0021-972X/90/7002-0810$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society
Vol. 70, No. 3 Printed in U.S.A.
Comments
T-Lymphoblast Cell Lines from Laron Dwarfs Augment Basal Colony Formation in Response to Extremely High Concentrations of Growth Hormone* MITCHELL E. GEFFNER, NOELLE BERSCH, BARBARA M. LIPPE, DAVID W. GOLDE Departments of Pediatrics (M.E.G., B.M.L.) and Medicine (N.B., D.W.G.), UCLA Medical Center, Los Angeles, California 90024
ABSTRACT. The clinical entity of Laron dwarfism is characterized by resistance to both endogenous and exogenous GH and may be due to a deficiency or absence of functional GH receptors. We previously showed that two types of hematopoietic cells derived from these patients are resistant to the in vitro growth-promoting action of GH at concentrations below 500 n%/ L. In the current study we found that Laron T-cell lines had a mean peak augmentation of basal colony formation of 22 ± 3.4% above baseline in response to a GH concentration of 10,000 ng/ L. Since cloned cDNAs for human and rabbit GH receptors and rat PRL receptors show a high degree of sequence homology, we undertook studies of PRL action in cells from patients with Laron dwarfism to determine if the Laron defect was also associated with PRL unresponsiveness. Quantitating the aug-
T
HE CLINICAL entity of Laron dwarfism appears to result from GH resistance due to a deficiency or absence of functional GH receptors (1). This concept is based in part on studies of GH binding to hepatic tissue from two affected individuals (2). Using erythroid progenitors and human T-cell leukemia virus (HTLV-II) transformed T-lymphoblasts derived from peripheral blood, we previously documented the presence of GH resistance in cells from two patients with Laron dwarfism based on lack of GH-induced augmentation of basal colony formation at concentrations of GH up to 500 ng/ L (3, 4). Because of the known structural homology between both GH and PRL (5) and between GH and PRL receptors (6, 7), we examined the growth responses of Laron dwarf T-cell lines to a broad concentration range of PRL and GH to determine the specificity of the resistance to GH.
Subjects and Methods In these studies we used HTLV-II-transformed T-lymphoblast cell lines from three Laron dwarfs. The clinical characReceived August 23, 1989. Address requests for reprints to: Mitchell E. Geffner, M.D., Department of Pediatrics, University of California Medical Center, Los Angeles, California 90024. * This work was supported in part by Grants CA-32737 and CA30388 from the NIH.
mentation of colony formation by T-lymphoblast cell lines established from three Laron dwarfs, we found normal responsiveness to PRL at concentrations of 25-10,000 Mg/L. It is, thus, possible that the responsiveness of Laron T-cell lines to very high concentrations of GH could be mediated through an intact PRL (or other lactogenic) receptor based on the known affinity of GH for these receptors in other systems. These data suggest that cells from patients with Laron dwarfism have normal in vitro responsiveness to PRL and that the defect in Laron dwarfism appears to be specific to the GH receptor-effector pathway. It remains to be determined whether intact alternative lactogenic receptor mechanisms subserve any clinical effects of GH in patients with Laron dwarfism. (J Clin Endocrinol Metab 70: 810,1990)
teristics and diagnostic serum GH levels of the patients have been reported previously (3, 4, 8, 9). Patient 3 had a serum PRL concentration of 1.5 ixg/L at age 17 yr (normal range, 318 Mg/L); the PRL level was not measured in the other two patients. In vitro GH studies were also performed using six Tcell lines obtained from normal individuals (two transformed by HTLV-I and four by HTLV-II). For the PRL studies, six normal HTLV-II-transformed T-cell lines were used (including four employed for the GH studies and two others). All studies were performed with the informed consent of the patient and/ or parents and with the approval of the UCLA Human Subject Protection Committee. The methodology for HTLV-I and -II transformation of peripheral blood lymphocytes has been described previously (10, 11). From 10 mL peripheral blood, low density mononuclear cells (5 x 105), obtained by Ficoll-Hypaque density gradient separation, were cocultivated with an equal number of lethally irradiated (12,000 rad) late passage Mo cells (HTLVII) or Me cells (HTLV-I) in Iscove's medium supplemented with 20% fetal bovine serum and interleukin-2. The Mo T-cell line was derived from the spleen of a patient with a variant of hairy cell leukemia (12). The HTLV-I-infected T-cell line (Me) was established from the peripheral blood of a patient with adult T-cell leukemia (13). In either case, a virally infected, immortalized T-cell line is produced in about 4 weeks. Cell lines were fed 2 days before clonogenic studies to ensure that all experiments were conducted with cells in an exponential
810 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 October 2016. at 12:24 For personal use only. No other uses without permission. . All rights reserved.
