Carcinogenesis vol.20 no.11 pp.2083–2088, 1999
Gain of chromosomes 15 and 19 is frequent in both mouse
hepatocellular carcinoma cell lines and primary tumors, but loss
of chromosomes 4 and 12 is detected only in the cell lines
Katsuhiro Ogawa2, Makoto Osanai, Masahiko Obata,
Kenichi Ishizaki and Kenji Kamiya1
Department of Pathology, Asahikawa Medical College, 4-5-3-11
Nishikagura, Asahikawa 078-8510 and 1Department of Developmental
Biology and Oncology, Division of Molecular Biology, Research Institute
for Radiation Biology and Medicine, Hiroshima University, Hiroshima,
Japan
2To
whom correspondence should be addressed
Email: [email protected]
Chromosomal alterations were investigated in hepatocellular carcinoma cell lines, primary tumors and liver epithelial
cell lines derived from normal livers of C57BL/6JHC3H/
HeJ F1 and C3H/HeJHC57BL/6J F1 mice. In the primary
tumors, non-random gain of chromosomes 15 and 19 was
found in seven and five of 14 hepatocellular carcinomas,
respectively. On the other hand, in the cases of both liver
epithelial and hepatocellular carcinoma cell lines, frequent
changes were loss of chromosomes 4 (4/9 cell lines) and
12 (3/9) as well as gain of chromosomes 15 (5/9) and
19 (4/9). These results indicate that the chromosomal
gain is associated with both in vivo carcinogenesis and
establishment of cell lines, while the loss is specific for the
latter. PCR analysis using polymorphic microsatellite DNA
markers revealed that the loss of chromosome 12 as well
as chromosome 4 was much more frequent for the C57BL/
6J hepatocarcinogenesis-resistant rather than the susceptible C3H/HeJ strain.
Introduction
been reported to be generally infrequent in primary HCCs
induced by chemical carcinogens (14,23,24), while it was
detected, with preferential involvement of chromosomes 1, 5,
7, 8 and 12, in HCCs of SV40 T antigen transgenic mice (18).
On the other hand, LOH has been reported to be very frequent
in mouse HCC and liver epithelial (LE) cell lines derived from
normal liver, most frequently involving chromosome 4 (25–
27). These observations indicate that some changes may occur
in primary tumors, but others may rather be associated with
the establishment of cell lines. To identify chromosomal
changes associated with in vivo carcinogenesis and in vitro
establishment, cytogenetic and allelic changes were investigated in cell lines of diethylnitrosamine (DEN)-induced HCCs
and primary tumors as well as in LE cell lines derived from
normal livers of C57BL/6J (B6)3C3H/HeJ (C3H) F1 (B6C3F1)
and C3H3B6 F1 (C3B6F1) mice.
Materials and methods
Primary HCCs and cell lines
Male B6C3F1 and C3B6F1 mice were used in this study. For induction of
HCCs, the mice were administered a single dose of DEN (5 mg/g body wt)
at the age of 3 weeks and killed 12–15 months after treatment. LE and HCC
cell lines were produced as described elsewhere (26). The culture medium
was Williams’ E medium supplemented with 10–7 M insulin, 10–7 M epidermal
growth factor, 10–3 M nicotinamide, 10–5 M dexamethasone, 10–7 M transferrin,
10–5 M aprotinin, 5 U/ml penicillin, 100 µg /ml streptomycin, 2.5 mg/ml
fungizone and 10% fetal bovine serum. The LE and HCC cell lines were used
at 6–10 population doubling levels.
