosis and reduced colony formation. Significantly, shRNA-mediated knockdown of hG9a attenuated p53-dependent activation of Puma. On the molecular level, hG9a interacted with histone acetyltransferase, p300/CBP, resulting in increased histone acetylation at the promoter of Puma. The bioinformatics data substantiated our findings showing that positive correlation between G9a and p53 expression is associated with better survival of lung cancer patients. Collectively, this study demonstrates that depending on the cellular and organismal context, orthologous proteins may exert both overlapping and opposing functions. Furthermore, this finding has important ramifications on the use of G9a inhibitors in combination with genotoxic drugs to treat p53-positive tumors.

The authors report a case of purine analogue–refractory hairy-cell leukemia with biallelic BRAF V600E mutations and a high leukemic burden that was treated successfully with vemurafenib, a BRAF inhibitor.

Oxygen availability has important effects on cell physiology. Although hyperoxic and hypoxic stresses have been well characterized, little is known about cellular functions in the oxygen levels commonly found in vivo. Here, we show that p53-dependent apoptosis in response to different DNA-damaging agents was reduced when normal and cancer cells were cultured at physiological oxygen tensions instead of the usual atmospheric levels. Different from what has been described in hypoxia, this was neither determined by decreases in p53 induction or its transactivation activity, nor by differences in the intracellular accumulation of reactive oxygen species. At these physiological oxygen levels, we found a constitutive activation of the ERK1/2 MAPK in all the models studied. Inhibition of this signaling pathway reversed the protective effect in some but not all cell lines. We conclude that a stress-independent constitutive activation of prosurvival pathways, including but probably not limited to MAPK, can protect cells in physiological oxygen tensions against genotoxic stress. Our results underscore the need of considering the impact of oxygen levels present in the tissue microenvironment when studying cell sensitivity to treatments such as chemotherapy and radiotherapy.

Tumor suppressor p53 is activated by several stimuli, including DNA damage and oncogenic stress. Previous studies (Takaoka, A., S. Hayakawa, H. Yanai, D. Stoiber, H. Negishi, H. Kikuchi, S. Sasaki, K. Imai, T. Shibue, K. Honda, and T. Taniguchi. 2003. Nature. 424:516–523) have shown that p53 is also induced in response to viral infections as a downstream transcriptional target of type I interferon (IFN) signaling. Moreover, many viruses, including SV40, human papillomavirus, Kaposi's sarcoma herpesvirus, adenoviruses, and even RNA viruses such as polioviruses, have evolved mechanisms designated to abrogate p53 responses. We describe a novel p53 function in the activation of the IFN pathway. We observed that infected mouse and human cells with functional p53 exhibited markedly decreased viral replication early after infection. This early inhibition of viral replication was mediated both in vitro and in vivo by a p53-dependent enhancement of IFN signaling, specifically the induction of genes containing IFN-stimulated response elements. Of note, p53 also contributed to an increase in IFN release from infected cells. We established that this p53-dependent enhancement of IFN signaling is dependent to a great extent on the ability of p53 to activate the transcription of IFN regulatory factor 9, a central component of the IFN-stimulated gene factor 3 complex. Our results demonstrate that p53 contributes to innate immunity by enhancing IFN-dependent antiviral activity independent of its functions as a proapoptotic and tumor suppressor gene.

Classically, the nucleotide excision repair (NER) of cyclobutane pyrimidine dimers (CPD) is a lengthy process (t1/2 > 48 h). Using the T4 endonuclease V-modified comet assay, we uniquely found a far more rapid repair of UVA-induced CPD (t1/2 = 4.5 h) in human skin keratinocytes. The repair of UVB-induced CPD began to slow within 1 h of irradiation, causing damage to persist for over 36 h. A similar trend was noted for the repair of oxidatively-modified purine nucleobases. Supportive of this differential repair, we noted an up-regulation of key genes associated with NER in UVA-irradiated cells, whereas the same genes were down regulated in UVB-irradiated cells. There were no significant differences in cell viability between the two treatments over the first 6 h post-irradiation, but after 24 h apoptosis had increased significantly in the UVB-irradiated cells. The role of apoptosis was confirmed using a pan-caspase inhibitor, which increased CPD repair, similar to that seen with UVA. These data indicate that the cellular ‘decision’ for apoptosis/DNA repair occurs far earlier than previously understood, and that the induction of apoptosis leads to lesion persistence, and not vice versa. This also highlights a new, potential increased carcinogenic risk from UVA-induced DNA damage as, rather than undergoing apoptosis, high levels of damage are tolerated and repaired, with the attendant risk of mutation.

