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Induction of differentiation of friend erythroleukemia cells with L histidinol
Life Sciences, Vol. 29, pp. 1203-1205 Printed in the U.S.A.
INDUCTION OF DIFFERENTIATION OF FRIEhD E R Y T H R O ~ I A
Pergamon Press
C R I ~ WITH L HISTIDINOL
E. Kundahl and R.A. Flickinger Department of Biological Sciences State University of New York Buffalo, New York 14260 (Received in final form July 8, 1981)
summary Addition of an analog of histidine, histidinol, together with lowering the level of histidine in the medium, can induce hemoglobin synthesis in murine erythroleukemia cells. Differentiation is triggered by starvation in Dictyosteli uml , Chlan~domonas 2, certain fungi 3 and some prokaryotes4,5. One aspect of starvation is that the synthesis of ribosomal RNA (r-RNA) is reduced. During starvation in bacteria the synthesis of r-RNA is restricted severly with less effect upon messenger RNA synthesis 6. Furthermore, low levels of actinomycin D primarily inhibit the synthesis of r-RNA7, as does restriction of required amino acids in cultures of mammalian cells 8, 9. It has been shown that low levels of actinc~ycin D stimulate melanin synthesis in rapidly growing_ chick retinal pigment cells I0, neuron formation in n~use neuroblastcma cells II and formation of hemoglobin in cultured Friend erythroleukemia cells 12 in vitro. If a low level of actinomycin D promotes differentiation in erythroleukemia cells by lowering tJ~e rate of r-RNA synthesis 7, and if lo~_r anino acid levels also reduce r-RNA synthesis, then it was considered possible that addition of an amino acid analog, together with reduction of the level of the normal acid, might induce differentiation, i.e., hemoglobin synthesis, in cultured Friend leukemia cells. Experiments were perfornmd with analogs of L-isoleucine (0methylthreonine), L-valine (e-ketobutyrate), L-arginine (Hcmoarginine) and Lhistidine (L-histidinol). Preliminary experiments indicated more success with histidinol and only these results are reported. Methods Friend leukemia cells (clone 745-GM86) 1 were grown in suspension culture in plastic culture dishes (60 mm x 15 nm) containing 5 ml of mediua. Stock cells were grown in Eagle's minim~n essential mediua (MI~4) containing 10% unheated foetal bovine se_rua (FBS) and 1% antibiotic-antimycotic solution containing penicillin, streptomycin, fungazone (PSF) at 37°C at 95% air-5% CO 2 . Stock cells were transferred twice each ~eek at an initial concentration of 1 x 105 cells per ml. Control cells were grown in medit~n containing either 10% unheated FBS or 10% unheated FBS which had been dialyzed against Earle's balanced saline medium to remove any endogenous amino acids. Experimental cells were cultured in medium containing 10% dialyzed FBS and lacking histidine or containing a reduced level of histidine. All othpr exogenously added amino acid levels were kept constant between the controls and experimental cultures. The experimental culture medium was made up using Earle's Balanced saline 1The cells were obtained frcm Dr. Arthor E. Green, the H ~ n a n ~ t a n t Repository, Institute for Medical Research, Camden, New Jersey. 0024-3205/81/121203-03502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
Cell
1204
Histidinol Induces Hemoglobin Synthesis
Vol. 29, No. 12, 1981
solution with 1% antibiotic-antimycotic, 10% dialyzed FBS, the normal level of vitanins found in M~4 and the addition of all essential amino acids at normal MHM levels except histidine. This m e d i ~ n w a s used t o d i l u t e the 5~94PSF-10% dialyzed FBS to achieve the desired final histidine concentration. All constituents of the mediumw~re obtained frcmGrand Island Biological Supply Co., Grand Island, New York. Control and experimental cultures were initially seeded at 2 x 105 cells per ml. Histidinol-containing medium was added 3-4 hours past the time of experimental seeding. The percent of benzidine-reactive cells containing hemoglobin was determined by the method of Friedman and Schildkraut 13. Four countings were made for each experiment and then averaged. Cells