[CANCER RESEARCH 32, 776-780, April 1972]
Sequential Changes in Spleen Cell Chromosomes during Friend
Virus Leukemia1
Stephen C. Elliott, Ricki M. Helm, and Michael E. Myszewski
Department of Biology, Drake University, Des Moines, Iowa 50311
SUMMARY
Infection of BALB/c mice with varying doses of Friend
virus resulted in the appearance of abnormal chromosome
numbers in spleen cells. Polyploid cells were found following
infection with all doses of virus used. Statistically, however,
polyploidy showed no correlation with virus dose, time after
infection, or their combined interaction. A total of 0.9% of all
infected metaphase figures examined showed the hyperdiploid
number of 41 chromosomes. This hyperdiploid number was
not clustered at any stage of the disease and was not higher
than that of the control mice. The number of abnormal cell
configurations in infected animals did differ significantly from
noninfected animals throughout the disease process, but no 1
abnormal
diploid number predominated.
A significant
correlation was found between the number of secondary
chromosomal constrictions and the progression of the disease.
As the spleen weight increased, the number of secondary
constrictions per metaphase figure increased. There was an
inverse relationship between increased spleen weight and the
number of normal 2N cells with no secondary constrictions.
Since an increased number of these secondary constrictions
appeared early in the disease process even with a low dose of
Friend virus, this change is considered to have significant
mutational importance.
INTRODUCTION
The rapid and extremely proficient transformation
of
normal spleen cells to malignant cells during infection of
susceptible mice with FV2 (3, 5) makes this system ideal for
studying virus-induced chromosomal aberrations. This system
provides a transition from the normal to the neoplastic state
under controlled conditions with a reproducible interval
between inoculation and the appearance of histologically
recognizable
tumors.
Previous reports (19, 20) have
documented the presence of chromosomal abnormalities in FV
disease but have not included critical information concerning
either the relationship of these changes to virus dose or the
rate of these changes through time. Since these previous
reports on FV-induced chromosome abnormalities, FV has
been further purified and its virulence increased by using
selected strains of mice, to the point where a large dose of
virus results in the death of almost all animals within 6 weeks
following infection. The present study was limited to 5 weeks
1Sponsored in part by funds from the Iowa Branch, American
Cancer Society, and the Drake University Research Council.
'The abbreviation used is: FV, Friend virus.
Received December 21, 1970; accepted December 28, 1971.
776
and not extended for the longer time period used in earlier
reports (19, 20). In these latter studies, animals had a longer
latent period before the viral leukemia resulted in animal
death.
This paper reports the quantitation of sequential changes in
spleen cell karyotype following the inoculation of 76 BALB/c
mice with serial dilutions of FV. Secondary constrictions have
been observed as a feature of normal mouse spleen cells (10).
The present paper indicates a significant difference between
the frequency of secondary constrictions in normal cells and
those from infected mice. With the well-established parameters
of the numbers and type of chromosome abnormalities in FV
infection with known virus doses, the relative role of these
abnormalities in the leukemic process can now be determined
by inhibiting the disease with a variety of antineoplastic
agents. On the basis of these findings, it is hoped to establish a
quantitative relationship between the frequency of secondary
constrictions through the disease process and the frequency of
these same aberrations during drug-induced remissions of
leukemia.
MATERIALS AND METHODS
Mice. Female BALB/c mice were obtained
from
Cumberland View Farms, Clinton, Tenn. The BALB/c strain
was chosen because of its demonstrated susceptibility to FV,
96 to 98% of infected mice showing evidence of FV disease
(3). The mice were kept in groups of 6 to 8 during the
experimental period and given food and water ad libitum.
Virus. The origin and preparation of the stock FV pool used
in this investigation has been previously reported (1,2). The
virus pool of titer 104-0 i.e., that dose which infects 50% of the
animals per ml (2, 12), was maintained at —¿65°
as a 20%
suspension of infected BALB/c mice spleens in sucrose
stabilizer.
