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Induction of the ceruloplasmin mRNA in BalbC 3T3 cells after Rous-Sarcoma virus transfection
173
Cancer Letters, 35 (1987) 173.-179 Elsevier Scientific Publishers Ireland Ltd.
INDUCTION OF THE CERULOPLASMIN CELLS AFTER ROUS-SARCOMA VIRUS
SATYA
P. KUNAPULI
and ASHOK
mRNA IN Balb/C 3T3 TRANSFECTION
KUMAR
Division of Cell Biology, Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.) (Received 12 August 1986) (Revised version received 23 December (Accepted 6 January 1987)
1986)
SUMMARY
Ceruloplasmin is a copper containing serum glycoprotein. It is an acutephase protein and its levels are increased in inflammation and in a number of experimental and human tumors. It is normally synthesized in the liver and not in fibroblasts. In this paper we present evidence that ceruloplasmin mRNA is synthesized in Balb/C 3T3 cells and its levels are increased about 3-fold in Rous-Sarcoma virus (RSV) transformed cells. The ceruloplasmin mRNAs from rat liver and RSV 3T3 cells have the same electrophoretic mobility.
INTRODUCTION
Ceruloplasmin is a 1046 amino acid long a,-glycoprotein which is mainly synthesized in the liver and transported into the blood where it binds 9&95% of copper [9,14]. It is a multifunctional protein and is involved as oxidase in the regulation of hepatic iron mobilization [ 10,131, in the regulation of plasma and tissue levels of biogenic amines and phenols [16], as a serum antioxidant [8] and scavenger of the superoxide anion radicals [ 151, as an endogenous modulator of the inflammatory response [ 111 and as a copper transport protein for its incorporation into different apo-enzymes [5,13]. Ceruloplasmin is an acute-phase protein and its levels are increased in response to inflammation and tissue damage [24]. Its levels are also increased
Address
correspondence
to: Ashok Kumar.
0304.3835/87/$03.50 (11987 Elsevier Scientific Published and Printed in Ireland
Publishers
Ireland Ltd.
174
in rheumatoid arthritis [26] and hyperoxia [23]. In vivo experiments show that ceruloplasmin inhibits metal ion-induced lipid peroxidation [l] and also protects erythrocytes against metal ion stimulated lysis [al]. Its levels are increased as a result of hormones [ 12,27,29] and interleukin-I administration
[281. Ceruloplasmin has been shown to be involved in the induction of angiogenesis or neovascularization due to its copper-binding activity [22,25]. Serum ceruloplasmin levels are increased in various forms of animal and human cancers [ 19,201. In a recent paper Chu and Olden [7] have shown the presence of ceruloplasmin-like protein in Balb/C 3T3 cells by immunological techniques. They have also presented evidence that the synthesis of this ceruloplasmin-like protein is increased in RSV-transformed cells. In order to understand the role of ceruloplasmin in cancer cells and to study the regulation of its gene expression, we have recently isolated and partially sequenced its cDNA clone from a human liver cDNA library [ 181. We have also shown that ceruloplasmin mRNA is present in liver as well as in the heart (Kunapuli, S.P. and Ashok Kumar, Molecular cloning of human ceruloplasmin cDNA and evidence for ceruloplasmin in rat heart (submitted)). Recently Koschinsky et al., [ 171 reported the complete nucleotide sequence of human ceruloplasmin. In the present communication we provide evidence for the first time that ceruloplasmin mRNA is present in Balb/C cells and its levels are increased about 3-fold in R.SV-transformed cells. We also show by Northern hybridization that ceruloplasmin mRNA isolated from RSV-transformed cells has the same electrophoretic mobility as mRNA from untransformed cells. MATERIALSAND METHODS Cell culture RSV-transformed Balb/C 3T3 cells were obtained from Dr. Ira Pastan of the Laboratory of Molecular Biology, National Cancer Institute and were grown in Dulbecco’s minimal essential medium (DMEM) containing 10% heat-inactivated calf serum. The RSV3T3 cells were morphologically different from control Balb/C 3T3 cells. Isolation of the RNA and Northern blot hybridization Total RNA was prepared by guanidinium isothiocyanate-cesium chloride method [6] and poly(A +) RNA was isolated by oligo-dT cellulose column chromatography [2]. Poly(A + ) RNA was separated by 1.4% agaroseformaldehyde gel electrophoresis and transferred to nitrocellulose paper. The nitrocellulose paper was prehybridized with 6 x SSC, 5 x Denharts, 0.1% SDS and 0.1 mg/ml sheared and denatured Salmon sperm DNA for 2 h at 65’C and then hybridized with “‘P-labelled nick translated cDNA probe. The probe is a 438-bp EcoRI fragment of our cDNA clone, pHCP6, and its nucleotide sequence corresponds to 24162854 of the published human
175
ceruloplasmin 6 x SSC and washed with 30 min each film with an
cDNA sequence [17]. Hybridization was carried out in 5 x Denharts solution at 65’C for 1620 h. The filter was then 1 x SSC at room temperature (4 x 15 min each) and twice for at 52“C. The filters were then exposed to Kodak XAR-5 X-ray intensifier screen at - 70°C.
