Carcinogenesis vol.30 no.5 pp.879–885, 2009
Advance Access publication March 2, 2009
Predominant modifier of extreme liver cancer susceptibility in C57BR/cdJ female mice
localized to 6 Mb on chromosome 17
Stephanie E.-M.Peychal, Andrea Bilger, Henry C.Pitot and
McArdle Laboratory for Cancer Research, University of Wisconsin School of
Medicine and Public Health, 1400 University Avenue, Madison, WI 53706,
?To whom correspondence should be addressed. Tel: þ1 608 262 2177;
Fax: þ1 608 262 2824;
Sex hormones influence the susceptibility of inbred mice to liver
cancer. C57BR/cdJ (BR) females are extremely susceptible to
spontaneous and chemically induced liver tumors, in part due
to a lack of protection against hepatocarcinogenesis normally of-
fered byovarian hormones. BR males are also moderately suscep-
tible, and the susceptibility of both sexes of BR mice to liver
tumors induced with N,N-diethylnitrosamine relative to the re-
sistant C57BL/6J (B6) strain is caused by two loci designated
Hcf1 and Hcf2 (hepatocarcinogenesis in females) located on chro-
mosomes 17 and 1, respectively. The Hcf1 locus on chromosome
17 is the predominant modifier of liver cancer in BR mice. To
validate the existence of this locus and investigate its potential
interaction with Hcf2, congenic mice for each region were gener-
ated. Homozygosity for the B6.BR(D17Mit164-D17Mit2) region
resulted in a 4-fold increase in liver tumor multiplicity in females
and a 4.5-fold increase in males compared with B6 controls.
A series of 16 recombinants covering the entire congenic region
was developed to further narrow the area containing Hcf1. Suscep-
tible heterozygous recombinants demonstrated a 3- to 7-fold effect
in females and a 1.5- to 2-fold effect in males compared with B6
siblings. The effect in susceptible lines completely recapitulated the
susceptibility of heterozygous full-length chromosome 17 congenics
andfurthermore narrowed the locationofthe Hcf1locus toa single
region of the chromosome from 30.05 to 35.83 Mb.
Hepatocellular carcinoma is the fifth most common neoplasm world-
wide and the third most common cause of cancer-related death, caus-
ing .500 000 deaths per year (1). The incidence of hepatocellular
carcinoma is 2- to 5-fold higher in men than in women, and it is
uncertain if this difference is caused solely by the differing hormonal
environments or is also influenced by differences in exposure to risk
factors (2,3). Known risk factors for liver cancer include hepatitis B or
C virus infection, alcohol consumption and aflatoxin B1ingestion (4).
Similar to humans, male mice have a higher incidence than female
mice of both spontaneous liver tumors (5,6) and liver tumors follow-
ing perinatal treatment with a variety of carcinogens (7,8). The sexual
dimorphism in murine hepatocarcinogenesis is due to the contrasting
effects of sex hormones on liver tumor induction. Castrated male mice
develop fewer liver tumors than intact males, whereas ovariectomized
females develop more liver tumors than their intact counterparts (7,9–
11). Furthermore, gonadectomy results in a similar incidence of liver
tumors in B6C3F1males and females (7). These studies demonstrate
that androgens promote liver tumors, whereas ovarian hormones in-
hibit their development.
C57BR/cdJ (BR) male mice have an intermediate susceptibility to
liver tumors compared with males of other inbred strains, whereas BR
females are extremely susceptible, developing up to 50-fold more
tumors than females of all other strains tested (8,11). This high sus-
ceptibility is cell autonomous (12) and is due, in part, to a loss of the
ovarian hormones’ protection from liver tumor development. As a re-
sult, in contrast to other strains, ovariectomy in BR females does not
cause a significant change in liver tumor multiplicity after treatment
with N,N-diethylnitrosamine (DEN) (11). The loss of protection seen
in BR females is not due to a difference in the amount or affinity of
estrogen receptor in the liver of BR females (13). BR females, there-
fore, offer a unique opportunity to investigate how ovarian hormones
influence susceptibility to hepatocarcinogenesis.
Ovarian hormones may cause a decrease in the growth rate of pre-
neoplastic and neoplastic lesions. Preneoplastic lesions in the mouse
liver are indicated by an abnormal deficiency in glucose-6-phosphatase
(14). These lesions have similar growth rates in C57BL/6J (B6) and
C3H/HeJ (C3H) females, and the growth rates in these females are
lower than in the corresponding males (11,15). In contrast, preneoplas-
growth rates in BR males and BR females are similar. Furthermore,
ovariectomy increases the growth rate of preneoplastic lesions in B6,
C3H and B6C3F1females, but not in BR females (7,11,16–18). There-
fore, the high susceptibility of BR female mice to liver tumors may be
due to the inability of their ovarian hormones to inhibit the growth of
preneoplastic lesions in the liver (11).
