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L-ASPARAGINASE AND BLASTOGENESIS
423 the newly proposed " course " organised by the Worshipful Society of Apothecaries of London will be successful in sorting out the really able teachers of medical history. University Department of Child Health, Royal Hospital for Sick Children, Bristol 2.
A. V. NEALE.
L-ASPARAGINASE AND BLASTOGENESIS wondered whether L-asparaginase damages cells as such, or particular biological conditions of these cells-in other words, whether it is a specific agent against the " malignancy " of tumour cells, or whether intensely active normal cells could also be inhibited in their biological response and in their growth by deprivation of L-asparagine. A good model for such a study seemed to us to be human peripheral-blood lymphocytes, cultivated in a culture medium added to phytohaemagglutinin (P.H.A.), since such cells intensively synthesise R.N.A., proteins, and D.N.A., and undergo mitotic division.1 In a group of 9 subjects without any hxmatological or immunological disorders, 4 lots of peripheral-blood lymphocyte cultures per subject were set up. The lst lot was set up in a culture medium to which no extra agent was added (blank controls). The 2nd lot was set up in a medium to which P.H.A., 0-02 mg. per ml. medium, was added P.H.A.-controls). (This P.H.A. was a highly purified non-commercial product, kindly supplied by Dr. A. H. Griffith, Clinical Research Department, Wellcome Research Laboratory, Langley, Beckenham, Kent, U.K.) The 3rd lot was set up in a medium added to which L-asparaginase, 5 i.u. per ml. medium, was added. (This drug was of almost 95% purity, and had no L.D’50 level in rats; it was kindly supplied by Dr. J. Hill, Wadley Institutes of Molecular Medicine, Dallas, Texas, U.S.A.) The 4th lot was set up in a medium to which were added both P.H.A. and L-asparaginase, in the above-mentioned doses. Lymphocytes were obtained from heparinised cubital-vein blood and added to a 6% dextran solution, in the proportion of 20/1 by volume. The culture medium was composed of’N.C.T.C. 109’ (Microbiological Associates) 7-6 parts; the lymphocyte-donor’s plasma 2 parts; isotonic saline solution alone (1st lot), or added to P.H.A. (2nd lot), to L-asparaginase (3rd lot), or to P.H.A. + Lasparaginase (4th lot), 0-4 parts. After 72 hours’ incubation, smear preparations of the cultures were made, and treated with May-Grunwald-Giemsa stain. 500 cells of the all-lymphocytic cell population per culture were then examined, and the percentage of blast-like cells determined. In fact, morphological criteria for judging which lymphocytes undergo blastic transformation have been considered as reliable as,2 or in some instances even superior to,3 the criteria based on incorporation of [H3]-thymidine. Percentages of blasts 4:s.D. in the 4 different media after 3 days’ culture were as follows:
SIR,-We
tumour
In the accompanying figure, the blasts developed in the P.H.A./ L-asparaginase cultures of each case are graphically related to the values for blasts obtained in the P.H.A.-control cultures, which are taken as 100%. As expected, there was no significant blastic transformation in the lymphocytes cultured without P.H.A., either in those cultures set up in standard medium alone (blank controls), or in those set up in standard medium with L-asparaginase. On the other hand, there was normal blastic transformation (67-48:7-46%) in the lymphocytes cultured with P.H.A. (P.H.A.-controls), though there was low transformation when L-asparaginase was added to the lymphocyte/ 1.
2. 3.
Nowell, P. C. Cancer Res. 1961, 21, 1501. Cooper, E. H., Barkhan, P., Hale, A. J. Lancet, 1961, ii, 210. McIntyre, O. R., Ebaugh, F. G., Jr. Blood, 1962, 19, 443. Astaldi, G., Strosselli, E., Sauli, S. Hœmatologica, 1962, 47, suppl. p. 1. Winkelstein, A., Craddock, C. G. Blood, 1967, 29, 594. Schwarz, M. R. Proc. Soc. exp. Biol. Med. 1967, 125, 701.
Blasts
developed in P.H.A./L-asparaginase cultures (shaded columns) as percentages of blasts developed in P.H.A. cultures (hatched column-100%).
cultures (2-32:1-98%). The untransformed lymphocytes in the cultures to which both P.H.A. and L-asparaginase were added were morphologically well preserved. Again, the great majority of the untransformed lymphocytes were viable at least by the trypan-blue staining test, when examined periodically every 3 hours until 36 hours after the start of culture. These results, taken as a whole, show that L-asparaginase can intensely inhibit blastic transformation of human peripheralblood lymphocytes in the P.H.A.-culture system. Therefore, these data support the interpretation that L-asparaginase can act in cell culture, not only on tumour cells by inhibiting their growth but also on normal lymphocytes during their immunological activation. In other words, it seems logical to believe that human peripheral-blood lymphocytes need L-asparagine for their blastic transformation, and that they are not able to synthesise this aminoacid during such intense biological activities as are required for blastic transformation. It is assumed that lymphocytes in the P.H.A.-culture system cannot grow in the absence of L-asparagine. P.H.A.
