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The a Subunit of GTP-Binding Protein G0 in Neuroblastoma: Correlation with Advanced Disease Stage

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The a Subunit of GTP-Binding Protein G0 in Neuroblastoma: Correlation with Advanced Disease Stage [CANCER RESEARCH 54, 2334-2336, May 1, 19941 The a Subunit of GTP-binding Protein G0 in Neuroblastoma: Correlation with Advanced Disease Stage Yukio Ishiguro,' Kanefusa Kato, Tomiko Asano, Hiroshi Akatsuka, Hiroyuki Iwata, Fujio Ito, and Takahiro Ito Department ofSurgery, Branch Hospital ofNagoya University School ofMedicine, Daiko-minam4 Higashi-ku, Nagoya 461 [V. L, H. A., H. L, F. I., T. I.J, and Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kamiya-cyo, Kasugai [K K, T. A.J, Japan ABSTRACT chemotherapy with cyclophosphamide and vincristine for 3 to 12 months. Stage III patients without recurrences (n = 5) had postoperative chemotherapy Tissue levels of the a subunit of G protein G0 (G0 a) were measured in for 2 years. Nine of 24 patients with distant metastases or recurrences had bone solid tumors from pediatric patients by immunoassay.G0 a concentra marrow transplants after complete clinical remissions. Survival time was tions were determined in the supernatant obtained by centrifugatlon of measured from the start of treatment. Diagnoses were confirmed histologically. tissue homogenates prepared in the presence (total G0 a) or absence of 2% Preparation of Tissue Extracts. Tissues were homogenized at 0°Cin 10 sodium cholate (soluble G0 a). Mean G0 a concentrations (total G0 a and volumes of 10 mM Tris-HCI (pH 7.5) containing 1 mM EDTA, using a soluble G0 a) in neuroblastomas (7 ganglioneuromas,13 ganglioneuro Polytron-type homogenizer. One-half of each homogenate was centrifuged at blastomas, and 50 neuroblastomas) were over 50-fold higher than those in 4°Cat 125,000 X g for 40 mm, and the supernatant was saved for G0 a other solidtumors from pediatric patients (n 13).Mean total G0a and analysis of the soluble fraction (soluble G0 a). Ten % sodium cholate was soluble G0 a concentrations were 207.0 ±166.0(SD)ng/mg of cholate added to the other one-half of the homogenate to a final concentration of 2%, extractable protein and 58.6 ±47.0 ng/mg ofsoluble protein, respectively, and the tissue was further homogenized. This homogenate was sonicated for 1 in the neuroblastoma group (n 70). Total G0 a concentration decreased mm and then centrifuged as described above. The supernatant fraction was with disease stage and was strongly correlated with outcome in patients then used for G0a analysis of the cholate-extractable fraction (total G0 a) (9). with neuroblastoma. The mean total G0 a concentration in tumors from G0a andAntibodyPreparations.G0awaspurifiedfrombovinebrain, younger patients (<1 year old) was 297.0 ±137.0 ng/mg of cholate human brain, and human neuroblastoma by the method of Sternweis and extractable protein, significantly higher than in twuors from older pa Robishaw (4) with modifications. Antibodies were raised in rabbits with dents (140.0 ±155.0ng/mg cholate-extractable protein, P < 0.0001). These bovine G0 a as an immunogen and purified to be monospecific to G0 a as results suggest that total C0 a levels in neuroblastoma may indicate the described previously (9). The purified antibodies cross-reacted with human G0 degree of malignancy. a, but not with G. a or fry subunits (9). Enzyme Immunoassay of G@a. G@,a was assayed by a sandwich-type INTRODUCTION immunoassay for bovine G@,aas described previously (9). This assay system consists of a solid phase (jolystyrene ball) with immobilized purified antibod G0 is a member of a family of heterotrimeric GTP-binding proteins ies to bovine G0 a and the same antibodies labeled with @-D-galactosidase (G proteins) which function as signal transducers (1, 2). Recently, from Escherichia coli. The reactivity of human brain and neuroblastoma 0,, a many investigators have reported that G0 interacts with several recep in the immunoassay was identical and was approximately 80% that of bovine tors and regulates Ca2@ and K@ channels as well as phospholipase C Go a (7). Purified human G0 a was used as the standard for immunoassay of @ (1, 2). G@was initially isolated from bovine brain (3, 4); G0 a2 G0 a, and results were expressed as ng of human G0 a per mg of soluble protein, per mg of cholate-extractable protein, or per g of wet tissue. mainly localized to nervous tissues and neuroendocrine cells (5). We Proteins were determined by the method of Schaffer and Weissmann (11) have reported that G0 a is present in tumors derived from neuroen with bovine serum albumin as a standard. docrine cells (6, 7) and that serum G0 a is a useful marker for Statistics. Mann-Whitney-Wilcoxon (2-sample) rank sum test, a nonpara neuroblastoma (8), a tumor in the amine precursor uptake and decar metric method, was used for the analysis of the