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1.
J Tissue Eng Regen Med ; 12(3): e1337-e1348, 2018 03.
Article in English | MEDLINE | ID: mdl-28714568

ABSTRACT

Articular chondrocytes derived from osteoarthritic tissues (OA HAC) show a severely reduced chondrogenic commitment. This impairment undermines their use for tissue-engineered cartilage repair, which relies on cell proliferation and growth to meet therapeutic needs, but also on efficient cell plasticity to recover the chondrogenic phenotype. Reversine (Rev), a 2,6-disubstituted purine inhibitor of spindle-assembly checkpoints, was described to convert differentiated mesenchymal cells to their undifferentiated precursors. We hypothesized that Rev exposure could divert OA HAC to more plastic cells, re-boosting their subsequent commitment. HAC were enzymatically released from OA cartilage specimens, expanded for 2 weeks and treated with 5 µm Rev in dimethylsulphoxide (DMSO) or with DMSO alone for 6 days. Cell growth was assessed using the AlamarBlueTM assay. Cytoskeletal structure, endoproliferation and caspase-3-immunopositivity were assayed by epifluorescence microscopy. The OA HAC chondrogenic performance was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate dehydrogenase, Sox9, Aggrecan (Agg), type II collagen (Col2), Ki67, cyclinD1, transforming growth factor-ß1 (TGF-ß1), -2 and -3, interleukin-1ß (IL-1ß) and -6 , SMAD3 and -7, and vascular endothelial growth factor. Rev-treated OA HAC recovered polygonal morphology and reduced Ki67 expression and proliferation. Cell-cycle impairment accounted for altered cytoskeletal organization, endoproliferation and apoptosis, whereas a compensatory mechanism sustained the increased cyclinD1 transcript levels. Sox9, Agg and TGFs were overexpressed, but not Col2. IL transcripts were massively downregulated. These events were dose-related and transient. Overall, in spite of a higher Rev-induced transcriptional activity for extracellular matrix components and in spite of a Rev-treated cell phenotype closer to that of the three-dimensional native articular chondrocyte, Rev effects seem unleashed from a full regained chondrogenic potential.


Subject(s)
Cell Shape/drug effects , Chondrocytes/cytology , Morpholines/pharmacology , Purines/pharmacology , Aged , Aged, 80 and over , Cartilage, Articular , Cell Cycle Checkpoints/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Chondrocytes/drug effects , Chondrocytes/metabolism , Cyclin D1/genetics , Cyclin D1/metabolism , Dimethyl Sulfoxide/pharmacology , Female , Gene Expression Regulation/drug effects , Humans , Ki-67 Antigen/genetics , Ki-67 Antigen/metabolism , Male , Osteoarthritis/pathology , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Smad3 Protein/genetics , Smad3 Protein/metabolism
2.
Br J Cancer ; 100(10): 1608-16, 2009 May 19.
Article in English | MEDLINE | ID: mdl-19401687

ABSTRACT

Using proteomic analysis of the nuclear matrix (NM), we found that heterogeneous nuclear ribonucleoprotein K (hnRNP K), a member of the hnRNP family with pleiotropic functions, was differentially expressed in prostate cancer (PCa) tissues. This study aimed to characterise the expression of hnRNP K and its subcellular localisation in PCa, utilising immunohistochemical and quantitative western blot techniques. Furthermore, the hnRNP K expression was studied in human PCa cell lines in order to determine its modulation by bicalutamide, the anti-androgen widely used in PCa therapy. Immunohistochemical staining of paraffin-embedded tissues showed that hnRNP K was overexpressed in PCa, where it was localised both in the cytoplasm and in the nucleus. Staining of non-tumour tissues showed exclusively nuclear localisation and a less intense or absent signal. Immunoblot analysis demonstrated that the hnRNP K level within the NM was higher in PCa compared with non-tumour tissues and closely correlated with Gleason score (P=0.008). Higher expression within the NM was significantly (P=0.032) associated with poor prognosis. In two-dimensional western blot analysis hnRNP K presented several isoforms; the one with pI 5.1 was the most differently expressed between non-tumour and PCa tissues. Preliminary results indicate that hnRNP K can be modulated in vitro by a non-steroidal anti-androgen. Taken together, our findings suggest that hnRNP K has potential implications at the diagnostic, prognostic and therapeutic levels in PCa.


Subject(s)
Carcinoma/diagnosis , Carcinoma/metabolism , Heterogeneous-Nuclear Ribonucleoprotein K/metabolism , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/metabolism , Aged , Biomarkers, Tumor/metabolism , Carcinoma/pathology , Cell Line, Tumor , Humans , Male , Middle Aged , Neoplasm Metastasis , Phosphorylation , Prognosis , Prostatic Neoplasms/pathology , Protein Kinases/metabolism , Proteomics/methods , Tissue Distribution
3.
Bioelectromagnetics ; 25(7): 545-51, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15376242

ABSTRACT

Extremely low frequency electromagnetic fields (ELF-EMFs) of 75 Hz with amplitudes above a threshold of about 125 microT have a dramatic effect on the adenylate kinase (AK) activity of the rod outer segment (ROS) membranes. In fact, the ATP production by ROS membranes or by purified disk membranes placed in the field decreased by approximately 54%. The decrease in enzymatic activity was independent of the time of exposure to the field and was completely reversible. When disk membranes were solubilized with Triton or a soluble isoform of AK was used, negligible effects of the field were obtained on the enzymatic activity, suggesting that the membrane has an important role in determining the conditions for the enzyme inactivation.


Subject(s)
Adenylate Kinase/radiation effects , Electromagnetic Fields , Rod Cell Outer Segment/radiation effects , Adenosine Triphosphate/radiation effects , Adenylate Kinase/antagonists & inhibitors , Animals , Cattle , Cell Membrane/enzymology , Cell Membrane/radiation effects , Isoenzymes/radiation effects , Polyethylene Glycols/pharmacology , Rod Cell Outer Segment/enzymology , Solubility , Surface-Active Agents/pharmacology , Time Factors
4.
Bioelectrochemistry ; 63(1-2): 317-20, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15110295

ABSTRACT

Adenylate kinase activity in rod outer segment membranes of bovine retina decreased of about 55% when exposed to an extremely low frequency electromagnetic field of 75 Hz and 250 microT. The effect was independent of the time of permanence in the field. Negligible effects of the field were found on the enzymatic activity of a soluble isoform of adenylate kinase or of rod outer segment membranes solubilized with Triton, suggesting the importance of the membrane in determining the conditions of the enzyme inactivation.


Subject(s)
Adenosine Triphosphate/metabolism , Adenylate Kinase/metabolism , Adenylate Kinase/radiation effects , Cell Membrane/enzymology , Cell Membrane/radiation effects , Retinal Rod Photoreceptor Cells/enzymology , Retinal Rod Photoreceptor Cells/radiation effects , Adenylate Kinase/chemistry , Cells, Cultured , Dose-Response Relationship, Radiation , Electricity , Electromagnetic Fields , Enzyme Activation/radiation effects , Radiation Dosage
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