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Leukemia Research Vol. 17, No. 11, pp. 1017-1018, 1993. Printed in Great Britain.
0145-2126/93 $6(10 ÷ .00 O 1993 Pergamon Press Ltd
LETTERS TO THE EDITORS INDUCTION
OF DIFFERENTIATION OF HL-60 AND WEHI-3B CELLS BY LITHIUM CHLORIDE
THE RECENT article by Sokoloski et al. [1] overlooks our earlier study of the interaction of lithium and retinoic acid on HL-60 cells and the expression of c-myc and c-fms proto-oncogenes in these cells in the presence of lithium [2]; studies evidently of relevance to the issue of induced differentiation. Furthermore, these authors assert that we and others [3] 'did not monitor the effect of concentrations of lithium in the range of 10-20 m M on the induction of differentiation, the range of the cation that produced optimum differentiation in the present study'. In fact, such studies were conducted (10 -2 M = 10 raM, see Table 2, ref. [2] and also ref. [4]). It is of note that Sokoloski et al. observed no effect on HL-60 of lithium at <5 mM, a concentration already supra-therapeutic in the clinical context. Furthermore, the suggestion by these authors, that even higher concentrations should be contemplated in oioo, is decidedly dangerous. As they and we have observed, concentrations exceeding 10-20mM in vitro are cytotoxic for HL-60 cells; we have demonstrated reduced clonogenesis from normal human bone marrow in these circumstances [5]; and such concentrations are predictably toxic, and sometimes fatal [6], with associated leucopenia [6] in patients. Why are the findings of Sokoloski et al. apparently at variance with earlier, reported experience? As we [7] and others [3] have suggested, there may be true biological differences between HL-60 clones. At least as important are the differences between the effects of c/s-retinoic acid (as used in our study) and alltrans-retinoic acid (as used by Sokoloski et al.) on granulocyte differentiation. Although these isomers have been reported to exhibit comparable effects on HL-60 cells [8], all-trans-retinoic acid appears to be
REPLY
D + LEUKEMIA
more potent in the induction of differentiation in fresh promyelocytic leukemia cells [9]. REFERENCES 1. Sokoloski J. A., Li J., Nigam A. & Sartorelli A. C. (1993) Induction of differentiation of HL-60 and WEHI3B D ÷ leukemia cells by lithium chloride. Leukemia Res. 17, 403. 2. Knight S. C., Harnish D., Scheid E., Koekebakker M & Barr R. D. (1989) Lithium and hydrocortisone interactions on cell growth and gene expression in human promyelocytic leukemia (HL60). Leukemia Res. 13, 289. 3. Tyobeka E. M. & Becker R. W. (1990) Growth and morphological changes induced by lithium chloride treatment in HL-60 cells. Cell Biol. Int. Rep. 14, 667. 4. Becker R. W. & Tyobeka E. M. (1990) Lithium enhances the proliferation of HL-60 promyelocytic leukemia cells. Leukemia Res. 14, 879. 5. Barr R. D., Koekebakker M., Brown E. A. & Falbo M. C. (1987). Putative role for lithium in human hematopoiesis. J. Lab. Clin. Med. 109, 159. 6. Green S. T. & Dunn F. G. (1985) Severe leucopenia in fatal lithium poisoning. BMJ 290, 517. 7. Barr R. D. and Harnish D. (1991) The impact of lithium on human leukemic cells. Leukemia Res. 15, 766. 8. Koeffler H. P. (1983) Induction of differentiation of human acute myeiogenous leukemia cells: therapeutic implications. Blood 62, 709. 9. Chomienne C., Ballerini P., Balitrand N., Daniel M. T., Fenaux P., Castaigne S. & Degos L. (1990) All-trans retinoic acid in acute promyelocytic leukemias II. In vitro studies: structure-function relationship. Blood 76, 1710. RONALD D. BARR*t and DELSWORTHHARNISHt Departments of *Pediatrics and tPathology McMaster University Hamilton Ontario L8N 3Z5 Canada
TO THE
WE ARE appreciative of the opportunity to respond to the letter to the Editor written by R. D. Barr and D. Harnish. These individuals give the impression that we did not cite their report [2] on the interaction
COMMENTS of lithium and retinoic acid on HL-60 cells, when in fact, we did not 'overlook' their study; this manuscript was ref. [20] in our paper. Although changes in the expression of c-myc and c-fms proto-oncogenes
1017
1018
Letters to the Editors
