P-cadherin controls the differentiation of oral keratinocytes by regulating cytokeratin 1/10 expression via C/EBP-beta-mediated signaling

P-cadherin belongs to the family of Ca²⁺-dependent homophilic glycosylated cell adhesion molecules. In the normal oral epithelium it shows a strong expression in the basal cell layer which gradually decreases in the suprabasal cell layers. The exact role of P-cadherin during the development and homeostasis of the oral epithelium has not been elucidated, yet. Here, we show for the first time that P-cadherin controls differentiation by regulating cytokeratin (CK) 1/10 expression in primary oral keratinocytes (POK) from normal, but interestingly not in POKs from oral squamous cell carcinoma (OSCC) tissue. SiRNA knockdown of P-cadherin in normal POKs revealed a strong upregulation of CK1/10 expression on mRNA and protein level. In contrast, E-cadherin knockdown in normal oral keratinocytes did not show any influence on CK1/10 expression. Moreover, in comparison with normal control keratinocytes normal oral keratinocytes with reduced P-cadherin expression displayed an enhanced expression and a stronger nuclear staining of C/EBP-beta, a well-known regulator of CK1/10 expression in keratinocytes. Furthermore, after P-cadherin knockdown in normal POKs the promoter activity of a C/EBP-responsive luciferase construct was significantly higher than in normal POKs with regular P-cadherin expression. Additionally, we noticed a proliferation advantage in normal oral keratinocytes in contrast to keratinocytes with diminished P-cadherin expression. However, the inverted effect was seen in tumor derived primary oral keratinocytes. In summary, we show that P-cadherin contributes to the keratinocyte differentiation in the oral epithelium by influencing the CK1 and CK10 expression via C/EBP-beta-mediated signaling in normal but not in tumor derived oral keratinocytes from OSCC patients.     

Cytokeratin 5 and cytokeratin 20 inversely correlate with tumour grading in Ta non-muscle-invasive bladder cancer

Cytokeratin 5 is a marker of basal molecular subtypes of muscle-invasive bladder cancer (MIBC), which correlates with worse overall survival compared to luminal subtypes. Our observations have not confirmed CK5 as a marker of high-grade (HG) disease in Ta non-muscle-invasive bladder cancer (NMIBC). Therefore, to understand the basal-luminal immunohistochemistry profile in Ta NMIBC, we performed immunohistochemistry for CK5, P40, P63 (basal), GATA3 and CK20 (luminal) and studied the correlation with HG and clinical outcome in 109 patients with Ta NMIBC. HG and low-grade (LG) diseases were scored in each patient. Four different CK5 patterns were evaluated: absent (median 41.3%), normal (72.5%), rising (84.4%) and full thickness (23.9%). The median percentage of GATA3 was 100%. HG disease and CK5 expression and rising CK5 pattern had a significant inverse correlation, whereas HG disease and CK20 expression had a significant positive correlation. We also found a significant inverse correlation between CK5 expression and CK20 expression. Quantitative PCR confirmed that the presence of CK5 correlated with up-regulation of CK5 RNA. None of the markers could differentiate patients with regard to clinical outcome. Our results suggest a role for CK5 and CK20 in differentiating between LG and HG disease in Ta NMIBC.     

Inhibition of the Arp2/3 complex impairs early embryo development of porcine parthenotes

The Arp2/3 complex, which nucleates actin filaments, comprises a stable assembly of seven-protein subunits including two actin-related proteins (Arp2 and Arp3). Previous work showed that Arp2/3 binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. In the present study, we show that the Arp2/3 complex is critical for cytokinesis during early embryonic development in porcine parthenotes. The Arp2/3 complex is concentrated at the cortex of each cell at the 1-, 2-, and 4-cell stages, and at the periphery at the morula stage. The amount of Arp2/3 significantly decreased at the blastocyst stage in parthenogenetically activated porcine embryos. Inhibition of the Arp2/3 complex in the pig embryos by the Arp2/3-specific inhibitor CK666 resulted in abnormal cell division, a decrease in developmental rate and total cell numbers, and an increase in the ratio of trophectoderm cell number to inner cell mass number in blastocyst-stage embryos. In addition, 4-cell stage embryos subjected to CK666 treatment exhibited significantly decreased expression of ZGA genes (Pou5f1, Sox2, and Nanog), suggesting that the Arp2/3 complex plays an important role in early porcine embryo development. Thus, our data demonstrate that the Arp2/3 complex is required for early embryonic development in pigs and appears to regulate the expression of pluripotency genes.     

