Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 46, No. 6 Printed in U.S.A.
ALAN C. MOSES, KENNETH L. COHEN,f ROGER JOHNSONBAUGH, AND S. PETER NISSLEY Endocrinology Section, Metabolism Branch, National Cancer Institute, National Institutes of Health (A.C.M., K.L.C., S.P.N.); and the National Naval Medical Center (R.J.), Bethesda, Maryland 20014 ABSTRACT. Although purified human somatomedins and related peptides have been shown to stimulate growth-related processes in cultured human skin fibroblasts, it is unknown whether the serum macromolecules required for the routine growth of human fibroblasts in culture include the somatomedins. To evaluate this question we have obtained sera from five GH-deficient patients before and after GH treatment and have compared these pre- and post-GH sera for their ability to stimulate [3H]thymidine incorporation into DNA and cell multiplication in the patients' own fibroblasts in culture. Subconfluent human fibroblasts demonstrated a dose-dependent increase in thymidine incorporation and cell number when exposed to human sera. Pre- and post-GH sera were equipotent. Partial purification of somatomedin activity by boiling sera at pH 5.5 diminished the level of thymidine incorporation to 10-20% of the level achieved with unboiled sera but did not unmask a difference between pre- and post-GH sera. In
I
T HAS been recognized for some time that human skin fibroblasts require serum in order to multiply in culture (1). More recently, several investigators have recognized that the somatomedins, originally proposed as the peripheral mediators of GH action on skeletal tissue (2, 3), may contribute to the serum growth requirement of cells in culture (4-9). Growth-related processes in chick embryo fibroblasts and in human skin fibroblasts are stimulated by purified somatomedins in the absence of serum. This stimulation has been correlated with the presence of specific recepReceived August 23, 1976. Address requests for reprints to: Dr. Alan C. Moses, Clinical Associate, Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014. * This work was presented in part at the 58th Annual Meeting of the Endocrine Society, San Francisco, California, June 23-25, 1976. f Present address: Department of Medicine, Yale University, New Haven, Connecticut.
contrast to the results in human fibroblasts, boiled postGH sera were 2- to 5-fold more potent than pre-GH sera in stimulating thymidine incorporation in chick embryo fibroblasts. Mixing experiments failed to demonstrate inhibition of thymidine incorporation by boiled pre-GH sera. Boiled post-GH sera also were twice as active as boiled pre-GH sera in stimulating multiplication of chick embryo fibroblasts and confirmed that thymidine incorporation reflected DNA synthesis. GH added directly to chick embryo fibroblasts did not stimulate thymidine incorporation. We conclude that despite the presence of specific receptors for somatomedin-like peptides on human skin fibroblasts, somatomedins are not a major component of the serum macromolecules required for the routine growth of human fibroblasts in culture. In contrast, in chick embryo fibroblasts, somatomedins do appear to constitute an important part of the serum macromolecules supporting cell growth. (J Clin Endocrinol Metab 46: 937, 1978)
tors for the somatomedins on the surface membrane of the target cells (8, 9). The present study evaluates the contribution of human somatomedin activity to the serum growth requirement of human skin fibroblasts and chick embryo fibroblasts in culture. Two previous studies have examined this question in human fibroblasts and have yielded contradictory results. August et al. demonstrated that the generation time of human fibroblasts derived from embryonic lung (WI-38) was the same in human serum from normal volunteers, GH-deficient patients, and GH-deficient patients treated with GH (10). That is, the replication of human embryonic lung fibroblasts was not dependent on the presence of GH-dependent serum factors. On the other hand, MacGillivray and coworkers reported that human skin fibroblasts began to detach from the culture dish after a second consecutive passage in media containing sera 937
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Contribution of Human Somatomedin Activity to the Serum Growth Requirement of Human Skin Fibroblasts and Chick Embryo Fibroblasts in Culture*
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JCK & M . 1978 Vol46 No 6
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air and 5% CO2 at 37 C. Human fibroblasts were split 1:3 or 1:4. All human fibroblasts were shown to be free from mycoplasma contamination (Flow Laboratories). Fibroblasts were prepared for thymidine incorporation studies by the replicate plating of 2.25-3.00 X 105 cells/60-mm dish in 3 ml Eagles' MEM containing 0.4% FCS. The cells were maintained in 0.4% FCS 5-7 days before stimulation with Eagle's MEM containing the test serum samples. This 5to 7-day maintenance period in low serum concentration arrests the cells in the early Gi phase of the growth cycle and allows a synchronous wave of DNA synthesis upon addition of fresh serum or growth factors to these cells (6). Rechler et al. (16) have demonstrated previously that the peak of stimulation of thymidine incorporation into the DNA of human fibroblasts occurs 16 h after the addition of insulin, of the somatomedin nonsuppressible insulin-like activity (NSILA-S), or of serum to serum-deprived human fibroblasts. We have confirmed that the maximal incorporation of [3H]thymidine into DNA in cells from two hypopituitary patients occurs 16 h after the exposure of growth-arrested cells to 10% FCS (unpublished results). After 16 h of exposure to the test media, the Materials and Methods cells were pulsed for 1 h with [''H-raeJ/iyZJthyrnidine (0.4 juCi/ml in 2 ml media). [3H]Thymidine incorMedia, chemical, and hormones poration into DNA was measured as trichloroacetic Temin's modified Eagle's media (E media), Ea- acid (TCA) precipitable material by a method degle's minimal essential media (Eagle's MEM), and scribed previously (12). Samples from triplicate Ham's F-10 media were prepared by the Media dishes were counted in a Beckman LS 355 scintilUnit of the NIH. Fetal calf serum (FCS), penicillin, lation counter. Sera from each patient were tested and streptomycin were purchased from GIBCO. on his own fibroblasts; several patients' sera were [3H-metfiyZ]Thymidine (11-21 Ci/mmol) was ob- also tested on normal human fibroblasts. For cell multiplication studies, human skin fibrotained from Schwarz-Mann. Human GH was supblasts were plated in Eagle's MEM with 0.4% FCS plied by the National Pituitary Agency. at 2.25 X 105 cells/60-mm dish. At 24 h, when the cells were firmly attached to the dish, the media Human fibroblasts was aspirated and replaced with media containing All patients and a normal volunteer underwent the test sera. Cell counts were performed at the forearm punch skin biopsies for the initiation of time of application of test media, and 2 and 4-5 fibroblast cultures. A 3- or 4-mm skin biopsy was days after application of the test media. Media was minced under sterile conditions. Small sections of not changed during this incubation period. Cells the biopsy were secured to a 60-mm Petri dish were counted on a Particle Data cell counter after removal from the dish with 0.25% trypsin in Dulunder a glass coverslip in the presence of Eagle's becco's phosphate-buffered saline. Additional MEM and 10% FCS. Media was changed twice growth studies with human fibroblasts in Eagle's weekly until fibroblast growth was confluent under MEM and Ham's F-10 media were performed after the coverslip, at which time the cells were trypsin- plating cells by the method of MacGillivray (11), ized and plated in Falcon flasks. Fibroblasts were except that 5% human serum was used in the final passaged in Eagle's MEM supplemented with 10% two platings. FCS, penicillin, and streptomycin and were utilized for thymidine incorporation assays and growth Chick embryo fibroblasts studies between the third and 16th passages. They Tertiary cultures of chick embryo fibroblasts, were grown in a high humidity atmosphere of 95% from GH-deficient patients (11). The same cells demonstrated normal surface attachment when replated into medium containing normal human serum or serum from GH-deficient patients after GH treatment. These data suggested that the growth of human fibroblasts in vitro was dependent on GH-dependent serum factors. For the present study, sera from five GHdeficient patients were obtained before and after GH treatment and were tested for their ability to stimulate thymidine incorporation into DNA and cell multiplication in the patients' own fibroblasts and in skin fibroblasts derived from a normal volunteer. In addition, as it has been demonstrated previously that the growth of chick embryo fibroblasts in rat serum is dependent on the somatomedin activity of that serum (12), we have evaluated the growth response of chick embryo fibroblasts to human sera from GH-deficient individuals before and after GH treatment.