presumably are of importance to the induction of the differentiation of these cells, these changes had no direct bearing on the results of our study. We concur that Knight et al. [2] did use 10 mM lithium in their studies; however, in Table 2 only cellular proliferation was monitored; no data on cell differentiation are presented in this table. Although these authors state (p. 290) that lithium did not modulate cell growth, morphology or cytochemistry of HL-60 cells in the presence of retinoic acid, no information on the concentrations of lithium employed was presented. Becker and Tyobeka [3] did not measure the differentiation status of cells in their report. Becket and Tyobeka in a relatively recent paper [4], however, did state in results not shown that they evaluated concentrations of lithium of 0-50 mM and no induction of differentiation above basal levels was detected. The clone of HL-60 employed in their study gave 56% nitroblue tetrazolium positive cells in the absence of inducing agent, which is an exceedingly high basal level of induction of differentiation. Barr and Harnish are incorrect in their statement that we did not observe an effect of lithium on HL60 cells at <5 mM. Figure 1 of our report [1] demonstrates a small but statistically significant increase in Mol positive (differentiated) HL-60 cells at 3 mM lithium, and Figs 2 and 5 of this report present data that suggest that lithium enhances the retinoic acid induced differentiation of WEHI-3B D ÷ and HL-60 cells, respectively, at concentrations of lithium below 5 mM. With respect to the possible use of supra-therapeutic doses of lithium in patients, we obviously agree that levels of this agent that produce unacceptable toxicities to humans cannot be employed. We speculated (admittedly poorly presented) that short term administration of lithium might well allow a greater dose level than that employed chronically for the treatment of manic-depressive disorders. It is quite possible that the 5 mM concentration of lithium seen in the plasma of patients during acute toxicity may not completely reflect the true levels of lithium responsible for producing toxicity, since patients presumably will have entered the hospital several hours after ingestion of an acute dose of the agent, thereby
reducing the measured plasma levels. Furthermore, plasma levels may not be indicative of the degree of redistribution of lithium into tissues, which may accumulate significantly higher levels of lithium than those found in the plasma. These issues suggest that further pharmacokinetic studies will be necessary to determine possible therapeutic levels of this cation as a differentiating agent in combination with retinoic acid. Finally, we agree with Barr and Harnish that significant variability may well exist between clones of HL-60 cells used in different laboratories. It is for this reason that, in addition to HL-60, we employed the WEHI-3B D ÷ murine myelomonocytic cell line in our study to confirm the generality of our observations to other leukemic models. Furthermore, as we indicated previously, the concentration of lithium that we employed could be reduced to levels below 5 mM when employed with low concentrations (10100 riM) of retinoic acid. While Knight et al. [2] mentioned (p. 290) that lithium employed in combination with retinoic acid did not modulate the differentiation of HL-60 cells, no data were presented to support this statement. In addition, the concentration of retinoic acid employed in their studies (10/~M) results, in our hands, in maximum differentiation of HL-60 cells making it unlikely that a further increase in the number of mature cells could be discerned when used in combination with lithium. REFERENCES 1. Sokoloski J. A., Li J., Nigam A. & Sartoreili A. C. (1993) Leukemia Res. 17, 403. 2. Knight S. C., Harnish D., Scheid E., Koekebakker M. & Barr R. D. (1989) Leukemia Res. 13, 289. 3. Becket R. W. & Tyobeka E. M. (1990) Leukemia Res. 14, 879. 4. Becker R. W. & Tyobeka E. M. (1992) Leukemia Res. 16, 823. JOHN A. SOKOLOSKI and ALAN C. SARTORELLI
Department of Pharmacology & Developmental Therapeutics Program Comprehensive Cancer Center Yale University School of Medicine New Haven CT 06510 U.S.A.
