Thyroid Hormones Stimulate Erythropoiesis in Vitro D. W . GOLDE, N. BERSCH, I. J. CHOPRA A N D M. J. CLINE Department of Medicine, U C L A School of Medicine, Los Angeles, Cali$ornia, U.S.A. (Received 4 January 1977; accepted for publication 19 April 1977) SUMMARY. Thyroid hormones have important effects on erythropoiesis in man and animals. We performed in vitro culture studies with murine and human bone marrow in order to define the interaction of these hormones with erythroid and granulocyte-monocyte progenitor cells. The methylcellulose clonogenic assay was used with the appropriate addition of erythropoietin or colony-stimulating activity. L-Thyroxine, D-thyroxine and L-triiodothyronine potentiated erythropoietinstimulated erythroid colony formation in concentrations of so-roo ng/ml. These hormones had no effect on granulocyte-monocyte colony formation at concentrations up to 5 0 0 ng/ml. Testing of various thyroid analogues showed no clear correlation between potentiation of erythropoiesis and known calorigenic potency. Reverse triiodothyronine also had potentiating activity in this system. The active thyroid hormones stimulated erythroid colony formation at several concentrations of erythropoietin but could not substitute for erythropoietin. These data suggest that thyroid hormones have a direct effect on erythroid precursor proliferative capacity, a finding which may have relevance to the mechanism of erythropoietic dysfunction in human thyroid disease. Although erythropoietin is the primary humoral factor regulating mammalian erythropoiesis, other hormones may influence red blood cell production. Thyroid hormones in particular have been shown to increase erythropoiesis when administed to animals (Fisher & Crook, 1962; Malgor et al, 197s; Waldmann et al, 1962), and anaemia is regularly observed in hypothyroid man (Fein & Rivlin, 197s). It is not clear from the in vivo animal studies whether thyroid hormones directly effect stem cells or act primarily upon erythropoietin production (Das et al, 197s; Fein & Rivlin, 1975; Malgor et al, 197s; Peschle et al, 1971; Shalet et al, 1966). There is a documented effect of thyroid hormone on the levels of 2,3-diphosphoglycerate in mature erythrocytes (Snyder & Reddy, 1970). The recent development of in vitro cloning techniques for culturing erythroid progenitors provides a means of studying isolated effects ofhormones on erythropoiesis (Golde et al, 1976a; Iscove et al, 1974; Moriyama & Fisher, 1975; Stephenson et al, 1971). We report here that thyroid hormones potentiate erythropoiesis in vitro by a direct effect on precursor cells. METHODS Haematopoietic cells were isolated from bone marrow of young adult male Swiss-Webster Correspondence:Dr David W. Golde, Division of Hematology-Oncology, Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024, U.S.A.
I73
I74
D. W . Golde e t a1
mice (Golde et af, 1976a). Single-cell suspensions were prepared and plated at appropriate concentrations in 3 5-mm Petri dishes using the methylcellulose technique of Iscove et a1 (1974). All cultures were established with enriched a medium containing 30% fetal calf serum, antibiotics, and IO-, M a-thioglycerol (Golde et af, 1976a). Sheep plasma erythropoietin (Step 111) was obtained from Connaught Laboratories and human urinary erythropoietin (approximately 80 U/mg) from the Blood Resources Division of the National Institutes of Health, Bethesda, Maryland. Duplicate plates were incubated at 37°C in a humidlfied atmosphere of 7.5 YOCO, in air. Colonies were enumerated at 48 h with an inverted microscope, counting all clusters of eight cells or more. The erythroid nature of the colonies was confirmed by transferring them to slides and staining with benzidine (Golde et al, 1976a; Stephenson et al, 1971). Normal human bone marrow was obtained from healthy volunteers and plated under the same conditions as for mouse haematopoietic cells except that human urinary erythropoietin was used exclusively and colonies were enumerated after 8 d of culture. Assays for granulocyte-monocyte