[CANCER RESEARCH 32, 1476-1488,
July 1972]
The Effects of Cytosine Arabinoside upon Proliferating
Epithelial Cells1
Robert S. Verbin,2 Gloria Diluiso, Hilda Liang3 and Emmanuel Farber3
Departments of Pathology, University of Pittsburgh Schools of Medicine and Dental Medicine, Pittsburgh, Pennsylvania 15213
SUMMARY
Experiments were conducted to obtain additional insight
into the response patterns of the intestinal crypts to
1-0-D-arabinofuranosylcytosine,
a potent inhibitor of DNA
synthesis. The results indicate that this interference with
nucleic acid metabolism is not lethally toxic to the crypt
epithelial cells. This formulation is based on the following
observations: (a) the absence of definitive ultrastructural
manifestations of irreversible cell damage, particularly with
respect to the nucleus; (b) the presence of labeled thymidine
in virtually 100% of the mitotic figures occurring during
recovery from the inhibitory effects of the analog; (c) the
enhanced mitotic
activity which closely followed the
resumption of a considerable degree of DNA synthesis; (d) the
failure to find any indication of intestinal epithelial cell death
in animals killed 24 and 48 hr after the first of multiple
injections of l-(3-D-arabinofuranosylcytosine;
and (e) the
absence of histológica! evidence of cell death in a number of
other epithelial tissues programmed for continuous replication.
In contrast, however, stigmata of severe cytotoxic effects were
readily discernible in the intestinal lymphocytes. It is therefore
concluded that the apparent necrosis of the intestinal crypts
noted with conventional
microscopy actually represents
phagocytosis
of degenerating
lymphocytic
elements by
adjacent crypt epithelial cells.
INTRODUCTION
Several studies have demonstrated that ara-C,4 a potent
inhibitor of DNA synthesis (3-5, 7, 10, 12, 14, 16, 19, 25, 27,
35, 39-41, 43, 44, 47, 54, 55, 59, 62), induces severe damage
in the intestinal crypts of both mice (19, 44) and rats (43,47).
In order to study this phenomenon in greater detail, we
undertook an ultrastructural and autoradiographic investiga
tion in the hope of obtaining some insight into the mechanism
by which this antimetabolite might be exerting its necrogenic
' This research was supported in part by Grants CA-11390 and
CA-12218 from the National Cancer Institute, Grant AM-14882 from
the National Institute of Arthritis and Metabolic Diseases, and Grants
1N-581 and BC-7N from the American Cancer Society.
'Recipient of a Career Development Award (5 KO4 DE 35 155)
from the National Institute of Dental Research.
3Present address: Fels Research Institute, Temple University School
of Medicine, Philadelphia, Pa. 19140.
4The abbreviations used are: ara-C, 1-0-D-arabinofuranosylcytosine;
H & E, hematoxylin and eosin; ara-FC, l-/3-D-arabinofuranosyl-5fluorocystosine.
Received December 22, 1971; accepted March 29, 1972.
1476
effect. It was also anticipated that this approach would
provide the basis for future experiments designed to establish
the means by which 2 inhibitors of protein synthesis,
cycloheximide (cf. Ref. 73) and tenuazonic acid (58, 72),
prevent the ara-C-induced intestinal damage (47).
Examination by electron microscopy of the mucosal lesions
occurring in response to ara-C treatment revealed that the
damage to the crypt epithelial cells, seen so early with light
microscopy, was only apparent and that the target for the
lethal cytotoxic effect of ara-C in vivo was actually the
lymphocyte. The evidence supporting this conclusion is the
subject of this communication.
Subsequent papers in this
series will be concerned with the similarities and differences
between the effects of ara-C and of some alkylating agents on
the structure and function of the intestinal mucosa.
MATERIALS AND METHODS
Male white Wistar rats (Carworth Farms Inc., New
City, N. Y.), weighing between 160 and 200 g after a 16- to
18-hr period of fasting, were utilized in all experiments. ara-C
(Cancer Chemotherapy Service Center of the National Cancer
Institute, Bethesda, Md.), used in the hydrochloride form, was
freshly prepared in distilled water (50 mg/ml), adjusted to pH
5.5 to 6.0 with a few drops of 10% NaOH, and injected i.p. in
a dose of 250 mg/kg body weight. Control animals received
corresponding volumes of 0.9% NaCl solution by a similar
route. (Utilization of the 0.9% NaCl solution was considered
appropriate, since ara-C was supplied as the hydrochloride.)
All injections were given between 8:00 and 9:00 a.m. to
minimize diurnal variations.
The animals were killed by decapitation at various intervals
after the administration of the drug or 0.9% NaCl solution.
Selected for study were tissues of the skin, tongue, esophagus,
stomach, and small intestine, all of which consist in part of
epithelium containing populations of continuously dividing
cells (18). Fixation for light microscopic morphological studies
was routinely carried out in Stieve's solution. For histological
examination,
sections of paraffin-embedded
tissue were
stained with H & E as well as by the Feulgen method for DNA.
