The human placental growth hormone variant is mitogenic for rat lymphoma Nb2 cells.

0013-7227/90/1262-0971$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 126, No. 2 Printed in U.S.A.

The Human Placental Growth Hormone Variant Is Mitogenic for Rat Lymphoma Nb2 Cells* BARBARA E. NICKELj, ELISSAVET KARDAMIJ, AND PETER A. CATTIN^ Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3; and St. Boniface General Hospital Research Centre, Division of Cardiovascular Science, Winnipeg, Manitoba, Canada R2H 2A6 (E.K.)

ABSTRACT. Although there is evidence that human (h) placental GH variant (hGH-V) possesses a growth-promoting function, lactogenic activity by the hormone has not been demonstrated. Rat anterior pituitary tumor (GC) cells stably transfected with the hGH-V gene (GC[hGH-V] cells) synthesize and secrete hGH-V. This hormone shares considerable structural similarity with pituitary growth hormone (hGH-N) and chorionic somatomammotropin (hCS) at the nucleotide (>90%) and amino acid (>80%) levels. As expected, both hGH-N and hCS antibodies detect hGH-V by immunoblotting. However, hGH-V, but not hGH-N or hCS, cross-reacts with human or rat pituitary prolactin (PRL) antibodies. These data indicate that structural features shared by hGH-V and pituitary PRL are not present in

hGH-N or hCS. Comparison of amino acid sequences implicates two regions that may account for a common epitope between hGH-V and hPRL, and structural difference from hGH-N and hCS. The possible lactogenic activity by hGH-V was assessed in a rat lymphoma Nb2 cell bioassay. Conditioned medium from GC [hGH-V] cells permitted growth of lactogen-dependent Nb2 lymphoma cells in culture. This activity was blocked by antibodies raised to rat PRL but not hPRL or hGH-N. Comparison of the hGH-V amino acid sequence with those from 14 other lactogenic hormones, including hPRL, hCS and hGH-N, reveals 6 conserved amino acids. These data indicate a lactogenic as well as growth-promoting function for the secreted hGH-V protein in vivo. {Endocrinology 126: 971-976, 1990)

U

thought to replace hGH-N in controlling maternal metabolism during pregnancy (5). Experiments using transgenic mice carrying the hGH-V gene indicate that hGHV has growth-promoting activity (7). There are no reports however, that the hGH-V protein has functional lactogenic activity. The rat anterior pituitary tumor (GC) cell line is a subclone of GH3 cells that is reported to express endogenous rat GH (rGH) but not PRL (rPRL). Both pituitary and placental members of the hGH gene family are expressed in GC cells after gene transfer (8-11). Previously, we have described GC [hGH-N], GC[hCS-A], and GC [hGH-V] cells, which are stable cell lines derived from GC cells that synthesize and secrete hGH-N, hCS, and hGH-V, respectively (8,9,11). In this study, we examined secreted protein from these cells using polyclonal antibodies raised to members of the GH family and tested the lactogenic activity of the secreted protein using the Nb2 lymphoma cell bioassay. The presence of a lactogen is a requirement for growth of a number of cultured cell lines established from lymphomas that arose in Nb strain rats (12). The Nb2 cell line grows at a rate proportional to the concentration of PRL present in the culture medium (13). We show here that hGH-V is lactogenic and that, although its structure is related to hGH-N and hCS, it also shares features with pituitary prolactin not found in hGH-N or hCS.

ntil recently, the human GH (hGH) family was believed to include pituitary GH (hGH-N), placental chorionic somatomammotropin (hCS), encoded by either of two genes, hCS-A and B, and prolactin (hPRL). Although two additional genes, hGH-V and hCS-L, with greater than 90% sequence similarity to the hGH-N and hCS genes, were known, no products had been identified (1). The hGH gene family is believed to have evolved by gene duplication (2). All five hGH/hCS genes are clustered together in a single locus on chromosome 17 in the following 5' to 3' order: hGH-N, hCS-L, hCS-A, hGHV, hCS-B (1). These genes share considerable structural similarity, and their mRNAs are greater than 93% homologous (3). By comparison, the hPRL gene on chromosome 6 is more divergent, with its mRNA sharing only 40% homology to those of hGH-N or hCS (2). Expression of the hGH-V gene by the syncytiotrophoblast of the placenta has now been detected (4-6). The gene product, placental growth hormone variant, is Received August 22,1989. Address all correspondence and requests for reprints to: Peter A. Cattini, Department of Physiology, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, R3E 0W3, Canada. * This work was funded by the Medical Research Council of Canada (MRCC). t Recipient of an MRCC Fellowship. X Recipient of an MRCC Scholarship.

