Stem Cells, Vol. 15, No. 5, 364-367,
September 1997
© 1997 AlphaMed Press
Human Growth Hormone Antagonist (G120R) Delivered by a Murine Yolk Sac Cell-Derived Mini-Organ Decreases the Growth Rate of Mice
Yanzhang Wei,
Jinhua Li,
Wen Y. Chen,
Thomas E. Wagner
Edison Biotechnology Institute, Molecular and Cellular Biology Program and Department of Biological Sciences of Ohio University, Athens, Ohio, USA
Key Words. Mini-organ • G120R • Yolk sac cell • IGF-1 • Mice • Gene delivery
Dr. Thomas E. Wagner, Edison Biotechnology Institute of Ohio University, Athens, OH 45701, USA.
 |
Abstract
|
|---|
Long-term cultured murine embryonic yolk sac cells that are capable of forming capillary structures when cultured on base membrane proteins (Matrigel) were successfully transfected with a human growth hormone antagonist (G120R) gene. Cells that stably express relatively high levels of G120R were co-implanted s.c. with Matrigel into BALB/c mice. G120R can be detected in the sera of those implanted mice for more than 14 days at levels from 4 ng/ml to 28 ng/ml. The insulin-like growth factor-1 levels in the sera of those implanted mice were significantly affected by the delivered G120R. One of the physiological effects of G120R delivered by this murine embryonic yolk sac cell-derived mini-organ system is to decrease the growth rate of the implanted mice. This gene delivery system can also be used as an alternative to transgenic animals to study protein function in vivo.
|
Introduction
|
|---|
Recently, we have established a systemic gene delivery system by mixing cultured embryonic yolk sac cells with Matrigel and implanting it s.c. as a mini-organ [1]. The reporter gene product, human growth hormone (HuGH), can be detected at a physiological level for more than seven months in syngeneic mice [1] and more than ten months in allogeneic mice [2]. HuGH delivered by this system showed biological function [1].
A mutant HuGH (G120R) has been recently generated by a single amino acid substitution in the third 
-helix. Instead of growth promotion, this mutant HuGH functions as a HuGH antagonist both in vitro [3] and in vivo [4, 5]. This HuGH antagonist is a potential protein drug for diseases such as acromegalies, diabetic retinopathy and nephropathy. It can also be used to prevent tumor cell proliferation.
In this study, we tested the possibility of using our recently established embryonic yolk sac cell-derived mini-organ gene delivery system to deliver the G120R and observe its physiological effects. Cultured murine yolk sac cells that stably express G120R were transplanted s.c. with Matrigel into BALB/c mice. The results presented in this study confirmed the HuGH antagonist function of G120R at physiological levels. It provided further evidence for using this gene delivery system as a quick and efficient way of studying therapeutic proteins in vivo.
|
Materials and methods
|
|---|
Cells and Animals
Cells used in this study were long-term cultured murine yolk sac cells, YS4 [1]. Cells were cultured in -minimal essential medium (-MEM) containing 18% fetal bovine serum (Hyclone; Logan, UT), 10% leukemia inhibitory factor conditioned medium, 50 µg/ml gentamicin and 0.2 mM of ß-mercaptoethanol at 37°C in a fully humidified atmosphere of 5% CO2. The culture wares were coated with 0.1% gelatin at least 20 min before cell inoculation. Adult mice (BALB/c) used in this study were purchased from Jackson Laboratory (Bar Harbor, ME), maintained in our facilities and used at 8 to 10 weeks of age.
Plasmids and Transfection
Our previous work showed that the cytomegalovirus (CMV) immediate early promoter functioned well in murine yolk sac cells [1, 2]. The BglII/SacI fragment within the wild type HuGH gene sequence in plasmid pCMV-HuGH was replaced with the BglII/SacI fragment containing the mutations from plasmid pIG-Mt-HuGH120R. The resultant plasmid (pCMVHuGH-G120R) is driven by the CMV promoter and terminated by a bovine growth hormone poly A signal.
