Proteomic analysis of telomerase inhibition by telomere specific ligands

in Anticancer Research (2008), 28(5c), 3257-3258

Telomeres consist of protein complexes and repeated ‘TTAGGG’ double strand DNA sequences ended by a 3’ single strand DNA of the same sequence. Progressive telomere shortening is observed in vitro upon ... [more ▼]

Telomeres consist of protein complexes and repeated ‘TTAGGG’ double strand DNA sequences ended by a 3’ single strand DNA of the same sequence. Progressive telomere shortening is observed in vitro upon cell divisions and with ageing in vivo. At a critical telomere length, shortened telomeres trigger a permanent growth arrest known as replicative senescence. Telomerase is an RNA-dependent DNA polymerase that extends telomeres by adding ‘TTAGGG’ repeats. It consists of a functional RNA component (hTR) which serves as template and a catalytic protein (hTERT) with reverse transcriptase activity. The expression of hTERT alone is sufficient for the immortalisation of cells. Telomerase is highly expressed in tumor cells but at very low level in most somatic cells. These observations make the telomerase an attractive target for anticancer strategies. One of these strategies relies on the use of drug candidates able to stabilize the particular telomere G-quadruplex DNA structures. The stabilization of these structures makes the telomere inaccessible for telomerase and thus inhibits telomerase activity. The effect of the hTERT transfection was first studied on the proteome of human WI38 fibroblast cells (1). Then, the proteome alteration response of hTERT transfected WI38 cells induced by the treatment of two G-quadruplexes ligands, telomestatin and TMPyP4, was analyzed. Both compounds can inhibit telomerase but have different selectivity for the different G-quadruplexes structures. Proteome analysis of the treated cells reveals that TMPyP4 induces much more protein expression alterations than telomestatin probably due to its poor selectivity. TMPyP4 induces especially a drastic down expression of the hnRNPs, a modulation of the proteasome pathway, an apparent decrease of the translation and an over expression of several molecular chaperones. Telomestatin induces in particular an over expression of the protein BCL2A1 which is involved in drug resistance of cancer cells and a probable increase of the translation. Both treatments have a common effect particularly on the molecular chaperone CCT (down expression), HSP90 alpha (over expression) and hnRNP D (down expression). The protein HSP90 alpha is also over expressed in hTERT transfected cells compared to parental cells. This protein is already a promising anticancer target protein due to its central role in oncogenesis and in telomerase activity regulation. 1 Mazzucchelli et al: Proteome Science 6: 12, 2008. [less ▲]

Proteome alteration induced by hTERT transfection of human fibroblast cells

in Proteome Science (2008), 6(1), 12

Background: Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human ... [more ▼]

Background: Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT) gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (W138). Cytosolic and nuclear fractions of W138 cells, empty vector transfected W138 (W138-HPV) and hTERT W138 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis) analysis. Only spots that had a similar abundance in W138 and W138-HPV, but were differentially expressed in W138 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control W138-HPV cells. The proteome alteration induced by hTERT W138 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results: 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion: We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest that telomerase expression enhances natural cell repair mechanisms and stress resistance probably required for long term resistance of immortalized cells. Thus, hTERT transfected cells can not be only consider as an immortal equivalent to parental cells but also as cells which are over-resistant to stresses. These findings are the prerequisite for any larger proteomics aiming to evaluate anti-telomerase drugs proteome alteration and thus therapeutics induced cell reactions. [less ▲]

Improved performances of intraocular lenses by poly(ethylene glycol) chemical coatings

in Biomacromolecules (2007), 8(8), 2379-2387

Cataract surgery is a routine ophthalmologic intervention resulting in replacement of the opacified natural lens by a polymeric intraocular lens (IOL). A main postoperative complication, as a result of ... [more ▼]

Cataract surgery is a routine ophthalmologic intervention resulting in replacement of the opacified natural lens by a polymeric intraocular lens (IOL). A main postoperative complication, as a result of protein adsorption and lens epithelial cell (LEC) adhesion, growth, and proliferation, is the secondary cataract, referred to as posterior capsular opacification (PCO). To avoid PCO formation, a poly(ethylene glycol) (PEG) chemical coating was created on the surface of hydrogel IOLs. Attenuated total reflectance Fourier transform infrared spectroscopy, “captive bubble” and “water droplet” contact angle measurements, and atomic force microscopy analyses proved the covalent grafting of the PEG chains on the IOL surface while keeping unchanged the optical properties of the initial material. A strong decrease of protein adsorption and cell adhesion depending on the molar mass of the grafted PEG (1100, 2000, and 5000 g/mol) was observed by performing the relevant in vitro tests with green fluorescent protein and LECs, respectively. Thus, the study provides a facile method for developing materials with nonfouling properties, particularly IOLs. [less ▲]

Global strategy for the development of estrogenic compounds detection screening test

Poster (2007, May)

MG-63 Osteoblast culture on P culture on PLA– based copolymers for bone tissue engineering applications

