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Chromosome aberrations induced by thymidine
Experimentul
130
CHROMOSOME
ABERRATIONS S.-J.
Department
YANG,
of Radiology,
G. M. Stanford
INDUCED HAHN
University Received
Cell Research
and School
September
M.
A.
BY
42, 130-135
(1966)
THYMIDINE’
BAGSHAW
of Medicine,
Palo
Alto,
Calif.,
U.SA.
14, 1965
CHHOMOSOMAL alterations in the mammalian cell have recently received increasing attention. Metabolic inhibitors are among the agents which produce mammalian chromosomal injury. Agents such as 5-fluorouracil [7], cytosine arabinoside [2, 81, 5-fluorodeoxyuridine [3], and 5-bromodeoxyuridine [S] have been shown to cause chromosomal irregularities. In addition, however, a normal deoxyriboside, deoxyadenosine, when present in excess concentration was shown to induce aberrations in human leucocyte cultures [6]. In this laboratory, extensive chromosomal injury has now been observed during attempts to synchronize Chinese hamster cells in vitro by the addition of large amounts (1 mM and greater) of non-radioactive thymidine (TdR) to the growth medium. This method of inducing synchrony of mammalian cells was first described by Xeros [ 131 and has been used subsequently by various investigators [l, 9, lo]. None of these authors reported the production of chromosome breaks or aberrations by thymidine treatment, although Bootsma et al. [l] observed an irregularity in the distribution of metaphase chromosomes in cells exposed to this agent. The present communication describes the chromosome aberrations found in Chinese hamster cells after treatment with thymidine.
MATERIALS
AND
METHODS
Chinesehamster cells from two different sourceswere used. The Don cell line was supplied through the courtesy of Dr T. C. Hsu, University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas. The other cell line, originally designated as CHDOCl subline, but hereafter referred to as HO, was obtained through the courtesy of Dr T. T. Puck, University of Colorado Medical School, Denver, Colorado. The Don line consistedpredominantly of euploid cellswith 22 chromosomes. Over 60 per cent of the HO line cells were aneuploid, with 33 + 2 chromosomesat the time of experimentation. Two recently cloned derivatives of the HO cells were also tested. These are designated as the HA1 and HE sublines, each containing 70-90 per cent of aneuploid cells with 22 and 59 &-2 chromosomesrespectively. 1 This work Service, under Experimental
was supported by the National Institutes grants CA 04542-07 and CA 05838-04. Cell
Research
42
of Health,
United
States
Public
Health
Chromosome
aberrations
induced by th ymidine
131
In general, the techniques of cell culture described by Puck [II] were followed, except that cells were grown in Eagle’s medium supplemented with 15 per cent fetal bovine serum. Exponentially growing cultures were treated with TdR either by adding concentrates of TdR to the cultures or by transferring them to medium containing the appropriate concentration of the nucleoside. Incubation continued for no less than the duration of G, + M + G, phases. Cells were released from the thymidine induced block by removing the TdR medium and rinsing twice with normal medium. Both TdR-treated and control cultures were then reincubated in fresh medium. In other experiments, IO-’ M deoxycytidine (CdR) was added to overcome the thymidine effect. At various times, colchicine or colcemid was added, and cytological preparations were made 2-4 hr later using the techniques of Hsu and Kellogg [4]. Aberration frequencies were obtained by analysing SO-100 metaphase cells which had approximately the modal number of chromosomesfor the given cell line, i.e., 33 for HO cells, 22 for HA1 and Don cells, and 59 for HE cells.
RESULTS
AND
DISCUSSION
The HO cells have an S phase of 6 + 0.2 hr and a doubling time of 20 k 2 hr as measured by r4C TdR uptake and cell counts (Coulter counter), respectively. As a convenient working schedule, cells were incubated in 1 or 2 mM TdR for 24 hr. Samples taken during the first 8 hr of treatment showed a gradual decline in mitotic activity. However, the aberration rate did not exceed that of the controls. By the 24th hr, the number of cells entering mitosis was drastically reduced. Chromatid breaks, gaps, and constrictions were often present in these dividing cells; chromosomal breaks were also seen, though less frequently. After release with normal medium, the number of dividing cells increased rapidly with time, reaching a peak by the 8th or 9th hr following rescue. The degree of synchrony varied from one experiment to another; a fourfold increase of mitotic index over the controls was the highest observed. In these cells secondary constrictions, gaps, extended centromeric regions, and true breaks were seen consistently in all experiments. The predominant types of aberrations were chromatid breaks, deletions, and interchanges (Figs. 1 and 2). Less commonly encountered were chromosomal breaks and exchanges. Cells fixed at 7, 8, and 9 hr after rescue were analyzed for the extent of damage. Each simple aberration was counted as one break, each exchange as two breaks. A small fraction, not more than l-2 per cent, of the mitotic cells could not be scored because of severely broken or fragmented chromosomes (Fig. 3). These were recorded as aberrant cells. Table I lists the average number of breaks per cell and the percentage of aberrant cells which exhibited at least one break. Values given do not include gaps or constrictions. It can be seen that despite the high rate of spontaneous aberraExperimental
Cell
Research
42
S.-J.
