Vol.
168,
No.
April
30,
1990
BIOCHEMICAL
2, 1990
PRODUCING
Akihiro Jimi4,
1Department 3First
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
PANCREASTATIN
Atsuo
AND
Funakoshil*, Hideyuki
CELL LINE FROM HUMAN PANCREATIC CELL TUMOR Kayoko Wakasugil,
of Gastroenterology Kyushu Cancer
Department
4Department
of
Tateishi3, Yurika
Michiyo TsuruI, Ikeda2 and Akira
and 2Research Center, Fukuoka,
Institute, Japan
of Biochemistry, School of University, Fukuoka, Japan First
741-746
Pathology,
Kurume
University,
ISLET
Kono 2 National
Medicine,Fukuoka Fukuoka,
Japan
Received March 12, 1990 SUMMARY: It has been characterized that cell line QGP-1 derived from human non-functioning pancreatic islet cell tumor produces human pancreastatin. Exponen ially growing cultures produced 5.7 ii cells/hr. fmol of pancreastatin/lU Human pancreastatin immunoreactivities in plasma and tumor after xenografting with QGP-1 into nude mouse were 92.7 fmol/ml and 160.2 pmol/g wet weight, respectively. Immunocytochemical study revealed both chromogranin A and pancreastatin immunoreactive cells in the Gel filtrations of culture medium and tumor extract tumor. identified heterogenous molecular forms of PST-L1 which eluted as large and smaller molecular species. These results suggest that plasma pancreastatin levels may be useful as a tumor marker of endocrine tumor of the pancreas, and the pancreastatin producing cell line may be useful for studies of the mechanism of secretions and processing of chromogranin A and pancreastatin. 01990
Academic
Press,
Inc.
Chromogranin A (CgA) endocrine tissues (1) pancreastatin (PST) (2,3). 52 residue peptide amide of human CgA (hCgA) as a It has been reported that and pancreatic exocrine sensitive radioimmunoassay *Address correspondence Cancer Center, Notame Abbreviations: hPST, like immunoreactivity;
is widely distributed in the neuroand proposed to be a precursor of Human PST (hPST) was deduced to be a which is contained at residues 250-301 sequence homologous to porcine PST (4). hPST could suppress insulin release (5) secretion We have developed a (6). (RIA) for hPST-like immunoreactivity
to: 3-1-1,
Dr. A. Funakoshi, Minami-ku, Fukuoka
National Kyushu 815, Japan.
human pancreastatin; PST-LI, pancreastatinhCgA, human chromogranin A. COO6-291X/90$1.50 741
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in my form reserved.
Vol.
168,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(hPST-LI) using the antiserum (R711) produced against a synthetic C-terminal fragment hPST (24-52) (7,8). In vitro and in vivo experiments are required to study the mechanism of PST secretion by use of the RIA. However, there is no reports of PST-producing cell lines. This work includes preliminary characterization of a (QGP-1N) derived from human nonPST-producing cell line functioning pancreatic islet cell tumor, hPST-LI levels in plasma and tumor after xenografting with QGP-1 into nude mouse, and immunocytochemical studies of the tumor. MATERIALS
AND METHODS
Pathology of the parent tumor The tumztissueused to initiate the culture was obtained from a subtotal pancreatectomy specimen removed from a 61-yearold male with an elevated serum carcinoembryonic antigen (-A) level, who was admitted to National Kyushu Cancer Center. The tumor was a well-circumscribed nodular lesion in the tail of the pancreas. Vascular invasion and metastases of tumor cells to the peri-pancreatic lymph nodes and to the liver were also apparent. Light microscopic examination of the tumor showed it to be a pancreatic carcinoma of islet origin. The bulk of the tumor was composed of atypical cells which showed a cord-like arrangement around many capillary vessels with slight intermingling of fibroblasts. Each cell had round nuclei and slightly eosinophilic cytoplasm. This tumor did not demonstrably secrete insulin and glucagon clinically and immunochemistry. Culture QGP-1 was established from a human pancreatic islet cell carcinoma cell line above mentioned as a CEA secreting cell line The cells were maintained with Daigo's T medium (Nihon (9). Seiyaku Co. Ltd., Tokyo, Japan) supplemented with 10 % fetal calf serum (FCS) (Gibco Lab., Grand island, N.Y.) at 37OC in a humidified atmosphere of 5 % CO2 in air. Confluent cell monolayers were passaged by treatment with trypsin-EDTA solution (0.25 % trypsin and 0.075 % EDTA in phosphate-buffered saline:PBS). Xenograft to nude mouse and establishment of line ---- the cell - WQGP-~N"
