ANALYTICAL BIOCHEMISTRY 93, 286--294 (1979)
Radioreceptor Assay for Epidermal Growth Factor ROGER L. L A D D A , * LESLIE P. BULLOCK,~" TERILEE GIANOPOULOS,* AND LYNN MCCORMICK*
*Division of Genetics, Department of Pediatrics, and ?Division of Endocrinology, Department of Medicine, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 Received November 7, 1977 An established cell line of human lung fibroblasts with a high number of surface receptors for mouse epidermal growth factor (mEGF) was used to develop a simple and highly sensitive radioreceptor assay for EGF. 125I-Labeled mEGF competed mole for mole with unlabeled mEGF for specific receptors. Optimal range for discriminating EGF concentrations in body fluids and tissue extracts by a competitive binding assay was between 5 and 100 ng/ml. Interassay correlation of variation was 8.47% and the recovery of highly purified mEGF added to serum and urine samples was greater than 95%. Human serum and amniotic fluids contained about 24 and 4 ng/ml, respectively, of mEGF equivalents. Concentrations of mEGF in mouse urine and serum were highly variable and were 2- to 10-fold greater than that previously detected by radioimmune assay. Hypophysectomy nearly abolished submaxillary mEGF content in both male and female mice, but testosterone treatment of hypophysectomized animals restored normal concentrations of mEGF to the glands, mEGF added to culture medium disappeared with time as a function of the number of cellular EGF receptors indicating cellular degradation of the growth factor. The radioreceptor assay for EGF is based on the close biologic relationship between the cell receptor site and the native hormone and should prove to be a useful complementary tool to characterize the physiological role of EGF.
Mouse epidermal growth factor (mEGF) 1 is a small polypeptide with a molecular weight of 6045 originally isolated from submaxillary glands of mice (1). mEGF binds to specific receptors associated with the cell surface and thereby stimulates a potent mitogenic response in a variety of mammalian cell types both in vitro and in vivo (2-6). Human EGF (hEGF) has been isolated from urine (4) and appears to be similar to urogastrone, a small polypeptide isolated from human urine which inhibits gastric acid secretion (7-8). mEGF and hEGF have essentially identical binding 1 Abbreviations used: EGF, epidermal growth factor (mEGF, mouse; hEGF, human); RIA-EGF, radioimmune assay for EGF; RRA, radioreceptor assay; HF, human fibroblasts; HBSS, Hanks" balanced salt solution; BSA, bovine serum albumin; NRK, normal rat kidney cells; KNRK, transformed NRK. 0003-2697/79/040286-09502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved.
properties and compete mole for mole for specific EGF receptors (8). These accumulating data suggest that EGF may have important physiological functions and thus, sensitive methods to measure EGF concentration in animals is essential. Bioassay for EGF requires multiple injections of suckling mice with large amounts of test material and provides only semiquantitative values (1,9). Radioimmune assay (RIA) for EGF has been used to isolate hEGF and to estimate EGF content of extracts of various tissues (10-14). In addition, a simple and highly sensitive radioreceptor assay (RRA) has been used to measure EGF receptors on cells (15,16). We have developed the RRA-EGF to measure the concentration of EGF in animal tissues and fluids and have found that some values differ from those reported by RIA-EGF. It is possible that
286
RADIORECEPTOR ASSAY FOR EPIDERMAL GROWTH FACTOR
RRA-EGF and RIA-EGF may reflect different functional aspects of the EGF molecule. MATERIALS AND METHODS
Cells. Normal human fibroblasts (HF) derived from neonatal foreskin and its SV40-transformed derivative (SV40HF), 3T3, SV40 3T3, normal rat kindey cells, and a Kirsten sarcoma virus-transformed derivative, normal WI-38, and SV-40-transformed human fetal lung cells (SV40 WI-26) were routinely grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum.
mEGF isolation, mEGF was isolated from adult male mouse submaxillary glands according to Savage and Cohen (17). 125I-Labeling o f EGF. Iodination reaction (18) was carried out in a 10 × 75-mm glass vial at room temperature. Carrier-free Na~25I (Amersham/Searle), 0.5 mCi, in 5/zl was added to the vial and the pH was adjusted to 7.5 by the addition of 25/zl of 0.05 M phospate buffer (pH 6) and 100 /zl of 0.05 M phosphate buffer (pH 7.5). Ten micrograms of mEGF were added to the tube and the contents were gently mixed. Twenty-five microliters of chloramine-T (2 mg/ml) were added and the reaction carried out for 60 s; 25 /~1 of Na metabisulfite (2.5 mg/ml) was added to stop the reacton. ~25I-Labeled mEGF was separated from unbound 125I by passing the reaction mixture through a 20 x l-cm column of Sephadex G-25 (medium grain size). Specific activity of l~SI-labeled EGF ranged from 25-54/zCi//zg. Characterization of EGF binding. For binding and radioreceptor assays, 1 × 106 SV40 WI-28 cells were plated in 60-mm plastic culture dishes and allowed to adapt to culture conditions for 24 h. Binding reaction was carried out in 1.5 ml of conditioned culture medium, pH 7.4; serum in standard culture medium did not interfere with the assays. Increasing concentrations
287
of 12~I-labeled mEGF (0.1 to 750 ng/ml) were added to cultures in order to estimate the number of EGF receptors per cell. Nonspecific binding was determined by adding an excess of mEGF (6 ~g) to each of an additional set of culture dishes. Cells were incubated at 37°C for 55 min and the reaction stopped by washing each dish × 4 with cold Hanks' balanced salt solution (HBSS) containing 1 mg/ml of bovine serum albumin (BSA). Reaction supernatants were removed before washing and saved for determination of free 125I-labeled mEGF. To determine bound 12~I-labeled mEGF, cells were removed by adding 1 ml of 0.25% trypsin to each dish; cell suspension was transferred to a counting vial and each dish was rinsed x2 with 0.5 ml of a lysing buffer (0.5% sodium dodecyl sulfate, 10 mM Tris at pH 7.4, and 1 mM EDTA). Rinse fluid was added to vials and 3/radiation determined with a Beckman Biogamma-II counter. Competition assay. Competition between 125I-labeled mEGF and unlabeled mEGF for cell receptors was carried out on monolayers of cells (0.5-2 × 106 cells per 60-mm culture dish). Final reaction volume was 1.5 ml of conditioned growth medium. Increasing concentrations of unlabeled mEGF (0.01 to 2500 ng/ml) were added to duplicate dishes. A standard amount of 125I-labeled mEGF (2 to 4 ng/ml; 90,000 to 350,000 cpm) was added to each dish. Reaction mixture was incubated for 55 min at 37°C and discarded. Cells were rinsed x 4 with cold HBSS with BSA, trypsinized, and collected in lysing buffer for determination of bound radioactivity. Determination o f EGF in human~mouse body fluids/tissue extracts. Human amniotic fluids were obtained from pregnant women undergoing prenatal screening between 1618 weeks gestation. Fluid samples were frozen and stored at -20°C until assayed. Serum was collected from pregnant women between 15-21 weeks gestation and compared to serum from nonpregnant womem Normal and hypophysectomized male
288
L A D D A ET AL.