Mitotic activity, expressed as percentage of mitoses, was
established in each animal by counting the number of dividing
and nondividing nuclei in at least 30 longitudinally sectioned
intestinal crypts. Comparative DNA synthesis, as measured by
precursor incorporation
and corrected for changes in the
precursor pool, was determined as previously described (70).
For electron microscopic studies, 1 control and at least 2
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ara-C and Cell Proliferation
experimental animals were killed at 0.5, 1.5, 2.5, 3.5, and 8 hr
after the administration of either 0.9% NaCl solution or ara-C.
The procedures used for the general selection and processing
of intestinal tissue were similar to those reported elsewhere
(71, 72). Ideally, it would have been best to examine cells
known to be in each phase of the cell cycle. However, this can
be done with a degree of specificity in vivo only with
autoradiography and then only for cells in the S phase. For a
study of cells in all phases of the cycle, tissue obtained from a
minimum of 5 blocks from each ara-C-treated animal was
examined at each predetermined time interval. This approach
provided a sufficient number of sections to assure the
inclusion of cells in different compartments of the life cycle in
a random sampling.
Autoradiographs were prepared from tissue obtained from
animals given injections of thymidine-3 H, l /LtCi/gbody weight
(New England Nuclear, Boston, Mass), 6.76 X IO3 mCi/
mmole, 1000juCi/ml. A 15-cm segment of proximal intestine
was fixed in 10% formalin and embedded in paraffin. Four- to
5-m/j sections were placed on glass slides and dipped into
NTB3 liquid emulsion (Eastman Kodak Co., Rochester, N. Y.).
After an exposure period of from 1 to 3 weeks at 4°the
autoradiograms were developed with Kodak D-19 and stained
with H & E. From each animal an average of approximately
100 mitotic figures, selected at random and without regard to
their stage in division, were scored as being either labeled or
unlabeled according to the criteria established by Pederson and
Gelfant (53).
RESULTS
Effects of ara-C on DNA Synthesis. The 1st series of
experiments was conducted to ascertain both the degree and
duration of inhibition of DNA synthesis induced by ara-C
under our experimental conditions. As can be seen in Chart 1,
the uptake of the precursor fell to 2% of control levels within
0.5 hr following the injection of ara-C. After remaining at
essentially this same low level for an additional 4.5 hr, the rate
of DNA synthesis returned to about 75% of control values by
lOhr.
Light Microscopic Observations. For a study of the
histológica! response of a variety of proliferating epithelial
cells to a single injection of ara-C, 1 control and 2
experimental animals were killed at each of the following
intervals: 0.5, 1.5, 2.5, 3.5, 8, 12, 14, 16, and 24 hr
subsequent to the administration
of the antimetabolite.
Although actively engaged in both DNA replication and
mitotic division (18), epithelial cells in the mucosa of the
esophagus and stomach, as well as those in the basal layer of
the epithelium of the tongue and skin, revealed no light
microscopic evidence indicative of a cytotoxic reaction to the
ara-C treatment. This paucity of obvious degenerative changes
in these particular tissues was in marked contrast to the
striking alterations noted in the crypts of the small intestine.
As reported previously in both mice (19, 44) and rats (43, 47),
stigmata of apparent crypt cell damage were readily discernible
within 2.5 to 3.5 hr after administration of ara-C. In H &
E-stained sections, the lesion was manifested by the
development of numerous intracellular eosinophilic inclusions.
0
2
4
6
8
10
HOURS AFTER ADMINISTRATION OF oro-C
Chart 1. Effects of ara-C on in vivo incorporation
of
thymidine-2-1 4C into DNA of the small intestine. Points, mean of 3
experimental animals; vertical bars. S.D. Two control animals were
utilized at each time interval. The mean specific activity of the
acid-insoluble fractions of control rats was 746 cpm/mg.
These occasionally contained fragments of basophilic debris
(Fig. 1) which, in some instances, yielded a strongly positive
reaction when stained with the Feulgen method for DNA. The
epithelial cell nuclei, however, appeared to be normal. By 8 hr
only a few intracellular inclusions were still present. Recovery,
as measured by the disappearance of intracellular debris,
continued to progress so that beyond 16 hr no cytological
differences could be detected between the control and
ara-C-treated animals (47).
This apparent damage to the intestinal crypts was also
accompanied by an interruption of cell division (Chart 2).
Mitotic activity, markedly reduced at 1.5 hr, was completely
abolished within 2.5 hr. Subsequently, mitoses began to
reappear and were appreciable but were still below the normal
value at 8 hr, i.e., at the time of disappearance of the
intracellular inclusions. After this time they progressively
increased in number, reaching a peak at 12 hr when they
showed a 3-fold increase over control values. Cell division
subsequently decreased and returned to the range of the
controls by 16 to 24 hr (Chart 2).
