Protein CoAlation and antioxidant function of coenzyme A in prokaryotic cells.

In all living organisms, coenzyme A (CoA) is an essential cofactor with a unique design allowing it to function as an acyl group carrier and a carbonyl-activating group in diverse biochemical reactions. It is synthesized in a highly conserved process in prokaryotes and eukaryotes that requires pantothenic acid (vitamin B5), cysteine and ATP. CoA and its thioester derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. A novel unconventional function of CoA in redox regulation has been recently discovered in mammalian cells and termed protein CoAlation. Here, we report for the first time that protein CoAlation occurs at a background level in exponentially growing bacteria and is strongly induced in response to oxidizing agents and metabolic stress. Over 12% of Staphylococcus aureus gene products were shown to be CoAlated in response to diamide-induced stress. In vitro CoAlation of S. aureus glyceraldehyde-3-phosphate dehydrogenase was found to inhibit its enzymatic activity and to protect the catalytic cysteine 151 from overoxidation by hydrogen peroxide. These findings suggest that in exponentially growing bacteria, CoA functions to generate metabolically active thioesters, while it also has the potential to act as a low-molecular-weight antioxidant in response to oxidative and metabolic stress.

Independent overexpression of the subunits of translation elongation factor complex eEF1H in human lung cancer.

BACKGROUND: The constituents of stable multiprotein complexes are known to dissociate from the complex to play independent regulatory roles. The components of translation elongation complex eEF1H (eEF1A, eEF1Bα, eEF1Bß, eEF1Bγ) were found overexpressed in different cancers. To gain the knowledge about novel cancer-related translational mechanisms we intended to reveal whether eEF1H exists as a single unit or independent subunits in different human cancers. METHODS: The changes in the expression level of every subunit of eEF1H in the human non-small-cell lung cancer tissues were examined. The localization of eEF1H subunits was assessed by immunohistochemistry methods, subcellular fractionation and confocal microscopy. The possibility of the interaction between the subunits was estimated by co-immunoprecipitation. RESULTS: The level of eEF1Bß expression was increased more than two-fold in 36%, eEF1Bγ in 28%, eEF1A in 20% and eEF1Bα in 8% of tumor specimens. The cancer-induced alterations in the subunits level were found to be uncoordinated, therefore the increase in the level of at least one subunit of eEF1H was observed in 52% of samples. Nuclear localization of eEF1Bß in the cancer rather than distal normal looking tissues was found. In cancer tissue, eEF1A and eEF1Bα were not found in nuclei while all four subunits of eEF1H demonstrated both cytoplasmic and nuclear appearance in the lung carcinoma cell line A549. Unexpectedly, in the A549 nuclear fraction eEF1A lost the ability to interact with the eEF1B complex. CONCLUSIONS: The results suggest independent functioning of some fraction of the eEF1H subunits in human tumors. The absence of eEF1A and eEF1B interplay in nuclei of A549 cells is a first evidence for non-translational role of nuclear-localized subunits of eEF1B. We conclude the appearance of the individual eEF1B subunits in tumors is a more general phenomenon than appreciated before and thus is a novel signal of cancer-related changes in translation apparatus.

Unbalanced expression of the translation complex eEF1 subunits in human cardioesophageal carcinoma.

BACKGROUND: The signalling role of individual subunits released from some stable translation multi-molecular complexes under unfavourable circumstances is known. The disease-related role of the translation elongation factor 1 complex (eEF1) as a whole is never researched; however, its subunits possess apparent regulatory potency. Whether the individual eEF1 subunits can exist and function in cell beyond the complex is not known. MATERIALS AND METHODS: The protein and mRNA levels of the A1, Bα, Bß or Bγ subunits of eEF1 were analysed by Western and Northern blot techniques in the same specimens of cardioesophageal carcinoma and correspondingly paired normal tissues. Cancer-induced changes in localization patterns of the eEF1 subunits were examined immunohistochemically. RESULTS: Changes in different eEF1 subunits expression were found to be unbalanced, indicating cancer-related emergence of individual components of the eEF1 complex. Independent overexpression of at least one eEF1 component was observed in 72% clinical samples. Noncomplexed eEF1B subunits were also detected by immunohistochemical analysis. In the normal tissue, localization of the Bα, Bß and Bγ subunits was nuclear-cytoplasmic while in the cancer tissue the only Bγ subunit stayed in nucleus. CONCLUSIONS: Our data are first to indicate that the individual subunits can exist separately from the eEF1B complex in cancer tissues and that disintegration of eEF1B could be an important sign of cancer development. Nuclear localization of Bγ both in normal and in cancer tissues suggests its previously unknown nucleus-specific role in human cells.

Functional model of rat thyroid follicles cultured in Matrigel.