and female Swiss-Webster mice were obtained from Charles River, Inc. Animals were kept for at least 6-8 weeks before testing to follow growth to confirm pituitary ablation. Spontaneously voided urine was collected from animals prior to sacrifice, chilled, and neutralized to pH 7.4 and stored at -20°C. Blood was collected by cardiac or by medial canthal puncture and serum obtained. Mice were killed by cervical dislocation and submaxillary glands removed, wet weighed, and immediately frozen in liquid nitrogen for storage. Tissue extracts were prepared by hand homogenization (Dounce homogenizer) of single glands in 1.5 ml cold reagent grade water. Homogenates were centrifuged at 40,000g for 30 min at 4°C. Extracts were frozen until assayed for EGF. Each extract was serially diluted with HBSS and aliquots (10 to 200/xl) were added to culture dishes as described above for competition assay. mEGF was added to growing cultures to a final concentration of 25 ng/ml. Conditioned medium was collected from replicate cultures every 2 to 4 days and frozen until assayed. RESULTS
mEGF Binding Characteristics 125I-Labeled mEGF binding to SV40 WI-26 cells was affected by temperature (Fig. 1) and pH (data not shown). Maximal binding was achieved in 50-70 min at 37°C and at pH 7.4. With increasing concentration of ~25I-labeled mEGF, the binding rate was linear to about 25 ng/ml and then began to plateau indicating maximal binding capacity (Fig. 2). Scatchard plot of the binding data showed a single class of receptors (Ko - 2 x 10-9 M) and number of receptors was estimated at 450,000 per cell (Fig. 2, insert; Table 1). Table 1 lists the results of similar binding assays performed on a variety of cell types under the same conditions. Each cell type had a characteristic number of receptors which varied only
~1/
21o
r2 20 40 60 80 100 120 140 I60 180 200 TIME OF INCUBATION(minutes) FIG. 1. Effect of t e m p e r a t u r e on E G F binding to SV40 WI-26. Cells, 5 x 105, were plated in 60-ram culture dishes and 48 h later 4 ng x25I-labeled m E G F (about 120,000 cpm) were added to 1.5 ml of conditioned medium. At specific times duplicate dishes were rinsed x 4 with cold H B S S and cells r e m o v e d by trypsin and lysing buffer and counted in a B i o g a m m a II counter.
slightly under our culture conditions. Transformation of NRK with Kirsten sarcoma virus, an RNA virus, abolished mEGF binding to the transformed derivative KNRK. In contrast, transformation of human and mouse cells by simian virus (SV40 DNA virus) did not eliminate mEGF binding although number of EGF receptors may be increased or decreased compared to the normal cell type. Human and mouse erythrocytes and lymphocytes exhibited no EGF receptors.
Competition Assay Displacement of 125I-labeled mEGF by unlabeled mEGF is shown in Fig. 3. Standard curves were essentially superimposable from assay to assay with the SV40 WI-26. At 100 ng/ml of mEGF, 96-97% of 125I-labeled mEGF was displaced; 50% competition was found at about 10 ng/ml and 0.15 ng/ml displaced 2-4% of the control binding. Interassay coefficient of variation was 8.47%; intraassay coefficient of correlation was 6.07%. The assay was highly reproducible at concentrations from 5 to 100 ng/ml (correlation coefficients,
289
RADIORECEPTOR ASSAY FOR EPIDERMAL GROWTH FACTOR
I
I
I
~q
I
I
~..80 ~.60 rn
.40
LIA ,
.20 V
~
t
i
i
i
l
'EG~' ~~U;'D~"~°"/r"~' ~' ~
I
I
I
I
25
50
100
I
//
200
I
750
[1251] EGF (ng/ml) FIG. 2. Effect of 125I-labeled m E G F c o n c e n t r a t i o n on binding to SV40 WI-26. Insert: Scatchard plot of binding data. Binding was carried out as d e s c r i b e d unde r Materials and Methods.
2.67 to 6.50); at lower concentrations 0.1 to 1 ng/ml coefficients of variation ranged from 10.92-14.15%. Thus, results with concenTABLE 1 BINDING CHARACTERISTICS OF m E G F TO HUMAN, MOUSE, AND RAT CELLS
Cell H u m a n fibroblast (HF) SV40 H F 3T3 (BAL B) SV40 3T3 (BALB) NRK KNRK WI-38 SV40 WI-26 Erythrocytes Human Mo use Lymphocytesa Human Mo use
N u m b e r of receptors per cell 150,000 100,000 45,000 45,000 350,000 None 110,000 450,000
Ko 2 2 5 3 1
× 10 -9 × 10 -9 × 10 -9 × 10 -9 x 10 -9 -4 × 10 -9 2 × 10-9
None None
---
None None
---
a P h y t o h e m a g g l u t i n i n - s t i m u l a t e d l y m p h o c y t e cultures s h o w e d no E G F binding.
trations in the lower range of the competition curve were highly variable. Serial dilutions of mouse submaxillary gland extract competed parallel to the standard curve (Fig. 3). Recovery of mEGF Added to Culture Medium Purified m E G F was added to culture medium to final concentrations of 5, 25, and 75 ng/ml. Table 2 shows that the RRA accurately detected the calculated m E G F concentrations, m E G F added to human and mouse serum and urine was detected with >95% accuracy. m E G F in Mouse Fluids Table 3 shows the concentration of m E G F in mouse serum and urine. Males and females showed similar control levels; these were slightly increased with testosterone treatment, but the differences were not significant. Serial dilutions of the serum and urine samples produced proportional dis-
290
LADDA ET AL.
placement of 12~I-labeled mEGF parallel to the standard displacement curve.
mEGF in Mouse Submaxillary Gland mEGF concentration in normal male mouse glands was 1174 _+ 734 ng/mg wet wt and remained about the same after subcutaneous injections of testosterone enanthate (in sesame seed oil) 1 mg/animal/day for 8 days (Table 4). In the female mouse, normal submaxillary glands contained 370 _ 221 ng/mg wet wt. The mean value was about twofold greater than that reported by RIA-EGF (10,11), but our values are for single glands whereas previous workers have pooled glands from several animals. EGF concentrations in individual glands were highly variable. Following daily testosterone enanthate injections as above, the mEGF concentration in submaxillary glands of females increased about fivefold to male levels. As little as 1 /xl of the undiluted supernatant of a homogenized male gland produced 89-96% displacement of 12sIlabeled mEGF essentially identical to purified mEGF (Fig. 3: points a-d).
