- Email: [email protected]
Sensitivity of cultured human lymphoblasts (CCRF-CEM cells) to inhibition by thymidine
512
D. 0. Schachtschabel
et al.
Results and Conclusions.--The data in Figs. 2 and 3 demonstrate that although the maximum cell density attained was the same in both vessels, the growth rate in 50 ml spinners was greater than in 10 ml spinners. At the highest fetal calf serum protein concentration tested, this difference was maximal. Thus, the rate of growth and the maximum cell density attained are independent parameters in the description of the growth response to fetal calf serum. The results show that the 10 ml spinner was not conducive to the best growth response possible for a given concentration of fetal calf serum protein. However, these minispinners may be used successfully with small amounts of material with the limitation that if a material is found to be inactive in the IO ml spinners it should not be considered inactive until confirmed with 50 ml spinners. On the other hand, if an unknown material shows growth stimulation in the IO ml spinners it may be considered with certainty to have this activity.
The authors are grateful to Mr Peter Hasenpusch for constructing the minispinners. This work was supported in part by grants AM-07787 and CA-08204 from the United States Public Health Service and by the Helen W. Dutcher Memorial Grant for Cancer Research (E-284) from the American Cancer Society. REFERENCES 1. 2. 3. 4. 5. 6. 7.
DELUCA, C., RAKOWSKI, DELUCA, C., HABEEB, A. GORNALL, A. G., BARDAWILL, MCLIMANS, W. F., DAVIS, MOORE, G. E., MOUNT, D., MOORE, G. E. and ULRICH, TRITSCH, G. L., FLOSS, D.
P. F. and TRITSCH, G. L., Biochim. Biophgs. Acta 86, 346 (1964). F. S. A. and TRITSCH, G. L., Ezpptl Cell Res. 43, 98 (1966). C. J. and DAVID, M. M., J. Biol. Chem. 177, 751 (1949). E. V., GLOWER, F. L. and RAKE, G. W., J. Immunol. 79,428 (1957). TARA, G. and SCHWARTZ, N., J. Nafl Cancer Inst. 31, 1217 (1963). K., J. SurgicaI Res. 5, 270 (1965). R. and MOORE, G. E., Exptl Cell Res. 42, 523 (1966).
SENSITIVITY HUMAN
OF CULTURED
LYMPHOBLASTS INHIBITION
D.
0.
SCHACHTSCHABEL,
The Children’s
H.
(CCRF-CEM BY
LAZARUS,
THYMIDINE S. FARBER,
Cancer Research Foundation, School, at The Children’s
and Department Hospital, Boston,
Received
May 3, 1966
CELLS)
TO
l and
of Pathology, Mass., U.S.A.
G. E. FOLEY Harvard
Medical
T HE
continuous cultivation of human lymphoblasts (CCRF-CEM cells) directly in suspension cultures from the peripheral blood buffy coat of a child with acute lymphoblastic leukemia [2] and the karyological characteristics of these cells [3] have 1 These Institute, Research of Health.
Experimental
studies National Career
Cell
were supported in part by research grant C-6516 from the National Cancer Institutes of Health, United States Public Health Service. G. E. F. holds Award l-K6-CA-22,150 from the National Cancer Institute, National Institutes
Research
43
Human
lymphoblasts
and inhibition
513
by thymidine
been described elsewhere. The CCRF-CEM cells have been maintained in Eagle’s minimal essential medium modified for suspension culture [I] and supplemented with 10 per cent whole fetal calf serum for more than 16 months; and grow as monodispersed suspensions in spinner cultures in which an average density of 3 -4 x 106 cells/ml can be maintained. The generation time in such cultures is ca 40 hr. TABLE
I. Effects
of
thymidine
and deoxycytidine lymphoblasts.