HUMAN SOMATOMEDIN ACTIVITY AND FIBROBLAST GROWTH
Patients Five patients had GH deficiency documented by combined arginine infusion and insulin hypoglycemia. The clinical characteristics and growth response to GH of these patients are listed in Table 1. Patients had blood drawn before the initiation of GH therapy or after a 1-month rest from GH. They then were treated with 2.5 U human GH im every 12 h for 7 days. Within 12 h of the final GH injection, repeat blood samples were drawn and jtilized as the post-GH samples. The GH treatment protocol has been shown to result in approximately 3-fold increases in somatomedin activity in patients vith hypopituitarism (13). Blood was allowed to clot at 4 C and the serum vas removed after a 10-min centrifugation at 2500 < g. Serum was stored at —20 C. Before testing on PABLE
Patient
human fibroblasts or chick embryo fibroblasts, sera were diluted 1:3 with sterile Dulbecco's phosphatebuffered saline. Diluted sera were mixed with the incubation media for ['Hjthymidine assays or cell multiplication studies. These untreated serum dilutions are referred to as "straight" serum samples. An aliquot of diluted sera was acidified to pH 5.5 with 1 N HC1 and was boiled for 15 min. Precipitated protein was removed by centrifugation at 2500 X g for 10 min and the clear supernatant was mixed with the appropriate media and utilized as boiled, acidified serum. Pooled normal human serum that was boiled under acid conditions retained 50-70% of the activity of unboiled serum in the chick embryo fibroblast thymidine incorporation assay. When the activity of boiled serum was expressed per milligram of serum protein, there was a 10- to 15-fold increase in the specific activity of boiled serum as compared to the unboiled serum (unpublished data). This method of partially purifying somatomedin activity from rat serum previously has been shown to retain significant amounts of somatomedin activity in the hypophysectomized rat costal cartilage sulfate incorporation assay (14). To document that somatomedin activity increased in these GH-deficient patients with GH treatment, stimulation of [35S]sulfate incorporation into hypophysectomized rat costal cartilage produced by these sera was measured by Dr. Eckehart Wiedemann [(15) Table 1]. Sera from these same patients, before and after GH therapy, also were assayed for somatomedin C in the RIA of Drs. Furlanetto, Underwood, VanWyk, and D'Ercole [(16) Table 1]. Insulin levels in boiled serum ex-
1. Clinical data at onset of hGH therapy
Diagnosis
Sex
Age
Height (cm)
1 2 3
Craniopharyngioma Craniopharyngioma .Idiopathic hypopituitarism
M M F
lOWa HWfa 3-VI-J
108.2 117.1 75.5
4
Malignant teratoma, 3rd ventricle
F
16%2
141.4
5
Idiopathic G H deficiency
M
6%>
91.8
Hormone replacement
hGH hGH hGH Cortisol T, hGH Cortisol T, hGH
Rat costal cartilage somatomedin activity
Somatomedin C RIA (U/ml)
Before hGH
After hGH
Before hGH
After hGH
9.3 6.5 10.2
0.92 0.30 0.00
1.96 0.82 1.26
0.13 0.14 0.00''
1.18 1.52 0.17
0.4
0.97
1.06
0.18' 0.12
2.42 2.44
0.04
0.24
Growth before hGH
Growth (cm/yr) after hGH
2.58 1.61 3.8"
0.83''
9.6
T h e 95% fiducial limits for age-matched normal subjects in t h e somatomedin activity assay were: age 2-10, 0.3-1.3; age 11-16, .45-2.05. T h e range of somatomedin C for normal adults is 1.5 ± 0.5 U / m l . A normal sample from this laboratory was 1.63 U/ml. T h e erum samples assayed in the r a t costal cartilage assay and somatomedin C RIA were not identical. " Growth per 11 months. * Below assay limits. ' Before and after G H serum samples from two separate GH treatment periods. '' Growth per 5 months.
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derived from 12-day-old embryos, were plated at 1 X 106 cells/60-mm dish in 3 ml E media containing 20% (vol/vol) tryptose phosphate (ET media) and 0.4% FCS (12). Cells were maintained at low serum concentration for 4-5 days before incubation for 12 h with test sera in E media. Thymidine incorporation into DNA was measured after a 1-h pulse of (3H-metfiy/)thymidine (0.4 jttCi/ml) in media (12). For cell multiplication studies, tertiary cultures of chick embryo fibroblasts were plated at 0.5 X 106 cells/60-mm dish in ET media containing 0.4% FCS. Twenty-four hours later, the ET media was changed to E media containing test sera. Media incubations and cell counts were performed as described for the human fibroblasts.
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tracts were determined by RIA by Carla Hendricks, Diabetes Branch, NIAMDD. Sephadex chromatography of serum
Results Human fibroblasts When a patient's fibroblasts were exposed to media containing his own serum, the incorporation of [3H]thymidine into DNA was dose dependent from 1-5% final serum concentration (Fig. 1). Results from a fourth patient were similar to the results for the three patients shown in Fig. 1. Despite the ability of post-GH treatment serum to stimulate thymidine incorporation into DNA, there was no consistent difference between the response of human fibroblasts to serum obtained before or after GH therapy. This failure to differentiate pre- and post-GH sera in the thymidine incorporation assay was not due to a failure of
the GH treatment protocol to increase somatomedin levels, as all five patients had a 5- to 10-fold increase in their somatomedin content, as measured by the somatomedin C RIA (Table 1). Three of the four patients tested also demonstrated an increase in somatomedin activity, as measured in the hypophysectomized rat costal cartilage assay (Table 1). Partially purifying somatomedin activity in the patients' sera by boiling sera under acid conditions resulted in a significant and consistent decrease in the ability of these samples to stimulate [3H]thymidine incorporation into DNA when compared to unboiled sera (Fig. 1). Again, there was no difference between the pre-GH treatment and the post-GH treatment samples. Dialysis of boiled serum samples from one individual failed to increase the ability of the post-GH treatment sample to stimulate thymidine incorporation into DNA in human fibroblasts relative to the pre-GH treatment sample (data not shown), thereby demonstrating the absence of a dialyzable inhibitor for this assay in boiled post-GH treatment serum. To demonstrate that [JH]thymidine incorporation reflected actual DNA synthesis, cell multiplication experiments were undertaken with human fibroblasts and human serum. As suggested by the thymidine incorporation
Straight, Pre GH
Straight, Post GH
1.0
2.5
5.0
1.0
2.5
5.0
1.0
2.5
% FINAL SERUM CONCENTRATION
FIG. 1. [;iH]Thymidine incorporation into human fibroblast DNA as stimulated by serum from GH-deficient patients before and after GH treatment. Serum concentrations from 1-5% are plotted on a log scale on the abscissa. Boilec samples refer to serum adjusted to pH 5.5 and boiled for 15 min. Each point represents the mean of triplicate samples the SEM is indicated by the vertical bars when these exceed the height of the symbol used to represent the point.
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Human serum (0.5 ml) was mixed with 0.5 ml 2 M acetic acid and chromatographed over Sephadex G-50 (1.5 X 90 cm) equilibrated with 1 M acetic acid. Six-milliliter fractions were collected and 1.5ml aliquots were lyophilized to dryness before mixing in E media. Samples were incubated with chick embryo fibroblasts, as described above, to determine [3H]thymidine incorporation into DNA.