colony-forming cells (CFU-C) were performed with the same methylcellulose culture technique using a gravid mouse uterus extract as the source of colony-stimulating activity (CSA) in the mouse and leucocyte-conditioned medium in man (Golde et af, 1976b). Leucocytic colonies greater than 50 cells were counted after 7-10 d of culture. Thyroid hormones (Sigma Chemicals) were solubilized in 0.I N NaOH and diluted in phosphate-buffered saline (pH 9.3). Apprbpriate solutions were prepared without hormone and added to cultures to control for possible effects of the diluent materials. The concentration of thyroxine (T,, 3,3’,5-triiodothyronine (T,) and 3 ~’,~’-triiodothyronine (reverse T,) in the fetal calf serum was determined by radioimmunoassays as previously described (Chopra, 1972, 1974; Chopra et af, 1972). RESULTS T,, D-T, or T, was added to cultures of adult mouse bone marrow cells in concentrations ranging from 25 to 500 ng/ml. T, was found to have the greatest stimulatory effect with peak activity at approximately 50 ng/ml of added hormone (Fig I ) . At 50 ng/ml of added T, there was a mean 50% increase in erythroid colonies (CFU-E) in cultures containing maximally stimulating doses of sheep erythropoietin (0.5 U/ml). Higher concentrations of T, resulted in reduced cloning efficiency. D-T, was only slightly less active than T, with peak stimulation occurring at approximately IOO ng/ml. T, was also less active than T,, with maximal stimulation of 39% at IOO ng/ml. Sodium iodide was ineffective in stimulating CFU-E (Table I). Triiodothyroacetic acid and tetraiodothyroacetic acid, however, exhibited prominent stimulatory capacity. Reverse T, (rT,) showed activity comparable to T, even though the fetal calf serum concentration of rT, was high ( I 1.5 nglml). The content of T, in undiluted fetal calf serum was 130 ng/ml and T, was 2.0 ng/ml. Each culture plate therefore contained endogenous hormones in amounts of about 40 ng/ml T,, less than I ng/ml T, and about 4 ng/ml rT,. Human bone marrow erythroid colony formation in vitro was also potentiated by thyroid hormones. There was a mean augmentation in cloning of 50% with T, (50 ng/ml) and 37% with T, (100 ng/ml) in two experiments. The degree of stimulation of erythropoiesis by thyroid hormone did not change appreciably over a range of erythropoietin concentrations from 0.05 to 0.5 U. The addition of rabbit
Thyroid Hormones and Erythropoiesis 160 155
UJ
150 145
- 140 2
c
U
135
130
3 125
-
120
4
115
M
UJ
Y
u 110 105 100 25
50
100
200
Added Thyroid Hormone ( n g / m l )
FIG
I . Effect of thyroid hormones on erythroid colony formation (CFU-E) in vitro in nine experiments performed with adult mouse bone marrow. Plates contain 0.5 U sheep plasma erythropoietin. Mean cloning efficiency in these cultures was 329f. 19 (SEM) per I x 105 cells.
TABLE I. Effect ofthyroid analogues on erythroid colony formation in vitro (CFU-E)*. 0.5 U erythropoietin,
I
x
105 mouse
,
CFU-E (Yo of control SE)
No. of experiments
II7f 3 t 139k 8 t
7
50
Iso,Iot
7
50 I00
I35
50 I00
130,
50
IOO* 100,
Analogue added (concentration in ng/ml) 50 I00
Na iodide
I00
Tetraiodothyroacetic acid
25
50
Trijodothyroacetic acid
bone marrow cells plated.
25
50
CFU-E-colony-forming
fI 3 t
* 1st
5
13 I39k 8 t
3
I43
I I
3
167, I o t I 3 7 f 13
3
1 4 7 f I4t I39+ I4
3
unit, erythroid.
t Significantly different from control, P < o . o ~ ,T test.
1 76
D. W . Golde et a1
anti-human erythropoietin antibody (a gift of Dr James Fisher) to the cultures resulted in quantitative neutralization of added human urinary erythropoietin. Although thyroid hormones augmented colony formation at all concentrations of erythropoietin tested, they could not substitute for erythropoietin when this hormone was neutralized by antibody. In tests of thyroid hormones on the clonal growth of other haematopoietic cells, no consistent effects on granulocyte-monocyte colony formation were observed in five experiments with mouse bone marrow and two experiments with human bone marrow using maximally stimulating concentrations of CSA.