Careful examination of the intestinal mucosa, especially of
the crypts, revealed obvious necrotic cells immediately
beneath the epithelial cells, as well as in the underlying lamina
propria. Although these cells were possibly lymphocyte-like in
appearance,
their
advanced
nuclear
and cytoplasmic
degeneration
precluded an exact identification
by light
microscopy.
However, subsequent
electron microscopic
observation
indicated
the likelihood
of their being
lymphocytes (see below).
In agreement with our previous findings (47), severe
necrosis was noted in the germinal centers of intestinal
lymphoid follicles. The peripheral zone of small lymphocytes,
however, was unaffected (Fig. 2).
Autoradiographic Studies. For establishment of the source
of the mitotic intestinal epithelial cells noted during recovery
from the inhibitory effects of ara-C, autoradiographic
procedures were used.
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1477
R. S. Verbin, G. Diluito, H. Liang, and E. Farber
presence of numerous free ribosomes together with occasional
mitochondria and solitary, irregular channels of endoplasmic
EXPERIMENTAL
14reticulum. Junctional complexes were not present between
these cells and the adjacent epithelial cells. While the exact
13nature of these cells cannot be established unequivocably,
12their ultrastructural
organization is compatible with their
being intraepithelial lymphocytes (1, 63, 64).
IIIn animals treated with ara-C, no definitive evidence of
10epithelial cell necrosis was observed at any of the intervals
studied. Preservation of the structural integrity of all the stem
cells was readily apparent even at 3.5 hr, the time at which
karyorrhexis and other stigmata of cell death, as interpreted
by conventional microscopy, were maximal (Fig. 9). The stem
^ 7cells frequently
contained
a considerable
number of
membrane-bound vacuoles enclosing degenerating organelles.
Especially prominent in these vacuoles were large and small
5pieces of fragmented nuclei (Figs. 9, 10, and 12), yet the
4epithelial cells in which these vacuoles were found uniformly
failed to show any indication of lethal damage. The integrity
3of the epithelial cell nuclei was particularly obvious in that the
2nuclear chromatin
was well dispersed,
the nucleolar
components
retained their usual relationships,
and the
Inormal-appearing, double nuclear membranes remained intact
(Figs. 9 and 10). These reproducible findings led us reluctantly
O 2 4 6 8 10 12 14 16 18 20 22 24
to the tentative conclusion that the nuclear and cytoplasmic
HOURS
fragments found within the epithelial cell vacuoles were of
Chart 2. Effects of ara-C on mitotic activity in the intestinal crypts. exogenous origin, presumably arising as a result of the
Points in the experimental groups, mean of 2 animals; vertical bars, phagocytosis
of dead or degenerating
cells. Indeed,
modifications in the nonepithelial, lymphocyte-like cells were
ranges.
readily seen. Although first detected at 1.5 hr after
On 2 separate occasions, 4 animals initially were given i.p. administration of ara-C, these alterations were much more
injections of thymidine-3H and, after a 1-hr incorporation
dramatic at both the 2.5- and 3.5-hr intervals. An increase in
period, ara-C. The rats were killed 12 hr after this latter the number of lymphocytes within the epithelial lining was a
injection, the interval at which recovery of cell division had characteristic and consistent finding. In tangential sections,
reached its peak (Chart 2). Examination of the autoradiograms
this phenomenon was manifested by numerous cytoplasmic,
revealed the presence of silver grains over virtually 100% of the finger-like protrusions which could be seen lying within and
mitotic figures (Fig. 3).
causing a dilation of the intercellular spaces (Fig. 11). In some
Electron Microscopic Observation. The ultrastructural
instances, the lymphocytes
projected into the adjacent
appearance of normal crypt undifferentiated epithelial cells epithelial cell, resulting in an indentation of its nucleus. Quite
has been described in detail by a number of investigators (63, commonly, the epithelial cell nucleus appeared to be partially
67), and our observations generally do not differ significantly
wrapped around portions of the lymphocyte (Fig. 12).
from those earlier reports. Therefore, only a brief discussion of Clumping of chromatin
along the lymphocytic
nuclear
the cytological features of these cells is included in this membrane, as well as apparent nuclear fragmentation, was also
communication.
frequently seen (Figs. 13 and 14). In addition, necrotic cells or
The hyaloplasm of the stem cell is of moderate electron portions thereof were noted deep within the lamina propria
density
and contains
numerous
free ribosomes
and (Fig. 15), as well as adjacent to or passing through the
mitochondria, as well as scattered dense bodies, strands of basement membrane (Figs. 16 and 17). Despite the striking
rough endoplasmic reticulum, and moderately well-developed
accumulation of cellular debris noted between 1.5 and 3.5 hr
Golgi complexes (Fig. 6). The nucleus is situated in the basal after the administration of ara-C, by 8 hr large cytolysomes
portion of the cell and contains 1 or 2 nucleoli. The lateral cell were observed in only widely scattered epithelial cells.
membranes of adjacent cells are relatively straight and display Moreover, as in the earlier intervals, the structural organization
junctional complexes in their apical regions. Membrane-bound
of the stem cells themselves showed no changes, compared
vacuoles (cytolysomes) which contain homogeneous dense with similar cells obtained from control animals.