TABLE 2 RECOVERY OF m E G F ADDED TO CULTURE MEDIUM Calculated E G F concentration (ng/ml)
E G F detected ~
5 25 75
5.1 -+ 0.7 (4-6) 25.8 +_ 2.7 (22-29) 75.0 +_ 4.5 (69-79)
Mean _+ SD with range indicated in p a r e n t h e s e s . Based on five determinations at each concentration.
detectable amounts of mEGF (Table 5). Four to six weeks after ablation of the hypophysis a testosterone pellet (10 mg) was inserted subcutaneously near the occiput. Animals were sacrificed daily and submaxillary mEGF content was determined. In female mice testosterone treatment over 14 days stimulated mEGF accumulation to levels considerably greater than that found in the normal adult female, and, in fact many females had considerably greater levels than normal males. Hypophysectomized females showed a wide range of responsiveness to testosterone pellets with a mean value exceeding that of normal androgen-treated females. Hypophysectomized males treated Hypophysectomy with testosterone pellets showed complete Submaxillary glands from hypophysec- recovery of submaxillary mEGF content on a tomized males and females contained just i
I
TABLE 3
i
'~100
90
m E G F IN MOUSE BODY FLUIDSa 'q \ ,,,
'~ 80
~ 70 z 60 50 40 tal
ControP (ng/ml) Mouse serum Male
30
20 10
F 1.0
Female 10 100 UNLABELLED EGF ( n g / r n l )
1000
FIG. 3. Competitive binding a s s a y of m E G F and 125I-labeled m E G F on SV40 WI-26 (O). A s s a y s were carried out as described under Materials and Methods. Points a - d (11) represent dilutions of m o u s e submaxillary extract: a, 1:1000; b, 1:100; c, 1:10; d, undiluted extract. N o other peptide h o r m o n e s completed for specific E G F receptors: fibroblastic growth factor (n); insulin (O); multiplication stimulating activity (~); d e x a m e t h a s o n e (A).
Mouse urine Male
Female
Testosterone t r e a t e & (ng/ml)
86 + 22 (55-120) n = 10
119 -+ 31 (91-158) n - 10
78 + 18 (58-115) n = 10
110 -+ 41 (85-164) n = 10
1941 -+ 773 (750-2800) n -6
2377 -+ 1219 (487-3600) n =6
1690 + 810 (800-2650)
1950 _+ 1120 (790-3200)
n =6
n =6
Serum and urine samples were obtained from same animals described in Table 4. Serum or urine, 100-200/xt, were added to binding solution with the total volume 1.5 ml. A standard amount of a2~l-labeled m E G F was added and the binding was carried out as described under Materials and Methods. b Mean -+ SD with range indicated in parentheses.
R A D I O R E C E P T O R A S S A Y FOR E P I D E R M A L G R O W T H F A C T O R TABLE 4 E F F E C T OF D A I L Y T E S T O S T E R O N E E N A N T H A T E I N JECTIONS ON
mEGF
C O N T E N T OF I N D I V I D U A L
MOUSE SUBMAXILLARY GLANDS a
Male (20 g)
Female (20 g)
ControP (ng/mg wet tissue)
Treated b,~ (ng/mg wet tissue)
1174 _+ 734 (450-2100) n = 20
1253 -+ 260 (1010-1610) n = 10
370 ÷ 221 (100-680) n = 20
1261 _+ 78 (950-t400) n = 10
Each value represents mEGF concentration of a single gland, n. b Mean -+ SD with range indicated in parentheses. Treatment, 1 mg/day for 8 days.
wet weight basis. Submaxillary glands from treated animals were larger than untreated hypophysectomized glands and were substantially smaller than normal animals indicating that testosterone specifically stimulated mEGF production and/or storage and not generalized hypertrophy of the glands. Animals treated with testosterone pellets showed greater and more consistent responses than those given subcutaneous injections. These data will be reported in detail elsewhere.
mEGF Equivalents in Human Fluids mEGF equivalents in human amniotic fluids between 16-18 weeks' gestation
291
ranged from 0.9-8.8 ng/ml (Table 6). Amniotic fluid samples showed 5 to 12% displacement of lzSI-labeled mEGF. As little as 0.01 ng/ml mEGF caused 2 to 6% displacement and thus, the shallow slope of the competition curve between 0.01 to 1 ng/ml may result in a relative large error if this portion of the curve is used. Of the 50 samples of amniotic fluid 15 gave values in the less reliable part of the standard curve, 0.5-1 ng/ml. The remainder were within a highly reproducible range, 1-10 ng/ml. Twoto four-fold dilutions of amniotic fluid samples with 5.4 and 8.8 ng/ml mEGF equivalents caused a proportional decrease in a2~I-labeled mEGF displacement. Sera from pregnant and nonpregnant women showed similar values for mEGF equivalents. In two samples of pregnant sera with values of 34 and 48 ng/ml, threefold serial dilutions caused a proportional decrease in the concentrations of the growth factor.
Consumption of mEGF by Cells in Culture Table 7 shows that normal and transformed cells reduced the concentration of mEGF in culture medium during the course of a single passage. The disappearance of
TABLE 5 m E G F C O N T E N T OF M A L E AND F E M A L E S U B M A X I L L A R Y G L A N D S BEFORE A N D A F T E R H Y P O P H Y S E C T O M Y AND FOLLOWING TESTOSTERONE TREATMENT
No treatment a (ng/mg wet tissue) Normal female (30 g)
285 ± 109 (153-422) n = 5
Testosterone treatment a,b ng/mg wet tissue (range) 3809 ± 463 (3685-4200) n = 5
Hypophysectomized female (22 g)c
2.45 ± 0.91 (0.45-3.1) n=6
4484 ± 4714 (666-13,278) n=10
Hypophysectomized male (20 g)
3.12 ___ 2.13 (0.5-6) n =6
2008 ___ 1862 (450-5600) n =6
a Mean ___ SD with range indicated in parentheses. b Treatment, 10 mg pellet for 14 days. c Determinations were made about 6 weeks after hypophysectomy; original weight of animals was similar to normal untreated animals.
292
LADDA
mEGF from the medium was related to the rate of cell division and the number of EGF receptors. Receptorless cells such as KNRK showed essentially no degradation of mEGF. These studies tend to confirm the reports of internalization and degradation of mEGF by cells in culture (5).
DISCUSSION The simplicity of the radioreceptor assay for EGF makes it readily available to any laboratory with tissue culture capabilities. Although any healthy proliferating cell type with specific receptors for EGF may be used, an established cell line may be preferred so as to provide a continuous source of ceils with well-defined binding characteristics. The assay may be performed in conditioned medium or in serumfree balanced salt solution and identical standard curves are obtained. Although most of the assays reported here were performed on confluent monolayers in 60-mm culture dishes in 1.5 ml, the assay may be run efficiently in 16-mm wells of multidish disposo-trays (Linbro Chemical Co., Inc.). Discrimination is optimal between 5 and 100 ng/ml of mEGF. Sensitivity may be increased to 0.5 ng/ml by reducing the amount of standard 125I-labeled mEGF and limiting the number of cells per dish to 2 - 5 × 105. The total number of mEGE receptors per cell was density dependent and decreased slightly at confluence; however, this has no effect on the assay for the competition curve is expressed as the percentage of maximal binding of izsI-labeled mEGF in the absence of unlabeled mEGF. A critical point is to keep the specific activity of 125I-labeled mEGF below 50 /zCi//xg in order to prevent rapid loss of biological activity. At 35 /xCi//xg or less, izsI-labeled mEGF may remain stable for 6 to 8 weeks at -20°C. The RIA-mEGF assay has been described as showing significant displacement of ~25I-labeled mEGF at 0.03 ng of m E G F and
ET AL.
