Namiko Kuroiwa

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.

Specific recognition of cytosolic thymidine kinase in the human lung tumor by monoclonal antibodies raised against recombinant human thymidine kinase

Anti-TK monoclonal antibodies (mAbs) were raised against recombinant human cytosolic thymidine kinase (rhTK) and characterized by Western immunoblotting, enzyme-linked immunosorbent assay (ELISA) and immunostaining of tumor cells. Twenty-three clones of TK mAbs were characterized to recognize specifically not only rhTK produced by Escherichia coli but also TK subunit of 25 kDa in human lung cancer. The anti-TK mAbs reacted specifically with cytosolic TK but not with mitochondrial TK. Only one clone of the mAbs inhibited the catalytic activity of TK. By solid phase sandwich enzyme immunoassay using these mAbs, we could quantitate the cytosolic TK content in tissues. Immunohistochemical staining analysis using one of the TK mAbs showed that human lung adenocarcinoma and squamous cell carcinoma exhibited much higher staining intensity than stromal cells. These mAbs are useful for biochemical studies on the regulation of human TK in proliferating cells such as tumor cells and for diagnosis of highly proliferating tumors.

Characterization of nicotinamide methyltransferase in livers of mice bearing Ehrlich ascites tumors: preferential increase of activity

There was a 2- to 7-fold increase in nicotinamide methyltransferase activity in the livers of mice and rats bearing seven different kinds of tumors compared with the respective control normal livers, while activity in the tumors themselves was hardly detectable. The activity in the liver started to increase markedly 3-7 days after i.p. transplantation of Ehrlich ascites tumors into the mice, maintaining a plateau up to death. Metabolic conversion of 14C-nicotinamide to 14C-N1-methylnicotinamide was 3-fold higher in the slices of the ascites tumor host liver than in the normal liver, but the conversion to other radioactive metabolites was not significantly different. Nicotinamide methyltransferase was finally purified 20,000-fold with a yield of 4% from the cytosolic fraction of the ascites tumor host liver by means of five purification steps. At every purification step, only one enzyme fraction was detected. The enzyme finally isolated exhibited a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a molecular weight of 26,000. As for the compounds investigated, including the substrates for methyltransferases other than nicotinamide methyltransferase, only quinoline could be the substrate for enzyme activity. It is suggested that the increase in enzyme activity in the tumor host liver probably derived from the endogenous enzyme preexisting in the liver before tumor transplantation.

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 IκBα superrepressor and dominant‐negative IKKβ 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.

Characterization of the purified cytosolic thymidine kinase from murine Ehrlich ascites tumor: Interconversion of two different relative molecular weight forms

Cytosolic thymidine kinase was purified 18,000‐fold of the homogenate from murine Ehrlich ascites tumor, using the [p‐aminophenyl 3′‐dTMP]‐CH‐Sepharose affinity column. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band of molecular weight 26,000. Two different forms, relative molecular weight 50,000 and 70,000, were found by gel filtration, depending on the existence of dithiothreitol, ATP and other nucleotides. These agents also stabilize and stimulate the enzyme activity. The existence of two forms was also manifested by DEAE‐Sephacel column chromatography, where the 50,000 form was eluted by 50 mM NaCl and the 70,000 form by 400 mM NaCl.