[CANCER RESEARCH 36, 2268-2273, July 1976]

Developmental Phase-specific Alkaline Phosphatase Isoenzymes of Human Placenta and Their Occurrence in Human Cancer

Lillian Fishman, 2 Haruhiko Miyayama, Shirley G. Driscoll, and William H. Fishman 3

Tufts Cancer Research Center and the Department of Pathology, Tufts University School of Medicine [L. F., H. M., W. H. F.]; Department of Pathology, Harvard Medical School IS. G. D.]; and Boston Hospital for Women, Boston, Massachusetts 02111 IS. G. D.]

SUMMARY

Alkal ine phosphatase electrophoret ic patterns character- istic of three phases in early human trophoblast develop- ment are described in this prel iminary communicat ion. Phase 1 (6 to 10 weeks) consists entirely of two heat-sensi- tive, L-homoarginine- inhibi ted bands, the slower one of which possesses ant igenic determinants of l iver-bone-type alkaline phosphatase, whereas the fast band lacks any of the known alkaline phosphatase antigenic determinants. Phase 2 pattern (11 to 13 weeks) is that of a mixture of Phase 1 and Phase 3 isozyme components, the latter ex- hibit ing two isozyme bands with the characterist ics of term placental alkaline phosphatase. These three patterns of de- velopmental phase-specif ic placental alkal ine phospha- tases correspond in order to non-Regan isoenzyme, a mix- ture of Regan and non-Regan isozymes and Regan isoen- zyme in a variety of human cancer tissues. The biochemical prof i le characterist ic of t rophoblast developmental Phase 1 alkaline phosphatase is expressed as 78.5% heat-sensitive inhibi t ion (5 rain at 65~ 66.3% L-homoarginine inhibi t ion, and 17.3% L-phenylalanine inhibi t ion where n = 12. It is hypothesized that the alkaline phosphatase of human tumor tissues reflects the expression of placental genes corre- sponding to one or more phases of t rophoblast ic develop- ment.

INTRODUCTION

Antigens normal ly present in early embryonic and fetal life are produced by mal ignant cells. For instance, o~-feto- protein of the yolk sac (11) is detectable in yolk sac tumors (17) and in human and experimental hepatoma and terato- carc inoma (1). Also, human chorionic gonadotropin is pro- duced as one would expect in chor iocarc inoma, but also by nontrophoblast ic neoplasms such as cancers of the lung, testes, etc. (2). Another developmental protein antigen, pla- cental-type alkaline phosphatase [Regan isoenzyme, (8, 9)]

' Supported in part by Grants CA-13332 and CA-12924 from the National Cancer Institute, NIH, USPHS, Bethesda, Md. The support of the Council for Tobacco Research (Grant 935-M) is acknowledged. This paper was pre- sented at the 66th Annual Meeting of the American Association for Cancer Research in San Diego, Calif., May 8 to 11, 1975 (6).

" To whom requests for reprints should be addressed, at 136 Harrison Avenue, Boston, Mass. 02111.

:~ Recipient of Cancer Research Award K6-18453 of the National Cancer Institute. Part of this study was carried out at the University of California at Los Angeles during the tenure of a Visiting Professorship of Pathology in the Department of Pathology.

Received August 25, 1975; accepted March 15, 1976.

has been reported in bronchogenic carcinoma, cancer of the ovary, and other mal ignant neoplasms.

When such proteins appear in tumors, they may indicate the reexpression of a gene set characteristic of a particular event in early development. This information may be valua- ble in classifying stages in neoplastic progression.

The original purpose of this investigation has been to identify the phase in trophoblast development that elicits isoenzymes of the term placental type such as Regan isoen- zyme. However, in the earliest (8 to 10 weeks) placentas, the isoenzyme pattern as examined by biochemical, electropho- retic, and immunological criteria revealed the presence of a species that we had observed earlier as non-Regan (nonplao cental) alkaline phosphatase in ovarian cancer (15). This isoenzyme species was localized on the microvi l l i of the early placentas on the basis of enzyme histochemical and immunohistochemical studies. Subsequently, the term pla- cental isoenzyme appeared and increased in activity while the early placental type diminished. Most interesting was the appearance in early placenta of an unusual alkaline phosphatase isoenzyme that did not have any of the anti- genic determinants known for placental, intestinal, liver, and bone preparations.

