Kinase Phosphatase Research Articles

A comparative Study of Some Biochemical Characteristics of Common carp (Cyprinus carpio L.) in Earthen ponds at Different Locations of the Tigris and Euphrates Rivers / Middle of Iraq.

This study was conducted with the aim of showing the effect of different locations and water quality on some blood enzyme properties of common carp (Cyprinus carpio L.). This study was carried out in four different locations in central Iraq for the period from June 1, 2022 to November 30, 2022. The Dujail and Suwayra sites on the Tigris River were used, and the Fallujah and Mahaweel sites on the Euphrates River were used. 120 fish were collected from the four sites, 30 fish for each site, and some biochemical traits were measured, including total protein, albumin, kinase phosphatase, glucose and total cholesterol. The results of the current study showed that the values of blood enzymes increased with the rise in temperatures during the hot months and decreased with the decrease in temperatures in the cold months. It was also found that there were highly significant differences between the Tigris and Euphrates rivers in the values of total protein, kinase phosphatase, glucose and total cholesterol, as the Tigris River was significantly superior to the Euphrates River in the values of these four enzymes, and this indicates that the Euphrates River is better than the Tigris River. As for the value of the two albums, there are no significant differences between the Tigris and Euphrates rivers. The current results also showed that there were significant differences in the values of total protein and kinase phosphatase, as these values were significantly higher in the Dujail site than in the Fallujah site, while no significant differences were observed between the Suwayrah and Al-Mahaweel sites. The current results also showed that there were no significant differences between the four sites in the value of the two albums. The current results showed higher values of blood enzymes in the Dujail site compared to the Fallujah site, and this indicates that the Fallujah site is one of the best sites in the values of enzymes, being close and within the natural limits, and this is due to the availability of an appropriate healthy environment in the Fallujah site compared to other sites.

Biochemical Changes Occurring in Yellowfin Tuna Eggs during Embryonic Development

Changes in nutritional constituents and enzyme activities were clarified in yellowfin tuna (YFT, Thunnus albacares) eggs during embryonic development, from eggs immediately after fertilization to hatching. The protein levels in the eggs gradually increased with development until the completion of hatching. In contrast, the triglyceride (TG) and free amino acid (FAA) levels in the eggs gradually declined with embryonic development until hatching was complete, although the energy composition of the FAAs was lower than that of the TGs throughout embryonic development. These results indicate that endogenous TGs are preferentially expended as an energy source during embryonic development. Overall, changes in the activities of aspartate aminotransferase, alanine aminotransferase, creatine kinase, and alkaline phosphatase showed similar patterns throughout development. First, the enzyme levels diminished; then, they remained at constant, low levels just before hatching, when they rapidly increased. This rapid increase was consistent with the protein content, suggesting that organ differentiation and functionalization were promoted during this period. These results will contribute to the establishment of mass-seeding production of YFT.

Influence of age and sex on the blood biochemical constituent values of broiler breeders during the egg-laying stage.

This study aimed to evaluate the influence of age and sex on the blood biochemical constituents of broiler breeders during the egg production stage. The analysis was performed in an industrial broiler breeder farm, and blood samples were collected from males and females at five different ages. Biochemical elements analysed in each serum sample were total proteins, albumin, globulins, uric acid, total cholesterol and triglycerides, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), creatine kinase (CK), alkaline phosphatase (PAL), calcium and phosphorus, beside the glycaemic status. At most ages, females had higher values of total proteins, albumin, globulins, triglycerides, cholesterol, glucose, calcium, phosphorus, Ca/P (calcium and phosphorus) ratio and gamma-glutamyltransferase (GGT). The values of uric acid, aspartate aminotransferase (AST), creatine kinase (CK) and alkaline phosphatase (PAL) were higher in males. The lowest protein values were found at 28 and 60 weeks old. The mean albumin values were significantly higher at 44 and 52 weeks old in males and females. A trend of increasing globulin values with increasing age up to 52 weeks old was observed. Although calcium and phosphorus did not vary according to age, the Ca/P ratio was lowest at 36 weeks old. Comparing the means of both sexes, the AST and GGT values were significantly higher in 60-week-old birds. The highest serum levels of CK occurred at 28 and 52 weeks old. The physiological levels of serum biomarkers presented in this work are important for evaluating productivity performance, welfare and disease indication in breeding flocks.

