[CANCER RESEARCH 27 Part 1, 242-250,February 19671
Cell Morphology of a Human Diploid Cell Strain (WI-38) after Treat
ment with Arabinosylcytosine1
WAHEEB K. HENEEN2AND WARREN W. NICHOLS
South Jersey Medical Research Foundation,
Camden, New Jersey
Eagle's medium with 30 % newborn calf serum in Petri dishes
Summary
Cell morphology was studied in a human diploid cell strain
(WI-38) treated with various concentrations of arabinosylcytosine
for different periods. Mitotic inhibition followed most of these
treatments except for the short treatments with the lowest con
centration, which allowed mitosis in the postreatment growth
period. Paired acentric fragments were seen in anaphases after
these treatments.
The treated cells exhibited certain morphologic characteris
tics, such as clustering of cells, appearance of oval, elongated
nuclei and cells, increased cytoplasmic granularity, increased size
of nuclei and nucleoli, fusion and vacuolation of nucleoli which
became darker, and organization of new nucleoli and decreased
stainability of the reticulum. The changes in the appearance of
the nucleoli may indicate increased metabolic activities. Many of
these morphologic characteristics resemble changes seen in trans
formed cells.
Introduction
Arabinosylcytosine (l-/3-D-arabinofuranosylcytosine, ara-C) is
an analog of deoxycytidine and cytidine and has been observed
to interfere with DNA synthesis and cell reproduction by many
authors (3, 5-7, 13,14). It has also been demonstrated that ara-C
causes breakage of chromosomes in 2 tissue culture systems, viz.,
human leukocytes and a human cell line (2, 14, 19), as well as
in vivo (1). These breaks resembled those occurring after virus
infection (18, 20-22).
Nichols and Heneen (19) reported initial comparative studies
between long-term effects of ara-C on cell cultures and cell trans
formation following virus infection (9, 16, 23, 26).
The present study extends these initial observations to more
detailed cell morphology and pattern of growth in a human di
ploid cell strain (WI-38) treated with ara-C and subjected to a
differential stain technic.
Materials
and Methods
A diploid cell strain (WI-38) originally derived from a female
human embryo lung was used. These cells were grown in Hanks1 This work was supported in part by Grants CA-03845 and
CA-04953 from the NIH and by Research Career Development
Award 5-K3-CA-16.749.
* Institute of Genetics, University of Lund, Lund, Sweden.
Received January 17, 1966; accepted September 1, 1966.
242
containing coverslips. The different treatments were applied on
cells growing on coverslips when in Passages 23-32.
Arabinosylcytosine in final concentrations of 10~5 M, 10~6 M,
and 10~7Mwas used. The treatments were started 1-2 days after
transfer before the formation of a dense sheet of cells on the coverslip. In one series, ara-C treatments were continuous for 1, 3, 24,
and 48 hr before fixation. In another, the analog was removed
after such treatments and fresh medium was added. In this series,
fixations were made at intervals of 2 days for a period of 8 days,
after removing the analog. During the posttreatment period the
cells were refed every 2nd day. Controls for all treatments were
fixed or refed at similar times or intervals.
Before fixation, the coverslips were dipped for a few sec in NaCl
solution (0.85%) to take away remnant medium. Kahle's modi
fied fixative [formula given by Smith (25)], composed of 95%
ethyl alcohol-formalin-glacial acetic acid (15:6:1), was used for
10 min. The coverslips were then passed in an alcohol series before
hydrolysis for 10 min in l NHC1 at 60°Cand differentially stained
for chromatin and nucleoli with Feulgen's procedure and light
green (Östergren, Koopmans, and Reitalu; see Ref. 24).
Results
Frequency of Mitosis
The frequency of mitosis after continuous treatment with araC and during the period of posttreatment growth in ara-C-free
medium is presented in Table 1. Mitosis in frequencies less than
the control level were observed after a short treatment of 1 or 3
hr with all the concentrations (10~5, 10~6, or 10~7 M) of ara-C
used except for the 1-hr lü~7
M treatment, which had about the
same mitotic index as in the control. After 24- and 48-hr treat
ments, no mitoses were seen except for a low frequency after the
24-hr treatment at the lowest concentration (10~7 M). Dead cells
were of frequent occurrence after the 24- and 48-hr treatments,
and the appearance of their chromatin material indicated death
while in division.
During the posttreatment period (4 analyses at 2-day inter
vals) in ara-C-free medium, no mitosis was observed after the
10~5 M treatment. This concentration of ara-C, even if applied
only for 1 hr, caused complete inhibition of mitosis in the subse
quent period of observation. The 10~6 M concentration caused
strong inhibition of mitosis, but in this case a few mitoses oc
curred in the first 2-4 days after a short treatment of 1 or 3 hr.