From this information, it is now possible to show the patterns characteristic of 3 phases in t rophoblast ic develop- ment with respect to alkaline phosphatase. In each phase, we present an example of its occurrence in human cancer that provides a guide for interpreting tumor alkaline phos- phatases as reflections of the activation of phase-specific t rophoblast ic genes.

MATERIALS AND METHODS

Reagents. The suppliers, addresses, and chemicals are: Dajac, Philadelphia, Pa.: variamine blue 4-amino diphenyla- mine diazonium salt No. 5331. Fisher Scienti f ic Co., Bos- ton, Mass.: potassium ferr icyanide ACS, d isodium phenyl phosphate. Gelman Instrument Co., Ann Arbor, Mich.: high- resolution buffer, pH 8.8 (Tris barbital-sodium barbital). Nutrit ional Biochemicals Corp., Cleveland, Ohio: (x-naph- thyl acid phosphate (sodium), L-homoarginine (HCI). Schwarz/Mann, Orangeburg, N. Y.: Tris-base ultra pure. Sigma Chemical Co., St. Louis, Mo.: L-phenylalanine (Sigma grade), naphthol AS-MX phosphoric acid, disodium salt, glycerol gelatin. Winthrop Laboratories, New York, N. Y.: 4-aminoantipyrene.

Early human placentas from 8 to 16 weeks of gestation were washed free of blood with cold 0.9% NaCI solution and

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were homogenized individual ly by hand in 0.05 M Tris-HCI buffer, pH 8.6 (approximately 30% w/v), using a Duall ho- mogenizer. The homogenates were centri fuged in an Inter- national cl inical centr i fuge for 2 min at 2000 rpm. The supernatant was assayed for alkaline phosphatase (pH 9.8) activity according to the methods of Fishman and Ghosh (7) and Lin and Fishman (13). Condit ions for measuring inhibi- t ion by L-phenylalanine and L-homoarginine are those used previously (3). Heat inactivation was carried out for 5 min at 65 ~ . Units are defined as ~g phenol per ml per 15 min released from phenyl phosphate. Isoenzyme electrophore- sis was done on microzone, cellulose acetate membranes according to the method of Inglis et al. (12) using Gelman high-resolut ion buffer, pH 8.8, as the chamber buffer and naphthol AS-MX phosphoric acid as the substrate. The uni- form range of enzyme units applied in the experiments il lustrated in Figs. 3, 4, and 5 are shown in the tabulated portion of Fig. 3.

Histochemistry. The condit ions for evaluating the nature of the alkaline phosphatases in placental tissue sections were those of Sasaki and Fishman (15) who used the L- phenylalanine inhibi tor for conf i rming Regan isoenzyme and L-homoarginine for non-Regan, except that the sub- strate concentrat ion was 4.4 mM and variamine blue was substituted for fast blue BBN.

Immunohistochemistry. Peroxidase-labeled antiserum to term alkaline phosphatase was applied to fixed frozen sec- tions of early placenta as described by Miyayama et al. (14). These were then examined for the presence of the term placental isoenzyme.

RESULTS

Histochemical Evidence. The objective was to demon- strate the cell locus of the liver-bone-type alkaline phospha- tase in an 8-week placenta that had no biochemical indica- tion of term placental alkaline phosphatase but did exhibit heat-sensitive, homoarginine- inhibi ted activity. Fig. 1 shows that the isoenzyme activity is exclusively associated

Phase-specific Placental Alkaline Phosphatase Isoenzymes

with the microvi l l i by azo staining technique and that it exhibited complete inhibi t ion by L-homoarginine, whi le still exhibi t ing substantial activity in the presence of L-phenylal- anine. No evidence in this placenta of term placental alka- line phosphatase was obtained with peroxidase-labeled an- t ibody to term placental alkaline phosphatase under condi- t ions in which this reagent gave positive results with term placenta (Fig. 2).

Biochemical Studies. From 8 to 16 weeks of gestation (n = 8), the concentrat ion of total placental alkal ine phospha- tase increases l inearly from 150 to a value of approximately 1000 units/g, wet weight, which is about one-twenty-f i f th the concentrat ion value of term placenta. Data on 6- to 10- week placental tissue in Table 1 are given for heat stabil i ty and L-phenylalanine and L-homoarginine inhibi t ion.