A linkage analysis of crosses between the resistant B6 and sensitive
BR strains identified just two hepatocarcinogenesis in females (Hcf) loci
that are responsible for the majority of the difference in susceptibility to
DENinboth malesand females(19). Hcf1is located on chromosome 17
and Hcf2 is found on chromosome 1. These two loci together accounted
for ?85–90% of the susceptibility in the BR strain compared with B6.
The Hcf1 locus alone was responsible for roughly two-thirds of the
effect. The Hepatocarcinogen sensitivity 7 (Hcs7) locus has previously
been identified on chromosome 1 in a linkage analysis of crosses be-
tween the C3H and B6 strains (20). This locus accounted for ?85% of
C3H males compared with resistant B6 males. The Hcf2 locus from BR
and the Hcs7 locus in C3H may be identical (20).
Congenic lines for each Hcf locus from BR on a B6 background
were generated and their susceptibilities to DEN-induced hepatocar-
cinogenesis were evaluated toverify the existence of these loci as well
as to investigate any potential interaction. In addition, a series of 16
recombinant lines spanning the majority of chromosome 17 were bred
and similarly evaluated to further reduce the region known to contain
Hcf1, the locus responsible for the majority of the susceptibility of the
BR strain. Finally, the variation among B6, BR and C3H mice was
studied by analyzing genotypes at known single nucleotide polymor-
phisms (SNPs) and sequencing non-coding regions of genes to gen-
erate haplotype maps of the identified region.
Materials and methods
B6 and BR mice were purchased from the Jackson Laboratory (Bar Harbor,
ME) and bred in our facilities. Mice were housed in plastic cages on corncob
Abbreviations: B6, C57BL/6J; BR, C57BR/cdJ; C3H, C3H/HeJ; DEN, N,N-
diethylnitrosamine; IL-6, interleukin-6; PCR, polymerase chain reaction; SNP,
single nucleotide polymorphism; TNF, tumor necrosis factor.
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bedding (Bed O’Cobs, Anderson Cob Division, Maumee, OH). Mouse Diet 9F
5020 (LabDiet, Madison, WI) and acidified tap water were provided ad libi-
tum. Mice were inspected daily. All experimental protocols were approved by
the Animal Care and Use Committee of the University of Wisconsin School of
Medicine and Public Health.
Full-length congenic B6.BR(D1Mit5-D1Mit17) (B6.BR-Ch1) mice were gen-
erated as described previously (21). Full-length congenic B6.BR(D17Mit164-
D17Mit2) (B6.BR-Ch17) micewere generated as follows: B6 and BR micewere
mated to generate B6BRF1animals and the F1males were backcrossed to B6
from D17Mit164 at 3.92 Mb through D17Mit2 at 80.98 Mb, were selected and
used for subsequent backcrossing. Markers D17Mit10, D17Mit23 and D17Mit27
within this region were also genotyped to ensure that the entire segment would
be preserved. Repeated backcrossing and selection was continued for each sub-
then intercrossed to yield animals homozygous for the BR chromosome 17 re-
gion on a B6 background.
Animals recombinant within the chromosome 17 congenic region were
generated as follows: B6.BR-Ch17 males were crossed to B6 females to pro-
duce heterozygous F1animals; F1males were backcrossed to B6 females and
the male progeny were genotyped to identify new recombinations. Males with
the desired recombinations were bred to B6 females, and heterozygous prog-
eny were intercrossed to yield homozygous recombinant lines.
Liver tumor induction and analysis
Heterozygous chromosome 17 recombinant mice were initially bred by cross-
ing heterozygous recombinant males to B6 females, and progeny were geno-
typed to identify those mice with one copy of the entire desired region and
those that were B6 at all genotyping markers. For each line, two of these B6
genotype males and females were kept as B6 sibling control mice. All mice
with the entire desired recombinant region were also kept. This breeding pro-
cedure was followed until the recombinant line was bred to homozygosity and
then subsequent mice were generated by crossing homozygous recombinant
males to B6 females and genotyping progeny to ensure that all were hetero-
zygous for the desired recombinant region.
Liver tumors were induced by a single intraperitoneal injection of DEN
(Sigma, St Louis, MO; 0.1 lmol/g body wt) dissolved in trioctanoin (Sigma;
0.01 ml/g body wt) at 12 ± 1 days of age. As male mice are more susceptible
than females to both spontaneous (5,6) and chemically induced (7,8) liver tu-
mors, regardless of strain,males were killedat32weeks of age and femalesat50
weeks by CO2asphyxiation. Livers were removed and weighed and all tumors
.1 mm in diameter and visible on the surface of the liver were counted. Mean
tumor multiplicities of full-length congenics were compared with inbred B6
mice, and chromosome 17 recombinants were compared with B6 sibling mice
using the Wilcoxon rank sum test (22). For the congenic analysis, portions of
liver were sampled at random for histology and for the recombinant analysis,
liver tumors were randomly sampled. The selected tissues were fixed in buffered
formalin, embedded and sections were stained with hematoxylin and eosin.
