Modified properties of serum cholinesterases in primary carcinomas
11
Modified Properties of Serum Cholinesterases in Primary Carcinomas HAlM ZAKUT, MD,**t LEA EVEN, MD$§ SHLOMO BIRKENFELD, MD,’ GUSTAVO MALINGER, MD,’ RlVKA ZISLING, MSc,* AND HERMONA SOREQ, PHD*~II Cholinesterases were characterized in the serum of 77 treated and 1 I untreated patients having primary carcinomas of various tissue origins and 21 healthy volunteers which served as controls. In most of the samples, pseudocholinesterase (BuChE) accounted for almost all cholinesterase (ChE) activity and was inhibited by the organophosphorous poison tetraisopropyl pyrophosphoramide (iso-OMPA). In sam- ples from the tumor-bearing patients, ChE degraded 733 f 59 nmole acetylcholine/h/mg protein, lower than the 960 +. 175 nmole/hour/mg levels measured in controls. Tumor serum ChE exhibited elevated sensitivity to 1,5-bis-(4-allyldimethyl ammonium phenylkpentan3-one dibromide (BW), the selective bisquaternary inhibitor of “true” acetylcholinesterase (AChE), with no correlation to age, sex, staging of tumor, presence of metastases or the specific treatment protocol, and with a different distribution pattern from the decrease in ChE specific activity or the sensitivity to iso-OMPA. In sucrose gradients, ChE sedimented as 12s in controls whereas in tumor serum samples from treated patients an additional component of 6 to 7 S, inhibited by both iso-OMPA and BW, also was detected. However, the ChE activity in serum of patients with diagnosed carcinomas before surgery and medical treatment appeared to be nondistinguishable from controls. These findings suggest that the modified properties of serum cholinesterases in carcinoma patients are not the result of the tumor itself, but that the common therapy protocols used in the treatment of primary carcinomas may cause the appearance of soluble ChE activity with properties of both AChE and BuChE, which accumulates in the serum. Cancer 61:727-737, 1988. N ADDITION to their functional site at neuromuscular I junctions and cholinergic synapses, cholinesterases (ChE) are noted for appearance at various other loci and also for their unexplained transient expression in prolif- erating tissues of epithelial and mesenchymal origin.‘.’ Appearance of ChE early in the development of various embryonic tissues has been correlated with regulation of cell proliferation’ and with morphogenetic cell migra- tion,’~~ and it occurs also in tissues that do not express synaptic ChE, such as the chondrogenic core of the chicken limb bud’ or rat prornegakaryocyte~.~ Expres- sion of ChE is an early differentiation event in the entire From the $Department of Biological Chemistry, The Life Sciences Institute, The Hebrew University, Jerusalem, and *Department of Ob- stetrics and Gynecology at the Edith Wolfson Medical Center, Holon. and The Sackler Faculty of Medicine, Tel Aviv University, Israel. Supported by the Israeli Health Ministry (to H.S. and H.Z.) and by the Hermann and Lilly Schilling Foundation for Medical Research (to H.S.). 11 Recipient of contract DAMD 17-8543025 from the US Army Medical Research and Development Command, of a grant from the Silbermann Foundation for Research and of a grant from the Israeli Health Ministry (with H.Z.). 8 Participated in this research as part of the requirements towards an brain of chick^,^ of the macaque m ~ n k e y , ~ and of High levels of ChE also have been detected in various types of primary tumors, including ovarian car- cinomas,’ gli~blastomas~~~ and meningiomas.’ The ChE expressed in mature and embryonic tissues are highly polymorphic at several levels. They may be classified by their substrate specificity into acetylcholin- esterase (acetylcholine hydrolase, EC 3.1.1.7 [AChE]) and pseudocholinesterase (acylcholine acyl hydrolase, EC 3.1.1.8 [WhE] reviewed in the articles of Silver’ and Massoulie and Bon”). Acetylcholinesterase and W h E generally are distinguished by their specific sensitivity to MD degree in the Medical School of the Israeli Institute for Techonol- ogy, Haifa, Israel. t Recipient ofsupport from the Research Fund ofthe Edith Wolfson Medical Center and of a grant from the Israeli Health Ministry (with H.S.). The authors thank Professor I. Silman for critically reviewing this manuscript, and Smadar Glick for her excellent assistance throughout this study. Address for reprints: Hermona Soreq, PhD, Department of Biologi- cal Chemistry, The Life Sciences Institute, The Hebrew University. Jerusalem 9 1904. Accepted for publication August 25, 1987. 727