COMMENTS growth phase. Fifty thousand transformed T-cells/mL (5 x 103/ well) were cultured in microtiter plates in 20% fetal bovine serum without the addition of a-thioglycerol. Ten microliters of either highly purified pituitary or recombinant [either methionine or N085AV nonmethionine (Genentech, Inc., South San Francisco, CA)], human GH in final concentrations of 25, 50,100, 250, 500,1,000, 2,500, 5,000,10,000, 25,000, 50,000, and 100,000 Mg/L; human PRL [hPRL-(l-7); obtained from the NIDDK], or recombinant PRL (kindly provided by Genentech, Inc.)] in final concentrations of 25, 50, 100, 250, 500, 1,000, 2,500,5,000, and 10,000 Mg/L; or phosphate-buffered saline (pH 7.4) was added to each well. All experiments were performed in triplicate, with a maximal replicate variability of less than 5%. After 7-10 days, colonies containing a minimum of eight cells were enumerated using an inverted microscope. The unstimulated number of colonies formed in incubation mixtures without added hormone is defined as 100%. The data are presented as the mean ± SE.
811 200 Controls
180
160
140
120
100
80
Results Since there were no significant differences in GH responsiveness of T-cell lines transformed by either HTLV-I or -II (4) (our unpublished observations), the resultant GH response data were pooled. Similarly, since there was no difference between the degree of colony augmentation with either highly purified pituitary (used in nine control and six Laron dwarf studies) or recombinant GH (methionine-GH used in one normal and one Laron study and N085AV GH used in three normal studies) or the NIDDK (used in six normal and four Laron studies) or recombinant (used in two normal and three Laron studies) PRL, the appropriate response data were combined. In normal subjects (mean of 13 studies of 6 cell lines), the mean peak response to GH concentrations of 500 /ug/ L or less was 59 ± 3.8% above baseline at 100 Mg/L. In 6 GH studies of 3 normal T-cell lines, we extended the studies of clonal responsiveness to GH concentrations as high as 100,000 /ug/L (Fig. 1). After the first peak, there was a partial diminution of responsiveness at 500 /ug/L GH, followed by a mean second peak response of 60 ± 8.0% above baseline at 10,000 /ug/L GH. All three Laron dwarf T-cell lines (mean of seven studies) were resistant to the stimulatory effects of GH at concentrations of 500 /ug/L or less. Four studies of the Laron T-cell lines were extended to GH concentrations as high as 100,000 /ug/L. The mean peak augmentation of basal colony formation was 22 ± 3.4% above baseline, occurring at a GH concentration of 10,000 /ug/L (Fig. 1). We next examined the responsiveness of six normal T-cell lines (mean of eight studies) to PRL (Fig. 2). At PRL concentrations of 500 yug/mL or less, the mean peak response was 52 ± 2.3% above baseline, occurring at 100 /ug/L. After a partial fall to baseline, a second mean peak response of 51 ± 9.3% above baseline was observed at 2500 /ug/L.
Laron dwarfs
PBS
100
10
1000
10000
100000
GH Concentration
FIG. 1. In vitro responses of transformed T-cell lines to human GH at concentrations ranging between 25-100,000 Mg/L- Normal T-cell lines showed a biphasic response to GH, with mean peak colony augmentation about 60% above baseline at both 100 and 10,000 Mg/L. Note that there was no augmentation of basal colony formation of the Laron Tcell lines by GH concentrations up to 500 Mg/L. At higher GH concentrations, Laron T-cell lines showed a 22% augmentation of basal colony formation above baseline. The unstimulated number of colonies (phosphate-buffered saline with no added growth factor) is defined as 100% (baseline). Each point of line curves represents the mean percentage of colony augmentation above or below control (±SE) in response to the concentration of growth factor added. Portions of these data have been previously published (4).
200
180
80
PBS
10
100
Prolactin
1000
10000
Concentration (Ml/L)
FIG. 2. Proliferative responses of T-cell lines to PRL concentrations ranging between 25-10,000 Mg/L. Note the equivalent biphasic responses of normal and Laron T-cell lines, with two peaks in each case.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 October 2016. at 12:24 For personal use only. No other uses without permission. . All rights reserved.