DNAwas isolated from ?1 mm toe or tail clippings or ?1 mm3spleen. Tissue
was digested in 500 ll lysis solution (1% sodium dodecyl sulfate, 150 mM
NaCl, 100 mM ethylenediaminetetraacetic acid and 20 mM Tris–HCl, pH 8.0)
and 15 ll (for toe or tail) or 25 ll (for spleen) proteinase K (Roche, Indian-
apolis, IN; 10 mg/ml, 0.3 mg/ml or 0.5 mg/ml, respectively, final) and incu-
bated at 37?C overnight. Cellular debris was precipitated using 200 ll 6.25 M
ammonium acetate and pelleted. DNAwas precipitated with 700 ll isopropa-
nol and washed with 500 ll 70% ethanol. The pellet was resuspended in 100 ll
(for toe or tail) or 250 ll (for spleen) sterile water.
Microsatellite markers were amplified using 1 ll or 2 ll DNA (?100 ng),
184 nM each primer, 46 lM dNTPs, 10? polymerase chain reaction (PCR)
buffer (Roche; 10 mM Tris–HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl) and 0.5
U Taq polymerase (Roche or enzyme purified in our lab by ammonium sulfate
precipitation) in a final reactionvolume of 21.7 ll. Reactions were incubatedin
an MJ PTC-200 thermal cycler (Bio-Rad, Hercules, CA) at 94?C for 3 min and
thenfor40 cyclesof94?C for30 sec,55?C for40 sec,72?C for60sec and72?C
for 5 min. The products were separated by electrophoresis through a 7% poly-
acrylamide gel. To generate B6.BR-Ch17 congenics, backcross progeny were
genotyped at the following microsatellite markers: D17Mit2; -10; -23; -27 and
-164. Markers used to identify new recombinants along the chromosome 17
congenic region are listed in supplementary Table 1 (available at Carcinogen-
Recombinant breakpoint determination
Additional genotyping was performed in several recombinant lines to more
precisely determine the location of breakpoints. Genotyping initially utilized
additional polymorphic microsatellite markers, following the procedure de-
scribed above, and sequencing of known polymorphic SNPs identified from
the Mouse Phenome Database (www.jax.org/phenome). These results were
further supplemented by sequencing of non-coding portions of genes that were
determined to be polymorphic in the minimal susceptibility region. Primers
used for breakpoint sequencing are found in supplementary Table 2 (available
at Carcinogenesis Online).
SNP and sequencing analysis
SNPs from B6, BR and C3H along the entire minimal susceptibility region
were compiled from the Mouse Phenome Database. In addition, PCR primers
were designed to amplify 500–700 bp regions of the 3#-untranslated region of
chromosome 17 genes spanning the minimal susceptibility region. In some
genes, the length of the 3#-untranslated region was inadequate. Consequently,
500–700 bp segments of the most 3#-intron of sufficient length were amplified.
Supplementary Table 3 (available at Carcinogenesis Online) lists all non-
coding primers that were used in this sequence analysis. DNA from B6, BR
and C3H strains was initially amplified either exactly as for genotyping or with
FailSafe PCR 2? PreMixes (Epicentre, Madison, WI) in place of Roche 10?
PCR buffer and dNTPs, and a portion of the reaction was analyzed by elec-
trophoresis in a 1% agarose gel. Successful reactions were purified using
QIAquick PCR Purification Kit (Qiagen, Valencia, CA) using its standard
protocol, except the final elution was performed twice with 20 ll sterile water.
Approximately 35–50 ng of purified DNA was used in separate sequencing
reactions for forward and reverse primers with 90 nM primer, Big Dye
Terminator v3.1 (Applied Biosystems, Foster City, CA) and 5? buffer
(400 mM Tris pH 9.0 and 10 mM MgCl2, Applied Biosystems) in a final
volume of 11 ll. Reactions were precipitated using 80 ll of 75% isopropanol,
resuspended in 20 ll sterile water and electrophoresed in a 3730xl automated
DNA sequencing instrument (Applied Biosystems) using 50 cm capillary
arrays and POP-7 polymer. Sequences were analyzed and assembled with
Phred/Phrap software (21,23). All reactions were performed in duplicate to
confirm sequence differences.
Generation and susceptibility of BR full-length congenics
The extremely high susceptibility of BR females and moderate sus-
ceptibility of BR males to DEN-induced hepatocarcinogenesis have
been shown by linkage analysis to be primarily due to susceptibility
loci on chromosomes 1 and 17 (19). To verify the presence of these
loci and investigate any potential interaction between them, two sep-
arate full-length congenic lines were bred. Large portions of either
chromosome 1 or 17 from the susceptible BR strain were moved onto
a resistant B6 background. The generation of the B6.BR-Ch1 con-
genic has been described previously (20). To generate the full-length
congenic for chromosome 17, B6BRF1males were backcrossed to B6
females for nine generations to yield N10progeny. With each gener-
ation, males were genotyped at five microsatellite markers spanning
?77 Mb of chromosome 17 from D17Mit164 at 3.92 Mb to D17Mit2
at 80.98 Mb, and males heterozygous for this entire region were bred
to produce the next generation.