separation between two groups. boxylation group. In the bovine brain, more than 98% of 0,, a is The event-time distributions were estimated by the product limit method of membrane bound (9), while in neuroblastomas a significant amount of Kaplan and Meier. Differences between event-time distributions were tested G0 a is found in the soluble fraction (6). In this study, we measured for statistical significance by using the generalized Wilcoxon t test. G0 a concentrations in the soluble and cholate-extractable fractions of neuroblastomas and evaluated the relationship between G0 a tissue RESULTS levels and disease stage and outcome in patients with neuroblastoma. G0 a Concentrations in Pediatric SolidTumors. Table 1 shows the mean G0 a concentrations in solid tumors from pediatric patients. MATERIALS AND METHODS Mean G0 a concentrations (total G0 a and soluble G@,a) in the Tissues. Tumor tissues obtained at operation from 83 patients included 50 neuroblastoma group (ganglioneuroma, ganglioneuroblastoma, and neuroblastomas, 13 ganglioneuroblastomas, 7 ganglioneuromas, 4 Wilms' neuroblastoma) were over 50-fold higher than those in other solid tumors, 4 rhabdomyosarcomas, 1 hepatoblastoma, 1 dysgerminoma, and 3 tumors from pediatric patients. The mean total G0 a and soluble G0 a adrenal cortical tumors. All samples were rapidly frozen and kept at —80°Cconcentrations were 207.0 ±166.0 (SD) ng/mg of cholate-extractable until analysis. Neuroblastoma patients were classified into five groups (I, 17; protein and 58.6 ±47.0 ng/mg of soluble protein, respectively, in the II, 11; III, 17; IV, 12; IVs, 6) according to the pretreatment staging procedures neuroblastoma group (n = 70). The mean total G0 a concentration in of Evans CtaL (10). Nine of the 17 stage III neuroblastoma patients and 9 of the neuroblastoma group was approximately one-tenth the value re the 12 stage IV neuroblastoma patients received intensive preoperative che ported for human and bovine cerebral cortex (9, 12). On the other motherapy with cyclophosphamide, vincristine, doxorubicin, and cisplatin; tumor tissues from these patients were obtained within 6 months after begin hand, the mean total and soluble G0 a concentrations in other solid ning of chemotherapy. After surgery, stage I, H, and IVs patients had adjuvant tumors from pediatric patients were 3.34 ±3.20 ng/mg cholate extractable protein (P < 0.0001) and 0.93 ±0.54 ng/mg of soluble Received 7/19/93; accepted 3/2/94. protein (P < 0.0001), respectively. The costs of publication of this article were defrayed in part by the payment of page G0 a Concentrations and Clinical Stage in Neuroblastomas. charges. This article must therefore be hereby marked advertisement in accordance with Table 2 shows the mean total G0 a and soluble G0 a concentrations 18 U.S.C. Section 1734 solely to indicate this fact. I To whom requests for reprints should be addressed. in different disease stages of neuroblastoma. More extensive disease 2The abbreviation used is: G0 a, the a subunit of GTP-binding protein G0. was associated with a decreased total G0 a concentration. The mean 2334 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1994 American Association for Cancer Research. G0 a IN NEUROBLASTOMA value in advanced (stage III and IV) neuroblastomas was 118.0 ± 1.0 136.0 ng/mg of cholate-extractable protein and was markedly lower than that in localized (stages I and II) neuroblastomas (296.0 ±131.0 .0 0.8 11@ ng/mg of cholate-extractable protein, P < 0.0001). However, the mean total G@a concentration, 346.0 ±185.0 ng/mg of cholate-extractable protein, in stage P/s neuroblastomas (in which metastases were lim .@ 0.6 ited to liver, skin, and bone marrow), was significantly higher than that of stage N neuroblastomas (62.1 ±61.8 ng/mg of cholate Cl) 0.4 extractable protein, P < 0.005), and was similar to that of the most localized disease, stage I neuroblastoma (300.0 ±117.0 ng/mg of :@ 0.2 cholate-extractable protein). Soluble G0 a concentration (ng/mg sol In uble protein) also decreased with disease stages of neuroblastoma, but U.', the changes were not so remarkable compared with those of total G0 0 5@ 1000 1500 2000 2500 a concentration. Therefore, the mean percentage of G0 a in the SurvivalTime(days) soluble fraction, 37.3 ±21.2%, in advanced neuroblastomas was Fig. 1. Correlation between total tissue G0 a concentration J: >200 ng/mg protein; •: significantly higher than in stage I (mean, 16.7%) or stage II (mean, <200 ng/g wet tissue) and overall survival rate in children with neuroblastoma as 19.4%) tumors (P < 0.0001). calculated by the Kaplan-Meier method. Since almost one-half of advanced neuroblastoma patients (stage III and IV) received preoperative chemotherapy, we examined the effects G0 a concentration of >200 was associated with a statistically better of chemotherapy on G0 a concentrations in neuroblastomas.
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