Four to six week-old female nude mice were used. The animals were housed in shared cages and kept in a laminar flow facility. Free access was allowed to an autoclaved diet and autoclaved water. Approximately 5 million cells in 0.2 ml of PBS were injected subcutaneously into right shoulder region of mice. After 60 days animals were killed, and exsanguinated by cardiac puncture. Plasma was centrifuged and stored at -7OOC for PST-L1 assay. Excised tumor was weighed, and divided into three portions, one is for histological examination and the second for evaluation of PST content. Tumor tissue was extracted in boiling 1 M acetic acid or water (7). The third portion of tumor was minced into small pieces with scissors, and was dispersed with 0.1 % collagenase (Wako Chemical, Osaka, Japan), 0.005 % DNase (Sigma, St. Louis, MO) and 0.002 % hyaluronidase (Sigma, St. Louis, MO) in Daigo's T medium. The suspension was incubated for 30 min at 37OC. The cell pellets were suspended in 2 mM EDTA and kept for 15 min at 37OC. The cell pellets were incubated again in 0.2 % collagenase for 30 min at 742
Vol.
168, No. 2, 1990
Fig.
BIOCHEMICAL
1:Phase contrast (Magnification,
AND BIOPHYSICAL
photomicrograph X300).
of
RESEARCH COMMUNICATIONS
QGP-1
cell
line
37OC. The cells were suspended in the medium. One half volume of the medium was exchanged at every 3 to 7 days. The elimination of fibroblastic cells was accomplished chemically by brief exposure to 0.05 % trypsin and 0.1 % EDTA in PBS (-1 and by mechanical scrubbing with a silicon rubber policeman. Criteria for the establishment of a cell line in continuous culture were the growth of the tumor cells to semiconfluency in the absence of contaminating cell types such as fibroblasts or lymphocytes, and repeated successful subcultures. The cell line established from nude mouse tumor was designated as QGP-1N. Figure 1 is a photomicrograph of QGP-1N cells growing as a monolayer culture. Pancreastatin radioimmunoassay An antiserum was raised against a synthetic C-terminal fragment of hPST-24-52 coupled with keyhole limpet hemocyanin in The antiserum was specific for C-terminal glycine amide rabbits. of hPST, and cross-reacted with porcine PST l-49 (3.8 %), but did not cross-react with bovine PST, insulin, glucagon, somatostatin, pancreatic polypeptide, cholecystokinin, Arg-vasopressin and gastrin. Crossreactivity of the antiserum with hCgA was not determined since hCgA was not available. However, it seemed that antiserum did not cross-react with hCgA. hPST 1-52 and ;z% I]-tyr-hPST 24-52 were used as standard and tracer, respectively. RIA procedure was described previously (7,8). The intraand interassay coefficients of variation were less than 10 %, and the sensitivity of the RIA was 5 pmol/L. In the assay for plasma PST-LI, extraction with a Sep-Pak Cl8 cartridge was needed before RIA. The extraction procedure was described previously (7,8).
Gel filtration -PST-L1 in culture medium and tissue extract were applied to a Sephadex G-50 (fine) column (1.2 x 65.5 cm), equilibrated, and eluted with 1 M acetic acid containing 0.05 % gelatin at a flow rate of 5 ml/h. Fractions of 2 ml were collected, lyophilized and reconstituted in 0.5 ml assay buffer for RIA. The column was calibrated with blue dextraf25;;;ychrome C, synthetic hPST l-52, hPST 24-52, hPST 43-52 and Immunocytochemistry Tissue was fixed in 10 % formalin and embedded in paraffin. Sections of 5 pm were incubated with antiserum (Ri'll) to hPST at 743
Vol.