To assess the susceptibility of the full-length congenics to DEN-
induced hepatocarcinogenesis, resistant B6, susceptible BR, homozy-
gous congenic, heterozygous congenic and doubly heterozygous con-
genic mice were evaluated (Table I). The highly susceptible BR
females had an average of 92 liver tumors, 13-fold more than the
average of seven tumors seen in resistant B6 females (P 5 1 ? 10?10).
Homozygous B6.BR-Ch1 females showed a 3-fold increase,
whereas the B6.BR-Ch17 females had a 4-fold increase compared
with the B6 females. The BR males had a moderate susceptibility
with an average of 27 liver tumors, a 7-fold increase in tumor
multiplicity compared with the average of four tumors in resistant
B6 males (P 5 2 ? 10?7). B6.BR-Ch1 males showed a 7-fold in-
crease, whereas B6.BR-Ch17 males demonstrated a 4.5-fold increase
relative to B6 males. The increased susceptibility of both full-length
BR congenics to DEN-induced liver tumors confirms the presence of
susceptibility loci seen in this strain in the previous linkage analysis
(19). Portions of liver were randomly chosen from each group and
formalin fixed for histological analysis. In total, 155 tumors were
evaluated. With the exception of one lymphoma, all tumors observed
in liver sections were hepatocellular. Of these, 86% were adenomas or
S.E.-M.Peychal et al.
a mixture of adenoma and carcinoma, the rest were carcinomas. The
proportions of the subtypes did not depend on sex or genotype. A
small number of tumors in B6 and the BR congenics had vascular
invasion. Frequent fatty metamorphosis was seen in both normal and
tumor tissue of B6.BR-Ch1 ? B6.BR-Ch17 double heterozygotes.
The intermediatephenotyperepeatedlyseenin the B6? B6.BR-Ch1
mice heterozygous for the chromosome 1 region (Table I and A.Bilger
and N.R.Drinkwater, unpublished data) suggests that this susceptibility
locus acts semidominantly in males and females. The high susceptibil-
ity seen in B6 ? B6.BR-Ch17 mice heterozygous for the chromosome
17 region suggests that this locus acts semidominantly or dominantly
(Table I). The doubly heterozygous B6.BR-Ch1 ? B6.BR-Ch17 fe-
males, with one copy of each congenic region, demonstrated a 9-fold
increase in liver tumors compared with resistant B6 females, whereas
the B6.BR-Ch1 ? B6.BR-Ch17 males showed an 8-fold increase com-
pared with B6 males. These results indicate that the two susceptibility
loci act additively in both sexes. The high susceptibility of these double
heterozygotes reinforces the conclusion that these two susceptibility
loci are responsible for a majority of the increase in susceptibility seen
in the BR strain compared with B6. The effect of the chromosome 1
region was more significant than the effect of the chromosome 17 re-
gion in males, while the chromosome 17 region had a greater effect on
hepatocarcinogenesis in females.
Generation and susceptibility of BR chromosome 17 recombinants
To begin to map more precisely the location of the chromosome 17
susceptibility locus, homozygous full-length B6.BR-Ch17 congenic
males were crossed to B6 females and the resulting heterozygous F1
males were backcrossed to B6. Male progeny were genotyped at nine
microsatellite markers along the entire congenic region to identify
new recombinants. A series of 16 ordered recombinant males were
obtained that were heterozygous for new recombinations all along
chromosome 17 (Figure 1).
To assess the susceptibility of this set of recombinants to liver
tumors, inbred B6, inbred BR, B6 ? B6.BR-Ch17 and heterozygous
recombinants were generated and treated with DEN. During the
course of breeding the heterozygous recombinants, mice that were
B6 at all genotyping markers were also generated. Two of these mice
of each gender from each recombinant line were kept to serve as the
B6 sibling control group. All groups were compared with these B6
siblings. There was no difference in the susceptibility of these B6
sibling mice and the inbred B6 mice.
In females, one copy of the full-length chromosome 17 congenic
region resulted in an average of 26 liver tumors per animal (Table II),
a 4-fold increase compared with the average of six tumors seen in
resistant B6 sibling females (P 5 3 ? 10?5). Susceptible heterozy-
gous recombinant lines demonstrated between 16 and 45 liver tumors
on average or a 3- to 7.5-fold effect compared with B6 siblings,
completely recapitulating the increase seen in the full-length congen-
ic. In males, one copy of the full-length chromosome 17 congenic
region caused 31 liver tumors on average, a more moderate 2-fold
increase in livertumors compared with the average of 17 tumors in B6
sibling males. This 2-fold increase in the full-length chromosome 17
congenic males is slightly less than the 4.5-fold increase in liver
tumors observed when these heterozygotes were first evaluated due
to the elevated susceptibility of the B6 sibling control males. Males
from the recombinant lines that exhibited susceptibility in females
showed between 25 and 39 liver tumors on average, a 1.5- to 2-fold
increase in liver tumor multiplicity compared with B6 siblings, also
wholly recapitulating the effect of the full-length congenic region.
