Abstract Proteomic differential display analysis was performed on human renal cell carcinoma cell SN12C clones having different metastatic potentials by using 2‐DE and LC‐MS/MS. The SN12C cell clones were SN12C parent cell line, SN12C‐clone 2, SN12C‐clone 4, and SN12C‐PM6. The SN12C parent cell line was established from an HRCC surgical specimen. SN12C‐clone 4 has lower, and SN12C‐clone 2 and SN12C‐PM6 have higher metastatic potential than SN12C parent cells. We found eight protein spots whose expression level was different between low metastatic clones and high metastatic clones. The protein expression of three appeared to be higher in high metastatic clones than low metastatic clones, and that of other five protein spots appeared to be lower in high metastatic clones than low metastatic clones. These spots were selected, digested and analyzed by LC‐MS/MS analysis, and they were identified by peptide sequencing tag. In high metastatic potential clones, two isoforms of ubiquitin carboxyl‐terminal hydrolase isozyme L1 (UCH‐L1) were downregulated. These results suggest that UCH‐L1 expression seems to be associated with the metastatic potential of HRCC SN12C cell clones.
Two homologous serine proteinase inhibitors (serpins), squamous cell carcinoma (SCC) antigen-1 and -2 were separated by nondenaturing two-dimensional electrophoresis combined with immunostaining to acquire further information on these proteins under physiological conditions. Polymers of SCC antigen-2 were detected in cytosolic extracts prepared from tumor tissues. The polymer formation of SCC antigen-2 was apparently decreased and the SCC antigen-2-synthetic peptide binary complexes were newly formed by the addition of synthetic peptide with sequences corresponding to residues from P14 to P2 in the reactive center loop of SCC antigen-2. On the other hand, the incubation with synthetic peptides having the sequence of the reactive center loop of SCC antigen-1 or antithrombin had no effect on polymerization of SCC antigen-2. These data suggest that the polymerization of SCC antigen-2 may occur spontaneously in vivo by the loop-sheet mechanism of serpin.
Mouse antidextran monoclonal antibodies showed microheterogeneity which was analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Not only the heavy (H) chains but also the light (L) chains were heterogeneous in terms of isoelectric point (pI). The higher the pI, the more prominent the H chain spots. To demonstrate the cause of the microheterogeneity an IgG1 monoclonal antibody (mAb 35.8.2H) was examined especially for involvement of the sugar moiety in the microheterogeneity. The glycosylated region was determined in the Fc portion from serine 239 to methionine 309 by a glycan detection method using mild periodate oxidation, which confirms that the sugar chain is attached to the conserved glycosylation site of asparagine 297. However, charge heterogeneity of the H chain was not entirely attributed to the Fc because the papain digest of the antibody was separated into two Fc spots, a few Fd spots and two L chain spots by 2-D PAGE. This indicates that factors other than the sugar moiety are responsible for charge heterogeneity of IgG monoclonal antibody. On the other hand, the H chain isoforms of lower pI were shown to be more susceptible to V8 protease by peptide mapping. This result strongly suggests the occurrence of deamidation at glutamine or asparagine residues.
Abstract Hepatocellular carcinoma (HCC) is one of the most common fatal cancers, and chronic infection with hepatitis C virus (HCV) is thought to be one of the main causes in Japan. To identify diagnostic or therapeutic biomarkers for HCC associated with HCV (HCV‐HCC), we tried to elucidate the factors related to the products from cancerous tissues of HCV‐infected patients. From proteomic differential display analysis of liver tissue samples from HCV‐HCC cancerous tissues and corresponding non‐cancerous tissues from patients, three protein spots of the same molecular mass (42 kDa), whose expression increased in well‐differentiated cancerous tissues, were detected. Although their p I were different, they were identified as glutamine synthetase (GS) by PMF with MALDI‐TOF MS and by Western blotting using anti‐GS specific mAb. Immunohistochemical analysis showed that tumor tissue consists of two parts, GS‐positive cell and GS‐negative cell regions, suggesting that GS‐producing cells grew in the tumor tissue as a nodule in nodules. The tryptic peptides of the most acidic GS isoform lost the signal of 899.5 Da, corresponding a peptide of SASIRIPR, and gained a signal of 1059.5 Da, which was submitted to PSD analysis. PSD analysis showed the neutral loss by elimination of two phosphate groups, supposed to be on serine residues of the 899.5‐Da peptide, from serine 320 to arginine 327 in GS. PMF followed by PSD analysis is thought to be useful for the determination of phosphorylation sites of proteins showing molecular heterogeneity.
Two-dimensional gel electrophoresis was used to study the expression of intracellular proteins in adherent cells of human T lymphoblastic cell line, CCRF-CEM. The adherent cells grown in monolayer on a culture plate decreased the amount of proteins of M(r) 37,000 and pI 4.7-4.9, and of 17,000 and pI 5.7. The proteins were identified to be nucleophosmin for the 37,000 protein and stathmin for the 17,000 protein by microsequencing their CNBr fragments. The amount of proteins was increased in CCRF-CEM cells grown in floating mass to a comparable level of JURKAT cells which grew in floating mass throughout the culture. The adherent cells decreased their growth rate as compared with the cells in the floating mass. These results suggest that the adhesion of human T lymphoblastic cells modulates their morphology and proliferation via a concomitant decrease in the amount of nucleophosmin and stathmin.
While Gemcitabine (2'-deoxy-2'-difluorodeoxycytidine: Gemzar) (GEM) is thought to be most effective for pancreatic cancer, its clinical effects do not seem properly evaluated due to a high level of inherent and acquired resistance in the tumor cells. We analyzed GEM-resistant and GEM-sensitive human pancreatic cancer cell lines proteomically to identify the proteins related to the GEM-resistance. First, we separated cancer cell proteins by two-dimensional gel electrophoresis, and identified the protein spots whose expressions differed between GEM-sensitive and -resistant cell lines by liquid chromatography-tandem mass spectrometry (LC-MS / MS). It was recognized that heat shock protein 27 (HSP27) was high in GEM-resistant cell lines, and that KLM1-R cells, a cell line which has acquired resistance to GEM, restored sensitivity to it when HSP27 was knocked down by siRNA. These findings suggest that expression of HSP27 should be reduced in order that the antitumor effects of GEM may be well exerted. Since the expression of HSP27 was dramatically reduced in the KLM1-R cells treated with GEM and IFN-γ, improved therapeutic effects may be expected in the combination of IFN-γ with GEM.
