295
Clinica Chimica Acia, 186 (1989) 295-300 Hsevicr
CCA 04601
Short Communication
Placental-like alkaline phosphatase from ovary and ascites of a patient with a malignant ovarian tumour Iwao Koyama ‘, Ulf Stendahl 2 and Torgny St&brand ’ Deparrmenrs of ’ Physiological Chemistry and ’ Oncological Gynaecology, Umed (Sweden) (Raxived
University of lJme&
14 June 1989; revision received and accepted 17 August 1989)
Key words: Placental-like
alkaline phosphatase; Monoclonal antibody; Ova&m tumor; Gynecological cancer
Human ovary; Ascitcs;
Intmhxtion Human placental alkaline phosphatase (PLAP, EC 3.1.3.1) is present on the syncytiotrophoblast membrane from the 12th week of pregnancy. Several tumour cell lines also express PLAP or the closely related PLAP-like enzyme [l]. Trace expression of these isozymes is normally seen in some tissues such as testis, lung, and thymus [2,3]. The tumour-derived isozymes are known to differ biochemically somewhat from the placental isozyme, and the tumour-derived enzymes are referred to as PLAP-like enzymes. Recently, the cloning of cDNAs from the tissue-unspecific type (AP) [4], intestinal type (IAP) [5], and PLAP [6], and also the PLAP-like or the seminoma-derived Nagao isozyme were reported, revealing 98% homology between PLAP and the PLAP-like enzyme [7]. In patients with ovarian tumours, elevated levels of PLAP or the PLAP-like enzymes have been reported [l]. It is still, however, unclear which of these isozymes that are expressed. By use of mAbs with defined epitope specificity, possible immunochemical and conformational differences of PLAP and the PLAP-like isozymes may be identified (8,9]. Materials and methods Tissues and isozyme purification The ovaries, one normal and one with a serous cystadenocarcinoma,
omentum and ascites from a 35-yr-old patient with ovarian cancer were homogenized, extracted with 50% n-butanol, and the obtained waterphase was precipitated by 60% cold-acetone to remove lipids. The precipitates were dissolved in 50 mmol/l
Correspondence S-901 87 Urn&
to: Dr. T. Stigbrand, Sweden.
0009-8981/89/$03.50
Department
of Physiological
Chemistry,
0 1989 Elsevier Science Publishers B.V. (Biomedical
University
Division)
of Ume&
2%
Tris/HCl buffered saline (TBS), pH 7.5, and dialyzed against the same TBS. All purification steps were carried out at 4°C. The crude extract from the ovary was applied on a column of anti-PLAP mAb C2 [lo] coupled to CH-Sepharose.4B and equilibrated with TBS. The enzyme was eluted with 0.2 mol/l Na,CO, containing 0.5 mol/l NaCl. The eluted active fractions were neutralized, concentrated, and applied on a Sephadex G-200 column. The crude extract from the ascites was first subjected to DEAE-cellulose chromatography and the active fractions were then subjected to the same immunoaffinity chromatography and subsequent gel filtration. Immunochemical
tests
The immunochemical similarities between PLAP and the PLAP-like isozymes from the ovary and ascites were examined with different anti-PLAP mAbs. The tumour derived enzymes and the normal PLAP phenotype, the SS variant, were exactly adjusted to catalytic activities of 100 mu/ml as determined by the MICA technique [ll] and tested with each mAb. The four mAbs, H7, C2, DlO, and Fll (InRo Biomedtek, Sweden), are all reactive with PLAP, but not with AP or IAP [lo]. H7 and C2 are known to compete in binding to a C-terminal related epitope, whereas DlO and Fll bind to the N-terminal region of PLAP [13,14]. Phosphatase assays The enzyme levels were assayed by use of isozyme specific monoclonal
