A three immunologically distinct subunits, A, B and

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1 Imm unochemical and Imm unohistochemical Studies on Three Aldolase Isozymes in Human Lung Cancer Takeo Ojika, MD,* Munehisa Imaizumi, MD,* Toshio Abe, MD,* and Kanehusa Kato, MDt The aldolase isozymes A, B, and C in tumor tissues (63) and sera (14) of patients with lung cancer were determined with an enzyme immunoassay system, compared with normal lung tissues (13), and the sera of normal healthy subjects (1). Tissue aldolase A and C concentrations were enhanced in 83% (52/63) and 51% (32/63) of patients with lung cancer, respectively, regardless of histologic type or stage (P <.1). But aldolase B was not elevated in tissue levels. In the sera of patients with lung cancer, there were no significant elevations of the isozymes. Immunohistochemically aldolase A and C stained more intensely in the cytoplasm of lung cancer cells than those in normal tissues. These results indicate lung cancer cells contain enhanced tissue levels of aldolase A and C. Cancer 67: LDOLASE MOLECULES are tetramers composed of A three immunologically distinct subunits, A, B and C, and they are present not only as homotetramers (A4, B4, and C4) but also as various hybrid forms. However, the three aldolases (A, B, and C) show a characteristic tissue distribution. Aldolase A is predominantly distributed in muscle tissue, and aldolase B, in liver and kidney. Aldolase C is mainly in nervous tissue.' In addition, aldolase A is predominant in the undifferentiated tissues such as fetal organs and neoplastic tissues. There are some reports on the enzymatic activities of aldolase or aldolase isozymes in lung cancer tissues233 but none on the concentrations of the three aldolase isozymes in tissues and the sera of patients with lung cancer. In this report, to evaluate the three aldolase isozymes as tumor makers for lung cancer, we determined their concentrations in both sera and tissues and their immunohistochemical localizations using a highly sensitive enzyme immunoassay system and the peroxidase-labeled antibody method. From the *Department of Thoracic Surgery, Nagoya University School of Medicine, Nagoya, and the?department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi, Japan. The authors thank Dr. Munehisa Takashi, Dr. Hajime Haimoto, and Dr. Takashi Koshikawa for valuable suggestions in performing this study and Naomi Kurobe for technical assistance. Address for reprints: Takeo Ojika, MD, Department of Thoracic Surgery, Nagoya University School of Medicine, 65 Tsurumai-Cho, Showa- Ku, Nagoya 466, Japan. Accepted for publication October 18, 199. Materials and Methods Antibodies to Aldolase Isozymes The antibody to aldolase A was raised in sheep, and those to aldolase B and C in rabbits by intracutaneous injection of aldolase A4, B4, or C4 purified from human muscle, liver, and brain, respectively. Antibodies in antisera were purified by the use of the antigen-coupled sepharose column. The specificity of the antibodies to aldolase A, B, and C has been previously de~cribed.~-~ Antialdolase A antibody Fab' fragment labeled with horseradish peroxidase, which was used for immunoblotting test, was prepared as described by Yoshitake et al.' Assay of Aldolase Zsozymes Concentrations of the aldolase isozymes in the serum samples and tissue extracts were determined with a sandwich enzyme immunoassay system as previously re- ~orted.~-~ This assay system consisted of monospecific antibodies to the human aldolase A (A4), B (B4), or C (C,), and showed no cross-reactivity with other forms of homotetramer. However, it cross-reacted with hybrid types of aldolase containing the relevant subunit. For instance, the aldolase A assay system is specific for aldolase A4 and did not react with B4 and C4 but cross-reacted about 5% with aldolase A3C, 3% with A2C2, 3% with AC3. Since the &, B4, and C4 forms of aldolase were used as standards for the relevant assays, the results were expressed as the 2153