Electrophoretic Migration. In Fig. 3, the results of micro- zone cellulose acetate electrophoresis showing alkaline phosphatase zones can be correlated with biochemical in- format ion on the inhibi t ion by heat and amino acids ob- tained from early placental sonic extracts arranged in order of durat ion of gestation.

At 8 weeks, 2 bands were visible, A and B, representing predominant ly heat- and L-homoarginine-sensit ive alkaline phosphatase with low inhibi t ion by L-phenylalanine (Speci- men 1). This pattern is classified as Phase 1.

The later placentas of 10, 12, and 13 weeks (Specimens 2, 3, and 4) exhibited a 3rd band, C, which migrated most slowly. (Band A in Specimen 2 appeared to have 2 compo- nents.) Biochemical ly, the total alkal ine phosphatase is made up of components of intermediate heat stabil i ty and L- phenylalanine and L-homoarginine inhibi t ion, of which Band B is presumed to be entirely heat sensitive and L- homoargin ine inhibited (see below). Such patterns are rep- resentative of Phase 2.

Phase 3 was characterized by a migrat ion pattern of 2 bands (Specimen 5, 15.5 weeks), typical of the molecular weight Variants A and B of mature placenta and exhibi t ing their heat stabil ity and high inhibi t ion by L-phenylalanine.

Immunoelectrophoresis (Figs. 4 and 5; Tables 1 and 2).

Table 1 Heat inactivation and amino acid inhibition of early placental alkaline phosphatase

Alkaline phosphatase

Total" (/~g Heat inactivation Inhibition by 0.008 Inhibition by 0.005 phenol/ml/15 for 5 min at 65 ~ M L-homoarginine M L-phenylalanine

Wk of gestation min) (%) (%) (%) 6 178 87 74 10 6 248 84 70 19 6 170 81 70 14

7-8 101 73 65 18 8 128 69 55 19 8 230 82 71 20 8 258 88 70 6 8 214 76 71 32 9 185 70 59 15 9 110 90 80 16

10 90 62 51 28 10 350 80 60 10

Mean • S.D. 188.5 +- 76.6 78.5 +_ 8.6 66.3 +_ 8.5 17.3 +_ 7.4

" Containing 5 mM o-phenylalanine. Each of these 12 extracts exhibited Phase 1 isoenzyme pattern on microzone electrophoresis. All homogenates were sonicatly disrupted in the cold for 1 minute with the Biosonic set at "Lo," 60%.

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L. Fishman et al.

The pr inciple of retardation of antigen in the presence of specif ic ant ibody was used as il lustrated for Phase 1 alka- line phosphatase in Fig. 4. Thus, enzyme-containing super- natants of centr i fuged placental homogenates were mixed in the proper proport ion with specific antisera raised against various species of alkaline phosphatase (3, 4, 10). The combinat ion of antigen and antibody was marked by the formation of a complex the migrat ion of which was retarded as compared with that of the antigen alone. One notes that Band B was retarded by antiserum to liver alka- line phosphatase but not by antiserum to intestinal or pla- cental alkaline phosphatase.

In Phase 1 and Phase 2 preparations (Table 2), the fast band did not react with antisera produced against alkaline phosphatase of human liver, bone, intestine, and mature placenta as well as antisera to alkaline phosphatase of bone sarcoma and to 2 HeLa cell lines, TCRC-1 and TCRC-2, which were cloned from the wild type; the former contained Regan and the latter contained non-Regan alkaline phos- phatase (14).

The B band that appeared in extracts of Phase 1 and Phase 2 placentas cross-reacted with antiserum to bone, liver, bone sarcoma, and TCRC-2.

In Phase 1, no cross-reaction appeared with antiserum to mature placental alkaline phosphatase. In Phase 2, the C band was retarded by this antiserum and antiserum to TCRC-I . Thus, it was possible to characterize Bands B and C immunolog ica l ly in Phases 1 and 2, but Band A did not appear to be immunologica l ly similar to any type of alkaline phosphatase that we have tested thus far.

In Phase 3, all the alkaline phosphatase is retarded by ant iserum to mature placental alkaline phosphatase, the fast and slow bands now corresponding to the molecular weight variants A and B described by Fishman and Ghosh (7). (Similar f indings were seen with antiserum to TCRC-1 .)

