MKP-1 suppresses PARP-1 degradation to mediate cisplatin resistance.

Understanding the mechanisms of platinum compound resistance, including cisplatin resistance, has important implications for improving cancer treatments. Previous studies identified a potential role for mitogen-activated protein kinase phosphatase-1 (MKP-1) in cisplatin resistance. This work focuses on the regulation of poly(ADP-ribose) polymerase-1 (PARP-1) expression by MKP-1. We found that MKP-1 overexpression stimulates PARP-1 and poly(ADP-ribose) (PAR) protein expression and cisplatin resistance while its downregulation suppresses PARP-1 and PAR protein expression and cisplatin resistance. Silencing MKP-1 promoted PARP-1 ubiquitination, which decreased PARP-1 protein levels. We also found that silencing c-Jun N-terminal kinase 1/2 (JNK1/2) decreased PARP-1 ubiquitination while increasing total PARP-1 protein levels. Furthermore, we showed that acquired cisplatin-resistant ovarian cancer cells expressed high levels of MKP-1 and PARP-1 proteins, and that silencing MKP-1 or PARP-1 increased cisplatin sensitivity in resistant cells. Notably, the pharmacologic inhibition of PARP activity restored cisplatin sensitivity in MKP-1 overexpressing cells. Thus, this work indicates that suppression of JNK1/2 activity by MKP-1 maintains PARP-1 levels and suggests that MKP-1-mediated cisplatin resistance can be bypassed by PARP-1 inhibition.

JNKs function as CDK4-activating kinases by phosphorylating CDK4 and p21.

Cyclin D-CDK4/6 are the first cyclin-dependent kinase (CDK) complexes to be activated by mitogenic/oncogenic pathways. They have a central role in the cell multiplication decision and in its deregulation in cancer cells. We identified T172 phosphorylation of CDK4 rather than cyclin D accumulation as the distinctly regulated step determining CDK4 activation. This finding challenges the view that the only identified metazoan CDK-activating kinase, cyclin H-CDK7-Mat1 (CAK), which is constitutively active, is responsible for the activating phosphorylation of all cell cycle CDKs. We previously showed that T172 phosphorylation of CDK4 is conditioned by an adjacent proline (P173), which is not present in CDK6 and CDK1/2. Although CDK7 activity was recently shown to be required for CDK4 activation, we proposed that proline-directed kinases might specifically initiate the activation of CDK4. Here, we report that JNKs, but not ERK1/2 or CAK, can be direct CDK4-activating kinases for cyclin D-CDK4 complexes that are inactivated by p21-mediated stabilization. JNKs and ERK1/2 also phosphorylated p21 at S130 and T57, which might facilitate CDK7-dependent activation of p21-bound CDK4, however, mutation of these sites did not impair the phosphorylation of CDK4 by JNKs. In two selected tumor cells, two different JNK inhibitors inhibited the phosphorylation and activation of cyclin D1-CDK4-p21 but not the activation of cyclin D3-CDK4 that is mainly associated to p27. Specific inhibition by chemical genetics in MEFs confirmed the involvement of JNK2 in cyclin D1-CDK4 activation. Therefore, JNKs could be activating kinases for cyclin D1-CDK4 bound to p21, by independently phosphorylating both CDK4 and p21.

c-Jun N-terminal kinase 2 promotes enterocyte survival and goblet cell differentiation in the inflamed intestine.

c-Jun N-terminal kinases (JNKs) contribute to immune signaling but their functional role during intestinal mucosal inflammation has remained ill defined. Using genetic mouse models, we characterized the role of JNK1 and JNK2 during homeostasis and acute colitis. Epithelial apoptosis, regeneration, differentiation, and barrier function were analyzed in intestinal epithelium-specific (ΔIEC) or complete JNK1 and bone marrow chimeric or complete JNK2 deficient mice as well as double-knockout animals (JNK1ΔIECJNK2-/-) during homeostasis and acute dextran sulfate sodium (DSS)-induced colitis. Results were confirmed using human HT-29 cells and wild-type or JNK2-deficient mouse intestinal organoid cultures. We show that nonhematopoietic JNK2 but not JNK1 expression confers protection from DSS-induced intestinal inflammation reducing epithelial barrier dysfunction and enterocyte apoptosis. JNK2 additionally enhanced Atonal homolog 1 expression, goblet cell and enteroendocrine cell differentiation, and mucus production under inflammatory conditions. Our results identify a protective role of epithelial JNK2 signaling to maintain mucosal barrier function, epithelial cell integrity, and mucus layer production in the event of inflammatory tissue damage.

Apoptosis induced by the fungal pathogen gliotoxin requires a triple phosphorylation of Bim by JNK.