Quantitation of RNA leuels by dot-blot hybridization Total RNA (20 pg/ml) was denatured in 20 x SSC and formaldehyde at 65°C for 15 min and applied at different dilutions on nitrocellulose paper using BRL dot-blot apparatus. The filter was air-dried for l-2 h and baked for 3 h at 80°C in a vaccum oven. The filter was then hybridized with the nick-translated cDNA probe (lo6 cpm/ml, lo8 cpm/pg) at 65”C, washed in 1 x SSC four times at room temperature and twice at 52°C (30 min each) and exposed to Kodak XAR-5 X-Ray film for 16 h at -70°C. RESULTS
Total RNA prepared from primary cultures of mouse embryo fibroblasts, Balb/C 3T3 and RSV-transformed-3T3 were analyzed for the presence of ceruloplasmin mRNA using a dot-blot procedure. Whereas, the RNA from primary cultures of mouse fibroblasts did not show the presence of ceruloplasmin mRNA, the RNA from Balb/C 3T3 and RSV 3T3 cells indicated positive spots on autoradiography (Fig. 1). The ceruloplasmin mRNA levels were quantitated by scanning the intensity of the spots present on the X-ray film. The ceruloplasmin mRNA levels in RSV 3T3 cells were 3 times higher than in untransformed Balb/C 3T3 cells. The poly( A + ) RNA isolated from Balb/C 3T3 cells and from the RSVtransformed 3T3 cells was analyzed by Northern blot hybridization. The
Fig. 1. Dot-blot hybridization of total RNA from Balb/C 3T3 and RSV 3T3 cells. Total RNA (20 fgjml) from (1) Balb/C 3T3 cells and (2) RSV-transformed Balb/C 3T3 cells was blotted to nitrocellulose paper at different dilutions using BRL hybriblot apparatus. The filter was hybridized with nick-translated human ceruloplasmin cDNA probe at 65’ C for 16 h in 6 x SSC and 5 x Denharts solution, washed at 52 C in 1 x SSC and autoradiographed.
176
-
18s
Fig. 2. Northern hybridization of Balb/C 3T3 and RSV 3T3 poly(A+). Poly(A+) RNA from (1) Balb/C 3T3 cells and (2) RSV-transformed cells was electrophoressed on 1.4% agaroseformaldehyde gel, blotted to nitrocellulose paper and hybridized with nick-translated human ceruloplasmin cDNA clone at 65.C for 18 h in 6 x SSC and 5 x Denharts solution. The filter was washed at 52°C in 1 x SSC and autoradiographed. The position of ribosomal RNA markers is indicated on the side.