Transcript of Modified properties of serum cholinesterases in primary carcinomas
Modified Properties of Serum Cholinesterases in Primary Carcinomas
HAlM ZAKUT, MD,**t LEA EVEN, MD$§ SHLOMO BIRKENFELD, MD,’ GUSTAVO MALINGER, MD,’ RlVKA ZISLING, MSc,* AND HERMONA SOREQ, PHD*~II
Cholinesterases were characterized in the serum of 77 treated and 1 I untreated patients having primary carcinomas of various tissue origins and 21 healthy volunteers which served as controls. In most of the samples, pseudocholinesterase (BuChE) accounted for almost all cholinesterase (ChE) activity and was inhibited by the organophosphorous poison tetraisopropyl pyrophosphoramide (iso-OMPA). In sam- ples from the tumor-bearing patients, ChE degraded 733 f 59 nmole acetylcholine/h/mg protein, lower than the 960 +. 175 nmole/hour/mg levels measured in controls. Tumor serum ChE exhibited elevated sensitivity to 1,5-bis-(4-allyldimethyl ammonium phenylkpentan3-one dibromide (BW), the selective bisquaternary inhibitor of “true” acetylcholinesterase (AChE), with no correlation to age, sex, staging of tumor, presence of metastases or the specific treatment protocol, and with a different distribution pattern from the decrease in ChE specific activity or the sensitivity to iso-OMPA. In sucrose gradients, ChE sedimented as 12s in controls whereas in tumor serum samples from treated patients an additional component of 6 to 7 S, inhibited by both iso-OMPA and BW, also was detected. However, the ChE activity in serum of patients with diagnosed carcinomas before surgery and medical treatment appeared to be nondistinguishable from controls. These findings suggest that the modified properties of serum cholinesterases in carcinoma patients are not the result of the tumor itself, but that the common therapy protocols used in the treatment of primary carcinomas may cause the appearance of soluble ChE activity with properties of both AChE and BuChE, which accumulates in the serum.
Cancer 61:727-737, 1988.
N ADDITION to their functional site at neuromuscular I junctions and cholinergic synapses, cholinesterases (ChE) are noted for appearance at various other loci and also for their unexplained transient expression in prolif- erating tissues of epithelial and mesenchymal origin.‘.’ Appearance of ChE early in the development of various embryonic tissues has been correlated with regulation of cell proliferation’ and with morphogenetic cell migra- tion,’~~ and it occurs also in tissues that do not express synaptic ChE, such as the chondrogenic core of the chicken limb bud’ or rat prornegakaryocyte~.~ Expres- sion of ChE is an early differentiation event in the entire
From the $Department of Biological Chemistry, The Life Sciences Institute, The Hebrew University, Jerusalem, and *Department of Ob- stetrics and Gynecology at the Edith Wolfson Medical Center, Holon. and The Sackler Faculty of Medicine, Tel Aviv University, Israel.
Supported by the Israeli Health Ministry (to H.S. and H.Z.) and by the Hermann and Lilly Schilling Foundation for Medical Research (to H.S.).
11 Recipient of contract DAMD 17-8543025 from the US Army Medical Research and Development Command, of a grant from the Silbermann Foundation for Research and of a grant from the Israeli Health Ministry (with H.Z.).
8 Participated in this research as part of the requirements towards an
brain of chick^,^ of the macaque m ~ n k e y , ~ and of High levels of ChE also have been detected in
various types of primary tumors, including ovarian car- cinomas,’ gl i~blastomas~~~ and meningiomas.’