TABLE 6 m E G F EQUIVALENTS IN HUMAN FLUIDSa
EGFb (ng/ml)
Condition Pregnant s e r u m (5-21 w e e k s ' gestation)
24.8
Normal female s e r u m
20.4
_+ 9 . 0 ( 6 - 4 8 ) n ±
= 33 11.0 (5-52)
n=6
Amniotic fluid (16-19 weeks' gestation)
4.3
±
1.9 n
(0.9-8.8)
= 50
a Samples were stored at - 2 0 ° C until studied; 100- to 200-/~1 aliquots were added to binding m e d i u m with the final reaction volume 1.5 ml. b Mean - SD with range indicated in parentheses.
almost complete displacement at 10 ng (10-14). In contrast, the radioreceptor competitive displacement curve developed in the range of 1-100 ng/ml. RIA-mEGF assay presumably would be more sensitive than the radioreceptor assay. Starkey et al. (14) noted that RIA-mEGF was not a sensitive assay for hEGF since hEGF was less reactive to antibodies raised against mEGF. RIA-mEGF either did not detect or detected only trace amounts of hEGF in TABLE
mEGF
CONCENTRATION
A SINGLE
PASSAGE
HUMAN
7
IN CULTURE
OF NORMAL
AND RAT
MEDIUM
DURING
AND TRANSFORMED
FIBROBLASTS
IN CULTURE a
Human b
Day 1 2 3 4 6 10
Normal 25.5 25.0 19.5 19.0 17.0 7.2
± ± ± ± -±
3.5 1.4 3.5 2.0 0.5 0.8
Transformed EGF (ng/ml) 25.2 25.4 19.5 8.7 5.0 4.5
+_ 0.5 ± 1.6 ± 3.5 ± 1.1 ± 2.1 + 0.5
Rat b NRK c
KNRK a
25.3 ± 0.9 -17.7 ± 2.5 -5.5 ± 1.2 --
25.1 ± 1.3 -24.5 ± 2.5 -23.1 ± 3.5 --
a Culture medium was prepared with m E G F , 25 ng/ml. Supernatant was r e m o v e d from culture dishes o n day indicated and assayed for m E G F in triplicate. b Mean _+ SD. " N o r m a l " kidney cell culture. a Rat kidney cells transformed by Kirsten sarcoma virus ( K N R K ) have no detectable E G F receptors.
RADIORECEPTOR ASSAY FOR EPIDERMAL GROWTH FACTOR
human plasma, urine, and amniotic fluids (10,19). Yet, relatively high levels of hEGF were found in human milk and saliva (14). These same investigators briefly stated that RRA-mEGF detected considerably greater amounts of hEGF in human urine and serum than that found by RIA-mEGF (10). Our findings confirm those preliminary observations. In our experience with both assays, RRA-mEGF has consistenly indicated greater levels of EGF in almost all tissues and fluid samples than that detected by RIA-mEGF. Antibody against EGF from mouse or man may be directed against a portion of the EGF molecule unrelated to its binding specificity and thus, may significantly underestimate "functional" levels of EGF (4,6). It is possible that a variety of EGF-like molecules may exist in body fluids which are not immunoreactive to antibody for mEGF or hEGF, but are capable of binding to specific receptors. The RRA-mEGF was then used to quantitate EGF in a variety of sources from mouse and human with and without hormone treatment. The normal male mouse submaxillary gland had more than 5-fold greater amounts of mEGF compared to the female gland. Although 1 mg testosterone induced a 3- to 4-fold increase of mEGF in normal female submaxillary glands, it had little effect in the normal male. The relative lack of response of the male animals suggests that the male gland already is stimulated maximally by endogenous hormone; however, hypophysectomized males treated with testosterone pellets had a larger mean response than normal males treated with injections. Mouse serum and urine levels of mEGF were slightly increased in animals treated with androgen, but the mEGF values of normal and treated animals overlapped considerably and the differences were not significant. Serum levels were 10- to 20-fold greater than that reported by RIA-mEGF (10,11,13), but urine levels of mEGF were similar to RIA-mEGF (11). Hypophysectomy of male and female
293
mice reduced submaxillary mEGF content to trace amounts. The exquisite sensitivity of the gland to testosterone was underscored once again by the dramatic recovery of the hypophysectomized animal following testosterone treatment. In these experiments testosterone was delivered from a pellet placed subcutaneously and the responses of the animals were more consistent than those receiving daily injections. mEGF concentration of the hypophysectomized-testosterone-treated glands often was found to attain a twofold greater level than normal animals, indicating a greatly exaggerated response to treatment. The range of the responses was large. Byyny et al. (10) originally noted the effect of testosterone on the female gland and further found that castration of the male reduced mEGF content of male glands to female levels. In our study, hypophysectomy further reduced male submaxillary levels suggesting a direct humoral effect of the pituitary on the submaxillary. It is possible that the effect of hypophysectomy may be related to decreased adrenal androgen production. Mouse submaxillary gland EGF concentration displayed a circadian variation which was abolished by superior cervical ganglionectomy (13). Interestingly, superior cervical ganglionectomy did not abolish the stimulating effect of testosterone (13). Regulation of mouse submaxillary gland EGF concentration apparently involves a complex interplay of endocrine and neurohumoral factors. The physiological role of EGF remains unknown, but the availability of simple and sensitive methods to measure it will permit us to establish normal levels in man and other animals and to search for conditions associated with a deficiency or excess of EGF. It is of considerable interest that RRA-EGF and RIA-EGF appear to detect different biological properties of EGF and thus, these assays should prove to be useful complementary tools in the characterization of EGF in various biological systems.
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ACKNOWLEDGMENTS This work was supported by Grant AG-00421 from the National Institute on Aging and Research Career Development Award AG-00006 to RLL.
REFERENCES 1. Cohen, S. (1962)J. Biol. Chem. 237, 1555-1562. 2. HoUenberg, M., and Cuatrecasas, P. (1973)Proc. Nat. Acad. Sci. 70, 2964-2968. 3. Hollenberg, M., and Cuatrecasas, P. (1975)J. Biol. Chem. 250, 3845-3853. 4. Cohen, S., and Carpenter, G. (1975) Proc. Nat. Acad. Sci. 72, 1317-1321. 5. Carpenter, G., and Cohen, A. (1976) J. Cell Biol. 71, 159-171. 6. Starkey, R., Cohen, S., and Orth, D. (1975) Science 189, 800-802. 7. Gregory, H. (1975)Nature (London) 257, 325-327. 8. Hollenberg, M., and Gregory, H. (1976) Life Sci. 20, 267-274.
9. Blosse, P., and Fenton, E. (1974) Biochim. Biophys. Acta. 354, 57-60. 10. Byyny, R., Orth, D., and Cohen, S. (1972) Endocrinology 90, 1261-1266. 11. Byyny, R., Orth, D., Cohen, S., and Doyne, E. (1974) Endocrinology 95, 776-782. 12. Barthe, P., Bullock, L., Mowszowicz, I., Bardin, W., and Orth, D. (1974) Endocrinology 95, 991-997. 13. Krieger, D., Hauser, H., Liotta, A., and Zelentetz, A. (1976)Endocrinology 99, 1589-1596. 14. Starkey, R., and Orth, D. (1977) J. Clin. Endocrinol. Metabol. 45, 1144-1153. 15. Carpenter, G., Lembach, K., Morrison, M., and Cohen, S. (1975)J. Biol. Chem. 250, 4297-4304. 16. Frati, L., Cenci, G., Sbaraglia, G., Venza Teti, D., and Covelli, I. (1976) Life Sci. 18, 905-911. 17. Savage, C., and Cohen, S. (1972) J. Biol. Chem. 247, 7609-7611. 18. Greenwood, F., Hunter, W., and Glover, J. (1963) Biochem. J. 89, 114-123. 19. Ances, I. (1973)Amer. J. Obstet. Gynecol. 115, 357-362.