JCK&M • 1978 V0U6 • No 6
HUMAN SOMATOMEDIN ACTIVITY AND FIBROBLAST GROWTH
detach from the culture dish while being grown in the pre-GH serum. In addition, we were unable to demonstrate that human fibroblasts, passaged according to the method of MacGillivray (11), detached from the dish during multiple passages in media containing 5% GH-deficient serum (data not shown). Finally, the level of thymidine incorporation into DNA and cell multiplication of skin fibroblasts from a normal volunteer were identical to the response of cells from patients with GH deficiency when exposed to pre- and post-GH human sera or a purified somatomedin-like polypeptide (MSA; unpublished results). The finding that human skin fibroblasts from patients with GH deficiency and fibroblasts from normals responded equally in the [3H]thymidine incorporation assay agrees with the report of Jones and Addison (17). Chick embryo fibroblasts
Previous studies from this laboratory demonstrated that the stimulation of thymidine incorporation into DNA and multiplication of chick embryo fibroblasts by rat serum was dependent on the GH status of the rat and, therefore, was dependent on the somatomedin activity of rat serum (12). Bovine GH was inactive in stimulating DNA synthesis in chick embryo fibroblasts when added directly to the chick embryo fibroblasts (12). As the growth of human skin fibroblasts was not dependent 20 r in a major way on the somatomedin activity of the human serum to which they were exposed, we have evaluated the growth response of chick embryo fibroblasts to the same sera. 10 As in human fibroblasts, the stimulation of 8 thymidine incorporation into DNA of chick x
8
_CJ
4
2
3 PATIENT
ratio with that serum, thereby ruling out the presence of an inhibitor in sufficient concentration in boiled pre-GH treatment serum to explain its decreased potency in the thymidine incorporation assay. Finally, the human serum components capable of stimulating thymidine incorporation into the DNA of chick embryo fibroblasts eluted from a Sephadex G-50 column equilibrated with 1 M acetic acid in the same position as that expected for the somatomedin activity of rat serum previously demonstrated by this laboratory [(18)] Fig. 5]. To demonstrate that the stimulation of [3H]thymidine incorporation into chick embryo fibroblast DNA by human sera was a
I 1
5
reflection of actual DNA synthesis, cell multiplication experiments were performed utilizing human sera boiled at pH 5.5 (Fig. 6). As anticipated from the thymidine incorporation studies, post-GH treatment serum boiled at pH 5.5 was twice as potent at 2.5% concentration as similarly treated pre-GH treatment serum. FCS at 2.5% concentration was significantly more potent than boiled human serum. Sera from two other patients, before and after GH therapy, gave similar results in stimulating the growth of chick embryo fibroblasts. These experiments confirmed the growth-promoting effect of human somatomedin activity for chick embryo fibroblasts and demon-
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o o a.
MOSES ET AL.
944 b
2
SAL
t-
[Vo]
S i. o z o
1
CORPO
o AFTER GH
1 f
z
\*
10
i > -
•^A \
I BEFOREX GH \
5
I
V , . . i-t
6
8
10
\
B ^
Q -1?
• ^ • ^ /
o
\
J V° r ^-'o 12 14 16 FRACTION NO.
18
20
22
24
FIG. 5. [:lH]Thymidine incorporation into chick embryo fibroblasts by fractions of human serum before and after GH treatment, separated on a 1.5 X 90-cm Sephadex G50 column equilibrated in 1 M acetic acid. The void volume of the column is designated by Vo. Each point represents one dish of chick embryo fibroblasts.
2 . 5 % Fetal CaM
20
m
6
Z
1
2
3 DAYS
4
5
FIG. 6. Cell multiplication of chick embryo fibroblasts exposed to boiled human serum before and after GH treatment. Each point represents the mean of triplicate dishes; the SEM is indicated by the vertical bars.
strated that the measurement of [3H]thymidine incorporation into DNA reflected actual DNA synthesis. Discussion The ability of cultured human skin fibroblasts to bind somatomedin-like polypeptides specifically (8) suggested that these cells would be a convenient tool to evaluate clinical disturbances of growth at the cellular level. The binding of homogeneous somatomedin A
1978 No 6
and multiplication-stimulating activity (MSA) and partially purified NSILA-S to a specific receptor on human fibroblasts correlates with the ability of these peptides to stimulate the incorporation of [3H]thymidine into DNA in these cells (6, 8, 19). The identification of a "growth" receptor on human fibroblasts has prompted us to evaluate whether the somatomedins present in human serum constitute a major component of the serum macromolecules required for the routine growth of these cells in culture. Human sera from GH-deficient patients, both before and after GH treatment, were equipotent in stimulating thymidine incorporation into the DNA of human fibroblasts. Boiling human sera under acid conditions, a procedure previously demonstrated to retain significant amounts of somatomedin activity (14), markedly diminished the ability of these sera to stimulate thymidine incorporation into DNA and failed to unmask a difference between pre- and post-GH treatment sera in the thymidine incorporation assay. The results of cell multiplication experiments paralleled the thymidine incorporation assays. That is, human fibroblasts multiplied to the same extent in serum from patients before and after GH therapy and did not multiply above control levels (media without serum) in these same sera boiled under acid conditions. It is possible that concentrations of boiled human sera greater than 1% would stimulate the growth of human skin fibroblasts in culture, but the results of the thymidine incorporation studies suggest that there would be no difference between sera before or after GH treatment. These results suggest that human skin fibroblasts are not dependent solely on human somatomedin activity for routine growth in culture despite the presence of a specific receptor for somatomedin-like polypeptides on the cell surface. Our observations support the report of August et al. that the generation time of human embryonic lung fibroblasts (WI-38) was independent of GH-dependent serum factors (10). The inability of the boiled extract of human serum to support the multiplication of human skin fibroblasts (Fig. 2) is consistent with the failure to stimulate multi-
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JCK & M Vol46
HUMAN SOMATOMEDIN ACTIVITY AND FIBROBLAST GROWTH
lack of dependence of DNA synthesis on somatomedin activity in whole serum is not surprising considering the variety of serum components demonstrated to have growthpromoting activity (1). The small increment of stimulatory activity in post-GH treatment sera compared to pre-GH treatment sera at very low serum concentrations (0.5% and below) may be due to differences in the shape of the dose-response curves for somatomedin compared to other mitogens in untreated human serum. Serum boiled at pH 5.5 from GHdeficient individuals before GH treatment was much less potent than whole serum in stimulating thymidine incorporation into chick embryo fibroblast DNA. This loss of activity of boiled pre-GH serum is probably secondary to the denaturation of proteins capable of stimulating DNA synthesis that are present in whole serum. On the other hand, boiled serum from the same individual after GH treatment was much more potent in stimulating thymidine incorporation into DNA and cell multiplication than the boiled pre-GH treatment serum, and approached whole serum in its activity. This activity correlated in all cases with an increase in somatomedin content in these patients' sera, as measured by the somatomedin C RIA. There also was general agreement between the chick embryo fibroblast assay and somatomedin activity, as measured by sulfate incorporation into hypophysectomized rat costal cartilage. Although patient 1 demonstrated normal somatomedin activity in the rat costal cartilage assay before GH, he had a significant increase in the activity after GH treatment. The apparent discrepancy for patient 4 between the rat costal cartilage assay on the one hand and thymidine incorporation into chick embryo fibroblast DNA or the somatomedin C RIA on the other hand, remains unexplained and requires further investigation. The potency of boiled postGH treatment sera was not due to the presence of higher concentrations of GH or insulin in the sera. Although no attempt has been made in this study to quantitate human somatomedin activity as measured by [3H]thymidine incorporation into chick embryo fibroblast DNA, this assay may be useful as an-
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plication of human skin fibroblasts with a purified somatomedin-like peptide (MSA) in the absence of serum (20). Furthermore, we have been unable to demonstrate that human skin fibroblasts detach from the dish during successive passages in 5% serum from GHdeficient patients before GH treatment. It is possible that the somatomedins require other factors in order to exert a major effect on the multiplication of human skin fibroblasts. Our experiments have not explored this possibility. In any case, the study of the interaction of purified somatomedins with receptors on cultured human skin fibroblasts may yield important information concerning both growth at the cellular level and end organ resistance to somatomedin, as expressed phenotypically by certain genetic forms of short stature. Chick embryo fibroblasts also specifically bind somatomedin-like polypeptides (9, 21). The specific receptor for somatomedin-like polypeptides on chick embryo fibroblasts has been designated a growth receptor because for MSA there is a very close correlation between the concentration of peptide required for halfmaximal displacement of radiolabeled MSA from the receptor and the concentration of peptide that causes half-maximal stimulation of thymidine incorporation into DNA (9). MSA also can stimulate the multiplication of chick embryo fibroblasts in the absence of serum; this further supports the concept of somatomedin-like peptides interacting with a growth receptor on the chick embryo fibroblast surface membrane (22). As suggested by the above findings, the present study demonstrates that chick embryo fibroblasts, in part, are dependent for growth on the somatomedin activity of the human sera to which they are exposed. This extends previous observations on chick embryo fibroblasts, utilizing normal, hypophysectomized, and bovine GH-treated hypophysectomized rat serum (12), for the first time to human sera. As in human fibroblasts, there was no difference in the level of thymidine incorporation in chick embryo fibroblasts stimulated by straight human serum before or after GH treatment at 1-5% final concentration. This
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Acknowledgments The authors wish to thank Patricia Short and Annie Bell Cooper for their skillful technical assistance, and Patricia Short and James Phang for helpful suggestions in reviewing the manuscript. We also thank Dr. Eckehart Wiedemann for the measurements of somatomedin activity utilizing the rat costal cartilage assay, Dr. Richard Furlanetto for the somatomedin C RIA, and Carla Hendricks for performing the insulin RIA.