The ChE expressed in mature and embryonic tissues are highly polymorphic at several levels. They may be classified by their substrate specificity into acetylcholin- esterase (acetylcholine hydrolase, EC 3.1.1.7 [AChE]) and pseudocholinesterase (acylcholine acyl hydrolase, EC 3.1.1.8 [WhE] reviewed in the articles of Silver’ and Massoulie and Bon”). Acetylcholinesterase and WhE generally are distinguished by their specific sensitivity to
MD degree in the Medical School of the Israeli Institute for Techonol- ogy, Haifa, Israel.
t Recipient ofsupport from the Research Fund ofthe Edith Wolfson Medical Center and of a grant from the Israeli Health Ministry (with H.S.).
The authors thank Professor I. Silman for critically reviewing this manuscript, and Smadar Glick for her excellent assistance throughout this study.
Address for reprints: Hermona Soreq, PhD, Department of Biologi- cal Chemistry, The Life Sciences Institute, The Hebrew University. Jerusalem 9 1904.
Accepted for publication August 25, 1987.
727
TA
BL
E
I. C
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erum
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Prot
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Act
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(p
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Typ
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tum
or
Clin
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tre
atm
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Con
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ser
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I
Inhi
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ChE
act
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No.
A
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ex
Eth
nic origin
His
tolo
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type
M
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s Su
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Che
m
Hor
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Gyn
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a ov
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+ -
-
-
70
600
84
0 21
II
67
E
ur
Mut
inou
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+ -
-
79
600
95
15
49
10
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Cys
tade
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y 44
A
sia
Ca
of C
x +
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+ -
-
64
800
87
8 I6
-
+ +
-
- 69
86
6 76
40
27
12
13
77
Eu
r C
a of
14
41
Asi
a G
ranu
losa
cel
l -
+ +
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-
76
666
71
29
44
15
73
Asi
a A
deno
Ca
Cx
-
+ -
-
65
667
92
39
55
endo
met
rium
Cx o
vary
-
Nor
mal
1 2 3 4 5 6 7 8 9 10
11
12
13
14
I5
16
17
18
19
20
21
MI4
8 M
I66
F/62
M
I53
MI5
2 F/
58
FIN
M
I43
MI3
3 F/
62
F/55
MI4
4 F/
37
MI4
6 M
I42
F/52
F/
22
F/40
F/55
MI4
3 M
I50
us
Asi
a U
SSR
Eur
Asi
a Eu
r A
sia
No
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USS
R A
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Asi
a E
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No
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Eur
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a
Ova
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and
test
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fr E
ndom
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USS
R
Sero
us A
deno
4 55
U
SSR
Se
rous
Ade
no
Ca
Ade
no C
a
Ca
Ca
65
48
68
70
68
78
73
66
71
65
68
73
78
75
71
73
80
68
65
70
74
1267
I2
67
933
866
933
800
866
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93
3 93
3 10
66
933
866
1 I33
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6 66
7 13
33
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80
0
94
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78
85
86
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92
93
88
87
87
93
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91
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91
60
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30 1 17 3 19
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16
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1.
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23
33
47
40
29
25
54
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32
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83
62
21 4 87
59
71
1200
71
-1
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I 100
84
25
63
1115
74
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70
584
75
-7
No. 4 BW AND Iso-OMPA INHIBITED CHE FORMS IN CA Zakut et al. 73 1
selective inhibitors, with AChE being selectively blocked by the reversible bisquaternary inhibitor BW284CS 1, whereas $ChE is irreversibly inhibited by binding of the organophosphorous poison tetraisopropyl pyrophos- phamide (iso-OMPA) to the serine residue in its active site.".'* Both enzymes appear in multiple molecular forms with various levels of hydrophobicity, different numbers of catalytic subunits and distinct sedimenta- tion properties in sucrose gradients."
