201
Clinica Chimica Acta, 86 (1978) 201-215 @ Elsevier/North-Holland Biomedical Press
CCA 9393
PLACENTAL-LIKE ALKALINE PHOSPHATASE AND BENIGN OVARIAN TUMORS
FRANCES
J. BENHAM
*-**,
MRC Human Biochemical London (U.K.) (Received
December
M. SUSAN
Genetics
16th,
POVEY
IN MALIGNANT
and HARRY
Unit, Galton Laboratory,
HARRIS
University
*
College London,
1977)
Summary Alkaline phosphatase (ALP) components in extracts of seven malignant and eleven benign ovarian tumors were characterized using the criteria of electrophoretic mobility before and after neuraminidase treatment, heat stability, L-phenylalanine inhibition and reactivity against antiplacental ALP antiserum. Seven of the eighteen tumors had ALP components which most closely resembled the ALP isozyme normally found in placenta and were clearly distinguished from all other tissue ALPS. The proportion of tumors with the placental-like ALP in the malignant group (five out of seven) was significantly greater than the proportion in the benign group (two out of eleven). The fraction (78%) of the malignant tumors with the isozyme represents a larger percentage than has previously been found by examination of cancer patients’ sera. The electrophoretic mobilities of the placental-like ALPS in the tumors were in no case identical to the mobilities of any of the six common placental ALP phenotypes. The tumor ALPS may thus be determined by rare variant alleles at the ALP locus, or alternatively, the enzyme molecules may have been subject to structural modification. At least two of these tumors contained an electrophoretically slow. heat-stable, leucine-sensitive ALP, which may correspond to what has been termed the D-variant of placental ALP found in some other tumors.
Introduction Placental alkaline phosphatase is biochemically and genetically distinct from other human tissue alkaline phosphatases (ALPS) such as those found in liver, * Present Richards * * To
whom
address: Building,
Department
of
Philadelphia,
correspondence
should
Human
Pa. 19104,
Genetics, U.S.A.
be addressed.
University
of
Pennsvlvania.
School
of
Medicine.
202
bone, kidney and intestine [ 11. In normal development, the placental ALP isozyme, as the name implies, is expressed only in placental tissue, but in various malignant tumors of miscellaneous origin, an ALP with similar properties has been found [2-g]. This aberrant ALP is often called the Regan isozyme, named after the patient in which the phenomenon was first described [2]. The patient had a bronchogenic carcinoma, but subsequent examples of placentallike ALPS have been reported in a variety of other malignancies including tumors of ovary [4], breast [4], bile duct [ 51, and stomach [9]. It has been suggested that the synthesis of placental-like ALP by malignant cells of nontrophoblastic origin may represent derepression of the placental ALP structural gene locus in these cells 121. In many cases, tumor ALPS closely resemble placental ALP in heat stability, sensitivity to inhibition by L-phenylalanine, and immunological reactivity. These characteristics clearly differentiate the placent.al and the tumor ALPS from all other tissue ALPS. In one case it was shown that the peptide fingerprint pattern of the tumor ALP closely resembled that of the placental ALP [ 71. Recent studies have shown that the aberrant ALP in some tumors behaved like the placental ALP isozyme in the standard characterization tests, but it could be differentiated from the placental ALP by such properties as sensitivity to inhibition by L-leucine and slower electrophoretic mobility in starch has been called the Nagao isogel [ 3,5,8]. This placental-like ALP “variant” zyme. A third form of aberrant ALP, so far found only in primary hepatomas, behaves like placental ALP in terms of its inhibition by L-phenylalanine and cross-reaction with antiplacental ALP antiserum, but differs from placental and other known tissue ALPS in its faster electrophoretic mobility and heat stability [ lO,ll]. The ALP from placenta, unlike the ALP from other tissues, is genetically polymorphic with six electrophoretic phenotypes determined by three common alleles at the placental ALP locus [12,13]. In addition, a considerable number of rare allelic variants have been identified [13]. The electrophoretic phenotypes of the placental-like ALPS in tumors have been considered by only a few authors. However, it has been suggested that the L-leucine sensitive, electrophoretically slow ALP (Nagao isozyme) represents a rare phenotype of placbental ALP which has been referred to as the “D-variant” [8]. Various surveys have demonstrated raised levels of placental ALP in 8 to 25%’ of sera from patients with miscellaneous cancers [4,14-171, with somewhat higher proportions (14 to 35%)) in sera from patients with urogenital cancers 1171. Only very low levels of this ALP isozyme have been reported in the sera of healthy individuals [ 14,151. In the present study a series of eighteen ovarian tumors was examined for the presence of placental-like ALP using electrophoretic and other procedures which allow discrimination between the different tissue AL,P isozymes. This series included seven malignant and eleven benign tumors. Placental-like ALP occurred more frequently in the malignant than in the benign tumors. Electrophoretic mobilities of the placental-like ALPS from the tumors were compared wit,h those of the six common phenotypes of placental ALP and in no case were the tumor enzymes electrophoretically identical with a common placental isozyme.
203
Materials
and methods
Ovarian tumors were obtained as surgical specimens from University College Hospital, London, and Norfolk and Norwich Hospital, Norwich. Tumor tissue (up to approximately 3 g), and where possible, normal ovarian tissue, cyst fluid, and plasma were taken from each patient and stored at --20°C or in liquid N2 until needed. Placentae and a range of postmortem tissues which had been stored at -20°C were also available. To weighed portions of washed tissue, an equal volume of distilled water was added and the mixtures homogenized. Butanol extracts of the whole homogenates were made according to methods previously described [ 181. Neuraminidase treatment of the butanol extracts was carried out by adding one volume of neuraminidase (Clostridium perfringens, Sigma, 1. unit/ml made up in 10 mM citrate phosphate buffer, pH 5.0) to two volumes of extract, and incubating for 45 to 60 min at 37’C. Control samples were incubated with buffer only. Some tumor extracts appeared to require additional neuraminidase treatment and for these, neuraminidase was mixed with the extract in equal proportions. The mixture was then dialyzed for 18 h against the citrate phosphate buffer pH 6.0 to allow the liberated sialic acid residues to diffuse out from the sample and hence shift the equilibrium to maximize the neuraminidase activity. Control samples were treated similarly, but with buffer only. Starch gel electrophoresis was carried out in a Tris/borate discontinuous buffer system, pH 8.6, with a voltage gradient of 6 V/cm for six hours at room temperature [ 191. An additional buffer system consisting of citrate/NaOH/ borate, pH 6.0, was also used on occasion to investigate placental ALP phenotypes [19]. Zones of ALP activity were located by two methods. One used fl-naphthyl phosphate as substrate and Fast Blue RR, a coupling agent, to produce insoluble purple precipitate at the sites of enzyme activity [ 191. The second, more sensitive stain, used 4-methylumbelliferyl phosphate as substrate, and fluorescent methylumbelliferone was visualized under UV at the sites of enzyme activity [ 201. The heat stability of the ALP isozymes present in tissues and tumor specimens was assessed by two procedures. The first involved separating the ALP components by starch gel electrophoresis and then heating the gel for various lengths of time at 60°C prior to staining for enzyme activity, as previously described [21]. The second involved heating the tissue extracts (which had been dialysed against 5 mM Tris pH 7.4 plus 5 mM MgCl,) for various periods of time up to 30 min at 65°C. The heated extract was then subjected to electrophoresis and stained for ALP activity. Inhibition by L-phenylalanine (2.5, 5.0 and 10.0 mM) of the tumor and tissue ALP isozymes was assessed following electrophoresis, as previously described [ 211. The extent to which L-leucine inhibited the heat stable ALP components (i.e. stable to 30 min at 65°C) in the tumor and placental extracts was assessed using 4 mM methylumbelliferyl phosphate as substrate in a continuous, fluorimetric assay system in borate buffer, pH 10.0 [20]. Activity in the presence of various concentrations of L-leucine (0.5 to 10.0 mM) was expressed as a percentage of the activity in the absence of inhibitor.
