- Email: [email protected]
Human tumor cell lines express low levels of oncomodulin
Vol.
169,
No.
June
29,
1990
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
905-909
HUMAN TUMOR CELL LINES EXPRESS LOW LEVELS OF ONCOMODULIN S. Huber, M. Leuthold’, E.W. Sommei and C.W. Heizmann” Department of Pediatrics, Division of Clinical Chemistry, University of Zi.irich, Steinwiesstrasse 75, CH-8032 Zurich, Switzerland ‘Laboratory of Oncology, University Hospital, Zurich , Switzerland ‘Institute of Toxicology, ETH-Zurich, CH-8603 Schwerzenbach , Switzerland Received
May 14,
1990
Different human carcinoma cell lines were screened for the presence of Ca’+-binding oncomodulin. A specific polyclonal antibody was raised against a synthetic peptide (amino acids 99-l 08) of oncomodulin coupled to hemocyanin. Extracts of tumor cell lines (several human, one rat) were analyzed for the presence of oncomodulin by immunoblotting. A strong immunoreaction of oncomodulin was obtained in chemically transformed rat fibroblasts (T14c) in contrast to all human tumor cell lines investigated, where no immunoreaction was obtained. These results suggest that oncomodulin cannot be used in diagnostics of human tumors. 0 1990Academic Press,Inc. Oncomodulin belongs to the family of Ca*+-binding EF-hand proteins with highest sequence homology to pan/albumin (1). Oncomodulin was first detected in neoplastic liver and isolated from rat Morris hepatomas (2, 3). It was found in other rodent tumors, but never in normal fetal and adult tissues with the exception of placenta, where it is present in extraembryonic membranes. Based on these data, oncomodulin was suggested to be a valuable tool in tumor diagnostics. To test this hypothesis we raised polyclonal antibodies against a synthetic peptide, representing the C-terminus of oncomodulin and screened human tumor cell lines of different origin for the presence of oncomodulin and compared the oncomodulin levels with those in chemically transformed rat fibroblasts (T14c). MATERIAL AND METHODS Buffalo rats bearing Morris hepatoma 5123tc were obtained from Dr Slaughter, Howard University, Washington DC, USA. Tumors of 30-509 were dissected free of necrotic tissue and stored at -80°C before protein extraction (4). T14c cells were kindly supplied by J.K. Blum (4, 5). The human tumor cell lines
“To whom correspondence should be addressed. 0006-291XBO
905
$1.50
Copyright 0 I990 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOCHEMICAL
Vol. 169, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
were grown in RPM1 medium supplemented with 10% fetal calf serum, 1mM Lglutamine and antibiotics. Confluent cells were trypsinized and washed twice with cold PBS. After centrifugation, the cell pellet was stored at -70°C. Oncomodulin was isolated from rat Morris hepatomas as previously described (3, 6) with minor modifications. The final purification steps were carried out on HPLC using a C,-reverse phase column and a Tris/EGTA buffer system (7) followed by separation on the same column with 0.1% (v/v) trffluoroacetic acid (buffer A) and 90% acetonitrile in buffer A (buffer B). Gradient and flow conditions were as previously described (7). Oncomodulin eluted at 45% buffer B and its amino acid composition was identical to previously published data (8). A synthetic peptide consisting of the C-terminus of rat oncomodulin (amino acids 99-l 08) was coupled via a 8-malein-imido-propionyl-group to keyhole limpet hemocyanin. A rabbit was immunized with 1 mg of this conjugate and complete Freund adjuvans, and 13 booster injections with conjugate (1 mg each time) and incomplete Freund adjuvans were given at 3-4 weeks intervals. The anti-serum gave a titer of 1 :lOOO with 10 ng antigen when