Phosphorylation of recombinant human spermidine/spermine N(1)-acetyltransferase by CK1 and modulation of its binding to mitochondria: a comparison with CK2.

Cytosolic spermidine/spermine acetyltransferase (SSAT) catalyzes the acetylation of the N(1)-propylamino groups of spermine and spermidine. The enzyme has a very short half-life and is rapidly induced by various stimuli. Once acetylated, these polyamines are subjected to the action of polyamine oxidase, which, besides initiating polyamine catabolism, may produce reactive oxygen species that in turn trigger modifications in subcellular compartments such as mitochondria. The present work evaluates the ability of the cAMP-independent Ser/Thr-protein kinase CK1 to phosphorylate SSAT. Results demonstrate that SSAT is phosphorylated by CK1, in sites distinct from those phosphorylated by CK2. Moreover, both phosphorylation processes are involved in the uptake of SSAT into rat liver mitochondria. Although CK2 is less effective than CK1 in phosphorylating SSAT, CK2 phosphorylation is much more powerful in preventing binding of SSAT to mitochondrial structures. These results suggest the involvement of CK1- and CK2-mediated SSAT phosphorylation in regulating the contents of polyamines and SSAT itself within subcellular compartments and implicate SSAT and polyamines as indirect modulators of progression through the cell cycle.     

CKIP-1: 一种新型的凋亡促进因子

酪蛋白激酶2相互作用蛋白1(caseinkinase2interactionprotein1,CKIP-1)是近年来发现的一种重要分子,它通过与其他分子的相互作用在许多细胞行为中都发挥着重要的作用。最新研究发现,CKIP-1还具有促进细胞凋亡的作用。     

Routine enzymes in the monitoring of type 2 diabetes mellitus

We examined the relationships of glucose and HbA1c levels with the routinely screened serum enzyme activities in type 2 diabetes mellitus, and we designed an in vitro study to evaluate the direct effect of glucose levels on enzyme activities. The study was performed on a consecutive series of outpatients with type 2 diabetes who were followed up at Dicle University Medical Faculty Hospital from May 2009 to May 2010 for the first time. Effects of aspartate transaminase, aminotransferase, gamma‐glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) activities, glucose and HbA1c levels and in vitro glucose (492, 287, 184, 131, 82 mg dl⁻¹, respectively) on enzymes were determined. The patients were categorized on the basis of glucose and HbA1c levels and grouped according to a range of values. In patients with high HbA1c levels (>10.1%), ALP, GGT activities and creatine kinase (CK)‐MB/CK (p = 0.008, 0.026, 0.014) ratio were increased significantly when compared with those in the control group. In patients with high glucose levels (>200 mg dl⁻¹), ALP, GGT activities and CK‐MB/CK ratio (p = 0.003, 0.001, 0.001) were increased significantly when compared with those in the control group. Glucose, which was added to serum in different concentrations in vitro, did not directly affect enzyme activities such as ALP, GGT and CK. We concluded that increased glucose levels could damage the liver and the heart muscle cells. Monitoring of blood glucose levels is a more valuable parameter than monitoring HbA1c in the momentary evaluation of diabetes. Copyright © 2011 John Wiley & Sons, Ltd.     

Transient expression of bile-duct-specific cytokeratin in fetal mouse hepatocytes

The differentiation of hepatocytes and biliary epithelial cells has been histochemically analyzed with anti-calf cytokeratin antiserum in the fetal mouse liver. Almost all young fetal hepatocytes transiently express bile-duct-specific cytokeratin; subsequently, the strong staining of the cytokeratin is confined to progenitor cells of intrahepatic biliary epithelial cells around portal veins. These results suggest that all fetal hepatocytes are bi-potent in terms of the differentiation of mature hepatocytes and intrahepatic bile-duct cells, and that the microenvironment around portal veins plays an important role in bile-duct differentiation. Large periportal hepatocytes continue to stain weakly for cytokeratin until 2 weeks after birth, although the number of positive hepatocytes decreases with development. The differentiation of bile ducts from periportal hepatocytes may continue for 2 weeks after birth.     