Radioreceptor Assay for Epidermal Growth Factor ROGER L. L A D D A , * LESLIE P. BULLOCK,~" TERILEE GIANOPOULOS,* AND LYNN MCCORMICK*
*Division of Genetics, Department of Pediatrics, and ?Division of Endocrinology, Department of Medicine, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033 Received November 7, 1977 An established cell line of human lung fibroblasts with a high number of surface receptors for mouse epidermal growth factor (mEGF) was used to develop a simple and highly sensitive radioreceptor assay for EGF. 125I-Labeled mEGF competed mole for mole with unlabeled mEGF for specific receptors. Optimal range for discriminating EGF concentrations in body fluids and tissue extracts by a competitive binding assay was between 5 and 100 ng/ml. Interassay correlation of variation was 8.47% and the recovery of highly purified mEGF added to serum and urine samples was greater than 95%. Human serum and amniotic fluids contained about 24 and 4 ng/ml, respectively, of mEGF equivalents. Concentrations of mEGF in mouse urine and serum were highly variable and were 2- to 10-fold greater than that previously detected by radioimmune assay. Hypophysectomy nearly abolished submaxillary mEGF content in both male and female mice, but testosterone treatment of hypophysectomized animals restored normal concentrations of mEGF to the glands, mEGF added to culture medium disappeared with time as a function of the number of cellular EGF receptors indicating cellular degradation of the growth factor. The radioreceptor assay for EGF is based on the close biologic relationship between the cell receptor site and the native hormone and should prove to be a useful complementary tool to characterize the physiological role of EGF.
Mouse epidermal growth factor (mEGF) 1 is a small polypeptide with a molecular weight of 6045 originally isolated from submaxillary glands of mice (1). mEGF binds to specific receptors associated with the cell surface and thereby stimulates a potent mitogenic response in a variety of mammalian cell types both in vitro and in vivo (2-6). Human EGF (hEGF) has been isolated from urine (4) and appears to be similar to urogastrone, a small polypeptide isolated from human urine which inhibits gastric acid secretion (7-8). mEGF and hEGF have essentially identical binding 1 Abbreviations used: EGF, epidermal growth factor (mEGF, mouse; hEGF, human); RIA-EGF, radioimmune assay for EGF; RRA, radioreceptor assay; HF, human fibroblasts; HBSS, Hanks" balanced salt solution; BSA, bovine serum albumin; NRK, normal rat kidney cells; KNRK, transformed NRK. 0003-2697/79/040286-09502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved.
properties and compete mole for mole for specific EGF receptors (8). These accumulating data suggest that EGF may have important physiological functions and thus, sensitive methods to measure EGF concentration in animals is essential. Bioassay for EGF requires multiple injections of suckling mice with large amounts of test material and provides only semiquantitative values (1,9). Radioimmune assay (RIA) for EGF has been used to isolate hEGF and to estimate EGF content of extracts of various tissues (10-14). In addition, a simple and highly sensitive radioreceptor assay (RRA) has been used to measure EGF receptors on cells (15,16). We have developed the RRA-EGF to measure the concentration of EGF in animal tissues and fluids and have found that some values differ from those reported by RIA-EGF. It is possible that
286
RADIORECEPTOR ASSAY FOR EPIDERMAL GROWTH FACTOR
RRA-EGF and RIA-EGF may reflect different functional aspects of the EGF molecule. MATERIALS AND METHODS
Cells. Normal human fibroblasts (HF) derived from neonatal foreskin and its SV40-transformed derivative (SV40HF), 3T3, SV40 3T3, normal rat kindey cells, and a Kirsten sarcoma virus-transformed derivative, normal WI-38, and SV-40-transformed human fetal lung cells (SV40 WI-26) were routinely grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum.
mEGF isolation, mEGF was isolated from adult male mouse submaxillary glands according to Savage and Cohen (17). 125I-Labeling o f EGF. Iodination reaction (18) was carried out in a 10 × 75-mm glass vial at room temperature. Carrier-free Na~25I (Amersham/Searle), 0.5 mCi, in 5/zl was added to the vial and the pH was adjusted to 7.5 by the addition of 25/zl of 0.05 M phospate buffer (pH 6) and 100 /zl of 0.05 M phosphate buffer (pH 7.5). Ten micrograms of mEGF were added to the tube and the contents were gently mixed. Twenty-five microliters of chloramine-T (2 mg/ml) were added and the reaction carried out for 60 s; 25 /~1 of Na metabisulfite (2.5 mg/ml) was added to stop the reacton. ~25I-Labeled mEGF was separated from unbound 125I by passing the reaction mixture through a 20 x l-cm column of Sephadex G-25 (medium grain size). Specific activity of l~SI-labeled EGF ranged from 25-54/zCi//zg. Characterization of EGF binding. For binding and radioreceptor assays, 1 × 106 SV40 WI-28 cells were plated in 60-mm plastic culture dishes and allowed to adapt to culture conditions for 24 h. Binding reaction was carried out in 1.5 ml of conditioned culture medium, pH 7.4; serum in standard culture medium did not interfere with the assays. Increasing concentrations
287
of 12~I-labeled mEGF (0.1 to 750 ng/ml) were added to cultures in order to estimate the number of EGF receptors per cell. Nonspecific binding was determined by adding an excess of mEGF (6 ~g) to each of an additional set of culture dishes. Cells were incubated at 37°C for 55 min and the reaction stopped by washing each dish × 4 with cold Hanks' balanced salt solution (HBSS) containing 1 mg/ml of bovine serum albumin (BSA). Reaction supernatants were removed before washing and saved for determination of free 125I-labeled mEGF. To determine bound 12~I-labeled mEGF, cells were removed by adding 1 ml of 0.25% trypsin to each dish; cell suspension was transferred to a counting vial and each dish was rinsed x2 with 0.5 ml of a lysing buffer (0.5% sodium dodecyl sulfate, 10 mM Tris at pH 7.4, and 1 mM EDTA). Rinse fluid was added to vials and 3/radiation determined with a Beckman Biogamma-II counter. Competition assay. Competition between 125I-labeled mEGF and unlabeled mEGF for cell receptors was carried out on monolayers of cells (0.5-2 × 106 cells per 60-mm culture dish). Final reaction volume was 1.5 ml of conditioned growth medium. Increasing concentrations of unlabeled mEGF (0.01 to 2500 ng/ml) were added to duplicate dishes. A standard amount of 125I-labeled mEGF (2 to 4 ng/ml; 90,000 to 350,000 cpm) was added to each dish. Reaction mixture was incubated for 55 min at 37°C and discarded. Cells were rinsed x 4 with cold HBSS with BSA, trypsinized, and collected in lysing buffer for determination of bound radioactivity. Determination o f EGF in human~mouse body fluids/tissue extracts. Human amniotic fluids were obtained from pregnant women undergoing prenatal screening between 1618 weeks gestation. Fluid samples were frozen and stored at -20°C until assayed. Serum was collected from pregnant women between 15-21 weeks gestation and compared to serum from nonpregnant womem Normal and hypophysectomized male
288
L A D D A ET AL.