COMMENTS
812
The mean peak response of the three Laron T-cell lines (mean of seven studies) to PRL concentrations of 500 fig/L or less was 53 ± 6.2% above baseline, occurring at 100 Mg/L. In the higher concentration range (mean of three studies), the mean peak response was 64 ± 6.7% above baseline, occurring at 5000 yug/L. There was no significant difference between control and Laron T-cell responses at any PRL concentration (Fig. 2). Discussion With the recent cloning and expression of cDNA for rabbit and human GH receptors (6), the role of deficient or absent GH receptors in Laron dwarfism will soon be clarified. That GH receptor deficiency is the basic defect is suggested by the lack of any specific GH binding to membranes prepared from liver samples obtained from two Laron dwarfs (2). The absence of serum GH-binding protein, which corresponds to the extracellular hormonebinding domain of the membrane-bound GH receptor, in patients with Laron dwarfism (14, 15) is also consistent with this proposed defect as the pathogenetic mechanism. More recently, the cDNA encoding the rat PRL receptor has been molecularly cloned and found to have strong localized sequence homology with the GH receptor in both the extracellular and cytoplasmic domains (7). TCell lines established from normal individuals show similar degrees of clonal expansion in response to GH and PRL, demonstrating two peaks for each lactogen. It is presumed that the initial peak in response to concentrations of lactogen of 500 ixg/L or less is mediated through the ligand-specific receptor (since responsiveness is seen at physiological concentrations of ligand). The second peak, seen at supraphysiological concentrations of ligand, is presumed to be mediated via other lactogenic receptors. Our findings of normal growth of Laron T-cell lines in response to PRL, but not to physiological levels of GH, are consistent with a normal PRL receptoreffector mechanism and a highly specific defect in GH receptor function. Identical biphasic responses of normal T-cell lines were seen with both recombinant and pituitary forms of GH and PRL. Similarly, the response of Laron T-cell lines to high dose GH occurred to a similar degree with either recombinant or pituitary GH, suggesting that this response to pituitary GH was not the result of contamination of natural GH with a small amount of PRL. The reproducible second peak response of normal Tcell lines after stimulation with very high GH concentrations and its presence to a lesser degree in GH-resistant Laron dwarfs may represent PRL- or other lactogenic receptor-mediated GH action (e.g. that of human placental lactogen). Were only two lactogenic receptors (those
JCE & M • 1990 VoI70«No3
of PRL and GH) involved in the biphasic response to PRL, one would expect that the T-cells from Laron dwarfs would not demonstrate a second peak, since these cells are presumed to lack GH receptors. If, however, the Laron T-cell lines possessed hPL receptors, then the clonal responses to the very high PRL concentrations could be mediated through this lactogenic receptor. Indeed, the presence of receptors for all three lactogens is known to occur in many tissues, including rat Nb2 T-cell lymphoma cells (16) and rabbit mammary gland (17). Similarly, the second peak response to GH in either normal or Laron T-cell lines could be mediated by either the PRL and/or human placental lactogen receptor. The concentrations of GH that effect this second peak are approximately 50-100 times higher than the concentrations that induce the first peak in normal T-cell lines and are consistent with the known relative affinities of rat and bovine GH for somatogenic receptor and lactogenic receptor sites in isolated rat hepatocytes (18). These hypotheses can be specifically tested when blocking antibodies against lactogenic receptors are available. Since the PRL receptor-effector pathway appears intact in Laron dwarfs, and there is homology between both PRL and GH as well as between their receptors, it is possible that the high circulating levels of GH in these patients could result in some metabolic effect. The concept that one hormone may act through both its own receptor and a second structurally similar receptor has been termed spillover action (19). For example, this is the likely mechanism by which insulin exerts growthpromoting action in tissues such as skin, ovary, and heart (20) in various disorders associated with hyperinsulinemia and diminished insulin effect through the insulin receptor. In these situations insulin is believed to act via homologous insulin-like growth factor-I receptors. Likewise, it is postulated that the galactorrhea in some patients with acromegaly and normal serum PRL levels is mediated by GH action through the PRL receptor (19). Since the concentrations of GH capable of eliciting in vitro action in Laron T-cell lines are far higher than are ever present in serum of Laron dwarfs, and since Laron dwarfs fail to show clinical evidence of body growth in response to endogenous or exogenous GH, it remains unclear whether lactogenic receptors mediate any clinical effects of GH in patients with Laron dwarfism.
