Vol. March
167,
No.
30,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
PARATHYROID HORMONE RELATED PEPTIDE CAN FUNCTION AN AUTOCRINE GROWTH FACTOR IN HUMAN RENAL CELL CARCINOMA PAUL B. J. BURTONl,
CAJE MONIZ2
1134-1138
AS
AND DEREK E. KNIGHT1
‘Division of Biomedical Sciences, Kings College,London WC2R 2L,S, United Kingdom 2Dept. of ChemicalPathology, Kings College Hospital, London SE5 9RS, United Received
February
12,
Kingdom
1990
SUMMARY: Parathyroid hormone related peptide (PTHrP) has been implicated in the cause of the hypercalcemia associated with a number of malignant tumours. The data presented here suggests that PTHrP (in addition to its known role of mediating hypercalcemia) may be involved in the autocrine regulation of growth of some tumours. Polyclonal PTHrP antiserum almost totally inhibited the growth of a human renal cell carcinoma cell line, known to secrete PTHrP, in vitro and growth was significantly inhibited by the competitive PTH antagonist PTH (334)NHz. 0 1990Academic Press,Inc.
The initiation and perpetuationof carcinogenesisis a multifactorial process(1). While the ectopic and unregulated secretionof hormonesfrequently complicate the clinical managementof a numberof malignancies,the role they play in the autocrineregulation of tumour growth is becoming increasingly apparent (2). Throughout the last decade there hasbeenmuch interestin the syndromeof humoralhypercalcemiaof malignancy (SHHM). SHHM is associatedwith some20% of tumours, mostcommonly renal cell and lung smallcell carcinoma, and is characterisedby raisedserumlevels of calcium, increasedurinary excretion of CAMP and phosphaturia. Serum levels of bioactive parathyroid hormone(PTH) are found to be elevated, yet paradoxically serumlevels of immunoreactivePTH arefound to be normalor even suppressed (3). This suggeststhe presenceof a circulating factor with PTH like bioactivity but which is structurally and immunologicallydistinct from it. Recently, a peptide has been isolated from a variety of hypercalcemic tumours, which is composed of 141 amino acids, of which 8 out of the first 13 are common to PTH. This limited N-terminal amino acid homology with PTH is sufficient to confer upon the peptide PTH like bioactivity and has led to it being termed Parathyroid hormonerelated peptide or PTHrP (4). While PTHrP is now believed to be a major contributing factor to the aetiology of SHHM, the aim of this study was to determine 0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. AN rights of reproduction in an-y form reserved.
1134
Vol.
167,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
whether or not the protein plays any part in the autocrine regulation of growth of a hypercalcemic renal carcinoma cell line known to secrete PTHrP. MATERIALS
AND
METHODS
Muter-ids: PTHrP (l-34) was purchased from Peninsula Laboratories (Merseyside, UK). PTH (l-34) and PTH (3-34)NHz were obtained from Sigma. PTHrP (1-141) was kindly donated by Dr. R. G. Hammonds (Genentech, Inc, Calif, U.S.A.). Polyclonal PTHrP antiserum raised against PTHrP (l-34) was the generous gift of Drs. G.V. Segre and H. Jueppner (Massachusetts General Hospital, Boston, Mass). Cell Culture: SKRC-1 cells ( kindly supplied by Dr. N. H. Bander, New York, U.S.A.), were grown as previously described (5), with minor modifications, In all experiments cells were seeded into 24 substituting DMEM for Eagles MEM. well plates (Nunc) and grown in medium containing 10% FCS. The cells were fed daily for the desired time period with test substance (either peptide or antiserum), trypsinised and counted using an Elzone particle counter model 280PC. Data are expressed as mean +/- S.E.M. and statistical significance was determined by Students t-test, a value of P,
167,
No.
30,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
PARATHYROID HORMONE RELATED PEPTIDE CAN FUNCTION AN AUTOCRINE GROWTH FACTOR IN HUMAN RENAL CELL CARCINOMA PAUL B. J. BURTONl,
CAJE MONIZ2
1134-1138
AS
AND DEREK E. KNIGHT1
‘Division of Biomedical Sciences, Kings College,London WC2R 2L,S, United Kingdom 2Dept. of ChemicalPathology, Kings College Hospital, London SE5 9RS, United Received
February
12,
Kingdom
1990
SUMMARY: Parathyroid hormone related peptide (PTHrP) has been implicated in the cause of the hypercalcemia associated with a number of malignant tumours. The data presented here suggests that PTHrP (in addition to its known role of mediating hypercalcemia) may be involved in the autocrine regulation of growth of some tumours. Polyclonal PTHrP antiserum almost totally inhibited the growth of a human renal cell carcinoma cell line, known to secrete PTHrP, in vitro and growth was significantly inhibited by the competitive PTH antagonist PTH (334)NHz. 0 1990Academic Press,Inc.
The initiation and perpetuationof carcinogenesisis a multifactorial process(1). While the ectopic and unregulated secretionof hormonesfrequently complicate the clinical managementof a numberof malignancies,the role they play in the autocrineregulation of tumour growth is becoming increasingly apparent (2). Throughout the last decade there hasbeenmuch interestin the syndromeof humoralhypercalcemiaof malignancy (SHHM). SHHM is associatedwith some20% of tumours, mostcommonly renal cell and lung smallcell carcinoma, and is characterisedby raisedserumlevels of calcium, increasedurinary excretion of CAMP and phosphaturia. Serum levels of bioactive parathyroid hormone(PTH) are found to be elevated, yet paradoxically serumlevels of immunoreactivePTH arefound to be normalor even suppressed (3). This suggeststhe presenceof a circulating factor with PTH like bioactivity but which is structurally and immunologicallydistinct from it. Recently, a peptide has been isolated from a variety of hypercalcemic tumours, which is composed of 141 amino acids, of which 8 out of the first 13 are common to PTH. This limited N-terminal amino acid homology with PTH is sufficient to confer upon the peptide PTH like bioactivity and has led to it being termed Parathyroid hormonerelated peptide or PTHrP (4). While PTHrP is now believed to be a major contributing factor to the aetiology of SHHM, the aim of this study was to determine 0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. AN rights of reproduction in an-y form reserved.
1134
Vol.
167,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
whether or not the protein plays any part in the autocrine regulation of growth of a hypercalcemic renal carcinoma cell line known to secrete PTHrP. MATERIALS
AND
METHODS
Muter-ids: PTHrP (l-34) was purchased from Peninsula Laboratories (Merseyside, UK). PTH (l-34) and PTH (3-34)NHz were obtained from Sigma. PTHrP (1-141) was kindly donated by Dr. R. G. Hammonds (Genentech, Inc, Calif, U.S.A.). Polyclonal PTHrP antiserum raised against PTHrP (l-34) was the generous gift of Drs. G.V. Segre and H. Jueppner (Massachusetts General Hospital, Boston, Mass). Cell Culture: SKRC-1 cells ( kindly supplied by Dr. N. H. Bander, New York, U.S.A.), were grown as previously described (5), with minor modifications, In all experiments cells were seeded into 24 substituting DMEM for Eagles MEM. well plates (Nunc) and grown in medium containing 10% FCS. The cells were fed daily for the desired time period with test substance (either peptide or antiserum), trypsinised and counted using an Elzone particle counter model 280PC. Data are expressed as mean +/- S.E.M. and statistical significance was determined by Students t-test, a value of P,
