[CANCER
RESEARCH
26, 898-902, May 1966]
The Effect of Necrosis and Tumor
on the Cell Kinetics of Small Bowel1
ANTHONY
BETTS, ELIZABETH L. SEWALL,
AND
ROBERT TANGUAY
Maine Medical Center, Portland, Maine
Summary
Generation time and S phase time of the jejunum in the normal
golden hamster were estimated by the autoradiographic technic
with thymidine-3H. These times were compared with the genera
tion and S phase times of a group of hamsters with transplanted
fibrosarcomas and a group with turpentine abscesses. The
generation time was shorter in animals with necrotic lesions. It
is suggested that the common factor of tissue necrosis in both
inflammatory lesions and tumors causes acceleration of cell turn
over in other regenerating tissues; or, both tissue necrosis and
neoplasia can produce this effect. This generalized increased
synthesis of tissue may contribute to the cachectic state.
Introduction
There have been various studies on generation times of bowel
epithelium in man in an attempt to establish the normal turn
over rate (3, 5). These studies were made on patients with ad
vanced disease, often of a malignant nature. Although it has been
conceded in these reports that the neoplastic disease might have
affected the cell generation time in the normal tissue (3, 4), we
have found no subsequent report to investigate this issue. (Of
course, the limiting factor in any such study in man is the
hazardous exposure of healthy adults to the radioactive thymidine used in the technic.) Similarly, normal turnover cycles and
generation times have been established for some rodents, but we
have found no comparison between bowel kinetics in healthy
normal animals and in diseased animals of the same species.
The possibility that animals with necrotic lesions might have
a decreased generation time in bowel epithelium was suggested
by reports that patients with advanced malignancy and chronic
infections, and animals with necrotic lesions, showed a rapid red
cell turnover (2, 8, 9). The kinetics of the erythropoietic system
have been thoroughly explored in numerous conditions, in con
trast to the relatively little work devoted to other tissue cells.
We decided to compare the bowel kinetics of a group of normal,
healthy hamsters and 2 groups of animals with induced necrotic
lesions, using the autoradiographic technic. The following is a
brief outline of the rationale of this method.
Sufficient tritiated thymidine (thymidine-3H) is given to an
animal, and this is incorporated into all cells (S phase) forming
DNA at that moment and for approximately the next hr. There
1This investigation was supported by Cirant ÇA08100from the
U. S. Department of Health, Education and Welfare.
Received for publication May 3, 1965; revised November 5,
19G5.
898
after, the thymidine-3H is excreted or catabolized (1) and is un
available to any cell.2 Those labeled cells subsequently under
going mitosis will have tritiated mitotic figures, which can be
detected by autoradiograph; these figures can be counted. In a
short while all mitotic figures will be labeled, giving a reading of
approximately 100% labeled mitoses. The percentage of labeled
cells becomes reduced as those cells which had not been syn
thesizing DNA when thymidine-3H was available enter mitosis.
The percentages of labeled mitoses may be plotted against time
(Chart 1).
Materials
and
Methods
One hundred and fourteen adult golden hamsters were used
in this study. They were maintained on water and Purina chow
ad libitum.
Xormal Controls
Thirty normal adult animals received 75 nc each of thymidine3H (thymidine-methyl-3H, specific activity 6.7 c/mmole, New
England Nuclear), or approximately 0.67 M<Vgmof body weight.
The thymidine-3H was administered i.p. without anesthesia.
Animals were killed at various intervals between 0.5 and 24 hr
with an overdose of Nembutal (Abbott). Immediately after
death a segment of small bowel was taken at a point 2 cm distal
to the pylorus. This segment of bowel was fixed in buffered forma
lin and paraffin sections cut at 3 p. Slides were dipped in NTB3
emulsion (Kodak), exposed for 3 weeks, and then developed and
stained with hematoxylin and eosin. The sections were examined
for mitotic figures in the crypt area of each segment of jejunal
epithelium, and 50 mitoses were counted by each worker. Only
those mitotic cells having more than 10 autoradiographic dots
were deemed "labeled cells." Percentage mitotic labeling was
determined (Table 1).
Turpentine-induced
Necrosis
Thirty-one animals received 0.2 ml of sterile turpentine into
the left thigh muscle under Nembutal anesthesia. After 4 days
all animals had an obvious necrotic abscess of the left thigh, but
1 In a pilot experiment to substantiate this, 2 hamsters were
given the usual dose of thymidme-3H and killed at 5 and 8 hr and
bled. Bowel epithelium by autoradiograph showed heavy labeling
of nuclei. One ml of the removed sera was given to 2 normal ham
sters; 14 hr later these hamsters were killed. Autoradiographs of
bowel showed no labeled cells.
CANCER RESEARCH VOL. 26
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Cell Kinetics of Small Bowel
TABLE 1
PERCENT LABELING OF MITOSES IN NORMALHAMSTERS"
TABLE 2
PERCENT LABELINGOF MITOSES IN HAMSTERS\VITHTURPENTINEINDUCEDNECROSIS"
LABELED
S.D.+
MITOSES ± 2 X
TIME OF SACRIFICE
LABELED
(hr)0.51.02.02.252.52.753.03.253.53.754.04.254.55.05.5G.O6.57.08.09.010.013.014.015.010.