Spleen Cell Chromosome Preparations. A modification of
the method of Fox and Zeiss (4) was used in preparing spleen
cells for chromosome analysis. Ninety min before animals
were sacrificed they were given i.p. injections of 0.75 ml of
0.2% Colcemid (Ciba Corporation, Summit, N. J.). A total of 6
to 10 slides was made from each suspension of isolated, fixed
spleen cells. Cells were affixed to the slides by a freeze-flame
dry technique.
Cells were stained with synthetic acetoorcein, dehydrated,
and mounted in Euparol Vert. After initial scanning at X 200,
a total of 30 metaphase figures for each animal was critically
examined under oil immersion at X 1250. Cells which
appeared under low magnification to be complete, with
nonoverlapping, well-spread chromosomes, were selected for
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Chromosome Changes in FV Leukemia
counting. For several mice, 30 countable cells could not be
found.
The chromosome data included counting the number of
chromosomes, assessment of polyploidy, and counting the
number of abnormalities found in each metaphase cell. The
primary abnormality was achromatic zones or secondary
constrictions (18) noted near the centromere. The change in
the number of these secondary constrictions was noted for
virus dose and elapsed time. No other chromosome
abnormalities observed appeared with any appreciable number
or in any consistent pattern in any of the mice tested.
Questionable figures were examined independently by 2
people. Cells, once chosen, were not rejected because of
chromosome number, size, shape, or degree of staining.
Selected cells were photographed with Kodak high contrast
copy film.
Experimental Design. Groups of 30 mice received i.p. 0.2 ml
of either a IO"1, IO"3, or 10"s dilution of the stock virus
prepared in sucrose stabilizer. Uninfected mice with and
without Colcemid treatment served as controls, and 14 of
these control animals were sacrificed after the same time
interval as experimental mice. A total of 6 Colcemid-treated,
virus-infected mice from each dilution was sacrificed weekly
for 5 weeks.
During the experiment, control mice did not demonstrate
any deviation from the normal spleen weight of an adult
mouse. In contrast, the infected mice developed splenomegaly
through the course of the experiment, giving a reliable
indicator of the degree of infection.
Statistical Analysis. Standard methods for computerized
statistical analysis were used for analysis of variance (Anova)
in the different data categories. Where needed, various
transformations of the data were carried out to fulfill the
assumptions underlying the analysis of variance techniques
used. Other bivariate analysis correlations were examined by
computer routines found in the book by Sokal and RohJf (16).
RESULTS
Spleen Weights. All mice infected with the Friend leukemia
virus exhibited hyperplasia of the erythroid elements of the
spleen with a resultant
splenomegaly
that increased
progressively during the 5-week course of study. The average
spleen weights with their standard error for all control and
infected animals are shown in Table 1. The spleen weight
increases proportionately both with time and with virus dose
when compared with the control values. The analysis of
variance for the transformed spleen weights V spleen weights
showed significant interaction at the 99% confidence level for
dose, weeks, and dose X weeks.
Chromosome Numbers. Among the 2149 metaphase cells
examined from infected animals, 21 or 0.9% exhibited 41
chromosomes. This is contrasted to 3 of 131 cells (2.3%)
among the control animals showing this abnormality. Only 3
cells from the infected animals and none from control animals
fell between counts of 41 chromosomes and the cells which
were interpreted to be polyploid. In many instances, a
chromosome number of less than 40 was noted (Table 1).
A significant
difference
was shown between
the
chromosome numbers in the various virus doses and for the
different time intervals. This difference was established
through analysis of variance (Anova) of cells from infected
animals that did not have a count of 40 chromosomes. The
raw data were prepared for this analysis by adding a constant
(0.01) to remove zero values and then transforming the data to
6 = arcsine \/p + 0.01, where p is the proportion of cells not
having a count of 40 chromosomes. The arcsine and square
root transformations were utilized to satisfy the underlying
assumptions required for analysis of variance ( 16). Significance
was shown with respect to dose, time following infection, and
the interaction between dose and time.