and female Swiss-Webster mice were obtained from Charles River, Inc. Animals were kept for at least 6-8 weeks before testing to follow growth to confirm pituitary ablation. Spontaneously voided urine was collected from animals prior to sacrifice, chilled, and neutralized to pH 7.4 and stored at -20°C. Blood was collected by cardiac or by medial canthal puncture and serum obtained. Mice were killed by cervical dislocation and submaxillary glands removed, wet weighed, and immediately frozen in liquid nitrogen for storage. Tissue extracts were prepared by hand homogenization (Dounce homogenizer) of single glands in 1.5 ml cold reagent grade water. Homogenates were centrifuged at 40,000g for 30 min at 4°C. Extracts were frozen until assayed for EGF. Each extract was serially diluted with HBSS and aliquots (10 to 200/xl) were added to culture dishes as described above for competition assay. mEGF was added to growing cultures to a final concentration of 25 ng/ml. Conditioned medium was collected from replicate cultures every 2 to 4 days and frozen until assayed. RESULTS
mEGF Binding Characteristics 125I-Labeled mEGF binding to SV40 WI-26 cells was affected by temperature (Fig. 1) and pH (data not shown). Maximal binding was achieved in 50-70 min at 37°C and at pH 7.4. With increasing concentration of ~25I-labeled mEGF, the binding rate was linear to about 25 ng/ml and then began to plateau indicating maximal binding capacity (Fig. 2). Scatchard plot of the binding data showed a single class of receptors (Ko - 2 x 10-9 M) and number of receptors was estimated at 450,000 per cell (Fig. 2, insert; Table 1). Table 1 lists the results of similar binding assays performed on a variety of cell types under the same conditions. Each cell type had a characteristic number of receptors which varied only
~1/
21o
r2 20 40 60 80 100 120 140 I60 180 200 TIME OF INCUBATION(minutes) FIG. 1. Effect of t e m p e r a t u r e on E G F binding to SV40 WI-26. Cells, 5 x 105, were plated in 60-ram culture dishes and 48 h later 4 ng x25I-labeled m E G F (about 120,000 cpm) were added to 1.5 ml of conditioned medium. At specific times duplicate dishes were rinsed x 4 with cold H B S S and cells r e m o v e d by trypsin and lysing buffer and counted in a B i o g a m m a II counter.
slightly under our culture conditions. Transformation of NRK with Kirsten sarcoma virus, an RNA virus, abolished mEGF binding to the transformed derivative KNRK. In contrast, transformation of human and mouse cells by simian virus (SV40 DNA virus) did not eliminate mEGF binding although number of EGF receptors may be increased or decreased compared to the normal cell type. Human and mouse erythrocytes and lymphocytes exhibited no EGF receptors.
Competition Assay Displacement of 125I-labeled mEGF by unlabeled mEGF is shown in Fig. 3. Standard curves were essentially superimposable from assay to assay with the SV40 WI-26. At 100 ng/ml of mEGF, 96-97% of 125I-labeled mEGF was displaced; 50% competition was found at about 10 ng/ml and 0.15 ng/ml displaced 2-4% of the control binding. Interassay coefficient of variation was 8.47%; intraassay coefficient of correlation was 6.07%. The assay was highly reproducible at concentrations from 5 to 100 ng/ml (correlation coefficients,
289
RADIORECEPTOR ASSAY FOR EPIDERMAL GROWTH FACTOR
I
I
I
~q
I
I
~..80 ~.60 rn
.40
LIA ,
.20 V
~
t
i
i
i
l
'EG~' ~~U;'D~"~°"/r"~' ~' ~
I
I
I
I
25
50
100
I
//
200
I
750
[1251] EGF (ng/ml) FIG. 2. Effect of 125I-labeled m E G F c o n c e n t r a t i o n on binding to SV40 WI-26. Insert: Scatchard plot of binding data. Binding was carried out as d e s c r i b e d unde r Materials and Methods.
2.67 to 6.50); at lower concentrations 0.1 to 1 ng/ml coefficients of variation ranged from 10.92-14.15%. Thus, results with concenTABLE 1 BINDING CHARACTERISTICS OF m E G F TO HUMAN, MOUSE, AND RAT CELLS
Cell H u m a n fibroblast (HF) SV40 H F 3T3 (BAL B) SV40 3T3 (BALB) NRK KNRK WI-38 SV40 WI-26 Erythrocytes Human Mo use Lymphocytesa Human Mo use
N u m b e r of receptors per cell 150,000 100,000 45,000 45,000 350,000 None 110,000 450,000
Ko 2 2 5 3 1
× 10 -9 × 10 -9 × 10 -9 × 10 -9 x 10 -9 -4 × 10 -9 2 × 10-9
None None
---
None None
---
a P h y t o h e m a g g l u t i n i n - s t i m u l a t e d l y m p h o c y t e cultures s h o w e d no E G F binding.
trations in the lower range of the competition curve were highly variable. Serial dilutions of mouse submaxillary gland extract competed parallel to the standard curve (Fig. 3). Recovery of mEGF Added to Culture Medium Purified m E G F was added to culture medium to final concentrations of 5, 25, and 75 ng/ml. Table 2 shows that the RRA accurately detected the calculated m E G F concentrations, m E G F added to human and mouse serum and urine was detected with >95% accuracy. m E G F in Mouse Fluids Table 3 shows the concentration of m E G F in mouse serum and urine. Males and females showed similar control levels; these were slightly increased with testosterone treatment, but the differences were not significant. Serial dilutions of the serum and urine samples produced proportional dis-
290
LADDA ET AL.
placement of 12~I-labeled mEGF parallel to the standard displacement curve.
mEGF in Mouse Submaxillary Gland mEGF concentration in normal male mouse glands was 1174 _+ 734 ng/mg wet wt and remained about the same after subcutaneous injections of testosterone enanthate (in sesame seed oil) 1 mg/animal/day for 8 days (Table 4). In the female mouse, normal submaxillary glands contained 370 _ 221 ng/mg wet wt. The mean value was about twofold greater than that reported by RIA-EGF (10,11), but our values are for single glands whereas previous workers have pooled glands from several animals. EGF concentrations in individual glands were highly variable. Following daily testosterone enanthate injections as above, the mEGF concentration in submaxillary glands of females increased about fivefold to male levels. As little as 1 /xl of the undiluted supernatant of a homogenized male gland produced 89-96% displacement of 12sIlabeled mEGF essentially identical to purified mEGF (Fig. 3: points a-d).
