Serological identification of TROP2 by recombinant cDNA expression cloning using sera of patients with esophageal squamous cell carcinoma.

We applied serological analysis of recombinant cDNA expression libraries (SEREX) to cases of esophageal squamous cell carcinoma (SCC) to identify tumor antigens. One of the clones identified was TROP2, which is known as calcium signal transducer. To evaluate the clinical significance of serum anti-TROP2 antibodies (s-TROP2-Abs) in patients with esophageal SCC, the presence of s-TROP2-Abs was analyzed by Western blotting using bacterially expressed TROP2 protein. We found that 23 of 75 (31%) patients were positive for s-TROP2-Abs. Positivity in terms of s-TROP2-Abs showed a significant association with tumor size but not with other clinicopathological features. The protein expression levels of TROP2 were much higher in esophageal SCC cell lines as compared to those in normal esophageal mucosa and its immortalized cells although the mRNA expression levels were not necessarily elevated in malignant cell lines and tissues. Immunohistochemical studies showed that the expression of TROP2 protein in esophageal SCC specimens was noticeably higher than that found in mild hyperplasia of esophageal mucosae. Thus, s-TROP2-Abs seemed useful in the diagnosis of SCC and may be a candidate for serum tumor markers.

Drug-sensitivity pattern analysis for study of functional relationship between gene products.

We have developed a method that we call 'drug-sensitivity pattern analysis', or DSPA, for analysis of protein function. Cells are transfected with cDNA of the test molecule, followed by analysis of the sensitivity of the transfected cells to multiple growth-inhibitory drugs. If two cDNA products have similar functions, their transfected cells should show similar drug-sensitivity patterns. The cDNAs of some signaling molecules were transfected into NIH3T3 or Ha-ras-transformed NIH3T3 (ras-NIH) cells and stable transfectants, which expressed high amounts of the gene product, were isolated. Chemosensitivity of the transfected clone was compared with the parental cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method using more than 40 drugs. The chemosensitivity changes caused by the transfected gene were calculated and expressed numerically as 'drug chemosensitivity index' (DCI). When the DCI values were analyzed by regression analysis, a significant positive relationship between IkappaBalpha superrepressor and dominant-negative IKKbeta and an inverse relationship between p53 and Mdm2 were consistent with previous reports. Thus, the DSPA method is useful for identifying functional similarities between gene products.

Continuous increase in phosphorylation of cytosolic thymidine kinase during proliferation of rat hepatoma JB1 cells.

Thymidine kinase (TK) is a protein closely associated with DNA replication. Here we show that, in contrast with the variation of TK activity, which changed parallel with S phase cell distribution during asynchronous culture of rat hepatoma JB1 cells, the serine residues of cytosolic TK protein was phosphorylated continuously in response to increasing G0/G1 phase cell distribution. The shifting of phosphorylation of TK protein during different cell growth conditions was further confirmed with the examination of the elution profile of cytosolic TK activity by anion-exchange high performance liquid chromatography (HPLC). HPLC analysis revealed that the rapid proliferating (62 h after asynchronous culture) rat hepatoma JB1 cells showed only the hyperphosphorylated form of cytosolic TK activity, while synchronously M phase-arrested JB1 cells showed hypo- and hyper-phosphorylated forms of cytosolic TK activity. These results suggested that the modulation of phosphorylation of cytosolic TK protein was cell cycle-dependent, and that the phosphorylation of cytosolic TK protein might be involved in the negative regulation of TK activity in vivo.

Expression and regulation of the gene for arginase I in mouse salivary glands: requirement of CCAAT/enhancer-binding protein alpha for the expression in the parotid gland.

Arginase in salivary glands is potentially involved in the synthesis of proline, glutamate, and polyamines that play specific physiological roles in the glands, and also in depletion of arginine in the oral cavity to protect teeth from microorganisms. We detected protein and mRNA for the type I isoform of arginase in mouse salivary glands. Enzymes of the arginine-biosynthetic pathway were also detected. Immunohistochemical analysis revealed that arginase I was enriched in the striated duct, and was also present in the acinus, demilune and granulated duct. Mice with targeted disruption of the gene for C/EBPalpha, which is a transcription factor essential for expression of the arginase I gene in the liver, showed dramatically reduced immunoreactivity for arginase I in the parotid gland but not in the submandibular and sublingual glands. Therefore, C/EBPalpha is specifically required for expression of the arginase I gene in the parotid gland.