We previously reported that gliotoxin (GT), the major virulence factor of the mold Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised patients, induces apoptosis in a Bak-dependent manner. The signaling pathway leading to Bak activation and subsequent mitochondrial outer membrane permeabilization (MOMP) is elusive. Here, we show that GT and the supernatant of A. fumigatus (but not its GT-defective mutant) activate the JNK pathway and require a co-operative JNK-mediated BimEL phosphorylation at three sites (S100, T112 and S114) to induce apoptosis in mouse fibroblasts, human bronchial and mouse alveolar epithelial cells. Cells (i) treated with the JNK inhibitor SP600125, (ii) deleted or knocked down for JNK1/2 or Bim or (iii) carrying the BimEL triple phosphomutant S100A/T112A/S114A instead of wild-type BimEL are similarly resistant to GT-induced apoptosis. Triple-phosphorylated BimEL is more stable, redistributes from a cytoskeletal to a membrane fraction, better interacts with Bcl-2 and Bcl-xL and more effectively activates Bak than the unphosphorylated mutant. These data indicate that JNK-mediated BimEL phosphorylation at S100, T112 and S114 constitutes a novel regulatory mechanism to activate Bim in response to apoptotic stimuli.

Epizootics of sudden death in tammar wallabies (Macropus eugenii) associated with an orbivirus infection.

Epizootics of sudden death in tammar wallabies (Macropus eugenii) occurred at six research facilities and zoological gardens in New South Wales, Australia, in late 1998 and at one Queensland research facility in March 1999. There were 120 confirmed tammar wallaby deaths during this period; however, population censuses indicated that up to 230 tammar wallabies may have died. The majority of animals died without premonitory signs. A small proportion of wallabies exhibited increased respiratory rate, sat with a lowered head shortly before death or were discovered in lateral recumbency, moribund and with muscle fasciculations. Gross postmortem findings consistently included massive pulmonary congestion, mottled hepatic parenchyma and subcutaneous oedema throughout the hindlimbs and inguinal region. Approximately 30% of the animals examined also had extensive haemorrhage within the fascial planes and skeletal muscle of the hindlimb adductors, inguinal region, ventral thorax, dorsal cervical region and perirenal retroperitoneal area. The tissues of affected animals became autolytic within a short period after death. Bacteriological examination of tissues from 14 animals did not provide any significant findings. Toxicological examination of the gastric and colonic contents of four animals did not reveal evidence of brodifacoume or other rodenticides. Viruses from the Eubenangee serogroup of the Orbivirus genus were isolated from the cerebral cortex of nine, and the myocardium of two, tammar wallabies and the liver and intestine of another tammar wallaby. A similar orbivirus was also isolated from the cerebrospinal fluid of another tammar wallaby that died suddenly. The disease agent appears to be a previously unrecognised orbivirus in the Eubenangee serogroup. This is the first report of epizootics of sudden deaths in tammar wallabies apparently associated with an orbivirus infection.

Alternative splicing of Bim and Erk-mediated Bim(EL) phosphorylation are dispensable for hematopoietic homeostasis in vivo.

The pro-apoptotic BH3-only protein Bim has a major role in hematopoietic homeostasis, particularly in the lymphocyte compartment, where it strongly affects immune function. The three major Bim isoforms (Bim(EL), Bim(L) and Bim(S)) are generated by alternative splicing. Bim(EL), the most abundant isoform, contains a unique sequence that has been reported to be the target of phosphorylation by several MAP kinases. In particular, Erk1/2 has been shown to interact with Bim(EL) through the DEF2 domain of Bim(EL) and specifically phosphorylate this isoform, thereby targeting it for ubiquitination and proteasomal degradation. To examine the physiological importance of this mechanism of regulation and of the alternative splicing of Bim, we have generated several Bim knock-in mouse strains and analyzed their hematopoietic system. Although mutation in the DEF2 domain reduces Bim(EL) degradation in some circumstances, this mutation did not significantly increase Bim's pro-apoptotic activity in vivo nor impact on the homeostasis of the hematopoietic system. We also show that Bim(EL) and Bim(L) are interchangeable, and that Bim(S) is dispensable for the function of Bim. Hence, we conclude that physiological regulation of Bim relies on mechanisms independent of its alternative splicing or the Erk-dependent phosphorylation of Bim(EL).

A prospective longitudinal study of naturally infected horses to evaluate the performance characteristics of rapid diagnostic tests for equine influenza virus.