References 1. Laron Z. Laron-type dwarfism (hereditary somatomedin deficiency): a review. Adv Intern Med Pediatr. 1984;51:117-50. 2. Eshet R, Laron Z, Pertzelan A, Arnon R, Dintzman M. Defect of human growth hormone receptors in the liver of two patients with Laron-type dwarfism. Isr J Med Sci. 1984;20:8-ll. 3. Golde DW, Bersch N, Kaplan SA, Rimoin DL, Li CH. Peripheral unresponsiveness to human growth hormone in Laron dwarfism. N Engl J Med. 1980;303:1156-9. 4. Geffner ME, Golde DW, Lippe BM, Kaplan SA, Bersch N, Li CH.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 October 2016. at 12:24 For personal use only. No other uses without permission. . All rights reserved.
COMMENTS
5.
6. 7. 8. 9. 10. 11.
Tissues of the Laron dwarf are sensitive to insulin-like growth factor I but not to growth hormone. J Clin Endocrinol Metab. 1987;64:1042-7. Niall HD, Hogan ML, Sauer R, Rosenblum IY, Greenwood FC. Sequences of pituitary and placental lactogenic and growth hormones: evolution from a primordial peptide by gene reduplication. Proc Natl Acad Sci USA. 1971;68:866-9. Leung DW, Spencer SA, Cachianes G. Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature. 1987;330:537-43. Boutin J-M, Jolicoeur C, Okamura H. Cloning and expression of the rat prolactin receptor, a member of the growth hormone/ prolactin receptor gene family. Cell. 1988;53:69-77. Merimee TJ, Hall J, Rabinowitz D, McKusick VA, Rimoin DL. An unusual variety of endocrine dwarfism: subresponsiveness to growth hormone in a sexually mature dwarf. Lancet. 1968;2:191-3. New MI, Schwartz E, Parks GA, Landey S, Wiedermann E. Pseudopituitary dwarfism with normal plasma growth hormone and low serum sulfation factor. J Pediatr. 1972;80:620-6. Chen ISY, Quan SG, Golde D. Human T-cell leukemia virus type II transforms normal human lymphocytes. Proc Natl Acad Sci USA. 1983;80:7006-9. Chen ISY, McLaughlin J, Gasson JC, Clark SC, Golde DW. Molecular characterization of genome of a novel human T-cell leukemia virus. Nature. 1983;305:502-5.
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12. Saxon A, Stevens RH, Golde DW. T-Lymphocyte variant of hairy cell leukemia. Ann Intern Med. 1978;88:323-6. 13. Koeffler HP, Chen ISY, Golde DW. Characterization of a novel HTLV-infected cell line. Blood. 1984;64:482-90. 14. Daughaday WH, Trivedi B. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism). Proc Natl Acad Sci USA. 1987;84:463640. 15. Baumann G, Shaw MA, Winter RJ. Absence of the plasma growth hormone-binding protein in Laron-type dwarfism. J Clin Endocrinol Metab. 1987;65:814-6. 16. Shiu RPC, Elsholtz HP, Tanaka T. Receptor-mediated mitogenic action of prolactin in a rat lymphoma cell line. Endocrinology. 1983;113:159-65. 17. Wallis M. Receptors for growth hormone, prolactin, and the somatomedins. In: Schulster D, Levitzki A, eds. Brighton: Wiley and Sons; 1980;174. 18. Ranke MB, Stanley CA, Tenore A, Rodbard D, Bongiovanni AM, Parks JS. Characterization of somatogenic and lactogenic binding sites in isolated rat hepatocytes. Endocrinology. 1976;99:1033-45. 19. Roth J, Grunfeld C. Mechanism of action of peptide hormones and catecholamines. In: Wilson JD, Foster DW, eds. Williams textbook of endocrinology. Philadelphia: Saunders; 1985;96. 20. Geffner ME, Golde DW. Selective insulin action on skin, ovary, and heart in insulin-resistant states. Diabetes Care. 1988;ll:5005.