132
Yang, G. M. Hahn
Figs. 1 to 3.-HO cells fixed with normal medium. Fig.
l.-Two
Fig.
2.-Two
Fig.
3.--Severely
8 hr
chromatid
breaks
terminal
chromatid
damaged
following
and a terminal deletions,
release
and M. A. Bagshaw
from
TdR-induced
chromatid
deletion.
a chromatid
break
and
block
by
a chromatid
replacement
interchange.
chromosomes.
Figs. 4 and 5.-Don cells fixed block, showing numerous breaks
6& hr following and fragmented
release with chromosomes.
normal
medium
from
TdR-induced
tions in the controls, a definite increase in the number of damaged cells and in the average number of breaks per cell was found in the TdR-treated cultures. Furthermore, the data indicated a correlation between the number of aberrations and the concentration of TdR to which the cells were exposed. Cells of the HAI and HE sublines, with doubling times of 25 and 23 hr, respectively, were treated with 2mM TdR for 24 hr and fixed 8 hr after release. Experimental
Cell Research
42
Chromosome The lines. their show
induced by thymidine
133
first experiment was performed shortly after the isolation of these subAs seen in Table I, the aberration frequencies were higher than that of parental HO cell line. It was interesting to note that HA1 cells did not more breaks than the H E cells, although the latter had more than twice
TABLE
1. Aberration
Column a, average In parentheses,
Cell line HO
aberrations
frequencies
in control
and thymidine
number of breaks per cell; Column b, percentage percentage of mitotic cells with severely damaged
Interval between removal from high TdR medium and fixation (hr)
Thymidine 0
1 m&f
a
b
a
b
7 8 9
0.12 0.29 0.16
9.9 18.3 13.8
0.49 0.54 0.48
25.0’ 23.8 32.0**
8 8
0.17 0.06
8 7
64
2mM a
treated cultures, of aberrant chromosomes.
cells.
concentration 5 m&l b
a
20 m&f a
b
b
(S=33$2)
HAL (S = 22) Exp. 1 Exp. 2 HE (S=59+2) Exp. 1 Exp. 2 Don (S = 22)
* Significantly
different
0.81 0.98 1.03
37.5** 33.3* 45.0**
12.4 4.0
1.56 0.09
50.0** 6.0 0.13
0.16 0.06
9.6 4.0
1.20 0.09
51.3”’ 6.0
0.00
0.0
0.00
0.0
from
control
at 5 oh level,
0.09
9.0
5.0 (lP+
and ** at 1 y0 level by xa
0.15
11.0(5.4)**
test.