DISCUSSION These studies provide data on the fundamental problem of whether thyroid hormones act directly on erythroid cell progenitors or act exclusively by potentiating erythropoietin elaboration. They indicate that thyroid hormones can stimulate erythropoiesis directly in vitro in systems where hormonal effects on the production of erythropoietin are eliminated. The observations suggest that the erythroid progenitor cell capable of colony formation in vitro has a thyroid receptor mechanism which when activated results in an augmented response to erythropoietin. An unexpected finding was the observation that T, was somewhat more active in this system than T,. Similarly, D-T,, which has little calorigenic activity relative to T,, is also an active stimulator of erythropoiesis in vitro. The erythropoietic activity of thyroid hormone analogues did not closely correlate to known calorigenic potency (Money et al, 1960; Stasilli et al, 1959). Our results are consistent with the in vivo studies of Malgor et al(1975) who showed erythropoietic stimulation by T, and T, that was not dependent on the stimulation of erythropoietin production. Similarly, the dextrorotary isomer of T, has been demonstrated to have erythropoietic activity in vivo (Bozzini et al, 1969; Donati et al, 1966). There is also other evidence for a noncalorigenic effect of thyroxine on erythropoiesis (Meineke & Crafts, 1964). There are few in vitro systems for studying the biological effects of thyroid hormones. A rat pituitary cell line responds to physiological concentrations of thyroid hormones (Samuels et al, 1973) and human kidney T-I cells are also responsive (Siegel & Tobias, 1966). In the T-I system, diiodotyrosine and D-T, were found to be active (Siegel & Tobias, 1966) and recent studies have shown effects of T, on nuclear and extranuclear DNA in these cells (Siegel & Siegel, 1974). Effects of thyroid hormones on bone marrow cell protein synthesis in vitro have also been reported (Necheles, 1962). Our results in the clonogenic assay for erythroid progenitors indicate that both T, and T, are active stimulators of erythropoiesis as are some other thyroid analogues that are calorigenically inert in other model systems. The thyroid hormones and their analogues appear to have a direct effect on erythroid progenitors with no appreciable stimulation of the granulocyte-monocyte series.
ACKNOWLEDGMENT
This work was supported by U.S. Public Health Service grants CA 15688, CA 15619 and RR 00865.
Thyroid Hormones and Erythropoieris
I77
REFERENCES BOZZINI, C.E., NIOTTI,H.F. & BARRIO RENDO,M.E. (1969)The erythropoietic and calorigenic effects of isomeres of triiodothyronine. Acta Physiologica Latino-Americana, 19,309 CHOPRA, I.J. (1972)A radioimmunoassay for measurement of thyroxine in unextracted serum. Journal of Clinical Endocrinology and Metabolism, 34, 938. CHOPRA, I.J. (1974)A radioimmunoassay for measurement of 3,3’,5’-triiodothyronine(reverse T3).Journal of Clinical Investigation, 54, 583. CHOPRA, I.J., H o , R.S. & LAM,R. (1972)An improved radioimmunoassay of triiodothyronine in serum: its application to clinical and physiological studies. Journal ofLaboratory and Clinical Medicine, 80, 729. DAS,K.C., MUKHERJEE, M., SARKAR, T.K., DASH, R.J. & RASTOGI, G.K. (1975)Erythropoiesis and erythropoietin in hypo- and hyperthryroidism. Journal of Clinical Endocrinology and Metabolism, 40, 21I. DONATI,R.M., WARNECKE, M.A. & GALLAGHER, N.I. (1966)Effect of isomeres of triiodothyronine on erythrokinetics. Proceedings of the Society f o r Experimental Biology and Medicine, 122, I 199. FEIN,H.G. & RIVLIN, R.S. (197s)Anemia in thyroid diseases. Medical Clinics ofNorth America, 59, I 133. FISHER, J.W. & CROOK, J.J. (1962)Influence of several hormones on erythropoiesis and oxygen consumption in the hypophysectomized rat. Blood, 19,$57. GOLDE,D.W., BERSCH,N. & CLINE, M.J. (1976a) Potentiation of erythropoiesis in vitro by dexamethasone. Journal of Clinical Investigation, 57, 57. GOLDE, D.W., BERSCH, N., QUAN,S.G. & CLINE,M.J. (1976b)Inhibition of murine granulopoiesis in vitro by dexamethasone. American Journal of Hematology, I, 369. ISCOVE,N.N., SIEBER,F. & WINTERHALTER, K.H. (1974)Erythroid colony formation in cultures of mouse and human bone marrow: analysis of the requirement for erythropoietin by gel filtration and affinity chromatography on agarose-concanavalin A. Journal of Cellular Physiology, 83, 309. MALGOR,L.A., BLANC,C.C., KLAINER, E., IRIZAR, L. (197s)Direct S.E., TORALES, P.R. & BARRIOS, effects of thyroid hormones on bone marrow erythroid cells of rats. Blood, 45, 671. MEINEKE, H.A. & C R A ~R.C. S , (1964)Evidence for a non-calorigenic effect of thyroxin on erythropoiesis as judged by radioiron utilization. Proceedings of tbe Society for Experimental Biology and Medicine, 117,5 2 0 .