In the human blood, soluble iso-OMPA-sensitive tet- ramers of W h E reside in the serum" whereas hydro- phobic, 1,5-bis-(Callyldimethyl ammonium pheny1)- pentan-3-one dibromide (BW)-sensitive dimers of AChE" are covalently attached to the membrane of erythrocytes through phospholipid moieties. l4 Minor activities of AChE (ca 0.1% of $ChE levels) also were detected in the serum." In malignant and embryonic tissues, both BW-sensitive and iso-OMPA sensitive ChE were detected, with independent patterns of expression. The term "embryonic cholinesterase" has been pro- posed for these activities by Drew? and later supported by Layer.4 However, it is not known yet whether the heterogeneity of ChE in general stems from different structural genes or from posttranscriptional and post- translational processing.16 Thus it has also remained unclear whether the term "embryonic ChE" refers to a biochemically distinct entity and what are its character- istic properties. Furthermore, it has not been clarified whether the enhanced expression of ChE in particular tumors is related to tumorogenetic processes or whether it stems from antitumor therapy. In previous studies, we found indications for the expression of a ChE form which is sensitive to both iso-OMPA and BW in primary brain tumors and in fetal human brain.'.'' To further examine this finding, we characterize the ChE which accumulate in the serum of patients who have carci- nomas of different tissue origins, with various rates of cell proliferation and differentiation levels. Our findings indicate that in patients under treatment for primary carcinomas, a modified route of expression for ChE genes results in the accumulation in the serum of embry- onic type of ChE activity, susceptible to both BW and iso-OMPA inhibition.
Materials and Methods
Serum Samples
Serum samples were drawn from patients having car- cinomas of different tissue origins, who were either hos- pitalized or under follow-up as outpatients. These in- cluded 28 patients with breast carcinoma, seven with lung carcinoma, 17 with various carcinomas of the di- gestive tract, ten with urinary tract carcinomas, and 15 with gynecological carcinomas. A second group of 1 1
732 CANCER February I5 1988 Vol. 61
samples was taken from 11 patients with diagnosed ovarian carcinomas, before the surgical removal of these localized resectable tumors and before medical treat- ment. This particular type of primary carcinomas was selected as the control group of patients (Table 1) tested before treatment because all of these tumors originate from one tissue, namely, the ovary, and could be matched with ten other samples from ovarian carci- noma patients, that were all treated (Table 1). Serum from 2 1 healthy volunteers served for controls. Details regarding these patients, as well as the male/female ratio in each subcategory of tumors, are brought in Table 1. For preparation of serum samples, fresh human blood (5 ml) was drawn without anticoagulant, and centrifuged in a clinical centrifuge at 1000 X g. The supernatant, in which no hemolysis could be detected, was retained for assay of serum ChE activity. Serum samples were kept frozen at -70°C until used, and were not thawed more than twice.
Cholinesterase Assays
[3H]-Acetylcholine hydrolyzing activity was measured according to the general procedure of Johnson and Rus- selli8 as previously detailed For inhibi- tion studies, a volume of a stock solution of the inhibitor was diluted so as to yield the appropriate final concen- trations employed. In the case of iso-OMPA, the reac- tion mixture was preincubated for 30 minutes at 25°C before initiation of the assay by addition of substrate.” Assays were run at 25”C, for 60 minutes, using 10 p1 of 1 : lO diluted serum per sample in 100 pl reaction mix- tures. Preliminary experiments ensured that under these conditions the reaction is linear with time and volume of sample added. Parallel spectrometric measurements of acetylthiocholine and butyrylthiocholine hydrolysis were carried out as detailed previously.’
Sucrose Density Gradient Centrijugation
Two hundred-microliter serum samples were centri- fuged in 5% to 20% linear sucrose gradients as previously de~cribed.~~’~” About 60 fractions were collected and aliquots were assayed as above, with or without the se- lective inhibitors. Beef liver catalase ( 1 1.4 S, Sigma, St. Louis, MO) was the principal marker.
Protein Measurements
Protein measurements were carried out according to Bradford,” using a Microelisa Auto Reader MR 580 (Dynatech, Denmark) and Nucattom 96F microwell plates (Nunc. Denmark).