References 1. TEMIN, H. M., R. W. PIERSON, and N. C. DULAK, The role of
serum in the control of multiplication of avian and mammalian cells in culture, In Rothblat, A., and V. J. Cristofalo (eds.), Growth, Nutrition and Metabolism of Cells in Culture, vol. 1, New York, Academic Press, 1972, p. 50.
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5.
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on fibroblasts in culture, Eur J Clin Invest 3: 119, 1973. 6. RECHLER, M. M., J. M. PODSKALNY, I. D. GOLDFINE, and C.
A. WELLS, DNA synthesis in human fibroblasts: stimulation by insulin and by non-suppressible insulin-like activity (NSILA-S), J Clin Endocrinol Metab 39: 512, 1974. 7. BASEMAN, J. B., and N. S. HAYES, Differential effect of hormones on macromolecular synthesis and mitosis in chick embryo cells, J Cell Biol 67: 492, 1975. 8. RECHLER, M. M., S. P. NISSLEY, J. M. PODSKALNY, A. C.
MOSES, and L. FRYKLUND, Identification of a receptor for somatomedin-like polypeptides in human fibroblasts, J Clin Endocrinol Metab 44: 820, 1977. 9. RECHLER, M. M., J. M. PODSKALNY, and S. P. NISSLEY,
Interaction of multiplication-stimulating activity with chick embryo fibroblasts demonstrates a growth receptor, Nature 259: 134, 1976. 10. AUGUST, G. N., R. F. CHENG, W. HUNG, and J. C. HOUCK,
Fibroblast proliferative activity in the sera of growth hormone deficient patients, Horm Metab Ren 5: 340, 1975. 11. MACGILLIVRAY, M. H., C. HASTINGS, and J. A. BROWN,
Growth hormone-dependent effects of human serum on the in vitro growth characteristics of human skin fibroblasts, J Clin Endocrinol Metab 40: 62, 1975. 12. COHEN, K. L., P. A. SHORT, and S. P. NISSLEY, Growth
hormone-dependent serum stimulation of DNA synthesis in chick embryo fibroblasts in culture, Endocrinology 96: 193, 1975. 13. DAUGHADAY, W. H., Z. LARON, A. PERTZELAN, and J. N.
HEINS, Defective sulfation factor generation: a possible etiologic link in dwarfism, Trans Assoc Am Physicians 82: 129, 1969. 14. SALMON, W. D., JR., Investigation with a partially purified preparation of serum sulfation factor: lack of specificity for cartilage sulfation, In Pecile, A., and E. Muller (eds.), Growth and Growth Hormone, Amsterdam, Excerpta Medica, 1971, p. 180. 15. WIEDEMANN, E., E. SCHWARTZ, and A. G. FRANTZ, Acute
and chronic estrogen effects upon serum somatomedin activity, growth hormone and prolactin in man. J Clin Endocrinol Metab 42: 942, 1976. 16. FURLANETTO, R. W., L. E. UNDERWOOD, J. J. VANWYK, and
A. J. D'ERCOLE, Estimation of somatomedin-C level in normals and patients with pituitary disease by radioimmunoassay, J Clin Invest 60: 648, 1977. 17. JONES, K. L., and J. ADDISON, Studies of human fibroblast growth factor, insulin, and serum, J Clin Endocrinol Metab 43: 721, 1976. 18. COHEN, K. L., and S. P. NISSLEY, Comparison of somatomedin activity in rat serum and lymph, Endocrinology 97: 654,1975. 19. RECHLER, M. M., L. FRYKLUND, S. P. NISSLEY, K. HALL, J. M. PODSKALNY, A. SKOTTNER, and A. C. MOSES, Purified
human somatomedin A and rat multiplication stimulating activity; mitogens for cultured fibroblasts that cross-react with the same growth peptide receptors, Eur J Biochem 82: SALMON, W. D., J R . , and W. H. DAUGHADAY, A hormonally 5, 1978. controlled serum factor which stimulates sulfate incorporation 20. NISSLEY, S. P., and M. M. RECHLER, Multiplication stimulatby cartilage in vitro, J Lab Clin Med 49: 825, 1957. ing activity (MSA): a somatomedin-like polypeptide from DAUGHADAY, W. H., K. HALL, M. S. RABEN, W. D. SALMON, cultured rat liver cells, Natl Cancer Inst Monogr 48: 167, J. L. VAN DEN BRANDE, and J. J. VAN WYK, Somatomedin: 1978. proposed designation for sulphation factor, Nature 235: 107, 21. ZAPF, J., M. MADER, M. WALDVOGEL, D. S. SCHALCH, and E. 1972. R. FROESCH, Specific binding of nonsuppressible insulin like DULAK, N. C, and H. M. TEMIN, A partially purified polyactivity to chicken embryo fibroblasts and to a solubilized peptide fraction from rat liver cell conditioned medium with fibroblast receptor, Arch Biochem Biophys 168: 630 1975. multiplication-stimulating activity for embryo fibroblasts, J Cell Physiol 81: 153, 1973. 22. NISSLEY, S. P., M. M. RECHLER, A. C. MOSES, P. A. SHORT, and J. M. PODSKALNY, Proinsulin binds to a growth peptide MORELL, B., and E. R. FROESCH, Fibroblasts as an experireceptor and stimulates DNA synthesis in chick embryo fimental tool in metabolic and hormone studies. II. Effects of broblasts, Endocrinology 101: 708, 1977. insulin and nonsuppressible insulin-like activity (NSILA-S)
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other bioassay for human somatomedin activity. These experiments show for the first time that human somatomedin activity is an important constituent of the serum macromolecules that support the growth of chick embryo fibroblasts in culture. Furthermore, the response of chick embryo fibroblasts to crudely purified human somatomedin activity supports recent studies that define a growth receptor on chick embryo fibroblasts that specifically binds purified somatomedins and somatomedin-like compounds (9, 21). Thus, although both human skin fibroblasts and chick embryo fibroblasts are useful tools in evaluating the interaction of purified somatomedins with a cell membrane receptor, chick embryo fibroblasts appear to be more sensitive than human fibroblasts in their growth response to the somatomedin activity of human serum.