antibodies, i.e. the anti PLAP mAb, HPMS-1, and anti-AP mAb, HLMS-1, respectively, as described previously [ 111. RC?SUltS
The content of the placental-type phosphatase and tissue-unspecific phosphatase (AP) in the extracts are shown in Table I. The normal ovary had low amounts not only of the PLAP-like isozyme but also of AP. The malignant ovary on the contrary contained high catalytic activities of both the PLAP-like phosphatase and AP. The ratio of the PLAP-like level compared to the total activity (PLAP-like + AP) was increased in the tumour in comparison to the normal ovary and amounted to 45%. Ascites from the same patients also contained high amounts of the PLAP-like isozyme. Since ascites contains large amounts of proteins, the specific activity was low (1.9 mU/mg), but the total activity of the PLAP-like activity in ascites was very high (147 mu/ml). The omentum obtained from the same patient did not contain significant levels of the PLAP-like isozyme, constituting only 6.0% of the total phosphatase activity. In order to further evaluate the immunochemical properties of the enzymes derived from the different sources, the isozymes were purified using immunoaffinity chromatography. The purified enzymes from the malignant ovary and ascites had high and similar specific activities of 130 and 110 U/mg protein, respectively. The purified enzymes reacted only with the anti-PLAP mAbs, but not with anti-AP or anti-IAP mAbs,
291 TABLE I Content of alkaline phosphatase isozymes in normal and tumour-related tissues a Enzyme source
Relative content of PLAP-like (W)
Activity (mU/mg protein) PLAP-like
AP
PLAP-like PLAP-like + AP
Normal ovary
1.27 1.44 12.9 0.57 (44.2 b) 34.2
0.14 0.13 10.6 1.90 (147 b) 2.2
Malignant ovary Ascites from malignant ovary Omentum
9.9 17.1 45.1 76.9 6.0
a The crude extract was incubated with the HPMS-1 or the HLMS-1 bound to a paper disc for 3 h at 37 a C, followed by washing three times with TBS. The activity of the PLAP-like isozyme and AP trapped on the paper disc was assayed using p-nitrophenylphosphate as substrate. One unit of activity was defined as 1 pmol of substrate hydrolyzed per min using the molar absorption of 1.87 x lo4 for p-nitrophenolate at 405 nm. b U; unit/ml ascites.
1.0
0.5
-z 8 ;
O
2 B :z 5
F 11
1.0
D 10 afA
8 h ! 0.5 w
0
I
0.01
0.1
1.0
10
Concentration
of antibody (Wml)
Fig. 1. Reactivities of PLAP and the PLAP-like isozyme from the ovarian tumour with different mAbs. Anti-mouse IgG antibodies were coated in a 96 well microtiter plate, followed by washing three times with TBS containing 0.05% Tween 20. 100 ~1 of serially diluted mAbs were added to each well and allowed to react for 3 h at 37 ’ C. After washing. 100 pl of PLAP (A),the PLAP-like enzyme derived from the tumour (O), or the ascites (0) adjusted to 100 mu/ml, was added to each well and incubated for 12 h at 4°C: After washing, the activity was determined in 200 pl of p-nitrophenylphosphate at 405 nm. Each point represents means of triplicate experiments.
