Test of Correlation between the Pink-Eye Gene and Sus
ceptibility to Induced Fibrosarcoma in Mice*
LEONELL C. STRONG
(From the Department
of Anatomy,
Yale University School of Medicine,
The x2 determination
of this difference is 24.15,
which is significant. This evidence indicated a cor
relation between pink eye and a lower fibrosar
coma susceptibility.
The present experiment continues observations
on the relationship between the gene for pink eye
and the rate at which fibrosarcoma
appears in
mice following the subcutaneous
injection of
methylcholanthrene.
Mice of inbred strains with pink eyes (I, JX)
show a lower incidence of methylcholanthrene
induced fibrosarcomas
than do mice with dark
eyes. Mice of thirteen other inbred strains, simi
larly treated with rnethylcholanthrene
(1 mg. in
jected subcutaneously
in 0.1 cc. of sesame oil at 60
days of age), showed a higher incidence of induced
tumors. Furthermore, the “screening―of
the fifteen
strains revealed correlations between five genetic
markers and fibrosarcoma
susceptibility,
as fol
lows : (a) brown versus black, (b) nonagouti versus
agouti, (c) piebald versus self, (d) pink eye versus
dark eye, and (e) female versus male (7). Mice
with multiple dominant genetic complexes, such as
CSH, with dark eye, intense, black, agouti, and
self show the highest incidence of chemically in
duced fibrosarcomas. Mice with multiple recessive
complexes, such as the I strain with five recessive
genes, pink eye, dilute, brown, nonagouti, and pie
bald have shown the greatest resistance to fibro
sarcomas. Between the two extremes are mice of
the other thirteen inbred strains with various com
binations of genetic markers and susceptibilities to
fibrosarcomas.
Since mice of the C81I strain possess wild type
characters, these facts cited above may mean that
mice with the fewest mutants have the highest
susceptibility to induced fibrosarcomas.
.
The
F2
pink-eye
segregates
of
a
cross
MATERIALS AND METHODS
Dark-eyed,
brown, nonagouti, long-eared, fe
male mouse No. 138,716 of the F, generation of the
r,m descent(5) wasoutcrossedto a maleof the
pink-eyed,
brown, nonagouti,
short-eared,
JK
strain. This mating produced three litters of six
teen young, all of which were dark-eyed, brown,
nonagouti,
and long-eared.
Male mouse No.
184,442 (dark-eyed, brown, nonagouti, long-eared)
of the NH strain was mated to a female mouse of
the JK strain, who produced four litters of 21
mice, all of which were dark-eyed, brown, non
agouti, and long-eared. Thus, it was concluded
that each of these F, mice was homozygous for
dark eyes, and they were then mated together and
the progeny continued for the Br (dark-eyed,
brown, nonagouti) descent. One mg. of methyl
cholanthrene,
dissolved in 0.1 cc. of sesame oil,
was
between
dark- and pink-eyed mice (Brs X I strains) showed
approximately
15 per cent fewer induced tumors at
325 days than their dark-eyed sibs. Thus, of 358
F2 pink-eyed mice injected with methyicholan
threne, 151, or 42.7 per cent, developed fibro
sarcomas by 32@5days; whereas of 1,204 F2 dark
eyed mice similarly treated, 697, or 57.9 per cent,
developed fibrosarcomas within the same period.
The experiment was terminated at 325 days fol
lowing the injection of the methylcholanthrene.
* This
experiment
has been
made
possible
by
grants
New Haven, Conn.)
injected
subcutaneously
into
both
parents
of
each generation at 60 days of life. During the en
suing ten generations of direct descent, 264 mice
were produced,
all of which were dark-eyed,
brown, and nonagouti.
In the collateral lines,
dark-eyed, brown, and nonagouti mice were ob
tained in excess of ten thousand. The mating of
mouse No. 219,009 female and No. 219,010 male
of the F13 generation produced 42 young, 38 of
which were dark-eyed and 4 pink-eyed. One of
these pink-eyed mutants
was outerossed
to a
mouse of the pink-eyed, brown, nonagouti, short
eared J@Kstrain and produced seventeen young, all
of which were pink-eyed, brown, nonagouti, and
long-eared. It was concluded that a new pink-eye
mutation had occurred at the pink-eye or “p―
from
The Anna Fuller Fund, The Jane Coffin Childs Memorial
Fund for Medical Research, and the Public Health Service,
acting through the National Advisory Cancer Council.
Received for publication August 18, 1950.
42
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1951 American Association for Cancer Research.
SmoNc@—Pink-Eye Gene and Fibrosarcom,a in Mice
43
locus. Thus, pink eye had occurred as a mutation
Brp mice, together with comparative data for the
somewhere in the preceding descent (between the
Br descent, are given in Table 1.
F4 and F12).