Phosphorylated synaphin/complexin found in the brain exhibits enhanced SNARE complex binding

The cytosolic protein synaphin/complexin critically regulates fast neurotransmitter release at the synapse by binding to SNARE complex. However, the exact mechanism of its action remains unclear, and very little is known about how it is physiologically regulated. Here we show that synaphins (Syps) 1 and 2 can be phosphorylated in vitro by protein kinase CK2 (CK2). The only phosphorylation site by CK2 was serine-115 (Ser-115) of Syps 1 and 2. Syps 1 and 2 exhibited higher affinities to native and recombinant SNARE complexes when phosphorylated at Ser-115. We found Ser-115-phosphorylated Syp 1 (pS115-Syp 1) in the cytosolic fraction of the rat brain using polyclonal antibody specific to pS115-Syps 1 and 2. These results suggest that the activity of Syp is regulated by CK2 phosphorylation of its Ser-115 in vivo. The phosphorylation may provide a new route for modulating fast neurotransmitter release.     

Cancer-associated cleavage of cytokeratin 8/18 heterotypic complexes exposes a neoepitope in human adenocarcinomas

The intermediate filament network in simple glandular epithelial cells predominantly consists of heterotypic complexes of cytokeratin 8 (K8) and cytokeratin 18 (K18). In contrast to other cytokeratins, K8 and K18 are persistently expressed during malignant transformation, but changes in cell morphology are accompanied by alterations in the intermediate filament network. To study molecular changes, K8 and K18 were purified from surgically removed colon cancer and normal epithelia tissues. Western blotting and amino acid sequencing revealed the presence of abundant K8 and K18 fragments, truncated at the N terminus, from cancerous, but not normal, epithelial cells. The fragmentation pattern indicates proteolysis mediated by several enzymes, including trypsin-like enzymes. The cancer-associated forms of K8 and K18 are specifically recognized by the human antibody, COU-1, cloned from the B cells of a cancer patient. We demonstrate that COU-1 recognizes a unique conformational epitope presented only by a complex between K8 and K18. The epitope is revealed after proteolytic removal of the head domain of either K8 or K18. A large panel of recombinant K8 and K18 fragments, deleted N- or C-terminally, allowed for the localization of the COU-1 epitope to the N-terminal part of the rod domains. Using surface plasmon resonance, the affinity of COU-1 for this epitope was determined to be 10(9) x m(-1), i.e. more than 2 orders of magnitude higher than for intact heterotypic K8/K18 complexes. The cellular distribution of truncated K8/K18 heterotypic complexes in viable adenocarcinomas cells was probed using COU-1 showing small fibrillar structures distinct from those of intact K8/K18 complexes. Previously we demonstrated the binding and subsequent internalization of recombinant Fab COU-1 to live cancer cells. We have thus characterized a cancer neoepitope recognized by the humoral immune system. The results have biological as well as clinical implications.     

The value of calretinin and cytokeratin 5/6 as markers for mesothelioma in cell block preparations of serous effusions