In many instances it was impossible to ascertain whether the
material and cellular debris in varying stages of dissolution are
occasionally found lying within the cytoplasm of the stem cell cytoplasmic vacuoles were really intracellular or were merely
(Fig. 7). Also noted within the epithelial layer of the crypts representative of extracellular material projecting into the
are cells which are cytologically distinct from the stem cells. adjacent epithelial cells. However, as demonstrated in Fig. 10,
As shown in Fig. 8, these cells are considerably smaller than some of the inclusions were enclosed by a single membrane and
the epithelial cells, are roughly oval, and demonstrate a contained readily identifiable nuclear material, even though
striking paucity of cytoplasmic organdÃ-es, except for the the epithelial cell nucleus appeared to be normal. Thus, there
15-
•¿
CONTROL
¡an
1478
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ara-C and Cell Proliferation
is some evidence that the epithelial cell had ingested
exogenous cellular debris.
Effects of Multiple Injections of ara-C. ara-C has been
reported to be rapidly metabolized and excreted (11, 15, 22,
38, 61,62). Therefore, it seemed important to conduct studies
in which sufficient levels of this agent were maintained to
ensure a maximal inhibition of DNA synthesis over a period of
time equivalent to the entire duration of the cycle of the
intestinal crypts, i.e., 10.5 to 12 hr (8). In addition to the
immediate effect on those cells already engaged in the
replication of their DNA, this approach would also permit the
eventual exposure to the drug of cells originally in G2, M, or
GÌ
as they traversed the cell cycle and entered or approached
the S phase.
Eight animals were given ara-C every 2 hr for 6 doses. Four
additional rats received corresponding injections of 0.9%NaCl
solution and served as controls. Two control and 4
experimental animals were killed either 24 or 48 hr after the
initial injections of either ara-C or 0.9% NaCl solution. At the
24-hr interval, the intestines of the ara-C-treated rats revealed
slightly atrophie crypts in which the goblet cells appeared to
be more prominent than those in control animals. Only
scattered mitotic figures and isolated damaged cells were
observed (Fig. 4). By 48 hr, the crypts were histologically
indistinguishable from those of the 0.9% NaCl solutioninjected animals (Fig. 5). In addition, no cytological
aberrations were noted at either interval in the epithelial
components of skin, tongue, esophagus, or gastric mucosa.
DISCUSSION
It is evident from this study that ara-C is not necrogenic to
epithelial cells of the intestinal crypts in vivo under conditions
in which the antimetabolite inhibits DNA synthesis by over
95% for several hr and in which certain cells in the lymphoid
follicles are readily destroyed. The evidence for this conclusion
is both structural and functional and can be summarized as
follows: (a) the absence of definitive ultrastructural
manifestations of irreversible damage, particularly with respect
to the nucleus; (b) the presence of labeled thymidine in
virtually 100% of the mitotic figures at 12 hr after the
administration
of ara-C and 13 hr after the injection of
thymidine-3 H (this particular observation precludes the
possibility of a selective destruction of S-phase epithelial cells
by the analog (36, 76) or of a mobilization of reserve G2 cells
(23, 53, 56) for entry into mitosis); (c) the enhanced mitotic
activity at 12 hr which closely followed the resumption of a
considerable degree of DNA synthesis in the intestinal crypts;
and (d) the failure to find any indication of intestinal
epithelial cell death in animals killed 24 and 48 hr after the 1st
of multiple injections of ara-C. This latter regimen was
sufficient to inhibit DNA synthesis maximally and continu
ously for well over 12 hr, i.e., the length of the entire cell
cycle (8). The slight atrophy of the crypts seen at 24 but not
at 48 hr could be due to the fact that cell migration onto and
cell extrusion from the villus proceeds independently of the
rate of cell formation in the proliferative compartment of the
intestinal mucosa (28). Thus, the uninterrupted maturation
and movement of cells onto the villi in the presence of the
block in cellular replication (as manifested by the pronounced
depression of mitosis at 24 hr) could easily account for the
transient atrophy noted at that time interval. However, our
observations relative to the effects of multiple injections of
ara-C in the rat contrast with those of Leach et al. (42). As
described above, the regimen used in the present study (i.e.,
the administration of ara-C to rats every 2 hr over a 12-hr
period) was not associated with necrogenic alterations of the
crypt epithelial cells. On the other hand, Leach et al. (42) have
demonstrated that mice treated with ara-C every 3 hr for 24,
72, or 96 hr developed severe karyorrhexis and distortion of
the intestinal mucosa. While this disparity of results may be
explained at least in part on the basis of species and/or dose
differences, it also seems likely that the crypt damage noted in
the studies of Leach et al. (42) represents a reflection of the
development of cellular aberrations occurring in response to
exposure to the antimetabolite for longer periods of time.
