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1.  NPM-ALK mediates phosphorylation of MSH2 at tyrosine 238, creating a functional deficiency in MSH2 and the loss of mismatch repair 
Blood Cancer Journal  2015;5(5):e311-.
The vast majority of anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ALCL) tumors express the characteristic oncogenic fusion protein NPM-ALK, which mediates tumorigenesis by exerting its constitutive tyrosine kinase activity on various substrates. We recently identified MSH2, a protein central to DNA mismatch repair (MMR), as a novel binding partner and phosphorylation substrate of NPM-ALK. Here, using liquid chromatography–mass spectrometry, we report for the first time that MSH2 is phosphorylated by NPM-ALK at a specific residue, tyrosine 238. Using GP293 cells transfected with NPM-ALK, we confirmed that the MSH2Y238F mutant is not tyrosine phosphorylated. Furthermore, transfection of MSH2Y238F into these cells substantially decreased the tyrosine phosphorylation of endogenous MSH2. Importantly, gene transfection of MSH2Y238F abrogated the binding of NPM-ALK with endogenous MSH2, re-established the dimerization of MSH2:MSH6 and restored the sensitivity to DNA mismatch-inducing drugs, indicative of MMR return. Parallel findings were observed in two ALK+ALCL cell lines, Karpas 299 and SUP-M2. In addition, we found that enforced expression of MSH2Y238F into ALK+ALCL cells alone was sufficient to induce spontaneous apoptosis. In conclusion, our findings have identified NPM-ALK-induced phosphorylation of MSH2 at Y238 as a crucial event in suppressing MMR. Our studies have provided novel insights into the mechanism by which oncogenic tyrosine kinases disrupt MMR.
PMCID: PMC4476014  PMID: 25978431
2.  Unexpectedly high affinity of a novel histamine H3 receptor antagonist, GSK239512, in vivo in human brain, determined using PET 
British Journal of Pharmacology  2014;171(5):1241-1249.
This study aimed to investigate the relationship between the plasma concentration (PK) of the novel histamine H3 receptor antagonist, GSK239512, and the brain occupancy of H3 receptors (RO) in healthy human volunteers.
PET scans were obtained after i.v. administration of the H3-specific radioligand [11C]GSK189254. Each subject was scanned before and after single oral doses of GSK239512, at 4 and 24 h after dose. PET data were analysed by compartmental analysis, and regional RO estimates were obtained by graphical analysis of changes in the total volumes of distribution of the radioligand, followed by a correction for occupancy by the high affinity radioligand. The PK/RO relationship was analysed by a population-modelling approach, using the average PK of GSK239512 during each scan.
Following administration of GSK239512, there was a reduction in the brain uptake of [11C]GSK189254 in all regions, including cerebellum. RO at 4 h was higher than at 24 h, and the PK/RO model estimated a PK associated with 50% of RO of 0.0068 ng·mL−1. This corresponds to a free concentration of 4.50 × 10−12 M (pK = 11.3).
The affinity of GSK239512 for brain H3 receptors in humans in vivo is much higher than that expected from studies in vitro, and higher than that observed in PET studies in pigs. The study illustrates the utility of carrying out PET studies in humans early in drug development, providing accurate quantification of GSK239512 RO in vivo as a function of time and dose.
PMCID: PMC3952801  PMID: 24670146
H3 receptors; drug development; pharmacokinetics; receptor occupancy; PET study
3.  Cross-validation study of class III beta-tubulin as a predictive marker for benefit from adjuvant chemotherapy in resected non-small-cell lung cancer: analysis of four randomized trials 
Annals of Oncology  2011;23(1):86-93.
Background: The IALT, JBR.10, ANITA and Cancer and Leukemia Group B 9633 trials compared adjuvant chemotherapy with observation for patients with resected non-small-cell lung cancer (R-NSCLC). Data from the metastatic setting suggest high tumor class III beta-tubulin (TUBB3) expression is a determinant of insensitivity to tubulin-targeting agents (e.g. vinorelbine, paclitaxel). In 265 patients from JBR.10 (vinorelbine–cisplatin versus observation), high TUBB3 was an adverse prognostic factor and was associated (nonsignificantly) with ‘greater’ survival benefit from chemotherapy. We explored this further in additional patients from JBR.10 and the other three trials.