Results
To find out whether changes in the biochemical prop- erties of serum ChE appear in patients under treatment for carcinoma tumors, we set out to determine inhibi- tion curves for the ChE in 88 serum samples drawn from patients experiencing carcinomas of various tissue ori- gins and in 21 serum samples from healthy volunteers, using selective inhibitors specific to particular types of ChE. Three compounds were used: iso-OMPA: an or- ganophosphorous poison with high specificity towards serum $ChE,’,’’ BW284C5 1 : a bisquaternary reversible inhibitor of “true” AChE; and succinylcholine, a sub- strate analog of acetylcholine which is rapidly hydro- lyzed by normal $ChE but cannot be degraded by the “atypic” or “silent” types of serum J/ChE.’.*’ Each in- hibitor was added at six different dilutions, covering c1
wide range of concentrations. Representative curves from the inhibition experiments are presented in Figure 1. The enzyme in all of the serum samples was clearly sensitive to iso-OMPA, as expected from human WhE, with 1 X M of the inhibitor sufficient for quantitative block of acetylcholine hydrolysis. The inhibition curves of the tumor serum samples could be divided to three major groups:
up to 1 X
1. curves which are indistinguishable from control ones, with high sensitivity towards iso-OMPA and con- siderably lower sensitivity to BW (for example: curve 5/3 in Fig. I ) ;
2. curves displaying high sensitivity to BW, and nor- mal inhibition by iso-OMPA (for example: curve 1/14 as compared with 6/10); and
3. curves with parallel levels of sensitivity towards both inhibitors (such as 1/19 and 1/13 in Fig. 1).
In some of the samples examined, low concentrations of particular inhibitors caused an increase in the rate of acetylcholine hydrolysis. This may be due to the block- ing action of these inhibitors on the competitive or in- hibitory activity of other serum proteins.*’ It should be noted that spectrophotometric measurements of ChE activities, using acetylthiocholine or butyrylthiocholine as substrate’ (not shown) yielded parallel changes. This indicates that inhibitor studies truly reflect the relative activities of AChE and BuChE in the examined samples.
About 75% of the samples were drawn postsurgery from patients under irradiation therapy, chemotherapy, or hormone treatment. Metastases were diagnosed, ei- ther by x-ray analysis, computerized tomography, ultra- sound, or biopsy in a few patients only, and the age group examined ranged between 22 and 86 years. To determine whether the properties of serum ChE are re- lated to the medical treatment protocols to which carci- noma patients are commonly subjected, a group of pa-
No. 4 BW AND Iso-OMPA INHIBITED CHE FORMS IN CA - Zakut el a/.
Succinylcholine concentration ,ng/ml
733
I n h i b i t o r concent ra t ion , M FIG. I . Inhibition curves of serum cholinesterase activities. Serum samples were drawn as detailed under “Materials and Methods” from patients
described in Table 1 (For example: the curve marked 3/ 10 refers to sample no. 10 in the third section of Table I , which includes patients who have digestive tract carcinomas). [3H]-acetylcholine hydrolysis was measured in the absence (empty squares) or in the presence ofthe noted concentra- tions of the following inhibitors: iso-OMPA (full circles), BW (empty circles), succinylcholine (full triangles). [’HI-acetate released was measured in cpm and background radioactivity, obtained in the absence ofserum samples, was subtracted. Each digit notes the release of 1 x lo4 cpm per hour, induced by the addition of I pl serum.
tients with diagnosed but untreated carcinomas of the ovary also was examined. Specific values from the above described inhibition curves (the total ChE specific activ- ity and the inhibitions caused by 50 ng/ml succinylcho- line, 1 X lo-’ M BW and 1 X M iso-OMPA) were selected as representative data and are brought for all of
the samples which were tested in Table 1, together with clinical description of the tumor types and the patients.