On the 2nd day, after the 10~7M concentration was applied for
CANCER
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VOL. 27
Cell Morphology after Arabinosylcyiosine Treatment
TABLE 1
FREQUENCYOF MITOSIS"IN WI-38 CELLSAFTERTREATMENTWITHARABINOSYLCYTOSINE
AND
DURING
THE
PERIOD
OF PoSTTREATMENT
GROWTH
(hr)11.551.202.202.2530.700.551.092.2524——0.352.9448———2.011324481324481324481-3POSTTREATMENT
OF TREATMENT
(DAYS)2———0.20——1.891.72——2.014——0.05——0.300.10—0.436—
PERIOD
MOLARCONCENTRATIONio-*10-«io-7ControlDURATION
ara-C
.0.20——0.14S——————0.02
0 Mitotic indices based on counts of 2000-0000cells in each treatment; dashes (—)indicate no mitosis.
1 or 3 hr, mitosis was close to the control level. This was followed
by a decreasing frequency in the analyses made on Days 4, 6, and
8. Twenty-four- or 48-hr treatments with 10~' Mara-C completely
inhibited mitosis in the posttreatment period.
As indicated in Table 1, mitosis in the control was frequent on
Day 2 and decreased successively on Days 4, 6, and 8. These were
periods corresponding to the days of growth in ara-C-free medium
in the treated material. Some mitotic figures can be seen in the
cells shown in Figs. 1-4, which represent the controls at various
periods of growth.
The chromosomes in most of the metaphase plates seen in the
treated material exhibited some degree of stickiness. This has been
observed both directly after ara-C treatments and 2-8 days fol
lowing these treatments. A metaphase figure from the control
and an ara-C treatment are shown in Figs. 5 and 6, respectively.
Persistent nucleolar material (arrows, Fig. 6) was of more common
occurrence in the treated cells than in the control. For example, on
Day 2 after a 3-hr 10~7Mtreatment, persistent nucleoli occurred
arms. In the case of the control, most of the aberrant anaphases
showed separation difficulties, giving rise to chromatid bridges
(Fig. 9).
Pattern of Growth and Morphology of Cells
The treatment with ara-C caused a marked decrease in the
number of cells that grew on the coverslip (Figs. 10-15). Control
cells at various periods of growth are seen in Figs. 1-4. The in
tensity of cell loss increased with increasing concentrations and
time of treatment. Proliferation of cells during the posttreatment
period occurred only in the 1- and 3-hr treatments, with the
lowest concentration, 10~7M, of ara-C.
A difference in growth pattern between cells treated with ara-C
and the controls could sometimes also be seen. Treated cells
tended to group together in clusters (Fig. 11). They also showed a
more elongated appearance than the controls (Fig. 14). This often
happened shortly before cells detached from the glass. A some
what similar, but less marked, effect was seen in control cells as
in 46% of the metaphase plates and in 22% of the controls (67
they became crowded on the glass.
and 151 metaphase analyses, respectively).
During the posttreatment period, especially after long treat
Besides stickiness and overcontraction of chromosomes at ments with high concentrations of ara-C, the cytoplasm exhibited
metaphase, aberrations at anaphase were also encountered.
more granularity and stainability with light green (Figs. 12-15)
These were clearly demonstrable in the following 4 treatments of
ara-C; 3 hr with 10~5 M or 10~6 M concentration, 24 hr with 10~7 and the cells often appeared to be degenerating.
M concentration, and day 2 posttreatment analysis of the IO"7 M
Appearance of Nuclei
3-hr treatment. A total of 675 anaphases studied from these treat
ments showed aberrations in a frequency of 25-29%. In 492 anaCompared with the control, there was a wider variation in
nuclear sizes after treatment with ara-C. Small, shrunken nuclei
phases analyzed in control material, the frequency of aberrations
ranged from 3 to 11%. Besides the differences in frequency of were seen immediately after the short treatments (1 and 3 hr).
After long treatments (24 and 48 hr) and during the posttreat
anaphase aberrations, there was a difference in the type of aber
ration most frequently observed in the treated versus control ment period, most of the nuclei were increased in size (Figs.
10-15). Some of these large nuclei were so much larger than the
material. The most common anaphase aberration after ara-C
treatment was a pair of acentric fragments (Figs. 7, 8) which rest of the cells that they probably represented polyploid cells
varied in size from dotlike to as large as whole chromosome
(Fig. 10).