Patients and methods: TUBB3 immunohistochemical staining was scored for 1149 patients on the four trials. The original JBR.10 cut-off scores were used to classify tumors as TUBB3 high or low. The prognostic and predictive value of TUBB3 on disease-free survival (DFS) and overall survival (OS) was assessed by Cox models stratified by trial and adjusted for clinical factors.
Results: High TUBB3 expression was prognostic for OS [hazard ratio (HR) = 1.27 (1.07–1.51), P = 0.008) and DFS [HR = 1.30 (1.11–1.53), P = 0.001). TUBB3 was not predictive of a differential treatment effect [interaction P = 0.20 (OS), P = 0.23 (DFS)]. Subset analysis (n = 420) on vinorelbine–cisplatin gave similar results.
Conclusions: The prognostic effect of high TUBB3 expression in patients with R-NSCLC has been validated. We were unable to confirm a predictive effect for TUBB3.
PMCID: PMC3276322  PMID: 21471564
chemotherapy; meta-analysis; predictive value; prognostic; randomized trial; tubulin
4.  Suppressor of cytokine signaling 6 (SOCS6) promotes mitochondrial fission via regulating DRP1 translocation 
Cell Death and Differentiation  2012;20(1):139-153.
Mitochondria are highly motile organelles that constantly undergo fission and fusion. Impairment of mitochondrial dynamics is associated with mitochondrial dysfunction and is frequently linked to the pathogenesis of neurodegenerative diseases and cancer. We have previously shown that biallelic inactivation of the suppressor of cytokine signaling 6 (SOCS6) gene is a frequent event in human gastric cancer. In this study, we recapitulated the event of SOCS6 loss using a Lentivirus-based knockdown approach, and demonstrated the linkage between SOCS6 depletion and the suppression of programmed cell death. SOCS6 promotes intrinsic apoptosis, with increased Bax conformational change, mitochondrial targeting, and oligomerization. Most importantly, SOCS6 is targeted to mitochondria and induces mitochondrial fragmentation mediated through an increase in DRP1 fission activity. Here, we show that SOCS6 forms complex with DRP1 and the mitochondrial phosphatase PGAM5, attenuates DRP1 phosphorylation, and promotes DRP1 mitochondrial translocation. Based on mutation analyses, SOCS6-mediated apoptosis is tightly coupled to its ability to induce mitochondrial fission. This study demonstrates an important role for SOCS6 in modulating mitochondrial dynamics and apoptosis.
PMCID: PMC3524647  PMID: 22955947
apoptosis; DRP1; mitochondrial dynamic; PGAM5; SOCS6
5.  Aberrant expression and biological significance of Sox2, an embryonic stem cell transcriptional factor, in ALK-positive anaplastic large cell lymphoma 
Blood Cancer Journal  2012;2(8):e82-.
Sox2 (sex-determining region Y-Box) is one of the master transcriptional factors that are important in maintaining the pluripotency of embryonic stem cells (ESCs). In line with this function, Sox2 expression is largely restricted to ESCs and somatic stem cells. We report that Sox2 is expressed in cell lines and tumor samples derived from ALK-positive anaplastic large cell lymphoma (ALK+ALCL), for which the normal cellular counterpart is believed to be mature T-cells. The expression of Sox2 in ALK+ALCL can be attributed to nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the oncogenic fusion protein carrying a central pathogenetic role in these tumors. By confocal microscopy, Sox2 protein was detectable in virtually all cells in ALK+ALCL cell lines. However, the transcriptional activity of Sox2, as assessed using a Sox2-responsive reporter construct, was detectable only in a small proportion of cells. Importantly, downregulation of Sox2 using short interfering RNA in isolated Sox2active cells, but not Sox2inactive cells, resulted in a significant decrease in cell growth, invasiveness and tumorigenicity. To conclude, ALK+ALCL represents the first example of a hematologic malignancy that aberrantly expresses Sox2, which represents a novel mechanism by which NPM-ALK mediates tumorigenesis. We also found that the transcriptional activity and oncogenic effects of Sox2 can be heterogeneous in cancer cells.