204
For immunodiffusion, rabbit antiserum raised against purified placental ALP by a method described [5] and kindly provided by the original authors was used. Immunodiffusion was allowed to proceed at 4°C for two days in Ouchterlony plates of 1.5% Ion Agar made up in 0.85% saline containing 5 mM MgCl, and 0.02% sodium azide as preservative. The plates were washed with saline, and then water for 48 h, and then stained for ALP activity. Extracts which contained very small amounts or no placental-like ALP were tested for cross-reacting material by an alternative method. The antiserum was mixed with dialysed tissue extract in varying proportions and the mixture incubated and then subjected to electrophoresis. Control samples were incubated with nonimmune serum. The gels were stained for ALP activity before and after heating for one hour at 60°C. Placental ALP and any cross-reacting enzyme did not migrate into the gel. Results A total of 18 different ovarian tumor specimens were collected: seven malignant and 11 classified as benign. From many of the patients, cyst fluid, normal ovary and serum samples were also obtained, as given in Table I, which also summarizes the histology of each tumor.
TABLE
I
DETAILS
OF THE EIGHTEEN Histology
TLlm0r
OVARIAN
TUMOR
summary
SPECIMENS
Tumor
cyst
fluid
COLLECTED Normal tissue
Plasma
Age of patient (Years)
Benign ovc ovc ovc ovc ovc ovc ovc ovc OVC ovc ovc
2 4 7 8 9 13 14 15 18 19 20
Malignant ovc 5 ovc
10
ovc ovc OVC ovc ovc
11 12 16 17 21
Mucinuos cystadenoma Endometriotic cyst Cystic atretic follicle Mutinous cystadenoma Oedematous cyst Serous. papillary cystadenoma Pseudomucinous cystadenoma Serous cystadenofibroma Serous cystoma Mutinous cystadenoma Cystic corpus luteum Serous, papillary cystadenocarcinoma Serous. papillary cystadenocarcinoma Mutinous adenocarcinoma Adenopapillary carcinoma “solid” adenocarcinoma Solid, papillary adenocarcinoma Endometrioid adenocarcinoma
* mm,no placental-like ALP detected; +, substantial like ALP detected; ++, high level of placental-like
x
_
x x
x
x
x
x
x
x
x
x
x
x x
x
x
x
x
x
x _ _
x
x x x
x x x x x
x _
x x
x
x
x
x
x
x x x
x _
Placent&like ALP *
62 31 30 41 23
_ _ _ _ _
44 58
++ _
53 49
+
62 24 47
+
-.
x
42
++
_ _
x x x x x
68 68
(+) _
36 51
++ +
x
x
placental-like ALP detected; ALP detected.
60 (+), low level of placental-
205
Fig. 1. ALP components in four normal human tissues as separated by starch gel electrophoresis (pH 8.6). The enzyme substrate was P-naphthyl phosphate. (a) Tissue extracts treated with neuraminidase prior to electrophoresis. (b) Tissue extracts untreated. The faster moving main mnes of activity are accompanied in all tissues by slow compact bands which do not migrate far from the origin. These latter bands have been shown to be aggregate forms of the faster moving enzymes. Note that in the intestinal sample. besides the main ALP band which is unaffected by neuraminidase, an additional faster moving neuraminidasesensitive, liver-like ALP component is also present. In the present paper the term “intestinal ALP” is used to refer to the main, neuraminidase-insensitive component. (c) ALP components in placenta (PL type Z-1). a benign ovarian tumor and a normal ovary as separated by starch gel electrophoresis (PH 8.6) before and after neuraminidase treatment. The enzyme substrate was 4methylumbelliferyl phosphate. 1. Sample untreated; 2. sample treated with neuraminidase. L. liver type ALP: LN, liver type ALP neuraminidase treated; I, intestinal type ALP.
206
Electrophoretic
and related studies
ALP activity was demonstrated in extracts of all normal and postmortem ovaries and ovarian tumors. Starch gel electrophoresis separated the ALP activity into multiple components. In all normal ovaries and ovarian tumors there was a band of ALP activity which migrated as the liver ALP in starch gel both before and after neuraminidase treatment, which removes accessible sialic acid residues and thereby reduces anodal mobility (Fig. 1). This component was heat labile (no activity remaining after 30 min at 65°C) and was not appreciably inhibited by low concentrations (2.5, 5.0 mM) of L-phenylalanine. In these properties it also closely resembled the liver ALP isozyme, and is thus most likely a product of the same ALP locus which is expressed in liver. In some of the normal ovaries and tumors there was also an ALP component which closely resembled the major intestinal ALP isozyme in it,s electrophoretic mobility before and after neuraminidase treatment (Fig. l), its inactivation by heating and its sensitivity to inhibition by L-phenylalanine. Since the liver type ALP appeared in all normal and postmortem ovaries (18 examined) and the intestinal type ALP appeared in at least ten of these specimens, these two isozymes, when they appeared in the tumor extracts were considered to be “normal ovarian” ALP components. Additional ALP components tiere found in many of the tumor extracts. Seven of the tumors contained ALP components which here heat stable (no loss of activity after one hour at 6O”C), inhibited by low concentrations (2.5, 5.0 mM) of L-phenylalanine, and whose anodal mobilities in starch gel were somewhat retarded by neuraminidase treatment. In these properties, these ALP component,s resembled placental ALP and were clearly differentiated from the other major tissue ALP forms (Fig. 2). However, the electrophoretic mobilities of these placental-like ALPS were -in no case precisely identical to the electrophoretic mobilities of any of the six common phenotypes of placental ALP. The diagram in Fig. 3 shows the electrophoretic mobilities of the ALP components in all the tumors relative to the mobilities of the isozymes found in normal placent,a and liver. The effect of neuraminidase treatment and results of t,he heat inactivation and L-phenylalanine inhibition studies on each component are also indicated. The seven tumors which were shown to have ALP component with placental-like ALP were OVC 5, 10, 11,13,16, 17 and 18. Of the remaining tumors, seven had only the liver type ALP (OVC 2,4,7, 14, 15,20 and 21) and four had additional components which resembled the liver form in the heat lability and insensitivity to L-phenylalanine inhibition but which showed electrophoretic mobility different from any normal tissue ALP (OVC 8,9,12 and 19).
Immgzodiffusion In double diffusion plates, extracts of placenta and of all tumors which contained readily detectable amounts of the placental-like ALP (OVC 5, 10, 13, 16 and 17) cross-reacted with antiplacental ALP antiserum to form enzymatically active preclpitin lines (Fig. 4a). The precipitin lines from these tumor extracts were continuous with the placental-antiplacental ALP precipitin line suggesting immunological identity of the tumor enzyme with the placental enzyme. Extracts of normal ovary, liver and the other tumors did not show cross-reactiv-
207 l-1
__
i
Fig. 2. (a) ALP components of OVC 13 (malignant tumor) and placenta (PL type 2-l) as separated by starch gel electrophoresis (pH 8.6). ‘Heated’ samples were heated at 65’C for 30 min prior to electrophoresis. 1, Sample untreated with neuraminidase; 2. sample treated with neuraminidase. (b) L-Phenylalanine inhibition studies of the ALP components of OVC 13 and placenta (PL type 2-l). Both the tumor and the placental extracts were neuraminidase treated. Prior to staining for enzyme activity strips of gel were incubated with various concentrations of L-phenylalanine as indicated. The placental ALP and the heat-stable components of the tumor ALP were increasingly inhibited by increasing concentrations of L-phenylalanine. In contrast. the liver-like ALP components in the tumor were not inhibited by L-phenylalanine.