The Effect of the Repair of Induced Articular Cartilage Defects in Pigs Using Calcium Phosphate Cement with Aminoacids on the Concentrations of Selected Inflammatory Markers and Serum Enzyme Activities

The repair of articular cartilage defects is an intensively developing area of research. Severe inflammatory reactions after surgical interventions on bones or their structures may lead to changes of bone or joint asymmetry. In laboratory diagnostics, some inflammatory biomarkers and serum enzymes are available for the evaluation of these inflammatory processes. A general understanding of inflammatory responses following the repair of cartilage defects is still lacking. The aim of this study was to describe the alterations in the values of five inflammatory markers and the activities of several enzymes in seven 5-month-old pigs within the first 30 days following the restoration of induced articular cartilage defects using the tetracalcium phosphate/nanomoneite cement powder enriched with amino acids (CAL). The reconstruction of surgically induced defects was accompanied by a significant increase of serum amyloid A (SAA, p < 0.05), haptoglobin (Hp, p < 0.001), C-reactive protein (CRP, p < 0.01), and pig major acute phase protein (pig-MAP, p < 0.001). Their concentrations decreased gradually within one-month post-surgery, the values recorded at the end of this period were higher than those obtained prior to surgery. The concentrations of α1-acid glycoprotein (AGP) showed no significant changes during the evaluated period. Alterations were also found in the enzyme activities of creatine kinase (p < 0.05), lactate-dehydrogenase and alkaline phosphatase (p < 0.01). These findings suggest that SAA, Hp, CRP and pig-MAP might be applicable biomarkers of acute phase response for the monitoring of postoperative period.

Characterization of Contractile Machinery of Vascular Smooth Muscles in Hypertension

Hypertension is a key risk factor for cardiovascular disease and it is a growing public health problem worldwide. The pathophysiological mechanisms of vascular smooth muscle (VSM) contraction contribute to the development of hypertension. Calcium (Ca2+)-dependent and -independent signaling mechanisms regulate the balance of the myosin light chain kinase and myosin light chain phosphatase to induce myosin phosphorylation, which activates VSM contraction to control blood pressure (BP). Here, we discuss the mechanism of the contractile machinery in VSM, especially RhoA/Rho kinase and PKC/CPI-17 of Ca2+ sensitization pathway in hypertension. The two signaling pathways affect BP in physiological and pathophysiological conditions and are highlighted in pulmonary, pregnancy, and salt-sensitive hypertension.

MKP-3 suppresses LPS-induced inflammatory responses in HUVECs via inhibition of p38 MAPK/NF-κB pathway

ABSTRACT Endothelial cell dysfunction and inflammatory responses play critical roles in the development of atherosclerosis. Recent data on the processes underlying atherogenesis indicate the substantial role of endotoxins (lipopolysaccharides; LPS) of the intestinal microflora in the initiation and progression of atherosclerosis. Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a cytoplasmic dual-specificity protein phosphatase that specifically binds to and inactivates MAP kinases in mammalian cells, but its biological function in endothelial cell dysfunction and inflammatory responses remains largely unknown. The aim of the present study was to investigate the role of MKP-3 in endotoxin-induced endothelial inflammation by western blotting, quantitative polymerase chain reaction, and immunofluorescence. The results of our study demonstrated that MKP-3 overexpression markedly inhibited the adhesion of human monocytic THP-1 cells to human umbilical vein endothelial cells (HUVECs) by downregulating the expression of vascular cell adhesion protein 1 (VCAM-1) and pro-inflammatory cytokines. In contrast, MKP-3-encoding gene knockdown by small interfering RNA (siRNA) exacerbated LPS-induced endothelial dysfunction. Additionally, we found that MKP-3 overexpression inhibited LPS-induced p38 MAPK phosphorylation and decreased the nuclear translocation of nuclear factor kappa B (NF-κB) after LPS treatment, suggesting its implication in the LPS/Toll-like receptor 4 (TLR4)/p38/NF-κB pathway. Overall, these observations suggest that MKP-3 plays a protective role in endothelial dysfunction and may be a therapeutic target.