JCK & M Vol46
Vol. 46, No. 6 Printed in U.S.A.
ALAN C. MOSES, KENNETH L. COHEN,f ROGER JOHNSONBAUGH, AND S. PETER NISSLEY Endocrinology Section, Metabolism Branch, National Cancer Institute, National Institutes of Health (A.C.M., K.L.C., S.P.N.); and the National Naval Medical Center (R.J.), Bethesda, Maryland 20014 ABSTRACT. Although purified human somatomedins and related peptides have been shown to stimulate growth-related processes in cultured human skin fibroblasts, it is unknown whether the serum macromolecules required for the routine growth of human fibroblasts in culture include the somatomedins. To evaluate this question we have obtained sera from five GH-deficient patients before and after GH treatment and have compared these pre- and post-GH sera for their ability to stimulate [3H]thymidine incorporation into DNA and cell multiplication in the patients' own fibroblasts in culture. Subconfluent human fibroblasts demonstrated a dose-dependent increase in thymidine incorporation and cell number when exposed to human sera. Pre- and post-GH sera were equipotent. Partial purification of somatomedin activity by boiling sera at pH 5.5 diminished the level of thymidine incorporation to 10-20% of the level achieved with unboiled sera but did not unmask a difference between pre- and post-GH sera. In
I
T HAS been recognized for some time that human skin fibroblasts require serum in order to multiply in culture (1). More recently, several investigators have recognized that the somatomedins, originally proposed as the peripheral mediators of GH action on skeletal tissue (2, 3), may contribute to the serum growth requirement of cells in culture (4-9). Growth-related processes in chick embryo fibroblasts and in human skin fibroblasts are stimulated by purified somatomedins in the absence of serum. This stimulation has been correlated with the presence of specific recepReceived August 23, 1976. Address requests for reprints to: Dr. Alan C. Moses, Clinical Associate, Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014. * This work was presented in part at the 58th Annual Meeting of the Endocrine Society, San Francisco, California, June 23-25, 1976. f Present address: Department of Medicine, Yale University, New Haven, Connecticut.
contrast to the results in human fibroblasts, boiled postGH sera were 2- to 5-fold more potent than pre-GH sera in stimulating thymidine incorporation in chick embryo fibroblasts. Mixing experiments failed to demonstrate inhibition of thymidine incorporation by boiled pre-GH sera. Boiled post-GH sera also were twice as active as boiled pre-GH sera in stimulating multiplication of chick embryo fibroblasts and confirmed that thymidine incorporation reflected DNA synthesis. GH added directly to chick embryo fibroblasts did not stimulate thymidine incorporation. We conclude that despite the presence of specific receptors for somatomedin-like peptides on human skin fibroblasts, somatomedins are not a major component of the serum macromolecules required for the routine growth of human fibroblasts in culture. In contrast, in chick embryo fibroblasts, somatomedins do appear to constitute an important part of the serum macromolecules supporting cell growth. (J Clin Endocrinol Metab 46: 937, 1978)
tors for the somatomedins on the surface membrane of the target cells (8, 9). The present study evaluates the contribution of human somatomedin activity to the serum growth requirement of human skin fibroblasts and chick embryo fibroblasts in culture. Two previous studies have examined this question in human fibroblasts and have yielded contradictory results. August et al. demonstrated that the generation time of human fibroblasts derived from embryonic lung (WI-38) was the same in human serum from normal volunteers, GH-deficient patients, and GH-deficient patients treated with GH (10). That is, the replication of human embryonic lung fibroblasts was not dependent on the presence of GH-dependent serum factors. On the other hand, MacGillivray and coworkers reported that human skin fibroblasts began to detach from the culture dish after a second consecutive passage in media containing sera 937
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Contribution of Human Somatomedin Activity to the Serum Growth Requirement of Human Skin Fibroblasts and Chick Embryo Fibroblasts in Culture*
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JCK & M . 1978 Vol46 No 6
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air and 5% CO2 at 37 C. Human fibroblasts were split 1:3 or 1:4. All human fibroblasts were shown to be free from mycoplasma contamination (Flow Laboratories). Fibroblasts were prepared for thymidine incorporation studies by the replicate plating of 2.25-3.00 X 105 cells/60-mm dish in 3 ml Eagles' MEM containing 0.4% FCS. The cells were maintained in 0.4% FCS 5-7 days before stimulation with Eagle's MEM containing the test serum samples. This 5to 7-day maintenance period in low serum concentration arrests the cells in the early Gi phase of the growth cycle and allows a synchronous wave of DNA synthesis upon addition of fresh serum or growth factors to these cells (6). Rechler et al. (16) have demonstrated previously that the peak of stimulation of thymidine incorporation into the DNA of human fibroblasts occurs 16 h after the addition of insulin, of the somatomedin nonsuppressible insulin-like activity (NSILA-S), or of serum to serum-deprived human fibroblasts. We have confirmed that the maximal incorporation of [3H]thymidine into DNA in cells from two hypopituitary patients occurs 16 h after the exposure of growth-arrested cells to 10% FCS (unpublished results). After 16 h of exposure to the test media, the Materials and Methods cells were pulsed for 1 h with [''H-raeJ/iyZJthyrnidine (0.4 juCi/ml in 2 ml media). [3H]Thymidine incorMedia, chemical, and hormones poration into DNA was measured as trichloroacetic Temin's modified Eagle's media (E media), Ea- acid (TCA) precipitable material by a method degle's minimal essential media (Eagle's MEM), and scribed previously (12). Samples from triplicate Ham's F-10 media were prepared by the Media dishes were counted in a Beckman LS 355 scintilUnit of the NIH. Fetal calf serum (FCS), penicillin, lation counter. Sera from each patient were tested and streptomycin were purchased from GIBCO. on his own fibroblasts; several patients' sera were [3H-metfiyZ]Thymidine (11-21 Ci/mmol) was ob- also tested on normal human fibroblasts. For cell multiplication studies, human skin fibrotained from Schwarz-Mann. Human GH was supblasts were plated in Eagle's MEM with 0.4% FCS plied by the National Pituitary Agency. at 2.25 X 105 cells/60-mm dish. At 24 h, when the cells were firmly attached to the dish, the media Human fibroblasts was aspirated and replaced with media containing All patients and a normal volunteer underwent the test sera. Cell counts were performed at the forearm punch skin biopsies for the initiation of time of application of test media, and 2 and 4-5 fibroblast cultures. A 3- or 4-mm skin biopsy was days after application of the test media. Media was minced under sterile conditions. Small sections of not changed during this incubation period. Cells the biopsy were secured to a 60-mm Petri dish were counted on a Particle Data cell counter after removal from the dish with 0.25% trypsin in Dulunder a glass coverslip in the presence of Eagle's becco's phosphate-buffered saline. Additional MEM and 10% FCS. Media was changed twice growth studies with human fibroblasts in Eagle's weekly until fibroblast growth was confluent under MEM and Ham's F-10 media were performed after the coverslip, at which time the cells were trypsin- plating cells by the method of MacGillivray (11), ized and plated in Falcon flasks. Fibroblasts were except that 5% human serum was used in the final passaged in Eagle's MEM supplemented with 10% two platings. FCS, penicillin, and streptomycin and were utilized for thymidine incorporation assays and growth Chick embryo fibroblasts studies between the third and 16th passages. They Tertiary cultures of chick embryo fibroblasts, were grown in a high humidity atmosphere of 95% from GH-deficient patients (11). The same cells demonstrated normal surface attachment when replated into medium containing normal human serum or serum from GH-deficient patients after GH treatment. These data suggested that the growth of human fibroblasts in vitro was dependent on GH-dependent serum factors. For the present study, sera from five GHdeficient patients were obtained before and after GH treatment and were tested for their ability to stimulate thymidine incorporation into DNA and cell multiplication in the patients' own fibroblasts and in skin fibroblasts derived from a normal volunteer. In addition, as it has been demonstrated previously that the growth of chick embryo fibroblasts in rat serum is dependent on the somatomedin activity of that serum (12), we have evaluated the growth response of chick embryo fibroblasts to human sera from GH-deficient individuals before and after GH treatment.