PMCID: PMC3432482  PMID: 22885405
Sox2; transcriptional activity; NPM-ALK; STAT3; tumorigenicity
7.  Human equilibrative nucleoside transporter 1 and carcinoma of the ampulla of Vater: expression differences in tumour histotypes 
The human equilibrative nucleoside transporter 1 (hENT1) is the major means by which gemcitabine enters human cells; recent evidence exists that hENT1 is expressed in carcinoma of the ampulla of Vater and that it should be considered as a molecular prognostic marker for patients with resected ampullary cancer. Aim of the present study is to evaluate the variations of hENT1 expression in ampullary carcinomas and to correlate such variations with histological subtypes and clinicopathological parameters. Forty-one ampullary carcinomas were histologically classified into intestinal, pancreaticobiliary and unusual types. hENT1 and Ki67 expression were evaluated by immunohistochemistry, and apoptotic cells were identified by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate biotin nick end labelling (TUNEL) method. hENT1 overexpression was detected in 63.4% ampullary carcinomas. A significant difference in terms of hENT1 and Ki67 expression was found between intestinal vs. pancreaticobiliary types (P=0.03 and P=0.009 respectively). Moreover, a significant statistical positive correlation was found between apoptotic and proliferative Index (P=0.036), while no significant correlation was found between hENT1 and apoptosis. Our results on hENT1 expression suggest that classification of ampullary carcinoma by morphological subtypes may represent an additional tool in prospective clinical trials aimed at examining treatment efficacy; in addition, data obtained from Ki67 and TUNEL suggest a key role of hENT1 in tumour growth of ampullary carcinoma.
PMCID: PMC3167316  PMID: 20839414
human equilibrative nucleoside transporter 1; histotypes; vater ampulla; cancer; immunohistochemistry.
8.  Enteropathy type T cell lymphoma with an unusually late relapse: a case report 
PMCID: PMC1860587  PMID: 17213361
9.  Safety enhanced photodynamic therapy with half dose verteporfin for chronic central serous chorioretinopathy: a short term pilot study 
To evaluate short term safety of an enhanced photodynamic therapy (PDT) protocol with half dose verteporfin for treating chronic central serous chorioretinopathy (CSC).
20 eyes of 18 patients with symptomatic chronic CSC underwent PDT using 3 mg/m2 verteporfin. Verteporfin was infused over 8 minutes followed by indocyanine green angiography guided laser application 2 minutes later. Serial optical coherence tomography (OCT) and multifocal electroretinography (mfERG) recordings were performed before PDT, at 4 days, 2 weeks, and 1 month after PDT. The best corrected visual acuity (BCVA), OCT central retinal thickness, and mean mfERG response amplitudes and peak latencies were compared longitudinally. Subgroup analysis was further performed for eyes with or without pigment epithelial detachment (PED).
At 1 month after PDT, the median BCVA improved from 20/40 to 20/30 (p = 0.001). The mean central retinal thickness also reduced from 276 μm to 158 μm (p<0.001) and 17 (85%) eyes had complete resolution of serous retinal detachment and/or PED. MfERG showed no significant changes in the mean N1 and P1 response amplitude and latency for all eyes. Subgroup analysis demonstrated that eyes without PED had a significant increase in the mean central mfERG P1 response amplitude with reduction in P1 peak latency at 1 month post‐PDT. For eyes with PED, transient reduction in the mean central P1 response amplitude was observed at 4 days post‐PDT.
The modified safety enhanced PDT protocol with half dose verteporfin appeared to be a beneficial treatment option for patients with chronic CSC, especially in eyes without serous PED. Further controlled study is warranted to demonstrate the long term safety and efficacy of this treatment option.
PMCID: PMC1857171  PMID: 16597666
central serous chorioretinopathy; photodynamic therapy; verteporfin; multifocal electroretinography; optical coherence tomography
10.  Proliferation, apoptosis, and intratumoral vascularity in multiple myeloma: correlation with the clinical stage and cytological grade 
Journal of Clinical Pathology  2002;55(7):530-534.