Cytogenetic analysis
For normal hepatocytes and primay HCCs, the isolated cells were seeded onto
hydrophobic plastic dishes (Becton Dickinson, Bedford, MA) with a diameter
of 10 cm at the density of 106 cells/dish and cultivated for 2–3 days. The
cells of either primary cultures and cells lines were harvested from plastic
dishes, re-seeded in 10 cm diameter collagen-coated dishes (Becton Dickinson)
at a density of 23105 cells per plate and cultivated for 1 day. These cells
were treated with 0.1 µg/ml colcemid for 30 min, harvested from the dishes
using 0.25% trypsin solution, treated with a 0.075 M KCl solution for 20 min
and fixed in Carnoy’s fixative. Metaphase spreads were trypsinized and stained
with the Giemsa solution. For analysis of chromosome counts, 100 metaphase
nuclei were examined and 10–19 metaphases with clear features were
karyotyped. The criteria used for mouse chromosomes followed those of
Nesbitt and Franke (28) and changes were described according to the
Guidelines for Cancer Cytogenetics: Supplement to an International System
for Human Cytogenetic Nomenclature (29). In this study, cells with 30–49
chromosomes were termed ‘diploid range cells’, those with 50–69 as ‘triploid
range cells’ and those with 70–89 as ‘tetraploid range cells’.
Cancer cells usually show a variety of chromosomal alterations,
some of which may be related to the mechanism of oncogenesis,
while the others may be simply consequential to neoplasia. In
mice, non-random chromosomal alterations were detected in
various tumors, such as plasmacytomas, lymphomas, leukemias
and skin tumors (1,2), which are related to activation of
oncogenes such as c-myc (3), mdm2 (4,5) and H-ras (6,7). On
the other hand, allelic loss has been reported in various mouse
neoplasias (8–21), indicating that functional loss of putative
tumor suppressor genes associated with allelic loss may be
related to the mechanisms of tumorigenesis. Chromosomal
regions involved in mouse tumors have frequently been identified as homologous to those involved in human tumors,
indicating that common genetic alterations may underlie mouse
and human tumors.
In mouse hepatic tumors, although gain of chromosomes 11
and 19 has been reported to be a non-random change in
adenomas (22), karyotypic characteristics of hepatocellular
carcinomas (HCC) have not been well documented. With
regard to allelic changes, loss of heterozygosity (LOH) has
FISH
Chromosome painting probes specific for chromosome 4 or 12 labeled with
biotin were purchased from Cambio (Cambridge, UK). Chromosome spreads
on slide glasses were denatured at 70°C in 70% formamide in 23 SSC for
2 min and hybridized with the probes for 1 or 2 days at 37°C. Hybridization
signals were detected by fluorescein isothiocyanate–avidin/anti-avidin sandwich amplification. Finally, the chromosomes were counterstained with
propidium iodide and examined under a Nikon fluorescence microscope
equipped with a cooled digital camera system (Hamamatsu Photo Co.,
Hamamatsu, Japan). At least 10 cells were analyzed for each cell line.
Abbreviations: B6, C57BL6/J; B6C3F1, C57BL6/J3C3H/HeJ F1; C3H, C3H/
HeJ; C3B6F1, C3H/HeJ3C57BL/6J F1; DEN, diethylnitrosamine; HCC,
hepatocellular carcinoma; LE, liver epithelial; LOH, loss of heterozygosity.
LOH analysis
DNA samples isolated from the primary cultures and the cell lines were
analyzed by PCR using primers for the polymorphic microsatellite DNA,
which were purchased from Research Genetics (Huntsville, AL). PCR was
© Oxford University Press
2083
K.Ogawa et al.
Table I. Karyotypes of primary cultures of normal hepatocytes and HCC cells, and LE and HCC cell lines
Cells
Mode
Range
No.