ity with the same antiserum. When the tumor extracts which cross-reacted with the antiserum were heated under conditions which completely destroy all nonplacental ALP (30 min at 65”C), enzymatically active precipitin lines were still formed (Fig. 4b). Thus in five of the tumors, the ALP components which resembled the placental ALP in heat stability and L-phenylalanine sensitivity also showed immunological identity with the placental ALP. However, two tumor extracts which did not show cross-reacting material in the double diffusion plates had been shown to contain small amounts of a heat-stable, L-phenyl-
208
209
Fig. 4. lmmunodiffusion plates stained for ALP activity using fl-naphthyl phosphate as substrate. Center well in plates contained antiplacental ALP antiserum. Surrounding wells contained butanol extracts of tissue or tumors. Where indicated, sample was heated for 30 min at 65°C prior to application into the well. A. 1, placenta; 2. OVC 17; 3. OVC 10; 4, no sample: 5. OVC 16; 6, OVC 13. B. 1, placenta (heated); 2, OVC 10 (heated); 3, OVC 16 (heated); 4, placenta; 5, OVC 13 (heated): 6. OVC 17 (heated). C. 1, placenta; 2. OVC 4; 3, no sample: 4, placenta: 5, OVC 7; 6, OVC 8. D. 1. placenta; 2. OVC 21; 3. OVC 21 (heated); 4. placenta; 5, ovary; 6, liver. Note than OVC 17 (A2 and B6) did show faint precipitin lines. although they are not evident in the photograph.
alanine sensitive ALP (OVC 11 and 18). When these extracts were mixed with the antiserum and then electrophoresed, the heat-stable, placental-like ALP components did not migrate into the gel, indicating that they had been selectively precipitated by the antiserum. The electrophoretic mobilities of nonplacental ALPS were unaffected or only slightly retarded. Non-immune serum did not significantly retard the mobilities of any of the ALPS.
Benign vs. malignant
tumors
Placental-like ALP activity, as characterized by heat stability, sensitivity to inhibition by L-phenylalanine, and cross-reaction with antiplacental ALP antiserum, was found in two out of the 11 benign, and five out of the seven malignant tumors (Table II). The proportion of tumors with the ALP in the malignant group was significantly greater than the proportion in the benign group at. the 5% level (p = 0.0387, Fisher’s extract treatment of a 2 X 2 contingency table). Thus in this tumor series, there was an increased incidence of the ALP in the malignant tumors as compared with the nonmalignant tumors. The presTABLE
II
SUMMARY OF ALP TO PLACENTALLIKE Tumor
type
COMPONENTS ALP Number
IN THE EIGHTEEN
examined
Number
Benign Malignant
11 7
2 5
Total
18
7
* As characterized by heat stability, anti-placental ALP antiserum.
sensitivity
to inhibition
OVARIAN
TUMORS.
with placental-like
WITI
REFERENCE
ALP *
by L-phenylananine,
and cross-reaction
with
210
i *;
7
?
f!CJ!i’
,
Fig. 5. ALP components in OVC 16 and placenta (PL type 2-1) as separated by starch gel electrophomis (pH 8.6) before and after neuraminidase treatment. Heated samples were heated for 30 min at 65OC. 1. sample untreated: 2, sample treated with neuraminidase. Fig. 6. ALP components of OVC 13 and placenta (PL type 2-l) as separated by starch gel electrophoresis (PH 8.6). All samples were neuraminidase treated. 1. Placenta PL type 2-1. 2, Placenta PL type 2-l extract mixed with OVC 13 extract in a ratio of one part to two parts. 3. Placenta PL type 2-l extract mixed with OVC 13 extract in a ratio of one part to five parts. 4, OVC 13.
ence of the placental-like pre- and post-menopausal tumors which contained
Phenotypes
ALP did not correlate ages were represented the placental-like ALP.
with age of the patient. in the groups of patients
Both with
of the platen tal-like ALP
The standard electrophoretic procedures used to determine the phenotypes of normal placental ALP did not achieve separation of the placental-like ALPS into unambigusously typeable components. The diffuse appearance of the placental-like ALP in four of the tumors (OVC 5, 10, 11, 16) made then untypeable; all the normal placental enzyme phenotypes separate into discrete, sharp bands [19] (Fig. 5). The placental-like ALPS of OVC 13, 17, and 18 separated into sharper bands and moved to normal placental positions in starch gel. After neuraminidase treatment, OVC 13 had four or five major bands of activity, OVC 17 had two or three major bands of activity, and OVC 18 had three major bands of activity (Fig. 3). The first two of these patterns thus showed more bands than would be expected for completed desialated placental ALP (since the enzyme is a dimer, the heterozygote pattern has three major bands, and the homozygote pattern has one major band). Since it seemed possible that all the accessible sialic acid groups had not been removed by the standard neuraminidase treatment, the tumor extracts were subjected to additional neuraminidase treatment. This reduced the activity in OVC 13 to three major bands of activity. Electrophoresis of a mixture containing an extract of a PL type 2-l placenta and the OVC 13 (both neuraminidase treated) showed that the three bands of activity in OVC 13 were slightly slower than the three bands
211
of PL type 2-l (Fig. 6). Thus if the ALP pattern in OVC 13 represents an unmodified placental phenotype, the individual might be heterozygous for two rare alleles at the PL locus. Alternatively, it is possible that the pattern represents a slightly modified common phenotype, probably type 2-l. The additional neuraminidase treatment reduced the activity in OVC 17 to one major ALP band plus two minor bands. This could represent a homozygous pattern, as minor secondary bands are frequently seen in the normal placenta [ 121. The mobility of the major band of OVC 17 was slightly slower than the common placental type 1, as shown by mixing the two extracts together and electrophoresing the mixed sample. Thus the individual might be homozygous for a rare allele at the PL locus, or this pattern could also represent a slightly modified common phenotype, type 1. In OVC 18 the fastest of the two ALP bands had approximately the same mobility as the common placental type 2; however, the two slower bands did not correspond with any common phenotype. Electrophoresis of these tumor extracts in the pH 6.0 system did not improve resolution of the placental-like ALPS or yield any further information about their phenotypes. L-Leucine inhibition The placental-like ALPS present in five of the seven tumors were tested for inhibition by L-leucine. Results given in Table III show that the heat-stable ALPS in extracts of OVC 10 and OVC 16 were much more inhibited than the normal placental ALP (PL type 1 and PL type 2) by all L-leucine concentrations tested (0.5 to 10 mM). The heat-stable ALPS of OVC 13, 17, 18 had inhibition profiles similar to that of the normal placental enzyme though they were somewhat more inhibited at the lower L-leucine concentrations. Thus the tumor ALPS (heat-stable) which had slow, smeary electrophoretic patterns (OVC 10, 16) were also very inhibited by L-leucine. None of the OVC 5 or 11 extracts, which also contained electrophoretically slow, smeary heat-stable ALPS were available to test. Plasma Plasma or serum samples were obtained from 15 of the 18 ovarian tumor patients (Table I). Sera was available from five of the seven patients with TABLE
III
L-LEUCINE PONENTS Values
given
L-Leucine
INHIBITION IN
FIVE
OF
OVARIAN
represent
PLACENTAL
the average Percent
ALP
(PL
TYPE
1)
AND
THE
HEAT
STABLE
ALP
of two
separate
experiments,
each
done
on duplicate
sample
aliquots.
inhibition
concentration (mM) 0.5
Placenta n.t.
ovc 49.3
10
ovc 8.9
13
OVC 44.5
16
ovc 5.8
17
ovc n.t.