The results obtained with different serum samples re- flect considerable variabilities, with extensive heteroge- neity of the inhibition patterns obtained with various inhibitors and high level of scattering in the specific ac-
7 34 CANCER February 15 1988 Vol. 61
TABLE 2. Properties of Cholinesterases in Serum of Carcinoma Patients
ChE Sp Act Type of No. of Age. Protein pmo~/pg P + 1.w4 M + I . I O - ~ M +50ng/ml
No. treatment patients and means (mg/ml) ( hr ) iso-OMPA BW284.CSI Suc Ch carcinoma under samples distribution
Breast 28 30-71 (54.9) 72.3 f 8.3 667 * 333 Lung 1 43-82 (61.0) 72.3 f 6.5 661 k 200
Urinary tract 10 70-86(73.6) 71 f 5.5 8 0 0 f 266 Gynecological 15 39-73 (57.0) 71.5 f 5 .5 733 f 133
Digestive tract 17 42-86 (67.2) 65.8 f 10 800 k 266
Average of all treated tumor
Untreated ovarian types - 30-86 (61.0) 70.7 f 2.2 733 f 59
carcinomas I 1 22-70(52.5) 70.2 f 1.8 799 f 58 Control 21 22-66 (47.8) 69.8 f 6.5 960 f 175
Total I09
83.9 ? 16.1 88.4 2 6.9 88.5 2 9.5 79.3 2 11.7 85.8 ? 9.3
85.2 f 3.4
73 2 10.1 83.0 5 19.0
24.1 f 21.4 39.6 ? 19.8 31.8 f 22.4 27.1 f 21.3 23.6 f 27.0
29.2 f 6.0
9.3 f 12.5 6.2 f 16.6
35.3 f 43.7 30.6 t 14.8 25.5 t 34.3 26.9 f 20.2 39.0 r 18.2
31.5 f 5.0
n.d. 39.2 t 20.6
Average values and standard deviations were calculated for each of the test groups separately and for all of the tumor samples together. Statistical significance (P) was measured by the Student’s f test and is noted for the decrease in ChE specific activity and for the enhanced
tivity measurements. However, in most of these mea- surements, the total ChE activity was lower and the sen- sitivity to BW higher in samples from tumor patients under treatment as compared with controls. In contrast, the sensitivity to BW in serum samples from nontreated carcinoma patients were nondistinguishable from con- trols. although the 1 1 women with ovarian carcinomas that were tested before treatment appear to be somewhat younger and, perhaps, of a less variable ethnic origin (Table 1).
Also, there were no differences in the total protein concentration or in the sensitivity to iso-OMPA or suc- cinylcholine in any of these groups (Table I) . The en- hanced sensitivity to BW was not significantly corre- lated to age, sex, ethnic origin, or mode of treatment, and did not differ between patients being treated for distinct types of carcinomas. Because of insufficient his- tologic information, it remained unclear whether the modified ChE properties are related to the state of dif- ferentiation of particular carcinomas. A summary of the biochemical observations, presented in Table 2, re- vealed that the total specific activity of ChE was signifi- cantly lower by about 25% (P I 0.001) in the serum of patients treated for carcinomas, in agreement with pre- vious findings of others.” This phenomenon generally is attributed to decreased functioning of the liver in pa- tients having carcinomas.20 The sensitivity to iso-OMPA was generally similar in tumor serum samples to that measured in control samples. In contrast, 1 X M of BW284C5 1 was sufficient to cause a 29 k 6% decrease in ChE activity in the serum samples oftreated patients but only 9.3 t- 12.5% in the serum of nontreated carcinoma patients and 6 * 16% in controls (P I 0.0005). The
sensitivity to BW. ChE Sp Act: cholinesterase specific activity; Conc: concentration;
BW: BW284 C5 1 1,5-bis-(4-allyldimethyl ammonium phenylkpen- tan-3-one dibromide.
controls included two samples in which BW inhibition was exceptionally high (Table 1 , normal no. 1,6). Succi- nylcholine, in turn, showed relatively low variability in the extent of inhibition in tumor serum samples. How- ever, there was no significant difference between the in- hibition observed in diseased and healthy serum, and 50 ng/ml of this analog were essentially sufficient to cause substantial block in all serum samples (Table 2).
