Expression of Aldolase C Isozyme In Renal Cell Carcinoma

Expression of Aldolase C Isozyme In Renal Cell Carcinoma MUNEHISA TAKASHI, M.D., HAJIME HAIMOTO, M.D., TAKASHI KOSHIKAWA, M.D., AND KANEFUSA KATO, M.D.

FRUCTOSE-1, 6-bisphosphate aldolase (EC 4.1.2.13), a glycolytic enzyme, has a tetrameric form15 with three immunologically distinct subunits: A, B, and C.' 8 The A subunit (aldolase A), a fetal form of aldolase, is present in large amounts in muscle and in much smaller amounts in the kidney. The B subunit (aldolase B) is found predominantly in liver and kidney.15 On the other hand, the C subunit (aldolase C) is localized mainly in the brain and is hybridized with aldolase A; thus, aldolases A and C form five members that could be separated by electrophoresis: A4, A3C, A2C2, AC3, and C4.15 Immunohistochemical studies showed that aldolase C is localized in cerebellar Purkinje cells, astrocytes, some large neurons in the cerebral cortex,13'26 and gliomas.' 3 Sato and associates suggested that change in aldolase isozyme patterns obtained by electrophoresis would be useful in diagnosis of brain tumors. 20 Recently, we developed a highly sensitive immunoassay of human aldolase C and reported that peripheral tissues other than brain, such as liver, testis, adrenal gland, and jejunum, contained considerable amounts of aldolase C but that only a small amount of this isozyme exists in the kidney.7 Previous studies, using electrophoretic,15 immunohistochemical,19 and immunochemical methods, 1 " showed that kidney contained mainly aldolases A and B.

Departments of Urology and Pathology, Nagoya University School of Medicine, Nagoya; Laboratory of Pathology, Aichi Cancer Center Research Institute, Nagoya; and Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi, Japan

Consequently, aldolase C in the kidney may be a fetal rather than adult form. Fetal forms of isozymes occur during carcinogenesis, and so these oncofetal isozymes could be useful as tumor biomarkers. We showed that yenolase, an isozyme of another glycolytic enzyme, is nearly absent in normal proximal tubules but is expressed at high levels during renal carcinogenesis.6 Our present study immunochemically and immunohistochemically elucidates the expression of aldolase C in renal cell carcinoma. Materials and Methods Antibodies

Received April 20, 1989; received revised manuscript and accepted for publication November 1, 1989. Address reprint requests to Dr. Takashi: Department of Urology, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan.

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Antibodies to human aldolase C4 were raised in New Zealand white rabbits by injecting the purified aldolase C4 with Freund's complete adjuvant as described elsewhere. 710 Antibodies monospecific to aldolase C were further purified by immunoaffinity column chromatography using aldolase C4-coupled Sepharose 4B® (Pharmacia Fine Chemicals, Uppsala, Sweden) as described previously.710 The specificity of the purified antibody IgG thus obtained was also reported.7 For secondary antibodies in immunohistochemistry, horseradish peroxidase (HRP)-labeled goat IgG Fab' fragments against rabbit IgG were prepared.5 Tissue and Serum Samples Tumor tissues were obtained at surgical operation from 43 patients with renal cell carcinoma, including one of sarcomatoid variant. Histologically normal renal tissues (n = 8) adjacent to the tumor tissues were also prepared. For immunohistochemistry, the tissues from the 43 patients were fixed in periodate-lysine-4% (w/v) paraformaldehyde (PLP)16 for six hours, washed in phosphatebuffered saline (PBS, pH 7.2) containing increasing concentrations of sucrose, embedded in OCT® compound (Tissue-Tek, Naperville, IL), and frozen quickly in dry ice and ethanol. For enzyme immunoassay, the tumor tissues (n = 26) and tissues of normal cortex (n = 8) and