168, No. 2, 1990
dilutions complex antiserum antiserum). RESULTS
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of l:lOOO, and stained by the avidin-biotin peroxidase method. Control sections were treated with specific final dilution of preabsorbed with hPST l-52 (10 yg/ml
AND DISCUSSION
The PST-L1 level was almost the same in the conditioned medium from QGP-1 and QGP-1N cells. The population-doubling time of QGP1 and QGP-1N calculated from the growth curve was about 37 hrs and 31 hrs, respectively. The modal chromosome number was 98. Exponentially growing cultures produce 5.7 fmol of PST/lo6 cells/hr. The plasma level of hPST-LI in the nude mouse with tumor implanted QGP-1 was 92.7 fmol/ml. HPST-LI was 160.2 pmol/g wet weight in the xenografted tumor. Immunocytochemical study cells in the tumor revealed both CgA- and PST-immunoreactive (Fig. 2). Gel filtrations of culture medium and acid-extract and water extract of the tumor identified heterogeneous molecular forms of PST-L1 (Fig. 3). Gel filtration of water-extract from the tumor showed two molecular forms corresponding to PST-L1 molecules which were determined as 186 amino acid residue and 48
Fig.
2:Immunocytochemical Chromogranin (Magnification,
localiz :ation of pancreastatin A (CgA) in the xenografted X100). 744
(PST) and tumor
Vol.
168, No. 2, 1990
va
Cyt
BIOCHEMICAL
PS52
PS29
PSlO
4
(8
((6
AND BIOPHYSICAL
vt
vo
60.
70
RESEARCH COMMUNICATIONS
Cvt
PS52
PS29
PSlO
4
+
vt 4
160.
QGP-1
-
culture
medium
140-
(A) 60
-
120z .g
3 .g E %
50-
ii 2
E c 7
100
-
E
‘, i
40-
60. I
k
i? 30
_
60 -
20
-
40 -
10
-
20 -
0 L,
A 10
25
30
35
40
25
Fractions
30
35
40
Fractions
Vo
Cyt
PS52
444
PS29 #
PS,O
Vt
4#
1
Fig.
residue
3:Elution profiles of PST-IS in culture medium (A), acid extract (B) and water extract (C) from the tumor on a Sephadex G-50 gel chromatography. Arrows indicate the elution volumes of markers: blue dextran (Vo), cytochrome human PS12$;:za \pVsz2), PST 24-52 c (CYt) I synthetic (PS29) I PST 43-52 (PSlO), and 1
from
insulinoma
molecule coeluted with and smaller molecules
extract
in
synthetic detected
previous
hPST 24-52 in culture 745
study in
(10).
The
the acid extract medium may be an
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
artifact produced during extraction with acid (11) and fragments produced by proteolytic cleavage during culture, respectively. The present report describes the high production of hPST-LI of a human cell line, QGP-1 established from a patient with nonfunctioning islet cell tumor. These results suggested that a high plasma level of PST-L1 in the inoculated mouse was derived from QGP-1 producing abundant PST and its precursor, and plasma PST levels may be useful as a tumor marker of non-functioning endocrine tumor of the pancreas. This cell line may be useful for studies of the mechanism of secretions and processing of CgA and PST. ACKNOWLEDGMENT This study was supported in Comprehensive lo-year Strategy of Health and Welfare.