Effect of aflatoxin B1 on digestibility and blood parameters in horses

The aim of this research was to evaluate the effect of mycotoxins on the digestion of nutrients and on the hematological profile of horses. Twelve horses were used in a completely randomized design, with three treatments and four replicates. Aliquots of 50 or 100 µg kg-1 of aflatoxin B1 (AFB1) were added to a basal diet naturally containing mycotoxins. The basal diet did not contain AFB1. The 40-day evaluation included a 12-day adaptation period and a 28-day experimental period. A digestion test was carried out at the end of the experimental period, using the partial stool collection method, using LIPE® as an indicator. Blood samples were collected once a week during 4 weeks of the essay for hematological and biochemical evaluations. The amounts of mycotoxins added did not influence the intake and digestibility of the nutrients (P>0.05). The aflatoxins in the diet influenced the white blood cell count, especially mature neutrophils and the creatine kinase and alkaline phosphatase (P <0.05), which had higher activity in horses fed diets with greater toxicity.

Drugging an “undruggable” phosphatase

Pharmacology Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) have roles in many diseases but have been considered “undruggable” because of their high degree of conservation. Gannam et al. identified and characterized a small molecule that inhibited MKP5 but did not affect the activity of related phosphatases. In muscle cells, the inhibitor blocked MKP5 from inactivating the MAPKs and inhibited a signaling pathway that promotes fibrosis in dystrophic muscle disease. These results demonstrate that it is possible to target MKPs with a high degree of specificity. Sci. Signal. 13 , eaba3043 (2020).

Interplay of MKP-1 and Nrf2 drives tumor growth and drug resistance in non-small cell lung cancer.

Alterations in KEAP1/ NF-E2 p45-related factor 2 (NFE2L2/Nrf2) signaling pathway have been reported in 23% lung adenocarcinoma patients, suggesting that deregulation of the pathway is a major cancer driver. Here we report that mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) drives tumor growth and drug resistance by up regulating transcription factor Nrf2. In non-small cell lung cancer (NSCLC) cells and xenografts, MKP-1 knockdown triggered the down-regulation of the metabolic enzymes and cytoprotective proteins, which are the target genes of Nrf2. Consequently, the cell growth was markedly inhibited with decrease of tumor metabolisms and GSH contents. Moreover, MKP-1 silencing sensitized NSCLC cells to cisplatin treatment. Mechanistically, MKP-1 inhibited the ubiquitylation of Nrf2 via a direct interaction with the transcription factor. Nrf2 was hence stabilized and its transcriptional program was activated. Notably, Nrf2 elevated MKP-1 expression at transcriptional level. In human lung adenoma tumor samples, high levels of expression of MKP-1, Nrf2, and its target gene heme oxygenase 1 were closely correlated. Thus, MKP-1 and Nrf2 form a forward feedback loop in lung cancer cells, which stabilizing and activating Nrf2 to promote anabolic metabolism and GSH biosynthesis. This study uncovers a novel role of MKP-1 in the malignant evolution of cancers.

Mitochondrial Protein PINK1 Positively Regulates RLR Signaling.

The serine/threonine kinase phosphatase and tensin homolog (PTEN)-induced putative kinase 1(PINK1) controls mitochondrial quality and plays a vital role in the pathogenesis of early-onset Parkinson's disease. However, whether PINK1 has functions in innate antiviral immunity is largely unknown. Here, we report that viral infection down regulates PINK1 expression in macrophages. PINK1 knockdown results in decreased cytokine production and attenuated IRF3 and NF-κB activation upon viral infection. PINK1 promotes the retinoic-acid-inducible gene I (RIG-I)-like receptors (RLR)-triggered immune responses in a kinase domain-dependent manner. Furthermore, PINK1 associates with TRAF3 via the kinase domain and inhibits Parkin-mediated TRAF3 K48-linked proteasomal degradation. In addition, PINK1 interacts with Yes-associated protein 1 (YAP1) upon viral infection and impairs YAP1/IRF3 complex formation. Collectively, our results demonstrate that PINK1 positively regulates RIG-I triggered innate immune responses by inhibiting TRAF3 degradation and relieving YAP-mediated inhibition of the cellular antiviral response.

