Metabolism and growth inhibition of four retinoids in head and neck squamous normal and malignant cells

Isotretinoin (13- cis -retinoic acid, 13cRA) has proven to be active in chemoprevention of head and neck squamous cell carcinoma (HNSCC). Moreover, both all-trans-retinoic acid (ATRA) and 13cRA induce objective responses in oral premalignant lesions. After binding of retinoids to retinoic acid receptors (RARs and RXRs) dimers are formed that are able to regulate the expression of genes involved in growth and differentiation. We compared the metabolism and level of growth inhibition of 13cRA with that of ATRA, 9cRA and retinol in four HNSCC cell lines and normal oral keratinocyte cultures (OKC). These retinoid compounds are known to bind with different affinities to the retinoic acid receptors. We observed that all retinoids were similar with respect to their capacity to induce growth inhibition. One HNSCC line could be ranked as sensitive, one as moderately sensitive and the remaining two were totally insensitive; OKC were moderately sensitive. The rate at which the cells were able to catabolize the retinoid was similar for all compounds. Retinoid metabolism in HNSCC cells resulted in a profile of metabolites that was unique for each retinoid. These metabolic profiles were different in OKC. Our findings indicate that differences in retinoid receptor selectivity of these retinoids do not influence the level of growth inhibition and rate of metabolism. © 2001 Cancer Research Campaign http://www.bjcancer.co

Visualization and Analysis Techniques for Three Dimensional Information Acquired by Confocal Microscopy

Confocal Scanning Laser Microscopy (CSLM) is particularly well suited for the acquisition of 3-dimensional data of microscopic objects. In the CSLM a specific volume in the object is sampled during the imaging process and the result is stored in a digital computer as a three-dimensional memory array. Optimal use of these data requires both the development of effective visual representations as well as analysis methods. In addition to the well known stereoscopic representation method a number of alternatives for various purposes are presented. When rendering in terms of solid-looking or semitransparent objects is required, an algorithm based on a simulated process of excitation and fluorescence is very suitable. Graphic techniques can be used to examine the 3-dimensional shape of surfaces. For (near-)real time applications a representation method should not require extensive previous data-processing or analysis. From the very extensive field of 3-D image analysis two examples are given

Retinoid metabolism and all-trans retinoic acid-induced growth inhibition in head and neck squamous cell carcinoma cell lines.

Retinoids can reverse potentially premalignant lesions and prevent second primary tumours in patients with head and neck squamous cell carcinoma (HNSCC). Furthermore, it has been reported that acquired resistance to all-trans retinoic acid (RA) in leukaemia is associated with decreased plasma peak levels, probably the result of enhanced retinoid metabolism. The aim of this study was to investigate the metabolism of retinoids and relate this to growth inhibition in HNSCC. Three HNSCC cell lines were selected on the basis of a large variation in the all-trans RA-induced growth inhibition. Cells were exposed to 9.5 nM (radioactive) for 4 and 24 h, and to 1 and 10 microM (nonradioactive) all-trans RA for 4, 24, 48 and 72 h, and medium and cells were analysed for retinoid metabolites. At all concentrations studied, the amount of growth inhibition was proportional to the extent at which all-trans-, 13- and 9-cis RA disappeared from the medium as well as from the cells. This turnover process coincided with the formation of a group of as yet unidentified polar retinoid metabolites. The level of mRNA of cellular RA-binding protein II (CRABP-II), involved in retinoid homeostasis, was inversely proportional to growth inhibition. These findings indicate that for HNSCC retinoid metabolism may be associated with growth inhibition

Changes in the status of p53 affect drug sensitivity to thymidylate synthase (TS) inhibitors by altering TS levels

Colorectal cancer (CRC) resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious clinical problem often associated with increased intracellular levels of TS. Since the tumour suppressor gene p53, which is mutated in 50% of CRC, regulates the expression of several genes, it may modulate TS activity, and changes in the status of p53 might be responsible for chemoresistance. Therefore, this study was aimed to investigate TS levels and sensitivity to TS inhibitors in wild-type (wt) and mutant (mt) p53 CRC cells, Lovo and WiDr, respectively, transfected with mt and wt p53. Lovo 175X2 cells (transfected with mt p53) were more resistant to 5-fluorouracil (5-FU; 2-fold), nolatrexed (3-fold), raltitrexed (3-fold) and pemetrexed (10-fold) in comparison with the wt p53 parental cells Lovo 92. Resistance was associated with an increase in TS protein expression and catalytic activity, which might be caused by the loss of the inhibitory effect on the activity of TS promoter or by the lack of TS mRNA degradation, as suggested by the reversal of TS expression to the levels of Lovo 92 cells by adding actinomycin. In contrast, Lovo li cells, characterized by functionally inactive p53, were 3-13-fold more sensitive to nolatrexed, raltitrexed and pemetrexed, and had a lower TS mRNA, protein expression and catalytic activity than Lovo 92. However, MDM-2 expression was significantly higher in Lovo li, while no significant differences were observed in Lovo 175X2 cells with respect to Lovo 92. Finally, mt p53 WiDr transfected with wt p53 were not significantly different from mt p53 WiDr cells with respect to sensitivity to TS inhibitors or TS levels. Altogether, these results indicate that changes in the status of p53, can differently alter sensitivity to TS inhibitors by affecting TS levels, depending on activity or cell line, and might explain the lack of clear correlation between mutations in p53 and clinical outcome after chemotherapy with TS inhibitors