part for
by a Grant-in Cancer Control
Aid from
for the Ministry
REFERENCES 1. Ehrhart,M., Grube,D., Bader,M.F., Aunis,D. & Gratzl, M. (1986) J. Histochem. Cytochem. 34, 1673-1682. 2. Iacangelo, A.L., Fischer-Colbrie, R., Koller, K-J., (1988) Endocrinology 122, 2339Brownstein, M.J. & Eiden, L.E. 2341. Funakoshi,A., Miyasaka,K., Ishida,K., Makk,G., 3. Nakano,I., Angwin,P., Chang, D. & Tatemoto, K (1989) Regul. Pept. 25, 207-213. 4. Konecki,D.S., Benedum, U.M., Gerdes,H.-H. & Huttner,W.B. (1987) J. Biol. Chem. 262, 17026-17030. 5. Funakoshi,A., Jimi,A., Yasunami,Y., Tateishi,K., Funakoshi,S., Tamamura,H. & Yajima,H. (1989) Biochem. Biophys. Res. Commun. 159,
913-918.
6.
Funakoshi,A., Miyasaka,K., Nakamura,R., Kitani,K., Funakoshi,S., Tamamura,H., Fujii,N. & Yajima,H. (1988) Biochem. Biophys. Res. Commun. 156, 1237-1242. 7. Tateishi,K., Funakoshi,A., Jimi,A., Funakoshi,S., Tamamura,H., Yajima,H. & Matsuoka,Y. (1989) Gastroenterology 97, 1313-1318. 8. Tateishi,K., Funakoshi,A., Wakasugi,H., Iguchi,H., Shinozaki,H., Abe,M., Funakoshi,S., Tamamura,H., Yajima,H. & Matasuoka,Y. (1989) J. Clin. Endocrinol. Metab. 69, 1305-1308. 9. Kaku,M., Nishiyama,T., Yagawa,K. & Abe,M. (1980) Gann 71, 596601. lO.Funakoshi,S., Tamamura,H., Ohta,M., Yoshizawa,K., Funakoshi,A., Miyasaka,K., Tateishi,K., Tatemoto,K., Nakano,I., Yajima,H. & Fujii,N. (1989) Biochem. Biophys. Res. Commun. 164, 141-148. ll.Sekiya,K., Ghatei,M.A., Minamino,N., Bretherton-Watt,D., Matsuo,H. & Bloom, S.R. (1988) FEBS Lett. 228, 153-156.
746
168,
No.
April
30,
1990
BIOCHEMICAL
2, 1990
PRODUCING
Akihiro Jimi4,
1Department 3First
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
PANCREASTATIN
Atsuo
AND
Funakoshil*, Hideyuki
CELL LINE FROM HUMAN PANCREATIC CELL TUMOR Kayoko Wakasugil,
of Gastroenterology Kyushu Cancer
Department
4Department
of
Tateishi3, Yurika
Michiyo TsuruI, Ikeda2 and Akira
and 2Research Center, Fukuoka,
Institute, Japan
of Biochemistry, School of University, Fukuoka, Japan First
741-746
Pathology,
Kurume
University,
ISLET
Kono 2 National
Medicine,Fukuoka Fukuoka,
Japan
Received March 12, 1990 SUMMARY: It has been characterized that cell line QGP-1 derived from human non-functioning pancreatic islet cell tumor produces human pancreastatin. Exponen ially growing cultures produced 5.7 ii cells/hr. fmol of pancreastatin/lU Human pancreastatin immunoreactivities in plasma and tumor after xenografting with QGP-1 into nude mouse were 92.7 fmol/ml and 160.2 pmol/g wet weight, respectively. Immunocytochemical study revealed both chromogranin A and pancreastatin immunoreactive cells in the Gel filtrations of culture medium and tumor extract tumor. identified heterogenous molecular forms of PST-L1 which eluted as large and smaller molecular species. These results suggest that plasma pancreastatin levels may be useful as a tumor marker of endocrine tumor of the pancreas, and the pancreastatin producing cell line may be useful for studies of the mechanism of secretions and processing of chromogranin A and pancreastatin. 01990
Academic
Press,
Inc.