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243
Waheeb K. Heneen and Warren W. Nichols
A general increase in size of nuclei that was of a lesser degree
than in these polyploid cells was also seen after treatment and
may have represented either a real increase in nuclear material
or a decreased compactness of the nucleus. Since there was a low
staining density of these big nuclei, the latter was considered
most likely. In degenerating cells, the faintly stained chromatin
reticulum condensed into darkly stained granules distributed all
over the nucleus (Figs. 14, 15). This was noticeable to a much
lesser extent in the late periods of growth in the control.
Appearance of Nucleoli
In the control material (Figs. 1-4), nucleoli were small and
varied in number and shape. From 2 to 4 nucleoli per nucleus
were most frequently observed. In the treated cells, nucleoli were
darker staining and appeared in a diffuse or branched condition
(Figs. 10,11). Attenuations of nucleoli appeared to be a condition
prior to their fusion in later stages of growth in ara-C-free medium
(Figs. 12-15). Vacuoles were frequently seen in large, fused nu
cleoli. The vacuoles were rounded, and varied in number from
few to many, covering most of the nucleolar area (Fig. 16). Small
vacuoles were seen much less frequently in nucleoli of the control.
These effects were accentuated by increase in concentration and
length of treatment.
In many of the treated cells, new, small nuclei appeared during
the growth period in the ara-C-free medium (Fig. 17).
The appearance of the nucleoli directly after the treatment and
during the posttreatment period might give an indication of an
increased metabolic activity. The nucleoli first appear in diffuse
branched condition and then fuse together, forming big, darkly
stained nucleoli. In the posttreatment period, new nucleoli are
sometimes organized. The appearance of vacuoles in nucleoli
may increase the area of the reactive surface (10). These changes
do not necessarily indicate increased metabolic activity, and, in
deed, in the protozoan Tetrahymena pyriformis, fusion of nucleoli
takes place in aged cultures at the end of the log phase and during
the stationary phase of growth (4). In this paper (4), the authors
referred to works demonstrating the effect of heat shocks in
causing nucleolar fusion. Also, vacuoles were seen in nucleoli of
green monkey kidney cells after infection with Simian Virus
40 (17), and they may represent a stage of pyknosis in these cells.
Many of the features seen in the ara-C-treated cells presented
here resemble those observed in transformed, or virus-treated,
cells (e.g., 16, 17, 23). Included in these similarities are the in
creased cytoplasmic granularity, more conspicuous large nucleoli,
enlargement of nuclei, and clustering of cells. Some of the ob
served morphologic characteristics are also seen in aged control
cells. Attempts are in progress to follow up the cells treated
with ara-C by repeated cloning and passing to detect cells that
fulfill all the requirements of transformation.
Acknowledgments
Discussion
The mitotic inhibition observed in WI-38 cells after arabinosylcytosine treatment was dependent on concentration and duration
of treatment. The 1- and 3-hr treatments with 10~7 M were the
only ones in which mitotic inhibition was not observed in the
posttreatment period of growth. Karon et al. (13) and Buthala (3)
reported similar observations in other systems, and the mitotic
inhibitory action of ara-C has also been pointed out by other
workers (6, 7, 11, 14, 19). The mitotic inhibitory effect of another
analog, 2'-deoxy-5-fluorouridine (FUDR), counteracted by the
addition of thymidine, was used as a method for synchronization
and accumulation of mitoses (see Hsu et al. 12).
The differences in frequency and type of aberrations found at
anaphase between treated and untreated cells further confirm
the chromosome-breaking effects of ara-C seen at metaphase
(2,14,19).
A prominent effect of ara-C is that it interferes with or blocks
DNA synthesis (3, 5-7, 14). The appearance of the treated cells
thus reflects morphologic characteristics of cells that have been
subjected to the interference of DNA synthesis by the analog, a
condition which may be compared to the unbalanced growth in
duced in a thymine-less state in bacteria (8) and observed in
HeLa S-3 cells after treatment with ara-C (15).
In short treatments with lower concentrations of ara-C, cells
started to replicate again after the removal of the analog, whereas
after long treatments with low or high concentrations, they re
mained in interphase.
The increase in nuclear volume of treated cells may represent
an actual increase in their DXA content, as found by Karon et al.
(13), or could also be explained by a decrease in the compactness
of these nuclei, as might be indicated by their fainter staining
reaction.
244
We would like to express thanks to Dr. S. S. Cohen for his
helpful discussions and criticism. We also wish to thank photogra
phy technician Miss Mary Federico and tissue culture technician
Mrs. Joan Lee for their endeavors.