177
RNA (2 pg) was separated by 1.4% agarose-formaldehyde gel electrophoresis, transferred to nitrocellulose paper, and hybridized with 32P-labelled nicktranslated human ceruloplasmin cDNA insert. The result of this experiment is shown in Fig. 2 where it is clear that Balb/C 3T3 and RSV 3T3 cells synthesize ceruloplasmin mRNA having the same electrophoretic mobility (about 28 S). An analysis of ceruloplasmin mRNA from RSV 3T3 and rat liver (Kunapuli, S.P. and Ashok Kumar, Molecular cloning of human ceruloplasmin cDNA and evidence for ceruloplasmin in rat heart (submitted)) indicated that they are of the same size as judged by their electrophoretic mobility. The quantitation of ceruloplasmin mRNA in RSV 3T3 cells by scanning the intensity of the band indicated that it is about 2.5-fold more abundant than the band of Balb/C 3T3 cells. Furthermore, the radioactive counts in the RSV 3T3 band were 2.5 times more than in the Balb/C 3T3 band. This analysis was repeated 3 times and the mean values were taken for quantitation. DISCUSSION
Chu and Olden [7] have recently reported that the angiogenic copper containing protein ceruloplasmin is synthesized by mouse embryonic fibroblasts and its synthesis is increased by RSV transformation. Ceruloplasmin is normally synthesized in the liver and its expression by non-hepatocytes is rather a rare event. Since ceruloplasmin levels have been shown to be increased in various forms of cancer and it has been implicated in the angiogenesis or neovascularization of the tissue, it is possible, that ceruloplasmin synthesis may be part of a cascade of events essential for tumor growth. The results presented in this communication show that ceruloplasmin mRNA is present in Balb/C 3T3 cells and its levels are increased about 3-fold by RSV transformation. Although, mouse 3T3 cell lines are generally regarded as normal cells in culture, it has been shown that highly vascularized malignant hemangioendotheliomas are produced from a subcutaneous inoculation of glass or plastic beads coated with Balb/C 3T3 cells [3,4] and ceruloplasmin may be required for the growth of these tumors. Since ceruloplasmin is the only hepatic protein expressed in these cells and other hepatic proteins such as albumin and transferrin are not expressed, we believe that the RSV 3T3 system will serve as an important model to understand the mechanism of the regulation of gene expression of a hepatic protein in a non-hepatic cell. ACKNOWLEDGEMENTS
We are thankful to Dr. Ira Pastan for RSV-transformed Balb/C 3T3 cells and to Dr. Preston Gadson for his help in the growth of cells. This work was supported by a grant from the American Heart Association (No. 84-864) to A.K.
178 REFERENCES 1 Al-Timimi,
2
3 4 5 6 7 8 9 10 11 12 13 14
15 16 17 18 19 20 21 22 23 24 25
D.J. and Dormandy, T.L. (1977) The inhibition of lipid autooxidation by human ceruloplasmin. Biochem. J., 168, 283. Aviv, H. and Leder. P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligo thymidylic acid-cellulose. Proc. Natl. Acad. Sci. (U.S.A.), 69, 1408. Boone, C.W. (1975) Malignant hemangioendotheliomas produced by subcutaneous inoculation of Balb/3T3 cells attached to glass beads. Science, 188, 68. Boone, C.W., Takeichi, N., Paranjpe, M. and Gilden, R. (1976) Vasoformative sarcomas arising from Balb/3T3 cells attached to solid substrates. Cancer Res., 36, 1626. Broman, L. (1967) The function of ceruloplasmin - a moot question. In: Molecular Basis of Some Aspects of Mental Actyvity. pp. 2. Editor: 0. Walas. Academic Press. Chirgwin, J.M., Przybyla, A.E., Macdonald, R.J. and Rutter, W.J. (1379) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18, 5294. Chu. F.