Wizard® Maxipreps (Promega; Madison, WI) purified pCMV-HuGH-G120R plasmids were used to transfect YS4 cells by lipofection. Twenty-four h before transfection 2 x 105 YS4 cells were seeded in each well of a six-well plate. One hundred µl -MEM containing 6 µg of plasmid DNA were mixed with 80 µl -MEM containing 20 µl Lipofectamine® (GIBCO BRL, Inc.; Gaithersburg, MD) solution, incubated at 37°C for 30 min, and then 800 µl -MEM were added to make 1 ml of transfection solution. The cell monolayer was washed twice with phosphate-buffered saline and covered with 1 ml transfection solution. The transfection reaction was performed at 37°C for two h. After that, the cells were washed twice with phosphate-buffered saline and once with normal medium and fed with normal medium. Forty-eight h later, the cells were trypsinized, transferred into a 100 mm petri dish and fed with -MEM containing G418 (400 µg/ml). The medium containing G418 was changed every other day until resistant colonies appeared. Single colonies were picked up and expanded. The G120R production by individual clones was measured by radioimmunoassay (RIA) using Nichols' HuGH RIA kit (Nichols Institute Diagnostics; San Juan Capistrano, CA). The clone that expressed the highest level of G120R, named GHAYS1, was chosen for the following implantation experiment.
Implantation
Both GHAYS1 and YS4 cells were grown to 95% confluence, trypsinized, and the single cell suspension was centrifuged at 200 g, 4°C for five min. The cell pellet was directly suspended with liquid Matrigel (Becton-Dickinson; Bedford, MA). The cell-Matrigel suspension was kept on ice prior to injection. Adult BALB/c mice were injected s.c. with 5 x 106 cells (either YS4 or GHAYS1). After implantation, serum samples were collected through tails for G120R assay and insulin-like growth factor-1 (IGF-1) assay. In order to test biological function of G120R delivered by this system, newborn BALB/c mice were each injected i.p. with 2 x 106 cells in 0.2 ml of Matrigel within the first 24 h after birth using a 1 ml syringe with a 27 gauge needle. The tip of the tails of those mice that had been injected with YS4 cells (control) was cut in order to distinguish them from those injected with GHAYS1 cells. At the age of two weeks, each injected mouse was reinjected s.c. with 5 x 106 cells (either YS4 or GHAYS1, respectively) in 0.5 ml of Matrigel at two sites under the abdomen. The body weights of the mice were monitored weekly.
Mutant HuGH RIA
The G120R levels of either medium samples from G418 resistant clones or serum samples from implanted mice were measured by Nichols' HuGH RIA kit (Nichols Institute Diagnostics) according to the procedures provided by the manufacturer.
IGF-1 RIA
Serum IGF-1 levels of implanted mice were determined using a heterologous RIA kit (Nichols Institute Diagnostics) after acid-ethanol extraction following manufacturer's recommendation.
|
Results
|
|---|
Stable Cell Lines Expressing G120R
After transfection and G418 selection, more than 50 G418 resistant clones were obtained. RIA results showed that all of the clones expressed G120R at levels around 500 ng/1 x 106 cells in 24 h. One of the clones (clone #1) that gave the highest G120R expression (650 ng/1 x 106 cells in 24 h) was named GHAYS1 and was chosen for a later implantation experiment.
G120R in Implanted Mice
After implantation, serum samples were collected from all injected mice. Figure 1 shows the G120R levels in those serum samples detected by RIA. Twenty-four h after implantation, all of the GHAYS1 cell-implanted mice expressed G120R at the level of 29.76 ± 1.63 ng/ml serum. At day 3 the average G120R level dropped to 22.31 ± 3.78 ng/ml serum. At day 7 the average G120R level was 8.95 ± 0.52 ng/ml serum. At day 10 and day 14 the average G120R levels were 4.47 ± 0.77 ng/ml serum and 4.38 ± 1.43 ng/ml serum, respectively. During this period of time no G120R was detected in the serum samples from YS4 cell-implanted mice.