on multiplication
of human
Cell counts ( x 108)a Compound added
y0 Inhibitionb 48 ;
0 hr
24 hr
2.5 2.5 2.5
2.14 2.46 2.66
Thymidine, 5 x 10-c M and Deoxycytidine, 5 x 10-s M
2.5
3.25
Deoxycytidine,
2.5
3.25
4.65
2.5
3.48
4.57
Thymidine,
None
5.0 x 10-s M 2.5 x 1O-5 M 1.0 x 10-S M
5 x 10-S A4
24 hr
48 hr
2.59 3.11 3.65
100 100 59
96 71 44
4.25
14
15
a In 100 ml spinner cultures in 250 ml Erlenmeyer flasks. b Net increase in cell count, experimental cultures - X100. Net increase in cell count, control cultures
In the course of studies concerned with the nucleic acid metabolism of these cells 161,it was observed that (deoxy)thymidine (Tdr) inhibited cell multiplication, even in concentrations reported to be non-inhibitory for other cells in culture [4, 5, 71. For example, the addition of 1 x 1O-5 M Tdr to cultures of actively multiplying cells resulted in ca 50 per cent inhibition, while 5 x 1O-5 M Tdr resulted in complete inhibition of cell multiplication (Table I) as adjudged by viable cell counts done as described previously [2]. This Tdr-induced inhibition can be reversed to a significant degree by the addition of 5 x 1O-6 M deoxycytidine (Cdr) to the cultures (Table I). Thymine, cytosine, cytidine, uracil, uridine, or deoxyuridine in concentrations of 5 x 1O-5 M are not inhibitory; and unlike Cdr, do not reverse or prevent inhibition by Tdr. The effects of Tdr on nucleic acid and protein synthesis were determined by incorporation studies with W-labeled thymidine, uridine, and leucine. DNA synthesis, as adjudged by the incorporation of %-2-thymidine, is inhibited by ca 50 per cent in the presence of 1 x 1O-6 M Tdr (Table II). Tdr in a concentration of 5 x 1O-5 M had no significant effect on protein synthesis, as adjudged by the incorporation of W-1-leucine during a 32 hr incubation period; and the incorporation of W-2-uridine was but slightly inhibited by the presence of 5 x 1O-6 M Tdr. Thus, small concentrations of exogenous Tdr induce a preferential inhibition of DNA synthesis in the CCRF-CEM cells. Its effective reversal or prevention by Cdr suggeststhat this Experimental
Cell Research 43
514
D. 0. Schachtschabel
et al.
inhibition probably is the result of interference with the synthesis of deoxycytidine nucleotides, as seems to be the case with other cell lines [4, 5, 71. It is of interest that these human lymphoblasts appear to be exquisitely sensitive to exogenous Tdr (e.g., ca 50 per cent inhibition of a population of 2.5 x lo* cells by 1 x 1O-5 M Tdr) as compared to the reported sensitivity of other cell lines of TABLE
II.
Effect of thymidine
Counts/minutes ( x 1000) after 30 hr at 37’C
df Thymidine 2.94 5.44 1.05 2.54 5.04
x x x x x
on DNA
10-G 10-B 10-S 10-G 10-s
644.2 301.1 92.9 23.7 9.5
synthesis
by human
p&f Thymidine incorporatedb 0.134 0.115 0.067 0.043 0.038
lymphoblasts.
y0 inhibitionc 0 14 50 68 72
a In 75 ml spinner cultures containing 1.0 PC W-2-thymidine and varying concentrations thymidine in 100 ml Erlenmeyer flasks. b Calculated from counts per minute data and corrected for dilution by the corresponding concentration of unlabeled thymidine. ’ Relative to culture containing 1.0 PC “C-2-thymidine (2.94 x 1O-B M) alone.
of
normal and neoplastic origins to Tdr. The present studies are being extended to include determination of the “enzyme profile” of the CCRF-CEM cells, as well as their responseto analogs of Tdr and related compounds to determine whether some chemotherapeutic advantage might be derived from this unusual Tdr sensitivity.
REFERENCES 1. EAGLE, 2. FOLEY, 3. 4. 5. 6. 7.
H., Science 130, 432 (1959). G. E., LAZARUS, H., FARBER, S., UZMAN, B. B. G., BOONE, B. A., and MCCARTHY, R. E., Cancer 18, 522 (1965). MCCARTHY, R. E., JUNIUS, V., FARBER, S., LAZARUS, H. and FOLEY, G. E., Exppfl Cell Res. 40, 197 (1965). MORRIS, N. R. and FISCHER, G. A., Biochim. Biophys. Ada 68, 84 (1963). MORSE, P. A. and POTTER, V. A., Cancer Res. 25, 499 (1965). SCHACHTSCHABEL, D. 0. et al., To be published. XEROS, N., Nature 194, 682 (1962).