0145-2126/93 $6(10 ÷ .00 O 1993 Pergamon Press Ltd
LETTERS TO THE EDITORS INDUCTION
OF DIFFERENTIATION OF HL-60 AND WEHI-3B CELLS BY LITHIUM CHLORIDE
THE RECENT article by Sokoloski et al. [1] overlooks our earlier study of the interaction of lithium and retinoic acid on HL-60 cells and the expression of c-myc and c-fms proto-oncogenes in these cells in the presence of lithium [2]; studies evidently of relevance to the issue of induced differentiation. Furthermore, these authors assert that we and others [3] 'did not monitor the effect of concentrations of lithium in the range of 10-20 m M on the induction of differentiation, the range of the cation that produced optimum differentiation in the present study'. In fact, such studies were conducted (10 -2 M = 10 raM, see Table 2, ref. [2] and also ref. [4]). It is of note that Sokoloski et al. observed no effect on HL-60 of lithium at <5 mM, a concentration already supra-therapeutic in the clinical context. Furthermore, the suggestion by these authors, that even higher concentrations should be contemplated in oioo, is decidedly dangerous. As they and we have observed, concentrations exceeding 10-20mM in vitro are cytotoxic for HL-60 cells; we have demonstrated reduced clonogenesis from normal human bone marrow in these circumstances [5]; and such concentrations are predictably toxic, and sometimes fatal [6], with associated leucopenia [6] in patients. Why are the findings of Sokoloski et al. apparently at variance with earlier, reported experience? As we [7] and others [3] have suggested, there may be true biological differences between HL-60 clones. At least as important are the differences between the effects of c/s-retinoic acid (as used in our study) and alltrans-retinoic acid (as used by Sokoloski et al.) on granulocyte differentiation. Although these isomers have been reported to exhibit comparable effects on HL-60 cells [8], all-trans-retinoic acid appears to be
REPLY
D + LEUKEMIA
more potent in the induction of differentiation in fresh promyelocytic leukemia cells [9]. REFERENCES 1. Sokoloski J. A., Li J., Nigam A. & Sartorelli A. C. (1993) Induction of differentiation of HL-60 and WEHI3B D ÷ leukemia cells by lithium chloride. Leukemia Res. 17, 403. 2. Knight S. C., Harnish D., Scheid E., Koekebakker M & Barr R. D. (1989) Lithium and hydrocortisone interactions on cell growth and gene expression in human promyelocytic leukemia (HL60). Leukemia Res. 13, 289. 3. Tyobeka E. M. & Becker R. W. (1990) Growth and morphological changes induced by lithium chloride treatment in HL-60 cells. Cell Biol. Int. Rep. 14, 667. 4. Becker R. W. & Tyobeka E. M. (1990) Lithium enhances the proliferation of HL-60 promyelocytic leukemia cells. Leukemia Res. 14, 879. 5. Barr R. D., Koekebakker M., Brown E. A. & Falbo M. C. (1987). Putative role for lithium in human hematopoiesis. J. Lab. Clin. Med. 109, 159. 6. Green S. T. & Dunn F. G. (1985) Severe leucopenia in fatal lithium poisoning. BMJ 290, 517. 7. Barr R. D. and Harnish D. (1991) The impact of lithium on human leukemic cells. Leukemia Res. 15, 766. 8. Koeffler H. P. (1983) Induction of differentiation of human acute myeiogenous leukemia cells: therapeutic implications. Blood 62, 709. 9. Chomienne C., Ballerini P., Balitrand N., Daniel M. T., Fenaux P., Castaigne S. & Degos L. (1990) All-trans retinoic acid in acute promyelocytic leukemias II. In vitro studies: structure-function relationship. Blood 76, 1710. RONALD D. BARR*t and DELSWORTHHARNISHt Departments of *Pediatrics and tPathology McMaster University Hamilton Ontario L8N 3Z5 Canada
TO THE
WE ARE appreciative of the opportunity to respond to the letter to the Editor written by R. D. Barr and D. Harnish. These individuals give the impression that we did not cite their report [2] on the interaction
COMMENTS of lithium and retinoic acid on HL-60 cells, when in fact, we did not 'overlook' their study; this manuscript was ref. [20] in our paper. Although changes in the expression of c-myc and c-fms proto-oncogenes
1017
1018
Letters to the Editors