971

The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 12:31 For personal use only. No other uses without permission. . All rights reserved.

HUMAN PLACENTAL GH VARIANT IS LACTOGENIC

972

Materials and Methods Cells and cell culture

GC cells were stably transfected with the 2.6-kilobase hGHV gene fragment (3) to generate GC[hGH-Vl cells as previously described (9,11). The production of GC[hGH-Nl and GC(hCSA] cells is described elsewhere (9, 11). Briefly, gene fragments were introduced into GC cells by calcium phosphate/DNA precipitation with cotransfection of a plasmid containing the gene for neomycin resistance (8, 11). Cells were selected in 0.5 mg/ml (active) G418 (Sigma), and pools of greater than 50 clones were grown to mass. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 2.5% fetal calf serum and 2.5% calf serum. GC[hGH-N], GC[hCS-A], GC[hGH-V], and GC cells were grown in monolayer culture to 70% to 80% confluency and washed extensively with phosphate-buffered saline, and serum-free Dulbecco's modified Eagle's medium was placed on the cells. After 24 h, the conditioned medium was removed and spun to remove any cells or debris. The conditioned medium was then used to generate samples for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or stored at -70 C for use later. For SDS-PAGE, the conditioned medium was dialysed against sterile distilled water to remove salt, lyophilized, and resuspended in sterile water. The protein concentration was determined by the method of Bradford (14). Immunoblotting Secreted protein samples of 0.1 mg from GC[hGH-N], GC[hCS-A], GC[hGH-V], and GC cells were each divided equally between four lanes (25 jig/lane) on 12.5% SDS-polyacrylamide gels. Electrophoresis, transfer of proteins to nitrocellulose, reaction with antibodies, and their detection with iodinated protein A were as described previously (15, 16). The hGH-N, hCS, and hPRL antibodies (rabbit) were obtained from Dako Corporation (California). The rPRL antibodies (rabbit) were a gift from Dr. M. Robertson in the Protein and Polypeptide Laboratories at the University of Manitoba. The hGH-N and hCS antibodies were used at a 1:5000 dilution and the hPRL and rPRL antibodies at a 1:2000 dilution. Nb2 assay Lactogenic activity in conditioned medium was determined using the Nb2 lymphoma cell bioassay (17) with the following modifications. Cells were transferred to Fishers medium with 10% horse serum, 10~4 M 2-mercaptoethanol, and antibiotics 24 h before use in a bioassay. For the assay, cells were resuspended at 1-2 x 105 cells/ml and 1 ml of cells was plated per well in 24-well dishes. The protein concentration of conditioned medium was determined, and 0.05-250 ng of protein was added in a maximum volume of 50 ^1 to the dish of cells. After 72 h, the contents of the wells (1 ml) was added to 8 ml of isoton, and the well was rinsed with an additional 1 ml to give a final volume of 10 ml. Cells were counted in duplicate (0.5 ml each) using a Coulter Counter. To test the blocking effect of antibodies, 50 n\ of a 1:100 dilution of antiserum (1:2000 final dilution) was added to the cultures simultaneously with the conditioned medium.

Endo• 1990 Vol 126 • No 2

Hydropathy plot Hydropathy profile of hGH-V was predicted by the Pustell Sequence Analysis Program (International Biotechnologies, Inc.) using the hydropathy analysis of Kyte and Doolittle (18) with a segment length of 9 amino acids.