Karyotypes
primary hepatocytes
primary HCC
#1
#2
#3
#4
#5
#6
#7
#8
#9
#10
#11
#12
#13
#14
LE cell lines
#1
#2
#3
#4
40
39–90
19
40, XY
40
38
40
40
39
39
40
40
40
40
40
40
40
40
38–90
37–90
35–90
36–85
39–90
36–82
39–90
37–90
38–90
40–90
37–90
39–90
39–90
38–90
17
18
10
16
14
10
11
14
14
13
14
11
15
11
39–40, XY, 119[7]
38–41, XY, 12[4], 115[3] [cp2]a
39–40, XY, tas(15;15)[3], 115[3], 119[3] [cp3]
39–42, XY, 115[4], 119[5] [cp3]
39–40, XY, –11[5], 115[4] [cp3]
41–42, XY, 115[5]
39–40, XY, 119[3]
39–40, XY, 119[3]
38–41, XY, –11[3], –13[4], 115[3], mar[7] [cp8]
40, XY, 115[3]
37–40, XY
39–40, XY
39–40, XY
38–40, XY
41
73
77
38
38–74
54–90
42–90
37–90
10
–
–
13
#5
#6
#7
#8
HCC cell lines
#1
#2
#3
#4
#5
39
40
40
40
38–90
38–89
38–90
39–90
13
10
13
10
36–41, XY, –12[4], 118[3], 119[4] [cp6]
–
–
37–41, XY, –Y[5], –4[7], –9[4], –13[6], 115[6], dic(15; 15)[12], –18[4],
119[4] [cp10]
38–40, XY, –Y[3], –4[4], 115[3] [cp5]
36–41, XY, –Y[8], –4[4], –13[3] [cp4]
40, XY
37–41, XY, –12[3], 115[7] [cp2]
39
39
63
40
64
37–90
37–90
57–90
38–90
56–90
19
10
–
12
–
40–43, 11[17], 115[11] [cp2]
37–39, XY, –4[10], –8[3], –12[10], –16[4], 119[5] [cp6]
–
38–43, XY, –Y[5], –11[3], 115[7], 115, 115[3], dic[15;19], 119[5] [cp6]
–
aComposite
karyotype (29). –; karyotyping was not done.
performed in 25 µl of solution containing 10 mM Tris–HCl, pH 8.4, 50 mM
KCl, 1.5 mM MgCl2, 100 ng genomic DNA, 100 µM dNTP, 0.2 µM primers
and 0.5 U Taq polymerase (Perkin Elmer, Norwalk, CT) for 35 cycles with
cycling times of 1 min at 95°C, 1 min at 55°C and 1 min at 72°C. The PCR
products were electrophoresed on 8% polyacrylamide gels and stained with
ethidium bromide. For evaluation of LOH, genomic DNA samples of C3H,
B6 and C3B6F1 mice were used as standards.
Statistical analysis
The differences in frequency of chromosomal changes and LOH between the
cell lines and primary tumors were statistically analyzed by Fischer’s exact
test. Comparison of strain preference of LOH was done by the χ2 test. The
significance level chosen was P , 0.05.
Results
Cytogenetic changes
The general data on chromosomal changes are presented in
Table I. For normal hepatocytes ~40% of cells had a diploid
range chromosomal count, while ~60% were in the triploid,
tetraploid or more hyperploid ranges. Of 19 diploid range
nuclei karyotyped, 13 showed the normal diploid configuration,
while the other six nuclei showed a gain or loss of various
chromosomes as well as marker chromosomes. For 14 primary
HCCs analyzed, most cells (60–90%) had diploid range
chromosomal numbers, in agreement with previous flow cytometric studies (30). Although gain and loss were noted in
various chromosomes, non-random changes (observed in more
than three cells) were limited to monosomy of chromosome
11 (2/14 cell lines) and trisomy of chromosomes 2 (1/14),
15 (7/14) and 19 (5/14) (Table I and Figure 1a). Non-random
structural alterations of chromosomes were generally rare in
2084
the primary HCCs, although telomere association of two
chromosomes 15 was observed in one HCC (Figure 1b).
Of the eight LE cell lines examined, six had 63–90% of
cells in the diploid range, while most cells were hyperploid in
the other two cell lines (Table 1). G-banding analysis of the
former six cell lines revealed prominent numerical alterations.