298
indicating that the purified isozymes were not contaminated by AP which is the dominating isozyme in the ovary. PLAP from placenta and the PLAP-like enzyme from tumours can be distinguished by their inhibition profiles by L-leucine {12]. The purified enzymes from the ovary and the ascites were both more inhibited than normal PLAP, in particular at low concentrations of L-leucine. Furthermore, the mobilities of the isozymes derived from the ovary were distinctly different from those of the common phenotypes of PLAP (SS and FF) (results not shown). The immunochemical reactivities of the purified isozymes were tested with the different monoclonal anti-PLAP antibodies. As shown in Fig. 1, the immunoreactivities of the PLAP-like enzymes from the ovary and ascites were lower than with the PLAP derived from placenta and especially with mAb DlO. Discussion The patterns of isozyme distribution of alkaline phosphatases were investigated in the normal ovary, a serous cystadenocarcinoma of the ovary, ascites and omentum from the same patient in order to evaluate the expression of the different isozymes as detected by isozyme specific monoclonal antibodies. The dominating isozyme in the normal ovary is AP (90%) with trace expression of the PLAP-like enzyme (10%). In the serous cystadenocarcinoma a lo-fold increase of the AP isozyme was seen and a lOO-fold increase in the PLAP-like expression as expressed in mU/mg protein. The tumour derived isozyme is also the PLAP-like isozyme as evidenced by leucine inhibition and by reactivity with the mAbs. This indicates that the appearance of the PLAP-like isozyme in the tumour is due to an enhanced eutopic expression of the isozyme found in the normal ovary. Ascites from the same patient was also found to contain high levels of PLAP amounting to almost 80% of the total catalytical activity, whereas the omentum was mainly expressing AP (94%). The immunochemical reactivities of the purified isozymes were compared to the placentally derived PLAP by monoclonal antibodies, which identified immunochemical differences between PLAP and the PLAP-like isozymes. In particular, DlO had poor reactivity with the PLAP-like enzyme, which is in agreement with the results obtained for the PLAFlike enzyme derived from seminomas [15]. The epitope recognized by DlO may be hidden or sterically distorted in the PLAP-like isozyme. Since DlO is reactive with an epitope in the N-terminal region of PLAP [ll], the structural differences between the isozymes may be confined to this part of the molecule. The reactivities of the isozyme from ascites with the mAbs were somewhat lower than those of the ovarian isozyme. The major findings, however, is that the PLAP-like isozymes derived from the tumour or the ascites exhibited similar reactivity profiles with the four mAbs, indicating no detectable immunochemical differences between these PLAP-like enzymes. The immunochemical reactivity of the PLAP-like isozyme digested with trypsin or bromelain which cause defined cleavages in the amino- and carboxy-terminal part of the molecule, respectively, in the presence of the mAbs were different from those of intact PLAP (results not shown). This suggests that the PLAP-like isozyme
299
appearing in ascites is not released from the tumour by proteolysis. The enzyme from ascites exhibited the same mobility as the enzyme from the tumour in SDS-polyacrylamide gel electrophoresis (results not shown). In order to obtain crude material for the purification of the PLAP-like enzyme from malignancies, ascites could thus serve as starting material. Furthermore, the relative content of the PLAP-like enzyme in the tumour was 45% but as much as 77% in the ascites (Table I) indicating that the PLAP-like enzyme is released from the tumour to a higher extent than the AP is. This observation is of importance regarding the significance of these antigens as tumour markers. Ascites furthermorr: is a convenient source of starting material for purification of the tumour deriveci isozyme. References 1 Fishman WH. Gncotrophoblast gene expression. Placental alkaline phosphatase. Adv Cancer Res 1987;48:1-35. 2 Chang CH, Angellis, D. Fishman WH. Presence of the rare D-variant heat stable placental type alkaline phosphatase in normal testis. Cancer Res 1984;40:1506-1510. 