Results Immunoreactivity of hGH- V with antibodies to hGH-N, hCS, hPRL, and rPRL Secreted proteins were prepared from conditioned medium from cultures of GC cells (untransfected controls) and from GC cells transfected with the hGH-V, hCS-A, and hGH-N genes. Samples containing 0.1 mg of total protein each were divided equally and run on four gels. Following electrophoresis, the proteins were transferred to nitrocellulose and immunostained with four different antibodies (Fig. 1). Antibodies to hGH-N reacted predominantly with a 22K protein band in samples containing hGH-V (lane b) or hGH-N, as expected (lane d). A larger protein corresponding in size to hCS (lane g) is seen in lane c. A less intense 22K protein is also seen in lanes a and c. Lane a corresponds to a sample from untransfected GC cells and reflects cross-reactivity between the hGH-N antibodies and endogenous GC cellsecreted protein, presumably rGH. Since the proportion of rGH in each of the 25-ixg samples will be greatest in that from untransfected GC cells, the increased signal observed in lanes b and d represent detection of hGH-V and hGH-N, respectively, by the hGH-N antibodies. In a similar manner, polyclonal antibodies against hCS detected hGH-V (lane f), hCS (lane g), and hGH-N (lane h). A background level of rGH is also detected by antibodies to hCS (lane e). By contrast, hPRL antibodies reacted with a protein band of 22K in only the GC [hGH-V] protein sample (lane j), even after prolonged exposure. Similarly, rPRL antibodies reacted predominantly with a 22K protein in the GC [hGH-V] sample (lane n), and to a much less extent a 22K protein in the untransfected GC cell sample (lane m). This is unlike the hPRL antibodies, which did not recognize any protein in the untransfected GC cell sample (lane i). This may indicate some cross-reactivity of the rPRL antibodies with rGH or suggest that GC cells, which are reported not to express rPRL, do release this hormone. Mitogenic activity of hGH- V for Nb2 cells Growth of Nb2 lymphoma cells in the presence of increasing concentrations of purified hPRL is shown in Fig. 2. Conditioned medium from untransfected GC cells and GC cells expressing hGH-N, hCS-A, and hGH-V was assayed for mitogenic activity in the Nb2 lymphoma


HUMAN PLACENTAL GH VARIANT IS LACTOGENIC FIG. 1. Detection of hGH-V with antibodies to members of the growth hormone family. Total secreted protein (25 /ug) from the medium of GC (lanes a, e, i, m), GC[hGH-V] (lanes b, f, j , n), GC[hCS-A] (lanes c, g, k, o), and GC[hGH-N] (lanes d, h, 1, p) cells were resolved by SDS-PAGE, transferred to nitrocellulose, and immunostained using the antibodies as indicated and iodinated protein A. Autoradiography was for 1 day except lanes i-1, which were for 4 days. The molecular weight markers are from Coomassie Blue-stained standards in the range 10-100K (Bio-Rad).

Anti-human GH-N

Anti-humanCS

973

Anti-rat PRL

Anti-human PRL

-310

-21-5

-14-4

e

f

g

h

i

j

k

I

GC(hGH-V)

i

m

n

o

p

GC(hGH-N)

o 10

23

1

3

3

SO

230

CONDITIONED MEDIUM

(ug PROTEIN)

SO

09

100

1

cr

jjj 3

I

GC(hCS-A)

GC

002 •

hPRL

(ng)

FIG. 2. Stimulation of Nb2 lymphoma cells by hPRL. Cells were incubated for 72 h with hPRL. Points are the mean values of duplicate wells.

cell system. Between 0.05 and 250 /xg of conditioned medium was assayed to ensure that the response was within the range of the standard curve obtained with purified hPRL (Fig. 3). A parallel rate of cell growth was obtained with medium from GC[hGH-V], GC[hGH-N], or GC[hCS-A] cells and purified hPRL, but not untransfected GC cells (Fig. 3). Results from two separate experiments are shown. Because the absolute quantity of hormone within each sample is not known, a direct comparison of lactogenic activity between hGH-N, hCS, and hGH-V cannot be made. However, the results show that hGH-V, like hGH-N and hCS, is mitogenic for Nb2 cells and, thus, is a lactogenic hormone. The stimulation of Nb2 cell growth with the highest level of untransfected GC cell-conditioned medium (250 jtg) indicates the presence of trace amounts of lactogen. Because rGH is not lactogenic, this suggests that some rPRL is produced by GC cells. The results in Fig. 1 suggest that PRL antibodies might neutralize the mitogenic activity of hGH-V but not that of hGH-N. To test this, the Nb2 assay was repeated in the presence of antibodies to rPRL, hPRL, or hGH-N at a final dilution of 1:2000 and with a quantity of medium

A'' SO

123

230

FIG. 3. Growth response curves of Nb2 cells to conditioned media from GC cells and GC cells stably transfected with hGH-V, hGH-N, and hCS-A in two separate experiments (•, A). The variation observed between experiments reflects the number of cells at the beginning of the assay. Points are the mean value of duplicate wells.