MONEY,W.L., KUMAOKA, S., RAWSON,R.W. & KROC,R.L. (1960)Comparative effects of thyroxine analogues in experimental animals. Part IV. Metabolic effects of thyroid hormones and their analogues. Annals of the N e w York Academy of Sciences, 86, 5 1 2 . MORIYAMA, Y. & FISHER,J.W. (1975)Effects of testosterone and erythropoietin on erythroid colony formation in human bone marrow cultures. Blood, 45,665. NECHELES, T.F. (1962)Peptide synthesis in bone marrow: insulin and thyroxin effects. American Journal of Physiology, 203, 693. PESCHLE, C., ZANJANI, E.D., GIDARI, A S . , MCLAURIN,W.D. & GORDON, A.S. (1971) Mechanism of thyroxine action on erythropoiesis. Endocrinology, 89, 609. SAMUELS, H.H., TSAI,J.S. & CINTRON, R. (1973) Thyroid hormone action: a cell-culture system responsive to physiological concentrations of thyroid hormones. Science, 181, 1253. SHALET,M., COE,D. & REISSMANN, K.R. (1966) Mechanism of erythropoietic action of thyroid hormone. Proceedings of the Societyfor Experimental Biology and Medicine, 123,443. SIEGEL,E. & SIEGEL,E.P. (1974)Triiodothyronine influences nuclear and extra-nuclear D N A in cultured human kidney cells. Nature, 249, 139. SIEGEL, E. & TOBIAS,C.A. (1966)Actions of thyroid hormones on cultured human cells. Nature, 212, 1318. SNYDER, L.M. & REDDY,W.J. (1970)Thyroid hormone control of erythrocyte ~,3-diphosphoglyceric acid concentrations. Science, 169, 879. STASILLI, N.R., KROC,R.L. & MELTZER, R.I. (1959) Antigoitrogenic and calorigenic activities of thyroxine analogues in rats. Endocrinology, 64, 62. STEPHENSON, J.R., AXELRAD, A.A., MCLEOD,D.L. & SHREEVE, M.M. (1971)Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proceedings ofthe National Academy ofSciences of the United States ofAmerica, 68, 1542. WALDMANN, T.A., WEISSMAN, S.M. & LEVIN,E.H. (1962)Effect of thyroid administration on erythropoiesis in the dog. Journal ofLaboratory and Clinical Medicine, 59, 926.