The percentage incidence of the three types of
The Brp subline of the NH descent consists of tumors (total tumors) in the mutant sublime is ap
pink-eyed and dark-eyed mice which were de
proximately the same as that for the mice of the
scended from one of the original pink-eyed mu
parental Br subline (79.9 per cent of total tumors
tants mated to a dark-eyed individual of the NH
line. The origin of the Brp descent is given in
33
F1
Chart 1. The pink-eyed mutant (“Brp―
subline)
has been continued for twelve generations (F@c— F2
22
F@) by mating a pink-eyed individual to a sib
heterozygous for dark eye. The “Brp―
descent has
5
Br
F3
produced 641 mice; 828 dark-eyed and 318 pink
eyed (expectation
of 1 : 1 ratio would be 320.5
24
Br
F4
dark-eyed :320.5 pink-eyed).
The sex ratio has
been 349 females to 292 males (54.5 per cent fe
36
Br
F5
males:45.5 per cent males).
One mg. of methylcholanthrene
dissolved in 0.1
22
Br
F6
cc. of sesame oil was injected in the right flank,
when the mice were 60 days old. The mice were ex
14
Br
F7
amined weekly for the appearance of tumors at the
site of injection and were killed as soon as the tu
I?
Br
F8
mors became firm and progressively increased in
size between two periods of observations. All mice
33
Br
F9
dying or killed during the experiment were ex
amined at autopsy, and lesions were fixed in
43
Br
0
Bouin's fixative and stained with hernatoxylin and
eosin.
34
Br
F11
RESULTS
Mice of the “Brp―
lineage injected with 1 mg. of
41
Br
2
methyicholanthrene
gave rise to three types of tu
264Brs
0
mors : (a) fibrosarcoma, arising at the site of the
38
Br@
4pBr
3
injection of methylcholanthrene;
(b) adenomas of
the lungs, and (c) a gastric lesion involving the
F14
glandular mucosa near the pylorus, similar to
lesions arising in mice of the Brs descent (8). The
CHART 1.—Lineal descent of a single pair of mice of a cross
between the CBAN and JK mice. Parentage
for the first
gastric lesion has also been discussed by McPeak
fourteen generations are given. The number of progeny pro
and Warren (3), Smith and Strong (4), Kaplan
(2),
and
Bagshaw
(1). The
tumors
were
similar
to
those appearing in mice of the ancestral Br sub
line. The incidence of the three types of tumors in
duced in each mating is given on the right. All mice from the
F2 generation onward were injected with methyicholanthrene.
Pink-eye mutants (pBr) appeared for the first time in the F1,
generation.
TABLE 1
PERCENTAGE OF TUMORS IN MICE'
Mice of the Br subline and of the two color classes of the pink-eye mu ant subline Brp. The sexes are separate, and the three
types of lesions and total tumors are given separately.
Per centPer
centL.P.Per
centNo.TotaltotalLocallocalfibro.LunglungStomachstomachtockColorSexinj.tumorstumorstumorstumorsS
centPer
.tumorstumorstumorstumorsBr917611263.610858.5228.1169.12413.6Brci'14713793.211074.8199.0138.87349.7BrppBr
145
118
205.2
81.4
66.9
pBr9
97
a―
51283.8 79.9129
Total:173
641145
48974.6 68.5224.0 218.421
23.7Br92225170876.5155770.0237.622910.826712.0BrBra―
Total:1509
37341257
* Symbols:
Br —dark-eyed,
296083.3
brown, nonagouti;
79.31071
pBr —pink-eyed,
262871.0
70.4211.4
brown, nonagouti;
226.9108
4.8
7
5712.1 8.921
8377.2
9.0513
84
28.4
15212.1
78084.0
20.9
S —sarcoma.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1951 American Association for Cancer Research.
Cancer Research
44
in mutants, compared with 79.2 per cent in the
parental mice) (Table 1). In each series the local
appearing fibrosarcoma is the predominant tumor,
68.5 per cent in mice of the combined Brp subline
and 70.4 per cent for mice of the Br sublime. This
difference is not significant. The incidence and rate
of appearance of tumors are presented in Charts 2,
3, 4, and 5. Lung adenomas appeared in 8.8 per
cent of mice of the Brp mutant line and in 9.0 per
cent of mice of the original Br subline. This differ
ence is also not significant.
The mucosal lesion appeared in 23.7 per cent of
the mice of the Brp descent and in 20.9 per cent of
mice of the parental Br stock(Table 1 and Chart 6).
The gastriclesion appeared more frequentlyin male
mice of all series. This lesion occurred in 12.8 per
cent of Brp females and in 36.6 per cent of males of
the same subline; the gastric lesion appeared in 12.0
per cent of females of the parental Br stock and in
33.3 per cent of males of the same stock. x' deter
mination on the sex difference for gastric lesions
for the Brp descent is 49.6, which gives a P value
< .001 ; x2 for the sex difference in the original Br
subline is 263.3 with a P value < .001. Both these
differences are significant.