tested with the dot immunobinding assay (9). The anti-serum was affinity-purified on an Affi-Prep 10 column (Bio-Rad Laboratories, Glattbrugg, Switzerland) to which the synthetic peptide was coupled. Cross-reactivity of the anti-serum was checked by immunoblotting (10) against other Ca”-binding proteins such as rat muscle parvalbumin, rat testis calmodulin, bovine brain S-100 proteins and recombinant rat brain calbindin D28K (11). Twenty millions of cells of each cell line were extracted in 0.5ml of extraction buffer (20mM Tris-HCI pH 7.4, 1 mM EGTA, 1 mM phenylmethylsulfonyl-fluoride, 0.1% (v/v) 2-mercapto-ethanol), heat-treated (10 min, 85°C) and centrifuged (10’000 x g, 15 min). The supernatant was analyzed for oncomodulin by immunoblotting and HPLC. Protein concentrations were determined by the Bio Radprotein assay using bovine serum albumin as a standard. Proteins and cell extracts were separated on Tricine-SDS-polyacrylamide gels in the presence of 2-mercaptoethanol (12). Proteins were visualized with Coomassie blue R-250. lmmunoblots were incubated with anti-oncomodulin serum (diluted 1:500) and developed with 4-chloronaphthol and H,O, (10). Prestained molecular weight markers (low range, bovine serum albumin 84 kD, ovalbumin 47 kD, bovine carbonic anhydrase 33 kD, soybean ttypsin inhibitor 24 kD, lysozyme 16 kD from Bio-Rad Laboratories) were used. RESULTS
AND DISCUSSION
Most studies were performed on rat oncomodulin were made to investigate oncomodulin is important
in human tumor diagnostics
and only a few attempts
in human tumors. However, if oncomodulin it should be present in sizeable amounts in
human tumor cell lines. The purification
of considerable
amounts of oncomodulin
from human solid
tumors and from first trimester human placenta according to published for rat tumors failed. No human oncomodulin oncomodulin
peak corresponding
procedures
to rat
was obtained on HPLC (data not shown). This was the first indication
that the level of oncomodulin A specific polyclonal
in human must be much lower than in rat. antibody against a synthetic peptide of rat oncomodulin
coupled to keyhole limpet hemocyanin
was prepared. The C-terminus 906
(amino acids
Vol.
169, No. 3, 1990
BIOCHEMICAL
99-108) of rat oncomodulin
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was chosen because of the greatest possible difference
of this sequence from rat and human parvalbumins. immunoblotting parvalbumin,
for cross-reactivity calmodulin,
against other Ca2’-binding
S-100 proteins, calbindin
reacted only against rat oncomodulin The anti-serum
The anti-serum
(M,=l2
was tested by
proteins, such as
D28K (Fig. 1). The anti-serum
kD).
was used to screen by immunoblotting
18 human carcinoma
cell lines of different origin (Fig. 2 and Table 1). None of these human cell extracts showed an oncomodulin
signal.
In contrast, the transformed
expresses a high amount of oncomodulin
rat cell line (T14c)
(Fig. 2, lane 7). Only two of the human
cell lines (SW2 and ZL5) are shown as an example (Fig. 2, lanes 1,4 and lanes 2,5). There was no signal at the site of oncomodulin bands in lane 4 and 5 disappeared Detailed examination
when using the affinity-purified
T14c cell line was submitted
However, when an extract of the
to HPLC, a prominent
observed and reacted with the anti-serum all examined
antibody.
of the human cell extracts by reverse phase HPLC showed
no peak eluting at the position of oncomodulin.
hydrophobicity
(Mp12 kD) and the faint
oncomodulin
peak was
(data not shown). A large difference in
between human and rat oncomodulin Ca2+-binding proteins (parvalbumin,
is not expected, since so far
S-100 proteins, calmodulin,
calcyclin) of various species did not show a large variation in hydrophobicity.