I73
I74
D. W . Golde e t a1
mice (Golde et af, 1976a). Single-cell suspensions were prepared and plated at appropriate concentrations in 3 5-mm Petri dishes using the methylcellulose technique of Iscove et a1 (1974). All cultures were established with enriched a medium containing 30% fetal calf serum, antibiotics, and IO-, M a-thioglycerol (Golde et af, 1976a). Sheep plasma erythropoietin (Step 111) was obtained from Connaught Laboratories and human urinary erythropoietin (approximately 80 U/mg) from the Blood Resources Division of the National Institutes of Health, Bethesda, Maryland. Duplicate plates were incubated at 37°C in a humidlfied atmosphere of 7.5 YOCO, in air. Colonies were enumerated at 48 h with an inverted microscope, counting all clusters of eight cells or more. The erythroid nature of the colonies was confirmed by transferring them to slides and staining with benzidine (Golde et al, 1976a; Stephenson et al, 1971). Normal human bone marrow was obtained from healthy volunteers and plated under the same conditions as for mouse haematopoietic cells except that human urinary erythropoietin was used exclusively and colonies were enumerated after 8 d of culture. Assays for granulocyte-monocyte colony-forming cells (CFU-C) were performed with the same methylcellulose culture technique using a gravid mouse uterus extract as the source of colony-stimulating activity (CSA) in the mouse and leucocyte-conditioned medium in man (Golde et af, 1976b). Leucocytic colonies greater than 50 cells were counted after 7-10 d of culture. Thyroid hormones (Sigma Chemicals) were solubilized in 0.I N NaOH and diluted in phosphate-buffered saline (pH 9.3). Apprbpriate solutions were prepared without hormone and added to cultures to control for possible effects of the diluent materials. The concentration of thyroxine (T,, 3,3’,5-triiodothyronine (T,) and 3 ~’,~’-triiodothyronine (reverse T,) in the fetal calf serum was determined by radioimmunoassays as previously described (Chopra, 1972, 1974; Chopra et af, 1972). RESULTS T,, D-T, or T, was added to cultures of adult mouse bone marrow cells in concentrations ranging from 25 to 500 ng/ml. T, was found to have the greatest stimulatory effect with peak activity at approximately 50 ng/ml of added hormone (Fig I ) . At 50 ng/ml of added T, there was a mean 50% increase in erythroid colonies (CFU-E) in cultures containing maximally stimulating doses of sheep erythropoietin (0.5 U/ml). Higher concentrations of T, resulted in reduced cloning efficiency. D-T, was only slightly less active than T, with peak stimulation occurring at approximately IOO ng/ml. T, was also less active than T,, with maximal stimulation of 39% at IOO ng/ml. Sodium iodide was ineffective in stimulating CFU-E (Table I). Triiodothyroacetic acid and tetraiodothyroacetic acid, however, exhibited prominent stimulatory capacity. Reverse T, (rT,) showed activity comparable to T, even though the fetal calf serum concentration of rT, was high ( I 1.5 nglml). The content of T, in undiluted fetal calf serum was 130 ng/ml and T, was 2.0 ng/ml. Each culture plate therefore contained endogenous hormones in amounts of about 40 ng/ml T,, less than I ng/ml T, and about 4 ng/ml rT,. Human bone marrow erythroid colony formation in vitro was also potentiated by thyroid hormones. There was a mean augmentation in cloning of 50% with T, (50 ng/ml) and 37% with T, (100 ng/ml) in two experiments. The degree of stimulation of erythropoiesis by thyroid hormone did not change appreciably over a range of erythropoietin concentrations from 0.05 to 0.5 U. The addition of rabbit
Thyroid Hormones and Erythropoiesis 160 155
UJ
150 145
- 140 2
c
U
135
130
3 125
-
120
4
115
M
UJ
Y
u 110 105 100 25
50
100
200
Added Thyroid Hormone ( n g / m l )
FIG
I . Effect of thyroid hormones on erythroid colony formation (CFU-E) in vitro in nine experiments performed with adult mouse bone marrow. Plates contain 0.5 U sheep plasma erythropoietin. Mean cloning efficiency in these cultures was 329f. 19 (SEM) per I x 105 cells.
TABLE I. Effect ofthyroid analogues on erythroid colony formation in vitro (CFU-E)*. 0.5 U erythropoietin,
I
x
105 mouse
,
CFU-E (Yo of control SE)
No. of experiments
II7f 3 t 139k 8 t
7
50
Iso,Iot
7
50 I00
I35
50 I00
130,
50
IOO* 100,
Analogue added (concentration in ng/ml) 50 I00
Na iodide
I00
Tetraiodothyroacetic acid
25
50
Trijodothyroacetic acid
bone marrow cells plated.