Similar analysis was made on the proportion of diploid
spleen cells from infected animals with normal mitotic
Table 1
Metaphase chromosome numbers and average spleen weights per control animal
and those infected with various FV dilutions
Time following
Dilution
animalsControlio-1
infection (wk)
i2345io-3
i234510's
No. of
(g)0.13 wt
0.01°0.38
+
0.0791.50
±
0.191.71
±
0.925.01
±
±0.367.5
0.00.26
±
0.110.53
±
0.041.55
±
±0.303.15
±0.416.9
0.00.15
±
1234514664616666166664Spleen
±0.020.15
0.020.30
±
0.031.10±
±
0.231.92
+ 0.64Total
cells counted131
no.<37043111010003233137012011225103293138866101021105101118165739138
>0000100100100000Polyploids008481120231135
)b168(28.3)180(30)97(17.5)180(30)28
(9.4
(28)180(30)171
(30)176
(29.3)180(30)30
(30)158(26.3)170(28.3)138
(23.0)180(30)108
(26.75)Chromosome
a Mean ±S.E.
6 No. in parentheses, average number per animal.
APRIL
1972
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777
S. C. Elliott, R. M. Helm, and M. E. Myszewski
patterns. Only at Week 2 did the proportion of normal cells
differ significantly between the 3 virus dilutions used and then
only between the highest and lowest doses of virus. The
proportion of cell configurations showing abnormal numbers
differed significantly from the controls after the 1st week.
Throughout the experiment, the infected animals always
exhibited a greater proportion of abnormal cells with respect
to diploid chromosome number.
Polyploid cells were observed during the course of infection
with all 3 doses of virus, but none were observed in any of the
control cells. In the infected animals the frequency of
polyploidy, as compared with the diploid number, showed no
dependency of the observed polyploid state upon dose, week,
or their combined interaction. However, in consideration of
polyploidy as a binomial function, the polyploidy occurring in
animals infected with a 10"1 dilution of FV differed
significantly at the 95% confidence limits in Weeks 2 and 4
from Week 1. Only a small overlap occurred at Week 3. The
occurrence of polyploidy among animals at the other virus
dilutions was too low to add anything significant in forming
trends during the sequence of the disease at these doses.
Table
1 summarizes the distribution
of metaphase
chromosome numbers per cell in animals receiving virus
dilutions of IO"1, 10~3, and 10~s, as well as results with
untreated
control animals with and without Colcemid
treatment. No significant differences were found between
these control groups with respect to chromosome number,
chromosome aberration, or any other parameter examined
because of the presence or absence of Colcemid treatment. For
this reason, all the controls have been analyzed as 1 group.
Chromosome
Aberrations.
Each metaphase figure was
carefully examined for the presence of chromosomal
aberrations. The number of secondary constrictions was
counted for each cell in which these constrictions were found,
and in these cells an average number of secondary
constrictions per cell was obtained for each animal where data
permitted. Those cells with abnormal chromosome counts
were included within the analysis; however, those that
contained no constrictions were ignored. Table 2 shows the
average number of constrictions per cell with the standard
error among those cells demonstrating constrictions. This table
also includes the proportion of such cells for each successive
week of the experiment. An increase was noted for each of the
virus dilutions through time with respect to the proportion of
cells showing 1 or more secondary constrictions. The
percentage of cells with constrictions and the average number
of constrictions per cell were greater in virus-infected animals,
with the sole exception of Week 1, 10~3 virus dose. As the
spleen weight increased so did the number of spleen cells with
these secondary chromosome constrictions and the average
number of secondary constrictions found in each cell.
A correlation of spleen weights and the average number of
constrictions per metaphase figure in those cells demonstrating
constrictions is shown in Chart 1. The 2 variables YI , spleen
weights transformed to V x + 0-5, and Y2, average number of
constrictions per figure, correlate in a population ellipse at the
95% confidence level. This ellipse demonstrates that, as the
spleen weight increases along the F-E axis, the average number
of chromosome
constrictions
in each cell increases
proportionately to the limits designated by the boundaries of
the ellipse.