-F. and Olden, K. (1985) The expression of ceruloplasmin, an angiogenic glycoprotein by mouse embryonic fibroblasts. Biochem. Biophys. Res. Commun., 126, 15. Cranfield, L.M., Gollan, J.L., White, A.G. and Dormandy, T.L. (1979) Serum antioxidant activity in normal and abnormal subjects. Adv. Clin. Biochem., 26, 299. Cousins, R.J. (1985) Absorption, transport and hepatic metabolism of copper and zinc: Special reference to metallothionein and ceruloplasmin. Physiol. Rev., 65, 238. Curzon. G. and O’Rilley, S. (1960) A coupled iron-ceruloplasmin oxidation system. Biochem. Biophys. Res. Commun., 2, 284. Denko, C.W. (1979) Protective role of ceruloplasmin in inflammation. Agents and Actions, 9, 333. Evans, G.W., Cornatzer, N.F. and Cornatzer, W.E. (1970) Hormonal induction of ceruloplasmin synthesis. Am. J. Physiol., 218, 613. Frieden, E. and Hsieh, H.S. (1976) Ceruloplasmin: The copper transport protein with essential oxidase activity. Adv. Enzyme, 46, 187. Frieden, E. (1981) Ceruloplasmin A Multifunctional metalloprotein of vertebrate plasma. In: Biological Roles of Copper, pp. 93124. ElsevierjNorth Holland, New York (Ciba Foundation Symposium). Goldstein I.M., Kaplan, H.B., Eldelson, H.S. and Weissman, G. (1979) Ceruloplasmin. A scavenger of superoxide anion radicals. J. Biol. Chem., 254, 4040. Holmberg, C.G. and Laurell, C.B. (1951) Investigations in serum copper III Ceruloplasmin as an enzyme. Acta Chem. Stand., 5, 475. Koschinsky, M.L., Funk, W.D.. Van Oost, B. A. and MacGillivray, R.T. (1986) Complete cDNA sequence of human preceruloplasmin. Proc. Natl. Acad. Sci. (U.S.A.), 83, 5086. Kunapuli, S., Varde, A., Riley, L.A. and Kumar, A. (1985) Isolation of cDNA clone for human ceruioplasmin. Fed. Proc., 44, 3411. Linder, M.C.. Bryant, R.R., Lim, S., Scott, L.E. and Moor J.R. (1979) Ceruloplasmin elevation and synthesis with transplantable tumors. J. E. Enzyme, 24, 85. Linder, M.C., Moor, J.R. and Wright, K. (1981) Ceruloplasmin assays in diagnosis and treatment of human lung, breast and gastrointestinal cancers. J. Natl. Cancer Inst., 67, 263. Lovstad, R.A. (1982) The protective action of ceruloplasmin on copper ion stimulated lysis of rat erythrocytes. Int. J. Biochem.. 14, 585. McAuslan. B.R., Reilley, W.G., Hannan, G.N. and Gole. G.A. (1983) Angiogenic factors and their assay. Microvas. Res., 26, 323. Moak, S.A. and Greenwald, R.A. (1984) Enchancement of rat serum ceruloplasmin levels by exposure to yperoxia. Proc. Sot. Exper. Biol, Med., 177, 97. Powanda, M.C. (1981) Systemic alterations in metal metabolism during inflammation as part of an integrated response to inflammation. Agents and Actions Suppl., 8, 121. Raju. K.S., Alexandri, G., Zinch, M. and Gullino, P.M. (1982) Ceruloplasmin, copper ions and angiogenesis. J. Natl. Cancer. Inst., 69, 1183.
179 26 Roeser, H.P., Lee, G.R. and Cartwright, G.E. (1973) Role of ceruloplasmin in hypoferremia associated with chronic inflammation and endotoxin. Proc. Sot. Exp. Biol. Med., 142, 1155. 27 Stavcher, B.C. and Hill, C.H. (1965) Hormonal induction of ceruloplasmin. Comp. Biochem. Physiol., 15, 429. 28 Wannemacher, R.W., Pekarek, R.S., Thompson, W.L., Curnow, R.T., Beall, F.A., Zenser, T.V., Derubertis, F.R. and Beisel, W.R. (1975) A protein from polymorphonuclear leukocytes which affects the rate of hepatic amino acid transport and synthesis of acute-phase globulins. Endocrinology, 98, 651. 29 Weiner, A.L. and Cousins, R.J. (1983) Hormonally produced changes in ceruloplasmin synthesis and secretion in primary cultured rat hepatocytes. Biochem. J., 212, 297.