Non-random changes were loss of chromosome Y (3/6 cell
lines), monosomy of chromosomes 4 (3/6), 9 (1/6), 12 (2/6),
13 (2/6) and 18 (1/6), trisomy of chromosomes 15 (3/6),
18 (1/6) and 19 (2/6) and centromere fusion between two
chromosomes 15 (1/6) (Table I). Of five HCC cell lines
examined, three contained relatively abundant diploid range
cells, while the other two mostly comprised hyperploid cells
(Table I). In the diploid range cell lines, non-random numerical
changes were loss of chromosomes Y (1/3), 4 (1/3), 8 (1/3),
11 (1/3), 12 (1/3) and 16 (1/3) and gain of chromosomes 1
(1/3), 15 (2/3) and 19 (2/3) (Table I and Figure 2a). In addition,
centromere fusion between chromosomes 15 and 19 was
detected in all cells in one case (Figure 2b). Thus loss of
chromosomes Y, 4 and 12 was relatively frequent for the cell
lines (comparison between primary HCC and cell lines,
P , 0.05), while gain of chromosomes 15 and 19 was seen
in both cell lines and primary HCCs.
Analysis of chromosomes 4 and 12 by FISH
Since complete karyotyping was difficult for the hyperploid
cell lines due to overlap of chromosomes, loss of chromosomes
4 and 12 was investigated by FISH in the two LE and two
HCC hyperploid cell lines. Two to four copies of chromosome
Chromosome changes in mouse HCC
Fig. 1. (a) Representative G-banding pattern for primary HCC 4. Note
trisomy of chromosomes 15 and 19. (b) Telomere association of two
chromosomes 15 in primary HCC 3.
Fig. 2. (a) Representative karyotype of HCC cell line 2. Note monosomy of
chromosomes 4 and 12 and trisomy of chromosome 19. (b) Centromere
fusion between chromosomes 15 and 19 in HCC cell line 4 with two copies
of intact chromosomes 15 and 19 in this particular cell.
Table II. FISH analysis of chromosomes 4 and 12 on hypotriploid and hypotetraploid cells
Cell lines
Chromosome 4
LE #2
LE #3
HCC#3
HCC#5
Chromosome 12
LE #2
LE #3
HCC#3
HCC#5
No. cells examined
Copy decreasea
Shorteningb
10
13
10
10
6
4
10
9
8
10
10
10
13
10
10
9
13
10
Elongationc
10
Translocationd
8
aNumbers of cells that show decrease in the copy numbers of chromosomes 4 and 12. The decrease is 2 and less for hypotriploid cells and 3 and less for
hypotetraploid cells.
b,c,dNumber of cells that show bshortening, celongation and dtranslocation of chromosome 4.
4 were detected in triploid and tetraploid range cells in two
LE cell lines (Figure 3a and b and Table II). In these cells,
the length of chromosome 4 was reduced to ~50% in one or
two copies (Figure 3a). In one of the two HCC cell lines
mainly consisting of hyperploid cells, all cells contained two
copies of chromosome 4 of normal length. In the other
hyperploid HCC cell line, the copy number of chromosome 4
was two to four, one being ~40% longer than the other,
indicating an increased segment in the copy, with a short
segment of chromosome 4 also translocated to the telomeric
portion of another chromosome (Figure 3b). Regarding chro-
mosome 12, the copy numbers were two in most cells in the
two LE cell lines and one of the two HCC cell lines (Figure
3c) and mainly one in the other HCC cell line (Figure 3d).
These FISH data demonstrate not only that copy numbers of
chromosomes 4 and 12 are decreased in the hyperploid cell
lines but also that chromosome 4 is structurally altered.
LOH analysis
To investigate which of the parental chromosomes were lost,
allelotype was analyzed using polymorphic microsatellite DNA
markers in LE and HCC cell lines. LOH on chromosome 12
2085
K.Ogawa et al.