The mechanisms of action of polycystin-1 (PC1) have been difficult to dissect because of its interaction with multiple factors, the heterogeneity of the genetic mutations, and the complexity of the experimental animal models. Here, stable knockdown of PC1 in MDCK epithelial cells was achieved by lentiviral-mediated delivery of a specific small interfering RNA for PKD1. The reduction of PC1 expression prevented tubulogenesis in three-dimensional collagen type I culture in response to hepatocyte growth factor and induced formation of cysts. PC1 knockdown created a condition of haploinsufficiency that led to hyperproliferation, increased adhesion to collagen type I, and increased apoptosis. It was shown that the suppression of PC1 was associated with the increased expression of integrin-alpha2beta1 and reduced apoptosis in cells grown on collagen type I. The engagement of integrin-alpha2beta1 seemed to be essential for the survival because PC1 knockdown cells were significantly less susceptible to anoikis by a mechanism that was reversible by anti-integrin-alpha2beta1 blocking antibodies. Overall, these data link integrin-alpha2beta1 to some of the biologic functions that are ascribed to PC1 and establish the potential of this approach for the direct study of PC1 functions in a genetically defined background. Furthermore, these findings indicate that reduction of PC1 expression levels, rather than the loss of heterozygosity, may be sufficient to induce cystogenesis.

The cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1/Sdi1) was identified initially as a gene induced in senescent cells and itself has been shown to cause permanent growth arrest/senescence. Reactive oxygen species (ROS), a byproduct of oxidative processes, can also induce an irreversible growth arrest similar to senescence. Here we show that p21 increased intracellular levels of ROS both in normal fibroblasts and in p53-negative cancer cells. N-acetyl-L-cysteine, an ROS inhibitor, rescued p21-induced senescence, showing that ROS elevation is necessary for induction of the permanent growth arrest phenotype. p16(Ink4a), a CDK4- and CDK6-specific inhibitor, failed to increase ROS levels, and cell cycle arrest induced by p16 was reversible following its down-regulation, demonstrating the specificity of this p21 effect. A p21 mutant that lacked the ability to bind proliferating cell nuclear antigen (PCNA) retained the ability to induce both ROS and permanent growth arrest. All of these findings establish that p21 mediates senescence by a mechanism involving ROS accumulation which does not require either its PCNA binding or the CDK inhibitory functions shared with p16.

HIF-1 is a transcription factor that mediates the cellular responses to low oxygen environments, mainly as a result of having an oxygen-labile subunit, HIF-1α. HIF-1α has been carefully studied in the context of severe hypoxic stresses (

Stra6 is the retinoic acid (RA)-inducible gene encoding the cellular receptor for holo-retinol binding protein. This transmembrane protein mediates the internalization of retinol, which then upregulates RA-responsive genes in target cells. Here, we show that Stra6 can be upregulated by DNA damage in a p53-dependent manner, and it has an important role in cell death responses. Stra6 expression induced significant amounts of apoptosis in normal and cancer cells, and it was also able to influence p53-mediated cell fate decisions by turning an initial arrest response into cell death. Moreover, inhibition of Stra6 severely compromised p53-induced apoptosis. We also found that Stra6 induced mitochondria depolarization and accumulation of reactive oxygen species, and that it was present not only at the cellular membrane but also in the cytosol. Finally, we show that these novel functions of Stra6 did not require downstream activation of RA signalling. Our results present a previously unknown link between the RA and p53 pathways and provide a rationale to use retinoids to upregulate Stra6, and thus enhance the tumour suppressor functions of p53. This may have implications for the role of vitamin A metabolites in cancer prevention and treatment.

p21Waf1/Cip1/Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs) and one of the best characterized p53 direct target genes. Moreover, it is one of the principal inducers of senescence, a permanent arrest phenotype related to ageing and the prevention of cell transformation. It was initially thought that p21 had tumour suppressor properties, due to its ability to stop cell cycle at different stages, either transitorily or permanently. However, recent evidence points to a much more complex picture. It is now well established that p21 itself can trigger apoptosis in certain situations, even independently of p53. On the other hand, p21-mediated cell arrest can actually limit the sensitivity to apoptotic stimuli. To complicate matters even further, p21 has been shown to have other direct pro-survival functions and could even be contributing to tumourigenesis through the secretion of growth factors by arrested cells. At the core of these antagonistic functions of p21 is the generation of Reactive Oxygen Species (ROS), which have been shown to be important both in the induction of apoptosis and the establishment of senescence, and could also participate in the negative effects on tissue homeostasis of the senescent cell secretome. p21 has the ability to stop cancer cell growth and is not mutated in cancer. However, without a better understanding of its pleiotropic functions we will not be able to harness its clinical potential. It is therefore important to investigate the mechanisms by which p21 affects cell physiology and its use of ROS as messengers and effectors.