Aims: Abnormalities involving proliferation, apoptosis, and angiogenesis are important in tumorigenesis. The purpose of this study was to examine these three biological processes, and their relation with the clinical stage and cytological grade in multiple myeloma (MM).
Methods: Fifty four newly diagnosed patients with MM were studied by immunohistochemistry using bone marrow clot sections. Proliferation and apoptosis were evaluated for the proportion of MM cells (indicated by morphology and CD138 reactivity) positive for the Ki67 antigen and single stranded DNA (ssDNA), respectively. Angiogenesis was evaluated by measuring the intratumoral microvessel density (IMVD) and by assessing the immunoreactivity of vascular endothelial growth factor (VEGF).
Results: There were 30 men and 24 women (median age, 65 years; range, 37–84). At initial presentation, 15 (28%) were in Durie stage I, 15 (28%) in stage II, and 24 (44%) in stage III. Advanced clinical stage correlated with high cytological grade (p < 0.03). The medians for Ki67, ssDNA, and IMVD were 4.4% (range, 0–15%), 0.2% (range, 0–2.8%), and 15.5 (range, 0–63), respectively. Among these three continuous parameters, the only significant correlation was that between Ki67 and IMVD (p < 0.0001). Both Ki67 and IMVD also correlated with the clinical stage, cytological grade, and VEGF positivity (p <0.05). No correlation was found between ssDNA and all of the other parameters.
Conclusions: These data suggest that proliferation is associated with angiogenesis in MM. Furthermore, proliferation and angiogenesis, but not apoptosis, may be important in disease progression. Lastly, increased production of VEGF may be one of the contributing factors to the increase in intratumoral vascularity seen in advanced MM.
PMCID: PMC1769685  PMID: 12101201
multiple myeloma; proliferation; apoptosis; angiogenesis; clinical stage; cytological grade
11.  Isolation measures in the hospital management of methicillin resistant Staphylococcus aureus (MRSA): systematic review of the literature 
BMJ : British Medical Journal  2004;329(7465):533.
Objective To evaluate the evidence for the effectiveness of isolation measures in reducing the incidence of methicillin resistant Staphylococcus aureus (MRSA) colonisation and infection in hospital inpatients.
Design Systematic review of published articles.
Data sources Medline, Embase, CINAHL, Cochrane Library, System for Information on Grey Literature in Europe (SIGLE), and citation lists (1966-2000).
Review methods Articles reporting MRSA related outcomes and describing an isolation policy were selected. No quality restrictions were imposed on studies using isolation wards or nurse cohorting. Other studies were included if they were prospective or employed planned comparisons of retrospective data.
Results 46 studies were accepted; 18 used isolation wards, nine used nurse cohorting, and 19 used other isolation policies. Most were interrupted time series, with few planned formal prospective studies. All but one reported multiple interventions. Consideration of potential confounders, measures to prevent bias, and appropriate statistical analysis were mostly lacking. No conclusions could be drawn in a third of studies. Most others provided evidence consistent with a reduction of MRSA acquisition. Six long interrupted time series provided the strongest evidence. Four of these provided evidence that intensive control measures including patient isolation were effective in controlling MRSA. In two others, isolation wards failed to prevent endemic MRSA.
Conclusion Major methodological weaknesses and inadequate reporting in published research mean that many plausible alternative explanations for reductions in MRSA acquisition associated with interventions cannot be excluded. No well designed studies exist that allow the role of isolation measures alone to be assessed. None the less, there is evidence that concerted efforts that include isolation can reduce MRSA even in endemic settings. Current isolation measures recommended in national guidelines should continue to be applied until further research establishes otherwise.
PMCID: PMC516101  PMID: 15345626
12.  Twin Pregnancy Achieved Through TESE in an Adult Male Exstrophy 
Bladder exstrophy is a rare anomaly, it compromises bladder functions, and in males it occurs with an impairment of reproductive functions, because of erectile and ejaculatory deficit. Advancements in the surgical treatment of bladder exstrophy have allowed an improvement of the bladder functions while spontaneous conception is still impaired. This is a case report of a pregnancy and subsequent birth of twins following testicular sperm extraction, on a man born with classical bladder exstrophy with infertility due to anejaculation.