01
1
12345678
2
34567
Fig. 1: The affinity-purified rat oncomodulin antibody is tested against other Ca’+-binding proteins. 1 D-SDS-PAGE stained with Coomassie blue (lanes l-5) and corresponding immununoblot (lanes 6-10). Lane 1,6: S-100 proteins (2.5 pg); lane 2,7: parvalbumin (3 pg); lane 3, oncomodulin (3 pg); lane 4,9: calmodulin (10 Kg); lane 510: calbindin D28K (5pg). Molecular weight markers are indicated on the left by arrowheads, from top to bottom: 64 kD, 47 kD, 33 kD, 24 kD and 16 kD. OM, oncomodulin (M,=l2 kD). Fig. 2: Extracts of human carcinoma cell lines and rat Tl4c cells. 1 D-SDS-PAGE (lanes 1-3) and immunoblot (lanes 4-7). Lanes 1,4: human tumor cell line SW2 (100 pg); lanes 2,5: human tumor cell line ZL5 (100 kg); lanes 3,6: purified rat oncomodulin (0.5, 2 pg) and lane 7: rat Tl4c cell-extract (50 pg). Molecular weight markers are indicated by arrowheads as in Fig. 1. 907
Vol. 169, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1: Human carcinoma cell lines Name
Origin
Ref.
ZL5 ZL 34 SPC 212 SPC 111 SLC 52 A 427 A 549 Calu 3 Calu 6 Calu 1 Hotz U 1752 SW2 NCI-H 249 NCI-H 526 NCI-N 417
Mesothelium I II I) Adenocarcinoma I 11 n II Squamous I 11 SCLC” SCLC classical SCLC variant 11
15 15 15 16 17 18 19 20 20 20 21 22 17 23 23 23
“SCLC, small cell lung cancer.
Our antibody therefore
was raised against a synthetic
it may not recognize
since the human sequence except that Glul 00 stands The concentration
peptide of rat oncomodulin,
the human counterpart.
(amino acids 99-108)
However
is identical to that of the rat
for Asp1 00 (D. Banville, personal of oncomodulin
this is unlikely
communication).
in human tumor cell lines is estimated
to
be at least 100 times lower than in the rat T14c cell line or in rat solid tumors. Therefore
oncomodulin
Our results
is not suitable as a marker
are sustained
gene of oncomodulin
in human tumor diagnostics.
by recently published
data, indicating
is under the control of a strong LTR promotor
raise to the high concentration in human and in mouse,
of rat oncomodulin.
explaining
This promoter
its much lower expression
that the rat
which gives element is absent
in these species
(13,
14). In future studies oncomodulin
it might be interesting,
in prenatal development,
already shortly
after fertilization
however,
to study the role of
since this protein is expressed
(in the rat)
(24).
ACKNOWLEDGMENTS This work was supported 9409.88),
EMDO-Stiftung,
Hartmann
Miiller-Stiftung
League (FOR 337.87.2).
by the Swiss
National Science Foundation
Geigy-Jubilaums-stiftung, fiir medizinische
Krebsliga
Forschung
We thank Dr. Gillessen, 908
des Kantons
and the Swiss
Hoffmann-La
(31Zurich,
Cancer
Roche, Basel, for
Vol.
169,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
the synthesis and coupling of the oncomodulin-peptide. Ursi Hiiriimann,
Erika Kijmin and Luisa Resenterra
RESEARCH
COMMUNICATIONS
The technical assistance of is acknowledged.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
MacManus, J.P., Brewer, L.M. and Gillen, M.F. (1987) in: The Role of Calcium in Biological Systems (Anghileri, L.J., ed.) vol. 4A, 1-19, CRC Press, Boca Raton. MacManus, J.P. (1979) Cancer Res. 39, 3000-3009. MacManus, J.P. (1980) Biochim. Biophys. Acta 621, 296-304. Sommer, E.W. and Heizmann, C.W. (1989) Cancer Res. 49, 899-905. Sommer, E.W., Blum, J.K., Berger, M.C. and Berchtold, M.W. (1989) FEBS Lett. 257, 307-310. Klee, C.B. and Heppel, L.A. (1984) Biochem. Biophys. Res. Commun. 125, 420-424. Pfyffer, G.E., Haemmerli, G. and Heizmann, C.W. (1984) Proc. Natl. Acad. Sci. USA 81, 6623-6636. MacManus, J.P., Watson, D.C. and Yaguchi, M. (1983) Eur. J. Biochem. 