This study was performed with a grant from A. Nattermann, Cologne-Braunsfeld, Germany. This Centre is supported by the Blood Research Foundation, Washington, D.C., U.S.A. Dr. J. Krc is from the lst Medical Clinic, Department of Internal Medicine, University Medical School, Olomouc, Czechoslovakia.
G. ASTALDI G. R. BURGIO Blood Research Foundation Centre, Tortona Hospital, and Pædiatric Clinic, University Medical School, Pavia, Italy.
J. KRČ R. GENOVA A. A. ASTALDI,
Jr.
L-ASPARAGINASE AND LOW-PROTEIN DIET IN ACUTE LEUKÆMIA SIR,-Papers on the effectiveness of L-asparaginase in acute leukxmia 1-3 prompt us to recall our communications on the beneficial action of low-protein diet in the treatment of this disease in children.4-6 Administration of L-asparaginase resulting in depletion of extracellular L-asparagine content has been successful in patients with acute lymphoblastic leukaemia, inducing remarkable destruction of leukxmic infiltrations in 1. 2.
3. 4. 5. 6.
Dolowy, W. C., Henson, D., Cornet, J., Sellin, H. Cancer, N.Y. 1966, 19, 1813. Old, L. J., Boyse, E. A., Campbell, H. A., Brodey, R. S., Fidler, J., Teller, J. D. Lancet, 1967, i, 447. Hill, J. M., Roberts, J., Loeb, E., Khan, A., Machellan, A., Hill, R. W. J. Am. med. Ass. 1967, 202, 882. Halikowski B., Armata, J., Garwicz, S. Lancet, 1965, i, 704. Halikowski, B., Armata, J., Garwicz, S. Br. med. J. 1966, i, 519. Garwicz, S., Halikowski, B., Armata, J., Wyszkowski, J. Przegl. Metod. Akad. Med., Kraków, 1967, 2, 93.
A. V. NEALE.
L-ASPARAGINASE AND BLASTOGENESIS wondered whether L-asparaginase damages cells as such, or particular biological conditions of these cells-in other words, whether it is a specific agent against the " malignancy " of tumour cells, or whether intensely active normal cells could also be inhibited in their biological response and in their growth by deprivation of L-asparagine. A good model for such a study seemed to us to be human peripheral-blood lymphocytes, cultivated in a culture medium added to phytohaemagglutinin (P.H.A.), since such cells intensively synthesise R.N.A., proteins, and D.N.A., and undergo mitotic division.1 In a group of 9 subjects without any hxmatological or immunological disorders, 4 lots of peripheral-blood lymphocyte cultures per subject were set up. The lst lot was set up in a culture medium to which no extra agent was added (blank controls). The 2nd lot was set up in a medium to which P.H.A., 0-02 mg. per ml. medium, was added P.H.A.-controls). (This P.H.A. was a highly purified non-commercial product, kindly supplied by Dr. A. H. Griffith, Clinical Research Department, Wellcome Research Laboratory, Langley, Beckenham, Kent, U.K.) The 3rd lot was set up in a medium added to which L-asparaginase, 5 i.u. per ml. medium, was added. (This drug was of almost 95% purity, and had no L.D’50 level in rats; it was kindly supplied by Dr. J. Hill, Wadley Institutes of Molecular Medicine, Dallas, Texas, U.S.A.) The 4th lot was set up in a medium to which were added both P.H.A. and L-asparaginase, in the above-mentioned doses. Lymphocytes were obtained from heparinised cubital-vein blood and added to a 6% dextran solution, in the proportion of 20/1 by volume. The culture medium was composed of’N.C.T.C. 109’ (Microbiological Associates) 7-6 parts; the lymphocyte-donor’s plasma 2 parts; isotonic saline solution alone (1st lot), or added to P.H.A. (2nd lot), to L-asparaginase (3rd lot), or to P.H.A. + Lasparaginase (4th lot), 0-4 parts. After 72 hours’ incubation, smear preparations of the cultures were made, and treated with May-Grunwald-Giemsa stain. 500 cells of the all-lymphocytic cell population per culture were then examined, and the percentage of blast-like cells determined. In fact, morphological criteria for judging which lymphocytes undergo blastic transformation have been considered as reliable as,2 or in some instances even superior to,3 the criteria based on incorporation of [H3]-thymidine. Percentages of blasts 4:s.D. in the 4 different media after 3 days’ culture were as follows:
SIR,-We
tumour
In the accompanying figure, the blasts developed in the P.H.A./ L-asparaginase cultures of each case are graphically related to the values for blasts obtained in the P.H.A.-control cultures, which are taken as 100%. As expected, there was no significant blastic transformation in the lymphocytes cultured without P.H.A., either in those cultures set up in standard medium alone (blank controls), or in those set up in standard medium with L-asparaginase. On the other hand, there was normal blastic transformation (67-48:7-46%) in the lymphocytes cultured with P.H.A. (P.H.A.-controls), though there was low transformation when L-asparaginase was added to the lymphocyte/ 1.