25
50
CFU-E-colony-forming
fI 3 t
* 1st
5
13 I39k 8 t
3
I43
I I
3
167, I o t I 3 7 f 13
3
1 4 7 f I4t I39+ I4
3
unit, erythroid.
t Significantly different from control, P < o . o ~ ,T test.
1 76
D. W . Golde et a1
anti-human erythropoietin antibody (a gift of Dr James Fisher) to the cultures resulted in quantitative neutralization of added human urinary erythropoietin. Although thyroid hormones augmented colony formation at all concentrations of erythropoietin tested, they could not substitute for erythropoietin when this hormone was neutralized by antibody. In tests of thyroid hormones on the clonal growth of other haematopoietic cells, no consistent effects on granulocyte-monocyte colony formation were observed in five experiments with mouse bone marrow and two experiments with human bone marrow using maximally stimulating concentrations of CSA.
DISCUSSION These studies provide data on the fundamental problem of whether thyroid hormones act directly on erythroid cell progenitors or act exclusively by potentiating erythropoietin elaboration. They indicate that thyroid hormones can stimulate erythropoiesis directly in vitro in systems where hormonal effects on the production of erythropoietin are eliminated. The observations suggest that the erythroid progenitor cell capable of colony formation in vitro has a thyroid receptor mechanism which when activated results in an augmented response to erythropoietin. An unexpected finding was the observation that T, was somewhat more active in this system than T,. Similarly, D-T,, which has little calorigenic activity relative to T,, is also an active stimulator of erythropoiesis in vitro. The erythropoietic activity of thyroid hormone analogues did not closely correlate to known calorigenic potency (Money et al, 1960; Stasilli et al, 1959). Our results are consistent with the in vivo studies of Malgor et al(1975) who showed erythropoietic stimulation by T, and T, that was not dependent on the stimulation of erythropoietin production. Similarly, the dextrorotary isomer of T, has been demonstrated to have erythropoietic activity in vivo (Bozzini et al, 1969; Donati et al, 1966). There is also other evidence for a noncalorigenic effect of thyroxine on erythropoiesis (Meineke & Crafts, 1964). There are few in vitro systems for studying the biological effects of thyroid hormones. A rat pituitary cell line responds to physiological concentrations of thyroid hormones (Samuels et al, 1973) and human kidney T-I cells are also responsive (Siegel & Tobias, 1966). In the T-I system, diiodotyrosine and D-T, were found to be active (Siegel & Tobias, 1966) and recent studies have shown effects of T, on nuclear and extranuclear DNA in these cells (Siegel & Siegel, 1974). Effects of thyroid hormones on bone marrow cell protein synthesis in vitro have also been reported (Necheles, 1962). Our results in the clonogenic assay for erythroid progenitors indicate that both T, and T, are active stimulators of erythropoiesis as are some other thyroid analogues that are calorigenically inert in other model systems. The thyroid hormones and their analogues appear to have a direct effect on erythroid progenitors with no appreciable stimulation of the granulocyte-monocyte series.
ACKNOWLEDGMENT
This work was supported by U.S. Public Health Service grants CA 15688, CA 15619 and RR 00865.