1.0
6.5
62.6
19.8
60.5
18.0
2.5
19.2
81.9
29.8
77.1
25.6
21.5
5.0
24.5
88.3
39.5
83.0
34.3
32.6
7.0
36.7
91.4
47.4
86.7
40.0
36.7
39.1
94.2
51.1
87.5
53.5
39.4
10.0
COM-
TUMORS
17.4
18
212
tumors which had placental-like ALP. Electrophoresis of the sera showed that in addition to the normal serum ALP components (present in all samples and in 15 samples from healthy individuals), three of the samples had extra ALP components. These sera were from patients, 10, 16 and 17, all of whom had malignant tumors which contained placental-like ALP. In each of these cases, the extra ALP components were heat-stable and they corresponded in electrophoretic mobility with the heat-stable components found in the respective tumors. Placental-like ALP was not detected (by electrophoresis) in two of the five sera available from patients with this ALP isozyme in their tumors. Cyst fluid Cyst fluid was available from 11 of the tumor patients (Table I). Nine of these fluids had ALP activity detectable by starch gel electrophoresis. All these nine samples contained liver and smaller amounts of intestinal ALP. Cyst fluids were obtained from two of the tumors (one malignant, one benign) which had been found to contain a placental-like ALP (OVC 10, 13). Both of these fluid samples contained the heat-stable ALP components which were present in the respective tumor. However, good resolution of these components in the fluid was not achieved because of the high viscosity of the samples. Discussion Characterization of the electrophoretically separated ALP components in the 18 ovarian tumors demonstrated that all the tumors retained the ALP present in normal ovary, i.e. liver type and small amounts of intestinal type. Seven of the tumors had additional, different ALP components which most closely resembled the product of the placental ALP locus, as shown by their heatstability, sensitivity to inhibition by low concentrations of L-phenylalanine, and cross-reaction with antiplacental ALP antiserum. These placental-like ALPS found in the tumor extracts did, however, show modified electrophoretic mobilities both before and after neuraminidase treatment when compared with the normal placental isozymes. This investigation, which involved looking at both benign and malignant ovarian tumors has shown that there was a significantly increased incidence of placental-like ALP in the malignant tumor group as compared with the benign tumor group. In a small second series of 13 ovarian tumors collected after the main work for this report was completed, ALP was characterized by electrophoresis and heat-stability only. Heat-stable ALP was seen in three out of four malignant tumors (three cystadenocarcinemas and one adenocarcinoma), but it was not detected in any of the benign tumors. These data suggest further a significant association between ovarian malignancy and the presence of placental-like ALP. The number of tumors so far studied thoroughly was small, and it would be useful to extend the survey to obtain a more accurate estimate of the association between malignancy and placental-like ALP. The proportion of malignant tumors which had placental-like ALP (five out of seven) was greater than reported proportions of cancer patients’ sera which contained placental-like ALP (8 to 35%). Our investigation of tumor specimens would thus imply that some tumors produce placental-like ALP which does not
213
get into the circulation in significant amounts. In one study where cancer tissue was examined, eight out of 23 (35%) gastric tumors were shown to contain placental-like ALP [ 91. Although ectopically synthesized placental-like ALP is associated with malignancy, this association is not exclusive; the enzyme was also discovered in two tumors classified as benign. It is thought that of all adenocarcinomas of the ovary, over half are derived from benign cystoadenomas [22]. It is therefore possible that the presence of the placental-like enzyme in apparently benign tumors may represent a pre-neoplastic change. An unexpected finding was that none of the placental-like ALPS found in the tumor extracts corresponded in electrophoretic mobility with any of the common electrophoretic phenotypes of placental ALP. It has been reported that the placental-like ALP in a lung carcinoma had a slightly slower mobility in acrylamide gel than the normal placental enzyme [7], Inglis et al. [ 81 have reported that in the sera of some cancer patients there was a placental-like ALP which exhibited slower electrophoretic mobility than the common phenotypes of placental ALP, and the authors suggested that these tumor ALPS represent the rare D-variant phenotype of placental ALP described by Beckman and Beckman [23]. The tumor ALPS which resembled the D-variant in electrophoretic mobility were also inhibited by L-lcucine. (This ALP was found in approximately 50% of all the sera which contained placent,al-like ALP). In the present study the placental-like ALP in four of the tumors had slow, diffuse electrophoretic mobility, and in two cases (the others were not tested), the enzymes were more inhibited by L-leucine than the common placental ALP types. These tumor ALPS may be very similar to what Inglis et al. [B] have described as the D-variant in other tumors, but their identity as the D-variant is still in question [24,25]. It would be useful to compare a D-type placental ALP with these tumor enzymes by electrophoresis in the same starch gel. The placental-like ALPS in the other tumors (OVC 13,17,18) did not resemble the D-variant of placental ALP. They had faster electrophoretic mobility than D, very discrete banding patterns and did not show the same inhibition by L-leutine. However, their electrophoretic patterns could represent, other rare variant phenotypes of placental ALP. A large number of rare alleles (more than 14) have been described at this enzyme locus [ 131. If the individuals concerned did have two rare alleles at this locus, it would imply that the locus is derepressed very much more frequently in tumor cells which carry the rare alleles than in cells which carry the common alleles. Alternatively, the derepressed ALPS in the tumors may have been subject to abnormal postranslational modifications by the unusual cell environment of the enzyme, e.g. resulting in a change of glycosylation of the enzyme molecule. In a previous report [21] we examined the electrophoretic phenotypes of the derepressed, placental-like ALP present in lines of HeLa cells cultured separately for many generations. The results showed that the placental-like ALP differed electrophoretically from line to line, and in one case only, was the mobility equal to that of a common placental ALP phenotype. The unusual electrophoretic patterns of these placental-like ALPS may have resulted from post-translational structural modification, or from mutations at the placental ALP locus. In contrast to the abnormal properties displayed by the tumor ALPS,
214
numerous other enzymes in tumor cells have not been shown to exhibit abnormal phenotypes [ 261. Most of the enzymes which have been studied are not “derepressed” enzymes, so the derepression process, or factors which promote it, may also promote defective transcription and translation, or even mutation. Recently, human chorionic gonadotropin, a glycoprotein hormone, was found to be expressed in a malignant cell line (HeLa) and the cu-subunit of this “derepressed” hormone had properties slightly different from those of the native placental hormone [ 271. Electrophoretic and related investigations of the tumor patients’ plasma suggest that placental-like ALP from the tumor had been released into the circulation in easily detectable amounts in three cases. The tumors of all these patients were malignant. The placental-like ALP from the one benign tumor which had had high levels of it (OVC 13, the other benign tumor with the ALP isozyme, OVC 18, had only trace amounts) was not detectable in the circulation. Thus although the methods used in this study for the detection of placental-like ALP in serum were perhaps less sensitive than some other methods used for sera surveys [14--161, a higher percentage of tumors may synthesize placental-like ALP than has previously been inferred on the basis of sera surveys. Acknowledgements We should like to thank professor D. Fairweather, Dr. P.C. Hughesdon and colleagues at University College Hospital, London, and also Mr. J. CarronBrown, Dr. J.H. Rack and colleagues at the Norfolk and Norwich Hospital, Norwich, for their permission to obtain ovarian tumor specimens and for kindly supplying us with histology reports. We are also grateful to Mr. Thompson for handling the collections of material and the documentation at Norwich. We thank Ms. G. Bulsara, Ms. A. Tanyar for skilled technical assistance. This work was in part supported by a grant from the American Cancer Society, No. VC-231. References 1
Fishman,
W.H.
2
Fishman,
W.H..
and
(1974)
Stolbach.
L.L.
Nakayama.
T..
4
Nathanson,
L. and
5
Jacoby.