Conceivably, the inhibition of DNA synthesis for extended
periods might activate secondary responses which, in turn, lead
to necrosis. Alternatively, certain aspects of intermediary
metabolism of ara-C that are of minor importance in a 12-hr
experiment may assume greater biological significance in
experiments of longer duration and could lead to qualitatively
new phenomena such as cell death.
Evidence that the conclusion concerning intestinal epithelial
cell viability with ara-C might pertain to other tissues
programmed for continuous replication, such as the epithelial
components of the tongue, skin, esophagus, and stomach (18),
is also presented. However, this aspect of the study is not as
well documented
and is based solely on histological
observations.
The contention that ara-C is not necrogenic to proliferating
epithelial cells is in agreement with that of at least 2 other
groups of investigators.
Hirschman et al. (27) have
demonstrated that ara-C almost completely inhibited DNA
synthesis in cultured 3T3 cells, and yet excellent recovery and
growth were obtained even after an exposure to the drug of
from 6 to 8 hr. Graham and Whitmore (25) found that
concentrations of ara-C sufficient to inhibit DNA synthesis in
mouse L-cells by more than 97% for nearly 2 generation times
were without a significant effect on cell viability. Only much
higher concentrations of the analog were lethal to those cells.
Although ara-C apparently is not necrogenic to crypt
epithelial cells, little doubt remains that the drug is cytotoxic
to certain other intestinal cells, including lymphocyte-like cells
in the mucosa of the crypts. With conventional microscopy,
numerous intracytoplasmic inclusion bodies were noted within
the epithelial cells lining the Lieberkühn crypts. In many
instances,
these inclusions
contained
Feulgen-positive
fragments, thus indicating the presence of nuclear material.
Similar structures have been observed in the intestine
following irradiation (24, 45, 47), as well as after the
administration of alkylating agents (47), methotrexate (65), or
aminopterin
(49); and these apparently
represent the
karyolytic bodies originally described by Montagna and Wilson
(51). Recent electron microscopic studies (24, 31, 33, 34, 49.
66) have revealed that these karyolytic bodies actually consist
of highly complex structures composed of degenerating
nuclear and cytoplasmic constituents. The demonstration of
acid phosphatase (29) and thiamine pyrophosphatase activities
(30) in these bodies indicates a relationship to cytolysomes
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1479
R. S. Verbin, G. Diluiso, H. Liang, and E. Farber
or autophagic vacuoles, both of which are involved in the
process of intracellular digestion. After examining the
ultrastructural
appearance of mouse intestine at several
intervals after exposure to irradiation, Hugon and Borgers (31)
suggested 3 possible sources of the cellular debris found within
these cytoplasmic vacuoles: focal cytoplasmic degradation of
injured but still viable epithelial cells, phagocytosis of dead
stem cells, or phagocytosis of dead lymphoid cells. We cannot
totally rule out the possibility that at least some of the
inclusions noted in the epithelial cells of ara-C-treated animals
represent an attempt by the stem cells to segregate portions of
injured cytoplasm and that these structures are therefore
indicative of areas of focal cytoplasmic degradation. However,
although many of the vacuoles in the crypt epithelial cells
contained nuclear fragments, we were unable to detect any
alterations in the epithelial cell nuclei (e.g., clumping of the
chromatin) that would suggest injury to these particular cells.
Therefore, it can be concluded that these inclusions are not
formed as the result of the phagocytosis of necrotic epithelial
cells. Accordingly, it seems most likely that the karyolytic
bodies are produced
by the incorporation
of dead
lymphocytes into adjacent crypt epithelial cells. A source of
such cells was readily apparent in our study, since dead and
dying lymphocytes were frequently present in both the crypt
epithelial lining and the lamina propria of the intestines of
animals treated with ara-C.
Although an occasional lymphocyte was noted between the
epithelial cells in control rats, the frequency with which they
were encountered was markedly increased in the ara-C-treated
animals. While the cell membranes of the lymphocytes and
neighboring
epithelial
cells remained intact, numerous
lymphocyte pseudopods were seen distending the intercellular
spaces, as well as projecting into the epithelial cell cytoplasm.