136, 9-17. Hawkes, R., Niday, E. and Gordon, J. (1982) Anal. Biochem. 119, 142-147. Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. Pinol, M.R., Kagi, U., Heizmann, C.W., Vogel, B., Sequier, J-M., Haas, W. and Hunziker, W. (1990) J. Neurochem., in press. Schaegger, H. and von Jagow, G. (1987) Anal. Biochem. 166, 368-370. Banville, D. and Boie, Y. (1989) J. Mol. Biol. 207, 481-490. Furter, C.S., Heizmann, C.W. and Berchtold, M.W. (1989) J. Biol. Chem. 264, 18276-l 8279. Established in R.A. Stahel’s Laboratory, University Hospital Zurich, manuscript in preparation. Stahel, R-A., O’Hara, C.J., Waibel, R. and Alexander, A. (1988) Int. J. Cancer 41, 212-223. Stahel, R.A., Speak, J.A. and Bernal, S.D. (1985) Int. J. Cancer 35, 11-17. Giard, D.J. (1973) J. Nat. Cancer Inst. 51, 1417-1423. Lieber, M., Smith, B., Szakal, A., Nelson-Rees, W. and Todaro, G. (1976) Int. J. Cancer 17, 62-70. Fogh, J. and Trempe, G. (1975) in: Human Tumor Cells in vitro (Fogh, J., ed.), 115-l 59, Plenum Press, New York. Peter, H.J. and Kistler, G.S. (1979) Schweiz. Med. Wochenschr. 109, 830832. Bergh, J., Nilsson, K., Zech, L. and Giovanella, B. (1981) Anticancer Res. 1, 317-322. Carney, D.N., Gazdan, A.F., Bepler, G., Guccion, J.G., Marangos, P.J., Moody, T.W., Zweig, M.H. and Muina, J.D. (1985) Cancer Res. 45, 29132923. MacManus, J.P. and Brewer, L.M. (1990) in: Calcium Binding Proteins in Normal and Transformed Cells (Pochet, R., Lawson, D.E. and Heizmann, C.W., eds.) Plenum Press, New York, in press.
169,
No.
June
29,
1990
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
905-909
HUMAN TUMOR CELL LINES EXPRESS LOW LEVELS OF ONCOMODULIN S. Huber, M. Leuthold’, E.W. Sommei and C.W. Heizmann” Department of Pediatrics, Division of Clinical Chemistry, University of Zi.irich, Steinwiesstrasse 75, CH-8032 Zurich, Switzerland ‘Laboratory of Oncology, University Hospital, Zurich , Switzerland ‘Institute of Toxicology, ETH-Zurich, CH-8603 Schwerzenbach , Switzerland Received
May 14,
1990
Different human carcinoma cell lines were screened for the presence of Ca’+-binding oncomodulin. A specific polyclonal antibody was raised against a synthetic peptide (amino acids 99-l 08) of oncomodulin coupled to hemocyanin. Extracts of tumor cell lines (several human, one rat) were analyzed for the presence of oncomodulin by immunoblotting. A strong immunoreaction of oncomodulin was obtained in chemically transformed rat fibroblasts (T14c) in contrast to all human tumor cell lines investigated, where no immunoreaction was obtained. These results suggest that oncomodulin cannot be used in diagnostics of human tumors. 0 1990Academic Press,Inc. Oncomodulin belongs to the family of Ca*+-binding EF-hand proteins with highest sequence homology to pan/albumin (1). Oncomodulin was first detected in neoplastic liver and isolated from rat Morris hepatomas (2, 3). It was found in other rodent tumors, but never in normal fetal and adult tissues with the exception of placenta, where it is present in extraembryonic membranes. Based on these data, oncomodulin was suggested to be a valuable tool in tumor diagnostics. To test this hypothesis we raised polyclonal antibodies against a synthetic peptide, representing the C-terminus of oncomodulin and screened human tumor cell lines of different origin for the presence of oncomodulin and compared the oncomodulin levels with those in chemically transformed rat fibroblasts (T14c). MATERIAL AND METHODS Buffalo rats bearing Morris hepatoma 5123tc were obtained from Dr Slaughter, Howard University, Washington DC, USA. Tumors of 30-509 were dissected free of necrotic tissue and stored at -80°C before protein extraction (4). T14c cells were kindly supplied by J.K. Blum (4, 5). The human tumor cell lines