An outbreak of equine influenza (EI) occurred in Australia in 2007. During the laboratory support for this outbreak, real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assays and a blocking enzyme linked immunosorbent assay (bELISA) were used as testing methods to detect infection with the virus. The qRT-PCR and bELISA tests had not been used for EI diagnosis before, so it was not known how soon after infection these tests would yield positive results, or for how long these results would remain positive. To answer these questions, nasal swabs and blood samples were collected daily from a group of 36 naturally infected horses. EI viral RNA was detected in all horses by qRT-PCR from the first to tenth day after clinical signs were evident, and was detected in some horses for up to 34 days. Antibody was detected in the bELISA in some horses by day 3, with a median time to seroconversion of 5 days. The results from this study indicate that viral RNA can be detected from nasal swabs for much longer than infectious virus is thought to be shed from horses. The bELISA detected antibodies against EI virus in all horses for 139 days following infection, but only detected approximately 50% of horses 12 months following infection. Haemagglutination inhibition testing detected antibodies against H3 antigens in all horses for 28 days following infection, but 2 were negative by 35 days following infection.

The first identified case of pandemic H1N1 influenza in pigs in Australia.

A 300-sow farrow-to-finish herd in New South Wales was infected with influenza pandemic (H1N1) 2009 (H1N1/09) virus in July 2009 and became the first recorded case of influenza in pigs in Australia. The outbreak resulted from human-to-pig transmission. Clinical signs in affected pigs were mild compared with overseas reports of 'classical' swine influenza virus and included coughing and decreased appetite in a small proportion of non-lactating breeding stock, weaners, growers and finishers. A diagnosis of H1N1/09 influenza virus infection was confirmed using a combination of serology (haemagglutination inhibition, blocking enzyme-linked immunosorbent assay) and real-time reverse transcription polymerase chain reaction. Attempts at virus isolation were unsuccessful. Results of a longitudinal study of pigs on this farm suggested that the virus continued to circulate for 9 weeks after the onset of infection, but was not present 6 months later. This report highlights the difficulties in preventing transmission of H1N1/09 influenza virus from infected humans to pigs during a human pandemic.

The first five days: field and laboratory investigations during the early stages of the equine influenza outbreak in Australia, 2007.

Until August 2007, Australia was one of only three countries internationally recognised to be free of equine influenza (EI). This report documents the diagnosis of the first cases of EI in Australian horses and summarises the investigations that took place over the next 5 days. During that time, a multifocal outbreak was identified across eastern New South Wales and south-eastern Queensland. The use of an influenza type A pan-reactive real-time reverse transcription polymerase chain reaction allowed rapid confirmation of suspect cases of EI.

Application of real-time PCR and ELISA assays for equine influenza virus to determine the duration of viral RNA shedding and onset of antibody response in naturally infected horses.

During the equine influenza (EI) outbreak, two assays were used in parallel to diagnose the disease, to demonstrate freedom from infection in disease control zones and ultimately to demonstrate that EI virus had been eliminated from the Australian horse population. A longitudinal study of a population of naturally infected horses was established to determine the performance characteristics of these assays.

Performance of an automated solid-phase red cell adherence system compared with that of a manual gel microcolumn assay for the identification of antibodies eluted from red blood cells.

IgG antibodies coating red blood cells (RBCs) can be removed by elution procedures and their specificity determined by antibody identification studies. Although such testing is traditionally performed using the tube agglutination assay, prior studies have shown that the gel microcolumn (GMC) assay may also be used with comparable results. The purpose of this study was to compare an automated solid-phase red cell adherence (SPRCA) system with a GMC assay for the detection of antibodies eluted from RBCs. Acid eluates from 51 peripheral blood (PB) and 7 cord blood (CB) samples were evaluated by both an automated SPRCA instrument and a manual GMC assay. The concordance rate between the two systems for peripheral RBC samples was 88.2 percent (45 of 51), including cases with alloantibodies (n = 8), warm autoantibodies (n = 12), antibodies with no identifiable specificity (n = 2), and negative results (n = 23). There were six discordant cases, of which four had alloantibodies (including anti-Jka, -E, and -e) demonstrable by the SPRCA system only. In the remaining 2 cases, anti-Fya and antibodies with no identifiable specificity were demonstrable by the GMC assay only. All seven CB specimens produced concordant results, showing anti-A (n = 3), -B (n = 1), maternal anti-Jka (n = 2), or a negative result (n = 1). Automated SPRCA technology has a performance that is comparable with that of a manual GMC assay for identifying antibodies eluted from PB and CB RBCs.

Infertility and venereal disease in cattle inseminated with semen containing bovine herpesvirus type 5.