In this latter location, they were frequently found in close
proximity to and compressing the nucleus of the epithelial
cells, thus imparting the impression of an early phagocytic
process (Fig. 12). Pycnosis and karyorrhexis
of the
lymphocytic nuclei were also evident (Figs. 13 and 14). These
cytological changes are comparable to those occurring in
lymphocytes at the end of their life-span or in those killed by
a variety of experimental procedures (69). Inasmuch as
approximately 2 to 3% of the intraepithelial lymphocytes
replicate their DNA in situ (21), it is conceivable that the
nuclear alterations noted in the present study reflect an
ara-C-induced disruption of nucleic acid synthesis within the
intraepithelial lymphocytes. However, approximately 50 to
55% of the proliferating crypt epithelial cells are also actively
engaged in synthesizing DNA at any given time (46). It
therefore
becomes
difficult
to explain
the greater
susceptibility
of the lymphocyte
to the ara-C-induced
metabolic imbalance. In an attempt to explain this inherent
vulnerability of lymphocytes, Trowell (68) has suggested that
their poor resistance to a number of toxic agents, e.g.,
irradiation, cortisone, and nitrogen mustard, might be related
to a disturbance of mitochondria! function. Since lymphocytes
contain a paucity of these organdÃ-es, any interference with
their physiochemical activities could result in the inability of
these cells to provide the internal environment required for the
maintenance of homeostasis. In this respect, it is interesting
1480
that the mitochondrial
changes noted in the intestinal
epithelial cells in animals exposed to irradiation (33) or
methotrexate
(66), both of which disrupt nucleic acid
metabolism (48, 60) and induce regressive changes in the
epithelial cells of the Lieberkühncrypts (31, 34, 47, 51, 65),
were not observed in animals treated with ara-C.
An abundance of necrotic cells or portions thereof were also
seen in the lamina propria, often adjacent to or in the process
of passing through the basement membrane (Figs. 15, 16 and
17). The advanced state of degenerative changes of this
material precludes the establishment of its origin with any
degree of certainty, neither is it possible to determine the
direction of movement from static micrographs. However, this
study, as well as our earlier investigations (47), disclosed that
ara-C caused extensive necrosis of the germinal centers of
intestinal lymphoid follicles. Consequently,
it is highly
probable that most if not all of this cellular debris was derived
from the necrotic germinal centers and that it subsequently
migrated into the crypt epithelial cells where it underwent
further degradation. Such an interpretation would suggest that
the intestinal epithelium might play a major role in the
disposal of lymphocytes. Some support for this contention can
be derived from the studies of Bryant (6). Following the
transfusion into isogenic, partially hepatectomized mice of cell
suspensions of lymphocytes that had been previously exposed
to thymidine-3H, the highest concentrations of the radioactive
isotype were found in the epithelial cells of Lieberkühn's
crypts. It was therefore concluded that the donor lymphocytes
were broken down and their DNA reutilized in association
with the renewal of the intestinal epithelium of the recipient
animal. While it is conceivable that this reutilization of
lymphocyte DNA by the crypt epithelial cells might account
for the labeled mitotic figures observed in animals recovering
from the effects of ara-C, certain parameters ascribed to the
cell cycle argue against this possibility. Cairnie et al. (8) have
established that the duration of S and G2 in Lieberkühn's
crypts are 6.5 to 8 hr and 1 hr, respectively. Therefore, if
S-phase cells were selectively killed by ara-C and if G, cells
eventually incorporated labeled nucleotides derived from the
degenerating lymphocytes into their own genome, the least
amount of time required for the detection of these G[ cells as
labeled mitoses would be 7.5 hr after the reinitiation of DNA
synthesis. Since the peak of mitotic recovery occurred 12 hr
subsequent to the administration
of ara-C (Chart 2), a
significant restitution of DNA synthesis would have had to
develop at the 4.5-hr interval. However, as shown in Chart 1,
nucleic acid synthesis was still almost totally suppressed at
that time period.
A number of other observations regarding the origin and
fate of intestinal lymphocytes also support the idea that the
intestinal crypt epithelial cells play a role in the disposal of
lymphocytes.
In untreated animals, cells morphologically
indistinguishable from small lymphocytes are often found in
considerable numbers in lamina propia (1). Many of these
lymphoid cells pass through the basement membrane (1, 52,
63, 64), after which time they either occupy the intercellular
spaces (1, 63, 64) or penetrate into the cytoplasm of the stem
cells (1, 2) where they frequently undergo degeneration in
large vacuoles (1, 2). In some instances, the lymphocytes
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ara-Cand Cell Proliferation
either migrate through the epithelium and are eventually shed
into the lumen (75) or remain intact within the epithelium for
indefinite periods of time (1, 26). Following irradiation, the
number
of lymphocytes
migrating
through
intestinal
epithelium increases considerably (45). Electron microscopic
studies conducted
in irradiated
mice (31, 32) have
demonstrated
that many of the lymphoid cells present
evidence of acute injury and that they are ultimately engulfed
by the crypt epithelial cells. The observations derived from the
present study suggest that a similar exaggeration of the
normally occurring processes of migration and degradation of
intestinal lymphoid elements might take place in animals
treated with ara-C and that this phenomenon could be a
reflection of the unusual susceptibility of some lymphocytes
to interferences with many metabolic processes (20). The
interpretations and conclusions derived from our results seem
to be in conflict with those of Lenaz et al. (44) in mice treated
with ara-FC, a compound similar in many of its effects to
ara-C. By means of autoradiographic
procedures, these
investigators found that, following the administration of
ara-FC, 97% of the epithelial cells of the intestinal crypts
considered by light microscopy to be necrotic were labeled.