Objective: To determine the value of calretinin and cytokeratin (CK) 5/6 in discriminating mesothelioma from adenocarcinoma in serous effusion specimens. Methods: A total of 101 recent, histologically or clinically confirmed malignant effusions with immunostained cell block preparations were reviewed. The cases consisted of 34 mesotheliomas and 67 adenocarcinomas. This included 17 ascitic fluid and 84 pleural fluid samples. The adenocarcinomas included metastatic carcinomas from the breast (12), lung (19), stomach (3), colon (1), pancreas (2), ovary (6) endometrium (1) and 23 histologically confirmed metastases from unknown primary sites. The cases were assessed as negative or positive (>5% of cells stained). The staining pattern was recorded as cytoplasmic, cell membrane, nuclear or cytoplasmic and nuclear staining. Results: Calretinin staining was present in 97% (33/34) of the mesothelioma cases with a majority of them showing both cytoplasmic and nuclear staining (29/33). Only 3% (2/67) of adenocarcinomas were positive for calretinin, one being a lung adenocarcinoma and the other an adenocarcinoma of unknown primary site in an ascitic fluid. Cytokeratin 5/6 staining was also present in 33/34 (97%) of mesothelioma cases. Six (9%) adenocarcinomas were positive, including metastases from the lung (1), breast (1), ovary (2) and unknown primary site (2). Four of the six adenocarcinoma cases positive for CK5/6 were in ascitic fluids. No cases of mesothelioma were negative for both calretinin and CK5/6. Only one adenocarcinoma case, (which was from unknown primary site in an ascitic fluid sample), was positive for both markers. Conclusions: The results confirm that calretinin and CK 5/6 are useful markers for mesothelioma in effusion specimens. CK5/6 staining may be less useful for peritoneal fluid specimens where metastatic adenocarcinomas may be more likely to express the antigen. Further study of ascitic/peritoneal specimens is warranted. However, positive staining, particularly for both antigens, is highly indicative of a mesothelial origin for cells. The two markers make a useful addition to EMA and the panel of adenocarcinoma markers routinely applied to effusion specimens.     

Discrimination between the activity of protein kinase CK2 holoenzyme and its catalytic subunits

The acronym CK2 denotes a highly pleiotropic Ser/Thr protein kinase whose over-expression correlates with neoplastic growth. A vexed question about the enigmatic regulation of CK2 concerns the actual existence in living cells of the catalytic (alpha and/or alpha') and regulatory beta-subunits of CK2 not assembled into the regular heterotetrameric holoenzyme. Here we take advantage of novel reagents, namely a peptide substrate and an inhibitor which discriminate between the holoenzyme and the catalytic subunits, to show that CK2 activity in CHO cells is entirely accounted for by the holoenzyme. Transfection with individual subunits moreover does not give rise to holoenzyme formation unless the catalytic and regulatory subunits are co-transfected together, arguing against the existence of free subunits in CHO cells.     

PTEN、p57／Kip2和CK19在非小细胞肺癌中的表达及意义研究

探讨非小细胞肺癌中PTEN、p57/Kip2和CK19的表达情况和临床意义。选取58例非小细胞肺癌手术切除标本,采用SP法进行免疫组织化学染色。PTEN、p57/Kip2和CK19的阳性表达率分别为44.8%、56.9%和98.3%。PTEN和p57/Kip2在低分化肿瘤中表达显著降低或缺失,在腺癌中的表达强度高于鳞癌;CK19几乎在所有的肺癌组织中均表达,且在腺癌中的表达强度高于鳞癌。PTEN和p57/Kip2的低表达参与肿瘤的生长分化和进展,并提示预后不良。     

The Protein Kinase CK2 Holoenzyme Structure Supports Proposed Models of Autoregulation and Trans-Autophosphorylation

Eukaryotic protein kinases are typically strictly controlled by second messenger binding, protein/protein interactions, dephosphorylations or similar processes. None of these regulatory mechanisms is known to work for protein kinase CK2 (former name “casein kinase 2”), an acidophilic and constitutively active eukaryotic protein kinase. CK2 predominantly exists as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) complexed to a dimer of non-catalytic subunits (CK2β). One model of CK2 regulation was proposed several times independently by theoretical docking of the first CK2 holoenzyme structure. According to this model, the CK2 holoenzyme forms autoinhibitory aggregates correlated with trans-autophosphorylation and driven by the down-regulatory affinity between an acidic loop of CK2β and the positively charged substrate binding region of CK2α from a neighboring CK2 heterotetramer. Circular trimeric aggregates in which one-half of the CK2α chains show the predicted inhibitory proximity between those regions were detected within the crystal packing of the human CK2 holoenzyme. Here, we present further in vitro support of the “regulation-by-aggregation” model by an alternative crystal form in which CK2 tetramers are arranged as approximately linear aggregates coinciding essentially with the early predictions. In this assembly, the substrate binding region of every CK2α chain is blocked by a CK2β acidic loop from a neighboring tetramer. We found these crystals with CK2^Andante that contains a CK2β variant mutated in a CK2α-contact helix and described to be responsible for a prolonged circadian rhythm in Drosophila. The increased propensity of CK2^Andante to form aggregates with completely blocked active sites may contribute to this phenotype.     