A group of 20 cows and heifers experienced poor conception rates and probable ovarian dysfunction after being artificially inseminated. When first examined, some showed signs of vulvovaginitis, with pustular, ulcerative lesions consistent with a herpesvirus infection. They had had no contact with bulls during the current breeding season. Bovine herpesvirus type 5 (BHV-5) was isolated from samples of frozen semen from the batch that had been used for the artificial insemination programme. BHV-5 was also isolated from the semen of a second apparently healthy bull during routine screening of its semen.

BGC20-1531, a novel, potent and selective prostanoid EP receptor antagonist: a putative new treatment for migraine headache.

BACKGROUND AND PURPOSE: Prostanoid EP(4) receptor antagonists may have therapeutic utility in the treatment of migraine since EP(4) receptors have been shown to be involved in prostaglandin (PG)E(2)-induced cerebral vascular dilatation, which may be an important contributor to migraine pain. This study reports the pharmacological characterization of BGC20-1531, a novel EP(4) receptor antagonist. EXPERIMENTAL APPROACH: BGC20-1531 was characterized in radioligand binding and in vitro functional assays employing recombinant and native EP(4) receptors. Changes in canine carotid haemodynamics were used to assess the pharmacodynamic profile of BGC20-1531 in vivo. KEY RESULTS: BGC20-1531 exhibited high affinity at recombinant human EP(4) receptors expressed in cell lines (pK(B) 7.6) and native EP(4) receptors in human cerebral and meningeal artery (pK(B) 7.6-7.8) but showed no appreciable affinity at a wide range of other receptors (including other prostanoid receptors), channels, transporters and enzymes (pKi < 5). BGC20-1531 competitively antagonized PGE(2)-induced vasodilatation of human middle cerebral (pK(B) 7.8) and meningeal (pK(B) 7.6) arteries in vitro, but had no effect on responses induced by PGE(2) on coronary, pulmonary or renal arteries in vitro. BGC20-1531 (1-10 mg.kg(-1) i.v.) caused a dose-dependent antagonism of the PGE(2)-induced increase in canine carotid blood flow in vivo. CONCLUSIONS AND IMPLICATIONS: BGC20-1531 is a potent and selective antagonist at EP(4) receptors in vitro and in vivo, with the potential to alleviate the symptoms of migraine that result from cerebral vasodilatation. BGC20-1531 is currently in clinical development for the treatment of migraine headache.

Role of MKK3-p38 MAPK signalling in the development of type 2 diabetes and renal injury in obese db/db mice.

AIMS/HYPOTHESIS: Obesity and diabetes are associated with increased intracellular p38 mitogen-activated protein kinase (MAPK) signalling, which may promote tissue inflammation and injury. Activation of p38 MAPK can be induced by either of the immediate upstream kinases, MAP kinase kinase (MKK)3 or MKK6, and recent evidence suggests that MKK3 has non-redundant roles in the pathology attributed to p38 MAPK activation. Therefore, this study examined whether MKK3 signalling influences the development of obesity, type 2 diabetes and diabetic nephropathy. METHODS: Wild-type and Mkk3 (also known as Map2k3) gene-deficient db/db mice were assessed for the development of obesity, type 2 diabetes and renal injury from 8 to 32 weeks of age. RESULTS: Mkk3 (+/+) db/db and Mkk3 (-/-) db/db mice developed comparable obesity and were similar in terms of incidence and severity of type 2 diabetes. At 32 weeks, diabetic Mkk3 (+/+) db/db mice had increased kidney levels of phospho-p38 and MKK3 protein. In comparison, kidney levels of phospho-p38 in diabetic Mkk3 ( -/- ) db/db mice remained normal, despite a fourfold compensatory increase in MKK6 protein levels. The reduced levels of p38 MAPK signalling in the diabetic kidneys of Mkk3 ( -/- ) db/db mice was associated with protection against the following: declining renal function, increasing albuminuria, renal hypertrophy, podocyte loss, mesangial cell activation and glomerular fibrosis. Diabetic Mkk3 ( -/- ) db/db mice were also significantly protected from tubular injury and interstitial fibrosis, which was associated with reduced Ccl2 mRNA expression and interstitial macrophage accumulation. CONCLUSIONS/INTERPRETATION: MKK3-p38 MAPK signalling is not required for the development of obesity or type 2 diabetes, but plays a distinct pathogenic role in the progression of diabetic nephropathy in db/db mice.

Infection with Menangle virus in flying foxes (Pteropus spp.) in Australia.