as many chromosomes per cell. A second experiment carried out three months later with the two sublines yielded much lower frequencies of chromosomal damage (Table I). The reduced sensitivity to excess TdR is not surprising in view of the ever evolving characteristics of such in vitro cultures. The Don cells, which show a doubling time of 12 hr in our hands, were blocked with thymidine for 12 hr and fixed 6+ hr following release. As listed in Table I, the effect of high concentrations of TdR on the chromosomes is again obvious. At a TdR concentration of 2 m&Z, these cells showed no irregularity. The extent of damage increased as the thymidine concentration was raised from 5 to 20 mA4 (Table I). After treatment with these high concentrations of TdR, the percentage of mitotic cells with severely damaged chromosomes (Figs. 4 and 5) was obtained by counting at least 500 metaExperimental
Cell Research
42
134
S.--J. Yang, G. M. Hahn
and M. A. Bagshaw
phase cells. The values were entered in parentheses in Table I. Although TdR affected mitotic activity, there was not a clear cut dependence on concentration comparable to that for chromosome aberration. At 2 mM, there was no departure from the activity of the controls. At 5 and 20 mM the mitotic index increased about threefold over that of the controls. It appeared that in the effective concentration range, changes in TdR molarity did not alter the extent of synchrony significantly. Petersen and Anderson [9] also found no appreciable differences in the quality of synchrony in their material within the range of 2 to 25 mM. Experiments were carried out to determine whether the chromosome aberrations were caused by a block of DNA synthesis or by synthesis taking place in a highly unbalanced nucleotide pool. It has been suggested that excess TdR causes a deficiency of deoxycytidine phosphate in the nucleotide pool [13], based on the finding that thymidine triphosphate inhibits the enzymatic conversion of cytidine diphosphate to deoxycytidine diphosphate [ 121. The presence of CdR in the medium should circumvent the TdR-induced block since CdR can be phosphorylated via a “salvage” pathway. HA1 cells blocked with 2 mM TdR resumed mitotic activity upon addition of 10e4 M CdR without the addition of fresh medium. The number of chromosome breaks observed in the cells released by CdR was not significantly different from the corresponding data for cells released by fresh medium. In another experiment, 5 x 1O-5 M CdR and 2 mM TdR were added to the medium simultaneously. No mitotic block could be observed. Harvest at 24 hr following addition of the nucleosides showed an intermediate number of damaged chromosomes, i.e., higher than the control and lower than that of medium containing 2 mM TdR alone. Growth experiments demonstrated that cells did not proliferate in medium containing 2 mM TdR. However, in medium containing 2 mM TdR and 1O-4 M CdR, cell number increased at approximately the same rate as in the controls. In one such experiment, the relative cell number increased over a 72 hr period from 1100 to 18,000 (TdR + CdR) as compared to 20,000 for the controls. Thus, the presence of a small amount of CdR in the presence of large amounts of TdR permits growth to continue at a normal rate, and simultaneously reduces the extent of chromosome damage. It appears that not only interruption of DNA synthesis, but also growth in the presence of an unbalanced pool of deoxyribonucleosides causes chromosome aberrations.
Experimental
Cell Research
42
Chromosome
aberrations
induced by thymidine
135
SUMMARY
Chromosome aberrations were observed in several lines of Chinese hamster cells treated with high concentrations (1 mM or greater) of thymidine. Gaps, constrictions, chromatid exchanges and breaks were most frequent, although chromosome breaks and exchanges were also seen. The effect of thymidine was concentration dependent, at least in the range tested (1 to 20 mM). Cells incubated in medium containing 2 mM thymidine did not grow; in medium containing 2 mM thymidine and 1O-4 M deoxycytidine, growth was normal. Chromosome aberrations were still observed although at a reduced rate. Chromosome damage appears to be related to interruption of DNA synthesis as well as to DNA synthesis in the presence of an unbalanced pool of nucleosides. REFERENCES BOOTSMA, D., BUDKE, L. and Vos, O., Expfl Cell Res. 33,301 (1964). BREWEN, J. G., Cyfogenetics 4, 28 (1965). Hsu, T. C., HUMPHREY, R. M. and SOMERS, C. E., J. Nat2 Cancer Inst. 32, 839 (1964). Hsu, T. C. and KELLOGG, D. S., J. Nat1 Cancer Inst. 25,221 (1960). Hsu, T. C. and SOMERS, C. E., Proc. Nat1 Acad. Sci. 47, 396 (1961). KIHLMAN, B. A., NICHOLS, W. W. and LEVAN, A., Heredifas 50, 139 (1963). LINDNER, A., KUTKAM, T., SANKARANARAYANAN, K., RUCKER, R. and ARRADONDO, Expfl Cell Res. Suppl. 9, 485 (1963). 8. NICHOLS, W. W., LEVAN, A. and KIHLMAN, B. A., Symp. Intern. Cell Biol. 3, 255 (1964). 9. PETERSEN, D. E. and ANDERSON, E. C., Nature 203, 642 (1964). 10. PUCK. T. T.. Science 144, 565 (1964). 11. PUCK; T. T.; CIECIURA, s. J. and F~HER, H. W., J. Expfl Med. 106, 145 (1957). 12. REICHARD. P.. J. Biol. Chem. 237, 3513 (1962). 13. XEROS, N.; Nhfure 194, 682 (1962j. 1. 2. 3. 4. 5. 6. 7.
Note added in proof:
KIM
that treatment of HeLa biochemical parameters.
et al., Biochem. S3
cells
with
Pharmacol. thymidine
14,182l introduces
(1965) have unbalance
J.,
recently demonstrated in a variety of cellular
Experimental
Cell Research
42
130
CHROMOSOME
ABERRATIONS S.-J.