Cancer Letters, 35 (1987) 173.-179 Elsevier Scientific Publishers Ireland Ltd.
INDUCTION OF THE CERULOPLASMIN CELLS AFTER ROUS-SARCOMA VIRUS
SATYA
P. KUNAPULI
and ASHOK
mRNA IN Balb/C 3T3 TRANSFECTION
KUMAR
Division of Cell Biology, Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.) (Received 12 August 1986) (Revised version received 23 December (Accepted 6 January 1987)
1986)
SUMMARY
Ceruloplasmin is a copper containing serum glycoprotein. It is an acutephase protein and its levels are increased in inflammation and in a number of experimental and human tumors. It is normally synthesized in the liver and not in fibroblasts. In this paper we present evidence that ceruloplasmin mRNA is synthesized in Balb/C 3T3 cells and its levels are increased about 3-fold in Rous-Sarcoma virus (RSV) transformed cells. The ceruloplasmin mRNAs from rat liver and RSV 3T3 cells have the same electrophoretic mobility.
INTRODUCTION
Ceruloplasmin is a 1046 amino acid long a,-glycoprotein which is mainly synthesized in the liver and transported into the blood where it binds 9&95% of copper [9,14]. It is a multifunctional protein and is involved as oxidase in the regulation of hepatic iron mobilization [ 10,131, in the regulation of plasma and tissue levels of biogenic amines and phenols [16], as a serum antioxidant [8] and scavenger of the superoxide anion radicals [ 151, as an endogenous modulator of the inflammatory response [ 111 and as a copper transport protein for its incorporation into different apo-enzymes [5,13]. Ceruloplasmin is an acute-phase protein and its levels are increased in response to inflammation and tissue damage [24]. Its levels are also increased
Address
correspondence
to: Ashok Kumar.
0304.3835/87/$03.50 (11987 Elsevier Scientific Published and Printed in Ireland
Publishers
Ireland Ltd.
174
in rheumatoid arthritis [26] and hyperoxia [23]. In vivo experiments show that ceruloplasmin inhibits metal ion-induced lipid peroxidation [l] and also protects erythrocytes against metal ion stimulated lysis [al]. Its levels are increased as a result of hormones [ 12,27,29] and interleukin-I administration
[281. Ceruloplasmin has been shown to be involved in the induction of angiogenesis or neovascularization due to its copper-binding activity [22,25]. Serum ceruloplasmin levels are increased in various forms of animal and human cancers [ 19,201. In a recent paper Chu and Olden [7] have shown the presence of ceruloplasmin-like protein in Balb/C 3T3 cells by immunological techniques. They have also presented evidence that the synthesis of this ceruloplasmin-like protein is increased in RSV-transformed cells. In order to understand the role of ceruloplasmin in cancer cells and to study the regulation of its gene expression, we have recently isolated and partially sequenced its cDNA clone from a human liver cDNA library [ 181. We have also shown that ceruloplasmin mRNA is present in liver as well as in the heart (Kunapuli, S.P. and Ashok Kumar, Molecular cloning of human ceruloplasmin cDNA and evidence for ceruloplasmin in rat heart (submitted)). Recently Koschinsky et al., [ 171 reported the complete nucleotide sequence of human ceruloplasmin. In the present communication we provide evidence for the first time that ceruloplasmin mRNA is present in Balb/C cells and its levels are increased about 3-fold in R.SV-transformed cells. We also show by Northern hybridization that ceruloplasmin mRNA isolated from RSV-transformed cells has the same electrophoretic mobility as mRNA from untransformed cells. MATERIALSAND METHODS Cell culture RSV-transformed Balb/C 3T3 cells were obtained from Dr. Ira Pastan of the Laboratory of Molecular Biology, National Cancer Institute and were grown in Dulbecco’s minimal essential medium (DMEM) containing 10% heat-inactivated calf serum. The RSV3T3 cells were morphologically different from control Balb/C 3T3 cells. Isolation of the RNA and Northern blot hybridization Total RNA was prepared by guanidinium isothiocyanate-cesium chloride method [6] and poly(A +) RNA was isolated by oligo-dT cellulose column chromatography [2]. Poly(A + ) RNA was separated by 1.4% agaroseformaldehyde gel electrophoresis and transferred to nitrocellulose paper. The nitrocellulose paper was prehybridized with 6 x SSC, 5 x Denharts, 0.1% SDS and 0.1 mg/ml sheared and denatured Salmon sperm DNA for 2 h at 65’C and then hybridized with “‘P-labelled nick translated cDNA probe. The probe is a 438-bp EcoRI fragment of our cDNA clone, pHCP6, and its nucleotide sequence corresponds to 24162854 of the published human
175
ceruloplasmin 6 x SSC and washed with 30 min each film with an
cDNA sequence [17]. Hybridization was carried out in 5 x Denharts solution at 65’C for 1620 h. The filter was then 1 x SSC at room temperature (4 x 15 min each) and twice for at 52“C. The filters were then exposed to Kodak XAR-5 X-ray intensifier screen at - 70°C.