HUMAN SOMATOMEDIN ACTIVITY AND FIBROBLAST GROWTH
Patients Five patients had GH deficiency documented by combined arginine infusion and insulin hypoglycemia. The clinical characteristics and growth response to GH of these patients are listed in Table 1. Patients had blood drawn before the initiation of GH therapy or after a 1-month rest from GH. They then were treated with 2.5 U human GH im every 12 h for 7 days. Within 12 h of the final GH injection, repeat blood samples were drawn and jtilized as the post-GH samples. The GH treatment protocol has been shown to result in approximately 3-fold increases in somatomedin activity in patients vith hypopituitarism (13). Blood was allowed to clot at 4 C and the serum vas removed after a 10-min centrifugation at 2500 < g. Serum was stored at —20 C. Before testing on PABLE
Patient
human fibroblasts or chick embryo fibroblasts, sera were diluted 1:3 with sterile Dulbecco's phosphatebuffered saline. Diluted sera were mixed with the incubation media for ['Hjthymidine assays or cell multiplication studies. These untreated serum dilutions are referred to as "straight" serum samples. An aliquot of diluted sera was acidified to pH 5.5 with 1 N HC1 and was boiled for 15 min. Precipitated protein was removed by centrifugation at 2500 X g for 10 min and the clear supernatant was mixed with the appropriate media and utilized as boiled, acidified serum. Pooled normal human serum that was boiled under acid conditions retained 50-70% of the activity of unboiled serum in the chick embryo fibroblast thymidine incorporation assay. When the activity of boiled serum was expressed per milligram of serum protein, there was a 10- to 15-fold increase in the specific activity of boiled serum as compared to the unboiled serum (unpublished data). This method of partially purifying somatomedin activity from rat serum previously has been shown to retain significant amounts of somatomedin activity in the hypophysectomized rat costal cartilage sulfate incorporation assay (14). To document that somatomedin activity increased in these GH-deficient patients with GH treatment, stimulation of [35S]sulfate incorporation into hypophysectomized rat costal cartilage produced by these sera was measured by Dr. Eckehart Wiedemann [(15) Table 1]. Sera from these same patients, before and after GH therapy, also were assayed for somatomedin C in the RIA of Drs. Furlanetto, Underwood, VanWyk, and D'Ercole [(16) Table 1]. Insulin levels in boiled serum ex-
1. Clinical data at onset of hGH therapy
Diagnosis
Sex
Age
Height (cm)
1 2 3
Craniopharyngioma Craniopharyngioma .Idiopathic hypopituitarism
M M F
lOWa HWfa 3-VI-J
108.2 117.1 75.5
4
Malignant teratoma, 3rd ventricle
F
16%2
141.4
5
Idiopathic G H deficiency
M
6%>
91.8
Hormone replacement
hGH hGH hGH Cortisol T, hGH Cortisol T, hGH
Rat costal cartilage somatomedin activity
Somatomedin C RIA (U/ml)
Before hGH
After hGH
Before hGH
After hGH
9.3 6.5 10.2
0.92 0.30 0.00
1.96 0.82 1.26
0.13 0.14 0.00''
1.18 1.52 0.17
0.4
0.97
1.06
0.18' 0.12
2.42 2.44
0.04
0.24
Growth before hGH
Growth (cm/yr) after hGH
2.58 1.61 3.8"
0.83''
9.6
T h e 95% fiducial limits for age-matched normal subjects in t h e somatomedin activity assay were: age 2-10, 0.3-1.3; age 11-16, .45-2.05. T h e range of somatomedin C for normal adults is 1.5 ± 0.5 U / m l . A normal sample from this laboratory was 1.63 U/ml. T h e erum samples assayed in the r a t costal cartilage assay and somatomedin C RIA were not identical. " Growth per 11 months. * Below assay limits. ' Before and after G H serum samples from two separate GH treatment periods. '' Growth per 5 months.
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derived from 12-day-old embryos, were plated at 1 X 106 cells/60-mm dish in 3 ml E media containing 20% (vol/vol) tryptose phosphate (ET media) and 0.4% FCS (12). Cells were maintained at low serum concentration for 4-5 days before incubation for 12 h with test sera in E media. Thymidine incorporation into DNA was measured after a 1-h pulse of (3H-metfiy/)thymidine (0.4 jttCi/ml) in media (12). For cell multiplication studies, tertiary cultures of chick embryo fibroblasts were plated at 0.5 X 106 cells/60-mm dish in ET media containing 0.4% FCS. Twenty-four hours later, the ET media was changed to E media containing test sera. Media incubations and cell counts were performed as described for the human fibroblasts.
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tracts were determined by RIA by Carla Hendricks, Diabetes Branch, NIAMDD. Sephadex chromatography of serum
Results Human fibroblasts When a patient's fibroblasts were exposed to media containing his own serum, the incorporation of [3H]thymidine into DNA was dose dependent from 1-5% final serum concentration (Fig. 1). Results from a fourth patient were similar to the results for the three patients shown in Fig. 1. Despite the ability of post-GH treatment serum to stimulate thymidine incorporation into DNA, there was no consistent difference between the response of human fibroblasts to serum obtained before or after GH therapy. This failure to differentiate pre- and post-GH sera in the thymidine incorporation assay was not due to a failure of
the GH treatment protocol to increase somatomedin levels, as all five patients had a 5- to 10-fold increase in their somatomedin content, as measured by the somatomedin C RIA (Table 1). Three of the four patients tested also demonstrated an increase in somatomedin activity, as measured in the hypophysectomized rat costal cartilage assay (Table 1). Partially purifying somatomedin activity in the patients' sera by boiling sera under acid conditions resulted in a significant and consistent decrease in the ability of these samples to stimulate [3H]thymidine incorporation into DNA when compared to unboiled sera (Fig. 1). Again, there was no difference between the pre-GH treatment and the post-GH treatment samples. Dialysis of boiled serum samples from one individual failed to increase the ability of the post-GH treatment sample to stimulate thymidine incorporation into DNA in human fibroblasts relative to the pre-GH treatment sample (data not shown), thereby demonstrating the absence of a dialyzable inhibitor for this assay in boiled post-GH treatment serum. To demonstrate that [JH]thymidine incorporation reflected actual DNA synthesis, cell multiplication experiments were undertaken with human fibroblasts and human serum. As suggested by the thymidine incorporation
Straight, Pre GH
Straight, Post GH
1.0
2.5
5.0
1.0
2.5
5.0
1.0
2.5
% FINAL SERUM CONCENTRATION
FIG. 1. [;iH]Thymidine incorporation into human fibroblast DNA as stimulated by serum from GH-deficient patients before and after GH treatment. Serum concentrations from 1-5% are plotted on a log scale on the abscissa. Boilec samples refer to serum adjusted to pH 5.5 and boiled for 15 min. Each point represents the mean of triplicate samples the SEM is indicated by the vertical bars when these exceed the height of the symbol used to represent the point.
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Human serum (0.5 ml) was mixed with 0.5 ml 2 M acetic acid and chromatographed over Sephadex G-50 (1.5 X 90 cm) equilibrated with 1 M acetic acid. Six-milliliter fractions were collected and 1.5ml aliquots were lyophilized to dryness before mixing in E media. Samples were incubated with chick embryo fibroblasts, as described above, to determine [3H]thymidine incorporation into DNA.