Chromogranin A (CgA) endocrine tissues (1) pancreastatin (PST) (2,3). 52 residue peptide amide of human CgA (hCgA) as a It has been reported that and pancreatic exocrine sensitive radioimmunoassay *Address correspondence Cancer Center, Notame Abbreviations: hPST, like immunoreactivity;
is widely distributed in the neuroand proposed to be a precursor of Human PST (hPST) was deduced to be a which is contained at residues 250-301 sequence homologous to porcine PST (4). hPST could suppress insulin release (5) secretion We have developed a (6). (RIA) for hPST-like immunoreactivity
to: 3-1-1,
Dr. A. Funakoshi, Minami-ku, Fukuoka
National Kyushu 815, Japan.
human pancreastatin; PST-LI, pancreastatinhCgA, human chromogranin A. COO6-291X/90$1.50 741
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in my form reserved.
Vol.
168,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(hPST-LI) using the antiserum (R711) produced against a synthetic C-terminal fragment hPST (24-52) (7,8). In vitro and in vivo experiments are required to study the mechanism of PST secretion by use of the RIA. However, there is no reports of PST-producing cell lines. This work includes preliminary characterization of a (QGP-1N) derived from human nonPST-producing cell line functioning pancreatic islet cell tumor, hPST-LI levels in plasma and tumor after xenografting with QGP-1 into nude mouse, and immunocytochemical studies of the tumor. MATERIALS
AND METHODS
Pathology of the parent tumor The tumztissueused to initiate the culture was obtained from a subtotal pancreatectomy specimen removed from a 61-yearold male with an elevated serum carcinoembryonic antigen (-A) level, who was admitted to National Kyushu Cancer Center. The tumor was a well-circumscribed nodular lesion in the tail of the pancreas. Vascular invasion and metastases of tumor cells to the peri-pancreatic lymph nodes and to the liver were also apparent. Light microscopic examination of the tumor showed it to be a pancreatic carcinoma of islet origin. The bulk of the tumor was composed of atypical cells which showed a cord-like arrangement around many capillary vessels with slight intermingling of fibroblasts. Each cell had round nuclei and slightly eosinophilic cytoplasm. This tumor did not demonstrably secrete insulin and glucagon clinically and immunochemistry. Culture QGP-1 was established from a human pancreatic islet cell carcinoma cell line above mentioned as a CEA secreting cell line The cells were maintained with Daigo's T medium (Nihon (9). Seiyaku Co. Ltd., Tokyo, Japan) supplemented with 10 % fetal calf serum (FCS) (Gibco Lab., Grand island, N.Y.) at 37OC in a humidified atmosphere of 5 % CO2 in air. Confluent cell monolayers were passaged by treatment with trypsin-EDTA solution (0.25 % trypsin and 0.075 % EDTA in phosphate-buffered saline:PBS). Xenograft to nude mouse and establishment of line ---- the cell - WQGP-~N"
Four to six week-old female nude mice were used. The animals were housed in shared cages and kept in a laminar flow facility. Free access was allowed to an autoclaved diet and autoclaved water. Approximately 5 million cells in 0.2 ml of PBS were injected subcutaneously into right shoulder region of mice. After 60 days animals were killed, and exsanguinated by cardiac puncture. Plasma was centrifuged and stored at -7OOC for PST-L1 assay. Excised tumor was weighed, and divided into three portions, one is for histological examination and the second for evaluation of PST content. Tumor tissue was extracted in boiling 1 M acetic acid or water (7). The third portion of tumor was minced into small pieces with scissors, and was dispersed with 0.1 % collagenase (Wako Chemical, Osaka, Japan), 0.005 % DNase (Sigma, St. Louis, MO) and 0.002 % hyaluronidase (Sigma, St. Louis, MO) in Daigo's T medium. The suspension was incubated for 30 min at 37OC. The cell pellets were suspended in 2 mM EDTA and kept for 15 min at 37OC. The cell pellets were incubated again in 0.2 % collagenase for 30 min at 742
Vol.
168, No. 2, 1990
Fig.