2 2154 CANCER April Vol. 67 relevant homotetrametric aldolase equivalent nanograms per milliliter serum (ng/ml) or per milligram protein (ng/ mg protein). The immunoassay can detect amounts as small as 1 pg/assay tube of aldolase A, and 3 pg/assay tube of aldolase B and C. Tissue Samples Samples were taken from 63 primary lung cancer tissue specimens (26 squamous cell s, 22 adenos, six large cell s, eight small cell s, and one adenosquamous ), and 13 histologically normal lung tissues. The two small cell tissues were obtained at autopsy and the other samples, during surgical operations. All specimens were obtained from patients prior to chemotherapy and radiotherapy except four cases of small cell s which were resected after chemotherapy. All the samples were immediately frozen and kept at -8 C. For assay of aldolase concentrations, each tissue (approximately.5g) was homogenized at C with a glass homogenizer in 1 volumes (vol/wt) of 5 mmol/l tris-hydrochloric acid (HCl) buffer (ph 7.5) containing 5 mmol/l magnesium chloride (MgCI2). The homogenate was centrifuged at 4 C at 15 X g for 2 minutes, and the soluble fraction was used for immunoassay of aldolase subunits. Protein concentrations of the extracts were estimated by Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA), which utilizes a principle of protein dye binding.* Serum Samples Serum samples were taken from 14 patients with primary lung cancer (34 squamous cell s, 48 adenos, five large cell s, 12 small cell s, three adenosquamous s, one adenoid cystic, one alveolar cell ). All samples were obtained from patients before chemotherapy or radiotherapy, and at various clinical stages. There were 66 male and 38 female patients, ranging in age from 27 to 79 years (mean, 62 years). Serum samples were also taken from 1 healthy subjects (5 male and 5 female), ages 16 to 66 (mean, 4 years) for use as controls. All samples were kept frozen at -8 C until analysis. When measurements were taken, the samples were thawed and IO-pI aliquots of the serum samples were subjected in duplicate to the immunoassay of aldolase subunits. Immunohistochemicaf Methods The study subjects were eight lung cancer tissue specimens (two squamous cell s, three adenos, three small cell s), and three histologically normal lung tissue specimens. Each tissue sample was promptly fixed in periodate lysine-4% paraformal- dehyde (PLP) solution at 4 C for 6 hours, rinsed in.1 mol/l phosphate-buffered saline (PBS, ph 7.2) containing increased concentrations of sucrose, embedded in optimum cutting temperature (OCT) compound (Lab-Tek Products, Miles Laboratories Inc., Naperville, IL), and frozen quickly in dry ice ethanol. The indirect peroxidaselabeled antibody method was used for immunostaining as previously reported.' The sections were incubated for 2 hours with the horseradish peroxidase-labeled anti-sheep IgG antibodies (for aldolase A), or anti-rabbit IgG antibodies (for aldolase B and c). The sections were reacted with.25% diaminobenzidine solution containing 1 mmol/l hydrogen peroxide (H22), and counterstained with methylgreen. Electrophoresis and Immunoblot Study Sodium dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) was performed according to Laemmli's method" in 1% polyacrylamide slab gel, and proteins were visualized with Coomassie blue. Immunoblotting was performed by a method similar to that of Towbin et a/.'' (Figs. 1A and 1B). The 15-pl aliquots of soluble fractions of squamous cell and adeno, each containing 2, 32 pg/ml of aldolase A and 5.1, 6.4 mg/ml of protein, respectively, were boiled with Laemmli's sample buffer together with.3 pg of purified aldolase A4 as a standard, and subjected to SDS-PAGE. The purified anti-human aldolase A antibody Fab' fragments labeled with horseradish peroxidase (.1 pg/ml) were used. The peroxidase activity on the nitrocellulose FIGS. 1A AND 1B. Immunoblots of squamous cell and adeno of the lung with purified anti-aldolase A antibodies. Crude extracts, each containing 77. pg of protein of squamous cell ( I), 95.3 pg of protein of adeno(2), and.3 pg of purified aldolase A(3) were boiled with Laemmli's sample buffer, and subjected to SDS-PAGE, followed by immunoblots. (A) Polyacrylamide gel stained with Coomassie blue. (B) Immunoblots with anti-aldolase A. Lane 1 and 2 contained.3 pg and.48 pg of human aldolase A, respectively.