However, parallel quantitative studies of the persistence of
prelabeled DNA of the entire intestinal wall showed only a
50% decrease in radioactivity 16 hr after the injection of
ara-FC. To explain this unanticipated finding, Lena/, et al. (44)
suggested that there is reutili/.ation of the labeled DNA
products released from the degenerating epithelial cells.
An equally plausible explanation and one in basic accord
with our observations is as follows. Certain lymphoid as well as
crypt epithelial cells are labeled in vivo following the injection
of radioactive thymidine. The destruction of the lymphoid
cells by ara-C or ara-FC and their phagocytosis by the crypt
epithelial cells would account for the large number of labeled,
damaged cells noted in Lieberkuhn's crypts (44). Similarly, the
ingestion of fragments of lymphoid cells would, on a light
microscopic level, simulate actual karyorrhexis of the crypt
epithelial cells. The observed 50% decrease in DNA
radioactivity (44) could represent the loss of label from
lymphoid cells as a result of digestion within and ultimate
elimination from the crypt epithelial cells. Retention of the
residual radioactivity (50%) could reflect the continued
presence of labeled DNA within the intact, undamaged
epithelial cells. Thus, in actuality, no discrepancy may exist
between the results of our study and those of Lenaz et al.
(44).
It has been demonstrated that the ara-C-induced inhibition
of DNA synthesis is dependent upon the capacity of the target
cell to convert the drug, via phosphorylation by deoxycytidine
kinase, to its active derivatives, ara-C diphosphate and ara-C
triphosphate (10, 11, 13, 38, 57). Since Kress (quoted in Ref.
61) found that 70% of the drug was excreted unchanged in the
urine of the rat and since the antitumor activity in the rat was
reported to be limited (74), it appeared that the drug was not
activated in that species (61). However, more recent evidence
has demonstrated that deoxycytidine kinase is indeed present
in the rat, although its concentrations in various tissues varies
considerably. Kessel et al. (37) demonstrated that the capacity
for drug phosphorylation was highest in mature lymphocytes.
Mitchell et al. (50) also found proportionally high levels of the
activating enzyme in lymphoid tissue. Similarly, Durham and
Ivés(17) reported that lymph nodes, spleen, and thymus in
the rat contained high levels of deoxycytidine kinase while
other nonlymphoid tissues, including the intestine, contained
much lower levels. However, the pronounced block in the
incorporation of radioactive precursor into DNA noted in this
study, as well as in other investigations (43, 47), clearly
indicate that the intestine of the rat must contain
concentrations of the activating enzyme that are high enough
to permit the requisite conversion of ara-C into its
metabolically
active forms. Conversely, the subsequent
enzymatic transformation
of ara-C to physiological inert
1-0-D-arabinofuranosyluraciI
(9) by intestinal pyrimidine
nucleoside deaminase activity (17) presumably accounts for
the eventual recovery of DNA synthesis. This pattern of
suppression and restitution of DNA synthesis results, first, in a
blockage of cells that were in the S phase at the time of drug
administration and, then, in a wave-like movement of these
cells out of S, through G2 and, ultimately, into mitosis. The
partial synchrony thus obtained is manifested morphologically
by the mitotic overshoot of control levels observed 12 hr after
the injection of ara-C. A similar phenomenon has been
detected in vitro in HeLa cells (39), as well as in vivo in murine
melanoma and Ehrlich tumors (5) and mouse intestine (44)
treated with the antimetabolite.
The rapidity with which
mitotic activity initially disappeared and the subsequent
abrupt increase in the number of dividing cells noted between
10 and 12 hr in the present study indicate that a block
induced by ara-C occurs at or near the end of the S phase. The
finding that the total interruption and eventual resumption of
cell division in rat intestine occurred at time intervals different
from those previously reported in mice (44) may be due to
species differences and/or to the smaller dose of ara-C utilized
by these latter investigators.
The results of our experiments pertaining to the resistance
of epithelial cells to severe pertubations of DNA synthesis by
ara-C raise a very important practical problem concerning the
basis for use of the analog in the treatment of nonepithelial
neoplasms. Is ara-C acting as a lethal cytotoxic agent or is its
effect more subtle? For example, since it is often used with
other chemotherapeutic drugs, could its action, at least in part,
be due to some induced synchronization of the neoplastic
cells? If this were the case, perhaps regimens designed to
synchronize
the target cells more efficiently might be
explored.
ACKNOWLEDGMENTS
We wish to express our gratitude for the invaluable assistance and
advice provided by Dr. H. Shinozuka during the course of this
investigation.
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1483
R. S. Verbin, G. Diluiso, H. Liang, and E. Farber
Fig. 1. Intestinal mucosa 3.5 hr after the administration of ara-C. Numerous eosinophilic inclusions, some of which contain fragments of
basophilic debris (arrows), arc present in the crypt epithelial cells. H & E, X 400.