Quantitation of RNA leuels by dot-blot hybridization Total RNA (20 pg/ml) was denatured in 20 x SSC and formaldehyde at 65°C for 15 min and applied at different dilutions on nitrocellulose paper using BRL dot-blot apparatus. The filter was air-dried for l-2 h and baked for 3 h at 80°C in a vaccum oven. The filter was then hybridized with the nick-translated cDNA probe (lo6 cpm/ml, lo8 cpm/pg) at 65”C, washed in 1 x SSC four times at room temperature and twice at 52°C (30 min each) and exposed to Kodak XAR-5 X-Ray film for 16 h at -70°C. RESULTS
Total RNA prepared from primary cultures of mouse embryo fibroblasts, Balb/C 3T3 and RSV-transformed-3T3 were analyzed for the presence of ceruloplasmin mRNA using a dot-blot procedure. Whereas, the RNA from primary cultures of mouse fibroblasts did not show the presence of ceruloplasmin mRNA, the RNA from Balb/C 3T3 and RSV 3T3 cells indicated positive spots on autoradiography (Fig. 1). The ceruloplasmin mRNA levels were quantitated by scanning the intensity of the spots present on the X-ray film. The ceruloplasmin mRNA levels in RSV 3T3 cells were 3 times higher than in untransformed Balb/C 3T3 cells. The poly( A + ) RNA isolated from Balb/C 3T3 cells and from the RSVtransformed 3T3 cells was analyzed by Northern blot hybridization. The
Fig. 1. Dot-blot hybridization of total RNA from Balb/C 3T3 and RSV 3T3 cells. Total RNA (20 fgjml) from (1) Balb/C 3T3 cells and (2) RSV-transformed Balb/C 3T3 cells was blotted to nitrocellulose paper at different dilutions using BRL hybriblot apparatus. The filter was hybridized with nick-translated human ceruloplasmin cDNA probe at 65’ C for 16 h in 6 x SSC and 5 x Denharts solution, washed at 52 C in 1 x SSC and autoradiographed.
176
-
18s
Fig. 2. Northern hybridization of Balb/C 3T3 and RSV 3T3 poly(A+). Poly(A+) RNA from (1) Balb/C 3T3 cells and (2) RSV-transformed cells was electrophoressed on 1.4% agaroseformaldehyde gel, blotted to nitrocellulose paper and hybridized with nick-translated human ceruloplasmin cDNA clone at 65.C for 18 h in 6 x SSC and 5 x Denharts solution. The filter was washed at 52°C in 1 x SSC and autoradiographed. The position of ribosomal RNA markers is indicated on the side.