3 No. 8 ALDOLASE A, B, AND C IN LUNG CANCER - Ojika el al TABLE 1. Aldolase Concentrations in Tissues of Lung Cancer and Normal Lung Tissue concentrations (ng/mg protein) No. of Histologic type samples Aldolase A Aldolase B Aldolase C Squamous cell f 21 1*.77 f f 21.5 Adeno C f ? 24.1 Large cell 6 367k f f 6.91 Small cell 8 3 f t f 14.8 Adenosquamous Normal lung IT f f 4.32 * Mean f standard deviation. M. 1 F I U m H I Normal Pathological stage 1 lung sheet was visualized with 3,3'-diaminobenzidine and hydrogen peroxide. Statistical Analysis The data were expressed as mean k standard deviation. Statistical analysis of the results was made by paired and unpaired Welch's t test, and statistical significance was assumed when P was less than.1. Results Aldolase Isozymes in Tissues Tissue concentrations of aldolase isozymes were determined and expressed as ng/mg protein. As shown in Table 1, the aldolase A, and C concentrations in lung cancer were two or five times higher than those in normal lung tissues, regardless of histologic type (P <.1 ), but aldolase B concentrations in tissues were not elevated significantly compared with those in normal lung tissues. Aldolase A was elevated in 83% (52/63 cases) of the tissue specimens FIG. 3. Relationship between aldolase A concentrations in tissues of lung cancer and pathologic stages of lung cancers. Symbols have the same meaning as in Figures 2A and 2B. when the normal upper limit was set at mean + two standard deviations ( 17 ng/mg protein) (Figs. 2A and 2B). Aldolase C was elevated in 5 1 % (32/63 cases) of the tissues examined when the normal limit was set at 16 ng/mg protein (mean + 2SD). There was no relationship between aldolase A and C values and pathologic stages (Figs. 3 and 4). Aldolase Isozymes in Sera The serum concentrations of aldolase A, B, and C in healthy subjects of various ages are shown in Table 2. The aldolase A concentrations ranged from 134 to 724 ng/ml and the mean values from 325 to 375 ng/ml(358 -t 136 ng/ml), and aldolase B concentrations, from 4.2 to r - 3 =I v) i2 2. O.. 8 t o A o 1 O O A. A n O8 normal l""l SpvrrnDur Cllll ca Llrp cell rr I U BI H I Normal - Idmaqumour ca LdcnoId cfrlv el ; lung cs d hall cell C8 Pathological stage I. I "ts x Llreolar cell ca I A A FIGS. 2A AND 2B. Aldolase A concentrations in tissues of (A) histologically normal lung tissue and lung cancer, and in (B) sera of healthy subjects and patients with lung cancer. FIG. 4. Relationship between aldolase C concentrations in tissues of lung cancer and pathologic stages of lung cancers. Symbols have the same meaning as in Figures 2A and 2B.

4 ~~ 2156 CANCER April Vol. 67 TABLE 2. Serum Aldolase Concentrations in Healthy Subjects Serum concentrations (ng/ml) No. of Age (yr) samples Aldolase A Aldolase B Aldolase C k127* llof32 11 f k f f f f 46 1 f f f f f k k If f 4.1 Total 1 358? k49 1 k3.7 * Mean? standard deviation. ng/ml and mean values, 12 to 125 ng/ml (1 17 -t 49 ng/ ml). The aldolase C concentrations were 1 -t 3.7 ng/ml ranging from 3.4 to 19 ng/ml, and the mean values, from 9.2 to 12 ng/ml. They showed no statistical significance among age-related and sex-related differences. The upper limits of the normal serum aldolase A, B, and C, defined as the mean plus two standard deviations, were 63 ng/ ml, 215 ng/ml, and 17.4 ng/ml, respectively. Any value greater than that was considered as positive. As shown in Table 3, the serum aldolase A, B, and C concentrations in patients with lung cancer were not significantly higher than those in normal healthy subjects. The positive rates of aldolase A, B, and C were 12%, 15%, and 3% respectively. The positive rates of aldolase B being higher than that of aldolase A was not related to the tissue concentrations of aldolase B, but was due perhaps to other tissues except lung cancer, possibly the liver. Immunohistochemical Localization of A ldolase A, B, and C In histologically normal tissues, aldolase A and C were localized in most bronchiolar epithelial cells, smooth muscles, type I, I1 alveolar epithelial cells, and nerve endings (Figs. 5A and 5C). However, aldolase B stained almost negatively (Fig. 5B). In the tumor cells of cancer tissues, aldolase A and C stained stronger in their cytoplasm than in normal lung tissues (Figs. 5D and 5F), and aldolase B stained faintly (Fig. 5E). This is consisted with the tissue concentrations of aldolase isozymes. Aldolase A and C were positively stained in the cytoplasm of most cancer cells; adeno, squamous cell, and small cell were also positive. But, aldolase B stained faintly in the cytoplasm of most cancer cells, irrespective of its histologic type. Discussion It is generally accepted that anaerobic glycolysis is enhanced in malignant tumor tissues" and that the activities of glycolytic enzymes in tumor cells or tissues are en- hanced, accompanied by increase of anaerobic glycolysis. Aldolase is a glycolytic and tetrametric enzyme, composed of immunologically distinct three subunits, A, B, and C, whose immunological specificity and properties were reported by Rutter et all3 In the current study, we found that aldolase isozymes A and C concentrations are also enhanced in all histologic types of lung cancer tissues more than in normal lung tissues, using an enzyme immunoassay system. The relations of aldolase isozymes with many kinds of cancerous tissues have been rep~rted,'~-'~ but regarding lung cancer, few reports are available on measurements of concentrations or enzyme activities.