were stained with 0.1% trypan blue to estimate the percent of living cells. Cell counts were made with a Neubar cell counter grid and are an average of eight separate countings. The cell counts for cell number, percent viable cells and percent benzidine-positive cells in any one experiment, and cfmparing different experiments, did not vary more than 5% frmm the average for any particular experimental regime. Results and Discussion Omission of L-histidine from the medium inhibits cell proliferation so that the analog, L-histidinol, was added to the experimental medit~n (Earle's saline containing the normal M~4 levels of vitamins and all amino acids except L-histidine, 10% dialyzed FBS and 1% PSF), together with l o ~ r than normal M194 levels of histidine. After five days of culture the cell n~mlber, percent viability and percent benzidine-positive cells were determined. The results are presented in Table 1 and are the averages of 4 different experiments. TABLE 1 Effect of L-Histidinol upon Cell Growth and Hemoglobin Formation
Percent Viability 95 95
Percentbenzidine positive cells < 1.0 < 1.0
41.0
80
77.0
Experimental L-Histidinol (0.4 ~M) + 0.15niM L-histidine + 0.10raM L-histidine
13.6 5.4
72 64
5.6 ll.0
L-Histidinol (0.3 mM) + 0.15raM L-histidine + 0. l~aM L-histidine
17.0 7.6
80 75
7.8 14.0
L-Histidinol (0.2 n%M) + 0.15n~ L-histidine + 0.10ram L-histidine + 0.05raM L-histidine
24.0 20.4 16.4
86 84 80
12.0 14.5 22.4
Controls ~/~4, 10% M~4, 10% M194, 10% +200 n~4
FBS, PS dialyzed FBS, PSF dialyzed FBS, PSF dimethylsulfoxide
Cell ntmlber x 105 cell/ml 44.2 27.6
The normal level of L-histidine in Eagle's MI94 is 0.2 mM and the most effective means of inducing hemoglobin formation was to reduce L-histidine to 1/4 its normal level in M194 (0.05 n~4) and add the L-histidine analog, Lhistidinol, at the normal level of L-histidine in the median. Increasing levels of L-histidinol were toxic as seen by decreased cell number and viability
Vol. 29, No. 12, 1981
Histidinol Induces Hemoglobin Synthesis
1205
at higher concentrations. With even lower levels of L-histidine (0.025 n~4), which is 1/8 the normal level in MI~4, little cell growth occurred. It is known that omission of leucine from the median can cause Friend cells to arrest in the G1 phase of the cell cycle 13. No cell-cycle experiments were carried out in this study, but it would be interesting to repeat these experiments with synchronized cells to see if the histidinol effect is related to the cell cycle. Yet another extension of this work will be to ascertain the means by which dimethylsulfoxide prevents the inhibition of growth in dialyzed fcetal calf serum. L-histidinol is a potent, reversible inhibitor of protein synthesis in cultured human cells 14 . In cultures of meuse ~nbryo fibroblasts containing L-histindinol, and with L-histidine depletion, both protein and ribosomal RNA synthesis are ~ i t e d , probably because about 40% of the t-RNA his is uncharged 15. It is likely that L-histidinol stimulates hemoglobin synthesis by inhibition of ribosomal RNA synthesis due to the uncharged t-RNA, followed by a restriction of protein synthesis. No measurements of ribosomal RNA synthesis were performed in this study, but since histidinol inhibits growth it is likely that r-RNA synthesis is inhibited8,9. However, there is no proof that ~ition of r-RNA synthesis induces differentiation. L-ethionine also induces differentiation in murine erythroleukemia cells 16 In this study about 25-30% of the cells in the culture were stimulated to form hanoglobin after 4-5 days of culture. These workers suggested that ethionineinduced Friend cell differentiation was due to an inhibition of the methylation of DNA and t-RNA. Among the inducers of differentiation of murine ervthroleukemia cells are a number of inhibitors of DNA, RNA and protein synthesis 12. The fact that the analog of L-histidine, L-histidinol, also has inducing activity suggests that induction of the final steps of differentiation in Friend tt~nor cells involves some form of metabolic starvation, just as is true in lo~_r organisms I-5 . References i.