A NEW PUBLICATION FROM THE ENDOCRINE SOCIETY! Introduction to Endocrine Investigation 1990 Syllabus* Is Now Available Topics include Cell and Molecular Biology, Receptors, and Basics—all or the edge of the field covered by the leaders in the field of Endocrinology. ° Signal Transduction ° Cell culture ° DNA Structure and ° Peptide Isolation Cloning ° Receptors and Binding $35 for each book includes shipping and handling (delivery 4-6 weeks), per book for international air mail. US currency or equivalent only. Order from: The Endocrine Society 9650 Rockville Pike Bethesda, MD 20814 (301) 571-1802 Phone (301) 571-1869 Fax course co-sponsored by The Endocrine Society and Serono Symposia, USA
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Vol. 70, No. 3 Printed in U.S.A.
Comments
T-Lymphoblast Cell Lines from Laron Dwarfs Augment Basal Colony Formation in Response to Extremely High Concentrations of Growth Hormone* MITCHELL E. GEFFNER, NOELLE BERSCH, BARBARA M. LIPPE, DAVID W. GOLDE Departments of Pediatrics (M.E.G., B.M.L.) and Medicine (N.B., D.W.G.), UCLA Medical Center, Los Angeles, California 90024
ABSTRACT. The clinical entity of Laron dwarfism is characterized by resistance to both endogenous and exogenous GH and may be due to a deficiency or absence of functional GH receptors. We previously showed that two types of hematopoietic cells derived from these patients are resistant to the in vitro growth-promoting action of GH at concentrations below 500 n%/ L. In the current study we found that Laron T-cell lines had a mean peak augmentation of basal colony formation of 22 ± 3.4% above baseline in response to a GH concentration of 10,000 ng/ L. Since cloned cDNAs for human and rabbit GH receptors and rat PRL receptors show a high degree of sequence homology, we undertook studies of PRL action in cells from patients with Laron dwarfism to determine if the Laron defect was also associated with PRL unresponsiveness. Quantitating the aug-
T
HE CLINICAL entity of Laron dwarfism appears to result from GH resistance due to a deficiency or absence of functional GH receptors (1). This concept is based in part on studies of GH binding to hepatic tissue from two affected individuals (2). Using erythroid progenitors and human T-cell leukemia virus (HTLV-II) transformed T-lymphoblasts derived from peripheral blood, we previously documented the presence of GH resistance in cells from two patients with Laron dwarfism based on lack of GH-induced augmentation of basal colony formation at concentrations of GH up to 500 ng/ L (3, 4). Because of the known structural homology between both GH and PRL (5) and between GH and PRL receptors (6, 7), we examined the growth responses of Laron dwarf T-cell lines to a broad concentration range of PRL and GH to determine the specificity of the resistance to GH.
Subjects and Methods In these studies we used HTLV-II-transformed T-lymphoblast cell lines from three Laron dwarfs. The clinical characReceived August 23, 1989. Address requests for reprints to: Mitchell E. Geffner, M.D., Department of Pediatrics, University of California Medical Center, Los Angeles, California 90024. * This work was supported in part by Grants CA-32737 and CA30388 from the NIH.
mentation of colony formation by T-lymphoblast cell lines established from three Laron dwarfs, we found normal responsiveness to PRL at concentrations of 25-10,000 Mg/L. It is, thus, possible that the responsiveness of Laron T-cell lines to very high concentrations of GH could be mediated through an intact PRL (or other lactogenic) receptor based on the known affinity of GH for these receptors in other systems. These data suggest that cells from patients with Laron dwarfism have normal in vitro responsiveness to PRL and that the defect in Laron dwarfism appears to be specific to the GH receptor-effector pathway. It remains to be determined whether intact alternative lactogenic receptor mechanisms subserve any clinical effects of GH in patients with Laron dwarfism. (J Clin Endocrinol Metab 70: 810,1990)
teristics and diagnostic serum GH levels of the patients have been reported previously (3, 4, 8, 9). Patient 3 had a serum PRL concentration of 1.5 ixg/L at age 17 yr (normal range, 318 Mg/L); the PRL level was not measured in the other two patients. In vitro GH studies were also performed using six Tcell lines obtained from normal individuals (two transformed by HTLV-I and four by HTLV-II). For the PRL studies, six normal HTLV-II-transformed T-cell lines were used (including four employed for the GH studies and two others). All studies were performed with the informed consent of the patient and/ or parents and with the approval of the UCLA Human Subject Protection Committee. The methodology for HTLV-I and -II transformation of peripheral blood lymphocytes has been described previously (10, 11). From 10 mL peripheral blood, low density mononuclear cells (5 x 105), obtained by Ficoll-Hypaque density gradient separation, were cocultivated with an equal number of lethally irradiated (12,000 rad) late passage Mo cells (HTLVII) or Me cells (HTLV-I) in Iscove's medium supplemented with 20% fetal bovine serum and interleukin-2. The Mo T-cell line was derived from the spleen of a patient with a variant of hairy cell leukemia (12). The HTLV-I-infected T-cell line (Me) was established from the peripheral blood of a patient with adult T-cell leukemia (13). In either case, a virally infected, immortalized T-cell line is produced in about 4 weeks. Cell lines were fed 2 days before clonogenic studies to ensure that all experiments were conducted with cells in an exponential
810 The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 24 October 2016. at 12:24 For personal use only. No other uses without permission. . All rights reserved.