Experimental
Cell Research
43
D. 0. Schachtschabel
et al.
Results and Conclusions.--The data in Figs. 2 and 3 demonstrate that although the maximum cell density attained was the same in both vessels, the growth rate in 50 ml spinners was greater than in 10 ml spinners. At the highest fetal calf serum protein concentration tested, this difference was maximal. Thus, the rate of growth and the maximum cell density attained are independent parameters in the description of the growth response to fetal calf serum. The results show that the 10 ml spinner was not conducive to the best growth response possible for a given concentration of fetal calf serum protein. However, these minispinners may be used successfully with small amounts of material with the limitation that if a material is found to be inactive in the IO ml spinners it should not be considered inactive until confirmed with 50 ml spinners. On the other hand, if an unknown material shows growth stimulation in the IO ml spinners it may be considered with certainty to have this activity.
The authors are grateful to Mr Peter Hasenpusch for constructing the minispinners. This work was supported in part by grants AM-07787 and CA-08204 from the United States Public Health Service and by the Helen W. Dutcher Memorial Grant for Cancer Research (E-284) from the American Cancer Society. REFERENCES 1. 2. 3. 4. 5. 6. 7.
DELUCA, C., RAKOWSKI, DELUCA, C., HABEEB, A. GORNALL, A. G., BARDAWILL, MCLIMANS, W. F., DAVIS, MOORE, G. E., MOUNT, D., MOORE, G. E. and ULRICH, TRITSCH, G. L., FLOSS, D.
P. F. and TRITSCH, G. L., Biochim. Biophgs. Acta 86, 346 (1964). F. S. A. and TRITSCH, G. L., Ezpptl Cell Res. 43, 98 (1966). C. J. and DAVID, M. M., J. Biol. Chem. 177, 751 (1949). E. V., GLOWER, F. L. and RAKE, G. W., J. Immunol. 79,428 (1957). TARA, G. and SCHWARTZ, N., J. Nafl Cancer Inst. 31, 1217 (1963). K., J. SurgicaI Res. 5, 270 (1965). R. and MOORE, G. E., Exptl Cell Res. 42, 523 (1966).
SENSITIVITY HUMAN
OF CULTURED
LYMPHOBLASTS INHIBITION
D.
0.
SCHACHTSCHABEL,
The Children’s
H.
(CCRF-CEM BY
LAZARUS,
THYMIDINE S. FARBER,
Cancer Research Foundation, School, at The Children’s
and Department Hospital, Boston,
Received
May 3, 1966
CELLS)
TO
l and
of Pathology, Mass., U.S.A.
G. E. FOLEY Harvard
Medical
T HE
continuous cultivation of human lymphoblasts (CCRF-CEM cells) directly in suspension cultures from the peripheral blood buffy coat of a child with acute lymphoblastic leukemia [2] and the karyological characteristics of these cells [3] have 1 These Institute, Research of Health.
Experimental
studies National Career
Cell
were supported in part by research grant C-6516 from the National Cancer Institutes of Health, United States Public Health Service. G. E. F. holds Award l-K6-CA-22,150 from the National Cancer Institute, National Institutes
Research
43
Human
lymphoblasts
and inhibition
513
by thymidine
been described elsewhere. The CCRF-CEM cells have been maintained in Eagle’s minimal essential medium modified for suspension culture [I] and supplemented with 10 per cent whole fetal calf serum for more than 16 months; and grow as monodispersed suspensions in spinner cultures in which an average density of 3 -4 x 106 cells/ml can be maintained. The generation time in such cultures is ca 40 hr. TABLE
I. Effects
of
thymidine
and deoxycytidine lymphoblasts.
