[Show abstract][Hide abstract] ABSTRACT: The lipid raft protein Flotillin-1 was previously shown to be required for cell proliferation. Here we show that it is critical
for the maintenance of the levels of the mitotic regulator Aurora B. Knockdown of Flotillin-1 induced aberrant mitotic events
similar to those produced by Aurora B depletion and led to a marked decline in Aurora B levels and activity. Transfection
of wild-type full-length Flotillin-1 or forms directed to the nucleus increased Aurora B levels and activity. Flotillin-1
interacted with Aurora B directly through its SPFH domain in a complex distinct from the chromosomal passenger protein complex,
and the two proteins co-purified in nuclear, non-raft fractions. These observations are the first evidence for a function
of Flotillin-1 outside of lipid rafts and suggest its critical role in the maintenance of a pool of active Aurora B.
[Show abstract][Hide abstract] ABSTRACT: HER3 (ERBB3) is a catalytically inactive pseudokinase of the HER receptor tyrosine kinase family, frequently overexpressed in prostate and other cancers. Aberrant expression and mutations of 2 other members of the family, EGFR and HER2, are key carcinogenic events in several types of tumors, and both are well- validated therapeutic targets. In this study, we show that HER3 is required to maintain the motile and invasive phenotypes of prostate (DU-145) and breast (MCF-7) cancer cells in response to the HER3 ligand neuregulin-1 (NRG-1), epidermal growth factor (EGF) and fetal bovine serum. Although MCF-7 breast cancer cells appeared to require HER3 as part of an autocrine response induced by EGF and FBS, the response of DU-145 prostate cancer cells to these stimuli, while requiring HER3, did not appear to involve autocrine stimulation of the receptor. DU-145 cells required the expression of HER3 for efficient clonogenicity in vitro in standard growth medium and for tumorigenicity in immunodeficient mice. These observations suggest that prostate cancer cells derived from tumors that overexpress HER3 are dependent on its expression for the maintenance of major attributes of neoplastic aggressiveness, with or without cognate ligand stimulation.
International Journal of Cancer 07/2009; 125(11):2565-75. DOI:10.1002/ijc.24651 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The heterodimeric ubiquitin conjugating enzyme (E2) UBC13-UEV mediates polyubiquitylation through lysine 63 of ubiquitin (K63), rather than lysine 48 (K48). This modification does not target proteins for proteasome-dependent degradation. Searching for potential regulators of this variant polyubiquitylation we have identified four proteins, namely RNF8, KIA00675, KF1, and ZNRF2, that interact with UBC13 through their RING finger domains. These domains can recruit, in addition to UBC13, other E2s that mediate canonical (K48) polyubiquitylation. None of these RING finger proteins were known previously to recruit UBC13. For one of these proteins, RNF8, we show its activity as a ubiquitin ligase that elongates chains through either K48 or K63 of ubiquitin, and its nuclear co-localization with UBC13. Thus, our screening reveals new potential regulators of non-canonical polyubiquitylation.
[Show abstract][Hide abstract] ABSTRACT: The combination of Raman spectroscopy and Optical Tweezers has been used to trap living cells and collect information about their biochemical state. Cells can continue living in such traps for periods of hours, allowing acquisition of time resolved Raman spectra. However no spatial information can be acquired as the cells continue to rotate and move in the single beam trap. Here we describe the development of Holographic Optical Tweezers (HOT) for the controlled movement of floating cells in order to construct their Raman images. Instead of a single trap, rapidly programmable multiple trapping points can be produced around the periphery of the cells to impede the rotational motion of the cell. By trapping and scanning the cell using HOT relative to a fixed Raman exciting laser, a point by point image of the cell can be constructed. We use an interactive program that permits us to position the trapping points relative to the live image feed we see from the microscope, using point and click. To demonstrate the possibilities of this technique images are shown of floating Jurkat cells.
Proceedings of SPIE - The International Society for Optical Engineering 01/2006; DOI:10.1117/12.679360 · 0.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Raman imaging can yield spatially resolved biochemical information from living cells. To date there have been no Raman images published of cells in suspension because of the problem of immobilizing them suitably to acquire space-resolved spectra. In this paper in order to overcome this problem the use of holographic optical tweezers is proposed and implemented, and data is shown for spatially resolved Raman spectroscopy of a live cell in suspension.
[Show abstract][Hide abstract] ABSTRACT: Multiple beam tweezers are demonstrated for Raman spectroscopy of living cells. Results are presented for time resolved Raman. Data for spatially resolved Raman is shown and holographic tweezers for cell imaging is proposed.
Lasers and Electro-Optics, 2005. (CLEO). Conference on; 06/2005
[Show abstract][Hide abstract] ABSTRACT: The introduction of high-throughput techniques is increasingly providing abundant information on molecular alterations requiring validation at the posttranscriptional level. Protein expression is now efficiently evaluated in large series of tumors included in tissue microarrays. We propose, describe and validate a technique to elaborate paraffin-embedded cell line microarrays (PECLIMA) from fixed cell cultures, which can be processed like standard surgical pathology biopsies prior to immunophenotyping. Our results show a reliable protein immunoexpression profiling in six widely used cell lines under different fixation conditions. This technique permits the simultaneous analysis of multiple antigens in multiple cell lines under different experimental conditions. Additional features of these arrays are long-term storage, their suitability for a variety of techniques including immunocytochemistry and in situ hybridization and their low cost.
[Show abstract][Hide abstract] ABSTRACT: We use an approach based on Factor Analysis to analyze datasets generated for transcriptional profiling. The method groups samples into biologically relevant categories, and enables the identification of genes and pathways most significantly associated to each phenotypic group, while allowing for the participation of a given gene in more than one cluster. Genes assigned to each cluster are used for the detection of pathways predominantly activated in that cluster by finding statistically significant associated GO terms. We tested the approach with a published dataset of microarray experiments in yeast. Upon validation with the yeast dataset, we applied the technique to a prostate cancer dataset.
Two major pathways are shown to be activated in organ-confined, non-metastatic prostate cancer: those regulated by the androgen receptor and by receptor tyrosine kinases. A number of gene markers (HER3, IQGAP2 and POR1) highlighted by the software and related to the later pathway have been validated experimentally a posteriori on independent samples.
Using a new microarray analysis tool followed by a posteriori experimental validation of the results, we have confirmed several putative markers of malignancy associated with peptide growth factor signalling in prostate cancer and revealed others, most notably ERRB3 (HER3). Our study suggest that, in primary prostate cancer, HER3, together or not with HER4, rather than in receptor complexes involving HER2, could play an important role in the biology of these tumors. These results provide new evidence for the role of receptor tyrosine kinases in the establishment and progression of prostate cancer.