A similar correlation was carried out with the use of the
variables YI , the spleen weight transformed to V* + 0.5, and
Y2, the arcsine \Jp + 0.01, where p is the proportion of
normal diploid cells with no constrictions among those ex
amined. Chart 2 exhibits the plotted points of the 95% con
fidence ellipse demonstrating the strong and significant correla
tion coefficient of these 2 variants. This analysis indicates that,
as the spleen weight increases, the probability of finding
metaphase figures with no secondary constrictions decreases.
DISCUSSION
Statistically valid alterations from the normal diploid
chromosome number in FV-infected BALB/c mice were found
Table 2
Proportion, mean, and standard error of secondary constrictions among control animals and those infected with FV
Time after
Dilution
(wk)ControlIO'1infection
12345IO'3
12345IO'5
12345No.
no. of
no. of
constrictions in cells
no. of
cells with
ofanimals14664616666166664Total
with
constrictions/cell1.88
constrictions591011608717327821351661733077105118160101%
constrictions45.759.488.993.596.196.445.675.094.396.1100.048.761.886.188.995.3T
showing constrictions12123246321548064169360428512991521
cells129170180931802818018017618030158170137180107Total
±0.50°2.22
0.942.89
±
1.172.40
±
1.232.77+
±
1.132.37
0.02.06
±
0.782.67
±
1.142.58
±
1.052.90
±
1.183.30+
±
0.01.97
0.802.05
±
0.822.17
±
0.922.41
±
0.992.7+
±
1.02
0 Mean ±S.E.
778
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Chromosome Changes in FV Leukemia
present study might have its basis in the strain differences
between mice or between virus stocks. Each experiment
reporting the high incidence of 41 chromosomes observed this
result in very few animals. This observation may have been due
to a chance fluctuation not observed in the present experi
ment. Tsuchida and Rich observed their finding in the "late
disease group" comprised of animals infected for 50 to 65
days. The present study comprised a 35-day period.
Polyploid cells were not found in increased numbers except
in the IO"1 virus dose and then only after the 1st 2 weeks of
1.6
1.5
1•¿
i
1
= 1.3
^3
TA
2.5
2.6
«VERACE NUMBER OF CONSTRICTIONS
Chart 1. Correlation of spleen weight and average number of
chromosomal constrictions in metaphase figures of spleen cells
containing chromosomal constrictions during FV leukemia. Coordinates
A to H show the 95% confidence ellipse limits.
r~i
1.50
1
1.10
til
SI
ARCSINE VTHToT
¡WHERE P = PROPORTION OF
DIPLOID CELLS WITH NO CONSTRICTION
Chart 2. Correlation of spleen weights to proportion of normal
diploio cells among mitotic figures examined during FV leukemia.
Coordinates A to H show the 95% confidence ellipse limits.
in this study. However, it does not seem possible to attach any
absolute significance to these differences as no consistent
abnormal number or pattern was obtained. Only a small
number of infected mice, 0.9%, exhibited the hyperdiploid
number of 41 chromosomes, and this abnormality was neither
clustered in any virus dose nor at any specific time. This is in
marked contrast to the increase in the number of cells with 41
chromosomes during the Friend disease reported by other
authors (19, 20). Tsuchida and Rich (19), using 3 ICR/Ha
Swiss mice, reported that 9% of 147 cells examined from these
animals late in the disease period showed the 41-chromosome
number. Wakonig-Vaartaja (20) reported that 1 of 3 mice
infected with FV had cells with a high incidence of 41
chromosomes. In this mouse, 42% of the 50 cells were
analyzed to be carrying this higher number of chromosomes.