to give a level of lactogenic response on the standard curve (Fig. 3). Mitogenic activity seen in the presence of GC[hGH-V], GC[hCS-A], and GC cell-conditioned medium was blocked by rPRL antibodies but not by antibodies against hPRL or hGH-N (Fig. 4). By contrast, the bioactivity observed with GC [hGH-N] cell conditioned medium was blocked by antibodies raised to hGHN but not rPRL or hPRL (Fig. 4). Some blocking of hPRL activity could be seen with antibodies to hPRL, but rPRL antibodies did not block the mitogenic activity of hPRL to the same extent (Fig. 4). These data indicate that folded hGH-V and hCS share common epitopes with rPRL in a region which interferes with lactogenic activity of the hormone. Discussion Our data show that GC cells stably transfected with the hGH-V gene will synthesize and secrete hGH-V,


974


E n d o • 1990 Vol 126 • No 2

control •orPRL •ohPRL •ahGH-N

100

FIG. 4. Effect of polyclonal antibodies to rPRL, hPRL, and hGH-N diluted 1:2000 on mitogenic activity obtained with conditioned media from GC cells stably transfected with hGH-V (25 ng), hCS-A (125 Mg), hGH-N (2 ng), untransfected GC cells (250 fig), and purified hPRL (62.5 pg). Each bar represents the average of four determinations, and error from the mean is indicated.

GC UJ CD

50 UJ

O

GC(hGH-V)

which is lactogenic. As expected, hGH-V will cross-react with antibodies raised to hGH-N or hCS (Fig. 1). However, after electrophoresis and transfer to nitrocellulose, hGH-V is also detected by immunostaining with antibodies to hPRL and rPRL (Fig. 1). No cross-reactivity is observed between hGH-N or hCS protein and rPRL or hPRL antibodies under the same conditions. These data indicate that, while hGH-V resembles hGH-N and hCS, it also shares some structural homology with hPRL. A comparison of hGH-V amino acid sequence (19) with members of the human and rat GH family is shown in Fig. 5, which was been adapted from Luck et al. (20). The hGH-V protein shares 94% and 82% similarity with hGH-N and hCS, respectively, without considering conservative amino acid changes (Fig. 5). When hGH-V amino acid sequence is compared with that of hPRL and rPRL, a 42% and 38% similarity is observed, allowing for conservative amino acid changes (Fig. 5). Although short regions of hGH-V sequence can be found with greater homology to either hPRL or rPRL, as in the carboxyl terminus, the similarity is invariably shared by hGH-N and/or hCS. Thus, it is difficult to identify regions unique to hGH-V and PRL, which may be recognized by immunoblotting. However, an analysis that predicts secondary structure from primary amino acid sequence reveals some unique secondary structures as well as several that are shared by hGH-N, hCS, and PRL (21). It is possible that, in our study, hGH-V as well as hGH-N and hCS have regained part of their native

GC(hCS-A)

GC (hGH-N)

hPRL

structure during their transfer to nitrocellulose for immunostaining. Many proteins transferred from SDScontaining gels are able to renature partially or completely following removal of the SDS by methanol during transfer (22). This is a possibility in our study because the transfer buffer contained 20% methanol in 25 mM Tris/192 mM glycine and no SDS. Assuming that some renaturation occurs, we re-examined the amino acid sequences to identify any features shared by hPRL and hGH-V, but not found in hGH-N or hCS, that could give rise to a change in tertiary structure and thus result in potentially unique epitopes. Possible candidates are indicated by asterisks (*) 1 to 3 in Fig. 5. Proline and asparagine residues correlate statistically with disruption of secondary structure (23). These residues are found at positions *1 and *3 in hGHN and hCS but not in the equivalent positions of hGHV or hPRL. By contrast, position *2 identifies a disruptive proline or asparagine residue found in hPRL and hGH-V that is absent in the other genes. A hydropathy plot shows that these three changes marked by asterisks in Fig. 5 are contained within hydrophilic regions of hGH-V (Fig. 6). Hydrophilic regions are most likely to be exposed on the outside of the hormone and thus, may play a role in PRL antibody binding. The ability of hGH-V to be mitogenic for Nb2 cells is dependent on hGH-V binding to the lactogen receptor on the cell membrane (24). Thus, hGH-V shares the lactogenic property common to hPRL, hGH-N, and hCS.