Thyroid Hormones and Erythropoieris
I77
REFERENCES BOZZINI, C.E., NIOTTI,H.F. & BARRIO RENDO,M.E. (1969)The erythropoietic and calorigenic effects of isomeres of triiodothyronine. Acta Physiologica Latino-Americana, 19,309 CHOPRA, I.J. (1972)A radioimmunoassay for measurement of thyroxine in unextracted serum. Journal of Clinical Endocrinology and Metabolism, 34, 938. CHOPRA, I.J. (1974)A radioimmunoassay for measurement of 3,3’,5’-triiodothyronine(reverse T3).Journal of Clinical Investigation, 54, 583. CHOPRA, I.J., H o , R.S. & LAM,R. (1972)An improved radioimmunoassay of triiodothyronine in serum: its application to clinical and physiological studies. Journal ofLaboratory and Clinical Medicine, 80, 729. DAS,K.C., MUKHERJEE, M., SARKAR, T.K., DASH, R.J. & RASTOGI, G.K. (1975)Erythropoiesis and erythropoietin in hypo- and hyperthryroidism. Journal of Clinical Endocrinology and Metabolism, 40, 21I. DONATI,R.M., WARNECKE, M.A. & GALLAGHER, N.I. (1966)Effect of isomeres of triiodothyronine on erythrokinetics. Proceedings of the Society f o r Experimental Biology and Medicine, 122, I 199. FEIN,H.G. & RIVLIN, R.S. (197s)Anemia in thyroid diseases. Medical Clinics ofNorth America, 59, I 133. FISHER, J.W. & CROOK, J.J. (1962)Influence of several hormones on erythropoiesis and oxygen consumption in the hypophysectomized rat. Blood, 19,$57. GOLDE,D.W., BERSCH,N. & CLINE, M.J. (1976a) Potentiation of erythropoiesis in vitro by dexamethasone. Journal of Clinical Investigation, 57, 57. GOLDE, D.W., BERSCH, N., QUAN,S.G. & CLINE,M.J. (1976b)Inhibition of murine granulopoiesis in vitro by dexamethasone. American Journal of Hematology, I, 369. ISCOVE,N.N., SIEBER,F. & WINTERHALTER, K.H. (1974)Erythroid colony formation in cultures of mouse and human bone marrow: analysis of the requirement for erythropoietin by gel filtration and affinity chromatography on agarose-concanavalin A. Journal of Cellular Physiology, 83, 309. MALGOR,L.A., BLANC,C.C., KLAINER, E., IRIZAR, L. (197s)Direct S.E., TORALES, P.R. & BARRIOS, effects of thyroid hormones on bone marrow erythroid cells of rats. Blood, 45, 671. MEINEKE, H.A. & C R A ~R.C. S , (1964)Evidence for a non-calorigenic effect of thyroxin on erythropoiesis as judged by radioiron utilization. Proceedings of tbe Society for Experimental Biology and Medicine, 117,5 2 0 .
MONEY,W.L., KUMAOKA, S., RAWSON,R.W. & KROC,R.L. (1960)Comparative effects of thyroxine analogues in experimental animals. Part IV. Metabolic effects of thyroid hormones and their analogues. Annals of the N e w York Academy of Sciences, 86, 5 1 2 . MORIYAMA, Y. & FISHER,J.W. (1975)Effects of testosterone and erythropoietin on erythroid colony formation in human bone marrow cultures. Blood, 45,665. NECHELES, T.F. (1962)Peptide synthesis in bone marrow: insulin and thyroxin effects. American Journal of Physiology, 203, 693. PESCHLE, C., ZANJANI, E.D., GIDARI, A S . , MCLAURIN,W.D. & GORDON, A.S. (1971) Mechanism of thyroxine action on erythropoiesis. Endocrinology, 89, 609. SAMUELS, H.H., TSAI,J.S. & CINTRON, R. (1973) Thyroid hormone action: a cell-culture system responsive to physiological concentrations of thyroid hormones. Science, 181, 1253. SHALET,M., COE,D. & REISSMANN, K.R. (1966) Mechanism of erythropoietic action of thyroid hormone. Proceedings of the Societyfor Experimental Biology and Medicine, 123,443. SIEGEL,E. & SIEGEL,E.P. (1974)Triiodothyronine influences nuclear and extra-nuclear D N A in cultured human kidney cells. Nature, 249, 139. SIEGEL, E. & TOBIAS,C.A. (1966)Actions of thyroid hormones on cultured human cells. Nature, 212, 1318. SNYDER, L.M. & REDDY,W.J. (1970)Thyroid hormone control of erythrocyte ~,3-diphosphoglyceric acid concentrations. Science, 169, 879. STASILLI, N.R., KROC,R.L. & MELTZER, R.I. (1959) Antigoitrogenic and calorigenic activities of thyroxine analogues in rats. Endocrinology, 64, 62. STEPHENSON, J.R., AXELRAD, A.A., MCLEOD,D.L. & SHREEVE, M.M. (1971)Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro. Proceedings ofthe National Academy ofSciences of the United States ofAmerica, 68, 1542. WALDMANN, T.A., WEISSMAN, S.M. & LEVIN,E.H. (1962)Effect of thyroid administration on erythropoiesis in the dog. Journal ofLaboratory and Clinical Medicine, 59, 926.