Tothom té una idea més o menys clara del que representa un càncer: un conjunt de cèl·lules que es divideixen sense control. Darrera aquesta imatge simplista s’amaga un dels problemes de salut més complexes i més greus que existeixen. És gràcies als avenços biomèdics dels últims vint anys que finalment estem reconeixent les grans diferències que hi ha entre els tipus de càncers coneguts, un descobriment que resultarà clau per a dissenyar teràpies més efectives en un futur proper.

p21(Waf1/Cip1/Sdi1) is a cyclin-dependent kinase inhibitor that mediates cell cycle arrest. Prolonged p21 up-regulation induces a senescent phenotype in normal and cancer cells, accompanied by an increase in intracellular reactive oxygen species (ROS). However, it has been shown recently that p21 expression can also lead to cell death in certain models. The mechanisms involved in this process are not fully understood. Here, we describe an induction of apoptosis by p21 in sarcoma cell lines that is p53-independent and can be ameliorated with antioxidants. Similar levels of p21 and ROS caused senescence in the absence of significant death in other cancer cell lines, suggesting a cell-specific response. We also found that cells undergoing p21-dependent cell death had higher sensitivity to oxidants and a specific pattern of mitochondrial polarization changes. Consistent with this, apoptosis could be blocked with targeted expression of catalase in the mitochondria of these cells. We propose that the balance between cancer cell death and arrest after p21 up-regulation depends on the specific effects of p21-induced ROS on the mitochondria. This suggests that selective up-regulation of p21 in cancer cells could be a successful therapeutic intervention for sarcomas and tumors with lower resistance to mitochondrial oxidative damage, regardless of p53 status.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenetic disease predominantly caused by alteration or dysregulation of the PKD1 gene, which encodes polycystin-1 (PC1). The disease is characterized by the progressive expansion of bilateral fluid-filled renal cysts that ultimately lead to renal failure. Individual cysts, even within patients with germline mutations, are genetically heterogeneous, displaying diverse chromosomal abnormalities. To date, the molecular mechanisms responsible for this genetic heterogeneity remain unknown. Using a lentiviral-mediated siRNA expression model of Pkd1 hypomorphism, we show that loss of PC1 function is sufficient to produce centrosome amplification and multipolar spindle formation. These events lead to genomic instability characterized by gross polyploidism and mitotic catastrophe. Following these dramatic early changes, the cell population rapidly converges toward a stable ploidy in which centrosome amplification is significantly decreased, though cytological abnormalities such as micronucleation, chromatin bridges and aneuploidy remain common. In agreement with our in vitro findings, we provide the first in vivo evidence that significant centrosome amplification occurs in kidneys from conditional Pkd1 knockout mice at early and late time during the disease progression as well as in human ADPKD patients. These findings establish a novel function of PC1 in ADPKD pathogenesis and a genetic mechanism that may underlie the intrafamilial variability of ADPKD progression.

Reactive oxygen species (ROS), the principal mediators of oxidative stress, induce responses such as apoptosis or permanent growth arrest/senescence in normal cells. Moreover, p53 activation itself contributes to ROS accumulation. Here we show that treatment of p53-null cancer cells with sublethal concentrations of ROS triggered an arrest with some morphological similarities to cellular senescence. Different from a classical senescent arrest in G(1), the ROS-induced arrest was predominantly in the G(2) phase of the cell cycle, and its establishment depended at least in part on an intact Chk1-dependent checkpoint. Chk1 remained phosphorylated only during the repair of double strand DNA breaks, after which Chk1 was inactivated, the G(2) arrest was suppressed, and some cells recovered their ability to proliferate. Inhibition of Chk1 by an RNAi approach resulted in an increase in cell death in p53-null cells, showing that the Chk1-dependent G(2) checkpoint protected cells that lacked a functional p53 pathway from oxidative stress. It has been proposed that the induction of a senescent-like phenotype by antineoplastic agents can contribute therapeutic efficacy. Our results indicate that oxidative stress-induced growth arrest of p53-null tumor cells cannot be equated with effective therapy owing to its reversibility and supports the concept that targeting Chk1 may enhance the effects of DNA-damaging agents on cancer progression in such tumors.