“To whom correspondence should be addressed. 0006-291XBO
905
$1.50
Copyright 0 I990 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOCHEMICAL
Vol. 169, No. 3, 1990
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
were grown in RPM1 medium supplemented with 10% fetal calf serum, 1mM Lglutamine and antibiotics. Confluent cells were trypsinized and washed twice with cold PBS. After centrifugation, the cell pellet was stored at -70°C. Oncomodulin was isolated from rat Morris hepatomas as previously described (3, 6) with minor modifications. The final purification steps were carried out on HPLC using a C,-reverse phase column and a Tris/EGTA buffer system (7) followed by separation on the same column with 0.1% (v/v) trffluoroacetic acid (buffer A) and 90% acetonitrile in buffer A (buffer B). Gradient and flow conditions were as previously described (7). Oncomodulin eluted at 45% buffer B and its amino acid composition was identical to previously published data (8). A synthetic peptide consisting of the C-terminus of rat oncomodulin (amino acids 99-l 08) was coupled via a 8-malein-imido-propionyl-group to keyhole limpet hemocyanin. A rabbit was immunized with 1 mg of this conjugate and complete Freund adjuvans, and 13 booster injections with conjugate (1 mg each time) and incomplete Freund adjuvans were given at 3-4 weeks intervals. The anti-serum gave a titer of 1 :lOOO with 10 ng antigen when tested with the dot immunobinding assay (9). The anti-serum was affinity-purified on an Affi-Prep 10 column (Bio-Rad Laboratories, Glattbrugg, Switzerland) to which the synthetic peptide was coupled. Cross-reactivity of the anti-serum was checked by immunoblotting (10) against other Ca”-binding proteins such as rat muscle parvalbumin, rat testis calmodulin, bovine brain S-100 proteins and recombinant rat brain calbindin D28K (11). Twenty millions of cells of each cell line were extracted in 0.5ml of extraction buffer (20mM Tris-HCI pH 7.4, 1 mM EGTA, 1 mM phenylmethylsulfonyl-fluoride, 0.1% (v/v) 2-mercapto-ethanol), heat-treated (10 min, 85°C) and centrifuged (10’000 x g, 15 min). The supernatant was analyzed for oncomodulin by immunoblotting and HPLC. Protein concentrations were determined by the Bio Radprotein assay using bovine serum albumin as a standard. Proteins and cell extracts were separated on Tricine-SDS-polyacrylamide gels in the presence of 2-mercaptoethanol (12). Proteins were visualized with Coomassie blue R-250. lmmunoblots were incubated with anti-oncomodulin serum (diluted 1:500) and developed with 4-chloronaphthol and H,O, (10). Prestained molecular weight markers (low range, bovine serum albumin 84 kD, ovalbumin 47 kD, bovine carbonic anhydrase 33 kD, soybean ttypsin inhibitor 24 kD, lysozyme 16 kD from Bio-Rad Laboratories) were used. RESULTS
AND DISCUSSION
Most studies were performed on rat oncomodulin were made to investigate oncomodulin is important
in human tumor diagnostics
and only a few attempts
in human tumors. However, if oncomodulin it should be present in sizeable amounts in
human tumor cell lines. The purification
of considerable
amounts of oncomodulin
from human solid
tumors and from first trimester human placenta according to published for rat tumors failed. No human oncomodulin oncomodulin
peak corresponding
procedures
to rat
was obtained on HPLC (data not shown). This was the first indication
that the level of oncomodulin A specific polyclonal
in human must be much lower than in rat. antibody against a synthetic peptide of rat oncomodulin
coupled to keyhole limpet hemocyanin
was prepared. The C-terminus 906
(amino acids
Vol.