Pseudophosphatase MK-STYX Alters Histone Deacetylase 6 Cytoplasmic Localization, Decreases Its Phosphorylation, and Increases Detyrosination of Tubulin.

The catalytically inactive mitogen-activated protein (MAP) kinase phosphatase, MK-STYX (MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein) interacts with the stress granule nucleator G3BP-1 (Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding protein-1), and decreases stress granule (stalled mRNA) formation. Histone deacetylase isoform 6 (HDAC6) also binds G3BP-1 and serves as a major component of stress granules. The discovery that MK-STYX and HDAC6 both interact with G3BP-1 led us to investigate the effects of MK-STYX on HDAC6 dynamics. In control HEK/293 cells, HDAC6 was cytosolic, as expected, and formed aggregates under conditions of stress. In contrast, in cells overexpressing MK-STYX, HDAC6 was both nuclear and cytosolic and the number of stress-induced aggregates significantly decreased. Immunoblots showed that MK-STYX decreases HDAC6 serine phosphorylation, protein tyrosine phosphorylation, and lysine acetylation. HDAC6 is known to regulate microtubule dynamics to form aggregates. MK-STYX did not affect the organization of microtubules, but did affect their post-translational modification. Tubulin acetylation was increased in the presence of MK-STYX. In addition, the detyrosination of tubulin was significantly increased in the presence of MK-STYX. These findings show that MK-STYX decreases the number of HDAC6-containing aggregates and alters their localization, sustains microtubule acetylation, and increases detyrosination of microtubules, implicating MK-STYX as a signaling molecule in HDAC6 activity.

Biochemical parameters of cerebrospinal fluid in patients with acute viral meningitis and meningoencephalitis

The aim. Determination of the diagnostic value of lactate, lactate dehydrogenase, cholinesterase, acid phosphatase and cholinesterase in CSF for early diagnosis and prognosis of acute viral meningitis and meningoencephalitis.Materials and methods. 92 patients with a confirmed viral etiology of the disease were examined. Among them – 20 patients with HSV 1,2 neuroinfection, 19 patients with EBV, 15 with VZV, 14 with HHV-6 and 24 patients with enterovirus neuroinfection. Patients were divided into groups depending on the etiology and severity of the disease. In addition to analyzing the clinical course of the disease, we conducted a CSF study to determine the level of lactate, lactate dehydrogenase, creatinine kinase, cholinesterase and acid phosphatase on admission to hospital and after 10-12 days of treatment.Results of the study. The highest mean age was observed in patients with VZV meningitis – 38.27±18.24 years, the youngest were patients with enterovirus infection – 24.05±5.72 (р˂0.001). The number of women and men was the same in almost all groups, but among patients with HSV 1, 2 neuroinfection women were significantly prevalent – 16 (80 %) out of 20 cases. The most severe course was observed in groups of EBV and HHV-6 neuroinfections. Neuroinfections of enterovirus etiology had the most favorable course.The obtained data indicate the dependence of the levels of indicators, which were determined primarily from the severity of the disease. So the level of creatinine kinase and acid phosphatase in patients with moderate severity was significantly higher in comparison with severe patients (p<0.05). The lactate level was higher in patients with severe neuroinfection (p<0.05). The highest levels of lactate were detected in patients with HHV-6 meningoencephalitis (p<0.05). The level of cholinesterase was significantly lower in severe patients.Conclusions. The obtained data confirm the presence of deep metabolic disturbances in the brain tissues in all patients with acute viral neuroinfections both at the onset of the disease and in the dynamics of treatment. Determination of levels of creatinine kinase, acid phosphatase, lactate and cholinesterase in CSF of patients with acute viral neuroinfection has a high diagnostic value, but cannot be used to predict an unfavorable course of the disease

Efeitos de um salgante isento de sódio sobre marcadores sanguíneos bioquímicos hepáticos, renais e cardíacos de ratos normo e hipertensos