OBJECTIVE: To examine flying foxes (Pteropus spp.) for evidence of infection with Menangle virus. DESIGN: Clustered non-random sampling for serology, virus isolation and electron microscopy (EM). PROCEDURE: Serum samples were collected from 306 Pteropus spp. in northern and eastern Australia and tested for antibodies against Menangle virus (MenV) using a virus neutralisation test (VNT). Virus isolation was attempted from tissues and faeces collected from 215 Pteropus spp. in New South Wales. Faecal samples from 68 individual Pteropus spp. and four pools of faeces were examined by transmission EM following routine negative staining and immunogold labelling. RESULTS: Neutralising antibodies (VNT titres > or = 8) against MenV were detected in 46% of black flying foxes (P. alecto), 41% of grey-headed flying foxes (P. poliocephalus), 25% of spectacled flying foxes (P. conspicillatus) and 1% of little red flying foxes (P. scapulatus) in Australia. Positive sera included samples collected from P. poliocephalus in a colony adjacent to a piggery that had experienced reproductive disease caused by MenV. Virus-like particles were observed by EM in faeces from Pteropus spp. and reactivity was detected in pooled faeces and urine by immunogold EM using sera from sows that had been exposed to MenV. Attempts to isolate the virus from the faeces and tissues from Pteropus spp. were unsuccessful. CONCLUSION: Serological evidence of infection with MenV was detected in Pteropus spp. in Australia. Although virus-like particles were detected in faeces, no viruses were isolated from faeces, urine or tissues of Pteropus spp.

Role of mitogen-activated protein kinase kinase 4 in cancer.

Mitogen-activated protein (MAP) kinase kinase 4 (MKK4) is a component of stress activated MAP kinase signaling modules. It directly phosphorylates and activates the c-Jun N-terminal kinase (JNK) and p38 families of MAP kinases in response to environmental stress, pro-inflammatory cytokines and developmental cues. MKK4 is ubiquitously expressed and the targeted deletion of the Mkk4 gene in mice results in early embryonic lethality. Further studies in mice have indicated a role for MKK4 in liver formation, the immune system and cardiac hypertrophy. In humans, it is reported that loss of function mutations in the MKK4 gene are found in approximately 5% of tumors from a variety of tissues, suggesting it may have a tumor suppression function. Furthermore, MKK4 has been identified as a suppressor of metastasis of prostate and ovarian cancers. However, the role of MKK4 in cancer development appears complex as other studies support a pro-oncogenic role for MKK4 and JNK. Here we review the biochemical and functional properties of MKK4 and discuss the likely mechanisms by which it may regulate the steps leading to the formation of cancers.

Induction of activating transcription factor 3 by anoxia is independent of p53 and the hypoxic HIF signalling pathway.

Solid tumors often have an inadequate blood supply, which results in large regions that are subjected to hypoxic or anoxic stress. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates much of the transcriptional response of cells to hypoxia. Activating transcription factor 3 (ATF3) is another transcription factor that responds to a variety of stresses and is often upregulated in cancer. We investigated the regulation of ATF3 by oxygen deprivation. ATF3 induction occurred most robustly under anoxia, is common, and it is not dependent on presence of HIF-1 or p53, but is sensitive to the inhibition of c-Jun NH2-terminal kinase activation and the antioxidant N-acetylcystein. ATF3 could also be induced by desferrioxamine but not by the mitochondrial poison cyanide or the nonspecific 2-oxoglutarate dioxygenase inhibitor dimethyloxalylglycine. We also show that anoxic ATF3 mRNA is more stable than normoxic mRNA providing a mechanism for this induction. Thus, this study demonstrates that the regulation of ATF3 under anoxia is independent of 2-oxoglutarate dioxygenase, HIF-1 and p53, presumably involving multiple regulatory pathways.

An asymmetric shock wave in the 2006 outburst of the recurrent nova RS Ophiuchi.

Nova outbursts take place in binary star systems comprising a white dwarf and either a low-mass Sun-like star or, as in the case of the recurrent nova RS Ophiuchi, a red giant. Although the cause of these outbursts is known to be thermonuclear explosion of matter transferred from the companion onto the surface of the white dwarf, models of the previous (1985) outburst of RS Ophiuchi failed to adequately fit the X-ray evolution and there was controversy over a single-epoch high-resolution radio image, which suggested that the remnant was bipolar rather than spherical as modelled. Here we report the detection of spatially resolved structure in RS Ophiuchi from two weeks after its 12 February 2006 outburst. We track an expanding shock wave as it sweeps through the red giant wind, producing a remnant similar to that of a type II supernova but evolving over months rather than millennia. As in supernova remnants, the radio emission is non-thermal (synchrotron emission), but asymmetries and multiple emission components clearly demonstrate that contrary to the assumptions of spherical symmetry in models of the 1985 explosion, the ejection is jet-like, collimated by the central binary whose orientation on the sky can be determined from these observations.