6
Kang, (1972)
7
Greene,
8
In&.
9
Miki,
K.,
10
Warnock,
11
Higashino,
T.,
M.L.
S.,
Kitamura,
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Clinica Chimica Acta, 86 (1978) 201-215 @ Elsevier/North-Holland Biomedical Press
CCA 9393
PLACENTAL-LIKE ALKALINE PHOSPHATASE AND BENIGN OVARIAN TUMORS
FRANCES
J. BENHAM
*-**,
MRC Human Biochemical London (U.K.) (Received
December
M. SUSAN
Genetics
16th,
POVEY
IN MALIGNANT
and HARRY
Unit, Galton Laboratory,
HARRIS
University
*
College London,
1977)
Summary Alkaline phosphatase (ALP) components in extracts of seven malignant and eleven benign ovarian tumors were characterized using the criteria of electrophoretic mobility before and after neuraminidase treatment, heat stability, L-phenylalanine inhibition and reactivity against antiplacental ALP antiserum. Seven of the eighteen tumors had ALP components which most closely resembled the ALP isozyme normally found in placenta and were clearly distinguished from all other tissue ALPS. The proportion of tumors with the placental-like ALP in the malignant group (five out of seven) was significantly greater than the proportion in the benign group (two out of eleven). The fraction (78%) of the malignant tumors with the isozyme represents a larger percentage than has previously been found by examination of cancer patients’ sera. The electrophoretic mobilities of the placental-like ALPS in the tumors were in no case identical to the mobilities of any of the six common placental ALP phenotypes. The tumor ALPS may thus be determined by rare variant alleles at the ALP locus, or alternatively, the enzyme molecules may have been subject to structural modification. At least two of these tumors contained an electrophoretically slow. heat-stable, leucine-sensitive ALP, which may correspond to what has been termed the D-variant of placental ALP found in some other tumors.
Introduction Placental alkaline phosphatase is biochemically and genetically distinct from other human tissue alkaline phosphatases (ALPS) such as those found in liver, * Present Richards * * To
whom
address: Building,
Department
of
Philadelphia,
correspondence
should
Human
Pa. 19104,
Genetics, U.S.A.
be addressed.
University
of
Pennsvlvania.
School
of
Medicine.
202
bone, kidney and intestine [ 11. In normal development, the placental ALP isozyme, as the name implies, is expressed only in placental tissue, but in various malignant tumors of miscellaneous origin, an ALP with similar properties has been found [2-g]. This aberrant ALP is often called the Regan isozyme, named after the patient in which the phenomenon was first described [2]. The patient had a bronchogenic carcinoma, but subsequent examples of placentallike ALPS have been reported in a variety of other malignancies including tumors of ovary [4], breast [4], bile duct [ 51, and stomach [9]. It has been suggested that the synthesis of placental-like ALP by malignant cells of nontrophoblastic origin may represent derepression of the placental ALP structural gene locus in these cells 121. In many cases, tumor ALPS closely resemble placental ALP in heat stability, sensitivity to inhibition by L-phenylalanine, and immunological reactivity. These characteristics clearly differentiate the placent.al and the tumor ALPS from all other tissue ALPS. In one case it was shown that the peptide fingerprint pattern of the tumor ALP closely resembled that of the placental ALP [ 71. Recent studies have shown that the aberrant ALP in some tumors behaved like the placental ALP isozyme in the standard characterization tests, but it could be differentiated from the placental ALP by such properties as sensitivity to inhibition by L-leucine and slower electrophoretic mobility in starch has been called the Nagao isogel [ 3,5,8]. This placental-like ALP “variant” zyme. A third form of aberrant ALP, so far found only in primary hepatomas, behaves like placental ALP in terms of its inhibition by L-phenylalanine and cross-reaction with antiplacental ALP antiserum, but differs from placental and other known tissue ALPS in its faster electrophoretic mobility and heat stability [ lO,ll]. The ALP from placenta, unlike the ALP from other tissues, is genetically polymorphic with six electrophoretic phenotypes determined by three common alleles at the placental ALP locus [12,13]. In addition, a considerable number of rare allelic variants have been identified [13]. The electrophoretic phenotypes of the placental-like ALPS in tumors have been considered by only a few authors. However, it has been suggested that the L-leucine sensitive, electrophoretically slow ALP (Nagao isozyme) represents a rare phenotype of placbental ALP which has been referred to as the “D-variant” [8]. Various surveys have demonstrated raised levels of placental ALP in 8 to 25%’ of sera from patients with miscellaneous cancers [4,14-171, with somewhat higher proportions (14 to 35%)) in sera from patients with urogenital cancers 1171. Only very low levels of this ALP isozyme have been reported in the sera of healthy individuals [ 14,151. In the present study a series of eighteen ovarian tumors was examined for the presence of placental-like ALP using electrophoretic and other procedures which allow discrimination between the different tissue AL,P isozymes. This series included seven malignant and eleven benign tumors. Placental-like ALP occurred more frequently in the malignant than in the benign tumors. Electrophoretic mobilities of the placental-like ALPS from the tumors were compared wit,h those of the six common phenotypes of placental ALP and in no case were the tumor enzymes electrophoretically identical with a common placental isozyme.
203
Materials
and methods
Ovarian tumors were obtained as surgical specimens from University College Hospital, London, and Norfolk and Norwich Hospital, Norwich. Tumor tissue (up to approximately 3 g), and where possible, normal ovarian tissue, cyst fluid, and plasma were taken from each patient and stored at --20°C or in liquid N2 until needed. Placentae and a range of postmortem tissues which had been stored at -20°C were also available. To weighed portions of washed tissue, an equal volume of distilled water was added and the mixtures homogenized. Butanol extracts of the whole homogenates were made according to methods previously described [ 181. Neuraminidase treatment of the butanol extracts was carried out by adding one volume of neuraminidase (Clostridium perfringens, Sigma, 1. unit/ml made up in 10 mM citrate phosphate buffer, pH 5.0) to two volumes of extract, and incubating for 45 to 60 min at 37’C. Control samples were incubated with buffer only. Some tumor extracts appeared to require additional neuraminidase treatment and for these, neuraminidase was mixed with the extract in equal proportions. The mixture was then dialyzed for 18 h against the citrate phosphate buffer pH 6.0 to allow the liberated sialic acid residues to diffuse out from the sample and hence shift the equilibrium to maximize the neuraminidase activity. Control samples were treated similarly, but with buffer only. Starch gel electrophoresis was carried out in a Tris/borate discontinuous buffer system, pH 8.6, with a voltage gradient of 6 V/cm for six hours at room temperature [ 191. An additional buffer system consisting of citrate/NaOH/ borate, pH 6.0, was also used on occasion to investigate placental ALP phenotypes [19]. Zones of ALP activity were located by two methods. One used fl-naphthyl phosphate as substrate and Fast Blue RR, a coupling agent, to produce insoluble purple precipitate at the sites of enzyme activity [ 191. The second, more sensitive stain, used 4-methylumbelliferyl phosphate as substrate, and fluorescent methylumbelliferone was visualized under UV at the sites of enzyme activity [ 201. The heat stability of the ALP isozymes present in tissues and tumor specimens was assessed by two procedures. The first involved separating the ALP components by starch gel electrophoresis and then heating the gel for various lengths of time at 60°C prior to staining for enzyme activity, as previously described [21]. The second involved heating the tissue extracts (which had been dialysed against 5 mM Tris pH 7.4 plus 5 mM MgCl,) for various periods of time up to 30 min at 65°C. The heated extract was then subjected to electrophoresis and stained for ALP activity. Inhibition by L-phenylalanine (2.5, 5.0 and 10.0 mM) of the tumor and tissue ALP isozymes was assessed following electrophoresis, as previously described [ 211. The extent to which L-leucine inhibited the heat stable ALP components (i.e. stable to 30 min at 65°C) in the tumor and placental extracts was assessed using 4 mM methylumbelliferyl phosphate as substrate in a continuous, fluorimetric assay system in borate buffer, pH 10.0 [20]. Activity in the presence of various concentrations of L-leucine (0.5 to 10.0 mM) was expressed as a percentage of the activity in the absence of inhibitor.