View larger version (14K):
[in this window]
[in a new window]
|
Figure 1. G120R expression in implanted BALB/c mice. G120R levels in the sera of both GHAYS1 (n = 8) and YS4 (n = 4) implanted mice were measured by HuGH RIA. The data are presented in the form of mean ± SE.
|
|
Serum IGF-1 Levels in Implanted Mice
The IGF-1 levels in this study are presented as the ratio of the specific IGF-1 value to the starting IGF-1 value (or day 0 IGF-1 value) for a particular mouse. The average IGF-1 ratio for YS4 implanted mice was from 97.75 ± 2.08 to 104 ± 3.52 during the experimental period ( Fig. 2). Instead of decreasing as expected, the average IGF-1 ratio for GHAYS1 implanted mice at day 1 was increased to 123.125 ± 3.06, which is significantly higher than that of the control (p < 0.001). However, at day 10 the mean IGF-1 levels dropped to 81.875 ± 2.69, which is significantly lower than that of control (p < 0.01). The IGF-1 levels then gradually returned to normal at day 21.
View larger version (17K):
[in this window]
[in a new window]
|
Figure 2. IGF-1 levels in the sera of both GHAYS1 (n = 8) and YS4 (n = 4) implanted mice. Since the IGF-1 levels are dramatically different from individual to individual, the IGF-1 levels in this study are presented as the ratio of the specific IGF-1 value to the starting IGF-1 value for a particular mouse (or day 0 IGF-1 value). For example, if the IGF-1 value of an individual mouse is 600 ng/ml serum at day 0, i.e., before implantation, and the IGF-1 value of the same mouse is 400 ng/ml serum at day 3 after implantation, the IGF-1 ratio for this mouse at day 3 will be 400/600 x 100 = 66.7%. The data are presented as mean ± SE.
|
|
Growth Rate of Implanted Mice
For both males and females the body weights were not significantly different between YS4 implanted mice and GHAYS1 implanted mice until the mice reached the age of 39 days. At day 39 the average body weight of YS4-implanted male mice was 23.94 ± 1.87 g, while those of GHAYS1-implanted male mice was 16.05 ± 0.81 g (p < 0.001, as determined by Student's t test [ Fig. 3A]). Also, the mean body weight of YS4-implanted females was 20.77 ± 0.89 g, while those of GHAYS1 implanted females was 17.83 ± 1.30 g (p < 0.01). ( Fig. 3B).
View larger version (28K):
[in this window]
[in a new window]
|
Figure 3. Growth rate comparison between GHAYS1-implanted mice and YS4-implanted mice. At the age of less than one day, the mice were randomly selected and implanted with either GHAYS1 cells or YS4 cells suspended with liquid Matrigel. Since G120R can only be detected in the implanted mice for 14 days (as Fig. 1 shows), those mice were reinjected with cell-Matrigel suspension accordingly at the age of 14 days. The data were analyzed by Student's t test and presented by mean ± SD for both males (A) and females (B).
|
|
|
Discussion
|
|---|
In the present study, we successfully transfected murine yolk sac cell line YS4 with a vector containing G120R gene and the stable cell lines obtained after G418 selection expressed relatively high levels of G120R. One of the lines, named GHAYS1, expressed G120R at the level of 650 ng/1 x 106 cells in 24 h. Therefore, this cell line was used in the implantation experiments.
Twenty-four h after implantation s.c. of GHAYS1 cells in Matrigel in BALB/c mice, G120R was detected in the serum of implanted mice at the level of an average of 29.76 ng/ml serum. This result is comparable to that of wild type HuGH expression in our previous work [1]; however, this expression lasted for only 14 days. One of the reasons why the length of expression was shortened may be because of the mutation of HuGH. The mutation generated on the third -helix on the HuGH gene might render this protein (G120R) more immunogenic to the recipients than wild type HuGH. This explanation is supported by the presence of G120R expressing cells within the vascular network of the implant at day 21, when all of the eight mice no longer showed G120R reactive to the RIA (data not shown). This may be the result of high levels of neutralizing antibody against G120R in these mice. By contrast, our previous work showed that wild type HuGH did not stimulate a strong immune response [1].