on multiplication
of human
Cell counts ( x 108)a Compound added
y0 Inhibitionb 48 ;
0 hr
24 hr
2.5 2.5 2.5
2.14 2.46 2.66
Thymidine, 5 x 10-c M and Deoxycytidine, 5 x 10-s M
2.5
3.25
Deoxycytidine,
2.5
3.25
4.65
2.5
3.48
4.57
Thymidine,
None
5.0 x 10-s M 2.5 x 1O-5 M 1.0 x 10-S M
5 x 10-S A4
24 hr
48 hr
2.59 3.11 3.65
100 100 59
96 71 44
4.25
14
15
a In 100 ml spinner cultures in 250 ml Erlenmeyer flasks. b Net increase in cell count, experimental cultures - X100. Net increase in cell count, control cultures
In the course of studies concerned with the nucleic acid metabolism of these cells 161,it was observed that (deoxy)thymidine (Tdr) inhibited cell multiplication, even in concentrations reported to be non-inhibitory for other cells in culture [4, 5, 71. For example, the addition of 1 x 1O-5 M Tdr to cultures of actively multiplying cells resulted in ca 50 per cent inhibition, while 5 x 1O-5 M Tdr resulted in complete inhibition of cell multiplication (Table I) as adjudged by viable cell counts done as described previously [2]. This Tdr-induced inhibition can be reversed to a significant degree by the addition of 5 x 1O-6 M deoxycytidine (Cdr) to the cultures (Table I). Thymine, cytosine, cytidine, uracil, uridine, or deoxyuridine in concentrations of 5 x 1O-5 M are not inhibitory; and unlike Cdr, do not reverse or prevent inhibition by Tdr. The effects of Tdr on nucleic acid and protein synthesis were determined by incorporation studies with W-labeled thymidine, uridine, and leucine. DNA synthesis, as adjudged by the incorporation of %-2-thymidine, is inhibited by ca 50 per cent in the presence of 1 x 1O-6 M Tdr (Table II). Tdr in a concentration of 5 x 1O-5 M had no significant effect on protein synthesis, as adjudged by the incorporation of W-1-leucine during a 32 hr incubation period; and the incorporation of W-2-uridine was but slightly inhibited by the presence of 5 x 1O-6 M Tdr. Thus, small concentrations of exogenous Tdr induce a preferential inhibition of DNA synthesis in the CCRF-CEM cells. Its effective reversal or prevention by Cdr suggeststhat this Experimental
Cell Research 43
514
D. 0. Schachtschabel
et al.
inhibition probably is the result of interference with the synthesis of deoxycytidine nucleotides, as seems to be the case with other cell lines [4, 5, 71. It is of interest that these human lymphoblasts appear to be exquisitely sensitive to exogenous Tdr (e.g., ca 50 per cent inhibition of a population of 2.5 x lo* cells by 1 x 1O-5 M Tdr) as compared to the reported sensitivity of other cell lines of TABLE
II.
Effect of thymidine
Counts/minutes ( x 1000) after 30 hr at 37’C
df Thymidine 2.94 5.44 1.05 2.54 5.04
x x x x x
on DNA
10-G 10-B 10-S 10-G 10-s
644.2 301.1 92.9 23.7 9.5
synthesis
by human
p&f Thymidine incorporatedb 0.134 0.115 0.067 0.043 0.038
lymphoblasts.
y0 inhibitionc 0 14 50 68 72
a In 75 ml spinner cultures containing 1.0 PC W-2-thymidine and varying concentrations thymidine in 100 ml Erlenmeyer flasks. b Calculated from counts per minute data and corrected for dilution by the corresponding concentration of unlabeled thymidine. ’ Relative to culture containing 1.0 PC “C-2-thymidine (2.94 x 1O-B M) alone.
of
normal and neoplastic origins to Tdr. The present studies are being extended to include determination of the “enzyme profile” of the CCRF-CEM cells, as well as their responseto analogs of Tdr and related compounds to determine whether some chemotherapeutic advantage might be derived from this unusual Tdr sensitivity.
REFERENCES 1. EAGLE, 2. FOLEY, 3. 4. 5. 6. 7.
H., Science 130, 432 (1959). G. E., LAZARUS, H., FARBER, S., UZMAN, B. B. G., BOONE, B. A., and MCCARTHY, R. E., Cancer 18, 522 (1965). MCCARTHY, R. E., JUNIUS, V., FARBER, S., LAZARUS, H. and FOLEY, G. E., Exppfl Cell Res. 40, 197 (1965). MORRIS, N. R. and FISCHER, G. A., Biochim. Biophys. Ada 68, 84 (1963). MORSE, P. A. and POTTER, V. A., Cancer Res. 25, 499 (1965). SCHACHTSCHABEL, D. 0. et al., To be published. XEROS, N., Nature 194, 682 (1962).
Experimental
Cell Research
43