References
1. Bell, W. R., Whang, J. J., Carbone, P. P., Brecher, G., and
Block, J. B. Cytogenetic and Morphologic Abnormalities in
Human Bone Marrow Cells during Cytosine Arabinoside
Therapy. Blood, 37: 771-81, 1966.
2. Brewen, J. G. The Induction of Chromatid Lesions by Cyto
sine Arabinoside in Post-DNA-synthetic
Human Leukocytes.
Cytogenetics, 4: 28-36, 1965.
3. Buthala, D. A. Cell Culture Studies on Antiviral Agents. I.
Action of Cytosine Arabinoside and Some Comparisons with
5-Iodo-2-deoxyuridine.
Proc. Soc. Exptl. Biol. Med., 115:
69-77, 1964.
4. Cameron, I. L., and Guile, E. E. Nucleolar and Biochemical
Changes during Unbalanced Growth of Tetrahymena pyri
formis. J. Cell Biol., 26: 845-55, 1965.
5. Cardeilhac, P. T., and Cohen, S. S. Some Metabolic Properties
of Nucleotides
of l-/3-D-Arabinofuranosylcytosine.
Cancer
Res., 24: 1595-1603, 1964.
6. Chu, M. Y., and Fischer, G. A. A Proposed Mechanism of
Action of l-/3-D-Arabinofuranosyl-cytosine
as an inhibitor of
the Growth of Leukemic Cells. Biochem. Pharmacol,
11:
423-30, 1962.
7.
. Comparative Studies of Leukemic Cells Sensitive and
Resistant to Cytosine Arabinoside. Ibid., 14: 333-41, 1965.
8. Cohen, S.S., and Barner, H. D. Studies on Unbalanced Growth
in Escherichia coli. Proc. Nati. Acad. Sci. U.S., 40: 885-93,
1954.
9. Cooper, H. L., and Black, P. H. Cytogenetic Studies of Ham
ster Kidney Cell Cultures Transformed by the Simian Vacuolating Virus (SV 40). J. Nati. Cancer Inst., SO: 1015-43, 1963.
CANCER RESEARCH VOL. 27
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Cell Morphology after Arabinosylcytosine
10. Esper, H. Studies on the Nucleolar Vacuole in the Oogénesis
of Arabacia punctulata. Exptl. Cell Res., 38: 85-96, 1965.
11. Evans, J. S., Musser, E. A., Mengel, G. D., Forsblad, K. R.,
and Hunter, J. H. Antitumor Activity of l-/3-D-Arabinofuranosylcytosine Hydrochloride. Proc. Soc. Exptl. Biol. Med., 106:
350-53, 1961.
12. Hsu, T. C., Humphrey, R. M., and Somers, C. E. Response of
Chinese Hamster and L Cells to 2'-Deoxy-5-fluoro-uridine
and Thymidine. J. Nati. Cancer Inst., 3£:839-55, 1964.
13. Karon, M., Henry, P., Weissman, S., and Meyer, C. The Effect
of l-j3-D-Arabinofuranosylcytosine
on Macromolecular
Syn
thesis in KB Spinner Cultures. Cancer Res., ÃŽ6:166-71, 1966.
14. Kihlman, B. A., Nichols, W. W., and Levan, A. The Effect of
Deoxyadenosine and Cytosine Arabinoside on the Chromo
somes of Human Leukocytes in Vitro. Hereditas, 50: 139-43,
1963.
15. Kim, J. H., and Eidinoff, M. L. Action of l-/3-D-Arabinofuranosylcytosine
on the Nucleic Acid Metabolism and Via
bility of HeLa Cells. Cancer Res., 25: 698-702, 1965.
16. Koprowski, H., Ponten, J. A., Jensen, F., Ravdin, R. G.,
Moorhead, P., and Saksela, E. Transformation
of Cultures of
Human Tissue Infected with Simian Virus SV 40. J. Cellular
Comp. Physiol., 59: 281-92, 1962.
17. Love, R., and Fernandes, M. V. Cytological and Cytochemical
Studies of Green Monkey Kidney Cells Infected in Vitro
with Simian Virus 40. J. Cell Biol., 25: 529^3, 1965.
18. Nichols, W. W. Relationships of Viruses, Chromosomes and
Carcinogenesis. Hereditas, 50: 53-80, 1963.
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Treatment
19. Nichols, W. W., and Heneen, W. K. Chromosomal Effects of
Arabinosylcytosine
in a Human Diploid Cell Strain. Ibid.,
52: 402-10, 1964.
20. Nichols, W. W., Levan, A., Aula, P., and Norrby, E. Extreme
Chromosome Breakage Induced by Measles Virus in Differ
ent in Vitro Systems. Preliminary Communication. Ibid., 51:
380-82, 1964.