TABLE 2 RECOVERY OF m E G F ADDED TO CULTURE MEDIUM Calculated E G F concentration (ng/ml)
E G F detected ~
5 25 75
5.1 -+ 0.7 (4-6) 25.8 +_ 2.7 (22-29) 75.0 +_ 4.5 (69-79)
Mean _+ SD with range indicated in p a r e n t h e s e s . Based on five determinations at each concentration.
detectable amounts of mEGF (Table 5). Four to six weeks after ablation of the hypophysis a testosterone pellet (10 mg) was inserted subcutaneously near the occiput. Animals were sacrificed daily and submaxillary mEGF content was determined. In female mice testosterone treatment over 14 days stimulated mEGF accumulation to levels considerably greater than that found in the normal adult female, and, in fact many females had considerably greater levels than normal males. Hypophysectomized females showed a wide range of responsiveness to testosterone pellets with a mean value exceeding that of normal androgen-treated females. Hypophysectomized males treated Hypophysectomy with testosterone pellets showed complete Submaxillary glands from hypophysec- recovery of submaxillary mEGF content on a tomized males and females contained just i
I
TABLE 3
i
'~100
90
m E G F IN MOUSE BODY FLUIDSa 'q \ ,,,
'~ 80
~ 70 z 60 50 40 tal
ControP (ng/ml) Mouse serum Male
30
20 10
F 1.0
Female 10 100 UNLABELLED EGF ( n g / r n l )
1000
FIG. 3. Competitive binding a s s a y of m E G F and 125I-labeled m E G F on SV40 WI-26 (O). A s s a y s were carried out as described under Materials and Methods. Points a - d (11) represent dilutions of m o u s e submaxillary extract: a, 1:1000; b, 1:100; c, 1:10; d, undiluted extract. N o other peptide h o r m o n e s completed for specific E G F receptors: fibroblastic growth factor (n); insulin (O); multiplication stimulating activity (~); d e x a m e t h a s o n e (A).
Mouse urine Male
Female
Testosterone t r e a t e & (ng/ml)
86 + 22 (55-120) n = 10
119 -+ 31 (91-158) n - 10
78 + 18 (58-115) n = 10
110 -+ 41 (85-164) n = 10
1941 -+ 773 (750-2800) n -6
2377 -+ 1219 (487-3600) n =6
1690 + 810 (800-2650)
1950 _+ 1120 (790-3200)
n =6
n =6
Serum and urine samples were obtained from same animals described in Table 4. Serum or urine, 100-200/xt, were added to binding solution with the total volume 1.5 ml. A standard amount of a2~l-labeled m E G F was added and the binding was carried out as described under Materials and Methods. b Mean -+ SD with range indicated in parentheses.
R A D I O R E C E P T O R A S S A Y FOR E P I D E R M A L G R O W T H F A C T O R TABLE 4 E F F E C T OF D A I L Y T E S T O S T E R O N E E N A N T H A T E I N JECTIONS ON
mEGF
C O N T E N T OF I N D I V I D U A L
MOUSE SUBMAXILLARY GLANDS a
Male (20 g)
Female (20 g)
ControP (ng/mg wet tissue)
Treated b,~ (ng/mg wet tissue)
1174 _+ 734 (450-2100) n = 20
1253 -+ 260 (1010-1610) n = 10
370 ÷ 221 (100-680) n = 20
1261 _+ 78 (950-t400) n = 10
Each value represents mEGF concentration of a single gland, n. b Mean -+ SD with range indicated in parentheses. Treatment, 1 mg/day for 8 days.
wet weight basis. Submaxillary glands from treated animals were larger than untreated hypophysectomized glands and were substantially smaller than normal animals indicating that testosterone specifically stimulated mEGF production and/or storage and not generalized hypertrophy of the glands. Animals treated with testosterone pellets showed greater and more consistent responses than those given subcutaneous injections. These data will be reported in detail elsewhere.
mEGF Equivalents in Human Fluids mEGF equivalents in human amniotic fluids between 16-18 weeks' gestation
291
ranged from 0.9-8.8 ng/ml (Table 6). Amniotic fluid samples showed 5 to 12% displacement of lzSI-labeled mEGF. As little as 0.01 ng/ml mEGF caused 2 to 6% displacement and thus, the shallow slope of the competition curve between 0.01 to 1 ng/ml may result in a relative large error if this portion of the curve is used. Of the 50 samples of amniotic fluid 15 gave values in the less reliable part of the standard curve, 0.5-1 ng/ml. The remainder were within a highly reproducible range, 1-10 ng/ml. Twoto four-fold dilutions of amniotic fluid samples with 5.4 and 8.8 ng/ml mEGF equivalents caused a proportional decrease in a2~I-labeled mEGF displacement. Sera from pregnant and nonpregnant women showed similar values for mEGF equivalents. In two samples of pregnant sera with values of 34 and 48 ng/ml, threefold serial dilutions caused a proportional decrease in the concentrations of the growth factor.
Consumption of mEGF by Cells in Culture Table 7 shows that normal and transformed cells reduced the concentration of mEGF in culture medium during the course of a single passage. The disappearance of
TABLE 5 m E G F C O N T E N T OF M A L E AND F E M A L E S U B M A X I L L A R Y G L A N D S BEFORE A N D A F T E R H Y P O P H Y S E C T O M Y AND FOLLOWING TESTOSTERONE TREATMENT
No treatment a (ng/mg wet tissue) Normal female (30 g)
285 ± 109 (153-422) n = 5
Testosterone treatment a,b ng/mg wet tissue (range) 3809 ± 463 (3685-4200) n = 5
Hypophysectomized female (22 g)c
2.45 ± 0.91 (0.45-3.1) n=6
4484 ± 4714 (666-13,278) n=10
Hypophysectomized male (20 g)
3.12 ___ 2.13 (0.5-6) n =6
2008 ___ 1862 (450-5600) n =6
a Mean ___ SD with range indicated in parentheses. b Treatment, 10 mg pellet for 14 days. c Determinations were made about 6 weeks after hypophysectomy; original weight of animals was similar to normal untreated animals.
292
LADDA
mEGF from the medium was related to the rate of cell division and the number of EGF receptors. Receptorless cells such as KNRK showed essentially no degradation of mEGF. These studies tend to confirm the reports of internalization and degradation of mEGF by cells in culture (5).
DISCUSSION The simplicity of the radioreceptor assay for EGF makes it readily available to any laboratory with tissue culture capabilities. Although any healthy proliferating cell type with specific receptors for EGF may be used, an established cell line may be preferred so as to provide a continuous source of ceils with well-defined binding characteristics. The assay may be performed in conditioned medium or in serumfree balanced salt solution and identical standard curves are obtained. Although most of the assays reported here were performed on confluent monolayers in 60-mm culture dishes in 1.5 ml, the assay may be run efficiently in 16-mm wells of multidish disposo-trays (Linbro Chemical Co., Inc.). Discrimination is optimal between 5 and 100 ng/ml of mEGF. Sensitivity may be increased to 0.5 ng/ml by reducing the amount of standard 125I-labeled mEGF and limiting the number of cells per dish to 2 - 5 × 105. The total number of mEGE receptors per cell was density dependent and decreased slightly at confluence; however, this has no effect on the assay for the competition curve is expressed as the percentage of maximal binding of izsI-labeled mEGF in the absence of unlabeled mEGF. A critical point is to keep the specific activity of 125I-labeled mEGF below 50 /zCi//xg in order to prevent rapid loss of biological activity. At 35 /xCi//xg or less, izsI-labeled mEGF may remain stable for 6 to 8 weeks at -20°C. The RIA-mEGF assay has been described as showing significant displacement of ~25I-labeled mEGF at 0.03 ng of m E G F and
ET AL.