BIOCHEMICAL
1:Phase contrast (Magnification,
AND BIOPHYSICAL
photomicrograph X300).
of
RESEARCH COMMUNICATIONS
QGP-1
cell
line
37OC. The cells were suspended in the medium. One half volume of the medium was exchanged at every 3 to 7 days. The elimination of fibroblastic cells was accomplished chemically by brief exposure to 0.05 % trypsin and 0.1 % EDTA in PBS (-1 and by mechanical scrubbing with a silicon rubber policeman. Criteria for the establishment of a cell line in continuous culture were the growth of the tumor cells to semiconfluency in the absence of contaminating cell types such as fibroblasts or lymphocytes, and repeated successful subcultures. The cell line established from nude mouse tumor was designated as QGP-1N. Figure 1 is a photomicrograph of QGP-1N cells growing as a monolayer culture. Pancreastatin radioimmunoassay An antiserum was raised against a synthetic C-terminal fragment of hPST-24-52 coupled with keyhole limpet hemocyanin in The antiserum was specific for C-terminal glycine amide rabbits. of hPST, and cross-reacted with porcine PST l-49 (3.8 %), but did not cross-react with bovine PST, insulin, glucagon, somatostatin, pancreatic polypeptide, cholecystokinin, Arg-vasopressin and gastrin. Crossreactivity of the antiserum with hCgA was not determined since hCgA was not available. However, it seemed that antiserum did not cross-react with hCgA. hPST 1-52 and ;z% I]-tyr-hPST 24-52 were used as standard and tracer, respectively. RIA procedure was described previously (7,8). The intraand interassay coefficients of variation were less than 10 %, and the sensitivity of the RIA was 5 pmol/L. In the assay for plasma PST-LI, extraction with a Sep-Pak Cl8 cartridge was needed before RIA. The extraction procedure was described previously (7,8).
Gel filtration -PST-L1 in culture medium and tissue extract were applied to a Sephadex G-50 (fine) column (1.2 x 65.5 cm), equilibrated, and eluted with 1 M acetic acid containing 0.05 % gelatin at a flow rate of 5 ml/h. Fractions of 2 ml were collected, lyophilized and reconstituted in 0.5 ml assay buffer for RIA. The column was calibrated with blue dextraf25;;;ychrome C, synthetic hPST l-52, hPST 24-52, hPST 43-52 and Immunocytochemistry Tissue was fixed in 10 % formalin and embedded in paraffin. Sections of 5 pm were incubated with antiserum (Ri'll) to hPST at 743
Vol.
168, No. 2, 1990
dilutions complex antiserum antiserum). RESULTS
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of l:lOOO, and stained by the avidin-biotin peroxidase method. Control sections were treated with specific final dilution of preabsorbed with hPST l-52 (10 yg/ml
AND DISCUSSION
The PST-L1 level was almost the same in the conditioned medium from QGP-1 and QGP-1N cells. The population-doubling time of QGP1 and QGP-1N calculated from the growth curve was about 37 hrs and 31 hrs, respectively. The modal chromosome number was 98. Exponentially growing cultures produce 5.7 fmol of PST/lo6 cells/hr. The plasma level of hPST-LI in the nude mouse with tumor implanted QGP-1 was 92.7 fmol/ml. HPST-LI was 160.2 pmol/g wet weight in the xenografted tumor. Immunocytochemical study cells in the tumor revealed both CgA- and PST-immunoreactive (Fig. 2). Gel filtrations of culture medium and acid-extract and water extract of the tumor identified heterogeneous molecular forms of PST-L1 (Fig. 3). Gel filtration of water-extract from the tumor showed two molecular forms corresponding to PST-L1 molecules which were determined as 186 amino acid residue and 48
Fig.
2:Immunocytochemical Chromogranin (Magnification,
localiz :ation of pancreastatin A (CgA) in the xenografted X100). 744
(PST) and tumor
Vol.
168, No. 2, 1990
va
Cyt
BIOCHEMICAL
PS52
PS29
PSlO
4
(8
((6
AND BIOPHYSICAL
vt
vo
60.
70
RESEARCH COMMUNICATIONS
Cvt
PS52
PS29
PSlO
4
+
vt 4
160.
