Inhibition of N-linked glycosylation of P-glycoprotein by tunicamycin results in a reduced multidrug resistance phenotype.

Characterisation of altered glycosylation of P-glycoprotein (P-gp) found associated with the absence of a multidrug resistance (MDR) phenotype in cell lines prompted an investigation to assess the role of post-translational processing in establishing P-gp efflux pump functionally. The clone A cell line used in this study displays a strong MDR phenotype mediated by high constitutive levels of expression of P-gp. Incubation of clone A cells with tunicamycin for different periods resulted in a time-dependent increase in daunorubicin accumulation, reflecting a reduction in P-gp function. Parallel experiments conducted with verapamil resulted in no loss of P-gp functionality in clone A cells. Reduction in surface-associated P-gp following exposure to tunicamycin was established by FACS analysis, Western blot analysis and immunoprecipitation of surface-iodinated P-gp. In addition, immunoprecipitation of P-gp from 32P-orthophosphate-labelled cells demonstrated reduced phosphorylation of P-gp associated with tunicamycin exposure. From these studies we conclude that glycosylation of P-gp is required to establish the cellular MDR phenotype

Clinical significance of altered nm23-H1, EGFR, RB and p53 expression in bilharzial bladder cancer

<p>Abstract</p> <p>Background</p> <p>Clinical characterization of bladder carcinomas is still inadequate using the standard clinico-pathological prognostic markers. We assessed the correlation between <it>nm23-H1</it>, <it>Rb, EGFR </it>and <it>p53 </it>in relation to the clinical outcome of patients with muscle invasive bilharzial bladder cancer (MI-BBC).</p> <p>Methods</p> <p><it>nm23-H1</it>, <it>Rb, EGFR and p53 </it>expression was assessed in 59 MI-BBC patients using immunohistochemistry and reverse transcription (RT-PCR) and was correlated to the standard clinico-pathological prognostic factors, patient's outcome and the overall survival (OS) rate.</p> <p>Results</p> <p>Overexpression of <it>EGFR </it>and <it>p53 </it>proteins was detected in 66.1% and 35.6%; respectively. Loss of <it>nm23-H1</it>and <it>Rb </it>proteins was detected in 42.4% and 57.6%; respectively. Increased <it>EGFR and </it>loss of <it>nm23-H1 </it>RNA were detected in 61.5% and 36.5%; respectively. There was a statistically significant correlation between <it>p53 </it>and <it>EGFR </it>overexpression (<it>p </it>< 0.0001), <it>nm23 </it>loss (protein and RNA), lymph node status (<it>p </it>< 0.0001); between the incidence of local recurrence and <it>EGFR </it>RNA overexpression (p= 0.003) as well as between the incidence of metastasis and altered <it>Rb </it>expression (<it>p </it>= 0.026), <it>p53 </it>overexpression (<it>p </it>< 0.0001) and mutation (<it>p </it>= 0.04). Advanced disease stage correlated significantly with increased <it>EGFR </it>(protein and RNA) (<it>p </it>= 0.003 & 0.01), reduced <it>nm23-H1 </it>RNA (<it>p </it>= 0.02), altered <it>Rb </it>(<it>p </it>= 0.023), and <it>p53 </it>overexpression (<it>p </it>= 0.004). OS rates correlated significantly, in univariate analysis, with <it>p53 </it>overexpression (<it>p </it>= 0.011), increased <it>EGFR </it>(protein and RNA, <it>p </it>= 0.034&0.031), <it>nm23-H1 RNA </it>loss (<it>p </it>= 0.021) and aberrations of ≥ 2 genes. However, multivariate analysis showed that only high <it>EGFR </it>overexpression, metastatic recurrence, high tumor grade and the combination of ≥ 2 affected markers were independent prognostic factors.</p> <p>Conclusion</p> <p><it>nm23-H1, EGFR </it>and <it>p53 </it>could be used as prognostic biomarkers in MI-BBC patients. In addition to the standard pathological prognostic factors, a combination of these markers (≥ 2) has synergistic effects in stratifying patients into variable risk groups. The higher is the number of altered biomarkers, the higher will be the risk of disease progression and death.</p

New interpretable machine-learning method for single-cell data reveals correlates of clinical response to cancer immunotherapy.

We introduce a new method for single-cell cytometry studies, FAUST, which performs unbiased cell population discovery and annotation. FAUST processes experimental data on a per-sample basis and returns biologically interpretable cell phenotypes, making it well suited for the analysis of complex datasets. We provide simulation studies that compare FAUST with existing methodology, exemplifying its strength. We apply FAUST to data from a Merkel cell carcinoma anti-PD-1 trial and discover pre-treatment effector memory T cell correlates of outcome co-expressing PD-1, HLA-DR, and CD28. Using FAUST, we then validate these correlates in cryopreserved peripheral blood mononuclear cell samples from the same study, as well as an independent CyTOF dataset from a published metastatic melanoma trial. Finally, we show how FAUST's phenotypes can be used to perform cross-study data integration in the presence of diverse staining panels. Together, these results establish FAUST as a powerful new approach for unbiased discovery in single-cell cytometry

Cyclooxygenase-2: a possible target in schistosoma-associated bladder cancer

Objective: To analyse and compare the expression of cyclooxygenase (COX) enzymes in schistosoma-associated bladder cancer, and to determine any association with tumour grade or stage. Materials and methods: Sixty paired samples of tumour and adjacent nonmalignant urothelium were identified. There were 25 squamous and 28 transitional cell carcinomas, and seven adenocarcinomas. Serial sections were obtained and a standard three-layer immunohistochemistry protocol, using COX-1- and COX-2-specific mouse monoclonal antibodies, applied. Results: COX-1 was expressed mostly in nonvascular smooth muscle with weak reactivity in malignant and nonmalignant urothelium. Nonmalignant urothelium expressed COX-2 weakly, notably in areas of dysplasia and squamous metaplasia whereas there was a significant increase in COX-2 (P < 0.001) with moderate to strong granular cytoplasmic expression in all three malignant histological types. The COX-2 reactivity was higher in transitional and adenocarcinomas than in squamous cell carcinoma (P < 0.001). Areas of carcinoma in situ showed COX-2 reactivity comparable with that in invasive areas and more intense than that detected in dysplastic or metaplastic urothelium (P < 0.001). There was a statistically significant positive correlation between COX-2 expression and tumour grade (P = 0.0052). Conclusion: COX-2 is over-expressed in schistosoma-associated bladder cancer, consistent with a potential role for COX-2 inhibitors in the prevention and management of this disease