The results in the present study clearly indicated that IGF-1 production in the recipient mice was affected by G120R delivered by the mini-organ although it was expressed for only two weeks. Chen et al. demonstrated that G120R functions as a HuGH antagonist and decreases the IGF-1 production in transgenic mice [3]. In the present study, however, the IGF-1 level, instead of being decreased as we expected, was increased in all of the GHAYS1-implanted mice in the first three days and, at the highest point (day 1), was significantly different from that of the control (p < 0.001). Then, decreasing gradually, at day 10 it reached the lowest point which was also significantly different from that of the control. The IGF-1 production in G120R-producing mice showed a clear biphase pattern. The explanation for this biphase phenomenon might be very complicated. First, the results of Chen et al. were obtained from transgenic mice in which G120R was expressed at the very beginning of the embryogenesis and at very high levels (from 0.05 to 5 µg/ml serum) [3]. Therefore, it is impossible to investigate what happened at the very beginning of the embryogenesis when G120R was first expressed. Second, a very recent study by Mode et al. [6] showed that G120R, when introduced into rats by i.v. infusion at a physiological level, worked as a partial agonist and stimulated growth of Hx rats. Their explanation for this is that G120R can bind to prolactin receptors on liver cells as a partial agonist. Because their infusion experiment ended in six days, and because their work was on rats, not mice, they could not see the prolonged effect of the G120R treatment. On the other hand, Murphy et al. [7] noted that much less stimulation of growth or serum IGF-1 was seen after more prolonged treatment of the prolactin receptor. Finally, transgenic mice expressing GH antagonist tend to show increased hepatic GH receptor expression, no matter whether the accompanying phenotype is of increased, normal or decreased growth [4]. More work needs to be done to explain this biphasic production of IGF-1 induced by HuGH antagonist at physiological levels.
Although the production of IGF-1 induced by G120R, delivered by the mini-organ gene delivery system, showed a biphasic pattern, the resulting growth rates in neonatal mice indicate a phenotype of decreased growth rates ( Figs. 3A and 3B).
In conclusion, the present study demonstrated that: A) protein products other than wild type HuGH can be delivered by this cultured murine yolk sac cell mini-organ system at physiological levels; B) IGF-1 production induced by G120R at physiological levels showed a biphasic pattern, and C) G120R delivered by our gene delivery system decreased the growth rate of the treated mice.
|
Acknowledgments
|
|---|
This research was funded by the Ohio Department of Development's Thomas Edison program, a grant from the National Institute of Allergy and Infectious Disease (5-RO1-AI33280) and by a research contract sponsored by Progenitor, Inc.
|
References
|
|---|
-
Wei YZ, Quertermous T, Wagner TE. Directed endothelial differentiation of cultured embryonic yolk sac cells in vivo provides a novel cell-based system for gene therapy. STEM CELLS 1995;13:541-547.[Abstract]
-
Wei YZ, Li JH, Wagner TE. Long-term expression of human growth hormone (hGH) in mice containing allogeneic yolk sac cell derived neovascular implants expressing hGH. STEM CELLS 1996;14:232-238.[Abstract]
-
Chen NY, Chen WY, Bellush L et al. Effects of streptozotocin treatment in growth hormone (GH) and GH antagonist transgenic mice. Endocrinology 1995;136:660-667.[Abstract]
-
Chen WY, Chen NY, Yun J et al. In vitro and in vivo studies of antagonistic effects of human growth hormone analogs. J Biol Chem 1994;269:15892-15897.[Abstract/Free Full Text]
-
Chen WY, White ME, Wagner TE et al. Functional antagonism between endogenous mouse growth-hormone (GH) and a GH analog results in dwarf transgenic mice. Endocrinology 1991;129:1402-1408.[Abstract]
-
Mode A, Tollet P, Wells T et al. The human growth hormone (hGH) antagonistG120R hGH does not antagonize GH in the rat, but has paradoxical agonist activity, probably via the prolactin receptors. Endocrinology 1996;137:447-454.[Abstract]
-
Murphy LJ, Tachibana K, Friesen HG. Stimulation of hepatic insulin-like growth factor-I gene expression by ovine prolactin: evidence for intrinsic somatogenic activity. Endocrinology 1988;122:2027-2033.[Abstract]
accepted for publication June 4, 1997.
Top
Abstract
Introduction