The distribution of the modified ChE properties in various samples from treated tumor patients is pre- sented in Figure 2. It is clear from this figure that the enhanced sensitivity to BW was not related to the de- crease in total ChE activity or to the stable susceptibility to iso-OMPA. This implied that the increased sensitivity to BW could reflect the appearance of other forms of ChE in the serum samples of patients under antitumor therapy. To further test this possibility, we subjected several of these serum samples to sucrose gradient cen- trifugation and determined the sedimentation profile of ChE forms in these samples in the absence and presence of BW and iso-OMPA. Four such profiles (of a total of 12 gradients which were run) are presented in Figure 3. The major ChE form in all of these gradients displayed a sedimentation value of ca 125, as expected from soluble $ChE tetramers and in agreement with previous investi- gators.’5*’7 The 12s form was the principal one in con- trol serum samples. In contrast, we detected an addi- tional minor but reproducible peak of activity sediment- ing as ca 6 to 7 S in gradient profiles of serum samples from patients having various tumor types. This tumor- characteristic peak of ChE activity was quantitatively blocked by both BW and iso-OMPA (Fig. 3). This im- plied that it differed from both AChE and +ChE, but
No. 4 BW AND ISO-OMPA INHIBITED CHE FORMS IN CA - Zakur et ai. 735
L l l [ f [ I ' 1 ' 1 - Specific activity, - O/O of Avemge control ~
20 - . could constitute of either another form of ChE with combined properties of both, or of two tumor-charac- teristic forms of AChE and WhE which migrate simi- larly on sucrose gradients. This ChE activity could be responsible for the considerable enhancement in sensi- tivity to BW in serum ChE which also displayed normal sensitivity to iso-OMPA.
Discussion
Biochemical characterization of ChE properties in the serum of 88 patients being treated for primary carci- nomas of different tissue origins as compared with con- trol serum of 2 1 healthy volunteers, revealed the appear- ance of a new type of ChE activity. The study was based on the analysis of a single serum sample from each pa- tient, and in most cases serum was drawn postsurgery from patients under surveillance and various treatment protocols according to the tissue origin of the tumor. The wide range of tumor types and the large age group of these patients further complicate the interpretation of our findings and are probably responsible for the consid- erable variability in ChE measurements.2o However, the reproducibility of inhibition patterns and gradient pro- files, supported by the statistical analysis of a rather large group of samples, indicates that the modified properties of ChE in the serum of these patients reflects a true in vivo phenomenon.
The new soluble serum ChE was found to be suscepti- ble to inhibition by both BW and iso-OMPA, hydro- lyzed both acetylthiocholine and butyrylthiocholine, and exhibited a sedimentation coefficient of 6 to 7 S in sucrose gradients. Thus its properties differ both from those of the well-characterized soluble serum $ChE, which is insensitive to BW and sediments as 12s tetra- mers,'0*'2 and from the properties of the dimeric eryth- rocyte AChE, which under normal conditions is not re- leased to the serum in a soluble form and is not sensitive to iso-OMPA inhibition."S2' To the best of our knowl- edge, partial proteolysis of various ChE forms does not alter their sensitivity to selective inhibitors. 1 ~ 1 0 9 2 1 - 2 4 It is therefore unlikely that this tumor-characteristic type of serum ChE results from a disease-related release of AChE from the erythrocyte membranes or from break- down of WhE tetramers into dimers. Furthermore, the appearance of this new ChE was not related to the aver- age decrease in total serum ChE activity or to its general level of sensitivity to iso-OMPA and succinylcholine. This may imply that these two serum ChE activities originate from different pools of nascent polypeptides. There is a single report in which the sensitivity of Serum ChE to succinylcholine increased in a case of carci- noma.26 However, in view of our current analysis, this seems to be an exception. In addition, it has recently
O / , inhibition 2 2o F by I x 2
M BW
100 80 60 40 20 0 Percent
FIG. 2. Distribution of ChE properties in tumor serum samples. Determinations of ChE were as detailed under "Materials and Methods." Values follow the data presented in Table I and Table 2. The figure presents percent of tumor serum samples (of a total of 77) which displayed certain ChE activities calculated as percent of average control activities (top), as well as percent of tumor serum samples which exhibited certain inhibition by I X lo-' M BW (middle) or I X M isPOMPA (bottom).
been reported that normal human serum contains dimers and tetramers of AChE.25 However, the amount of true AChE in normal serum was estimated to be as low as 0.1% of the WhE level, whereas the tumor serum measurements display much higher levels of AChE ac- tivity.