A summary of the f indings relating developmental phase with the biochemical and antigenic propert ies of placental and human tumor alkaline phosphatase appears in Table 2.

D I S C U S S I O N

Using biochemical , immunological , and electrophoret ic techniques on extracts of 8 early placentas, we found the

alkaline phosphatase of 8- to 10-week placentas to be differ- ent from that of mature placenta. At about 11 to 13 weeks, the latter isoenzyme type appears and by about 14 to 16 weeks all the alkaline phosphatase exhibits the properties of term placenta. The earliest placental isoenzyme on elec- trophoresis contained a fast-migrating moiety which did not resemble immunological ly the liver, intestinal, bone, or term placenta isoenzymes. Also, the total enzyme activity was all heat sensitive and was inhibited by L-homoarginine, but not by L-phenylalanine, indicating nonterm placental type. By the end of the 1st trimester, the enzyme activity had become heat stable and could be inhibited by L-phenylala- nine, properties of term placental-type alkaline phospha- tase.

The init iation of alkaline phosphatase product ion in the early trophoblast and the subsequent change to another form characteristic of mature placenta could be interpreted as the expression of 2 separate genes, one being "turned- off" whi le the other was being " turned-on," or the meta- bolic modif icat ion of a protein control led by 1 gene. Re- gardless of the ult imate explanation, we can profit from our abil i ty to dist inguish definite phases in the development of this isoenzyme during gestation.

Placental alkaline phosphatase is produced by certain neoplasms. An isoenzyme of alkaline phosphatase indistin- guishable immunological ly and biochemical ly from term placenta was first detected in a cancer of the lung of a male patient named Regan. Subsequently, Regan alkaline phos- phatase (term placenta) has been found in a variety of tumors, mainly of the ovary, testis, pancreas, etc.

Many other tumors produce alkaline phosphatase with properties that are unlike the isoenzyme of mature placenta, the so-called non-Regan isoenzymes. For instance, chorio- carcinoma (5), some ovarian tumors (15), etc., contain alka- line phosphatase resembling the liver-bone type which is L- homoarginine and heat sensitive and cross-reacts with anti- serum raised against the liver isoenzyme. In addit ion, these tumor isoenzymes react with antiserum to TCRC-2 HeLa cells (non-Regan), as well as to antiserum to bone sarcoma.

This similari ty of the early placental alkal ine phosphatase to that l iver-bone type was noted in that it reacts with antiserum to liver and is L-homoarginine sensitive. In addi- t ion, the former contains antigen sites in common with

Table 2 Immunological characteristics of 1st-trimester placental alkaline phosphatase

Reaction a with antiserum to alkaline phosphatase of

HeLa cell HeLa cell Microzone Term pla- line TCRC- line TCRC- Bone sar-

bands Intestine Liver" centa 1" 2 c coma

Phase 1,n = 3 8-10 wk

Phase 2, n = 3 11-13 wk

Phase 3, n = 2 14-16 wk

A 0 0 0 0 0 0 B 0 + 0 0 + +

A 0 0 0 0 0 0 B 0 + 0 0 + + C 0 0 + + 0 0

A 0 0 + + 0 0 C 0 0 + + 0 0

" Antigen retardation electrophoretic test. b The antigenic determinants for liver alkaline phosphatase cannot be distinguished from bone-type alkaline phos-

phatase in this system (3). ' See Ref. 16.

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some tumors as it comb ines with an t i se rum to TCRC-2 HeLa cells and an t i se rum raised aga ins t bone sarcoma. The above ev idence leads us to bel ieve that t u m o r a lkal ine phos- phatase wh ich shows l iver-bone- l ike charac ter is t i cs is of the early p lacenta l type in cont ras t to the Regan or mature placental type.

A l though ear ly p lacenta l a lkal ine phospha tase is s imi lar to liver i m m u n o l o g i c a l l y and in its sensi t iv i ty to inh ib i t ion by L -homoarg in ine , it is c lear f rom Table 1 that , un l ike bone and liver a l ka l ine phosphatases , a m ino r c o m p o n e n t is re- s istant to hea t ing for 5 min at 65 ~ Rabbi t ant isera to " l i ve r " alkal ine phospha tase cross-reacts wi th bone- type isoen- zyme.