In the set of 16 recombinant groups,lines 1, 3,5, 7,8, 10,12, 14 and
16 showed no difference in liver tumor susceptibility compared with
B6 sibling mice in either females or males. All other recombinant
lines were significantly more susceptible in females, even after
P-values were adjusted for multiple comparisons (P , 0.015). P-values
Table I. Susceptibility of female and male BR full-length congenicsa
No. of miceMean liver tumorsP-valueb
No. of miceMean liver tumorsP-value
B6 ? B6.BR-Ch1
B6 ? B6.BR-Ch17
7 ± 7
92 ± 47
20 ± 21
8 ± 8
26 ± 18
26 ± 22
54 ± 29
1 ? 10?10
2 ? 10?6
8 ? 10?6
5 ? 10?10
4 ± 5
27 ± 24
28 ± 25
12 ± 13
18 ± 15
18 ± 15
31 ± 21
2 ? 10?7
3 ? 10?8
1 ? 10?3
8 ? 10?8
2 ? 10?6
2 ? 10?12
aMice received an intraperitoneal injection of DEN (0.1 lmol/g body wt) at 12 ± 1 days of age. Females were killed at 50 weeks of age and males at 32 weeks of
age and tumors .1 mm in diameter on the surface of the liver were counted.
bWilcoxon rank sum test was used to determine the P-value of each group compared with resistant inbred B6 mice. P-values are not adjusted for multiple
Fig. 1. Ordered series of 16 recombinant lines for chromosome 17. Sixteen
ordered recombinant lines were bred, each containing a segment of
chromosome 17 from the susceptible BR strain on a resistant B6 background.
Microsatellite markers used for genotyping and their positions are indicated
at the top. Additional sequencing markers used to further refine breakpoint
locations are indicated at the bottom. Black regions indicate an area inherited
from the B6 strain, white regions indicate a portion inherited from the BR
strain and gray regions are areas of unknown genotypewhere recombinations
Mouse chromosome 17 liver cancer modifier gene
for males from these same recombinant lines were marginally signif-
icant when adjusted for multiple comparisons, suggesting that the
same region of chromosome 17 that caused the 3- to 7.5-fold increase
in females also caused an increase in liver tumor multiplicity in
males. Tumors were randomly chosen from each group and formalin
fixed for histological analysis. In total, 77 tumors were analyzed, and
?75% were hepatocellular adenomas and the remaining 25% were
mixed adenomas and carcinomas, independent of either strain or sex.
Additional genotyping was performed to further restrict the break-
point regions in several recombinant lines and this information is
included in Figure 1. This genotyping initially utilized polymorphic
microsatellite markers. Further genotyping analyzed known polymor-
phic SNPs and non-coding regions of genes determined to be poly-
morphic during the haplotype analysis of the minimal susceptibility
region. The ends of the minimal susceptibility region are set by the
breakpoints in resistant lines 7 and 8, which have the largest portion of
chromosome 17 from the BR strain that does not affect liver tumor
susceptibility. The breakpoints in these lines were determined as pre-
cisely as possible and further supported by refinement of the break-
points in susceptible lines 6 and 9, which have the smallest portion of
chromosome 17 from the BR strain that causes an increase in liver
tumor susceptibility. This additional mapping led to the conclusion
that the chromosome 17 susceptibility locus must be between
218RSFD13 and 218RSFD14 or between 30.05 and 35.83 Mb. This
single region accounts for the entire increase in susceptibility to liver
tumors seen in the full-length chromosome 17 congenic compared
with resistant B6 sibling controls.
Haplotype analysis of minimal susceptibility region
All of the SNPs with known alleles in the B6, C3H and BR strains in
the minimal susceptibility region identified by the recombinant lines
were assembled from the Mouse Phenome Database. The C3H strain
was included because this strain does not have any loci that confer
liver tumor susceptibility relative to B6 on chromosome 17. Thus, for
the minimal susceptibility region, C3H is in essence an additional
resistant strain. If the Hcf1 susceptibility alleles were the result of
an ancestral mutation, then it would be more likely to be found in
regions where the BR strain has inherited an ancestral haplotype that
is different from both the B6 and C3H strains. The proximal two-
thirds of the minimal region is virtually non-polymorphic in the BR
and B6 strains (Figure 2A), while the BR and C3H strains are known
to have the same H2 haplotype, which is located in the distal portion
of the minimal region from ?34.08 to 37.62 Mb. In addition, we
assessed sequence variation specifically in non-coding portions of
genes along the minimal susceptibility region among B6, C3H and
BR mice. One hundred thirty-two segments of 500–700 bp from 3#-
untranslated regions or introns of 120 genes throughout this region
were sequenced in the resistant B6 and susceptible BR strains (sup-
plementary Table 3 is available at Carcinogenesis Online). Any re-
gions that were polymorphic in these two strains were also sequenced
in the C3H strain. These sequence comparison studies determined that
the proximal two-thirds of the minimal region were virtually identical
in the B6 and BR strains (Figure 2B). In the remaining portion of the
region, the B6 and BR strains were highly polymorphic. However, the
C3H strain was virtually identical to the BR strain in this interval.