204
For immunodiffusion, rabbit antiserum raised against purified placental ALP by a method described [5] and kindly provided by the original authors was used. Immunodiffusion was allowed to proceed at 4°C for two days in Ouchterlony plates of 1.5% Ion Agar made up in 0.85% saline containing 5 mM MgCl, and 0.02% sodium azide as preservative. The plates were washed with saline, and then water for 48 h, and then stained for ALP activity. Extracts which contained very small amounts or no placental-like ALP were tested for cross-reacting material by an alternative method. The antiserum was mixed with dialysed tissue extract in varying proportions and the mixture incubated and then subjected to electrophoresis. Control samples were incubated with nonimmune serum. The gels were stained for ALP activity before and after heating for one hour at 60°C. Placental ALP and any cross-reacting enzyme did not migrate into the gel. Results A total of 18 different ovarian tumor specimens were collected: seven malignant and 11 classified as benign. From many of the patients, cyst fluid, normal ovary and serum samples were also obtained, as given in Table I, which also summarizes the histology of each tumor.
TABLE
I
DETAILS
OF THE EIGHTEEN Histology
TLlm0r
OVARIAN
TUMOR
summary
SPECIMENS
Tumor
cyst
fluid
COLLECTED Normal tissue
Plasma
Age of patient (Years)
Benign ovc ovc ovc ovc ovc ovc ovc ovc OVC ovc ovc
2 4 7 8 9 13 14 15 18 19 20
Malignant ovc 5 ovc
10
ovc ovc OVC ovc ovc
11 12 16 17 21
Mucinuos cystadenoma Endometriotic cyst Cystic atretic follicle Mutinous cystadenoma Oedematous cyst Serous. papillary cystadenoma Pseudomucinous cystadenoma Serous cystadenofibroma Serous cystoma Mutinous cystadenoma Cystic corpus luteum Serous, papillary cystadenocarcinoma Serous. papillary cystadenocarcinoma Mutinous adenocarcinoma Adenopapillary carcinoma “solid” adenocarcinoma Solid, papillary adenocarcinoma Endometrioid adenocarcinoma
* mm,no placental-like ALP detected; +, substantial like ALP detected; ++, high level of placental-like
x
_
x x
x
x
x
x
x
x
x
x
x
x x
x
x
x
x
x
x _ _
x
x x x
x x x x x
x _
x x
x
x
x
x
x
x x x
x _
Placent&like ALP *
62 31 30 41 23
_ _ _ _ _
44 58
++ _
53 49
+
62 24 47
+
-.
x
42
++
_ _
x x x x x
68 68
(+) _
36 51
++ +
x
x
placental-like ALP detected; ALP detected.
60 (+), low level of placental-
205
Fig. 1. ALP components in four normal human tissues as separated by starch gel electrophoresis (pH 8.6). The enzyme substrate was P-naphthyl phosphate. (a) Tissue extracts treated with neuraminidase prior to electrophoresis. (b) Tissue extracts untreated. The faster moving main mnes of activity are accompanied in all tissues by slow compact bands which do not migrate far from the origin. These latter bands have been shown to be aggregate forms of the faster moving enzymes. Note that in the intestinal sample. besides the main ALP band which is unaffected by neuraminidase, an additional faster moving neuraminidasesensitive, liver-like ALP component is also present. In the present paper the term “intestinal ALP” is used to refer to the main, neuraminidase-insensitive component. (c) ALP components in placenta (PL type Z-1). a benign ovarian tumor and a normal ovary as separated by starch gel electrophoresis (PH 8.6) before and after neuraminidase treatment. The enzyme substrate was 4methylumbelliferyl phosphate. 1. Sample untreated; 2. sample treated with neuraminidase. L. liver type ALP: LN, liver type ALP neuraminidase treated; I, intestinal type ALP.
206
Electrophoretic
and related studies
ALP activity was demonstrated in extracts of all normal and postmortem ovaries and ovarian tumors. Starch gel electrophoresis separated the ALP activity into multiple components. In all normal ovaries and ovarian tumors there was a band of ALP activity which migrated as the liver ALP in starch gel both before and after neuraminidase treatment, which removes accessible sialic acid residues and thereby reduces anodal mobility (Fig. 1). This component was heat labile (no activity remaining after 30 min at 65°C) and was not appreciably inhibited by low concentrations (2.5, 5.0 mM) of L-phenylalanine. In these properties it also closely resembled the liver ALP isozyme, and is thus most likely a product of the same ALP locus which is expressed in liver. In some of the normal ovaries and tumors there was also an ALP component which closely resembled the major intestinal ALP isozyme in it,s electrophoretic mobility before and after neuraminidase treatment (Fig. l), its inactivation by heating and its sensitivity to inhibition by L-phenylalanine. Since the liver type ALP appeared in all normal and postmortem ovaries (18 examined) and the intestinal type ALP appeared in at least ten of these specimens, these two isozymes, when they appeared in the tumor extracts were considered to be “normal ovarian” ALP components. Additional ALP components tiere found in many of the tumor extracts. Seven of the tumors contained ALP components which here heat stable (no loss of activity after one hour at 6O”C), inhibited by low concentrations (2.5, 5.0 mM) of L-phenylalanine, and whose anodal mobilities in starch gel were somewhat retarded by neuraminidase treatment. In these properties, these ALP component,s resembled placental ALP and were clearly differentiated from the other major tissue ALP forms (Fig. 2). However, the electrophoretic mobilities of these placental-like ALPS were -in no case precisely identical to the electrophoretic mobilities of any of the six common phenotypes of placental ALP. The diagram in Fig. 3 shows the electrophoretic mobilities of the ALP components in all the tumors relative to the mobilities of the isozymes found in normal placent,a and liver. The effect of neuraminidase treatment and results of t,he heat inactivation and L-phenylalanine inhibition studies on each component are also indicated. The seven tumors which were shown to have ALP component with placental-like ALP were OVC 5, 10, 11,13,16, 17 and 18. Of the remaining tumors, seven had only the liver type ALP (OVC 2,4,7, 14, 15,20 and 21) and four had additional components which resembled the liver form in the heat lability and insensitivity to L-phenylalanine inhibition but which showed electrophoretic mobility different from any normal tissue ALP (OVC 8,9,12 and 19).