presumably are of importance to the induction of the differentiation of these cells, these changes had no direct bearing on the results of our study. We concur that Knight et al. [2] did use 10 mM lithium in their studies; however, in Table 2 only cellular proliferation was monitored; no data on cell differentiation are presented in this table. Although these authors state (p. 290) that lithium did not modulate cell growth, morphology or cytochemistry of HL-60 cells in the presence of retinoic acid, no information on the concentrations of lithium employed was presented. Becker and Tyobeka [3] did not measure the differentiation status of cells in their report. Becket and Tyobeka in a relatively recent paper [4], however, did state in results not shown that they evaluated concentrations of lithium of 0-50 mM and no induction of differentiation above basal levels was detected. The clone of HL-60 employed in their study gave 56% nitroblue tetrazolium positive cells in the absence of inducing agent, which is an exceedingly high basal level of induction of differentiation. Barr and Harnish are incorrect in their statement that we did not observe an effect of lithium on HL60 cells at <5 mM. Figure 1 of our report [1] demonstrates a small but statistically significant increase in Mol positive (differentiated) HL-60 cells at 3 mM lithium, and Figs 2 and 5 of this report present data that suggest that lithium enhances the retinoic acid induced differentiation of WEHI-3B D ÷ and HL-60 cells, respectively, at concentrations of lithium below 5 mM. With respect to the possible use of supra-therapeutic doses of lithium in patients, we obviously agree that levels of this agent that produce unacceptable toxicities to humans cannot be employed. We speculated (admittedly poorly presented) that short term administration of lithium might well allow a greater dose level than that employed chronically for the treatment of manic-depressive disorders. It is quite possible that the 5 mM concentration of lithium seen in the plasma of patients during acute toxicity may not completely reflect the true levels of lithium responsible for producing toxicity, since patients presumably will have entered the hospital several hours after ingestion of an acute dose of the agent, thereby
reducing the measured plasma levels. Furthermore, plasma levels may not be indicative of the degree of redistribution of lithium into tissues, which may accumulate significantly higher levels of lithium than those found in the plasma. These issues suggest that further pharmacokinetic studies will be necessary to determine possible therapeutic levels of this cation as a differentiating agent in combination with retinoic acid. Finally, we agree with Barr and Harnish that significant variability may well exist between clones of HL-60 cells used in different laboratories. It is for this reason that, in addition to HL-60, we employed the WEHI-3B D ÷ murine myelomonocytic cell line in our study to confirm the generality of our observations to other leukemic models. Furthermore, as we indicated previously, the concentration of lithium that we employed could be reduced to levels below 5 mM when employed with low concentrations (10100 riM) of retinoic acid. While Knight et al. [2] mentioned (p. 290) that lithium employed in combination with retinoic acid did not modulate the differentiation of HL-60 cells, no data were presented to support this statement. In addition, the concentration of retinoic acid employed in their studies (10/~M) results, in our hands, in maximum differentiation of HL-60 cells making it unlikely that a further increase in the number of mature cells could be discerned when used in combination with lithium. REFERENCES 1. Sokoloski J. A., Li J., Nigam A. & Sartoreili A. C. (1993) Leukemia Res. 17, 403. 2. Knight S. C., Harnish D., Scheid E., Koekebakker M. & Barr R. D. (1989) Leukemia Res. 13, 289. 3. Becket R. W. & Tyobeka E. M. (1990) Leukemia Res. 14, 879. 4. Becker R. W. & Tyobeka E. M. (1992) Leukemia Res. 16, 823. JOHN A. SOKOLOSKI and ALAN C. SARTORELLI
Department of Pharmacology & Developmental Therapeutics Program Comprehensive Cancer Center Yale University School of Medicine New Haven CT 06510 U.S.A.