The average latent period for the appearance of
fibrosarcomas is approximately the same in mice of
all series (Table 1). However, there is a slight dif
ference between the sexes, the tumors appearing
somewhat earlier in males of two of the three series
(the parental Br and the Br segregates of the
mutant Brp descent) than in the three series of
females.
DISCUSSION
In the B! subline, made up of mice descended
from a mouse characterized by a dominant muta
tion at the Black versus Brown locus, there oc
curved two changes in tumor susceptibilities.
One
of these changes (susceptibility
to induced fibro
p,,cs,,o,.
,‘S
--——PB,,
DAYS
@
460
5@
1W
DAYS
CHART 4.—Dataforfemaleand
CHART 2.—Rate of appearance of fibrosarcomas at the site
of injection of methyicholanthrene
in (a) Br female (solid
line) and (6) pBr female of the mutant Brp sublime (dash
line). Time expressed in days is plotted along the base line;
percentage of fibrosarcomas on the vertical line.
malepBrmice
of the mutant
Brp sublimeare added together (dash line) and comparable data
for Br female and male mice are also combined
Time in days is given on base line and percentage
induced fibrosarcomas on the vertical line.
(solid line).
incidence of
p.,.e,,o,.
p—s's
DAYS
,00
CHART 3.—Rate
ZOO
300
of appearance
400
DAYS
500
of induced
500
700
fibrosarcomas
in
Caawr 5.—Induced fibrosarcomas in (a) original or ances
tral Br sublime (solid line) and (b) the combined data for all
(a) Br male mice (solid line) and (b) pBr male mice (dash line).
mice
Time in days is given on the base line; percentage
sarcomas on the vertical line.
base line; percentage
vertical line.
of fibro
of the
mutant
Brp
sublime.
Time
is given
in days
incidence of induced fibrosarcomaa
on
the
on the
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1951 American Association for Cancer Research.
STRONG—Pink-Eye Gene and Fibrosarcoma in Mice
sarcoma) was in the direction of an increased sus
ceptibility, whereas the other change (a suscepti
bility to the gastric lesion) was in the opposite
direction (6).
In the present experiment, the average latent
period for fibrosarcomas for females of the original
Br subline was 237.6 days and for males of the
same subline, 211.4 days. The average latent pe
nod for fibrosarcomas for dark-eyed females of the
Brp subline was 223.1 days and for pink-eyed fe
males, 224.0 days; for dark-eyed males, 199.0 days
and for pink-eyed males, 205.2 days. Thus, there
appears to be no change in susceptibility to fibro
sarcomas in the descendants of the pink-eyed mu
tants and in their litter-mate dark-eyed sibs.
Pvc..ts's
45
tological types occurred in 2,860 tumor-bearing
mice, or 1.26 tumors per tumor-bearing
mouse.
The present data do not give much information
on the original nature of an association between
pink eye and fibrosarcoma susceptibility. This as
sociation appeared to be in the nature of linkage
(twogenesorentitiescarriedonthesamechromo
some), but this conclusion may not be the final
one. Certainly, it is clear by the present evidence
that a mutation may occur at the pink-eye locus
without affecting susceptibility
to (a) fibrosar
coma, (b) lung adenoma, and (c) a gastric lesion
involving the mucosa just anterior to the pylorus.
The present evidence does not indicate whether
the new pink-eye mutation should be considered
identical to the old pink eye (pp) or to some new
allele.
SUMMARY
A mutation at the pink-eye locus occurred in @he
descendants
of dark-eyed
mice injected with
methylcholanthrene
(mutation = P to p). Mice
showing the recessive mutation and their dark
eyed sibs continued to develop (a) fibrosarcomas
at the site of injection of methylcholanthrene,
(b) lung adenomas, and (c) a mucosal lesion just
anterior to the pylorus, at the same rate and inci
dence as mice of their dark-eyed ancestry.
DAYS
CHART 6.—Data on the rate of appearance of gastric lesions
in (a) original Br sublime females (circle and solid line), (b)
the original Br sublimemales (solidline), (c)the femalemiceof
the mutant Brp sublime(circle and dash line), and (d) male
mice
of the
mutant
Brp
sublime
(dash
line).
Time
is plotted
in
days on the base line and percentage incidence of gastric
lesions on the vertical line.
The incidence of lung adenomas and the gastric
lesion also appear to be unaffected.by the mutation
at the pink-eye locus. The original sex difference
between the incidence of the gastric lesion also
appears to be unaffected by the mutation at the
“p―
locus.
The number of mice developing multiple tu
mors is the same in the two series. In the Brp
descent, 648 lesions (fibrosarcorna, lung adenomas,
and gastric lesions) developed in 512 mice, or 1.26
tumors per tumor-bearing
mouse, whereas in the
parental Br sublime 3,745 lesions of the same his
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1951 American Association for Cancer Research.
Test of Correlation between the Pink-Eye Gene and
Susceptibility to Induced Fibrosarcoma in Mice
Leonell C. Strong
Cancer Res 1951;11:42-45.
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