QGP-1
-
culture
medium
140-
(A) 60
-
120z .g
3 .g E %
50-
ii 2
E c 7
100
-
E
‘, i
40-
60. I
k
i? 30
_
60 -
20
-
40 -
10
-
20 -
0 L,
A 10
25
30
35
40
25
Fractions
30
35
40
Fractions
Vo
Cyt
PS52
444
PS29 #
PS,O
Vt
4#
1
Fig.
residue
3:Elution profiles of PST-IS in culture medium (A), acid extract (B) and water extract (C) from the tumor on a Sephadex G-50 gel chromatography. Arrows indicate the elution volumes of markers: blue dextran (Vo), cytochrome human PS12$;:za \pVsz2), PST 24-52 c (CYt) I synthetic (PS29) I PST 43-52 (PSlO), and 1
from
insulinoma
molecule coeluted with and smaller molecules
extract
in
synthetic detected
previous
hPST 24-52 in culture 745
study in
(10).
The
the acid extract medium may be an
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
artifact produced during extraction with acid (11) and fragments produced by proteolytic cleavage during culture, respectively. The present report describes the high production of hPST-LI of a human cell line, QGP-1 established from a patient with nonfunctioning islet cell tumor. These results suggested that a high plasma level of PST-L1 in the inoculated mouse was derived from QGP-1 producing abundant PST and its precursor, and plasma PST levels may be useful as a tumor marker of non-functioning endocrine tumor of the pancreas. This cell line may be useful for studies of the mechanism of secretions and processing of CgA and PST. ACKNOWLEDGMENT This study was supported in Comprehensive lo-year Strategy of Health and Welfare.
part for
by a Grant-in Cancer Control
Aid from
for the Ministry
REFERENCES 1. Ehrhart,M., Grube,D., Bader,M.F., Aunis,D. & Gratzl, M. (1986) J. Histochem. Cytochem. 34, 1673-1682. 2. Iacangelo, A.L., Fischer-Colbrie, R., Koller, K-J., (1988) Endocrinology 122, 2339Brownstein, M.J. & Eiden, L.E. 2341. Funakoshi,A., Miyasaka,K., Ishida,K., Makk,G., 3. Nakano,I., Angwin,P., Chang, D. & Tatemoto, K (1989) Regul. Pept. 25, 207-213. 4. Konecki,D.S., Benedum, U.M., Gerdes,H.-H. & Huttner,W.B. (1987) J. Biol. Chem. 262, 17026-17030. 5. Funakoshi,A., Jimi,A., Yasunami,Y., Tateishi,K., Funakoshi,S., Tamamura,H. & Yajima,H. (1989) Biochem. Biophys. Res. Commun. 159,
913-918.
6.
Funakoshi,A., Miyasaka,K., Nakamura,R., Kitani,K., Funakoshi,S., Tamamura,H., Fujii,N. & Yajima,H. (1988) Biochem. Biophys. Res. Commun. 156, 1237-1242. 7. Tateishi,K., Funakoshi,A., Jimi,A., Funakoshi,S., Tamamura,H., Yajima,H. & Matsuoka,Y. (1989) Gastroenterology 97, 1313-1318. 8. Tateishi,K., Funakoshi,A., Wakasugi,H., Iguchi,H., Shinozaki,H., Abe,M., Funakoshi,S., Tamamura,H., Yajima,H. & Matasuoka,Y. (1989) J. Clin. Endocrinol. Metab. 69, 1305-1308. 9. Kaku,M., Nishiyama,T., Yagawa,K. & Abe,M. (1980) Gann 71, 596601. lO.Funakoshi,S., Tamamura,H., Ohta,M., Yoshizawa,K., Funakoshi,A., Miyasaka,K., Tateishi,K., Tatemoto,K., Nakano,I., Yajima,H. & Fujii,N. (1989) Biochem. Biophys. Res. Commun. 164, 141-148. ll.Sekiya,K., Ghatei,M.A., Minamino,N., Bretherton-Watt,D., Matsuo,H. & Bloom, S.R. (1988) FEBS Lett. 228, 153-156.
746