BACKGROUND: Long-term maintenance of functional activity of thyroid cells is an essential requirement for basic in vitro studies on the physiology and pathology of the thyroid. An important prerequisite of thyrocytes' functional activity in vivo and in vitro is their follicle organization. AIM: This study aimed at developing a method of cultivation of functionally active rat thyroid follicles in Matrigel under three-dimensional conditions. METHODS: Undamaged rat thyroid follicles were isolated by enzymatic digestion with collagenase/dispase, then embedded into Matrigel, and cultivated for 2 weeks. Thyroglobulin, thyroxine and zonula occludens-1 (ZO-1) localization were revealed by immunofluorescence analysis. Iodide organification was tested by protein-bound 125I (PBI) measurement. RESULTS: Integrity of the follicles was preserved during the whole period of cultivation and was confirmed by 3D reconstruction of ZO-1 localization. Thyroglobulin was detected in the thyrocyte cytoplasm, as well as in the intrafollicular lumen. Thyroxine was observed predominantly at the apical side of thyrocytes. Also, generated cultures were characterized by a high level of iodide organification: PB125I represented 39% of the total radioactivity in the Matrigel drop embedding the follicles; at the same time, methimazole almost totally inhibited this process (0.2% of total radioactivity). CONCLUSION: The method of rat thyrocyte cultivation in Matrigel, as described here allows to maintain the structural integrity and the functional activity of thyroid follicles in vitro and could be used for wide ranges of basic and applied researches in thyroidology.

Nucleocytoplasmic distribution of S6K1 depends on the density and motility of MCF-7 cells in vitro.

Background: The ribosomal protein S6 kinase 1 (S6K1) is one of the main components of the mTOR/S6K signal transduction pathway, which controls cellular metabolism, autophagy, growth, and proliferation. Overexpression of S6K1 was detected in tumors of different origin including breast cancer, and correlated with the worse disease outcome. In addition, significant accumulation of S6K1 was found in the nuclei of breast carcinoma cells suggesting the implication of kinase nuclear substrates in tumor progression. However, this aspect of S6K1 functioning is still poorly understood. The main aim of the present work was to study the subcellular localization of S6K1 in breast cancer cells with the focus on cell migration. Methods: Multicellular spheroids of MCF-7 cells were generated using agarose-coated Petri dishes. Cell migration was induced by spheroids seeding onto adhesive growth surface and subsequent cultivation for 24 to 72 hours. The subcellular localization of S6K1 was studied in human normal breast and cancer tissue samples, 2D and 3D MCF-7 cell cultures using immunofluorescence analysis and confocal microscopy. Results: Analysis of histological sections of human breast tissue samples revealed predominantly nuclear localization of S6K1 in breast malignant cells and its mainly cytoplasmic localization in conditionally normal cells. In vitro studies of MCF-7 cells demonstrated that the subcellular localization of S6K1 depends on the cell density in the monolayer culture. S6K1 relocalization from the cytoplasm into the nucleus was detected in MCF-7 cells migrating from multicellular spheroids onto growth surface. Immunofluorescence analysis of S6K1 and immunocoprecipitation assay revealed the colocalization and interaction between S6K1 and transcription factor TBR2 (T-box brain protein 2) in MCF-7 cells. Conclusions: Subcellular localization of S6K1 depends on the density and locomotor activity of the MCF-7 cells.

Monoclonal antibodies to Ki-67 protein suitable for immunohistochemical analysis.

Detection of cell proliferation index is widely used in experimental and clinical research. Earlier it was shown that nuclear Ki-67 protein expression is strictly related to cell proliferation. It was revealed during all active phases of the cell cycle in mammals but was absent in G0 phase, so Ki-67 presence in cell nuclei reflects a potential growth fraction of whole cell population. The main area of Ki-67 antibody application is in immunocytochemical and immunohistochemical analyses. The aim of our work was to generate mouse monoclonal antibodies for Ki-67 antigen detection in mammalian tissues and in cultured cells. His-tagged fragment of Ki-67 expressed in bacteria was used as an antigen. Antibody-producing hybridoma cells were generated by standard procedure by fusing SP2/0 myeloma cells with splenocytes of immunized mice. Monoclonal antibodies were analyzed using paraffin-embedded human melanoma tissue samples and breast cancer cell line MCF-7. It was shown that generated anti-Ki-67 antibodies revealed proliferating cells in MCF-7 culture and after heat-induced epitope retrieval on paraffin sections of human melanoma tissue. In summary, generated antibodies could be useful for detection of proliferating cells in immunohistochemical and immunofluorescence studies of mammalian cells and tissues.

The small leucine-rich proteoglycan lumican inhibits melanoma progression.

Lumican is a member of the small leucine-rich proteoglycan (SLRP) family. It contributes to the organisation of the collagen network and plays an important role in cell migration and tissue repair. The present study aimed to determine the influence of lumican expression on adhesion, anchorage-dependent and -independent growth, migration, in vitro invasion and in vivo melanoma growth. For that purpose, B16F1 mouse melanoma cells were stably transfected with an expression plasmid containing the complete lumican cDNA. Lumican expression by tumor cells did not change the proliferative activity of mouse melanoma cells in monolayer culture and did not influence either cell adhesion to extracellular matrix gel or type I collagen or cell spreading on these substrates. In contrast, lumican-transfected cells were characterized by a strong reduction of their anchorage-independent proliferation in agarose gel and capacity to invade extracellular matrix gel. After subcutaneous injections of transfected B16F1 cells in syngenic mice, lumican expression significantly decreased subcutaneous tumor formation in vivo, with a concomitant decrease of cyclin D1 expression. Lumican induced and/or increased the apoptosis of B16F1 cells. The results suggest that lumican is involved in the control of melanoma growth and invasion and may be considered, like decorin, as an anti-tumor factor from the extracellular matrix.