Introduo: A hipertenso arterial um dos fatores de risco para doenas cardiovasculares, estando diretamente associada ao elevado consumo de sdio. Objetivo: Avaliar os nveis de marcadores de leso heptica, renal e cardaca em ratos hipertensos comparados aos seus controles normotensos, tratados com um salgante isento de sdio, gua ou NaCl. Mtodos: Ratos hipertensos (SHR) e seus controles normotensos (NWR) foram divididos em 3 grupos (n=7): G1 (gua); G2 (soluo aquosa contendo NaCl 70 mg/kg/dia); G3 (soluo aquosa contendo salgante sem sdio 70 mg/kg/dia). Aps 30 dias, o sangue dos animais foi processado. Resultados: No houve diferena entre os nveis sricos de creatina quinase total, creatina quinase-MB, lactato desidrogenase, cido rico, aspartato aminotransferase e fosfatase alcalina tanto nos NWR como nos SHR tratados com NaCl ou Salgante. Houve diminuio da creatinina nos NWR e SHR tratados com NaCl e Salgante em relao aos controles (p0,005). Concluses: A suplementao diria com o Salgante e NaCl diminuiu os nveis sricos de creatinina nos grupos NWR e SHR. Contudo, no houve modificao nos nveis sricos de marcadores de leso cardaca e heptica.

Abstract 2360: Regulation of the MAPK phosphatase MKP-1 by the E3 ligase GP78 in cancer cells

Abstract Mitogen-activated protein kinase (MAPK) phosphatase 1 (DUSP1/MKP-1) is a member of the threonine-tyrosine dual-specificity phosphatase family that is endogenous inhibitor for extracellular signal-regulated protein kinases (ERKs), p38 MAPKs, and JNKs signaling. MKP-1 plays a key role in determining the magnitude and duration of MAPK activation in response to extracellular stimuli. In human cancers, abnormal expression of DUSP1 is associated with tumor development, chemoresistance and prognosis. However, the mechanism by which MKP-1 is regulated is not fully understood. Here, we show that GP78, an E3 ligase, plays an important role in regulation of MKP-1 protein levels. Specifically, we found that MKP-1 interacts with GP78 both in vitro and in vivo. Their interaction occurs between the N-terminal, Rhodanese domain of MKP-1 and C-terminal E3 ligase domain of GP78. We also found that K230, 280, and 289 residues on MKP-1 are responsible for its ubiquitination. Importantly, this interaction causes MKP-1 ubiquitination and subsequent degradation. Furthermore, we showed by overexpression and knockdown studies that GP78 downregulates the level of MKP-1 protein in cancer cells. Therefore, we identify a novel mechanism by which GP78 controls MKP-1 protein levels and suggest that this regulatory mechanism may play an important in cancer. Citation Format: Dhonghyo Kho, Gen Sheng Wu. Regulation of the MAPK phosphatase MKP-1 by the E3 ligase GP78 in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2360. doi:10.1158/1538-7445.AM2017-2360

Protein Tyrosine Phosphatase-Prospective Target against Cancer: A Mini Review

Contemporary quantum leap on functional characterization of Protein Tyrosine Phosphatase (PTP) superfamily provides an incipient perspective on regulating signal transduction. PTPs are required in the regulation of several cellular processes; especially under stressed and pathogenic conditions leading to sundry human diseases. Concrete inhibition of PTP by oxyanions and active-site directed inhibitors (alkylating agents) may provide implements for human disease treatment involving them. The physiological paramountcy for the advancement of Protein Tyrosine Phosphatase, Non-receptor Type 1 (PTP1B) -predicated therapeutics is a prominent target for diabetes and inordinate corpulence treatment. PTPs are exhilarating quarry for active-site-mediated inhibitors generation. There, proneness to oxidation often create problem on high throughput screens, further the propensity for highly charged potent inhibitors, like non-hydrolysable pTyr mimetics, test with reverence to bioavailability. Subsequent preliminary concerns about specificity and quandaries with deference to hydrophilicity of phosphormimetics, promising successes attained by structure-predicated drug design, mainly the one exploit identical surface topology circumventing the catalytic pocket of each PTP. In PTP1B, it was found that a particular pTyr binding site could be habituated to succeed highly concrete bidentate inhibitors that bind both sites. This conventional approach can avail us to target highly categorical and efficacious inhibitors. Tyrosine phosphorylation increases 1-2% of total protein phosphorylation in tumorigenic transformation or magnification factor simulation essential for a controlled cellular event. This event is controlled by two molecular switches of enzymes protein tyrosine kinase and protein tyrosine phosphatase. Eccentric tyrosine phosphorylation is considered as one of the hallmarks of cancer. PTP has been recommended as next generation drug targets and a sum of PTP have been embroiled in sundry human disease, like cancer. The catalytic mechanism of PTP was demystified by site-directed mutagenesis then kinetic analyses with structural information. They have loss/ gain of function in cancer signaling events leading to dearth of inhibitors to control gain of function including modification of loss of function of PTP cognate genes. This review is about the consequentiality of tyrosine phosphatase enzyme and its role in the mundane cellular event and how it modifies the active site to agonise substrate and alter its action ultimately leading to tumorigenesis.