3 Goldstein DJ, Rogers CE, Harris H. A research for trace expression of placental-like alkaline phosphatase in non-malignant human tissue: Demonstration of its occurrence in lung, cervix, testis, and thymus. Clin Chim Acta 1982;125:63-75. 4 Weiss MJ, Henthom PS, Lafferty MA, Slaughter C, Raducha M, Harris H. Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase. Proc Nat1 Acad Sci USA 1986;83:7182-7186. 5 Berger J, Garanttini E, Hua J-C, Udenfriend S. Cloning and sequence of human intestinal alkaline phosphatase cDNA. Proc Nat1 Acad Sci USA 1987;84:695-698. 6 Mill&n JL. Molecular cloning and sequence analysis of human placental alkaline phosphatase. J Biol Chem 1986;261:3112-3115. 7 Mill&n JL, Manes T. Seminoma-derived nagao isozyme is encoded by a germ-cell alkaline phosphatase gene. Proc Nat1 Acad Sci USA 1988;85:3024-3028. 8 Mill&n JL, Stigbrand T, Ruoslahti E, Fishman WH. Characterization and use of an allotype-specific monoclonal antibody to placental alkaline phosphatase in the study of cancer-related phosphatase polymorphism. Cancer Res 1982;42:2444-2449. 9 Stigbrand T, Jemmerson R, Mill&n JL, Fishman WH. A hidden antigenic determinant on membranebound human placental alkaline phosphatase. Tumor Biol 1987;8:34-44. 10 Mill&n JL, Stigbrand T. Antigenic determinants of human placental and testicular placental-like alkaline phosphatases as mapped by monoclonal antibodies. Eur J Biochem 1983;136:1-7. 11 Hirano K, Matsumoto H, Tanaka T, Iino S, Domar U, Stigbrand T. Specific assays for human alkaline phosphatase isozymes. Clin Chim Acta 1987;166:265-273. 12 Nakayama T, Kitamura M. L-Leucine sensitive alkaline phosphatase isozyme from cancer tissue. Ann NY Acad Sci 1975;259:325-336. 13 Jemmerson R, Stigbrand T. Monoclonal antibodies block the trypsin cleavage site on human placental alkaline phosphatase. FEBS Lett 1984;173:357-359. 14 Jemmerson R, Mill&n JL, Kher FG, Fishman WH. Monoclonal antibodies block the bromelain-mediated release of human placental alkaline phosphatase from cultured cancer cells. FEBS Lett 1985;179:316-320. 15 Hirano K, Domar UM, Y amamoto H, Brehmer-Andersson EE, Wahren BE, Stigbrand TI. Levels of alkaline phosphatase isozymes in human seminoma tissue. Cancer Res 1987;47:2543-2546.
Clinica Chimica Acia, 186 (1989) 295-300 Hsevicr
CCA 04601
Short Communication
Placental-like alkaline phosphatase from ovary and ascites of a patient with a malignant ovarian tumour Iwao Koyama ‘, Ulf Stendahl 2 and Torgny St&brand ’ Deparrmenrs of ’ Physiological Chemistry and ’ Oncological Gynaecology, Umed (Sweden) (Raxived
University of lJme&
14 June 1989; revision received and accepted 17 August 1989)
Key words: Placental-like
alkaline phosphatase; Monoclonal antibody; Ova&m tumor; Gynecological cancer
Human ovary; Ascitcs;
Intmhxtion Human placental alkaline phosphatase (PLAP, EC 3.1.3.1) is present on the syncytiotrophoblast membrane from the 12th week of pregnancy. Several tumour cell lines also express PLAP or the closely related PLAP-like enzyme [l]. Trace expression of these isozymes is normally seen in some tissues such as testis, lung, and thymus [2,3]. The tumour-derived isozymes are known to differ biochemically somewhat from the placental isozyme, and the tumour-derived enzymes are referred to as PLAP-like enzymes. Recently, the cloning of cDNAs from the tissue-unspecific type (AP) [4], intestinal type (IAP) [5], and PLAP [6], and also the PLAP-like or the seminoma-derived Nagao isozyme were reported, revealing 98% homology between PLAP and the PLAP-like enzyme [7]. In patients with ovarian tumours, elevated levels of PLAP or the PLAP-like enzymes have been reported [l]. It is still, however, unclear which of these isozymes that are expressed. By use of mAbs with defined epitope specificity, possible immunochemical and conformational differences of PLAP and the PLAP-like isozymes may be identified (8,9]. Materials and methods Tissues and isozyme purification The ovaries, one normal and one with a serous cystadenocarcinoma,
omentum and ascites from a 35-yr-old patient with ovarian cancer were homogenized, extracted with 50% n-butanol, and the obtained waterphase was precipitated by 60% cold-acetone to remove lipids. The precipitates were dissolved in 50 mmol/l
Correspondence S-901 87 Urn&
to: Dr. T. Stigbrand, Sweden.