COMMENTS growth phase. Fifty thousand transformed T-cells/mL (5 x 103/ well) were cultured in microtiter plates in 20% fetal bovine serum without the addition of a-thioglycerol. Ten microliters of either highly purified pituitary or recombinant [either methionine or N085AV nonmethionine (Genentech, Inc., South San Francisco, CA)], human GH in final concentrations of 25, 50,100, 250, 500,1,000, 2,500, 5,000,10,000, 25,000, 50,000, and 100,000 Mg/L; human PRL [hPRL-(l-7); obtained from the NIDDK], or recombinant PRL (kindly provided by Genentech, Inc.)] in final concentrations of 25, 50, 100, 250, 500, 1,000, 2,500,5,000, and 10,000 Mg/L; or phosphate-buffered saline (pH 7.4) was added to each well. All experiments were performed in triplicate, with a maximal replicate variability of less than 5%. After 7-10 days, colonies containing a minimum of eight cells were enumerated using an inverted microscope. The unstimulated number of colonies formed in incubation mixtures without added hormone is defined as 100%. The data are presented as the mean ± SE.
811 200 Controls
180
160
140
120
100
80
Results Since there were no significant differences in GH responsiveness of T-cell lines transformed by either HTLV-I or -II (4) (our unpublished observations), the resultant GH response data were pooled. Similarly, since there was no difference between the degree of colony augmentation with either highly purified pituitary (used in nine control and six Laron dwarf studies) or recombinant GH (methionine-GH used in one normal and one Laron study and N085AV GH used in three normal studies) or the NIDDK (used in six normal and four Laron studies) or recombinant (used in two normal and three Laron studies) PRL, the appropriate response data were combined. In normal subjects (mean of 13 studies of 6 cell lines), the mean peak response to GH concentrations of 500 /ug/ L or less was 59 ± 3.8% above baseline at 100 Mg/L. In 6 GH studies of 3 normal T-cell lines, we extended the studies of clonal responsiveness to GH concentrations as high as 100,000 /ug/L (Fig. 1). After the first peak, there was a partial diminution of responsiveness at 500 /ug/L GH, followed by a mean second peak response of 60 ± 8.0% above baseline at 10,000 /ug/L GH. All three Laron dwarf T-cell lines (mean of seven studies) were resistant to the stimulatory effects of GH at concentrations of 500 /ug/L or less. Four studies of the Laron T-cell lines were extended to GH concentrations as high as 100,000 /ug/L. The mean peak augmentation of basal colony formation was 22 ± 3.4% above baseline, occurring at a GH concentration of 10,000 /ug/L (Fig. 1). We next examined the responsiveness of six normal T-cell lines (mean of eight studies) to PRL (Fig. 2). At PRL concentrations of 500 yug/mL or less, the mean peak response was 52 ± 2.3% above baseline, occurring at 100 /ug/L. After a partial fall to baseline, a second mean peak response of 51 ± 9.3% above baseline was observed at 2500 /ug/L.
Laron dwarfs
PBS
100
10
1000
10000
100000
GH Concentration
FIG. 1. In vitro responses of transformed T-cell lines to human GH at concentrations ranging between 25-100,000 Mg/L- Normal T-cell lines showed a biphasic response to GH, with mean peak colony augmentation about 60% above baseline at both 100 and 10,000 Mg/L. Note that there was no augmentation of basal colony formation of the Laron Tcell lines by GH concentrations up to 500 Mg/L. At higher GH concentrations, Laron T-cell lines showed a 22% augmentation of basal colony formation above baseline. The unstimulated number of colonies (phosphate-buffered saline with no added growth factor) is defined as 100% (baseline). Each point of line curves represents the mean percentage of colony augmentation above or below control (±SE) in response to the concentration of growth factor added. Portions of these data have been previously published (4).