FIG. 5. Amino acid sequences of rPRL, hPRL, hGH-V, hGH-N, and hCS. The numbers indicate the approximate amino acid position due to an unequal number of space insertions (:) to generate the best alignment. Similarity to the hPRL amino acid sequence is shown by uppercase characters, which indicate identical amino acids or conserved changes. The plus (+) or minus (-) signs indicate residues conserved in 14 lactogenic hormones (20). The asterisks (*) indicate residues common to hGH-V and hPRL that are distinct from those in hGH-N and might give rise to a structural difference. Standard abbreviations for the amino acids are shown. Conserved amino acid changes are as follows; acidic, D or E; basic, K or R; aromatic, F or Y; hydrophobic, I=L=M=V or T=S.

rPRL - LPVCsGG::d CQtpLpELFD R:VVaL:SHY IHtLyTDMFi hPRL - LPICPGGAAR CQVTLROLFD RAW: L: SHY IHNLSSEMFS hGH-V f ptlpLsrpFD nAM::Lrarr LyqLayOtYq hGH-N f ptlpLsrpFD nAN::LraHr LHqLayDtYq hCS-A v qtVpLsrlFD HAN::LqaHr aHqLqiDtYq

rPRL hPRL hGH-V hGH-N hCS-A

-

• INd:CpTSSL INS:CHTSSL qtSlCfSeSI qtSlCfSeSI qtSfCfSdSI

+ ATPEDKEQAQ ATPEDKEQAQ pTPsnRvktQ pTPsnREetQ pTPsnaEetQ

90 kVppeviL:: QMNQKDFL:: QksnlEILri QksnlElLri QksnlElLH


-

•f IISRAkEIEE I1SKAVEIEE sdSn:Vyrhl sdSn:Vydll sdSd:dyhll

QnKRLLEGIE QTKRLLEGNE ::KdLeEGIq ::KdLeEGIq ::KdLeEGIq

150 kllSQayPEa LIVSQVHPET tLMwrLedgS tLMgrLedgS tLMgrLedgS


-

YNnlrCLRRD YNLLHCLRRD rgLLyCfRKD :gLLyCfRKD zgLLyCfRKD

SHKVDNYLKf SHKIDNYLKL •dKVEtFLRI •dKVEtFLRI •dKVEtFLRM

210 LRCqIVHkNN LKCRIIHNNN VqCRsVEGS: VqCRsVEGS: VqCRsVEGS:

975

EFDKqYvqdR EFDKRYTHGR EFEeaY11:K EFEeaYip:K EFEetY1p:K •1

nLILSLVhSU SLIVSILRSU SLLL:I:qSU SLLL:I:qSU SLLL:I:eSW

NOPLFqLITg NEPLYHLVTE lEPVqUrS: lEPVqfLrS: lEPVrfLrS:

:KgNEIY1:V :KENEIYP:V prtgqIFnqs prtgqIFkqt rrtgqlikqt •2

WSqlPSLQgv WSGLPSLQMA ySkfdTkshn ySkfdTnshn ySkfdTnshn •3

e::::FIaKA G::::FITKA eqkysFLqnp eqkysFLqnp dqkysFLhds 120 LgGIhEAPDA VRGHQEAPEA VfansivygA Vfans?vygA MfonnlvyDt 180 DEESKDL:AF OEESR:LSAY DD::a1LknY D0::a1LknY hD::a1LknY

C C CGF CGF CGF

3r-

FiG. 6. Hydropathy profile of hGH-V. Positive and negative hydropathy scores indicate that residues are hydrophilic and exterior (below zero) or hydrophobic and interior (above zero), respectively. The arrows indicate the positions of L37, N140, and K149, which correspond to the amino acids marked by asterisks in Fig. 5 that may generate structural differences between hGH-V and hGH-N.

50

Recently, the amino acid sequences of 14 lactogenic hormones were compared (20). A total of 32 amino acids was conserved between these proteins. However, 25 of these residues were also present in the nonlactogenic bovine GH molecule. This leaves 7 amino acids that are unique to lactogenic hormones and are marked by plus (+) and minus (—) signs in Fig. 5. Six of the 7 amino acids are preserved in hGH-V (+), providing further evidence of their importance, but a glutamic acid residue is not maintained (—). Site-directed mutagenesis of bovine PRL by Luck et al. (20) indicates that it is tertiary

100 SEQUENCE NUMBER

structure, and not the simple inclusion of these amino acids, that is important for the binding and lactogenic activity of the hormone. The rPRL antibodies presumably block the mitogenic activity of GC[hGH-V] andGC[hCS-A] cell-conditioned medium (Fig. 4) by interfering with binding of the hGHV and hCS proteins to the Nb2 cell surface receptor (24). It is unlikely that this epitope is seen by immunoblotting because hCS is not detected by rPRL antibodies (Fig. 1, lane o). Interestingly, hGH-N was unaffected by rPRL antibodies, suggesting a structural difference in this func-