Recent in vivo studies suggest that hMPV is a poor inducer of inflammatory cytokines and that clinical symptoms may not be related to immune-mediated pathogenesis as it has been proposed for respiratory syncytial virus (RSV) and human parainfluenza 3 (HPF3). Dendritic cells (DCs) are specialized antigen presenting cells, and very effective at inducing specific CTLs after encountering invading viruses. Interactions of hMPV with DCs have not been characterized. We hypothesized that the relatively mild inflammatory responses observed in vivo after hMPV infection might be at least in part due to hMPV's poor ability to stimulate and activate DCs. hMPV actively infected immature monocyte-derived CD11c+/HLA-DR+ DCs. However, in contrast to RSV or HPF3, hMPV caused no gross cytopathic effects such as syncytia, lytic infection, or massive apoptosis. DCs exposed to hMPV show no cytopathic effects under tissue culture conditions permissive for viral replication. The surface maturation markers CD83 and CD86 were not significantly up-regulated in infected DCs as compared to uninfected controls, while expression of CD80 appeared increased. Stimulation of hMPV-infected DCs with LPS resulted in the enhanced expression of all these surface markers indicating that hMPV is not generally suppressing DC maturation. Overall, cytokine expression remained low. These results indicate that hMPV does not induce effective DC maturation in vitro and suggest that the weak stimulation of DCs may account for the overall low immunogenicity of this virus observed in vivo.

Mechanisms of dendritic cells (DCs) immunomodulation by parainfluenza viruses have not been characterized. We analyzed whether the human parainfluenza 3 (HPF3) virus hemagglutinin-neuraminidase glycoprotein (HN) might influence DC maturation. HN possesses a receptor binding function and a neuraminidase or desialidating activity. To assess whether the neuraminidase activity of HN affects DC maturation, human myeloid DCs were exposed to either live or UV-inactivated HPF3 viruses containing wild type or a mutated form of HN with decreased neuraminidase activity. Exposure of human DCs to either UV-inactivated or live virus induced up-regulation of CD83 and CD86 surface markers, morphological changes, and a cytokine expression pattern consistent with maturation. However, the level of maturation was found to be lower in DCs infected with the neuraminidase deficient variant as compared to the wild type. These results suggest that during the course of viral infection, HN's neuraminidase activity may play an important role contributing to maturation and activation of DCs.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to stimulate the growth of a variety of cells in an autocrine or paracrine manner. Although HB-EGF is widely expressed in tumors compared with normal tissue, its contribution to tumorigenicity is unknown. HB-EGF can be produced as a membrane-anchored form (pro-HB-EGF) and later processed to a soluble form (s-HB-EGF), although a significant amount of pro-HB-EGF remains uncleaved on the cell surface. To understand the roles of two forms of HB-EGF in promoting tumor growth, we have studied the effects of HB-EGF expression in the process of tumorigenesis using in vitro and in vivo systems. We demonstrate here that in EJ human bladder cancer cells containing a tetracycline-regulatable s-HB-EGF or pro-HB-EGF expression system, s-HB-EGF expression increased their transformed phenotypes, including growth rate, colony-forming ability, and activation of cyclin D1 promoter, as well as induction of vascular endothelial growth factor in vitro. Moreover, s-HB-EGF or wild-type HB-EGF induced the expression and activities of the metalloproteases, MMP-9 and MMP-3, leading to enhanced cell migration. In vivo studies also demonstrated that tumor cells expressing s-HB-EGF or wild-type HB-EGF significantly enhanced tumorigenic potential in athymic nude mice and exerted an angiogenic effect, increasing the density and size of tumor blood vessels. However, cells expressing solely pro-HB-EGF did not exhibit any significant tumorigenic potential. These findings establish s-HB-EGF as a potent inducer of tumor growth and angiogenesis and suggest that therapeutic intervention aimed at the inhibition of s-HB-EGF functions may be useful in cancer treatment.