Only a very small number of loci were unique in the BR strain, the
only strain containing a susceptibility locus on chromosome 17. Two
of these unique regions are located at ?30.05 and 35.74 Mb in both
comparisons (Figure 2). The non-coding region sequencing also iden-
If the Hcf1 mutation is the result of an ancestral mutation that the BR
strain inherited during its generation, then it would probably be found
inthese small uniqueregions.However,the overwhelming majorityof
sequence is not unique to the distinctively sensitive BR strain in the
minimal susceptibility region. This observation strongly suggests that
the susceptibility locus is not the result of an ancestral mutation, but is
most probably the result of a novel mutation that arose during gener-
ation of the BR strain. Consequently, all portions of the current min-
imal susceptibility region have an equal likelihood of containing the
The susceptibility of BR mice to hepatocarcinogenesis is primarily
caused by two susceptibility loci, Hcf1 and Hcf2, on chromosomes 17
and 1, respectively. These loci were initially identified through
Table II. Susceptibility of female and male heterozygous chromosome 17 recombinantsa
No. of mice Mean liver tumorsP-valuec
No. of mice Mean liver tumorsP-value
B6 ? B6.BR-Ch17
6 ± 7
3 ± 4
162 ± 54
26 ± 19
7 ± 6
4 ± 3
8 ± 12
12 ± 13
33 ± 26
30 ± 27
27 ± 23
16 ± 14
45 ± 32
26 ± 25
30 ± 28
9 ± 13
9 ± 25
3 ± 6
9 ± 11
4 ± 10
2 ? 10?10
3 ? 10?5
8 ? 10?7
2 ? 10?5
4 ? 10?5
1 ? 10?6
1 ? 10?4
9 ? 10?6
17 ± 21
16 ± 24
56 ± 37
31 ± 23
13 ± 19
15 ± 13
25 ± 24
15 ± 23
25 ± 17
25 ± 26
35 ± 41
29 ± 27
39 ± 29
34 ± 28
29 ± 24
15 ± 15
13 ± 11
12 ± 21
14 ± 16
17 ± 20
1 ? 10?4
aMice were treated with an intraperitoneal injection of DEN at 12 ± 1 days of age. Females were killed at 50 weeks of age and males at 32 weeks of age. Tumors
.1 mm in diameter on the surface of the liver were counted.
bAll mice were heterozygous for the indicated recombinant region.
cWilcoxon rank sum test was used to calculate the P-value of each group compared with B6 sibling mice. P-values are not adjusted for multiple comparisons.
dB6 sibling mice were B6 at all markers used for genotyping. Two such males and females from each recombinant line were kept for a B6 sibling control group.
S.E.-M.Peychal et al.
a linkage analysis of crosses between the susceptible BR and resistant
B6 strains and their existence has now been further validated through
the analysis of congenic mice. Homozygosity for the full-length BR
chromosome 1 region caused a 3-fold increase in liver tumor multi-
plicity in females and a 7-fold increase in males. The chromosome 17
region had a 4-fold effect in females and a 2- to 4.5-fold effect in
males. Both of these regions appear to act at least semidominantly in
both sexes. The high susceptibility seen in double heterozygotes, with
one copy of each region, indicates that these two loci are responsible
for the majority of the susceptibility in the BR strain. For the Hcf1
locus on chromosome 17, a set of 16 recombinant lines covering the
entire congenic region was generated, each containing a portion of
chromosome 17 from the BR strain on a B6 background. Susceptible
heterozygous chromosome 17 recombinants demonstrated between
a 3- and 7.5-fold effect in females and between a 1.5- and 2-fold effect
in males, completely recapitulating the increase seen in heterozygous
full-length congenics. The analysis of these recombinants indicates
that Hcf1 is located in a single region from 30.05 to 35.83 Mb that is
responsible for the increase in liver tumor sensitivity.
BR females are up to 50-fold more susceptible than females of all
other inbred strains that have been evaluated (8). This effect is partly
due to the loss of protection against liver tumor development typically
offered by ovarian hormones: ovariectomized and intact BR females
do not show a significant difference in liver tumor multiplicity (11).
However, the fact that the two susceptibility loci are the major cause
of susceptibility in BR females and males suggests that the same
underlying pathway must be at work in both sexes. Work from other
labs indicates that the protective effect of the ovaries is most probably
mediated by estrogen. Chronic administration of estrogen has been
shown to protect intact males (24) and ovariectomized females (25).
Furthermore, progesterone was seen to have no effect on liver tumor
incidence. As estrogen levels in females during diestrus have been
shown to be only approximately twice that measured in males (26),
estrogen could be exerting a protective effect in both sexes that is
overwhelmed in males by the promotion of liver tumors caused by
androgens (7,10,11,18,27). The increase in liver tumor multiplicity in
BR males and females due to the chromosome 17 locus could there-
fore be caused by the loss of the same protective effect in both sexes.