The strain of mice used in this latter experiment was not
reported. The difference between these 2 studies and the
infection. The sporadic finding of this alteration, as with the
occurrence of cells with 41 chromosomes, would be consistent
with the hypothesis that these abnormalities result from,
rather than initiate, the leukemic process. The increase in the
proportion of cells with these abnormal numbers would be a
secondary feature of these spleen cells that previously had
been transformed by FV.
The finding of an increased number of secondary
constrictions early in the disease process and with all 3 doses
of FV is of interest. There was a positive increase in secondary
constrictions as the spleen weight increased. This correlation is
in agreement with the results of Tsuchida and Rich (19). They
showed that infection with FV resulted in a 2- to 3-fold
increase in the number of secondary constrictions late in the
disease process (50 to 65 days after inoculation). Although we
were never able to find this large an increase in the number of
secondary constrictions, the proportion of cells that showed 1
or more such constrictions increased with time during the
experiment. Along with an increase over control values in the
average number of constrictions per cell, the total infected
cells that showed this abnormality increased to include almost
all of the cells studied by the end of the experiment.
Recent reports have demonstrated the presence of or an
increase in secondary constrictions in a wide variety of
oncogenic processes. Miles and O'Neill (11) have reported
secondary constrictions in tumor cells that showed no other
cytological abnormality. A marked increase in secondary
constrictions was found by Ito et al. (7) in 2 human cell lines
derived from embryonic cultures exposed to human leukemic
fluid in vitro. These cell lines showed not only this marked
increase of secondary constriction but also the presence of a
herpes-type
virus particle. This herpes-type particle is
morphologically similar to the agent known to exist in
Burkitt's lymphoma cell lines, and a similar increase in
secondary constrictions has been reported in these lymphoma
cells (6,8).
Secondary constrictions have been reported to be complete
breaks in DNA (14), but these breaks apparently do not
separate from the chromosome because of the presence of the
chromosome matrix. This type of chromosome aberration was
observed early in the disease in our studies, even with a low
dose of FV, and the aberrations progressively increased
through the course of the study. If secondary constrictions are
interpreted to be breaks and since they appear early in the
spleen cell transformation
process, they would likely be
considered of great mutational importance (13). We interpret
the secondary constriction to be a facet of FV disease that
produces DNA breaks. Once the initial lesion is produced,
other cellular factors may influence the relative observed
recovery of these constrictions. Since secondary constrictions
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779
S. C. Elliott, R. M. Helm, and M. E. Myszewski
are observed in control animals, some aspect of cell
differentiation or heterochromatization
may serve to fix these
aberrations in FV-infected cells. Alternatively, selection of
cells bearing secondary constrictions may occur during the
course of the leukemia. The observed quantitative relationship
between time-dose-aberration appears substantial regardless of
the specific interpretation applied to explain the process of
increasing the aberration through the course of the disease.
The frequency of chromosomal abnormalities found in
various human leukemias has a tendency to return closer to
normal values during periods of spontaneous or drug-induced
remissions (15). To determine
whether the increased
secondary constrictions that are induced by FV infection
would revert to near normal numbers, we retarded the
leukemic
process
with
7,12-dimethylbenz(a)anthracene.
Previous reports have shown this strongly carcinogenic
hydrocarbon to be an effective inhibitor of all parameters of
FV leukemia when used in small weekly doses (1, 2). Although
the 7,12-dimethylbenz(a)anthracene
in itself can induce a
range of chromosomal abnormalities (9, 17), our initial studies
show a significant retardation in the appearance of secondary
constrictions in spleen cells in FV-infected animals treated
with this carcinogen.
ACKNOWLEDGMENTS
We thank Dr. Lee Kelley, Department of Microbiology and Medical
Technology, University of Arizona, Tucson, Ariz., for assistance in
statistical analysis of data.
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CANCER
RESEARCH
VOL.
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32
Sequential Changes in Spleen Cell Chromosomes during Friend
Virus Leukemia
Stephen C. Elliott, Ricki M. Helm and Michael E. Myszewski
Cancer Res 1972;32:776-780.
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