177
RNA (2 pg) was separated by 1.4% agarose-formaldehyde gel electrophoresis, transferred to nitrocellulose paper, and hybridized with 32P-labelled nicktranslated human ceruloplasmin cDNA insert. The result of this experiment is shown in Fig. 2 where it is clear that Balb/C 3T3 and RSV 3T3 cells synthesize ceruloplasmin mRNA having the same electrophoretic mobility (about 28 S). An analysis of ceruloplasmin mRNA from RSV 3T3 and rat liver (Kunapuli, S.P. and Ashok Kumar, Molecular cloning of human ceruloplasmin cDNA and evidence for ceruloplasmin in rat heart (submitted)) indicated that they are of the same size as judged by their electrophoretic mobility. The quantitation of ceruloplasmin mRNA in RSV 3T3 cells by scanning the intensity of the band indicated that it is about 2.5-fold more abundant than the band of Balb/C 3T3 cells. Furthermore, the radioactive counts in the RSV 3T3 band were 2.5 times more than in the Balb/C 3T3 band. This analysis was repeated 3 times and the mean values were taken for quantitation. DISCUSSION
Chu and Olden [7] have recently reported that the angiogenic copper containing protein ceruloplasmin is synthesized by mouse embryonic fibroblasts and its synthesis is increased by RSV transformation. Ceruloplasmin is normally synthesized in the liver and its expression by non-hepatocytes is rather a rare event. Since ceruloplasmin levels have been shown to be increased in various forms of cancer and it has been implicated in the angiogenesis or neovascularization of the tissue, it is possible, that ceruloplasmin synthesis may be part of a cascade of events essential for tumor growth. The results presented in this communication show that ceruloplasmin mRNA is present in Balb/C 3T3 cells and its levels are increased about 3-fold by RSV transformation. Although, mouse 3T3 cell lines are generally regarded as normal cells in culture, it has been shown that highly vascularized malignant hemangioendotheliomas are produced from a subcutaneous inoculation of glass or plastic beads coated with Balb/C 3T3 cells [3,4] and ceruloplasmin may be required for the growth of these tumors. Since ceruloplasmin is the only hepatic protein expressed in these cells and other hepatic proteins such as albumin and transferrin are not expressed, we believe that the RSV 3T3 system will serve as an important model to understand the mechanism of the regulation of gene expression of a hepatic protein in a non-hepatic cell. ACKNOWLEDGEMENTS
We are thankful to Dr. Ira Pastan for RSV-transformed Balb/C 3T3 cells and to Dr. Preston Gadson for his help in the growth of cells. This work was supported by a grant from the American Heart Association (No. 84-864) to A.K.
178 REFERENCES 1 Al-Timimi,
2
3 4 5 6 7 8 9 10 11 12 13 14
15 16 17 18 19 20 21 22 23 24 25
D.J. and Dormandy, T.L. (1977) The inhibition of lipid autooxidation by human ceruloplasmin. Biochem. J., 168, 283. Aviv, H. and Leder. P. (1972). Purification of biologically active globin messenger RNA by chromatography on oligo thymidylic acid-cellulose. Proc. Natl. Acad. Sci. (U.S.A.), 69, 1408. Boone, C.W. (1975) Malignant hemangioendotheliomas produced by subcutaneous inoculation of Balb/3T3 cells attached to glass beads. Science, 188, 68. Boone, C.W., Takeichi, N., Paranjpe, M. and Gilden, R. (1976) Vasoformative sarcomas arising from Balb/3T3 cells attached to solid substrates. Cancer Res., 36, 1626. Broman, L. (1967) The function of ceruloplasmin - a moot question. In: Molecular Basis of Some Aspects of Mental Actyvity. pp. 2. Editor: 0. Walas. Academic Press. Chirgwin, J.M., Przybyla, A.E., Macdonald, R.J. and Rutter, W.J. (1379) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18, 5294. Chu. F.