2. 3.
Nowell, P. C. Cancer Res. 1961, 21, 1501. Cooper, E. H., Barkhan, P., Hale, A. J. Lancet, 1961, ii, 210. McIntyre, O. R., Ebaugh, F. G., Jr. Blood, 1962, 19, 443. Astaldi, G., Strosselli, E., Sauli, S. Hœmatologica, 1962, 47, suppl. p. 1. Winkelstein, A., Craddock, C. G. Blood, 1967, 29, 594. Schwarz, M. R. Proc. Soc. exp. Biol. Med. 1967, 125, 701.
Blasts
developed in P.H.A./L-asparaginase cultures (shaded columns) as percentages of blasts developed in P.H.A. cultures (hatched column-100%).
cultures (2-32:1-98%). The untransformed lymphocytes in the cultures to which both P.H.A. and L-asparaginase were added were morphologically well preserved. Again, the great majority of the untransformed lymphocytes were viable at least by the trypan-blue staining test, when examined periodically every 3 hours until 36 hours after the start of culture. These results, taken as a whole, show that L-asparaginase can intensely inhibit blastic transformation of human peripheralblood lymphocytes in the P.H.A.-culture system. Therefore, these data support the interpretation that L-asparaginase can act in cell culture, not only on tumour cells by inhibiting their growth but also on normal lymphocytes during their immunological activation. In other words, it seems logical to believe that human peripheral-blood lymphocytes need L-asparagine for their blastic transformation, and that they are not able to synthesise this aminoacid during such intense biological activities as are required for blastic transformation. It is assumed that lymphocytes in the P.H.A.-culture system cannot grow in the absence of L-asparagine. P.H.A.
This study was performed with a grant from A. Nattermann, Cologne-Braunsfeld, Germany. This Centre is supported by the Blood Research Foundation, Washington, D.C., U.S.A. Dr. J. Krc is from the lst Medical Clinic, Department of Internal Medicine, University Medical School, Olomouc, Czechoslovakia.
G. ASTALDI G. R. BURGIO Blood Research Foundation Centre, Tortona Hospital, and Pædiatric Clinic, University Medical School, Pavia, Italy.
J. KRČ R. GENOVA A. A. ASTALDI,
Jr.
L-ASPARAGINASE AND LOW-PROTEIN DIET IN ACUTE LEUKÆMIA SIR,-Papers on the effectiveness of L-asparaginase in acute leukxmia 1-3 prompt us to recall our communications on the beneficial action of low-protein diet in the treatment of this disease in children.4-6 Administration of L-asparaginase resulting in depletion of extracellular L-asparagine content has been successful in patients with acute lymphoblastic leukaemia, inducing remarkable destruction of leukxmic infiltrations in 1. 2.
3. 4. 5. 6.
Dolowy, W. C., Henson, D., Cornet, J., Sellin, H. Cancer, N.Y. 1966, 19, 1813. Old, L. J., Boyse, E. A., Campbell, H. A., Brodey, R. S., Fidler, J., Teller, J. D. Lancet, 1967, i, 447. Hill, J. M., Roberts, J., Loeb, E., Khan, A., Machellan, A., Hill, R. W. J. Am. med. Ass. 1967, 202, 882. Halikowski B., Armata, J., Garwicz, S. Lancet, 1965, i, 704. Halikowski, B., Armata, J., Garwicz, S. Br. med. J. 1966, i, 519. Garwicz, S., Halikowski, B., Armata, J., Wyszkowski, J. Przegl. Metod. Akad. Med., Kraków, 1967, 2, 93.