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The authors localized aldolase C in renal tubules and renal cell carcinoma by immunohistochemical study and quantitative analysis by an enzyme immunoassay. Aldolase C was localized in epithelial cells of loops of Henle and collecting ducts and in those of Bowman's capsules. In renal cell carcinoma, aldolase C was immunohistochemically demonstrated in 95% (41 of 43) of cases, including one sarcomatoid variant. The tissue concentrations of aldolase C in the renal cortex (n = 8) were 12.7 ± 6.2 Mg/g protein (mean ± standard deviation), and those of the medulla (n = 8) were 20.3 ± 6.9 Mg/g protein. On the other hand, the concentrations in renal cell carcinoma (n = 26) were 93.5 ± 95.9 jtg/g protein: about seven times higher than that in renal cortex (P < 0.001). These findings indicate that aldolase C was first expressed in renal cell carcinoma, which is derived from proximal renal tubules, because proximal renal tubules had aldolase B but not aldolase C. (Key words: Aldolase; Isozyme; Renal cell carcinoma; Peroxidase-labeled antibody method; Enzyme immunoassay) Am J Clin Pathol 1990;93:631-636

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medulla (n = 8) were promptly frozen and kept at - 80 °C until analysis. Thus, in 26 of the 43 patients, tissue samples were divided into two parts: one for immunohistochemistry and the other for quantitative analysis. Serum samples of patients with renal cell carcinoma (n = 20) were obtained before the operation by venipuncture. Serum samples were kept at — 80 °C or - 20 °C until analysis. Imm unoh istochemistry

Assay of Aldolase C Concentrations in the Tissue Extracts and Sera Frozen tissues were homogenized in a glass homogenizer at 0 °C with 10 volume (v/w) of 50 mmol/L sodium phosphate buffer (pH 7.0) containing 5 mmol/L magnesium sulfate. The homogenate was centrifuged at 4 °C at 20,000 X g for 20 minutes, and the soluble fraction was used for analysis. Concentrations of aldolase C in the extracts and sera were determined by the sandwich enzyme immunoassay system specific to human aldolase C.7 In brief, the extract was incubated with a piece of polystyrene ball with immobilized anti-aldolase C antibodies, and then the ball was incubated with the same antibodies labeled with /3-D-galactosidase from Escherichia coli1J0A2 The galactosidase activity bound to the ball was assayed with 4-methylumbelliferyl-|8-D-galactoside as a substrate. The purified human aldolase C4 was used as a standard, and the results were expressed as human aldolase-C4 equivalent microgram per gram soluble protein for tissues and microgram per liter for sera. Protein concentrations of the extracts were determined with the Bio-Rad® Protein Assay Kit (Bio-Rad Laboratories, Richmond, CA), which uses the principle of protein-dye binding.3

Electrophoresis and Immunoblot The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) was performed according to Laemmli's method14 in a 10% polyacrylamide slab gel, and proteins were visualized with Coomassie blue. Immunoblots were performed by a method similar to that of Towbin and associates.27 In brief, proteins were electrophoretically transferred from polyacrylamide gel to nitrocellulose sheet. The nitrocellulose sheet was incubated in buffers as described previously8 for 60 minutes at room temperature, and the purified anti-aldolase C antibodies (0.15 Mg/mL) were added for an additional 60 minutes incubation. After being washed, the sheet was incubated with HRP-labeled goat antirabbit IgG for 30 minutes, and then the peroxidase activity on the sheet was visualized with 3,3'-diaminobenzidine and hydrogen peroxide as described previously.8 Statistical Analysis Quantitative data were expressed as mean ± standard deviation (SD). The results were compared by Wilcoxon's rank-sum test. Cell types of tumors were classified into three group: clear, granular, and sarcomatoid.2 Two or three types were sometimes combined. When a predominant cell type of tumor constituted 80% or more of a section, the tumor was classified according to its main cell type. When the secondary or third components constituted more than 20%, the tumor was arbitrarily grouped as mixed type. Results lmmunohistochemical Localization of Aldolase C in Normal Renal Tubules and Renal Cell Carcinomas In the cortex, aldolase C was localized in the cytoplasm of epithelial cells of Bowman's capsules but not in that of convoluted and straight portions of proximal and distal tubules (Fig. I A). No staining was found in epithelial cells of cortical collecting ducts and connecting tubules. In the medulla, aldolase C was demonstrated in the cytoplasm of epithelial cells of outer medullary and inner medullary collecting ducts and thin limbs and a few thick limbs of loops of Henle (Fig. \B). Of 43 renal cell carcinomas studied, 41 (95%) were stained positively for aldolase C (Figs. 2A-C). The antigen was localized in the cytoplasm and occasionally in the nucleus of tumor cells. It was also found in the plasma membrane of clear cell type tumor cells. In one case of sarcomatoid variant, a few fusiform or pleomorphic tumor cells showed positive staining for aldolase C (Fig. 2C). Control sections of the tumor tissues treated with the antibody absorbed with aldolase C4 were uniformly negative (Fig. 2D).