21. Nichols, W. W., Levan, A., Hall, B., and Östergren, G. Measles
Associated Chromosome Breakage. Preliminary Communica
tion. Ibid., 48: 367-70, 1962.
22. Nichols, W. W., Levan, A., and Kihlman, B. A. Chromosome
Breakage Associated with Viruses and DNA Inhibitors. Cytogenetics of Cells in Culture. Symp. Intern. Soc. Cell Biol.,
3: 255-71, 1964.
23. Ponten, J., Jensen, F., and Koprowski, H. Morphological and
Virological Investigation of Human Tissue Cultures Trans
formed by SV 40. J. Cellular Comp. Physiol., 61: 145-63, 1963.
24. Reitalu, J. The Appearance of Nucleoli and Heterochromatin
in Mesothelial Cells and Cancer Cells of Ascites Tumours of
the Mouse. Acta Pathol. Microbiol. Scand., 41: 257-66, 1957.
25. Smith, S. G. Techniques for the Study of Insect Chromosomes.
Can. Entomologist, 75: 21-34, 1943.
26. Yerganian, G., Shein, H. M., and Enders, J. F. Chromosomal
Disturbances Observed in Human Fetal Renal Cells Trans
formed in Vitro by Simian Virus 40 and Carried in Culture.
Cytogenetics, 1: 314-24, 1962.
1967
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245
Waheeb K. Heneen and Warren W. Nichols
FIGS. l-i7. All figures are from material grown on coverslips, fixed in Kahle's modified fixative, and differentially stained with Feulgen's procedure and light green.
FIGS. 1-4. Human diploid cell strain (WI-38) control 1-2, 3-4, 5-6, and 7-8 days after passing, respectively. More mitoses in the
earlier days of growth. In an older state, cells form a dense sheet of cells, nuclei are lighter in color, and nucleoli are fewer in number.
X5CO.
FIGS. 5-11. Human diploid cell strain (WI-38) treated with arabinosylcytosine
(ara-C) (except cells in Figs. 5, 9).
FIG. 5. Metaphase or prometaphase in control. X 2000.
FIG. 6. A cell subjected to a 3-hr 10~7M treatment and 2 days growth in ara-C-free medium showing stickiness of chromosomes and
persistence of nucleolar material (arrows) in a metaphase or prometaphase. X 2600.
FIGS. 7, 8. Anaphases after 3-hr 10~6M treatment exhibiting paired acentric fragments.
X 3200.
FIG. 9. Anaphase with a chromatid bridge demonstrating the type of aberrations occasionally seen in control material. X 3200.
FIG. 10. Treatment with 10~7M for 2-1hr, exhibiting fewer cells, many of which seem to be polyploid. The diffuse or branched nature
of nucleoli is also seen. X 560.
FIG. 11. Treatment with 10~5 M for 24 hr, demonstrating clustering of cells. X 560.
FIGS. 12-15. Human diploid cell strain (WI-38) treated with ara-C and grown in ara-C-free medium after treatment. X 560.
FIG. 12. Treatment with 10~6 M for 3 hr and 4 days' posttreatment
period, exhibiting fused, rounded nucleoli.
FIG. 13. Treatment with 10~7Mfor 3 hr and 8 days' posttreatment
growth. Few dead cells with pyknotic chromatin material are seen.
FIG. 14. Treatment with 10"* M for 3 hr and 8 days' posttreatment
growth. Elongated nuclei in spindle-shaped fibroblasts and also
granularity of the chromatin material in some degenerating nuclei are seen.
FIG. 15. Treatment with 10~5Mfor 1 hr and 8 days' posttreatment
growth, demonstrating
246
degenerating
nuclei.
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Cell Morphology after Arabinosylcytosine
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Cell Morphology after Arabinosylcytosine Treatment
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249
Waheeb K. Heneen and Warren W. Nichols
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Fio. 16. Treatment with 10~s M for 24 hr and 8 days' posttreatment growth in ara-C-free medium, showing fusion of nucleoli
into 1 big nucleolus and presence of vacuoles. X 2600.
FIG. 17. Treatment with 10~6M for 24 hr and 8 days' posttreatment growth in ara-C-free medium. Fusion of nucleoli into 1
big nucleolus and the organization of many small new nucleoli
are seen. X 2600.
250
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VOL. 27
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Cell Morphology of a Human Diploid Cell Strain (WI-38) after
Treatment with Arabinosylcytosine
Waheeb K. Heneen and Warren W. Nichols
Cancer Res 1967;27:242-250.
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