From our l im i ted exper iences thus far, it is reasonable to expect a deg ree of b io log ica l var ia t ion f rom p lacenta to p lacenta, in pa r t due to the d i f f i cu l ty of f ix ing p lacenta l age with prec is ion and to the var iable rate of matura t ion of the placenta. It is a lso possib le that the isoenzyme mani fested at 8 weeks may later be shown to have appeared at an ear l ier stage o f deve lopment , just as ~x-fetoprotein usual ly at t r ibuted to fe ta l l iver has now been shown to have f irst appeared in the yo lk sac, an organ wh ich is the p recursor to liver. There fore , for the t ime being, we def ine ear ly p lacen- tal type as the heat-sensi t ive, L -homoarg in ine - inh ib i ted ac- t ivi ty which mig ra tes in 2 bands, the fas t -mov ing band on " m i c r o z o n e " e lec t rophores is lack ing known ant igen ic de- te rminants and the s lower band possess ing l iver (bone) ant igenic s i tes.

Ul t imately, w h e n it is proven that non-Regan a lka l ine phosphatase is the early p lacental form of the enzyme, then the p roduc t i on of a lkal ine phospha tase in human tumors would have a s ingle in terpre ta t ion . This postu la tes that t rophob las t ic genes for a lkal ine phospha tase are act ivated in cancer ce l ls wh ich may be those of the 6- to 10-week placenta, of t he term p lacenta, or of a mix ture of both .

A C K N O W L E D G M E N T S

We thank Dr. Julien Van Lancker for extending us the hospitality of his laboratory. We also thank Dr. R. M. Singer for making available antisera to HeLa TCRC-1 and TCRC-2.

P h a s e - s p e c i f i c P l a c e n t a / A l k a l i n e P h o s p h a t a s e I s o e n z y m e s

R E F E R E N C E S

1. Abelev, G. I. Alpha-Fetoprotein in Oncogenesis and Its Association with Malignant Tumors. Advan. Cancer Res., 14: 295-358, 1971.

2. Braunstein, G. D., Vaitukaitis, J. L., Carbone, P. P., and Ross, G. T. Ectopic Production of Human Chorionic Gonadotropin by Neoplasms. Ann. Internal Med., 78: 39-45, 1973.

3. Fishman, L., Inglis, N. R., and Fishman, W. H. Preparation of Two Antigens of Human Liver Isoenzymes of Alkaline Phosphatase. Clin. Chim. Acta, 34: 393-400, 1971.

4. Fishman, L., Inglis, N. R., and Fishman, W. H. Preparation and Charac- terization of Human Intestinal Alkaline Phosphatase Antigens. Clin. Chim. Acta, 38: 75-83, 1972.

5. Fishman, W. H. Discussion in "The Phosphohydrolases: Their Biology, Biochemistry and Clinical Enzymology." Ann. N. Y. Acad. Sci., 166: 808- 810, 1969.

6. Fishman, W. H., Driscoll, S., Miyayama, H., and Fishman, L. Human Tumor Non-Regan Alkaline Phosphatase Isoenzyme. Proc. Am. Assoc. Cancer Res., 16: 195, 1975.

7. Fishman, W. H., and Ghosh, N. K. Isoenzymes of Human Alkaline Phos- phatase. Advan. Clin. Chem., 10: 259-273, 1967.

8. Fishman, W. H., Inglis, N. R., Green, S., Anstiss, C. L., Ghosh, N. K., Reif, A. E., Rustigian, R., Krant, M. J., and Stolbach, L. L. Immunology and Biochemistry of Regan Isoenzyme of Alkaline Phosphatase in Hu- man Cancer. Nature, 219: 696-699, 1968.

9. Fishman, W. H., Inglis, N. R., Vaitukaitis, J., and Stolbach, L. L. Regan Isoenzymes and HCG in Ovarian Cancer. Natl. Cancer Inst. Monograph, 42: 63-73, 1975.

10. Fishman, W. H., Manning, J. P., Takeda, M., Angellis, D., and Green, S. Quantitation of Potency of Antisera to Placental Alkaline Phosphatase by an Automated Immunoenzyme Technique. Anal. Biochem., 51: 368-382, 1973.

11. Gitlin, D., Kitzes, J., and Boseman, M. Cellular Distribution of Serum a- Fetoproteins in Organs of the Fetal Rat. Nature, 215: 534, 1967.