200
180
80
PBS
10
100
Prolactin
1000
10000
Concentration (Ml/L)
FIG. 2. Proliferative responses of T-cell lines to PRL concentrations ranging between 25-10,000 Mg/L. Note the equivalent biphasic responses of normal and Laron T-cell lines, with two peaks in each case.
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COMMENTS
812
The mean peak response of the three Laron T-cell lines (mean of seven studies) to PRL concentrations of 500 fig/L or less was 53 ± 6.2% above baseline, occurring at 100 Mg/L. In the higher concentration range (mean of three studies), the mean peak response was 64 ± 6.7% above baseline, occurring at 5000 yug/L. There was no significant difference between control and Laron T-cell responses at any PRL concentration (Fig. 2). Discussion With the recent cloning and expression of cDNA for rabbit and human GH receptors (6), the role of deficient or absent GH receptors in Laron dwarfism will soon be clarified. That GH receptor deficiency is the basic defect is suggested by the lack of any specific GH binding to membranes prepared from liver samples obtained from two Laron dwarfs (2). The absence of serum GH-binding protein, which corresponds to the extracellular hormonebinding domain of the membrane-bound GH receptor, in patients with Laron dwarfism (14, 15) is also consistent with this proposed defect as the pathogenetic mechanism. More recently, the cDNA encoding the rat PRL receptor has been molecularly cloned and found to have strong localized sequence homology with the GH receptor in both the extracellular and cytoplasmic domains (7). TCell lines established from normal individuals show similar degrees of clonal expansion in response to GH and PRL, demonstrating two peaks for each lactogen. It is presumed that the initial peak in response to concentrations of lactogen of 500 ixg/L or less is mediated through the ligand-specific receptor (since responsiveness is seen at physiological concentrations of ligand). The second peak, seen at supraphysiological concentrations of ligand, is presumed to be mediated via other lactogenic receptors. Our findings of normal growth of Laron T-cell lines in response to PRL, but not to physiological levels of GH, are consistent with a normal PRL receptoreffector mechanism and a highly specific defect in GH receptor function. Identical biphasic responses of normal T-cell lines were seen with both recombinant and pituitary forms of GH and PRL. Similarly, the response of Laron T-cell lines to high dose GH occurred to a similar degree with either recombinant or pituitary GH, suggesting that this response to pituitary GH was not the result of contamination of natural GH with a small amount of PRL. The reproducible second peak response of normal Tcell lines after stimulation with very high GH concentrations and its presence to a lesser degree in GH-resistant Laron dwarfs may represent PRL- or other lactogenic receptor-mediated GH action (e.g. that of human placental lactogen). Were only two lactogenic receptors (those
JCE & M • 1990 VoI70«No3
of PRL and GH) involved in the biphasic response to PRL, one would expect that the T-cells from Laron dwarfs would not demonstrate a second peak, since these cells are presumed to lack GH receptors. If, however, the Laron T-cell lines possessed hPL receptors, then the clonal responses to the very high PRL concentrations could be mediated through this lactogenic receptor. Indeed, the presence of receptors for all three lactogens is known to occur in many tissues, including rat Nb2 T-cell lymphoma cells (16) and rabbit mammary gland (17). Similarly, the second peak response to GH in either normal or Laron T-cell lines could be mediated by either the PRL and/or human placental lactogen receptor. The concentrations of GH that effect this second peak are approximately 50-100 times higher than the concentrations that induce the first peak in normal T-cell lines and are consistent with the known relative affinities of rat and bovine GH for somatogenic receptor and lactogenic receptor sites in isolated rat hepatocytes (18). These hypotheses can be specifically tested when blocking antibodies against lactogenic receptors are available. Since the PRL receptor-effector pathway appears intact in Laron dwarfs, and there is homology between both PRL and GH as well as between their receptors, it is possible that the high circulating levels of GH in these patients could result in some metabolic effect. The concept that one hormone may act through both its own receptor and a second structurally similar receptor has been termed spillover action (19). For example, this is the likely mechanism by which insulin exerts growthpromoting action in tissues such as skin, ovary, and heart (20) in various disorders associated with hyperinsulinemia and diminished insulin effect through the insulin receptor. In these situations insulin is believed to act via homologous insulin-like growth factor-I receptors. Likewise, it is postulated that the galactorrhea in some patients with acromegaly and normal serum PRL levels is mediated by GH action through the PRL receptor (19). Since the concentrations of GH capable of eliciting in vitro action in Laron T-cell lines are far higher than are ever present in serum of Laron dwarfs, and since Laron dwarfs fail to show clinical evidence of body growth in response to endogenous or exogenous GH, it remains unclear whether lactogenic receptors mediate any clinical effects of GH in patients with Laron dwarfism.