Direct-Acting Antiviral Treatment for Hepatitis C Genotypes Uncommon in High-Income Countries: A Dutch Nationwide Cohort Study (vol 8, ofab006, 2021)

Cellular mechanisms in basic and clinical gastroenterology and hepatolog

Extended Field Laser Confocal Microscopy (EFLCM): Combining automated Gigapixel image capture with in silico virtual microscopy

<p>Abstract</p> <p>Background</p> <p>Confocal laser scanning microscopy has revolutionized cell biology. However, the technique has major limitations in speed and sensitivity due to the fact that a single laser beam scans the sample, allowing only a few microseconds signal collection for each pixel. This limitation has been overcome by the introduction of parallel beam illumination techniques in combination with cold CCD camera based image capture.</p> <p>Methods</p> <p>Using the combination of microlens enhanced Nipkow spinning disc confocal illumination together with fully automated image capture and large scale <it>in silico </it>image processing we have developed a system allowing the acquisition, presentation and analysis of maximum resolution confocal panorama images of several Gigapixel size. We call the method Extended Field Laser Confocal Microscopy (EFLCM).</p> <p>Results</p> <p>We show using the EFLCM technique that it is possible to create a continuous confocal multi-colour mosaic from thousands of individually captured images. EFLCM can digitize and analyze histological slides, sections of entire rodent organ and full size embryos. It can also record hundreds of thousands cultured cells at multiple wavelength in single event or time-lapse fashion on fixed slides, in live cell imaging chambers or microtiter plates.</p> <p>Conclusion</p> <p>The observer independent image capture of EFLCM allows quantitative measurements of fluorescence intensities and morphological parameters on a large number of cells. EFLCM therefore bridges the gap between the mainly illustrative fluorescence microscopy and purely quantitative flow cytometry. EFLCM can also be used as high content analysis (HCA) instrument for automated screening processes.</p

Long term survival following the detection of circulating tumour cells in head and neck squamous cell carcinoma

Background Techniques for detecting circulating tumor cells in the peripheral blood of patients with head and neck cancers may identify individuals likely to benefit from early systemic treatment. Methods Reconstruction experiments were used to optimise immunomagnetic enrichment and RT-PCR detection of circulating tumor cells using four markers (ELF3, CK19, EGFR and EphB4). This method was then tested in a pilot study using samples from 16 patients with advanced head and neck carcinomas. Results Seven patients were positive for circulating tumour cells both prior to and after surgery, 4 patients were positive prior to but not after surgery, 3 patients were positive after but not prior to surgery and 2 patients were negative. Two patients tested positive for circulating cells but there was no other evidence of tumor spread. Given this patient cohort had mostly advanced disease, as expected the detection of circulating tumour cells was not associated with significant differences in overall or disease free survival. Conclusion For the first time, we show that almost all patients with advanced head and neck cancers have circulating cells at the time of surgery. The clinical application of techniques for detection of spreading disease, such as the immunomagnetic enrichment RT-PCR analysis used in this study, should be explored further

DPHL: A DIA Pan-human Protein Mass Spectrometry Library for Robust Biomarker Discovery

To address the increasing need for detecting and validating protein biomarkers in clinical specimens, mass spectrometry (MS)-based targeted proteomic techniques, including the selected reaction monitoring (SRM), parallel reaction monitoring (PRM), and massively parallel data-independent acquisition (DIA), have been developed. For optimal performance, they require the fragment ion spectra of targeted peptides as prior knowledge. In this report, we describe a MS pipeline and spectral resource to support targeted proteomics studies for human tissue samples. To build the spectral resource, we integrated common open-source MS computational tools to assemble a freely accessible computational workflow based on Docker. We then applied the workflow to generate DPHL, a comprehensive DIA pan-human library, from 1096 data-dependent acquisition (DDA) MS raw files for 16 types of cancer samples. This extensive spectral resource was then applied to a proteomic study of 17 prostate cancer (PCa) patients. Thereafter, PRM validation was applied to a larger study of 57 PCa patients and the differential expression of three proteins in prostate tumor was validated. As a second application, the DPHL spectral resource was applied to a study consisting of plasma samples from 19 diffuse large B cell lymphoma (DLBCL) patients and 18 healthy control subjects. Differentially expressed proteins between DLBCL patients and healthy control subjects were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supports DIA and PRM MS pipelines for robust protein biomarker discovery. DPHL is freely accessible at https://www.iprox.org/page/project.html?id=IPX0001400000

Cerebral magnetic resonance imaging in quiescent Crohn’s disease patients with fatigue

FSW - Self-regulation models for health behavior and psychopathology - ou