976


tionally important region needed for receptor binding. The hPRL polyclonal antibodies had no effect on hGHV, hGH-N, or hCS lactogenic activity, but these antibodies only partially blocked growth in the presence of purified hPRL (Fig. 4). Thus, it is possible that one population of hPRL antibodies interferes with the binding of a second that, in the absence of the first, would block the interaction between hormone and receptor. However, hPRL antibodies did detect hGH-V by immunoblotting (Fig. 1). These data suggest that the epitope(s) recognized following immunoblotting are absent from the native structure or do not interfere with binding to the lactogenic receptor and its subsequent function. Thus, antibodies may bind to hormone (hGH-V, hGHN, or hCS), but this interaction is not detected in the Nb2 assay unless it interferes with the binding to the lactogen receptor. The ability of GC cell medium to induce some mitogenic activity at very high concentrations, and the neutralization of this activity with rPRL antibodies (Fig. 4) indicate that GC cells do produce low levels of rPRL. It is possible that hCS may represent a hormone unique to humans (1). However, it is more often considered the equivalent of placental lactogen from nonprimate mammals. In contrast to hCS, placental lactogens are structurally more related to PRL than GH (1). The results of this study indicate that both hGH-V and hCS share structural similarities with PRL. This raises the intriguing question of whether hGH-V or hCS, or even both, are equivalent to placental lactogen in nonprimates. Acknowledgments We are indebted to Drs. F. Croze for Nb2 cells, M. Robertson for rat prolactin antiserum, and H. Cosby for purified human prolactin. We thank M.E. Klassen for technical assistance.

References 1. Eberhardt NL, Hirt H, Cattini PA, Anderson TR, Peritz L, Baxter JD, Mellon P, Isaacs R, Slater EP, Barta A 1988 Human growth hormone genes: Structure evolution, expression and hormonal regulation. In: Lau Y-F (ed) Endocrine Genes. Oxford University Press, New York. 2. Miller WL, Eberhardt NL 1983 Structure and evolution of the growth hormone gene family. Endocr Rev 4:97 3. Chen EY, Liao Y-C, Smith DH, Barrera-Saldana HA, Gelinas RE, Seeburg PH 1989 The growth hormone locus: Nucleotide sequence, biology and evolution. Genomics 4:479 4. Cooke NE, Ray J, Emery JG, Liebhaber SA 1988 Two distinct species of human growth hormone variant mRNA in the human