-F. and Olden, K. (1985) The expression of ceruloplasmin, an angiogenic glycoprotein by mouse embryonic fibroblasts. Biochem. Biophys. Res. Commun., 126, 15. Cranfield, L.M., Gollan, J.L., White, A.G. and Dormandy, T.L. (1979) Serum antioxidant activity in normal and abnormal subjects. Adv. Clin. Biochem., 26, 299. Cousins, R.J. (1985) Absorption, transport and hepatic metabolism of copper and zinc: Special reference to metallothionein and ceruloplasmin. Physiol. Rev., 65, 238. Curzon. G. and O’Rilley, S. (1960) A coupled iron-ceruloplasmin oxidation system. Biochem. Biophys. Res. Commun., 2, 284. Denko, C.W. (1979) Protective role of ceruloplasmin in inflammation. Agents and Actions, 9, 333. Evans, G.W., Cornatzer, N.F. and Cornatzer, W.E. (1970) Hormonal induction of ceruloplasmin synthesis. Am. J. Physiol., 218, 613. Frieden, E. and Hsieh, H.S. (1976) Ceruloplasmin: The copper transport protein with essential oxidase activity. Adv. Enzyme, 46, 187. Frieden, E. (1981) Ceruloplasmin A Multifunctional metalloprotein of vertebrate plasma. In: Biological Roles of Copper, pp. 93124. ElsevierjNorth Holland, New York (Ciba Foundation Symposium). Goldstein I.M., Kaplan, H.B., Eldelson, H.S. and Weissman, G. (1979) Ceruloplasmin. A scavenger of superoxide anion radicals. J. Biol. Chem., 254, 4040. Holmberg, C.G. and Laurell, C.B. (1951) Investigations in serum copper III Ceruloplasmin as an enzyme. Acta Chem. Stand., 5, 475. Koschinsky, M.L., Funk, W.D.. Van Oost, B. A. and MacGillivray, R.T. (1986) Complete cDNA sequence of human preceruloplasmin. Proc. Natl. Acad. Sci. (U.S.A.), 83, 5086. Kunapuli, S., Varde, A., Riley, L.A. and Kumar, A. (1985) Isolation of cDNA clone for human ceruioplasmin. Fed. Proc., 44, 3411. Linder, M.C.. Bryant, R.R., Lim, S., Scott, L.E. and Moor J.R. (1979) Ceruloplasmin elevation and synthesis with transplantable tumors. J. E. Enzyme, 24, 85. Linder, M.C., Moor, J.R. and Wright, K. (1981) Ceruloplasmin assays in diagnosis and treatment of human lung, breast and gastrointestinal cancers. J. Natl. Cancer Inst., 67, 263. Lovstad, R.A. (1982) The protective action of ceruloplasmin on copper ion stimulated lysis of rat erythrocytes. Int. J. Biochem.. 14, 585. McAuslan. B.R., Reilley, W.G., Hannan, G.N. and Gole. G.A. (1983) Angiogenic factors and their assay. Microvas. Res., 26, 323. Moak, S.A. and Greenwald, R.A. (1984) Enchancement of rat serum ceruloplasmin levels by exposure to yperoxia. Proc. Sot. Exper. Biol, Med., 177, 97. Powanda, M.C. (1981) Systemic alterations in metal metabolism during inflammation as part of an integrated response to inflammation. Agents and Actions Suppl., 8, 121. Raju. K.S., Alexandri, G., Zinch, M. and Gullino, P.M. (1982) Ceruloplasmin, copper ions and angiogenesis. J. Natl. Cancer. Inst., 69, 1183.
179 26 Roeser, H.P., Lee, G.R. and Cartwright, G.E. (1973) Role of ceruloplasmin in hypoferremia associated with chronic inflammation and endotoxin. Proc. Sot. Exp. Biol. Med., 142, 1155. 27 Stavcher, B.C. and Hill, C.H. (1965) Hormonal induction of ceruloplasmin. Comp. Biochem. Physiol., 15, 429. 28 Wannemacher, R.W., Pekarek, R.S., Thompson, W.L., Curnow, R.T., Beall, F.A., Zenser, T.V., Derubertis, F.R. and Beisel, W.R. (1975) A protein from polymorphonuclear leukocytes which affects the rate of hepatic amino acid transport and synthesis of acute-phase globulins. Endocrinology, 98, 651. 29 Weiner, A.L. and Cousins, R.J. (1983) Hormonally produced changes in ceruloplasmin synthesis and secretion in primary cultured rat hepatocytes. Biochem. J., 212, 297.