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Leukemia Research Vol. 12. No. 8, p. 699, 1988. Printed in Great Britain.
0145 2126/88 $3.00 + .00 Pergamon Press plc
LETTERS TO THE EDITOR COMMENTS ON " T H E M O L E C U L A R BASIS F O R THE D I F F E R E N T I A L SENSITIVITY OF B AND T LYMPHOCYTES TO G R O W T H INHIBITION BY T H Y M I D I N E AND 5 - F L U O R O U R A C I L " *
THE WORKdescribed in the above paper was carried out by Dr Assouli while he was a post-doctoral trainee in the Department of Experimental Therapeutics, Roswell Park Memorial Institute. (1) It was stated that the enzyme from H R 1 K cells was thymidine phosphorylase. According to laboratory records, it was uridine phosphorylase (the enzyme used both thymidine and uridine as substrates), which has been suggested as a marker for mycoplasma contamination [1]. This enzyme was present in H R 1 K cells. These cells were known to be contaminated with mycoplasma at that time. (2) The values for thymidine phosphorylase given in Fig. 3 are puzzling. For example, values for MOLT-4 and RPMI-1788 are nearly comparable and peripheral blood lymphocytes, which are known, e.g. [2] and our findings, to be 4-6 fold higher in thymidine phosphorylase activity compared to B-cell lines and 20-40 fold higher compared to T-ALL cell lines, which are shown to be lowest in activity. (3) The cytotoxicity data in Fig. 2 is based on only one of four squares in the hematocytometer being counted. Moreover, high initial cell count (11.26 × 106/ml) at the time of drug addition not only
precluded cell growth, but also led to significant loss of cell viability already by day two even in controls. (4) The results in Figs 1 and 4 are based on experiments which were carried out by pelleting cells and resuspending in the fresh medium. The data for 8402, H R I K and K-562 in Fig. 1 are from the same experiment as in Fig. 2. In addition, values such as 140 and 170% are plotted as 100% in Fig. 1.
REFERENCES 1. Levine E. M. (1972) Mycoplasma contamination of animal cell cultures: a simple, rapid detection method. Expl Cell Res. 74, 99. 2. EngGan T., Hallam L., Pilkington G. R. & Van der Weyden M. B. (1981) A rapid and simple radiometric assay for thymidine phosphorylase of human peripheral blood cells. Clinica chim. Acta 116, 231. B. I. SAHAI SRIVASTAVA
Cancer Research Scientist Department of Laboratory Medicine Roswell Park Memorial Institute Buffalo NY 14263, U.S.A.
REPLY TO T H E COMMENTS To DEAL with Dr Srivastava's points in order: (1) I used two sources of H R 1 K cells. The enzymes from these behaved similarly and had the characteristics of thymidine phosphorylase. The first preparation was a gift from Dr J. Minowada and was to the best of my knowledge uncontaminated by mycoplasma. (2) Since B cells contain more thymidine phosphorylase than T cells, my finding that a B-cell line (RPMI-1788) contains more of the enzyme than peripheral blood lymphocytes (80% T cells) does not seem surprising. My finding that the T - A L L cell line M O L T - 4 contains more of the enzyme than
peripheral blood lymphocytes is clearly at variance with Dr Srivastava's own findings. (3) In practice the average of more than one square was taken, and experiments were done in duplicate. High initial cell counts were unavoidable but the cells in the control grew well compared to the cells treated with the drugs. (4) Cell numbers were adjusted by dilution and not by pelleting. S. M. EL-ASSOULI Head, Cancer Research Unit King Abdulaziz University College of Medicine & Allied Sciences King Fahd Medical Research Center Saudi Arabia
* This paper by Sufian M. E1-Assouli was published in Leukemia Res. 9, 391-398 (1985).
699
0145 2126/88 $3.00 + .00 Pergamon Press plc
LETTERS TO THE EDITOR COMMENTS ON " T H E M O L E C U L A R BASIS F O R THE D I F F E R E N T I A L SENSITIVITY OF B AND T LYMPHOCYTES TO G R O W T H INHIBITION BY T H Y M I D I N E AND 5 - F L U O R O U R A C I L " *
THE WORKdescribed in the above paper was carried out by Dr Assouli while he was a post-doctoral trainee in the Department of Experimental Therapeutics, Roswell Park Memorial Institute. (1) It was stated that the enzyme from H R 1 K cells was thymidine phosphorylase. According to laboratory records, it was uridine phosphorylase (the enzyme used both thymidine and uridine as substrates), which has been suggested as a marker for mycoplasma contamination [1]. This enzyme was present in H R 1 K cells. These cells were known to be contaminated with mycoplasma at that time. (2) The values for thymidine phosphorylase given in Fig. 3 are puzzling. For example, values for MOLT-4 and RPMI-1788 are nearly comparable and peripheral blood lymphocytes, which are known, e.g. [2] and our findings, to be 4-6 fold higher in thymidine phosphorylase activity compared to B-cell lines and 20-40 fold higher compared to T-ALL cell lines, which are shown to be lowest in activity. (3) The cytotoxicity data in Fig. 2 is based on only one of four squares in the hematocytometer being counted. Moreover, high initial cell count (11.26 × 106/ml) at the time of drug addition not only
precluded cell growth, but also led to significant loss of cell viability already by day two even in controls. (4) The results in Figs 1 and 4 are based on experiments which were carried out by pelleting cells and resuspending in the fresh medium. The data for 8402, H R I K and K-562 in Fig. 1 are from the same experiment as in Fig. 2. In addition, values such as 140 and 170% are plotted as 100% in Fig. 1.
REFERENCES 1. Levine E. M. (1972) Mycoplasma contamination of animal cell cultures: a simple, rapid detection method. Expl Cell Res. 74, 99. 2. EngGan T., Hallam L., Pilkington G. R. & Van der Weyden M. B. (1981) A rapid and simple radiometric assay for thymidine phosphorylase of human peripheral blood cells. Clinica chim. Acta 116, 231. B. I. SAHAI SRIVASTAVA
Cancer Research Scientist Department of Laboratory Medicine Roswell Park Memorial Institute Buffalo NY 14263, U.S.A.
REPLY TO T H E COMMENTS To DEAL with Dr Srivastava's points in order: (1) I used two sources of H R 1 K cells. The enzymes from these behaved similarly and had the characteristics of thymidine phosphorylase. The first preparation was a gift from Dr J. Minowada and was to the best of my knowledge uncontaminated by mycoplasma. (2) Since B cells contain more thymidine phosphorylase than T cells, my finding that a B-cell line (RPMI-1788) contains more of the enzyme than peripheral blood lymphocytes (80% T cells) does not seem surprising. My finding that the T - A L L cell line M O L T - 4 contains more of the enzyme than
peripheral blood lymphocytes is clearly at variance with Dr Srivastava's own findings. (3) In practice the average of more than one square was taken, and experiments were done in duplicate. High initial cell counts were unavoidable but the cells in the control grew well compared to the cells treated with the drugs. (4) Cell numbers were adjusted by dilution and not by pelleting. S. M. EL-ASSOULI Head, Cancer Research Unit King Abdulaziz University College of Medicine & Allied Sciences King Fahd Medical Research Center Saudi Arabia
* This paper by Sufian M. E1-Assouli was published in Leukemia Res. 9, 391-398 (1985).
699