J.T. BONNER, The Cellular Slime Molds. Princeton Univ. Press, Princeton, New Jersey (1967). 2. R. SAGER, S. GRANICK, J. Gen. Physiol. 37 729-742 (1954). 3. L.E. HAWKER, The Physiology of Reproduct-[on in Fungi. Hafner Pub. Co., New York (1971). 4. E. FREEZE, T. ICHIKA~, Y.K. OH, E.B. FREEZE, and C. PRASAD, Proc. Nat. Acad. Sci. U.S. 71 4188-4193 (1974). 5. W. WIP4~4AN, and M. DWORKIN, Sci. 189 516-523 (1975). 6. D.W. MORRIS and N.O. Kjeldgaard, J. Mol. Biol. 31 145-148 (1968). 7. R.P. PERRY, and D.E. K~IT~Y, J. Cell Physiol. 72-235-245 (1968). 8. B.E.H. M A D e , M.H. VALK~HAN, J.R. WARNER, and J.-E. DARNELL, J. Mol. Biol. 45 265-275 (1969). 9. M.H. VAUGHAN, R. SOEIRO, J.R. WARNER, and J.E. DARNRII., Proc. Nat. Acad. Sci. U.S. 58 1527-1534 (1967) i0. J. WHITtAKEr, J. Exp. Zool. 169 143-160 (1969). Ii. D. SCHUBERT, S. }K/MPHREYS, F. DEVITRY, and F. JACOB, Devel. Biol. 25 514-546 (1971). 12. P.S. EBERT, I. WARS, and D.N. BUELL, Cancer Res. 36 1809-1813 (1976). 13. E.A. FRIEDMAN, and C.L. SCHILDKRAUT, Cell 12 901-9---13 (1977). 14. B.S. HANS,, M.H. VAUGHAN, and L. WANG, J.-Biol. Chem. 247 3854-3857 (1972) 15. F. GRL~MMT, and I. G ~ , Eur. J. Biochem. 4 307-312 (i~6). 16. J.K. CHRIS~MAN, P. PRICE, L. P~DRINAN, and ~. ACS, Eur. J. Biochem. 81 53-61 (1977).
INDUCTION OF DIFFERENTIATION OF FRIEhD E R Y T H R O ~ I A
Pergamon Press
C R I ~ WITH L HISTIDINOL
E. Kundahl and R.A. Flickinger Department of Biological Sciences State University of New York Buffalo, New York 14260 (Received in final form July 8, 1981)
summary Addition of an analog of histidine, histidinol, together with lowering the level of histidine in the medium, can induce hemoglobin synthesis in murine erythroleukemia cells. Differentiation is triggered by starvation in Dictyosteli uml , Chlan~domonas 2, certain fungi 3 and some prokaryotes4,5. One aspect of starvation is that the synthesis of ribosomal RNA (r-RNA) is reduced. During starvation in bacteria the synthesis of r-RNA is restricted severly with less effect upon messenger RNA synthesis 6. Furthermore, low levels of actinomycin D primarily inhibit the synthesis of r-RNA7, as does restriction of required amino acids in cultures of mammalian cells 8, 9. It has been shown that low levels of actinc~ycin D stimulate melanin synthesis in rapidly growing_ chick retinal pigment cells I0, neuron formation in n~use neuroblastcma cells II and formation of hemoglobin in cultured Friend erythroleukemia cells 12 in vitro. If a low level of actinomycin D promotes differentiation in erythroleukemia cells by lowering tJ~e rate of r-RNA synthesis 7, and if lo~_r anino acid levels also reduce r-RNA synthesis, then it was considered possible that addition of an amino acid analog, together with reduction of the level of the normal acid, might induce differentiation, i.e., hemoglobin synthesis, in cultured Friend leukemia cells. Experiments were perfornmd with analogs of L-isoleucine (0methylthreonine), L-valine (e-ketobutyrate), L-arginine (Hcmoarginine) and Lhistidine (L-histidinol). Preliminary experiments indicated more success with histidinol and only these results are reported. Methods Friend leukemia cells (clone 745-GM86) 1 were grown in suspension culture in plastic culture dishes (60 mm x 15 nm) containing 5 ml of mediua. Stock cells were grown in Eagle's minim~n essential mediua (MI~4) containing 10% unheated foetal bovine se_rua (FBS) and 1% antibiotic-antimycotic solution containing penicillin, streptomycin, fungazone (PSF) at 37°C at 95% air-5% CO 2 . Stock cells