The indirect peroxidase-labeled antibody method was used for the immunostaining as described previously.517,24 In brief, 6-jim-thick cryostat sections were placed on albumin-coated slides and dried at room temperature. The sections were treated with 5 mmol/L periodic acid solution for 15 minutes to inactivate endogenous peroxidase. They were washed in PBS and then reacted with monospecific antihuman aldolase C antibodies IgG (1.6 Mg/mL) for 120 minutes at room temperature. For control sections, the antibodies absorbed with the purified human aldolase C antigen were substituted for the primary antibodies. After being washed in PBS, the sections were incubated with the HRP-labeled secondary antibodies for 60 minutes at room temperature. After another wash in PBS, they were reacted with 0.025% (w/v) 3,3'-diaminobenzidine solution containing 10 mmol/L hydrogen peroxide and 10 mmol/ L sodium azide and then counterstained with methyl green. Cryostat sections stained by hematoxylin and eosin were also histologically examined.

A.J.C.P.-May 1990

ALDOLASE C IN RENAL CELL CARCINOMA

Vol. 93 • No. 5

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%«, •**"«%£ FIG. 1. Localization of aldolase C in normal renal tissues. A (upper, left). In the cortex, aldolase C is localized in the cytoplasm of epithelial cells of Bowman's capsules (arrowheads), whereas it is scarce in epithelial cells of proximal and distal tubules. B (upper, right). In the medulla, aldolase C is localized in epithelial cells of collecting ducts (c) and thin limbs of loops of Henle (/). Indirect immunoperoxidase method, methyl green counterstain (XI80). FIG. 2. Localization of aldolase C in three types of renal cell carcinoma. Aldolase C is found in the cytoplasm, and occasionally in the nucleus of tumor cells in clear cell (A. center, left) and granular cell (B. center, right) types. Aldolase C also is stained in the plasma membrane of the tumor cells of clear cell type (A). In one case of sarcomatoid variant (C. lower, left), a few fusiform tumor cells show positive staining for aldolase C. Control section of the tumor is uniformly negative (D. lower, right). Indirect immunoperoxidase method, methyl green counterstain (XI80).

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Table J. Frequency of Positive Tumor Cells for Aldolase C in Renal Cell Carcinoma Histologic Type Clear cell type Granular cell type Mixed type Sarcomatoid type Total

-

+

++

+++

Positivity

1

5 4 2 1 12

20 2 4

26/26(100%) 8/9 (89%) 6/6 (100%) 1/2 (50%) 41/43(95%)

1 2 I 2

3

26

— = no positive cells; + = fewer than 10% positive cells; + + = from 10 to 50% positive cells; -f-f+ = more than 50% positive cells.

Tissue Concentrations of Aldolase C in Normal Kidneys and Renal Cell Carcinomas Table 2 shows the concentrations of aldolase C in soluble fractions from normal cortex and medulla and in those ofrenal cell carcinomas. A small amount of aldolase C was found in both cortex and medulla. In renal cell carcinoma, concentrations of aldolase C amounted to 93.5 ± 95.8 Mg/g protein (mean ± SD), ranging from 5.7 to 399 jtg/g protein. The average value was 7.4 and 4.6 times higher than in normal cortex and medulla, respectively (P < 0.001). Of the 26 cases ofrenal cell carcinoma, 14 (54%) had extremely high concentrations of aldolase C (more than 50 fig/g protein). Clear cell type tumors had significantly higher concentrations of aldolase C than granular cell type tumors (P < 0.01, Table 3). No relationship was found between the concentration of aldolase C and tumor grade or stage.

Table 2. Concentrations of Aldolase C in the Tissues of Normal Kidney and Renal Cell Carcinoma

Normal kidney Cortex Medullar Renal cell carcinoma

No. of Samples

Mean ± SD