0009-8981/89/$03.50
Department
of Physiological
Chemistry,
0 1989 Elsevier Science Publishers B.V. (Biomedical
University
Division)
of Ume&
2%
Tris/HCl buffered saline (TBS), pH 7.5, and dialyzed against the same TBS. All purification steps were carried out at 4°C. The crude extract from the ovary was applied on a column of anti-PLAP mAb C2 [lo] coupled to CH-Sepharose.4B and equilibrated with TBS. The enzyme was eluted with 0.2 mol/l Na,CO, containing 0.5 mol/l NaCl. The eluted active fractions were neutralized, concentrated, and applied on a Sephadex G-200 column. The crude extract from the ascites was first subjected to DEAE-cellulose chromatography and the active fractions were then subjected to the same immunoaffinity chromatography and subsequent gel filtration. Immunochemical
tests
The immunochemical similarities between PLAP and the PLAP-like isozymes from the ovary and ascites were examined with different anti-PLAP mAbs. The tumour derived enzymes and the normal PLAP phenotype, the SS variant, were exactly adjusted to catalytic activities of 100 mu/ml as determined by the MICA technique [ll] and tested with each mAb. The four mAbs, H7, C2, DlO, and Fll (InRo Biomedtek, Sweden), are all reactive with PLAP, but not with AP or IAP [lo]. H7 and C2 are known to compete in binding to a C-terminal related epitope, whereas DlO and Fll bind to the N-terminal region of PLAP [13,14]. Phosphatase assays The enzyme levels were assayed by use of isozyme specific monoclonal
antibodies, i.e. the anti PLAP mAb, HPMS-1, and anti-AP mAb, HLMS-1, respectively, as described previously [ 111. RC?SUltS
The content of the placental-type phosphatase and tissue-unspecific phosphatase (AP) in the extracts are shown in Table I. The normal ovary had low amounts not only of the PLAP-like isozyme but also of AP. The malignant ovary on the contrary contained high catalytic activities of both the PLAP-like phosphatase and AP. The ratio of the PLAP-like level compared to the total activity (PLAP-like + AP) was increased in the tumour in comparison to the normal ovary and amounted to 45%. Ascites from the same patients also contained high amounts of the PLAP-like isozyme. Since ascites contains large amounts of proteins, the specific activity was low (1.9 mU/mg), but the total activity of the PLAP-like activity in ascites was very high (147 mu/ml). The omentum obtained from the same patient did not contain significant levels of the PLAP-like isozyme, constituting only 6.0% of the total phosphatase activity. In order to further evaluate the immunochemical properties of the enzymes derived from the different sources, the isozymes were purified using immunoaffinity chromatography. The purified enzymes from the malignant ovary and ascites had high and similar specific activities of 130 and 110 U/mg protein, respectively. The purified enzymes reacted only with the anti-PLAP mAbs, but not with anti-AP or anti-IAP mAbs,
291 TABLE I Content of alkaline phosphatase isozymes in normal and tumour-related tissues a Enzyme source
Relative content of PLAP-like (W)
Activity (mU/mg protein) PLAP-like
AP
PLAP-like PLAP-like + AP
Normal ovary
1.27 1.44 12.9 0.57 (44.2 b) 34.2
0.14 0.13 10.6 1.90 (147 b) 2.2
Malignant ovary Ascites from malignant ovary Omentum
9.9 17.1 45.1 76.9 6.0
a The crude extract was incubated with the HPMS-1 or the HLMS-1 bound to a paper disc for 3 h at 37 a C, followed by washing three times with TBS. The activity of the PLAP-like isozyme and AP trapped on the paper disc was assayed using p-nitrophenylphosphate as substrate. One unit of activity was defined as 1 pmol of substrate hydrolyzed per min using the molar absorption of 1.87 x lo4 for p-nitrophenolate at 405 nm. b U; unit/ml ascites.