Endo • 1990 Vol 126 • No 2

placenta predict the expression of novel growth hormone proteins. J Biol Chem 263:9001 5. Frankenne F, Closset J, Scippo ML, Smal J, Hennen G 1988 The physiology of growth hormones (GHs) in pregnant women and partial characterization of the placental GH variant. J Clin Endocrinol Metab 66:1171 6. Liebhaber SA, Urbanek M, Ray J, Tuan RS, Cooke NE 1989 Characterization and histologic localization of human growth hormone-variant gene expression in the placenta. J Clin Invest 83:1985 7. Selden RF, Wagner TE, Blethen S, Yun JS, Rowe ME, Goodman HM 1988 Expression of the human growth hormone variant gene in cultured fibroblasts and transgenic mice. Proc Natl Acad Sci USA 85:8241 8. Cattini PA, Anderson TR, Mellon P, Baxter JD, Eberhardt NL 1986 The human growth hormone gene is negatively regulated by triiodothyronine when transfected into rat pituitary tumor cells. J Biol Chem 261:13367 9. Cattini, PA, Klassen ME, Nachtigal MW 1988 Regulation of human chorionic somatomammotropin gene expression in rat pituitary tumour cells. Mol Cell Endocrinol 60:217 10. Cattini PA, Eberhardt NL 1987 Regulated expression of chimaeric genes containing the 5'-flanking regions of human growth hormone related genes in transiently transfected rat pituitary tumor cells. Nucleic Acids Res 15:1297 11. Nickel BE, Kardami E, Cattini PA Differential expression of human placental growth hormone variant and chorionic somatomammotropin culture. Biochem J, in press. Program of the 71st Annual Meeting of the Endocrine Society, Seattle, 1989, p 36 (Abstract) 12. Gout PW, Noble RL, Beer CT 1986 Cultured Nb rat lymphoma cells in endocrine and cancer research. Biochem Cell Biol 64:659 13. Gout PW, Beer CT, Noble RL 1980 Prolactin-stimulated growth of cell cultures established from malignant Nb rat lymphomas. Cancer Res 40:2433 14. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248 15. Cattini PA, Kardami E, Eberhardt NL 1988 Effect of butyrate on thyroid-mediated gene expression in rat pituitary tumour cells. Mol Cell Endocrinol 56:263 16. Kardami E, Fandrich RR 1989 Basic fibroblast growth factor in atria and ventricles of the mammalian heart. J Cell Biol 109:1865 17. Tanaka T, Shiu RPC, Gout PW, Beer CT, Noble RL, Friesen HG 1980 A new sensitive and specific bioassay for lactogenic hormones: Measurement of prolactin and growth hormone in human sera. J Clin Endocrinol Metab 51:1058 18. Kyte J, Doolittle RF 1982 A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105 19. Seeburg PH 1982 The human growth hormone gene family: Nucleotide sequences show recent divergence and predict a new polypeptide hormone. DNA (NY) 1:239 20. Luck DN, Gout PW, Beer CT, Smith M 1989 Bioactive recombinant methionyl bovine prolactin: Structure function studies using site-directed mutagenesis. Mol Endocrinol 3:822 21. Nicoll CS, Mayer GL, Russel SM 1986 Structural features of prolactins and growth hormones that can be related to their biological properties. Endocr Rev 7:169 22. Bers G, Garfin D 1985 Protein and nucleic acid blotting and immunochemical detection. BioTechniques 3:276 23. Stryer L 1988 Molecular Design on Life, ed 1. WH Freeman Co, New York, p 37 24. Shiu RPC, Elsholtz HP, Tanaka T, Friesen HG, Gout PW, Beer CT, Noble RL 1983 Receptor-mediated mitogenic action of prolactin in a rat lymphoma cell line. Endocrinology 113:159


Binding of human growth hormone (GH)-variant (placental GH) to GH-binding proteins in human plasma.

Evidence for a rapid stimulation of tyrosine kinase activity by prolactin in Nb2 rat lymphoma cells.

Differential expression of human placental growth-hormone variant and chorionic somatomammotropin in culture.

Cross-linking of G-proteins to the prolactin receptor in rat NB2 lymphoma cells.

Identification of placental human growth hormone as the growth hormone-V gene expression product.

Interaction of human growth hormone with isolated rat liver cells.

Comparison of Nb2 lymphoma cell bioassay with immunoassay for human prolactin: the role of estrogen.

Human growth hormone variant produces insulin-like and lipolytic responses in rat adipose tissue.

The 20,000 Da variant of human growth hormone does not bind to growth hormone receptors in human liver.

Human growth hormone-releasing hormone analogues with much improved in vitro growth hormone-releasing potencies in rat pituitary cells.

Human growth hormone-variant is a biologically active somatogen and lactogen.

The role of G-proteins in the mitogenesis of rat lactogen-dependent and lactogen-independent Nb2 lymphoma cells.

Tissue-specific expression and thyroid hormone regulation of the endogenous placental growth hormone variant and chorionic somatomammotropin genes in a human choriocarcinoma cell line.

Effects of bovine growth hormone, human placental lactogen and ovine prolactin on intestinal fluid and ion transport in the rat.

Interleukin-7 is a growth factor for Sézary lymphoma cells.

Growth hormone (GH) receptors in clonal osteoblast-like cells mediate a mitogenic response to GH.

Mitogenic signaling pathways in the liver of growth hormone (GH)-overexpressing mice during the growth period.

Platelet-derived growth factor is mitogenic for O-2Aadult progenitor cells.

Proliferation of Nb2 lymphoma cells in vitro in response to interleukin-7.

Mitogenic response of rat Schwann cells to fibroblast growth factors is potentiated by increased intracellular cyclic AMP levels.

Introduction of exogenous growth hormone receptors augments growth hormone-responsive insulin biosynthesis in rat insulinoma cells.

Adenylate cyclase permanently uncoupled from hormone receptors in a novel variant of S49 mouse lymphoma cells.

Human placental lactogenic hormone as a parameter for placental function in renal transplanted women.

Multiple Effects of Growth Hormone in the Body: Is it Really the Hormone for Growth?