Evaluation of intraosseous sampling for measurements of alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, creatinine kinase, gamma-glutamyl transferase and lactate dehydrogenase

Background: Intraosseous (IO) access can be established faster than a venous or arterial access when there is an urgent need for rapid initiation of treatment. The access can also be used to draw marrow samples. The aim of the present study was to evaluate the potential use of IO samples for enzyme determinations using a porcine model.Materials and methods: Bilateral tibial intraosseous cannulae and an arterial catheter were used for blood sampling from five healthy anesthetized pigs. Samples were collected at baseline and thereafter hourly for 6 h and analyzed for alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, creatinine kinase, gamma-glutamyl transferase and lactate dehydrogenase.Results: Creatinine kinase, lactate dehydrogenase and alkaline phosphatase levels decreased over time. The differences between IO and arterial sampling were limited for all studied markers.Conclusion: The correlation between marrow and blood analysis for liver function tests and CK is sufficiently accurate in an emergency situation.

Tyrosine Kinase Inhibitors and Phosphatases: Overcoming the BCR-ABL T315I Mutation in CML with a Synergistic Combination of Ponatinib and Forskolin

Tyrosine Kinase Inhibitors and Phosphatases: Overcoming the BCR-ABL T315I Mutation in CML with a Synergistic Combination of Ponatinib and Forskolin

Identification of kinases, phosphatases, and phosphorylation sites in human and porcine spermatozoa

Multiple inter-connected signaling pathways, involving kinases and phosphatases, form a framework that controls sperm motility, function, and fertilizing ability. Methods that give a broad view of the proteomic landscape may prove valuable in uncovering new crosstalk connections, as well as in discovering new proteins within this regulatory framework. A multi-immunoblotting strategy was utilized to evaluate this concept on human and porcine spermatozoa samples. In human and porcine spermatozoa, a diversity of kinases were identified including protein kinase A (PKA), protein kinase B (PKB), isoforms of protein kinase C (PKC), calmodulin-dependent kinases (CAMK), casein kinase (CK), and isoforms of glycogen synthase kinase (GSK3). Several phosphatases, such as protein phosphatase (PP)-1, PP2A, PP2C, and mitogen activated protein kinase (MAPK) phosphatase (MKP-1), were identified in human spermatozoa. The phosphorylation epitopes recognized belonged to members of the MAPK family, in addition to α and β isoforms of GSK3 and cAMP response element binding protein (CREB). Proteomic approaches that allow a broad view may aid in understanding the crosstalk between signaling systems in spermatozoal physiology.

The Role of MKP-1 in the Anti-Proliferative Effects of Glucocorticoids in Primary Rat Pre-Osteoblasts.