Fig. 3. Painting of chromosomes 4 and 12 by FISH. (a) LE cell line 2, showing four copies of chromosome 4 with a 72 chromosome count. The length of
two copies of chromosome 4 (arrows) is clearly shorter than for the other two copies. (b) HCC cell line 4, showing two copies of chromosome 4, one (arrow)
of which is ~40% longer than the other. In addition, short translocated segments of chromosome 4 are apparent in the telomeric regions of two other
chromosomes (asterisks). (c) LE cell line 3 containing two copies of chromosome 12 with a 69 chromosome count. (d) HCC cell line 5 showing one copy of
chromosome 12 with a 50 chromosome count.
was detected in six of eight LE lines and four of five HCC
cell lines according to two to four markers (Figure 4). Of
these 10 LOH-positive cell lines, nine (six lines from C3B6F1
and three lines from B6C3F1) showed loss of the B6 allele,
with the C3H allele remaining (preference for B6 allele loss
is significant, 0.01, P , 0.02). For chromosome 4, loss of
the B6 chromosome dominated, as described previously (26,27;
data not shown).
Discussion
Cytogenetic analysis revealed that chromosomal alterations
were much more frequent in both LE and HCC cell lines than
the primary HCCs, suggesting that they are mainly associated
with establishment or maintenance of cell lines rather than
carcinogenesis in vivo. Furthermore, the fact that non-random
loss of chromosomes 4 and 12 was only detected in the cell
lines indicates that these changes may be more important for
establishment in vitro rather than for carcinogenesis in vivo.
On the other hand, gain of chromosomes 15 and 19 was
frequently detected in both primary HCC and cell lines,
indicating that it is presumably important to both phenomena.
The importance of chromosome 4 loss in HCC (25) and LE
cell lines (26,27) and primary tumors (13–15) has been
reported. The present study demonstrated that chromosome 12
2086
Fig. 4. LOH on chromosome 12 in LE and HCC cell lines. CB refers to a
C3B6F1 origin and BC to a B6C3F1 origin of the cell lines. Gray squares
represent cases where the C3H allele remains, closed squares represent the
cases where the B6 allele remains and open circles indicate no LOH.
Positions of mouse loci related to tumor susceptibility/resistance are
indicated: Ccs (35), Hcs3 (36) and Par3 (37).
Chromosome changes in mouse HCC
loss was also frequent in the LE and HCC cell lines. LOH on
chromosome 12 has been reported in mouse lung tumors (31)
and lymphomas (16,21). Mouse chromosome 12 has regions
homologous mainly to human chromosomes 2p and 14q and
LOH of these human chromosomes has been detected in
various cancers (32–35), suggesting that mouse chromosome
12 contains tumor suppressor genes. Mouse chromosome 12
also bears the genetic predisposition loci for colon cancer
susceptibility (Ccs) (36), hepatocarcinogenesis susceptibility
(Hsc3) (37) and the pulmonary adenoma resistance gene (Par3)
(38). It remains to be clarified whether the region(s) involved
in LOH in mouse hepatocyte lines may be related to those in
the human chromosomes and the mouse predisposition loci.
Fine mapping of the common deleted regions on chromosome
12 is now underway.
A noteworthy finding with the present LOH for chromosome
12 was the fact that the hepatocarcinogenesis-resistant B6
allele was lost, while the hepatocarcinogenesis-susceptible
C3H allele was retained, similarly to the case for chromosome
4 (26,27). Since B6-biased allelic loss was observed for both
B6C3F1 and C3B6F1 mice, genomic imprinting is presumably
not involved in the underlying mechanism. When normal
hepatocytes of C3H mice are cultivated for a long period,
hepatocyte colonies with indefinite growth capacity emerge at
a high incidence, while they are much fewer in the B6 mouse
case (39,40). It is thus possible that the putative growth
suppressor gene on B6 chromosome 12 may exert more potent
growth suppressive activity than the counterpart C3H gene
and loss of the B6 gene(s) may lead to a greater growth
advantage than loss of the C3H gene(s).