169, No. 3, 1990
BIOCHEMICAL
99-108) of rat oncomodulin
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was chosen because of the greatest possible difference
of this sequence from rat and human parvalbumins. immunoblotting parvalbumin,
for cross-reactivity calmodulin,
against other Ca2’-binding
S-100 proteins, calbindin
reacted only against rat oncomodulin The anti-serum
The anti-serum
(M,=l2
was tested by
proteins, such as
D28K (Fig. 1). The anti-serum
kD).
was used to screen by immunoblotting
18 human carcinoma
cell lines of different origin (Fig. 2 and Table 1). None of these human cell extracts showed an oncomodulin
signal.
In contrast, the transformed
expresses a high amount of oncomodulin
rat cell line (T14c)
(Fig. 2, lane 7). Only two of the human
cell lines (SW2 and ZL5) are shown as an example (Fig. 2, lanes 1,4 and lanes 2,5). There was no signal at the site of oncomodulin bands in lane 4 and 5 disappeared Detailed examination
when using the affinity-purified
T14c cell line was submitted
However, when an extract of the
to HPLC, a prominent
observed and reacted with the anti-serum all examined
antibody.
of the human cell extracts by reverse phase HPLC showed
no peak eluting at the position of oncomodulin.
hydrophobicity
(Mp12 kD) and the faint
oncomodulin
peak was
(data not shown). A large difference in
between human and rat oncomodulin Ca2+-binding proteins (parvalbumin,
is not expected, since so far
S-100 proteins, calmodulin,
calcyclin) of various species did not show a large variation in hydrophobicity.
01
1
12345678
2
34567
Fig. 1: The affinity-purified rat oncomodulin antibody is tested against other Ca’+-binding proteins. 1 D-SDS-PAGE stained with Coomassie blue (lanes l-5) and corresponding immununoblot (lanes 6-10). Lane 1,6: S-100 proteins (2.5 pg); lane 2,7: parvalbumin (3 pg); lane 3, oncomodulin (3 pg); lane 4,9: calmodulin (10 Kg); lane 510: calbindin D28K (5pg). Molecular weight markers are indicated on the left by arrowheads, from top to bottom: 64 kD, 47 kD, 33 kD, 24 kD and 16 kD. OM, oncomodulin (M,=l2 kD). Fig. 2: Extracts of human carcinoma cell lines and rat Tl4c cells. 1 D-SDS-PAGE (lanes 1-3) and immunoblot (lanes 4-7). Lanes 1,4: human tumor cell line SW2 (100 pg); lanes 2,5: human tumor cell line ZL5 (100 kg); lanes 3,6: purified rat oncomodulin (0.5, 2 pg) and lane 7: rat Tl4c cell-extract (50 pg). Molecular weight markers are indicated by arrowheads as in Fig. 1. 907
Vol. 169, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1: Human carcinoma cell lines Name
Origin
Ref.
ZL5 ZL 34 SPC 212 SPC 111 SLC 52 A 427 A 549 Calu 3 Calu 6 Calu 1 Hotz U 1752 SW2 NCI-H 249 NCI-H 526 NCI-N 417
Mesothelium I II I) Adenocarcinoma I 11 n II Squamous I 11 SCLC” SCLC classical SCLC variant 11
15 15 15 16 17 18 19 20 20 20 21 22 17 23 23 23
“SCLC, small cell lung cancer.
Our antibody therefore
was raised against a synthetic
it may not recognize
since the human sequence except that Glul 00 stands The concentration
peptide of rat oncomodulin,
the human counterpart.
(amino acids 99-108)
However
is identical to that of the rat
for Asp1 00 (D. Banville, personal of oncomodulin
this is unlikely
communication).
in human tumor cell lines is estimated
to
be at least 100 times lower than in the rat T14c cell line or in rat solid tumors. Therefore
oncomodulin
Our results
is not suitable as a marker
are sustained
gene of oncomodulin
in human tumor diagnostics.
by recently published
data, indicating
is under the control of a strong LTR promotor
raise to the high concentration in human and in mouse,
of rat oncomodulin.
explaining
This promoter
its much lower expression
that the rat
which gives element is absent
in these species
(13,
14). In future studies oncomodulin
it might be interesting,
in prenatal development,
already shortly
after fertilization
however,
to study the role of
since this protein is expressed
(in the rat)
(24).