MITOSES73179808089807482829486909591878467403410205562737319513024%
LABELED
S.D.+
MITOSES ± 2 X
TIMEOFSACRIFICE
(hr)0.50.751.01.52.02.252.52.753.03.253.754.254.55.50.58.09.511.012.012.012.513.013.514.015.016.0
MITOSES01445939898858369807052727262378241701040887367856940452845%
LABELED
152246949595104958997971091011051101061029982554925357077888834664539-is-816646565746559676779717580767269522519-5540475858436159
1515296010811311310098849585678787775223393215255510388821008455604360-15-
017.018.020.021.024.0%
" All animals received 75 MCthymidine-'II
data represents 1 specimen.
at 0 hr. Each line of
the abscess had not broken down or discharged. On the 4th day
each animal received 75 pc of thymidine-3H in a similar manner
to that described above for normal animals. The animals were
killed sequentially from 0.5 to 24 hr and segments of bowel were
made into autoradiographs and mitotic figures were counted
(Table 2).
Necrotic Tumor
Thirty adult hamsters had a fibrosarcoma (MCP1) trans
planted to the scapular region. This tumor grows rapidly and
becomes centrally necrotic in about 3 weeks. No tumor in this
series broke down at the surface. At 3 weeks the animals were
sacrificed at intervals over 21 hr after receiving thymidine-3H,
and segments of jejunum were removed and treated in a similar
manner to that described above in the other series (Table 3).
In order to evaluate the accuracy of the labeled mitotic
counts, a further 23 animals were studied. This group was com
prised of normal, turpentine abscess, and tumor animals. They
were given the usual dosage of thymidine-3H and sacrified 4 hr
later. Counts of the labeled mitotic figures were then made by
3 workers independently. From this study it was found that the
standard deviation was 7.5. Two standard deviations were then
MAY I960
«Allanimals received 75 /ic thymidine-3H
data represents 1 specimen.
at 0 hr. Each line of
added and subtracted from all mitotic counts in the 3 experi
mental groups (.Tables 1-3). To establish the S phase and
generation times, the time taken for each group to reach the
arbitrary figure of 50% labeling of mitotic figures was noted.
The longest times from the normal group for DNA synthesis and
generation were then compared with the shortest possible times
in the tumor and turpentine groups (Table 4). In contrast, the
shortest possible S phase and generation times were compared
with the longest in the tumor and turpentine groups (Table 5).
All mitotic counts were undertaken as a blind study by 3
workers. Attempts were made to count the labeled cells in the
crypts in order to establish a labeling index. Here there was great
variation in the counts by the workers even on the thinnest sec
tions and this approach to calculating a generation time was
abandoned.
Results
The normal time for DNA synthesis in the golden hamster is
between 5.25 and 8 hr, with a mean of 6| hr. The generation
SÕK)
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A. Belts, E. L. Sewall, and R. Tanguay
TABLE 5
TABLE 3
PEKCENTLABELINGOP MITOSESIN HAMSTERSWITHNECROTIC COMPARISON
OF SHORTESTPOSSIBLES PHASEANDGENERATION
TUMORS"
TIMESOFNORMALHAMSTERSANDLONGESTPOSSIBLE
TIMESOF EXPERIMENTAL
ANIMALS
LABELED
S.D.+
MITOSES ± 2 X
LABELED
TIMEOFSACRIFICE
AT WHICH 50% MITOTIC
MITOSES4296291909873787092909395749371446924203220270138375615047%
(hr)0.51.01.51.752.02.252.52.753.03.253.54.54.54.755.756.06.57.08.09.511.012.012.512.513.013.514.516.018.021.0%
OCCURS01st1.75112nd787.53rd13.513.2513.25S
LABELING
PHASE(hr)5.2576.5GENERA
1519447710610511388938510710510811089108865984393547354215289890766562-15-1114477675835863557775788059785629549517512-15-26860463532
GROUPNormalTurpentine
TION
(hr)11.7512.2512
necrosisTumorHR
" For graphic representation, see Chart 1.
TABLE 6
COMPARISON
OPTHEMEANS PHASEANDGENERATION
TIMES
PHASERange
GROUPNormalTurpentine
(hr)5.25-85.5-6.55.25-7Mean(hr)6ÃŽ6enGENERATIONRange
(hr)11.75-14.2511.5-1211.75-12.25Mean(
necrosisTumorS
NORMAL
100
HAMSTERS
90
PERCENT 80
LABELING 70
OF
MITOSES 60
50
4G
" All animals received 75 /tc thymidine-3H at 0 hr. Each line of
data represents 1 specimen.