There is no difference in the levels of estrogen in B6 and BR females
(M.H.Feld and N.D., unpublished data). The amounts and affinities of
estrogen receptor in the livers of B6 and BR mice are similar as well
(13), suggesting that the chromosome 17 locus acts downstream of
Estrogen may have either a direct or an indirect effect on hepato-
carcinogenesis. When chimeras between the BR and B6 strains were
treated with DEN, a majority of the tumors that developed originated
from the BR strain (12). This result indicates that the BR susceptibil-
ity genes collectively have a cell-autonomous effect. The chromo-
some 17 locus may act downstream of estrogen binding and so
Hcf1 could cause cell-autonomous effects in the liver in response to
estrogen. However, it is unknown whether only one locus or both Hcf
loci act cell autonomously. Chimeras between C3H and B6 have
similarly demonstrated the inherent susceptibility of the C3H strain
to both spontaneous (28) and DEN-induced (29) liver tumors. As the
C3H and BR strains appear to share a susceptibility locus on chro-
mosome 1, it is possible that the chromosome 1 locus acts cell auton-
omously in both of these strains, whereas the chromosome 17 locus
acts non-cell autonomously.
There is explicit evidence that estrogen may have an indirect effect
on hepatocarcinogenesis: subcutaneous implantation of estrogen pel-
lets decreases liver tumor incidence, whereas implantation of estrogen
pellets in the spleen, which drains directly into the liver, does not (25).
The change in expression of interleukin-6 (IL-6) in response to DEN
is greater in males and ovariectomized females than in intact females
(30), and the differences among these groups are reduced by the in-
jection of estrogen. Knocking out IL-6 confers on males a resistance
to hepatocarcinogenesis that is comparable with that of wild type and
IL-6 knockout females. Naugler et al. (30) have hypothesized that the
death of hepatocytes due to DEN causes Kupffer cells to release IL-6,
which promotes compensatory hepatocyte regeneration, increasing
the fixation of initiating mutations that result in liver tumors. Estrogen
might protect livers from tumors indirectly by inhibiting the produc-
tion of IL-6.
The multistage model of carcinogenesis, originally developed to
describe tumor formation in the skin (31), can also be appliedto tumor
formation in the liver (14). This model divides tumor development
into the three stages of initiation, promotion and progression. Ovarian
hormones exert much of their protection during the promotion phase.
Estrogen could act during the conversion stage of promotion (31),
when an initiated cell divides and begins to express its preneoplastic
Fig. 2. Haplotype analysis of chromosome 17 minimal susceptibility region. The top line in each panel indicates the positions of the SNPs. For each strain, SNP
alleles are indicated by both the shading of the vertical tic and its position with respect to the horizontal line. Sequences identical to those in B6 are indicated in
dark gray (abovethe horizontal line), sequences different from B6 but identical in C3H and BR are in light gray (across the line) and sequences unique to BR are in
black (below the line). Closely spaced SNPs may not be individually distinguishable in the representations for each strain. (A) SNPs with alleles in B6, C3H and
BR were compiled from the Mouse Phenome Database. The data show two areas unique to BR at ?30.05 and 35.74 Mb. (B) Segments of 3#-untranslated regions
or the most 3#-introns of genes were sequenced. These data identify two additional areas unique to BR at ?33.04 and 34.50 Mb.
Mouse chromosome 17 liver cancer modifier gene
phenotype, by inhibiting production of IL-6 (30). Estrogen may also
act later during the propagation stage of promotion, by inhibiting the
proliferation or enhancing apoptosis of preneoplastic cells. Preneo-
plastic lesions in the liver have similar growth rates in B6 and C3H
females, but grow more rapidly in BR females (11,15). These lesions
grow more slowly in B6 and C3H females than in the corresponding
males, but the growth rates of lesions in BR males and BR females are
similar. Moreover, ovariectomy performed weeks after carcinogen
treatment increases the growth rate of preneoplastic lesions in B6,
C3H and B6C3F1females, but not in BR females (7,11,16–18). Fur-
ther evidence that the modifiers act after initiation is provided by the
observation that BR males haves a high spontaneous incidence of liver
tumors (32) and that both sexes are susceptible tolivertumors induced
by the direct-acting carcinogen N-ethyl-N-nitrosourea (8).
The liver tumor susceptibility locus Hcf1 has so far been mapped to
a region of chromosome 17 from 30.05 to 35.83 Mb, which corre-
sponds to a section of human chromosome 6p21. Amplification of this
portion of the p arm of chromosome 6 has repeatedly been found in
human liver tumors. The proportion of tumors with this amplification
varied from 20 to 61% and was found in patients with hepatitis B or C
virus infection (33–36), as well as patients without hepatitis virus
infection (37–39). An increase in the copy number of this region
may indicate the presence of a dominantly acting oncogene. Addi-
tionally, this amplification was not associated with liver tumor grade,
indicating that it could be an early event (40–42). Interestingly, am-
plification of chromosome arm 6p was also seen specifically in he-
patic metastases from colorectal cancers (43,44), which are the
leading cause of colorectal cancer deaths. Genetic aberrations in pri-
mary tumors and metastases from Dukes’ stage C colorectal cancer
patients with lymph node metastasis were compared with Dukes’
stage D colorectal cancer patients with liver metastasis (44). Ampli-
fication of chromosome arm 6p was only significantly associated with
Dukes’ stage D and liver metastases. Consequently, identification of
the causative mutation in the Hcf1 locus could have wide-ranging
effects on other diseases in addition to liver cancer.