were transferred twice each ~eek at an initial concentration of 1 x 105 cells per ml. Control cells were grown in medit~n containing either 10% unheated FBS or 10% unheated FBS which had been dialyzed against Earle's balanced saline medium to remove any endogenous amino acids. Experimental cells were cultured in medium containing 10% dialyzed FBS and lacking histidine or containing a reduced level of histidine. All othpr exogenously added amino acid levels were kept constant between the controls and experimental cultures. The experimental culture medium was made up using Earle's Balanced saline 1The cells were obtained frcm Dr. Arthor E. Green, the H ~ n a n ~ t a n t Repository, Institute for Medical Research, Camden, New Jersey. 0024-3205/81/121203-03502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
Cell
1204
Histidinol Induces Hemoglobin Synthesis
Vol. 29, No. 12, 1981
solution with 1% antibiotic-antimycotic, 10% dialyzed FBS, the normal level of vitanins found in M~4 and the addition of all essential amino acids at normal MHM levels except histidine. This m e d i ~ n w a s used t o d i l u t e the 5~94PSF-10% dialyzed FBS to achieve the desired final histidine concentration. All constituents of the mediumw~re obtained frcmGrand Island Biological Supply Co., Grand Island, New York. Control and experimental cultures were initially seeded at 2 x 105 cells per ml. Histidinol-containing medium was added 3-4 hours past the time of experimental seeding. The percent of benzidine-reactive cells containing hemoglobin was determined by the method of Friedman and Schildkraut 13. Four countings were made for each experiment and then averaged. Cells were stained with 0.1% trypan blue to estimate the percent of living cells. Cell counts were made with a Neubar cell counter grid and are an average of eight separate countings. The cell counts for cell number, percent viable cells and percent benzidine-positive cells in any one experiment, and cfmparing different experiments, did not vary more than 5% frmm the average for any particular experimental regime. Results and Discussion Omission of L-histidine from the medium inhibits cell proliferation so that the analog, L-histidinol, was added to the experimental medit~n (Earle's saline containing the normal M~4 levels of vitamins and all amino acids except L-histidine, 10% dialyzed FBS and 1% PSF), together with l o ~ r than normal M194 levels of histidine. After five days of culture the cell n~mlber, percent viability and percent benzidine-positive cells were determined. The results are presented in Table 1 and are the averages of 4 different experiments. TABLE 1 Effect of L-Histidinol upon Cell Growth and Hemoglobin Formation
Percent Viability 95 95
Percentbenzidine positive cells < 1.0 < 1.0
41.0
80
77.0
Experimental L-Histidinol (0.4 ~M) + 0.15niM L-histidine + 0.10raM L-histidine
13.6 5.4
72 64
5.6 ll.0
L-Histidinol (0.3 mM) + 0.15raM L-histidine + 0. l~aM L-histidine
17.0 7.6
80 75
7.8 14.0
L-Histidinol (0.2 n%M) + 0.15n~ L-histidine + 0.10ram L-histidine + 0.05raM L-histidine
24.0 20.4 16.4
86 84 80
12.0 14.5 22.4
Controls ~/~4, 10% M~4, 10% M194, 10% +200 n~4
FBS, PS dialyzed FBS, PSF dialyzed FBS, PSF dimethylsulfoxide
Cell ntmlber x 105 cell/ml 44.2 27.6
The normal level of L-histidine in Eagle's MI94 is 0.2 mM and the most effective means of inducing hemoglobin formation was to reduce L-histidine to 1/4 its normal level in M194 (0.05 n~4) and add the L-histidine analog, Lhistidinol, at the normal level of L-histidine in the median. Increasing levels of L-histidinol were toxic as seen by decreased cell number and viability