Poster (2007)

Physical injury or pathological changes such as removal of a tumor can result in large bone defects, preventing the recovery of its original function. Autogenous bone grafting, which is the most common ... [more ▼]

Physical injury or pathological changes such as removal of a tumor can result in large bone defects, preventing the recovery of its original function. Autogenous bone grafting, which is the most common technique for bone defect repairing, is associated with serious limitations, e.g. limited supply and donor site morbidity. Since a few years bone tissue engineering by degradable biomaterials has been shown as a very promising avenue for providing bone substitutes. Among these materials, bioresorbable synthetic polymers such as poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly(acrylic acid) (PAA) and poly(lactic acid) (PLA) are very appealing because their chemistry and properties are controllable and reproducible. Cellular activity and proliferation of osteosarcoma cell lines (MG-63) on films were determined by the tetrazolium salt MTT assay and by phase contrast/fluorescence microscope observations. The cytotoxicity of the materials was found to be low or negligible. Cells viability variations were observed on the surface of the films. Long-term cell culture and degradability of PLA-PEOpolymer film was investigated by optical microscopy (Giemsa staining) and environmental scanning electron microscopy (ESEM). Hydrolysis of the PLLA ester linkages led to slow film degradation. After 113 days, optical microscope observations revealed the presence of large cracks on the surface, and even breaks of small polymer fragments, while MG-63 proliferation was still very important, showing a tissue-like aspect, with extracellular matrix (ECM) deposition. These results show that PLA-PEO copolymers are very interesting bioresorbable materials for long-term bone tissue engineering applications. [less ▲]

MG-63 osteoblasts culture on poly-(lactic acid) degradable textiles for bone tissue regeneration

Article for general public (2007)

This work shows that knitted PLA fabrics, seeded with human MG-63 osteoblasts can potentially be used as tissue engineered systems for in vitro to in vivo transition, for the treatment of bone defect

Study of the effect of exposition of MCF-7/bos cells to 17-ß-estradiol by 2D DIGE

Poster (2006, October)

The aim of this work is to identify biomarkers following exposition to 17ß-estradiol in MCF7/bos (hormonal dependent epithelial mammary cancer) cells. The application is to optimise screening tests that ... [more ▼]

The aim of this work is to identify biomarkers following exposition to 17ß-estradiol in MCF7/bos (hormonal dependent epithelial mammary cancer) cells. The application is to optimise screening tests that will enable the detection (and eventual quantification but not identification) of numerous compounds having an estrogenic activity in a single rapid test. [less ▲]

Antiprotozoal and cytotoxic triterpenoid saponins from the roots of Cephalaria gigantea

Poster (2006, July)

Induction of Apoptosis in Human Promyelocytic Leukemia Cells by a Natural Trachylobane Diterpene

in Anticancer Research (2005), 25(1A), 363-368

Background: Trachylobane diterpenes are secondary metabolites quite rare in nature and their bioactivities are poorly known. Recently we have described the cytotoxic activity of ent- trachyloban-3‚-ol ... [more ▼]

Background: Trachylobane diterpenes are secondary metabolites quite rare in nature and their bioactivities are poorly known. Recently we have described the cytotoxic activity of ent- trachyloban-3‚-ol isolated from the leaves of Croton zambesicus, a plant used in African folk medicine. Materials and Methods: Cell viability on several cell lines, cell morphology, DNA laddering, annexin V and caspase-3 activation experiments were undertaken in order to analyse the cytotoxicty of trachylobane diterpene and to determine if this compound is able to induce apoptosis. Results: ent-trachyloban-3‚-ol exerts a dose-dependent cytotoxic effect and which varies between cell lines. Induction of apoptosis in HL-60 cells could be detected at a concentration of 50 ÌM after 24-h treatment. Conclusion: We show here, for the first time, that a trachylobane diterpene is able to induce apoptosis in human promyelocytic leukemia cells via caspase-3 activation in a concentration-dependent manner. [less ▲]

Detection of biomarkers of pathogenic bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

in Lichtfouse, Eric; Schwarzbauer, Jan; Robert, Didier (Eds.) Environmental Chemistry : Green Chemistry and Pollutants in Ecosystems (2005)

In recent years, various mass spectrometry procedures has been developed for identifying bacteria. The accuracy and speed with which data can be obtained by Matrix-assisted laser Desorption/Ionization ... [more ▼]

In recent years, various mass spectrometry procedures has been developed for identifying bacteria. The accuracy and speed with which data can be obtained by Matrix-assisted laser Desorption/Ionization Time-of-flight Mass Spectrometry (MALDI-TOF-MS) make this an advantageous technique for environmental monitoring. However, minor variations in the sample preparation can influence the mass spectra significantly. In the present study, we have introduced a procedure to prepare bacteria by microextraction and we have optimized experimental parameters for rapid identification by MALDI-TOF-MS of whole bacterial cells isolated from environmental samples such as wastewater and soil. [less ▲]