(CANCER RESEARCH 43. 5390-5394, November 1983] Occurrence of the Tumor-specific, Calcium-binding Protein, Oncomodulin, in Virally Transformed Normal Rat Kidney Cells1 J. P. Durkin,2 L. M. Brewer, and J. P. MacManus Cell Physiology Group, Division ot Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6, Canada ABSTRACT of cells isolated from the kidney of a normal Osborne Mendel rat. The transformed ASV-NRK line was derived from infection of NRK cells with Oncomodulin, an apparently tumor-specific calcium-binding the B77 strain of ASV. Both cell lines were kindly supplied by Dr. P. K. protein, has been detected in many chemically induced rat hep- Vogt (University of Southern California, School of Medicine, Los Angeles, atomas. It is now possible to detect, by radioimmunoassay and Calif.). Spontaneously transformed lines (Spon 1-NRK and Spon 3-NRK) immunofluorescence, the presence of Oncomodulin in normal rat were isolated from NRK cultures which had been passaged repeatedly at high density. Stock cultures of all cells were maintained at 36° in kidney cells virally transformed by avian sarcoma virus. By medium containing 85% (v/v) Dulbecco's modified Eagle's medium contrast, it was not detected in uninfected, nonneoplastic normal (Grand Island Biological Co., Grand Island, N. Y.), 15% (v/v) bovine calf rat kidney cells. The protein was isolated and purified by a novel serum (Colorado Serum Co., Denver, Colo.), and the antibiotic gentami- high-performance liquid chromatography procedure and shown cin. ASV-NRK cells were highly tumorigenic: 107 of them produced large to be identical to that isolated previously from rat hepatoma. The (2 to 3 cm) fibrosarcomas within 5 weeks after s.c. injection into athymic cellular levels of oncomodulin approached the levels of calmo- BALB/c nude mice. Spon 1-NRK and Spon 3-NRK cells also formed dulin in avian sarcoma virus-transformed normal rat kidney cells, tumors in athymic mice, but the parent NRK line had failed to do so by suggesting that the total calcium-binding activity of the cell may 6 months after injection. play a role in expression of the transformed phenotype. Oncomodulin. Rat oncomodulin was purified to homogeneity from Morris hepatoma 5123tc by conventional means (10). The purity of 1- INTRODUCTION mg aliquots was shown to be greater than 99% by reverse-phase HPLC with monitoring at 220 nm. After elution of the major peak, only trace A unique calcium-binding protein found in the extracts of many amounts of 220-nm-absorbing material could be washed from the column chemically induced rat hepatomas, but not normal liver, has been support. Anti-Rat Hepatoma Oncomodulin IgG. The crude immunoglobuhn purified to homogeneity and shown to be an acidic protein with a molecular weight of 11,500, possessing 2 calcium-binding sites fraction obtained from an immunized goat has been described (11 ). Forty per molecule (9-11,13,15). The cellular function of this calcium- ml of IgG (20 mg/ml) in 20 mw KH2P04, pH 8.0, were further purified by passage through a column (1.5 x 30 cm) of DEAE-Affigel Blue equili binding protein remains unknown, but it is capable of modulating brated in 20 HIM KH2P04 buffer. The material passing through the column the activity of calmodulin-dependent phosphodiesterase in vitro was collected and dialyzed against 125 ITIMborate:75 mM NaCI, pH 8.4. (12) and of mimicking the effects of calmodulin by releasing T51B This IgG was further purified by antigen-affinity chromatography on an rat liver cells from a d-S transition block induced by low-calcium oncomodulin:Sepharose column (0.9 x 10 cm), prepared according to medium (1). The development of antisera to this protein purified the method of Klee and Krinks (7) and containing 0.8 mg of oncomodulin from rat hepatoma has enabled immunological methods to es per ml of gel. Antioncomodulin was desorbed by reverse flow with 100 tablish the presence of immunoreactive material in tumors arising m« glycine, pH 2.7. The eluted IgG was neutralized, desalted on a from a variety of chemical carcinogen-treated tissues and animal Sephadex G-25 column, and dialyzed against 100 mw NaCI:10 mw imidazole, pH 7.4. For immunocytology, rabbit anti-rat hepatoma onco species (13, 15). Such material has not been detected in normal modulin was produced and purified to the DEAE-Affigel Blue step as tissue or in nonneoplastic rodent and human cell lines by specific described above. radioimmunoassay (13, 15). Because of its similarity to calmo Radioimmunoassay. The oncomodulin radioimmunoassay using an dulin and its apparent tumor specificity, this calmodulin-binding tigen-purified goat anti-rat hepatoma oncomodulin IgG has been de protein has been named oncomodulin (1, 15). scribed (13, 15). The radioimmunoassay using either goat or rabbit IgG We now report the detection, by immunofluorescence and was not affected by the presence