Vol. 29, No. 12, 1981
Histidinol Induces Hemoglobin Synthesis
1205
at higher concentrations. With even lower levels of L-histidine (0.025 n~4), which is 1/8 the normal level in MI~4, little cell growth occurred. It is known that omission of leucine from the median can cause Friend cells to arrest in the G1 phase of the cell cycle 13. No cell-cycle experiments were carried out in this study, but it would be interesting to repeat these experiments with synchronized cells to see if the histidinol effect is related to the cell cycle. Yet another extension of this work will be to ascertain the means by which dimethylsulfoxide prevents the inhibition of growth in dialyzed fcetal calf serum. L-histidinol is a potent, reversible inhibitor of protein synthesis in cultured human cells 14 . In cultures of meuse ~nbryo fibroblasts containing L-histindinol, and with L-histidine depletion, both protein and ribosomal RNA synthesis are ~ i t e d , probably because about 40% of the t-RNA his is uncharged 15. It is likely that L-histidinol stimulates hemoglobin synthesis by inhibition of ribosomal RNA synthesis due to the uncharged t-RNA, followed by a restriction of protein synthesis. No measurements of ribosomal RNA synthesis were performed in this study, but since histidinol inhibits growth it is likely that r-RNA synthesis is inhibited8,9. However, there is no proof that ~ition of r-RNA synthesis induces differentiation. L-ethionine also induces differentiation in murine erythroleukemia cells 16 In this study about 25-30% of the cells in the culture were stimulated to form hanoglobin after 4-5 days of culture. These workers suggested that ethionineinduced Friend cell differentiation was due to an inhibition of the methylation of DNA and t-RNA. Among the inducers of differentiation of murine ervthroleukemia cells are a number of inhibitors of DNA, RNA and protein synthesis 12. The fact that the analog of L-histidine, L-histidinol, also has inducing activity suggests that induction of the final steps of differentiation in Friend tt~nor cells involves some form of metabolic starvation, just as is true in lo~_r organisms I-5 . References i.
J.T. BONNER, The Cellular Slime Molds. Princeton Univ. Press, Princeton, New Jersey (1967). 2. R. SAGER, S. GRANICK, J. Gen. Physiol. 37 729-742 (1954). 3. L.E. HAWKER, The Physiology of Reproduct-[on in Fungi. Hafner Pub. Co., New York (1971). 4. E. FREEZE, T. ICHIKA~, Y.K. OH, E.B. FREEZE, and C. PRASAD, Proc. Nat. Acad. Sci. U.S. 71 4188-4193 (1974). 5. W. WIP4~4AN, and M. DWORKIN, Sci. 189 516-523 (1975). 6. D.W. MORRIS and N.O. Kjeldgaard, J. Mol. Biol. 31 145-148 (1968). 7. R.P. PERRY, and D.E. K~IT~Y, J. Cell Physiol. 72-235-245 (1968). 8. B.E.H. M A D e , M.H. VALK~HAN, J.R. WARNER, and J.-E. DARNELL, J. Mol. Biol. 45 265-275 (1969). 9. M.H. VAUGHAN, R. SOEIRO, J.R. WARNER, and J.E. DARNRII., Proc. Nat. Acad. Sci. U.S. 58 1527-1534 (1967) i0. J. WHITtAKEr, J. Exp. Zool. 169 143-160 (1969). Ii. D. SCHUBERT, S. }K/MPHREYS, F. DEVITRY, and F. JACOB, Devel. Biol. 25 514-546 (1971). 12. P.S. EBERT, I. WARS, and D.N. BUELL, Cancer Res. 36 1809-1813 (1976). 13. E.A. FRIEDMAN, and C.L. SCHILDKRAUT, Cell 12 901-9---13 (1977). 14. B.S. HANS,, M.H. VAUGHAN, and L. WANG, J.-Biol. Chem. 247 3854-3857 (1972) 15. F. GRL~MMT, and I. G ~ , Eur. J. Biochem. 4 307-312 (i~6). 16. J.K. CHRIS~MAN, P. PRICE, L. P~DRINAN, and ~. ACS, Eur. J. Biochem. 81 53-61 (1977).