1.0
0.5
-z 8 ;
O
2 B :z 5
F 11
1.0
D 10 afA
8 h ! 0.5 w
0
I
0.01
0.1
1.0
10
Concentration
of antibody (Wml)
Fig. 1. Reactivities of PLAP and the PLAP-like isozyme from the ovarian tumour with different mAbs. Anti-mouse IgG antibodies were coated in a 96 well microtiter plate, followed by washing three times with TBS containing 0.05% Tween 20. 100 ~1 of serially diluted mAbs were added to each well and allowed to react for 3 h at 37 ’ C. After washing. 100 pl of PLAP (A),the PLAP-like enzyme derived from the tumour (O), or the ascites (0) adjusted to 100 mu/ml, was added to each well and incubated for 12 h at 4°C: After washing, the activity was determined in 200 pl of p-nitrophenylphosphate at 405 nm. Each point represents means of triplicate experiments.
298
indicating that the purified isozymes were not contaminated by AP which is the dominating isozyme in the ovary. PLAP from placenta and the PLAP-like enzyme from tumours can be distinguished by their inhibition profiles by L-leucine {12]. The purified enzymes from the ovary and the ascites were both more inhibited than normal PLAP, in particular at low concentrations of L-leucine. Furthermore, the mobilities of the isozymes derived from the ovary were distinctly different from those of the common phenotypes of PLAP (SS and FF) (results not shown). The immunochemical reactivities of the purified isozymes were tested with the different monoclonal anti-PLAP antibodies. As shown in Fig. 1, the immunoreactivities of the PLAP-like enzymes from the ovary and ascites were lower than with the PLAP derived from placenta and especially with mAb DlO. Discussion The patterns of isozyme distribution of alkaline phosphatases were investigated in the normal ovary, a serous cystadenocarcinoma of the ovary, ascites and omentum from the same patient in order to evaluate the expression of the different isozymes as detected by isozyme specific monoclonal antibodies. The dominating isozyme in the normal ovary is AP (90%) with trace expression of the PLAP-like enzyme (10%). In the serous cystadenocarcinoma a lo-fold increase of the AP isozyme was seen and a lOO-fold increase in the PLAP-like expression as expressed in mU/mg protein. The tumour derived isozyme is also the PLAP-like isozyme as evidenced by leucine inhibition and by reactivity with the mAbs. This indicates that the appearance of the PLAP-like isozyme in the tumour is due to an enhanced eutopic expression of the isozyme found in the normal ovary. Ascites from the same patient was also found to contain high levels of PLAP amounting to almost 80% of the total catalytical activity, whereas the omentum was mainly expressing AP (94%). The immunochemical reactivities of the purified isozymes were compared to the placentally derived PLAP by monoclonal antibodies, which identified immunochemical differences between PLAP and the PLAP-like isozymes. In particular, DlO had poor reactivity with the PLAP-like enzyme, which is in agreement with the results obtained for the PLAFlike enzyme derived from seminomas [15]. The epitope recognized by DlO may be hidden or sterically distorted in the PLAP-like isozyme. Since DlO is reactive with an epitope in the N-terminal region of PLAP [ll], the structural differences between the isozymes may be confined to this part of the molecule. The reactivities of the isozyme from ascites with the mAbs were somewhat lower than those of the ovarian isozyme. The major findings, however, is that the PLAP-like isozymes derived from the tumour or the ascites exhibited similar reactivity profiles with the four mAbs, indicating no detectable immunochemical differences between these PLAP-like enzymes. The immunochemical reactivity of the PLAP-like isozyme digested with trypsin or bromelain which cause defined cleavages in the amino- and carboxy-terminal part of the molecule, respectively, in the presence of the mAbs were different from those of intact PLAP (results not shown). This suggests that the PLAP-like isozyme