A gain of chromosomes 15 and 19 was observed for both
primary HCCs and cell lines in the present study, as well as
a telomeric and centromeric fusion between two chromosomes
15 and one case of centromeric fusion between chromosomes
15 and 19. An increase in chromosome 15 number may be of
importance with regard to c-myc and intracisternal A particles,
because they are located on chromosome 15 and their expression is frequently elevated in mouse HCC (41–43). It has also
been reported that rat HCC and hepatic adenomas show c-myc
amplification (44) and copy numbers of rat chromosome 7, on
which c-myc resides, are increased in rat HCC (45). Gain of
chromosome 19 has been reported in hepatic adenomas induced
by DEN in B6C3F1 mice (22) and although no oncogenes
have so far been found on this chromosome, it bears susceptibility genes for liver and lung tumors (46,47). However, the fact
that not all cells showed gain of chromosomes 15 and/or 19
indicates that this type of change may not be an essential
initial event for immortalization or carcinogenesis but rather
that it may increase the likelihood of the two phenomena.
It has been reported that rat HCC cells frequently demonstrate duplication of chromosome 1q (45), which is homologous
to mouse chromosome 7. This region contains a number of
genes related to rodent hepatocarcinogenesis, such as H-ras
(48,49), Igf2 (50,51) and H19 (52). However, we could not
identify any specific changes in mouse chromosome 7 in the
present series of primary HCC and cell lines. The question of
whether minor changes which cannot be detected by the usual
cytogenetic methods may be present remains to be clarified.
Acknowledgement
This study was supported by a grant-in-aid from the Japanese Ministry of
Education, Sciences, Sports and Culture.
References
1. Miller,D.A. and Miller,O.J. (1983) Chromosomes and cancer in the mouse:
studies in tumors, established cell lines, and cell hybrids. Adv. Cancer
Res., 39, 153–182.
2. Aldaz,C.M., Trono,D., Larcher,F., Slaga,T.J. and Conti,C.J. (1989)
Sequential trisomization of chromosomes 6 and 7 in mouse skin
premalignant lesions. Mol. Carcinog., 2, 22–26.
3. Mushinski,J.F. (1988) c-myc oncogene activation and chromosomal
translocation in Balb/c plasmacytomas. In Klein,G. (ed.) Cellular Oncogene
Activation. Marcel Dekker, New York, NY, pp. 181–222.
4. Cahilly-Snyder,L., Yang-Feng,T., Franke,U. and George,D.L. (1987)
Molecular analysis and chromosomal mapping of amplified genes isolated
from a transformed mouse 3T3 cell line. Somat. Cell Mol. Genet., 13,
235–244.
5. Berberich,S. and Cole,M. (1994) The mdm-2 oncogene is translocated and
overexpressed in a murine plasmacytoma cell line expressing wild-type
p53. Oncogene, 9, 1469–1472.
6. Bremner,R. and Balmain,A. (1990) Genetic changes in skin tumor
progression: correlation between presence of a mutant ras gene and loss
of heterozygosity on chromosomes 7. Cell, 61, 407–417.
7. Bianchi,A.B., Aldaz,C.M. and Conti,C.J. (1990) Nonrandom duplication
of the chromosome bearing a mutated Ha-ras-1 allele in mouse skin
tumors. Proc. Natl Acad. Sci. USA, 87, 6902–6906.
8. Kemp,C., Fee,F. and Balmain,A. (1993) Allelotype analysis of mouse skin
tumors using polymorphic microsatellites: sequential genetic alterations
on chromosomes 6, 7, and 11. Cancer Res., 53, 6022–6027.
9. Zenkelsen,J.C., Hodges,L.C. and Conti,C.J. (1997) Loss of heterozygosity
on murine chromosome 6 in two-stage carcinogenesis: evidence for a
conserved tumor suppressor gene. Oncogene, 14, 109–114.