30
20
TABLE 4
COMPARISON
OF LONGESTPOSSIBLES PHASE ANDGENERATION
TIMESOFNORMALHAMSTERSANDSHORTESTPOSSIBLE
TIMESOPEXPERIMENTAL
ANIMALS
AT WHICH 50% MITOTIC
OCCURS"1st11.25
LABELING
GROUPNormalTurpentine
1.52nd9(¡.573rd15.2513
13S
5.5GENERA
11.5
" For graphic representation, see Chart 1.
time is between 11.75 and 14.25 hr with a mean of 13 hr. These
times compare well with similar reports on other rodents (4, 7).
Table 4 shows the longest possible normal DNA synthesis time
and generation time compared with the shortest similar times for
the turpentine and tumor groups. It will be seen that the S phase
time and the generation time of the tumor and turpentine, ani900
0
468
'012
HOUR
14
16
18
20
22
24
CHART 1.—Percentage of labeled mitoses vs. time between
labeling and sacrifice, in normal hamster jejunum. Each dot
PHASE(hr)85.25
represents the count in 1 animal.
TION
(hr)14.2511.75
necrosis
TumorHR
10
mais are approximately 3 hr shorter than those of the normal
group of hamsters.
Table 5 shows the shortest possible normal times of DNA
synthesis and generation time compared with the longest possi
ble times of the tumor and turpentine groups. The S phase times
of the tumor and turpentine groups are 1.5 and 2 hr longer than
the normal S time, but the generation times are approximately
the same.
Chart 1 shows the distribution of percentage mitotic labeling
in the normal group of hamsters. Each line through a point rep
resents ±2X S.D. No attempt has been made to fit a curve to
these points by eye, as some have done, for as Mainland (6) has
quoted Snedecor, "Avoid drawing 'eye-fitted' curves. They are
CANCER RESEARCH VOL. 26
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Cell Kinetics of Small Bowel
highly subjective and are apt to be misleading to both the
perpetrator and the victim."
Discussion
The mean generation time for the 2 necrotic lesion groups ap
peared to be shortened by 1 hr (Table 6). The mean S phase
time is also possibly shortened. We attempted the method of
determining the generation time by the labeling index (7), or
ratio of labeled cells to all cells, but found excessive variation in
the labeled cell counts between each worker. This was mainly
attributed to the fact that even the thinnest section showed more
than 1 cell layer. By contrast, labeled mitotic figures were seldom
difficult to see because the cells projected into the center of the
villus and could be counted with ease (Fig. 1). The lack of cer
tainty that all labeled cells will mitose, and our own counting
difficulties, were the basis for rejecting this method as unreliable,
and we preferred to calculate generation time from the counts as
described above.
From our results in rodents it appears that necrotic lesions in
one area of the body may influence the generation time of other
regenerating tissues; possibly, a similar mechanism occurs in man.
If this is so, the conclusions reported of the normal cell turnover
of bowel in man, derived from patients who had neoplastic or
necrotic lesions, may not be valid.
The mechanism by which the generation time of the bowel
epithelium decreases is unknown. If the generation time and
DNA synthesis time are shortened, then perhaps this implies
earlier cell death. Or the adult cell may be directly affected by an
unrelated necrotic lesion and have shortened survival time analo
gous to the red cell in similar circumstances; the damaged, dying
cells may then locally stimulate crypt cells to replace them, and
this would cause a decreased generation time. In these studies,
all villi were examined for evidence of hyperplasia, both in the
MAY 1966
necrotic series and in the normal, and in none of them could any
hyperplasia be detected; nor could any other abnormality be
seen.
If indeed there is an increased proliferation of small bowel epi
thelium not immediately involved with a neoplastic and necrotic
lesion, it would seem that other regenerating tissue may be simi
larly accelerated. The shortened survival of the red cell is thought
to contribute to the state of anemia in malignancy. The general
ized increased synthesis of regenerating tissue in various parts of
the body, stimulated by necrosis, might contribute to the illdefined syndrome of cachexia.
¿References
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Technique in the Study of Cell Population Cytodynamics. Lab.
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2. Friedell, G. H. Anemia in Cancer. Lancet, 1: 356-59, 1965.
3. Lipkin, M., Bell, B., and Sherlock, P. Cell Proliferation Ki
netics in the Gastrointestinal Tract of Man. I. Cell Renewal
in Colon and Rectum. J. Clin. Invest., 42: 767-76, 19(13.
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8. Rigby, P. G., Betts, A., Friedell, G. H., and Emerson, C. P.
Kinetics and Loci of Destruction of Erythrocytes in Tumorbearing Hamsters. J. Lab. Clin. Med., 59: 638-44, 1962.
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901
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A. Betts, E. L. Sewall, and R. Tanguay
FIG. 1.—Microantoradiograph
into center of villas.
902
of normal hamster jejunum 1-2 hr after injection of thymidine-3H,
showing labeled mitoses projecting
CANCER
RESEARCH
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VOL. 26
The Effect of Necrosis and Tumor on the Cell Kinetics of Small
Bowel
Anthony Betts, Elizabeth L. Sewall and Robert Tanguay
Cancer Res 1966;26:898-902.
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