299
appearing in ascites is not released from the tumour by proteolysis. The enzyme from ascites exhibited the same mobility as the enzyme from the tumour in SDS-polyacrylamide gel electrophoresis (results not shown). In order to obtain crude material for the purification of the PLAP-like enzyme from malignancies, ascites could thus serve as starting material. Furthermore, the relative content of the PLAP-like enzyme in the tumour was 45% but as much as 77% in the ascites (Table I) indicating that the PLAP-like enzyme is released from the tumour to a higher extent than the AP is. This observation is of importance regarding the significance of these antigens as tumour markers. Ascites furthermorr: is a convenient source of starting material for purification of the tumour deriveci isozyme. References 1 Fishman WH. Gncotrophoblast gene expression. Placental alkaline phosphatase. Adv Cancer Res 1987;48:1-35. 2 Chang CH, Angellis, D. Fishman WH. Presence of the rare D-variant heat stable placental type alkaline phosphatase in normal testis. Cancer Res 1984;40:1506-1510. 3 Goldstein DJ, Rogers CE, Harris H. A research for trace expression of placental-like alkaline phosphatase in non-malignant human tissue: Demonstration of its occurrence in lung, cervix, testis, and thymus. Clin Chim Acta 1982;125:63-75. 4 Weiss MJ, Henthom PS, Lafferty MA, Slaughter C, Raducha M, Harris H. Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase. Proc Nat1 Acad Sci USA 1986;83:7182-7186. 5 Berger J, Garanttini E, Hua J-C, Udenfriend S. Cloning and sequence of human intestinal alkaline phosphatase cDNA. Proc Nat1 Acad Sci USA 1987;84:695-698. 6 Mill&n JL. Molecular cloning and sequence analysis of human placental alkaline phosphatase. J Biol Chem 1986;261:3112-3115. 7 Mill&n JL, Manes T. Seminoma-derived nagao isozyme is encoded by a germ-cell alkaline phosphatase gene. Proc Nat1 Acad Sci USA 1988;85:3024-3028. 8 Mill&n JL, Stigbrand T, Ruoslahti E, Fishman WH. Characterization and use of an allotype-specific monoclonal antibody to placental alkaline phosphatase in the study of cancer-related phosphatase polymorphism. Cancer Res 1982;42:2444-2449. 9 Stigbrand T, Jemmerson R, Mill&n JL, Fishman WH. A hidden antigenic determinant on membranebound human placental alkaline phosphatase. Tumor Biol 1987;8:34-44. 10 Mill&n JL, Stigbrand T. Antigenic determinants of human placental and testicular placental-like alkaline phosphatases as mapped by monoclonal antibodies. Eur J Biochem 1983;136:1-7. 11 Hirano K, Matsumoto H, Tanaka T, Iino S, Domar U, Stigbrand T. Specific assays for human alkaline phosphatase isozymes. Clin Chim Acta 1987;166:265-273. 12 Nakayama T, Kitamura M. L-Leucine sensitive alkaline phosphatase isozyme from cancer tissue. Ann NY Acad Sci 1975;259:325-336. 13 Jemmerson R, Stigbrand T. Monoclonal antibodies block the trypsin cleavage site on human placental alkaline phosphatase. FEBS Lett 1984;173:357-359. 14 Jemmerson R, Mill&n JL, Kher FG, Fishman WH. Monoclonal antibodies block the bromelain-mediated release of human placental alkaline phosphatase from cultured cancer cells. FEBS Lett 1985;179:316-320. 15 Hirano K, Domar UM, Y amamoto H, Brehmer-Andersson EE, Wahren BE, Stigbrand TI. Levels of alkaline phosphatase isozymes in human seminoma tissue. Cancer Res 1987;47:2543-2546.