Department
YANG,
of Radiology,
G. M. Stanford
INDUCED HAHN
University Received
Cell Research
and School
September
M.
A.
BY
42, 130-135
(1966)
THYMIDINE’
BAGSHAW
of Medicine,
Palo
Alto,
Calif.,
U.SA.
14, 1965
CHHOMOSOMAL alterations in the mammalian cell have recently received increasing attention. Metabolic inhibitors are among the agents which produce mammalian chromosomal injury. Agents such as 5-fluorouracil [7], cytosine arabinoside [2, 81, 5-fluorodeoxyuridine [3], and 5-bromodeoxyuridine [S] have been shown to cause chromosomal irregularities. In addition, however, a normal deoxyriboside, deoxyadenosine, when present in excess concentration was shown to induce aberrations in human leucocyte cultures [6]. In this laboratory, extensive chromosomal injury has now been observed during attempts to synchronize Chinese hamster cells in vitro by the addition of large amounts (1 mM and greater) of non-radioactive thymidine (TdR) to the growth medium. This method of inducing synchrony of mammalian cells was first described by Xeros [ 131 and has been used subsequently by various investigators [l, 9, lo]. None of these authors reported the production of chromosome breaks or aberrations by thymidine treatment, although Bootsma et al. [l] observed an irregularity in the distribution of metaphase chromosomes in cells exposed to this agent. The present communication describes the chromosome aberrations found in Chinese hamster cells after treatment with thymidine.
MATERIALS
AND
METHODS
Chinesehamster cells from two different sourceswere used. The Don cell line was supplied through the courtesy of Dr T. C. Hsu, University of Texas M. D. Anderson Hospital and Tumor Institute, Houston, Texas. The other cell line, originally designated as CHDOCl subline, but hereafter referred to as HO, was obtained through the courtesy of Dr T. T. Puck, University of Colorado Medical School, Denver, Colorado. The Don line consistedpredominantly of euploid cellswith 22 chromosomes. Over 60 per cent of the HO line cells were aneuploid, with 33 + 2 chromosomesat the time of experimentation. Two recently cloned derivatives of the HO cells were also tested. These are designated as the HA1 and HE sublines, each containing 70-90 per cent of aneuploid cells with 22 and 59 &-2 chromosomesrespectively. 1 This work Service, under Experimental
was supported by the National Institutes grants CA 04542-07 and CA 05838-04. Cell
Research
42
of Health,
United
States
Public
Health
Chromosome
aberrations
induced by th ymidine
131
In general, the techniques of cell culture described by Puck [II] were followed, except that cells were grown in Eagle’s medium supplemented with 15 per cent fetal bovine serum. Exponentially growing cultures were treated with TdR either by adding concentrates of TdR to the cultures or by transferring them to medium containing the appropriate concentration of the nucleoside. Incubation continued for no less than the duration of G, + M + G, phases. Cells were released from the thymidine induced block by removing the TdR medium and rinsing twice with normal medium. Both TdR-treated and control cultures were then reincubated in fresh medium. In other experiments, IO-’ M deoxycytidine (CdR) was added to overcome the thymidine effect. At various times, colchicine or colcemid was added, and cytological preparations were made 2-4 hr later using the techniques of Hsu and Kellogg [4]. Aberration frequencies were obtained by analysing SO-100 metaphase cells which had approximately the modal number of chromosomesfor the given cell line, i.e., 33 for HO cells, 22 for HA1 and Don cells, and 59 for HE cells.
RESULTS
AND
DISCUSSION
The HO cells have an S phase of 6 + 0.2 hr and a doubling time of 20 k 2 hr as measured by r4C TdR uptake and cell counts (Coulter counter), respectively. As a convenient working schedule, cells were incubated in 1 or 2 mM TdR for 24 hr. Samples taken during the first 8 hr of treatment showed a gradual decline in mitotic activity. However, the aberration rate did not exceed that of the controls. By the 24th hr, the number of cells entering mitosis was drastically reduced. Chromatid breaks, gaps, and constrictions were often present in these dividing cells; chromosomal breaks were also seen, though less frequently. After release with normal medium, the number of dividing cells increased rapidly with time, reaching a peak by the 8th or 9th hr following rescue. The degree of synchrony varied from one experiment to another; a fourfold increase of mitotic index over the controls was the highest observed. In these cells secondary constrictions, gaps, extended centromeric regions, and true breaks were seen consistently in all experiments. The predominant types of aberrations were chromatid breaks, deletions, and interchanges (Figs. 1 and 2). Less commonly encountered were chromosomal breaks and exchanges. Cells fixed at 7, 8, and 9 hr after rescue were analyzed for the extent of damage. Each simple aberration was counted as one break, each exchange as two breaks. A small fraction, not more than l-2 per cent, of the mitotic cells could not be scored because of severely broken or fragmented chromosomes (Fig. 3). These were recorded as aberrant cells. Table I lists the average number of breaks per cell and the percentage of aberrant cells which exhibited at least one break. Values given do not include gaps or constrictions. It can be seen that despite the high rate of spontaneous aberraExperimental
Cell
Research
42
S.-J.