Fig. 2. Intestinal lymphoid follicle 3.5 hr after the administration of ara-C. Note the extensive necrosis of cells in the germinal center (GC).
H & E, X 400.
Fig. 3. Autoradiogram of intestinal crypts 12 hr after the administration of ara-C and 13 hr after the injection of thymidine-3 H. Silver grains are
present over every mitotic figure (arrows). H & E, X 900.
Fig. 4. Intestinal mucosa from a rat given injections of ara-C every 2 hr for a total of 6 doses. The animal was killed 24 hr after the initial
injection. Note the prominence of the goblet cells within the somewhat atrophie crypts. Only an occasional mitotic figure is present (arrows).
H & E, X 200.
Fig. 5. Intestinal mucosa from a rat given injections of ara-C every 2 hr for a total of 6 doses. The animal was killed 48 hr after the initial
injection. Crypts reveal a normal histological appearance. H & E, X 125.
1484
CANCER RESEARCH VOL. 32
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Fig. 6. Survey view of several adjacent crypt undifferentiated epithelial cellsfrom a control rat. The hyaloplasm is of moderate electron density
and contains numerous free ribosomes (R) and mitochondria (M), as well as scattered dense bodies (DB) and strands of rough endoplasmic
reticulum (RER). The lateral cell membranes (CM)of adjacent cells are relatively straight and display junctional complexes (JO in their apical
regions.N, nucleus;NCL, nucleolus; G, Golgiapparatus; LP, lamina propria. X 7,600.
Fig. 7. A portion of a crypt stem cell from a control rat. Note the membrane-bound vacuole or cytolysome (arrow) containing cellular debris
in varying stages of dissolution. N, epithelial cell nucleus; LP, lamina propria. X 8,000.
Fig. 8. An intraepithelial lymphocyte (L) from a control rat. Note the paucity of cytoplasmic organelles except for ribosomal particles (R),
occasional
mitochondria
(M),
and
solitary,
irregular
channels
of endoplasmic
reticulum
(ER).
Note
also
the
absence
of junctional
complexes
between lymphocyte and adjacent epithelial cells (EC). LP, lamina propria. X 12,800.
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1972 American Association for Cancer Research.
R. S. Verbin, G. Diluiso, H. Liang, and E. Farber
N
EC
N
Figs. 9 and 10. Intestinal crypt cells 3.5 hi after administration of ara-C. Note the preservation of the structural integrity of the epithelial cells
(EC) and the normal chromatin pattern of their nuclei (N). Vacuoles (V) containing degenerating organdÃ-esand fragments of nuclear material
(arrows) are present in the cytoplasm of the epithelial cells. Note also that the vacuole demonstrated in Fig. 10 is surrounded by a distinct single
membrane. Fig. 9, X 6,100; Fig. 10, X 11,000.
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CANCER RESEARCH
VOL. 32
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'
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LP
N
A
N
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Fig. 11. Intestinal crypt 2.5 hi after administration of ara-C. Numerous lymphocytic cytoplasmic protrusions (arrows) can be seen lying within
the intercellular spaces. A', epithelial cell nuclei; LP, lamina propria. X 11,000.
Fig. 12. Intestinal crypts 3.5 hr after administration of ara-C. An indented epithelial cell nucleus (JV)appears to be wrapped around a portion of
a lymphocyte (/,). Note also the nuclear material (arrow) contained within the membrane-bound vacuole (K). X 7,600.
Fig. 13. An intraepithelial lymphocyte (/,) 1.5 hr after administration of ara-C. Note the clumping of chromatin (Cff) along the nuclear
membrane (arrows). EC, epithelial cells. X 6,700.
Fig. 14. Intraepithelial lymphocytes (L) 1.5 hr after administration of ara-C. Note the apparent nuclear fragmentation (arrows). LP, lamina
propria. X 12,400.
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\î
Fig. 15. Intestinal mucosa 2.5 hr after administration of ara-C. Portions of several necrotic cells (arrows) are present deep within the lamina
propria (LP). X 18,400.
Fig. 16. Intestinal mucosa 3.5 hr after administration of ara-C. A necrotic cell (arrow) can be seen lying adjacent to the basement membrane
(BM). EC, epithelial cell. X 15,600.
Fig. 17. Intestinal mucosa 2.5 hr after administration of ara-C. A portion of a necrotic cell (arrow) appears to be passing through the basement
membrane (BM). Additional necrotic debris is present in the lamina propria (LP) and within an epithelial cell cytoplasmic vacuole (K). X 13,400.
1488
CANCER RESEARCH VOL. 32
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The Effects of Cytosine Arabinoside upon Proliferating
Epithelial Cells
Robert S. Verbin, Gloria Diluiso, Hilda Liang, et al.
Cancer Res 1972;32:1476-1488.
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