Incomplete development of human spermatozoa is associated with increased creatine phosphokinase concentration and abnormal head morphology

Our previous creatine phosphokinase (CK) activity studies in human sperm revealed differences among men and among sperm populations within the same specimen. Samples with low sperm concentrations, high incidence of abnormal sperm morphology, and diminished fertility had higher per sperm CK activity. In the present work, we demonstrated, with ¹⁴C-FDNB covalent CK active site modification and with direct CK immunocytochemistry, that the higher CK activity is related to an increased content of CK and of other proteins in sperm. Also, sperm heads with higher CK content were significantly larger and rounder and showed a higher incidence of amorph configuration. We suggest that these biochemical and morphological irregularities are related and are due to a failure of spermatogenesis, more specifically, to a higher retention of cytoplasm, which in normal sperm development is lost to the Sertoli cells as residual bodies. Thus higher CK activity and larger or irregular head size in human sperm signify cellular immaturity and a failure to complete spermatogenesis. © 1993 Wiley-Liss, Inc.     

The multiple personalities of the regulatory subunit of protein kinase CK2: CK2 dependent and CK2 independent roles reveal a secret identity for CK2beta

Protein kinase CK2 (formerly casein kinase II), an enzyme that participates in a wide variety of cellular processes, has traditionally been classified as a stable tetrameric complex consisting of two catalytic CK2alpha or CK2alpha' subunits and two regulatory CK2beta subunits. While consideration of CK2 as a tetrameric complex remains relevant, significant evidence has emerged to challenge the view that its individual subunits exist exclusively within these complexes. This review will summarize biochemical and genetic evidence indicating that the regulatory CK2beta subunit exists and performs functions independently of CK2 tetramers. For example, unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumors. Furthermore, localization studies including live cell imaging have demonstrated that while the catalytic and regulatory subunits of CK2 exhibit extensive co-localization, independent mobility of the individual CK2 subunits can also be observed within cells. Identification of proteins that interact with CK2beta in the absence of catalytic CK2 subunits reinforces the notion that CK2beta has functions distinct from CK2 and begins to offer insights into these CK2-independent functions. In this respect, the discovery that CK2beta can interact with and modulate the activity of a number of other serine/threonine protein kinases including A-Raf, c-Mos and Chk1 is particularly striking. This review will discuss the interactions between CK2beta and these protein kinases with special emphasis on the properties of CK2beta that mediate these interactions and on the implications of these interactions in yielding new prospects for elucidation of the cellular functions of CK2beta.     

2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole: a novel powerful and selective inhibitor of protein kinase CK2

Protein kinase CK2 is a highly pleiotropic enzyme whose high constitutive activity is suspected to be instrumental to the enhancement of the tumour phenotype and to the propagation of infectious diseases. Here we describe a novel compound, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), which is superior to the commonly used specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in several respects. DMAT displays the lowest K(i) value ever reported for a CK2 inhibitor (40 nM); it is cell permeable and its efficacy on cultured cells, both in terms of endogenous CK2 inhibition and induction of apoptosis, is several fold higher than that of TBB. The selectivity of DMAT assayed on a panel of >30 protein kinases is comparable to that of TBB, with the additional advantage of being ineffective on protein kinase CK1 up to 200 microM. These properties make DMAT the first choice CK2 inhibitor for in vivo studies available to date.     