Glucocorticoid (GC)-induced osteoporosis has been attributed to a GC-induced suppression of pre-osteoblast proliferation. Our previous work identified a critical role for mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in mediating the anti-proliferative effects of GCs in immortalized pre-osteoblasts, but we subsequently found that MKP-1 null mice were not protected against the pathological effects of GCs on bone. In order to reconcile this discrepancy, we have assessed the effects of GCs on proliferation, activation of the MAPK ERK1/2 and MKP-1 expression in primary adipose-derived stromal cells (ADSCs) and ADSC-derived pre-osteoblasts (ADSC-OBs). ADSCs were isolated by means of collagenase digestion from adipose tissue biopsies harvested from adult male Wistar rats. ADSC-OBs were prepared by treating ADSCs with osteoblast differentiation media for 7 days. The effects of increasing concentrations of the GC dexamethasone on basal and mitogen-stimulated cell proliferation were quantified by tritiated thymidine incorporation. ERK1/2 activity was measured by Western blotting, while MKP-1 expression was quantified on both RNA and protein levels, using semi-quantitative real-time PCR and Western blotting, respectively. GCs were strongly anti-proliferative in both naïve ADSCs and ADSC-OBs, but had very little effect on mitogen-induced ERK1/2 activation and did not upregulate MKP-1 protein expression. These findings suggest that the anti-proliferative effects of GCs in primary ADSCs and ADSC-OBs in vitro do not require the inhibition of ERK1/2 activation by MKP-1, which is consistent with our in vivo findings in MKP-1 null mice.

Abstract 1171: Increased expression of PDK4 in bladder cancer cells

Abstract Introduction and Objectives. Cancer cells have a high rate of glucose metabolism due to an increased requirement for biomolecules and energy. In some solid tumors, cancer cells divert pyruvate to lactate even in the presence of oxygen (Warburg effect) providing a survival advantage in an acidic and anaerobic environment. Pyruvate dehydrogenase complex (PDC) catalyzes oxidative decarboxylation of pyruvate to acetyl-CoA and links glycolysis to energetic and anabolic functions of the tricarboxylic acid cycle. PDC activity is regulated by pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). In a laser capture, microarray pilot study we found PDK4 expression to be increased 33-fold in high-grade invasive vs low-grade bladder cancers. The aim of this study was to examine the expression of PDK and PDP genes in bladder cancer cells. Methods. Bladder cancer (HTB2, HTB4, HTB5, HTB9, HT1376) and benign urothelial (UROtsa) cell lines were cultured in defined media. Total RNA was extracted and quantitative PCR performed. Cell numbers were counted using a Coulter counter. Cell cycle and apoptosis analyses were performed using fluorescence-activated cell sorting. Transient transfections with PDK4 gene specific siRNAs were performed. Results. In the present study, PDK4 expression was increased (20-100 fold; p&amp;lt;0.01) in bladder tumor vs. benign cell lines. Expression of the other three PDK isoforms was increased 3-5 fold. Investigation of transcription factors known to regulate the expression of PDK4 revealed overexpression of PPARα (8-22 fold; p&amp;lt;0.05-0.001) and HIF1α (3.7 - 13.4 fold; p&amp;lt;0.01 only in HT1376). Expression of PDP1 and PDP2 was also assessed (0.4 - 2.2 fold, not significant). The ratio of PDK/PDP expression was found to be 101 (PDK4/PDP1) and 138 (PDK4/PDP2) for HTB9; 196 and 42 for HT1376; 94 and 50 for HTB5 and 57 and 26.3 for HTB4. To examine the impact of PDK4 inhibition, cells were treated with a known inhibitor, dichloroacetate (DCA), for 0-72 h. A significant decrease in cell number was detected in a dose dependent manner (10-50 mM). HTB2, a low grade cell line, showed a significant decline in cell number (70%) when incubated with 50 mM DCA for 72 h (p&amp;lt; 0.01). HTB5, a high-grade cell line with a higher PDK4 expression, had a greater decline (92%) with same treatment (p&amp;lt; 0.01). Further, we observed growth inhibition by cell count with siRNA knockdown of PDK4. Our preliminary data on cell cycle analysis showed an enhanced number of tumor cells in G1 phase and a decrease in S-phase. Conclusions. PDK4 gene expression was found to be higher in bladder cancer cell lines. Both inhibition and knock down of PDK4 resulted in impaired cell growth with cell cycle arrest at the G1 phase. Taken together these data suggest that PDK4 may prove to be a novel therapeutic target in the management of bladder cancer. Source of Funding. The Leo &amp; Anne Albert Charitable Trust Citation Format: Dharamainder Choudhary, Andrew Mikhalyuk, Carol Pilbeam, John Taylor. Increased expression of PDK4 in bladder cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1171. doi:10.1158/1538-7445.AM2015-1171