The current minimal region contains ?215 genes. Several genes in
this region are potential candidates for Hcf1. Cyp4F14 is a member of
the cytochromes P450 4F subfamily. These enzymes are involved in
arachidonic acid metabolism (45) and metabolize leukotriene B4into
biologically less active metabolites (46). Leukotriene B4is a powerful
promoter of inflammation, which can in turn lead to liver cancer (4).
The sequence of Cyp4F14 in mice has 95% sequence similarity with
Cyp4F1 in rats and both are expressed in the liver (45). The expres-
sion of this enzyme in rats is sex specific, with significantly higher
expression in females (47). Moreover, its expression was shown to
decrease after ovariectomy (47) and increase after exposure to afla-
toxin B1(48), a known risk factor for liver cancer (4). Cyp4F1 was
also the first P450 found to be constitutively overexpressed in rat
Another candidate for Hcf1, H2-Ke6, is a member of the nicotin-
amide adenine dinucleotide-dependent 17b-hydroxysteroid dehy-
drogenase family of enzymes. It is expressed in the ovaries, testes
and liver, in addition to other tissues (50). This family of enzymes
efficiently catalyzes the oxidation of estradiol, testosterone and
dihydrotestosterone as well as the reduction of estrone to form
estradiol (51). These enzymes carry out a key reaction in the syn-
thesis and metabolism of sex hormones and regulate the last step
required to form all androgens and estrogens in both gonadal and
non-gonadal tissues (52). Expression of H2-Ke6 has previously
been linked to the development of cysts in the livers and kidneys
A final candidate is tumor necrosis factor (TNF)-a, a proinflamma-
tory cytokine. TNF can trigger the acute phase response and a cascade
of other cytokines, and it also has a crucial role in the balance of
hepatocyte proliferation and death (53). The transcription factor nu-
clear factor-kappa B is at least partly responsible for cell proliferation
in response to TNF, and the activation of nuclear factor-kappa B in
liver regeneration is primarily due to IL-6 induction. As previously
stated, the production of IL-6 by non-parenchymal cells has been
hypothesized to cause the gender disparity in DEN-induced liver tu-
mors (30). In addition, TNF and IL-6 also have inhibitory effects on
each other (54). IL-6 can inhibit TNF expression and TNF can block
IL-6 induction of type II acute phase response genes and activation of
signal transducer and activator of transcription signaling (55).
All of the exons of Cyp4F14, H2-Ke6 and Tnfa and their splice sites
have been sequenced in BR, B6 and C3H strains to look for unique
mutations in the susceptible BR strain (S.Peychal and N.Drinkwater,
unpublished data). This sequencing did not identify any unique muta-
tions in either the exons or splice sites of these three candidate genes.
In addition, the hepatic expression levels of these genes at 10 weeks of
age were compared using microarrays. In B6, BR and BR chromo-
some 17 recombinant females, treated with DEN at 12 days and
ovariectomized or sham operated at 6 weeks of age, the expression
levels of the three genes were not significantly different among the
strains. However, as there could be a causative difference in the ex-
pression of these genes at another time, or in another tissue, that could
result in susceptibility to liver tumors, these genes cannot be excluded
as the Hcf1 locus.
Men have a 2- to 5-fold higher risk of developing liver cancer than
women (1). At least some of this difference is due to the different
hormonal environments, but it is not currently known what contribu-
tion, if any, is due to other complicating risk factors such as hepatitis
virus infection or aflatoxin B1exposure. Inbred mice offer a simpler
system in which to study liver cancer and the effect of sex hormones.
Work with these mice has shown the stimulatory effect of androgens
as well as the protective effect of ovarian hormones (7,9–11). The BR
females are highly susceptible to liver cancer due, in part, to their
unique lack of ovarian hormones’ protection (11). Therefore, they
offer a singular model to explore the suppressive pathways at work.
Elucidation of how these pathways are disrupted in this unique strain
may shed light on how these pathways function in all other strains and
may uncover information of relevance to women and liver cancer
development in humans as a whole.
Supplementary Tables 1–3 can be found at http://carcin.oxfordjournals.
National Institutes of Health, National Cancer Institute (CA96654,
The authors wish to acknowledge Rebecca Baus, Mei Finnerty, Kristin Liss,
Kimberley Luetkehoelter and McArdle animal care staff for their work with the
mice. Thanks also to Susan Schadewald for technical assistance and the McArdle
and Christopher Oberley for their helpful comments on the manuscript.
Conflict of Interest Statement: None declared.
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Received November 25, 2008; revised February 12, 2009;
accepted February 14, 2009
Mouse chromosome 17 liver cancer modifier gene