CK8寡糖链结构及翻译水平改变与人肝癌细胞转移相关性研究

糖组学方法筛查人肝癌细胞转移过程中发挥重要作用的核心岩藻糖基化蛋白质分子,解析比较筛出的差异蛋白——细胞角蛋白8(CK8)翻译及糖基化修饰改变与人肝癌细胞转移潜能的关系.应用双向电泳(2-DE)、凝集素亲和印迹、凝集素亲和沉淀联合质谱分析技术,筛查并验证与肝癌转移相关的核心岩藻糖基化蛋白;应用细胞免疫荧光和蛋白质免疫印迹检测CK8的蛋白质表达情况;应用免疫沉淀结合多种凝集素亲和印迹,推测其与肝癌转移相关的寡糖链结构改变.研究发现,3种不同转移潜能人肝癌细胞Hep3B、MHCC97-L和MHCC97-H的扁豆凝集素(LCA)亲和印迹表达谱中,分子质量55～60ku、等电点4～6区域处有核心岩藻糖基化蛋白呈差异表达,质谱鉴定为CK8.LCA亲和沉淀及蛋白质印迹进一步验证CK8异常核心岩藻糖基化与肝癌转移相关;研究发现,CK8分布于细胞浆内,在MHCC97-L和MHCC97-H细胞中蛋白质表达水平较Hep3B高,在MHCC97-H中与LCA和蓖麻凝集素(RCA-1)的亲和力较Hep3B强.以上结果提示,2-DE和凝集素印迹技术联合MALDI-TOF-MS/MS分析可用于筛查疾病过程相关的异常糖基化蛋白质分子,CK8蛋白水平、核心岩藻糖基化及β-1,4末端半乳糖基化的增加均与肝癌细胞转移潜能相关.     

Conformational plasticity of the catalytic subunit of protein kinase CK2 and its consequences for regulation and drug design

At the first glance CK2α, the catalytic subunit of protein kinase CK2, is a rigid molecule: in contrast to many eukaryotic protein kinases in CK2α the canonical regulatory key elements like the activation segment occur exclusively in their typical active conformations. This observation fits well to the constitutive activity of the enzyme, meaning, its independence from phosphorylation or other characteristic control factors. Most CK2α structures are based on the enzyme from Zea mays, supplemented by an increasing number of human CK2α structures. In the latter a surprising plasticity of important ATP-binding elements – the interdomain hinge region and the glycine-rich loop – was discovered. In fully active CK2α the hinge region is open and does not anchor the ATP ribose, but alternatively it can adopt a closed conformation, form hydrogen bonds to the ribose moiety and thus retract the γ-phospho group from its functional position. In addition to this partially inactive state human CK2α was recently found in a fully inactive conformation. It is incompatible with ATP-binding due to a combination of a closed hinge and a collapse of the glycine-rich loop into the ATP cavity. These conformational transitions are apparently correlated with the occupation state of a remote docking site located at the interface to the non-catalytic subunit CK2β: if CK2β blocks this site, the fully active conformation of CK2α is stabilized, while the binding of certain small molecule seems to favour the partially and fully inactive states. This observation may be exploited to design effective and selective CK2 inhibitors.     

胸腔镜术与右侧开胸术对中早期食管癌患者微转移相关因子水平影响的随机对照研究

目的:比较电视胸腔镜手术(VATS)与右侧开胸手术治疗中早期食管癌的临床疗效以及对患者血清微转移相关因子的影响。方法:选取60例食管癌患者作为研究对象,根据术式将其随机分为开胸对照组(30例)和VATS术组(30例),分析和比较两组患者的手术时间、术中出血量、下床活动时间及术后并发症发生率,并分别检测和比较两组手术前后胸腔灌洗液脱落肿瘤细胞标志CK19、CK20的m RNA水平以及血清中IL1-β和HMGB1的水平。结果:与开胸组相比,VTSA组的手术出血量、术后出院时间均明显少于或短于对照组(P0.05),且其术后24 h、72 h胸腔引流液及外周血术后3 d、7 d的IL-1β水平均低于开胸组(P0.05),而仅术后24 h的胸腔引流液HMGB1水平低于开胸组(P0.05),两组其它时点HMGB1水平比较均没有统计学差异(P0.05)。两组术后CK19、CK20的m RNA水平比较没有统计学差异(P0.05)。结论:与传统开胸手术相比,VATS辅助下的食管癌根治术不仅具有患者恢复快的特点,而且对于手术引起的转移相关因子较低,值得临床推广应用。