JCK&M • 1978 V0U6 • No 6
HUMAN SOMATOMEDIN ACTIVITY AND FIBROBLAST GROWTH
detach from the culture dish while being grown in the pre-GH serum. In addition, we were unable to demonstrate that human fibroblasts, passaged according to the method of MacGillivray (11), detached from the dish during multiple passages in media containing 5% GH-deficient serum (data not shown). Finally, the level of thymidine incorporation into DNA and cell multiplication of skin fibroblasts from a normal volunteer were identical to the response of cells from patients with GH deficiency when exposed to pre- and post-GH human sera or a purified somatomedin-like polypeptide (MSA; unpublished results). The finding that human skin fibroblasts from patients with GH deficiency and fibroblasts from normals responded equally in the [3H]thymidine incorporation assay agrees with the report of Jones and Addison (17). Chick embryo fibroblasts
Previous studies from this laboratory demonstrated that the stimulation of thymidine incorporation into DNA and multiplication of chick embryo fibroblasts by rat serum was dependent on the GH status of the rat and, therefore, was dependent on the somatomedin activity of rat serum (12). Bovine GH was inactive in stimulating DNA synthesis in chick embryo fibroblasts when added directly to the chick embryo fibroblasts (12). As the growth of human skin fibroblasts was not dependent 20 r in a major way on the somatomedin activity of the human serum to which they were exposed, we have evaluated the growth response of chick embryo fibroblasts to the same sera. 10 As in human fibroblasts, the stimulation of 8 thymidine incorporation into DNA of chick x
8
_CJ
4
2
3 PATIENT
ratio with that serum, thereby ruling out the presence of an inhibitor in sufficient concentration in boiled pre-GH treatment serum to explain its decreased potency in the thymidine incorporation assay. Finally, the human serum components capable of stimulating thymidine incorporation into the DNA of chick embryo fibroblasts eluted from a Sephadex G-50 column equilibrated with 1 M acetic acid in the same position as that expected for the somatomedin activity of rat serum previously demonstrated by this laboratory [(18)] Fig. 5]. To demonstrate that the stimulation of [3H]thymidine incorporation into chick embryo fibroblast DNA by human sera was a
I 1
5
reflection of actual DNA synthesis, cell multiplication experiments were performed utilizing human sera boiled at pH 5.5 (Fig. 6). As anticipated from the thymidine incorporation studies, post-GH treatment serum boiled at pH 5.5 was twice as potent at 2.5% concentration as similarly treated pre-GH treatment serum. FCS at 2.5% concentration was significantly more potent than boiled human serum. Sera from two other patients, before and after GH therapy, gave similar results in stimulating the growth of chick embryo fibroblasts. These experiments confirmed the growth-promoting effect of human somatomedin activity for chick embryo fibroblasts and demon-
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o o a.
MOSES ET AL.
944 b
2
SAL
t-
[Vo]
S i. o z o
1
CORPO
o AFTER GH
1 f
z
\*
10
i > -
•^A \
I BEFOREX GH \
5
I
V , . . i-t
6
8
10
\
B ^
Q -1?
• ^ • ^ /
o
\
J V° r ^-'o 12 14 16 FRACTION NO.
18
20
22
24
FIG. 5. [:lH]Thymidine incorporation into chick embryo fibroblasts by fractions of human serum before and after GH treatment, separated on a 1.5 X 90-cm Sephadex G50 column equilibrated in 1 M acetic acid. The void volume of the column is designated by Vo. Each point represents one dish of chick embryo fibroblasts.
2 . 5 % Fetal CaM
20
m
6
Z
1
2
3 DAYS
4
5
FIG. 6. Cell multiplication of chick embryo fibroblasts exposed to boiled human serum before and after GH treatment. Each point represents the mean of triplicate dishes; the SEM is indicated by the vertical bars.
strated that the measurement of [3H]thymidine incorporation into DNA reflected actual DNA synthesis. Discussion The ability of cultured human skin fibroblasts to bind somatomedin-like polypeptides specifically (8) suggested that these cells would be a convenient tool to evaluate clinical disturbances of growth at the cellular level. The binding of homogeneous somatomedin A
1978 No 6
and multiplication-stimulating activity (MSA) and partially purified NSILA-S to a specific receptor on human fibroblasts correlates with the ability of these peptides to stimulate the incorporation of [3H]thymidine into DNA in these cells (6, 8, 19). The identification of a "growth" receptor on human fibroblasts has prompted us to evaluate whether the somatomedins present in human serum constitute a major component of the serum macromolecules required for the routine growth of these cells in culture. Human sera from GH-deficient patients, both before and after GH treatment, were equipotent in stimulating thymidine incorporation into the DNA of human fibroblasts. Boiling human sera under acid conditions, a procedure previously demonstrated to retain significant amounts of somatomedin activity (14), markedly diminished the ability of these sera to stimulate thymidine incorporation into DNA and failed to unmask a difference between pre- and post-GH treatment sera in the thymidine incorporation assay. The results of cell multiplication experiments paralleled the thymidine incorporation assays. That is, human fibroblasts multiplied to the same extent in serum from patients before and after GH therapy and did not multiply above control levels (media without serum) in these same sera boiled under acid conditions. It is possible that concentrations of boiled human sera greater than 1% would stimulate the growth of human skin fibroblasts in culture, but the results of the thymidine incorporation studies suggest that there would be no difference between sera before or after GH treatment. These results suggest that human skin fibroblasts are not dependent solely on human somatomedin activity for routine growth in culture despite the presence of a specific receptor for somatomedin-like polypeptides on the cell surface. Our observations support the report of August et al. that the generation time of human embryonic lung fibroblasts (WI-38) was independent of GH-dependent serum factors (10). The inability of the boiled extract of human serum to support the multiplication of human skin fibroblasts (Fig. 2) is consistent with the failure to stimulate multi-
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JCK & M Vol46
HUMAN SOMATOMEDIN ACTIVITY AND FIBROBLAST GROWTH
lack of dependence of DNA synthesis on somatomedin activity in whole serum is not surprising considering the variety of serum components demonstrated to have growthpromoting activity (1). The small increment of stimulatory activity in post-GH treatment sera compared to pre-GH treatment sera at very low serum concentrations (0.5% and below) may be due to differences in the shape of the dose-response curves for somatomedin compared to other mitogens in untreated human serum. Serum boiled at pH 5.5 from GHdeficient individuals before GH treatment was much less potent than whole serum in stimulating thymidine incorporation into chick embryo fibroblast DNA. This loss of activity of boiled pre-GH serum is probably secondary to the denaturation of proteins capable of stimulating DNA synthesis that are present in whole serum. On the other hand, boiled serum from the same individual after GH treatment was much more potent in stimulating thymidine incorporation into DNA and cell multiplication than the boiled pre-GH treatment serum, and approached whole serum in its activity. This activity correlated in all cases with an increase in somatomedin content in these patients' sera, as measured by the somatomedin C RIA. There also was general agreement between the chick embryo fibroblast assay and somatomedin activity, as measured by sulfate incorporation into hypophysectomized rat costal cartilage. Although patient 1 demonstrated normal somatomedin activity in the rat costal cartilage assay before GH, he had a significant increase in the activity after GH treatment. The apparent discrepancy for patient 4 between the rat costal cartilage assay on the one hand and thymidine incorporation into chick embryo fibroblast DNA or the somatomedin C RIA on the other hand, remains unexplained and requires further investigation. The potency of boiled postGH treatment sera was not due to the presence of higher concentrations of GH or insulin in the sera. Although no attempt has been made in this study to quantitate human somatomedin activity as measured by [3H]thymidine incorporation into chick embryo fibroblast DNA, this assay may be useful as an-
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plication of human skin fibroblasts with a purified somatomedin-like peptide (MSA) in the absence of serum (20). Furthermore, we have been unable to demonstrate that human skin fibroblasts detach from the dish during successive passages in 5% serum from GHdeficient patients before GH treatment. It is possible that the somatomedins require other factors in order to exert a major effect on the multiplication of human skin fibroblasts. Our experiments have not explored this possibility. In any case, the study of the interaction of purified somatomedins with receptors on cultured human skin fibroblasts may yield important information concerning both growth at the cellular level and end organ resistance to somatomedin, as expressed phenotypically by certain genetic forms of short stature. Chick embryo fibroblasts also specifically bind somatomedin-like polypeptides (9, 21). The specific receptor for somatomedin-like polypeptides on chick embryo fibroblasts has been designated a growth receptor because for MSA there is a very close correlation between the concentration of peptide required for halfmaximal displacement of radiolabeled MSA from the receptor and the concentration of peptide that causes half-maximal stimulation of thymidine incorporation into DNA (9). MSA also can stimulate the multiplication of chick embryo fibroblasts in the absence of serum; this further supports the concept of somatomedin-like peptides interacting with a growth receptor on the chick embryo fibroblast surface membrane (22). As suggested by the above findings, the present study demonstrates that chick embryo fibroblasts, in part, are dependent for growth on the somatomedin activity of the human sera to which they are exposed. This extends previous observations on chick embryo fibroblasts, utilizing normal, hypophysectomized, and bovine GH-treated hypophysectomized rat serum (12), for the first time to human sera. As in human fibroblasts, there was no difference in the level of thymidine incorporation in chick embryo fibroblasts stimulated by straight human serum before or after GH treatment at 1-5% final concentration. This
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Acknowledgments The authors wish to thank Patricia Short and Annie Bell Cooper for their skillful technical assistance, and Patricia Short and James Phang for helpful suggestions in reviewing the manuscript. We also thank Dr. Eckehart Wiedemann for the measurements of somatomedin activity utilizing the rat costal cartilage assay, Dr. Richard Furlanetto for the somatomedin C RIA, and Carla Hendricks for performing the insulin RIA.