Immgzodiffusion In double diffusion plates, extracts of placenta and of all tumors which contained readily detectable amounts of the placental-like ALP (OVC 5, 10, 13, 16 and 17) cross-reacted with antiplacental ALP antiserum to form enzymatically active preclpitin lines (Fig. 4a). The precipitin lines from these tumor extracts were continuous with the placental-antiplacental ALP precipitin line suggesting immunological identity of the tumor enzyme with the placental enzyme. Extracts of normal ovary, liver and the other tumors did not show cross-reactiv-
207 l-1
__
i
Fig. 2. (a) ALP components of OVC 13 (malignant tumor) and placenta (PL type 2-l) as separated by starch gel electrophoresis (pH 8.6). ‘Heated’ samples were heated at 65’C for 30 min prior to electrophoresis. 1, Sample untreated with neuraminidase; 2. sample treated with neuraminidase. (b) L-Phenylalanine inhibition studies of the ALP components of OVC 13 and placenta (PL type 2-l). Both the tumor and the placental extracts were neuraminidase treated. Prior to staining for enzyme activity strips of gel were incubated with various concentrations of L-phenylalanine as indicated. The placental ALP and the heat-stable components of the tumor ALP were increasingly inhibited by increasing concentrations of L-phenylalanine. In contrast. the liver-like ALP components in the tumor were not inhibited by L-phenylalanine.
ity with the same antiserum. When the tumor extracts which cross-reacted with the antiserum were heated under conditions which completely destroy all nonplacental ALP (30 min at 65”C), enzymatically active precipitin lines were still formed (Fig. 4b). Thus in five of the tumors, the ALP components which resembled the placental ALP in heat stability and L-phenylalanine sensitivity also showed immunological identity with the placental ALP. However, two tumor extracts which did not show cross-reacting material in the double diffusion plates had been shown to contain small amounts of a heat-stable, L-phenyl-
208
209
Fig. 4. lmmunodiffusion plates stained for ALP activity using fl-naphthyl phosphate as substrate. Center well in plates contained antiplacental ALP antiserum. Surrounding wells contained butanol extracts of tissue or tumors. Where indicated, sample was heated for 30 min at 65°C prior to application into the well. A. 1, placenta; 2. OVC 17; 3. OVC 10; 4, no sample: 5. OVC 16; 6, OVC 13. B. 1, placenta (heated); 2, OVC 10 (heated); 3, OVC 16 (heated); 4, placenta; 5, OVC 13 (heated): 6. OVC 17 (heated). C. 1, placenta; 2. OVC 4; 3, no sample: 4, placenta: 5, OVC 7; 6, OVC 8. D. 1. placenta; 2. OVC 21; 3. OVC 21 (heated); 4. placenta; 5, ovary; 6, liver. Note than OVC 17 (A2 and B6) did show faint precipitin lines. although they are not evident in the photograph.
alanine sensitive ALP (OVC 11 and 18). When these extracts were mixed with the antiserum and then electrophoresed, the heat-stable, placental-like ALP components did not migrate into the gel, indicating that they had been selectively precipitated by the antiserum. The electrophoretic mobilities of nonplacental ALPS were unaffected or only slightly retarded. Non-immune serum did not significantly retard the mobilities of any of the ALPS.
Benign vs. malignant
tumors
Placental-like ALP activity, as characterized by heat stability, sensitivity to inhibition by L-phenylalanine, and cross-reaction with antiplacental ALP antiserum, was found in two out of the 11 benign, and five out of the seven malignant tumors (Table II). The proportion of tumors with the ALP in the malignant group was significantly greater than the proportion in the benign group at. the 5% level (p = 0.0387, Fisher’s extract treatment of a 2 X 2 contingency table). Thus in this tumor series, there was an increased incidence of the ALP in the malignant tumors as compared with the nonmalignant tumors. The presTABLE
II
SUMMARY OF ALP TO PLACENTALLIKE Tumor
type
COMPONENTS ALP Number
IN THE EIGHTEEN
examined
Number
Benign Malignant
11 7
2 5
Total
18
7
* As characterized by heat stability, anti-placental ALP antiserum.
sensitivity
to inhibition
OVARIAN
TUMORS.
with placental-like
WITI
REFERENCE
ALP *
by L-phenylananine,
and cross-reaction
with
210
i *;
7
?
f!CJ!i’
,
Fig. 5. ALP components in OVC 16 and placenta (PL type 2-1) as separated by starch gel electrophomis (pH 8.6) before and after neuraminidase treatment. Heated samples were heated for 30 min at 65OC. 1. sample untreated: 2, sample treated with neuraminidase. Fig. 6. ALP components of OVC 13 and placenta (PL type 2-l) as separated by starch gel electrophoresis (PH 8.6). All samples were neuraminidase treated. 1. Placenta PL type 2-1. 2, Placenta PL type 2-l extract mixed with OVC 13 extract in a ratio of one part to two parts. 3. Placenta PL type 2-l extract mixed with OVC 13 extract in a ratio of one part to five parts. 4, OVC 13.
ence of the placental-like pre- and post-menopausal tumors which contained
Phenotypes
ALP did not correlate ages were represented the placental-like ALP.
with age of the patient. in the groups of patients
Both with
of the platen tal-like ALP
The standard electrophoretic procedures used to determine the phenotypes of normal placental ALP did not achieve separation of the placental-like ALPS into unambigusously typeable components. The diffuse appearance of the placental-like ALP in four of the tumors (OVC 5, 10, 11, 16) made then untypeable; all the normal placental enzyme phenotypes separate into discrete, sharp bands [19] (Fig. 5). The placental-like ALPS of OVC 13, 17, and 18 separated into sharper bands and moved to normal placental positions in starch gel. After neuraminidase treatment, OVC 13 had four or five major bands of activity, OVC 17 had two or three major bands of activity, and OVC 18 had three major bands of activity (Fig. 3). The first two of these patterns thus showed more bands than would be expected for completed desialated placental ALP (since the enzyme is a dimer, the heterozygote pattern has three major bands, and the homozygote pattern has one major band). Since it seemed possible that all the accessible sialic acid groups had not been removed by the standard neuraminidase treatment, the tumor extracts were subjected to additional neuraminidase treatment. This reduced the activity in OVC 13 to three major bands of activity. Electrophoresis of a mixture containing an extract of a PL type 2-l placenta and the OVC 13 (both neuraminidase treated) showed that the three bands of activity in OVC 13 were slightly slower than the three bands
211
of PL type 2-l (Fig. 6). Thus if the ALP pattern in OVC 13 represents an unmodified placental phenotype, the individual might be heterozygous for two rare alleles at the PL locus. Alternatively, it is possible that the pattern represents a slightly modified common phenotype, probably type 2-l. The additional neuraminidase treatment reduced the activity in OVC 17 to one major ALP band plus two minor bands. This could represent a homozygous pattern, as minor secondary bands are frequently seen in the normal placenta [ 121. The mobility of the major band of OVC 17 was slightly slower than the common placental type 1, as shown by mixing the two extracts together and electrophoresing the mixed sample. Thus the individual might be homozygous for a rare allele at the PL locus, or this pattern could also represent a slightly modified common phenotype, type 1. In OVC 18 the fastest of the two ALP bands had approximately the same mobility as the common placental type 2; however, the two slower bands did not correspond with any common phenotype. Electrophoresis of these tumor extracts in the pH 6.0 system did not improve resolution of the placental-like ALPS or yield any further information about their phenotypes. L-Leucine inhibition The placental-like ALPS present in five of the seven tumors were tested for inhibition by L-leucine. Results given in Table III show that the heat-stable ALPS in extracts of OVC 10 and OVC 16 were much more inhibited than the normal placental ALP (PL type 1 and PL type 2) by all L-leucine concentrations tested (0.5 to 10 mM). The heat-stable ALPS of OVC 13, 17, 18 had inhibition profiles similar to that of the normal placental enzyme though they were somewhat more inhibited at the lower L-leucine concentrations. Thus the tumor ALPS (heat-stable) which had slow, smeary electrophoretic patterns (OVC 10, 16) were also very inhibited by L-leucine. None of the OVC 5 or 11 extracts, which also contained electrophoretically slow, smeary heat-stable ALPS were available to test. Plasma Plasma or serum samples were obtained from 15 of the 18 ovarian tumor patients (Table I). Sera was available from five of the seven patients with TABLE
III
L-LEUCINE PONENTS Values
given
L-Leucine
INHIBITION IN
FIVE
OF
OVARIAN
represent
PLACENTAL
the average Percent
ALP
(PL
TYPE
1)
AND
THE
HEAT
STABLE
ALP
of two
separate
experiments,
each
done
on duplicate
sample
aliquots.
inhibition
concentration (mM) 0.5
Placenta n.t.
ovc 49.3
10
ovc 8.9
13
OVC 44.5
16
ovc 5.8
17
ovc n.t.