PMCID: PMC3468235  PMID: 12099556
Anejaculation; bladder exstrophy; fertility; TESE
13.  CD44s as a surrogate marker for distinguishing intraductal papilloma from papillary carcinoma of the breast. 
Journal of Clinical Pathology  1999;52(11):862-864.
BACKGROUND: It has been shown that CD44 variants are differentially expressed in normal and neoplastic breast tissues. The diagnostic value of these markers in distinguishing benign from malignant breast lesions has not been well examined. AIMS: To evaluate the diagnostic value of CD44s in distinguishing between intraductal papillomas and papillary carcinomas of the breast, which may be difficult morphologically. METHODS: Expression of CD44s detected by immunohistochemistry was studied in a series of intraductal papillomas (11) and papillary carcinomas (10). The normal breast tissues surrounding the lesions of these cases served as a control. The number of CD44s positive epithelial cells was scored and categorised as < 10%, 10-70%, or > 70%. RESULTS: Normal breast epithelial cells and all intraductal papillomas (11 of 11) expressed CD44s in a high proportion of cells (> 70%). In contrast, the majority of papillary carcinoma cases (eight of 10) expressed this marker in < 10% of the cells. In the remaining two papillary carcinoma cases, positivity was seen in more than 10% but still less than 70% of the cells. CONCLUSIONS: CD44s detection by immunohistochemistry is useful in distinguishing intraductal papillomas from papillary carcinomas of the breast.
PMCID: PMC501606  PMID: 10690183
14.  Intrathoracic extramedullary haematopoiesis complicated by massive haemothorax in alpha-thalassaemia 
Thorax  1999;54(5):466-468.
Intrathoracic extramedullary haematopoiesis (EMH) is a rare entity that is usually asymptomatic. A 44 year old man with alpha-thalassaemia is described who developed dyspnoea and massive left sided haemothorax. The haemoglobin disorder was established by Hgb H staining and haemoglobin electrophoretic studies. The DNA analysis revealed it to be a case of double heterozygous terminal codon mutation with the genotype ααCS/ααT. Computed tomographic scanning and magnetic resonance imaging of the thorax showed multiple paravertebral masses which were found by thoracoscopic biopsy to be extramedullary haematopoiesis. Although no additional sclerosing pleurodesis or low dose radiation therapy was given, the lung expanded well and there has been no recurrence of haemothorax to date.

PMCID: PMC1763777  PMID: 10212116
15.  The methanogenic archaeon Methanosarcina thermophila TM-1 possesses a high-affinity glycine betaine transporter involved in osmotic adaptation. 
Methanogenic Archaea are found in a wide range of environments and use several strategies to adjust to changes in extracellular solute concentrations. One methanogenic archaeon, Methanosarcina thermophila TM-1, can adapt to various osmotic conditions by synthesis of alpha-glutamate and a newly discovered compatible solute, Ne-acetyl-beta-lysine, or by accumulation of glycine betaine (betaine) and potassium ions from the environment. Since betaine transport has not been characterized for any of the methanogenic Archaea, we examined the uptake of this solute by M. thermophila TM-1. When cells were grown in mineral salts media containing from 0.1 to 0.8 M NaC1, M. thermophila accumulated betaine in concentrations up to 140 times those of a concentration gradient within 10 min of exposure to the solute. The betaine uptake system consisted of a single, high-affinity transporter with an apparent K3 of 10 microM and an apparent maximum transport velocity of 1.15 nmol/min/mg of protein. The transporter appeared to be specific for betaine, since potential substrates, including glycine, sarcosine, dimethyl glycine, choline, and proline, did not significantly inhibit betaine uptake. M. thermophila TM-1 cells can also regulate the capacity for betaine accumulation, since the rate of betaine transport was reduced in cells pregrown in a high-osmolarity medium when 500 microM betaine was present. Betaine transport appears to be H+ and/or Na+ driven, since betaine transport was inhibited by several types of protonophores and sodium ionophores.
PMCID: PMC168517  PMID: 9172344

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