*s3 However, there was no report about the measurement of the concentrations of all three kinds of aldolase isozymes. In this study we made polyclonal antibodies specific to the three aldolase subunits, A, B and C, and measured their concentrations in tissues and the sera of patients with lung cancer. According to the literature, the enhancement of aldolase A concentration in tumor tissues was reported by Shapira et al.,17 and then by Endo et a1.i' and by Hatzfeld et al.,i9 using hepatocellular. It is said that in normal liver cells, aldolase B is dominant but in fetal liver or hepatoma, aldolase A and C increase." For lung cancer, Kellen et al. measured the activities of aldolase A, B, and C isozymes and concluded that total aldolase values were enhanced but the aldolase isozyme pattern in lung cancer could not be sub~tantiated.~ Greengard and Herzfeld reported that some enzymes in various metabolic pathways of human pulmonary tumors closely resemble those of the normal fetal lungs, but for aldolase it was not evident." In tissues of lung cancer, aldolase A and C (especially A) levels were significantly higher than those in normal lung tissues, as well as cases with hepatoma. But there was no correlation between the elevation of aldolase A, TABLE 3. Serum Aldolase Concentrations in Patients With Lung Cancer and in Healthy Subjects Histologic type Squamous cell Adeno Large cell Small cell Adenosquamous Adenoid cystic Alveolar cell Healthy subjects Serum concentrations (ng/ml) No. of samples Aldolase A Aldolase B Aldolase C I I * Mean f standard deviation. 461? 665* 284 +_ f f f k f f f I f 3.9 I1 & f3.6 1 f f f 3.7

5 No. 8 ALDOLASE A, B, AND C IN LUNG CANCER Ojika et al FIGS. 5A-5F. lmmunohistochemical stainings of aldolase A, B, and C in a normal lung tissue and lung cancer (X33). (A) In a normal lung tissue, aldolase A is localized in type I, 11 alveolar cells, smooth muscles, and most bronchiolar epithelial cells. (B) In a normal lung tissue, aldolase B stained almost negatively. (C) In a normal lung tissue, aldolase C also is localized in type I, I1 alveolar cells, smooth muscles, and most bronchiolar epithelial cells. (D) In a case of squamous cell, aldolase A is localized in the cytoplasm of most cancer cells. (E) In a case of squamous cell, aldolase B stained in the cytoplasm of cancer cells faintly. (F) In a case of squamous cell, aldolase C is localized in the cytoplasm of most cancer cells. In cancer cells, aldolase A and C stained stronger in their cytoplasm than in normal lung tissues and aldolase B stained faintly. The results were the same as other histologic types of lung cancer, adeno, or small cell.

6 2158 CANCER April Vol. 67 C, and pathologic stage or histologic type. These two isozymes were elevated in all histologic types of lung cancer in the tissue specimens, regardless of stage, or without distinction of small or non-small cell lung cancer. Aldolase C is rich in nervous tissues, but the increase of tissue aldolase C in small cell lung cancer was similar in degree to that of non-small cell lung cancer. The tissue concentration of aldolase A was much higher than that of aldolase C. Thus the elevation of aldolase C concentration in tissues may have been due to the enhancement of hybrid types of aldolase C and A. Aldolase isozymes in hepatic cells were all localized in the cytoplasm with aldolase B present at the highest level. Aldolase A and C increased in proportion to carcinogenesis as was also noticed in fetal liver.2 Asaka et al. reported that aldolase A was elevated in sera of 86% of patients with many kinds of malignant t~mors.~~,~~ Dalluge and Ziegenbei~~~~ or Schulz and reported that the serum aldolase values were increased in patients with lung cancer. However, with the current study, the serum concentrations of aldolase isozymes were not elevated in any histologic type, or at any stage, and there were no significant differences between cancer patients and normal healthy subjects. By immunohistochemical study, we demonstrated aldolase A and C to stain positively in the cytoplasm of lung cancer cells regardless of histologic type and aldolase B, to stain faintly. This result indicates that lung cancer cells contain increased levels of aldolase A and C, irrespective of its histologic type. To summarize, in tumor tissues of lung cancer where there is an acceleration of anaerobic glycolysis, both aldolase A and C isozyme levels increase, but not aldolase B. Aldolase A concentrations in tissues were enhanced in 83% of lung cancer specimens and aldolase C concentrations, in 5 1%. But in the sera of patients with lung cancer, they were not elevated and not useful as serum tumor makers. The changes in aldolase isozyme patterns in the tissues of lung cancer closely resemble those in the fetal types of aldolase patterns. This phenomenon is thought to be one of the specific metabolic activities of cancer cells, which is suitable for the anaerobic glycolysis. REFERENCES I. Eagles PA, Iqbal M. A comparative study of aldolase from human muscle and liver. Eiochem J 1973; 133: Greengard, Head JF, Goldberg SL, Kirschner PA. Enzyme pathology and the histologic categorization of human lung tumors. Cancer 1982; Kellen JA, Chan A, Caplan B, Malkin A. 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