Since drug resistance is a complex and multifactorial event involving activation/repression of multiple biochemical pathways, we used a proteomic approach to study cisplatin resistance and drug response in human tumor cell lines. The cervix squamous cell carcinoma cell line A431 and its cisplatin‐resistant subline, A431/Pt, were used as a model system. The experimental set‐up involved not just a two‐way comparison of the control vs. the drug‐resistant cell line, but also an acute cisplatin treatment of both cell lines, leading to a four‐way comparison, as follows: 1) A431 vs. A431/Pt cells; 2) A431 vs. A431 cisplatin exposed cells; 3) A431/Pt vs. A431/Pt cisplatin exposed cells; 4) A431 cisplatin exposed cells vs. A431/Pt cisplatin exposed cells. We found modulation of proteins, which could be classified under various categories, such as molecular chaperones (e.g. heat‐shock proteins HSP60, HSP90, HSC71, heat‐shock cognate 71 kDa protein), Ca2plus;‐binding proteins (e.g. calmodulin, calumenin), proteins involved in drug detoxification (such as peroxiredoxins PRX 2 and PRX 6, and glutathione‐S‐transferase, GST), anti‐apoptotic proteins (such as 14‐3‐3 switched on in cisplatin‐exposed cells) and ion channels (such as VDAC‐1, voltage‐dependent anion‐selective channel). In particular, the basal levels of HSC71 and HSP60 were increased in A431/Pt cells as compared to A431 cells, and cisplatin exposure resulted in up‐regulation of HSP60 and HSP90 only in A431 cells. Moreover, cisplatin exposure up‐regulated the anti‐apoptotic 14‐3‐3 protein in both cell lines, GST in sensitive cells and PRX6 in A431/Pt cells. These findings are consistent with a constitutive expression of defence factors by resistant cells and with activation by cisplatin of mechanisms acting to protect cells from drug‐induced damage. This pattern of response, also observed in parental cells, could reflect an intrinsic resistance of this tumor type.

The present review attempts to cover the vast array of methods which have appeared in the last few years for performing quantitative proteome analysis. These methods are divided into two classes: those applicable to conventional two-dimensional map analysis, coupling orthogonally a charge-based step (isoelectric focusing) to a size-based separation [sodium dodecylsulfate (SDS)-electrophoresis] and those applicable to two-dimensional chromatographic protocols. The first method, although being by and large the most popular approach, can offer differential display of paired samples with relatively few methods, the oldest one being based on statistical analysis performed on sets of gels via powerful software packages, such as the MELANIE, PDQuest, Z3 and Z4000, Phoretix and Progenesis. Recent developments comprise analysis performed on a single gel containing mixed samples differentially labeled, either with fluorophors (Cy3 and Cy5) or with d(0)/d(3) acrylamide. Conversely, chromatographic approaches, which mostly rely on analysis not of intact proteins but of their tryptic digests, offer a panoply of differential labeling protocols, most of which rely on stable isotope tagging. Essentially, all possible reactions have been described, such as those involving Lys, Asp, Glu, Cys residues, as well as a number of methods exploiting differential derivatization of amine and carboxyl groups generated during proteolysis. All such methods are described and evaluated.

No abstract

No abstract

A pancreatic adenocarcinoma cell line (PaCa44), which contains, among other alterations, a methylated p16 promoter, was treated with a chemoterapeutic agent, 5-aza-2'-deoxycytidine (DAC), in order to evaluate the effect of this drug on cell growth and protein expression. Cell proliferation was strongly inhibited by a 24 h DAC treatment and this inhibition lasted for at least 10 days. Master maps of control and treated PaCa44 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and downregulation of 45 polypeptide chains, of which 32 were downregulated and 13 upregulated, out of a total of 700 spots detected by a medium-sensitivity stain, micellar Coomassie Brilliant Blue. Fingerprinting by mass spectrometry analysis enabled the identification of 36 of these spots. Among the major changes in DAC-treated cells: cofilin and profilin 1 are silenced; coactosin, peptidyl-propyl cis-trans isomerase A and cystatin B are decreased by 22, 16- and 15-fold, respectively; stress-70 protein, superoxide dismutase and protein disulfide isomerase A3 are increased by 13-, 11-, and 5-fold, respectively. The significance of some of these major changes is discussed.

No abstract

A pancreatic adenocarcinoma cell line (Paca44) was treated with trichostatin-A (TSA), a potent inhibitor of histone deacetylases, in order to evaluate the effect of this drug on protein expression. Master maps of control and treated Paca44 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and downregulation of 51 polypeptide chains, out of a total of 700 spots detected by a medium-sensitivity stain, micellar Coomassie Brilliant Blue. Fingerprinting by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry analysis enabled the identification of 22 of these spots. Among these proteins, of particular interest are the two downregulated proteins nucleophosmin and translationally controlled tumor protein, as well as the upregulated proteins programmed cell death protein 5 (also designated as TFAR19) and stathmin (oncoprotein 18). The modulation of these four proteins is consistent with our observation that TSA is able to inhibit cell growth of Paca44 by causing cell cycle arrest at the G2 phase and apoptotic cell death.

The present research establishes standard two-dimensional (2-D) maps for control, reactive lymph node and non-Hodgkin's lymphoma (mantle cell lymphoma, MCL). Medium sensitivity, mass spectrometry compatible colloidal Coomassie has revealed a total of ca. 750 spots in each of the maps. Comparison of 2-D maps by statistical packages, such as the PDQuest, established up- and downregulation of a total of ca. 145 spots, with positive variations of up to 10-folds and negative variations of up to 13-folds in both MCL biopsies' protein extracts. Qualitative and quantitative variations in the two lymphoma samples are consistent. More than 20 proteins have been so far identified by matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry, with an additional five spots, which gave very good spectra but could not be matched to any of the presently available databases. Some of the spots, such as the 78 kDa glucose-regulated protein precursor and the glutathione S-transferase P, appear to be in common with other tumors, such as lung adenocarcinoma. Others may simply reflect overall changes in cellular metabolism and growth rate that occur during malignancy and thus might turn out to be in common with any cell population receiving any kind of stress. Some (notably T-cell leukemia/lymphoma protein 1A, TCL1, found to be 10-fold overexpressed) appear to be specific of the non-Hodgkin's lymphoma here studied. Western blot and immunohistochemical analyses were applied to obtain further information about stathmin (Op18) and TCL1, respectively.