132
Yang, G. M. Hahn
Figs. 1 to 3.-HO cells fixed with normal medium. Fig.
l.-Two
Fig.
2.-Two
Fig.
3.--Severely
8 hr
chromatid
breaks
terminal
chromatid
damaged
following
and a terminal deletions,
release
and M. A. Bagshaw
from
TdR-induced
chromatid
deletion.
a chromatid
break
and
block
by
a chromatid
replacement
interchange.
chromosomes.
Figs. 4 and 5.-Don cells fixed block, showing numerous breaks
6& hr following and fragmented
release with chromosomes.
normal
medium
from
TdR-induced
tions in the controls, a definite increase in the number of damaged cells and in the average number of breaks per cell was found in the TdR-treated cultures. Furthermore, the data indicated a correlation between the number of aberrations and the concentration of TdR to which the cells were exposed. Cells of the HAI and HE sublines, with doubling times of 25 and 23 hr, respectively, were treated with 2mM TdR for 24 hr and fixed 8 hr after release. Experimental
Cell Research
42
Chromosome The lines. their show
induced by thymidine
133
first experiment was performed shortly after the isolation of these subAs seen in Table I, the aberration frequencies were higher than that of parental HO cell line. It was interesting to note that HA1 cells did not more breaks than the H E cells, although the latter had more than twice
TABLE
1. Aberration
Column a, average In parentheses,
Cell line HO
aberrations
frequencies
in control
and thymidine
number of breaks per cell; Column b, percentage percentage of mitotic cells with severely damaged
Interval between removal from high TdR medium and fixation (hr)
Thymidine 0
1 m&f
a
b
a
b
7 8 9
0.12 0.29 0.16
9.9 18.3 13.8
0.49 0.54 0.48
25.0’ 23.8 32.0**
8 8
0.17 0.06
8 7
64
2mM a
treated cultures, of aberrant chromosomes.
cells.
concentration 5 m&l b
a
20 m&f a
b
b
(S=33$2)
HAL (S = 22) Exp. 1 Exp. 2 HE (S=59+2) Exp. 1 Exp. 2 Don (S = 22)
* Significantly
different
0.81 0.98 1.03
37.5** 33.3* 45.0**
12.4 4.0
1.56 0.09
50.0** 6.0 0.13
0.16 0.06
9.6 4.0
1.20 0.09
51.3”’ 6.0
0.00
0.0
0.00
0.0
from
control
at 5 oh level,
0.09
9.0
5.0 (lP+
and ** at 1 y0 level by xa
0.15
11.0(5.4)**
test.