References 1. Laron Z. Laron-type dwarfism (hereditary somatomedin deficiency): a review. Adv Intern Med Pediatr. 1984;51:117-50. 2. Eshet R, Laron Z, Pertzelan A, Arnon R, Dintzman M. Defect of human growth hormone receptors in the liver of two patients with Laron-type dwarfism. Isr J Med Sci. 1984;20:8-ll. 3. Golde DW, Bersch N, Kaplan SA, Rimoin DL, Li CH. Peripheral unresponsiveness to human growth hormone in Laron dwarfism. N Engl J Med. 1980;303:1156-9. 4. Geffner ME, Golde DW, Lippe BM, Kaplan SA, Bersch N, Li CH.
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COMMENTS
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Tissues of the Laron dwarf are sensitive to insulin-like growth factor I but not to growth hormone. J Clin Endocrinol Metab. 1987;64:1042-7. Niall HD, Hogan ML, Sauer R, Rosenblum IY, Greenwood FC. Sequences of pituitary and placental lactogenic and growth hormones: evolution from a primordial peptide by gene reduplication. Proc Natl Acad Sci USA. 1971;68:866-9. Leung DW, Spencer SA, Cachianes G. Growth hormone receptor and serum binding protein: purification, cloning and expression. Nature. 1987;330:537-43. Boutin J-M, Jolicoeur C, Okamura H. Cloning and expression of the rat prolactin receptor, a member of the growth hormone/ prolactin receptor gene family. Cell. 1988;53:69-77. Merimee TJ, Hall J, Rabinowitz D, McKusick VA, Rimoin DL. An unusual variety of endocrine dwarfism: subresponsiveness to growth hormone in a sexually mature dwarf. Lancet. 1968;2:191-3. New MI, Schwartz E, Parks GA, Landey S, Wiedermann E. Pseudopituitary dwarfism with normal plasma growth hormone and low serum sulfation factor. J Pediatr. 1972;80:620-6. Chen ISY, Quan SG, Golde D. Human T-cell leukemia virus type II transforms normal human lymphocytes. Proc Natl Acad Sci USA. 1983;80:7006-9. Chen ISY, McLaughlin J, Gasson JC, Clark SC, Golde DW. Molecular characterization of genome of a novel human T-cell leukemia virus. Nature. 1983;305:502-5.
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12. Saxon A, Stevens RH, Golde DW. T-Lymphocyte variant of hairy cell leukemia. Ann Intern Med. 1978;88:323-6. 13. Koeffler HP, Chen ISY, Golde DW. Characterization of a novel HTLV-infected cell line. Blood. 1984;64:482-90. 14. Daughaday WH, Trivedi B. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism). Proc Natl Acad Sci USA. 1987;84:463640. 15. Baumann G, Shaw MA, Winter RJ. Absence of the plasma growth hormone-binding protein in Laron-type dwarfism. J Clin Endocrinol Metab. 1987;65:814-6. 16. Shiu RPC, Elsholtz HP, Tanaka T. Receptor-mediated mitogenic action of prolactin in a rat lymphoma cell line. Endocrinology. 1983;113:159-65. 17. Wallis M. Receptors for growth hormone, prolactin, and the somatomedins. In: Schulster D, Levitzki A, eds. Brighton: Wiley and Sons; 1980;174. 18. Ranke MB, Stanley CA, Tenore A, Rodbard D, Bongiovanni AM, Parks JS. Characterization of somatogenic and lactogenic binding sites in isolated rat hepatocytes. Endocrinology. 1976;99:1033-45. 19. Roth J, Grunfeld C. Mechanism of action of peptide hormones and catecholamines. In: Wilson JD, Foster DW, eds. Williams textbook of endocrinology. Philadelphia: Saunders; 1985;96. 20. Geffner ME, Golde DW. Selective insulin action on skin, ovary, and heart in insulin-resistant states. Diabetes Care. 1988;ll:5005.
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