J. Exp.



1992, 168, 105-111

of Mn-Superoxide



Carcinogenesis NAOYUKI TANIGUCHI, MUTSUO ISHIKAWA*, TETSUO KAWAGUCHI,JUNICHI FUJII, KEIICHIRO SUZUKI and TOSHIYUKINAKATA Department of Biochemistry, Osaka University Medical School, Suita 565, and Department of Obstetrics and Gynecology, Asahikawa Medical College, Asahikawa 078

TANIGUCHI,N., ISHIKAWA, M., KAWAGUCHI, T., FUJII, J., SUZUKI,K, and NAKATA, T. Expression on Mn-Superoxide Dismutase in Carcinogenesis. Tohoku J. Exp. Med., 1992, 168 (2), 105-111 Human liver manganese superoxide dismutase (Mn-SOD) was highly purified by a simple procedure and crystallized. A monoclonal antibody against Mn-SOD, whose antigen-binding epitope is a 0-terminus peptide was developed. Using this antibody, an enzyme-linked immunosorbent assay (ELISA) was developed. We found that Mn-SOD is highly expressed in human ovarian cancer and the serum level of the enzyme is a useful marker for the diagnosis and monitoring of the epithelial type of ovarian cancer. Tumor necrosis factor-a (TNF), lipopolysaccharide, IL-1 and phorbol ester induced the m-RNA of Mn-SOD as well as protein levels in TNF-resistant cells. No such induction was observed in Cu, Zn-SOD. Studies on the induction mechanisms indicated that at least two separate signal-transducing pathways are involved in expression of the Mn-SOD gene. One is triggered by protein kinase C activation itself in the absence of new protein synthesis. The other can be activated by stimulations with various cytokines in which a protein factor that can be induced by phorbol ester treatments is involved. Mu-superoxide dismutase ; monoclonal antibody ; epithelial ovarian cancer ; Tumor necrosis factor, phorbol ester

Most aerobic cells have an enzyme system to eliminate active oxygen species. The SOD catalyzes the dismutation of superoxide anion (02-) to produce hydrogen peroxide (H2O2) and 02. The 02 is one of the reduced oxygen species generated in the cellular metabolism (McCord and Fridovich 1969). We have purified and crystallized human liver Mn-SOD (Matsuda et al. 1990 ; Deutsch et al. 1991) and developed polyclonal and monoclonal antibodies (Kawaguchi et al. 1989) to Mn-SODs and used these in an enzyme-linked immunosorbent assay (ELISA) for the SOD (Kawaguchi et al. 1990b). We found that serum Mn-SOD level of patients with several cancers including epithelial ovarian cancer gave high values (Ishikawa et al. 1990). Possible mechanisms by which the Mn-SOD is expressed in cancer tissues were discussed. Addressfor reprints : 2-2 Yamadaoka,Suita 565, Japan. 105


N. Taniguchi

et al.

Purification of Mn-SOD and its monoclonal antibody Mn-SODhas been purified and crystallized from human livers. The enzyme has hexagonal space group p6122or p6522 (Deutsch et al. 1991). Human Mn-SOD is composed of four identical subunits. Each monomer molecular weight is 22,000,as judged by the low laser angle light scattering method (Matsuda et al. 1990). Under denaturing conditions the enzyme is easily converted to monomers which form intra-disulfide bonds. Mn-SODhas two cysteine residues. Cys-196, located close to the C-terminus, is highly reactive to sulfhydryl reagents. The presence of the reactive sulfhydryl group Cys-196 also suggests that mixed disulfides are formed in vivo. A monoclonal antibody, PG 11, was raised in mice against the human liver Mn-SOD(Kawaguchi et al. 1989). The competition experiments using synthetic peptides revealed that the antibody-binding epitope was localized in the COON terminal peptides as described previously. Enzyme-linked immunosorbent assay (ELISA) for Mn-SOD An enzyme-linked immunosorbent assay (ELISA) has been developed using the monoclonal antibody (Kawaguchi et al. 1989). As described above, human Mn-SOD is a tetramer composed of identical subunits. Therefore, the same monoclonal antibody could be used in the sandwich immunoassay as both captor and detector ; in the case of monomeric enzymes, two different antibodies should be used. The ELISA offers a specific, sensitive and convenient means of measuring immunoreactive Mn-SOD in human sera. Under optimum conditions, the lower limit of detection was 2 ng/ml and the working range was 2-200 ng/ml. Serum Mn-SOD levels as a tumor marker for epithelial ovarian cancer The levels of Mn-SOD in sera from 194 male and 207 female healthy adult individuals were examined by ELISA. The mean level and s.D. for male and female were 99.8 ng/ml + 24.8 and 88.8+ 20.8 ng/ml, respectively. The upper limit of normal male was assumed to be 150 ng/ml (equivalent to the mean value for normal male subjects + 2s.D.) and that of normal female to be 130 ng/ml. Serum samples were taken within one week before surgery or radiation therapy from 119 patients with pelvic masses and gynecological malignancies, which included 21 benign masses, 2 borderline epithelial ovarian tumors, 33 ovarian carcinomas and 63 patients with other gynecologicaltumors. In our series of 308 patients, 158 proved to have invasive pelvic neoplasms. Only 7 of the 39 patients (17.9%) with benign ovarian tumors had Mn-SODlevels exceeding 130 ng/ml. In the non-ovarian gynecological malignancies group, 9 out of 40 patients (22.5%) with uterine cervical cancer patients an 8 of 40 patients (20.0%) with endometrial cancer had Mn-SODlevels above 130 ng/ml. When a

Expression TABLE 1.

of Mn-Sup eroxi de Dismutase

Positivity rate for gynecologicaldiseases

serum Mn-SOD

in Carcinogenesis an


107 with


serum Mn-SOD value greater than 130 ng/ml was utilized as the diagnostic criterion, the positive rate was 59.7% for patients with epithelial ovarian carcinomas and 0% for patients with non-epithelial carcinomas. Among the 40 patients with epithelial ovarian carcinomas, the Mn-SOD assay showed a significant difference (p

Expression of Mn-superoxide dismutase in carcinogenesis.

Human liver manganese superoxide dismutase (Mn-SOD) was highly purified by a simple procedure and crystallized. A monoclonal antibody against Mn-SOD, ...
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