TABLE 6 m E G F EQUIVALENTS IN HUMAN FLUIDSa
EGFb (ng/ml)
Condition Pregnant s e r u m (5-21 w e e k s ' gestation)
24.8
Normal female s e r u m
20.4
_+ 9 . 0 ( 6 - 4 8 ) n ±
= 33 11.0 (5-52)
n=6
Amniotic fluid (16-19 weeks' gestation)
4.3
±
1.9 n
(0.9-8.8)
= 50
a Samples were stored at - 2 0 ° C until studied; 100- to 200-/~1 aliquots were added to binding m e d i u m with the final reaction volume 1.5 ml. b Mean - SD with range indicated in parentheses.
almost complete displacement at 10 ng (10-14). In contrast, the radioreceptor competitive displacement curve developed in the range of 1-100 ng/ml. RIA-mEGF assay presumably would be more sensitive than the radioreceptor assay. Starkey et al. (14) noted that RIA-mEGF was not a sensitive assay for hEGF since hEGF was less reactive to antibodies raised against mEGF. RIA-mEGF either did not detect or detected only trace amounts of hEGF in TABLE
mEGF
CONCENTRATION
A SINGLE
PASSAGE
HUMAN
7
IN CULTURE
OF NORMAL
AND RAT
MEDIUM
DURING
AND TRANSFORMED
FIBROBLASTS
IN CULTURE a
Human b
Day 1 2 3 4 6 10
Normal 25.5 25.0 19.5 19.0 17.0 7.2
± ± ± ± -±
3.5 1.4 3.5 2.0 0.5 0.8
Transformed EGF (ng/ml) 25.2 25.4 19.5 8.7 5.0 4.5
+_ 0.5 ± 1.6 ± 3.5 ± 1.1 ± 2.1 + 0.5
Rat b NRK c
KNRK a
25.3 ± 0.9 -17.7 ± 2.5 -5.5 ± 1.2 --
25.1 ± 1.3 -24.5 ± 2.5 -23.1 ± 3.5 --
a Culture medium was prepared with m E G F , 25 ng/ml. Supernatant was r e m o v e d from culture dishes o n day indicated and assayed for m E G F in triplicate. b Mean _+ SD. " N o r m a l " kidney cell culture. a Rat kidney cells transformed by Kirsten sarcoma virus ( K N R K ) have no detectable E G F receptors.
RADIORECEPTOR ASSAY FOR EPIDERMAL GROWTH FACTOR
human plasma, urine, and amniotic fluids (10,19). Yet, relatively high levels of hEGF were found in human milk and saliva (14). These same investigators briefly stated that RRA-mEGF detected considerably greater amounts of hEGF in human urine and serum than that found by RIA-mEGF (10). Our findings confirm those preliminary observations. In our experience with both assays, RRA-mEGF has consistenly indicated greater levels of EGF in almost all tissues and fluid samples than that detected by RIA-mEGF. Antibody against EGF from mouse or man may be directed against a portion of the EGF molecule unrelated to its binding specificity and thus, may significantly underestimate "functional" levels of EGF (4,6). It is possible that a variety of EGF-like molecules may exist in body fluids which are not immunoreactive to antibody for mEGF or hEGF, but are capable of binding to specific receptors. The RRA-mEGF was then used to quantitate EGF in a variety of sources from mouse and human with and without hormone treatment. The normal male mouse submaxillary gland had more than 5-fold greater amounts of mEGF compared to the female gland. Although 1 mg testosterone induced a 3- to 4-fold increase of mEGF in normal female submaxillary glands, it had little effect in the normal male. The relative lack of response of the male animals suggests that the male gland already is stimulated maximally by endogenous hormone; however, hypophysectomized males treated with testosterone pellets had a larger mean response than normal males treated with injections. Mouse serum and urine levels of mEGF were slightly increased in animals treated with androgen, but the mEGF values of normal and treated animals overlapped considerably and the differences were not significant. Serum levels were 10- to 20-fold greater than that reported by RIA-mEGF (10,11,13), but urine levels of mEGF were similar to RIA-mEGF (11). Hypophysectomy of male and female
293
mice reduced submaxillary mEGF content to trace amounts. The exquisite sensitivity of the gland to testosterone was underscored once again by the dramatic recovery of the hypophysectomized animal following testosterone treatment. In these experiments testosterone was delivered from a pellet placed subcutaneously and the responses of the animals were more consistent than those receiving daily injections. mEGF concentration of the hypophysectomized-testosterone-treated glands often was found to attain a twofold greater level than normal animals, indicating a greatly exaggerated response to treatment. The range of the responses was large. Byyny et al. (10) originally noted the effect of testosterone on the female gland and further found that castration of the male reduced mEGF content of male glands to female levels. In our study, hypophysectomy further reduced male submaxillary levels suggesting a direct humoral effect of the pituitary on the submaxillary. It is possible that the effect of hypophysectomy may be related to decreased adrenal androgen production. Mouse submaxillary gland EGF concentration displayed a circadian variation which was abolished by superior cervical ganglionectomy (13). Interestingly, superior cervical ganglionectomy did not abolish the stimulating effect of testosterone (13). Regulation of mouse submaxillary gland EGF concentration apparently involves a complex interplay of endocrine and neurohumoral factors. The physiological role of EGF remains unknown, but the availability of simple and sensitive methods to measure it will permit us to establish normal levels in man and other animals and to search for conditions associated with a deficiency or excess of EGF. It is of considerable interest that RRA-EGF and RIA-EGF appear to detect different biological properties of EGF and thus, these assays should prove to be useful complementary tools in the characterization of EGF in various biological systems.
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LADDA ET AL.
ACKNOWLEDGMENTS This work was supported by Grant AG-00421 from the National Institute on Aging and Research Career Development Award AG-00006 to RLL.
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9. Blosse, P., and Fenton, E. (1974) Biochim. Biophys. Acta. 354, 57-60. 10. Byyny, R., Orth, D., and Cohen, S. (1972) Endocrinology 90, 1261-1266. 11. Byyny, R., Orth, D., Cohen, S., and Doyne, E. (1974) Endocrinology 95, 776-782. 12. Barthe, P., Bullock, L., Mowszowicz, I., Bardin, W., and Orth, D. (1974) Endocrinology 95, 991-997. 13. Krieger, D., Hauser, H., Liotta, A., and Zelentetz, A. (1976)Endocrinology 99, 1589-1596. 14. Starkey, R., and Orth, D. (1977) J. Clin. Endocrinol. Metabol. 45, 1144-1153. 15. Carpenter, G., Lembach, K., Morrison, M., and Cohen, S. (1975)J. Biol. Chem. 250, 4297-4304. 16. Frati, L., Cenci, G., Sbaraglia, G., Venza Teti, D., and Covelli, I. (1976) Life Sci. 18, 905-911. 17. Savage, C., and Cohen, S. (1972) J. Biol. Chem. 247, 7609-7611. 18. Greenwood, F., Hunter, W., and Glover, J. (1963) Biochem. J. 89, 114-123. 19. Ances, I. (1973)Amer. J. Obstet. Gynecol. 115, 357-362.