12. Inglis, N. R., Guzek, D. T., Kirley, S., Green, S., and Fishman, W. H. Rapid Electrophoretic Microzone Membrane Techniques for Regan Iso- enzyme (Placental Type Alkaline Phosphatase) Using a Fluorogenic Sub- strate. Clin. Chim. Acta, 33: 287-292, 1971.

13. Lin, C-W., and Fishman, W. H. L-Homoarginine: An Organ-Specific, Uncompetitive Inhibitor of Human Liver and Bone Alkaline Phosphohy- drolases. J. Biol. Chem., 247: 3082, 3087, 1972.

14. Miyayama, H., Doellgast, G., Memoli, V., Gandbhir, L., and Fishman, W. H. Direct Immunoperoxidase Staining for Regan Isoenzyme of Alkaline Phosphatase in Human Tumor Tissues. Cancer, in press.

15. Sasaki, M., and Fishman, W. H. Ultrastructural Studies on Regan and Non-Regan Isoenzymes of Alkaline Phosphatase in Human Ovarian Can- cer Cells. Cancer Res., 33: 3008-3018, 1973.

16. Singer, R. M., and Fishman, W. H. Characterization of Two Sublines: TCRC-1 Produces Regan Isoenzyme and TCRC-2, Non-Regan Isoen- zyme. J. Cell Biol., 60: 777-780, 1974.

17. Tsuchida, Y., Endo, Y., Urano, Y., and Ishida, M. Alpha-Fetoproteins in Yolk Sac Tumors. Ann. N. Y. Acad. Sci., 259: 221-233, 1975.

18. Weintraub, B. D., and Rosen, S. W. Ectopic Production of Human Chorionic Somatomammotropin by Non-Trophoblastic Cancers. J. Clin. Endocrinol. Metab., 32: 94-101, 1971.

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L. F ishman et al.

lb

..... : ,~,,

Fig. 1. Cytochemical staining for alkaline phosphatase of an 8-week (Phase 1 ) placenta, a, microvillar staining in the presence of L-phenylalanine, b, lack of reaction in the presence of L-homoarginine in the incubating medium.

Fig. 2. Positive immunohistochemical localization of term placental alkaline phosphatase (a) and negative reaction in an 8-week placenta (b).

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Phase-specif ic P lacenta/Alka l ine Phosphatase Isoenzymes

1 2 3 4 5

Fig. 3. Microzone cellulose acetate electrophoresis of alkaline phospha- tase in homogenates of placentas arranged in order of gestational age. From left to right they are 8, 10, 12, 13, and 16 weeks. The pattern at 8 weeks is defined as Phase 1 ; at 10, 12 and 13 weeks, Phase 2; and at 16 weeks, Phase 3.

% i nh ib i t i on A lka l ine

Gesta- phospha- L-phenyl- L-homo- Heat (5 Speci- t iona l age tase act iv- a lan ine a rg in ine min at

men (wk) ity (/~g/ml) (5 mM) (8 mM) 65 ~

1 8 110 16 80 90 2 10 75 25 25 33 3 12 123 51 39 16 4 13 153 57 34 27 5 15.5 246 70 11 8

Fig. 4. Antigen-retardation tests of a Phase 1 placental homogenate alka- line phosphatase. From left to right, the 1st slot is Phase 1 homogenate control; the 2nd slot is a combination of control plus antiserum to human liver alkaline phosphatase; the 3rd slot, control plus antiintestinal alkaline phosphatase; and the 4th slot, control plus antiserum to human placental alkaline phosphatase.

Fig. 5. Antigen retardation tests of a Phase 2 placental alkaline phospha- tase pattern in which bands A and B overlap. From left to right, the 1st pattern is Phase 2 homogenate control; the 2nd pattern represents the mixture of control with antiserum to human liver alkaline phosphatase; the 3rd pattern, control plus antiserum to term placental alkaline phosphatase; and the 4th pattern, control plus antiserum to bone sarcoma alkaline phos- phatase.

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1976;36:2268-2273. Cancer Res   Lillian Fishman, Haruhiko Miyayama, Shirley G. Driscoll, et al.   Human CancerIsoenzymes of Human Placenta and Their Occurrence in Developmental Phase-specific Alkaline Phosphatase

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