Increased levels of BW-sensitive ChE previously has been detected by histochemical techniques in tissue sec- tions derived from various types of primary carcinomas, as compared with the parallel benign tissues2 In tissue homogenates from primary glioblastomas and menin- giomas, we have found light forms of ChE which were sensitive to inhibition by both BW and ~W-OMPA.~ Mi- croinjection of mRNA from glioblastomas, menin- giomas and fetal brain into Xenopus oocytes induced the production of ChE activities which could be blocked by both inhibitors." Finally, we have recently isolated ChEcDNA clones from fetal brain origin26 which code
736 CANCER February 15 1988 Vol. 61
Gastpointestinal
N 0. FIG. 3. Sucrose gradient profiles showing molecular forms of ChEs in
diseased and control serum samples. Serum samples were centrifuged on 5% to 20% sucrose gradients, and analyzed for ChE activity in the presence and absence of appropriate inhibitors as described under “Methods.” The type of tumor and no. of sample are noted for each profile (see Table I for further details regarding these samples). T total ChE activity; BW: activity in the presence of 1 X lo-’ M BW284C51; i0: activity in the presence of I X M iso-OMPA. The tumor- characteristic peak of ChE activity susceptible to both inhibitors is marked by dotted lines on the top gradient profiles.
for a protein that interacts with anti-AChE antibodies and shares common sequences with human J.ChE.*’ AI- together, this evidence raises the possibility that the novel serum ChE fraction which we describe is similar to the less-characterized enzyme for which the term “embryonic ChE” has been p r o p ~ s e d . ~ . ~ In view of the fact that we only detect this modified activity in the serum of patients under antitumor treatment (either chemotherapy or irradiation and/or hormone therapy), it appears that the production of this novel ChE activity in the liver may be induced by the medical treatment. It
seems unlikely to assume that the new form of ChE is synthesized within and transported from the tumor tis- sue into the serum, since it does not appear in yet un- treated patients. However, due to the limitations de- tailed above, it cannot be concluded currently whether this putative embryonic ChE consists of one new form or of a mixture of AChE and K h E dimers.
To find out whether the production of embryonic ChE also takes place within the tumor cells, in sitir hy- bridization studies currently are being performed in fro- zen tumor sections using ChE cDNA probes in parallel with cytochemical staining. The primary structure of the soluble embryonic ChE and its relationship to other human ChEs, such as neuromuscular AChE or Serum WhE, is of particular interest. Understanding the regu- lation of this ChE may reveal the molecular control mechanisms leading to the tissue and cell type specificity of ChE polymorphism and shed light on the unknown physiological function of these serine hydrolases in pro- liferating and differentiating cells. In pheochromocy- toma cells, AChE biosynthesis is induced by nerve growth factor29 and in glioblastomas we found that en- hanced levels of AChE9 accompany the increase in epi- dermal growth factor receptor protein3’ and the amplifi- cation of the Erb-2 ~ncogene.~’
It would be interesting to examine whether the ex- pression of ChE genes in primary carcinomas and in the liver of patients subjected to antitumor therapy is coreg- dated with oncogenes and, if so, in what way. To a p proach this issue, heterogenous ChEcDNA clones of primary tumor origin currently are being characterized. The nucleotide sequence of such tumor-originated ChEcDNA clones will then be compared to the se- quence of human cholinesterase cDNA from nonmalig- nant tissue^.^'*^* These clones will subsequently be used as labeled probes to examine, also in longitudinal stud- ies, whether new types of ChEmRNA are transcribed from ChE genes in carcinoma tissues and in the liver of carcinoma patients under treatment.
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