1.0
6.5
62.6
19.8
60.5
18.0
2.5
19.2
81.9
29.8
77.1
25.6
21.5
5.0
24.5
88.3
39.5
83.0
34.3
32.6
7.0
36.7
91.4
47.4
86.7
40.0
36.7
39.1
94.2
51.1
87.5
53.5
39.4
10.0
COM-
TUMORS
17.4
18
212
tumors which had placental-like ALP. Electrophoresis of the sera showed that in addition to the normal serum ALP components (present in all samples and in 15 samples from healthy individuals), three of the samples had extra ALP components. These sera were from patients, 10, 16 and 17, all of whom had malignant tumors which contained placental-like ALP. In each of these cases, the extra ALP components were heat-stable and they corresponded in electrophoretic mobility with the heat-stable components found in the respective tumors. Placental-like ALP was not detected (by electrophoresis) in two of the five sera available from patients with this ALP isozyme in their tumors. Cyst fluid Cyst fluid was available from 11 of the tumor patients (Table I). Nine of these fluids had ALP activity detectable by starch gel electrophoresis. All these nine samples contained liver and smaller amounts of intestinal ALP. Cyst fluids were obtained from two of the tumors (one malignant, one benign) which had been found to contain a placental-like ALP (OVC 10, 13). Both of these fluid samples contained the heat-stable ALP components which were present in the respective tumor. However, good resolution of these components in the fluid was not achieved because of the high viscosity of the samples. Discussion Characterization of the electrophoretically separated ALP components in the 18 ovarian tumors demonstrated that all the tumors retained the ALP present in normal ovary, i.e. liver type and small amounts of intestinal type. Seven of the tumors had additional, different ALP components which most closely resembled the product of the placental ALP locus, as shown by their heatstability, sensitivity to inhibition by low concentrations of L-phenylalanine, and cross-reaction with antiplacental ALP antiserum. These placental-like ALPS found in the tumor extracts did, however, show modified electrophoretic mobilities both before and after neuraminidase treatment when compared with the normal placental isozymes. This investigation, which involved looking at both benign and malignant ovarian tumors has shown that there was a significantly increased incidence of placental-like ALP in the malignant tumor group as compared with the benign tumor group. In a small second series of 13 ovarian tumors collected after the main work for this report was completed, ALP was characterized by electrophoresis and heat-stability only. Heat-stable ALP was seen in three out of four malignant tumors (three cystadenocarcinemas and one adenocarcinoma), but it was not detected in any of the benign tumors. These data suggest further a significant association between ovarian malignancy and the presence of placental-like ALP. The number of tumors so far studied thoroughly was small, and it would be useful to extend the survey to obtain a more accurate estimate of the association between malignancy and placental-like ALP. The proportion of malignant tumors which had placental-like ALP (five out of seven) was greater than reported proportions of cancer patients’ sera which contained placental-like ALP (8 to 35%). Our investigation of tumor specimens would thus imply that some tumors produce placental-like ALP which does not
213
get into the circulation in significant amounts. In one study where cancer tissue was examined, eight out of 23 (35%) gastric tumors were shown to contain placental-like ALP [ 91. Although ectopically synthesized placental-like ALP is associated with malignancy, this association is not exclusive; the enzyme was also discovered in two tumors classified as benign. It is thought that of all adenocarcinomas of the ovary, over half are derived from benign cystoadenomas [22]. It is therefore possible that the presence of the placental-like enzyme in apparently benign tumors may represent a pre-neoplastic change. An unexpected finding was that none of the placental-like ALPS found in the tumor extracts corresponded in electrophoretic mobility with any of the common electrophoretic phenotypes of placental ALP. It has been reported that the placental-like ALP in a lung carcinoma had a slightly slower mobility in acrylamide gel than the normal placental enzyme [7], Inglis et al. [ 81 have reported that in the sera of some cancer patients there was a placental-like ALP which exhibited slower electrophoretic mobility than the common phenotypes of placental ALP, and the authors suggested that these tumor ALPS represent the rare D-variant phenotype of placental ALP described by Beckman and Beckman [23]. The tumor ALPS which resembled the D-variant in electrophoretic mobility were also inhibited by L-lcucine. (This ALP was found in approximately 50% of all the sera which contained placent,al-like ALP). In the present study the placental-like ALP in four of the tumors had slow, diffuse electrophoretic mobility, and in two cases (the others were not tested), the enzymes were more inhibited by L-leucine than the common placental ALP types. These tumor ALPS may be very similar to what Inglis et al. [B] have described as the D-variant in other tumors, but their identity as the D-variant is still in question [24,25]. It would be useful to compare a D-type placental ALP with these tumor enzymes by electrophoresis in the same starch gel. The placental-like ALPS in the other tumors (OVC 13,17,18) did not resemble the D-variant of placental ALP. They had faster electrophoretic mobility than D, very discrete banding patterns and did not show the same inhibition by L-leutine. However, their electrophoretic patterns could represent, other rare variant phenotypes of placental ALP. A large number of rare alleles (more than 14) have been described at this enzyme locus [ 131. If the individuals concerned did have two rare alleles at this locus, it would imply that the locus is derepressed very much more frequently in tumor cells which carry the rare alleles than in cells which carry the common alleles. Alternatively, the derepressed ALPS in the tumors may have been subject to abnormal postranslational modifications by the unusual cell environment of the enzyme, e.g. resulting in a change of glycosylation of the enzyme molecule. In a previous report [21] we examined the electrophoretic phenotypes of the derepressed, placental-like ALP present in lines of HeLa cells cultured separately for many generations. The results showed that the placental-like ALP differed electrophoretically from line to line, and in one case only, was the mobility equal to that of a common placental ALP phenotype. The unusual electrophoretic patterns of these placental-like ALPS may have resulted from post-translational structural modification, or from mutations at the placental ALP locus. In contrast to the abnormal properties displayed by the tumor ALPS,
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numerous other enzymes in tumor cells have not been shown to exhibit abnormal phenotypes [ 261. Most of the enzymes which have been studied are not “derepressed” enzymes, so the derepression process, or factors which promote it, may also promote defective transcription and translation, or even mutation. Recently, human chorionic gonadotropin, a glycoprotein hormone, was found to be expressed in a malignant cell line (HeLa) and the cu-subunit of this “derepressed” hormone had properties slightly different from those of the native placental hormone [ 271. Electrophoretic and related investigations of the tumor patients’ plasma suggest that placental-like ALP from the tumor had been released into the circulation in easily detectable amounts in three cases. The tumors of all these patients were malignant. The placental-like ALP from the one benign tumor which had had high levels of it (OVC 13, the other benign tumor with the ALP isozyme, OVC 18, had only trace amounts) was not detectable in the circulation. Thus although the methods used in this study for the detection of placental-like ALP in serum were perhaps less sensitive than some other methods used for sera surveys [14--161, a higher percentage of tumors may synthesize placental-like ALP than has previously been inferred on the basis of sera surveys. Acknowledgements We should like to thank professor D. Fairweather, Dr. P.C. Hughesdon and colleagues at University College Hospital, London, and also Mr. J. CarronBrown, Dr. J.H. Rack and colleagues at the Norfolk and Norwich Hospital, Norwich, for their permission to obtain ovarian tumor specimens and for kindly supplying us with histology reports. We are also grateful to Mr. Thompson for handling the collections of material and the documentation at Norwich. We thank Ms. G. Bulsara, Ms. A. Tanyar for skilled technical assistance. This work was in part supported by a grant from the American Cancer Society, No. VC-231. References 1
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