as many chromosomes per cell. A second experiment carried out three months later with the two sublines yielded much lower frequencies of chromosomal damage (Table I). The reduced sensitivity to excess TdR is not surprising in view of the ever evolving characteristics of such in vitro cultures. The Don cells, which show a doubling time of 12 hr in our hands, were blocked with thymidine for 12 hr and fixed 6+ hr following release. As listed in Table I, the effect of high concentrations of TdR on the chromosomes is again obvious. At a TdR concentration of 2 m&Z, these cells showed no irregularity. The extent of damage increased as the thymidine concentration was raised from 5 to 20 mA4 (Table I). After treatment with these high concentrations of TdR, the percentage of mitotic cells with severely damaged chromosomes (Figs. 4 and 5) was obtained by counting at least 500 metaExperimental
Cell Research
42
134
S.--J. Yang, G. M. Hahn
and M. A. Bagshaw
phase cells. The values were entered in parentheses in Table I. Although TdR affected mitotic activity, there was not a clear cut dependence on concentration comparable to that for chromosome aberration. At 2 mM, there was no departure from the activity of the controls. At 5 and 20 mM the mitotic index increased about threefold over that of the controls. It appeared that in the effective concentration range, changes in TdR molarity did not alter the extent of synchrony significantly. Petersen and Anderson [9] also found no appreciable differences in the quality of synchrony in their material within the range of 2 to 25 mM. Experiments were carried out to determine whether the chromosome aberrations were caused by a block of DNA synthesis or by synthesis taking place in a highly unbalanced nucleotide pool. It has been suggested that excess TdR causes a deficiency of deoxycytidine phosphate in the nucleotide pool [13], based on the finding that thymidine triphosphate inhibits the enzymatic conversion of cytidine diphosphate to deoxycytidine diphosphate [ 121. The presence of CdR in the medium should circumvent the TdR-induced block since CdR can be phosphorylated via a “salvage” pathway. HA1 cells blocked with 2 mM TdR resumed mitotic activity upon addition of 10e4 M CdR without the addition of fresh medium. The number of chromosome breaks observed in the cells released by CdR was not significantly different from the corresponding data for cells released by fresh medium. In another experiment, 5 x 1O-5 M CdR and 2 mM TdR were added to the medium simultaneously. No mitotic block could be observed. Harvest at 24 hr following addition of the nucleosides showed an intermediate number of damaged chromosomes, i.e., higher than the control and lower than that of medium containing 2 mM TdR alone. Growth experiments demonstrated that cells did not proliferate in medium containing 2 mM TdR. However, in medium containing 2 mM TdR and 1O-4 M CdR, cell number increased at approximately the same rate as in the controls. In one such experiment, the relative cell number increased over a 72 hr period from 1100 to 18,000 (TdR + CdR) as compared to 20,000 for the controls. Thus, the presence of a small amount of CdR in the presence of large amounts of TdR permits growth to continue at a normal rate, and simultaneously reduces the extent of chromosome damage. It appears that not only interruption of DNA synthesis, but also growth in the presence of an unbalanced pool of deoxyribonucleosides causes chromosome aberrations.
Experimental
Cell Research
42
Chromosome
aberrations
induced by thymidine
135
SUMMARY
Chromosome aberrations were observed in several lines of Chinese hamster cells treated with high concentrations (1 mM or greater) of thymidine. Gaps, constrictions, chromatid exchanges and breaks were most frequent, although chromosome breaks and exchanges were also seen. The effect of thymidine was concentration dependent, at least in the range tested (1 to 20 mM). Cells incubated in medium containing 2 mM thymidine did not grow; in medium containing 2 mM thymidine and 1O-4 M deoxycytidine, growth was normal. Chromosome aberrations were still observed although at a reduced rate. Chromosome damage appears to be related to interruption of DNA synthesis as well as to DNA synthesis in the presence of an unbalanced pool of nucleosides. REFERENCES BOOTSMA, D., BUDKE, L. and Vos, O., Expfl Cell Res. 33,301 (1964). BREWEN, J. G., Cyfogenetics 4, 28 (1965). Hsu, T. C., HUMPHREY, R. M. and SOMERS, C. E., J. Nat2 Cancer Inst. 32, 839 (1964). Hsu, T. C. and KELLOGG, D. S., J. Nat1 Cancer Inst. 25,221 (1960). Hsu, T. C. and SOMERS, C. E., Proc. Nat1 Acad. Sci. 47, 396 (1961). KIHLMAN, B. A., NICHOLS, W. W. and LEVAN, A., Heredifas 50, 139 (1963). LINDNER, A., KUTKAM, T., SANKARANARAYANAN, K., RUCKER, R. and ARRADONDO, Expfl Cell Res. Suppl. 9, 485 (1963). 8. NICHOLS, W. W., LEVAN, A. and KIHLMAN, B. A., Symp. Intern. Cell Biol. 3, 255 (1964). 9. PETERSEN, D. E. and ANDERSON, E. C., Nature 203, 642 (1964). 10. PUCK. T. T.. Science 144, 565 (1964). 11. PUCK; T. T.; CIECIURA, s. J. and F~HER, H. W., J. Expfl Med. 106, 145 (1957). 12. REICHARD. P.. J. Biol. Chem. 237, 3513 (1962). 13. XEROS, N.; Nhfure 194, 682 (1962j. 1. 2. 3. 4. 5. 6. 7.
Note added in proof:
KIM
that treatment of HeLa biochemical parameters.
et al., Biochem. S3
cells
with
Pharmacol. thymidine
14,182l introduces
(1965) have unbalance
J.,
recently demonstrated in a variety of cellular
Experimental
Cell Research
42