References 1. TEMIN, H. M., R. W. PIERSON, and N. C. DULAK, The role of
serum in the control of multiplication of avian and mammalian cells in culture, In Rothblat, A., and V. J. Cristofalo (eds.), Growth, Nutrition and Metabolism of Cells in Culture, vol. 1, New York, Academic Press, 1972, p. 50.
2.
3.
4.
5.
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on fibroblasts in culture, Eur J Clin Invest 3: 119, 1973. 6. RECHLER, M. M., J. M. PODSKALNY, I. D. GOLDFINE, and C.
A. WELLS, DNA synthesis in human fibroblasts: stimulation by insulin and by non-suppressible insulin-like activity (NSILA-S), J Clin Endocrinol Metab 39: 512, 1974. 7. BASEMAN, J. B., and N. S. HAYES, Differential effect of hormones on macromolecular synthesis and mitosis in chick embryo cells, J Cell Biol 67: 492, 1975. 8. RECHLER, M. M., S. P. NISSLEY, J. M. PODSKALNY, A. C.
MOSES, and L. FRYKLUND, Identification of a receptor for somatomedin-like polypeptides in human fibroblasts, J Clin Endocrinol Metab 44: 820, 1977. 9. RECHLER, M. M., J. M. PODSKALNY, and S. P. NISSLEY,
Interaction of multiplication-stimulating activity with chick embryo fibroblasts demonstrates a growth receptor, Nature 259: 134, 1976. 10. AUGUST, G. N., R. F. CHENG, W. HUNG, and J. C. HOUCK,
Fibroblast proliferative activity in the sera of growth hormone deficient patients, Horm Metab Ren 5: 340, 1975. 11. MACGILLIVRAY, M. H., C. HASTINGS, and J. A. BROWN,
Growth hormone-dependent effects of human serum on the in vitro growth characteristics of human skin fibroblasts, J Clin Endocrinol Metab 40: 62, 1975. 12. COHEN, K. L., P. A. SHORT, and S. P. NISSLEY, Growth
hormone-dependent serum stimulation of DNA synthesis in chick embryo fibroblasts in culture, Endocrinology 96: 193, 1975. 13. DAUGHADAY, W. H., Z. LARON, A. PERTZELAN, and J. N.
HEINS, Defective sulfation factor generation: a possible etiologic link in dwarfism, Trans Assoc Am Physicians 82: 129, 1969. 14. SALMON, W. D., JR., Investigation with a partially purified preparation of serum sulfation factor: lack of specificity for cartilage sulfation, In Pecile, A., and E. Muller (eds.), Growth and Growth Hormone, Amsterdam, Excerpta Medica, 1971, p. 180. 15. WIEDEMANN, E., E. SCHWARTZ, and A. G. FRANTZ, Acute
and chronic estrogen effects upon serum somatomedin activity, growth hormone and prolactin in man. J Clin Endocrinol Metab 42: 942, 1976. 16. FURLANETTO, R. W., L. E. UNDERWOOD, J. J. VANWYK, and
A. J. D'ERCOLE, Estimation of somatomedin-C level in normals and patients with pituitary disease by radioimmunoassay, J Clin Invest 60: 648, 1977. 17. JONES, K. L., and J. ADDISON, Studies of human fibroblast growth factor, insulin, and serum, J Clin Endocrinol Metab 43: 721, 1976. 18. COHEN, K. L., and S. P. NISSLEY, Comparison of somatomedin activity in rat serum and lymph, Endocrinology 97: 654,1975. 19. RECHLER, M. M., L. FRYKLUND, S. P. NISSLEY, K. HALL, J. M. PODSKALNY, A. SKOTTNER, and A. C. MOSES, Purified
human somatomedin A and rat multiplication stimulating activity; mitogens for cultured fibroblasts that cross-react with the same growth peptide receptors, Eur J Biochem 82: SALMON, W. D., J R . , and W. H. DAUGHADAY, A hormonally 5, 1978. controlled serum factor which stimulates sulfate incorporation 20. NISSLEY, S. P., and M. M. RECHLER, Multiplication stimulatby cartilage in vitro, J Lab Clin Med 49: 825, 1957. ing activity (MSA): a somatomedin-like polypeptide from DAUGHADAY, W. H., K. HALL, M. S. RABEN, W. D. SALMON, cultured rat liver cells, Natl Cancer Inst Monogr 48: 167, J. L. VAN DEN BRANDE, and J. J. VAN WYK, Somatomedin: 1978. proposed designation for sulphation factor, Nature 235: 107, 21. ZAPF, J., M. MADER, M. WALDVOGEL, D. S. SCHALCH, and E. 1972. R. FROESCH, Specific binding of nonsuppressible insulin like DULAK, N. C, and H. M. TEMIN, A partially purified polyactivity to chicken embryo fibroblasts and to a solubilized peptide fraction from rat liver cell conditioned medium with fibroblast receptor, Arch Biochem Biophys 168: 630 1975. multiplication-stimulating activity for embryo fibroblasts, J Cell Physiol 81: 153, 1973. 22. NISSLEY, S. P., M. M. RECHLER, A. C. MOSES, P. A. SHORT, and J. M. PODSKALNY, Proinsulin binds to a growth peptide MORELL, B., and E. R. FROESCH, Fibroblasts as an experireceptor and stimulates DNA synthesis in chick embryo fimental tool in metabolic and hormone studies. II. Effects of broblasts, Endocrinology 101: 708, 1977. insulin and nonsuppressible insulin-like activity (NSILA-S)
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other bioassay for human somatomedin activity. These experiments show for the first time that human somatomedin activity is an important constituent of the serum macromolecules that support the growth of chick embryo fibroblasts in culture. Furthermore, the response of chick embryo fibroblasts to crudely purified human somatomedin activity supports recent studies that define a growth receptor on chick embryo fibroblasts that specifically binds purified somatomedins and somatomedin-like compounds (9, 21). Thus, although both human skin fibroblasts and chick embryo fibroblasts are useful tools in evaluating the interaction of purified somatomedins with a cell membrane receptor, chick embryo fibroblasts appear to be more sensitive than human fibroblasts in their growth response to the somatomedin activity of human serum.
JCK & M Vol46