The p53 tumor suppressor gene can induce either apoptosis or a permanent growth arrest (also termed senescence) phenotype in response to cellular stresses. We show that the increase in intracellular reactive oxygen species (ROS) associated with the magnitude of p53 protein expression correlated with the induction of either senescence or apoptosis in both normal and cancer cells. ROS inhibitors ameliorated both p53-dependent cell fates, implicating ROS accumulation as an effector in each case. The absence of Bax or PUMA strongly inhibited both p53-induced apoptosis and ROS increase, indicating an important role these p53 targets affecting mitochondrial function genes in p53-mediated ROS accumulation. Moreover, physiological p53 levels in combination with an exogenous ROS source were able to convert a p53 senescence response into apoptosis. All of these findings establish a critical role of ROS accumulation and mitochondrial function in p53-dependent cell fates and show that other ROS inducers can collaborate with p53 to influence these fate decisions. Thus, our studies imply that therapeutic agents that generate ROS are more likely to be toxic for normal cells than p53-negative tumor cells and provide a rationale for identifying therapeutic agents that do not complement p53 in ROS generation to ameliorate the cytotoxic side effects in normal cells.

The apoptosis-associated speck-like protein (ASC) is an unusual adaptor protein that contains the Pyrin/PAAD death domain in addition to the CARD protein-protein interaction domain. Here, we present evidence that ASC can function as an adaptor molecule for Bax and regulate a p53-Bax mitochondrial pathway of apoptosis. When ectopically expressed, ASC interacted directly with Bax, colocalized with Bax to the mitochondria, induced cytochrome c release with a significant reduction of mitochondrial membrane potential and resulted in the activation of caspase-9, -2 and -3. The rapid induction of apoptosis by ASC was not observed in Bax-deficient cells. We also show that induction of ASC after exposure to genotoxic stress is dependent on p53. Blocking of endogenous ASC expression by small-interfering RNA (siRNA) reduced the apoptotic response and inhibited translocation of Bax to mitochondria in response to p53 or genotoxic insult, suggesting that ASC is required to translocate Bax to the mitochondria. Our findings demonstrate that ASC has an essential role in the intrinsic mitochondrial pathway of apoptosis through a p53-Bax network.

Cellular senescence is a terminal differentiation state that has been proposed to have a role in both tumour suppression and ageing. This view is supported by the fact that accumulation of senescent cells can be observed in response to oncogenic stress as well as a result of normal organismal ageing. Thus, identifying senescent cells in in vivo and in vitro has an important diagnostic and therapeutic potential. The molecular pathways involved in triggering and/or maintaining the senescent phenotype are not fully understood. As a consequence, the markers currently utilized to detect senescent cells are limited and lack specificity. In order to address this issue, we screened for plasma membrane-associated proteins that are preferentially expressed in senescent cells. We identified 107 proteins that could be potential markers of senescence and validated 10 of them (DEP1, NTAL, EBP50, STX4, VAMP3, ARMX3, B2MG, LANCL1, VPS26A and PLD3). We demonstrated that a combination of these proteins can be used to specifically recognize senescent cells in culture and in tissue samples and we developed a straightforward fluorescence-activated cell sorting-based detection approach using two of them (DEP1 and B2MG). Of note, we found that expression of several of these markers correlated with increased survival in different tumours, especially in breast cancer. Thus, our results could facilitate the study of senescence, define potential new effectors and modulators of this cellular mechanism and provide potential diagnostic and prognostic tools to be used clinically.

Overexpression of alphaB-crystallin is associated with numerous neurodegenerative diseases and abnormal cell growth patterns. To study the mechanisms involved in the control of the transcriptional activity of the gene we have characterized its expression in different chicken tissues. The sequence of the alphaB-crystallin cDNA isolated from chicken testis and 6-day-old chick embryo is highly homologous to the duck alphaB-crystallin cDNA and differs from the previously reported chicken lens alphaB-crystallin cDNA in the 5' untranslated region (5'-UTR) and in one amino acid of the coding sequence. Four forms of the alphaB-crystallin cDNA detected in chicken testes arise from the use of alternative transcription initiation sites and alternative polyadenylation signals. The two principal hybridizing bands found in lens and embryonic tissues possess a short 5'-UTR and differ in the length of the 3'-UTR. Forms with longer 5'-UTR are present in testis, muscle, and heart. The use of different start sites and polyadenylation signals could modulate transcriptional activity and the stability of the messages. The expression of the alphaB-crystallin gene decreases from day 6 to day 8 of chick embryogenesis, in parallel with the expression of the polyubiquitin gene UbII.