ACKNOWLEDGMENTS This work was supported 9409.88),
EMDO-Stiftung,
Hartmann
Miiller-Stiftung
League (FOR 337.87.2).
by the Swiss
National Science Foundation
Geigy-Jubilaums-stiftung, fiir medizinische
Krebsliga
Forschung
We thank Dr. Gillessen, 908
des Kantons
and the Swiss
Hoffmann-La
(31Zurich,
Cancer
Roche, Basel, for
Vol.
169,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
the synthesis and coupling of the oncomodulin-peptide. Ursi Hiiriimann,
Erika Kijmin and Luisa Resenterra
RESEARCH
COMMUNICATIONS
The technical assistance of is acknowledged.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
MacManus, J.P., Brewer, L.M. and Gillen, M.F. (1987) in: The Role of Calcium in Biological Systems (Anghileri, L.J., ed.) vol. 4A, 1-19, CRC Press, Boca Raton. MacManus, J.P. (1979) Cancer Res. 39, 3000-3009. MacManus, J.P. (1980) Biochim. Biophys. Acta 621, 296-304. Sommer, E.W. and Heizmann, C.W. (1989) Cancer Res. 49, 899-905. Sommer, E.W., Blum, J.K., Berger, M.C. and Berchtold, M.W. (1989) FEBS Lett. 257, 307-310. Klee, C.B. and Heppel, L.A. (1984) Biochem. Biophys. Res. Commun. 125, 420-424. Pfyffer, G.E., Haemmerli, G. and Heizmann, C.W. (1984) Proc. Natl. Acad. Sci. USA 81, 6623-6636. MacManus, J.P., Watson, D.C. and Yaguchi, M. (1983) Eur. J. Biochem. 136, 9-17. Hawkes, R., Niday, E. and Gordon, J. (1982) Anal. Biochem. 119, 142-147. Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. Pinol, M.R., Kagi, U., Heizmann, C.W., Vogel, B., Sequier, J-M., Haas, W. and Hunziker, W. (1990) J. Neurochem., in press. Schaegger, H. and von Jagow, G. (1987) Anal. Biochem. 166, 368-370. Banville, D. and Boie, Y. (1989) J. Mol. Biol. 207, 481-490. Furter, C.S., Heizmann, C.W. and Berchtold, M.W. (1989) J. Biol. Chem. 264, 18276-l 8279. Established in R.A. Stahel’s Laboratory, University Hospital Zurich, manuscript in preparation. Stahel, R-A., O’Hara, C.J., Waibel, R. and Alexander, A. (1988) Int. J. Cancer 41, 212-223. Stahel, R.A., Speak, J.A. and Bernal, S.D. (1985) Int. J. Cancer 35, 11-17. Giard, D.J. (1973) J. Nat. Cancer Inst. 51, 1417-1423. Lieber, M., Smith, B., Szakal, A., Nelson-Rees, W. and Todaro, G. (1976) Int. J. Cancer 17, 62-70. Fogh, J. and Trempe, G. (1975) in: Human Tumor Cells in vitro (Fogh, J., ed.), 115-l 59, Plenum Press, New York. Peter, H.J. and Kistler, G.S. (1979) Schweiz. Med. Wochenschr. 109, 830832. Bergh, J., Nilsson, K., Zech, L. and Giovanella, B. (1981) Anticancer Res. 1, 317-322. Carney, D.N., Gazdan, A.F., Bepler, G., Guccion, J.G., Marangos, P.J., Moody, T.W., Zweig, M.H. and Muina, J.D. (1985) Cancer Res. 45, 29132923. MacManus, J.P. and Brewer, L.M. (1990) in: Calcium Binding Proteins in Normal and Transformed Cells (Pochet, R., Lawson, D.E. and Heizmann, C.W., eds.) Plenum Press, New York, in press.