of a 2000-fold excess of other calcium- radioimmunoassay, of high levels of oncomodulin in NRK3 cells binding proteins such as calmodulin, troponin C, or parvalbumin (13,15). transformed by ASV. This protein has been isolated, purified by The preparation of extracts of cells and tumors for oncomodulin assay has been described (13-15). a novel and rapid method using HPLC, and shown to be identical Immunocytofluorescence. NRK and ASV-NRK cells were plated at a to that isolated previously from the chemically induced rat hep density of 3 x 103/sq cm on tissue culture chamber slides (Lab-Tek; atoma, 5123tc. Miles Laboratories, Inc., Naperville, III.) in stock medium and incubated at 36°for 48 hr. Logarithmically growing cells were rinsed in PBS, fixed MATERIALS AND METHODS in 3.7% (v/v) formaldehyde: 10% acetic acid in PBS for 15 min at room temperature, rinsed 3 times in distilled water, immersed in acetone at Cell Line. Nonneoplastic NRK fibroblast-like cells were descendants -20° for 10 min, and air dried. Apparent oncomodulin-specific immuno fluorescence was detected by the indirect antibody approach. Slides 1National Research Council of Canada No. 21315. supported in part by Grant were preincubated in 0.5% Triton X-100 in PBS for 20 min, flooded with X-ROI-CA-31898 of the National Cancer Institute, NIH. Affigel Blue-purified rabbit anti-rat hepatoma oncomodulin IgG (100 ^g 2 To whom requests for reprints should be addressed. 3 The abbreviations used are: NRK, normal rat kidney; ASV, avian sarcoma IgG per ml), and incubated for 90 min at room temperature in a humidified virus; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered atmosphere. This concentration of antibody was determined by serial saline. dilution to yield maximum specific staining while maintaining an accept Received March 1, 1983; accepted July 29, 1983. able nonspecific background fluorescence. The slides were washed in 5390 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1983 American Association for Cancer Research. Oncomodulin in Virally Transformed Cells 8 12 16 C.S's.-\ 1 0.3[ Il D^>-,.-*"À d/1/1y11\li Cz 0.2iVIjii KX)m'a1 „*540 0 ' 50 250S¡^¿. sh\*o ;3030 20m¿r \4Jt\ O5 ^§20 IOW x 'B77 O-1i50SoC"fi_-K s1 ~ ikâ„¢!™™Pf\ v*i\8 PTIO 5 '/V_"^"-•'•""' |4 20 3 j l"^^^") 1 1 —"M in iJ) ÃŒ0_12 19 |4 '0iU •— 4 8s. 20MINUTES1VI2¿-I-(V.t16 2)500ioo§ )MIINU1624MINUTES0.280.1¡iiJio(~ 18 20 22 4 8 12 162MINUTES,.',-'•'.--'"'y|20 28 32^—_^_136450 Chart 1. Purification of immunoreactive material from ASV-NRK solid tumors by HPLC methods. A, ion-exchange HPLC on AX300 at 1.0 ml/min. The hatched area in A indicates the immunoreactive fractions pooled and subjected to B, size exclusion HPLC on a TSK-125 column at 0.5 ml/min. The hatched area in B indicates immunoreactive fractions pooled and subjected to reverse-phase HPLC. C, calibration of TSK-125 column. Standards used were: 1, bovine serum albumin (M, 68,000); 2, ovalbumin (M, 45,000); 3, chymotrypsinogen A (M, 25,000); 4, myoglobin (M, 17,500); 5, cytochrome c (M, 12,500); 6, aprotinin (M, 6,800); 7, insulin chain B (M, 3,500); and 8, insulin chain A (M, 2,500). x, retention time of rat hepatoma oncomodulin (M, 11,500) running anomalously at 18 min; j, retention time of immunoreactive material from ASV-NRK tumors. D, reverse-phase HPLC on RP-P at 0.8 ml/min. Material from the single peak eluting at 35.5 min was collected, lyophilized, and subjected to amino acid analysis (see Table 2). PBS for 20 min and incubated 1 hr with fluorescein isothiocyanate- purified by HPLC methods using 2 Beckman 10OA pumps controlled by conjugated sheep anti-rabbit IgG (Cappel Laboratories, West Chester, a Model 420 programmer and monitored at 280 nm with a Beckman Pa.) diluted 1:100 in PBS. Cells were washed in PBS, mounted in antifade Model 165 UV detector. Ion exchange HPLC was performed first on an mounting medium (6), and examined on a Leitz Orthoplan microscope AX300 column (4.1 x 300 mm; Synchrom, Inc., Linden, Ind.) equilibrated equipped with a Pleompak 2 fluorescence incidence illuminator and a in 20 mw Tris-acetate, pH 8.0, and eluted with a gradient (0 to 500 mw) standard fluorescein filter system. Both preimmune rabbit serum (100 ¿¿gof sodium acetate. The relevant fractions (Chart 1/1) were pooled and IgG per ml) and oncomodulin-adsorbed serum served as immunological lyophilized. The dried material was dissolved in 1 ml of 20 mivi controls. Adsorbed serum was produced by the addition of purified rat NaH2PO4:100 mM Na2SO4 (pH 6.8), and 200-^1 aliquots were subjected hepatoma oncomodulin to Affigel Blue-purified rabbit anti-oncomodulin sequentially to size exclusion HPLC on a BioSil TSK-125 column (7.5 x IgG (340 n\, 7.5 mg/ml) to a final concentration of 2 x 10~6 M and 300 nm; Bio-Rad, Mississauga, Ontario, Canada) equilibrated in the incubation for 1 hr at room temperature and then overnight at 4°.