10. Wiseman,R.W., Cochran,C., Dietrich,W., Lander,W.E. and Soderkvist,P.
(1994) Allelotyping of butadiene-induced lung and mammary
adenocarcinomas of B6C3F1 mice: frequent losses of heterozygosity in
regions homologous to human tumor-suppressor genes. Proc. Natl Acad.
Sci. USA, 91, 3759–3763.
11. Radany,E.H., Hong,K., Kesharvarzi,S., Lander,E.S. and Bishop,M. (1997)
Mouse mammary tumor virus/v-Ha-ras transgene-induced mammary
tumors exhibit strain-specific allelic loss on mouse chromosome 4. Proc.
Natl Acad. Sci. USA, 94, 8664–8669.
12. Aldaz,C.M., Liao,Q.Y., Paladugu,A., Rehm,S. and Wang,H. (1996)
Allelotypic and cytogenetic characterization of chemically induced mouse
mammary tumors: high frequency of chromosome 4 loss of heterozygosity
at advanced stages of progression. Mol. Carcinog., 17, 126–133.
13. Herzog,C.R., Wang,Y. and You,M. (1995) Allelic loss of distal chromosome
4 in mouse lung tumors localize a putative tumor suppressor gene to
a region homologous with human chromosome 1p36. Oncogene, 11,
1811–1815.
14. Hegi,M.E., Devereux,T.R., Dietrich,W.F., Cochran,C.J., Lander,E.C.,
Foley,J.F., Maronpot,R.R., Anderson,M.W. and Wiseman,R.W. (1994)
Allelotype analysis of mouse lung carcinomas reveals frequent allelic
losses on chromosome 4 and an association between allelic imbalance on
chromosome 6 and K-ras activation. Cancer Res., 54, 6257–6264.
15. Santos,J., de Castro,I.P., Merranz,M., Pellicer,A. and Fernandez-Piqueras,J.
(1996) Allelic losses on chromosome 4 suggest the existance of a candidate
tumor suppressor region of about 0.6 cM in γ-radiation-induced mouse
primary thymic lymphomas. Oncogene, 12, 669–676.
16. Zhaung,S.-M., Eklund,L.K., Cochran,C., Rao,G.N., Wiseman,R.W. and
Söderkvist, P. (1996) Allelotypic analysis of 29,39-dideoxycytidine- and
1,3 butadiene-induced lymphomas in B6C3F1 mice. Cancer Res., 56,
3338–3343.
17. Dietrich,W.F., Radany,E.H., Smith,J.S., Bishop,J.M., Hanahan,D. and
Lander,E.S. (1994) Genome-wide search for loss of heterozygosity in
transgenic mouse tumors reveals candidate tumor suppressor genes on
chromosomes 9 and 16. Proc. Natl Acad. Sci. USA, 91, 9451–9455.
18. Held,W.A., Pazik,J., O’Brien,J.G., Kerns,K., Gobey,M., Meis,R., Kenny,L.
and Rustum,Y. (1994) Genetic analysis of liver tumorigenesis in SV40 T
antigen transgenic mice implies a role for imprinted genes. Cancer Res.,
54, 6489–6495.
19. Ritland,S.R., Rowse,G.J., Chang,Y. and Gendler,S.J. (1997) Loss of
heterozygosity analysis in primary mammary tumors and lung metastasis
of MMTV-MTAg and MMTV-neu transgenic mice. Cancer Res., 54,
3520–3525.
20. Clark,D.J., Meijne,E.I.M., Bouffler,S.D., Huiskamp,R., Skidmore,C.J.,
Cox,R. and Silver,A.R.J. (1996) Microsatellite analysis of recurrent
chromosome 2 deletions in acute myeloid leukemia induced by radiation
in F1 hybrid mice. Genes Chromosomes Cancer, 16, 238–246.
2087
K.Ogawa et al.
21. Matsumoto,Y., Kosugi,S., Shinbo,T. et al. (1998) Allelic loss analysis of
γ-ray-induced mouse thymic lymphomas: two candidate tumor suppressor
gene loci on chromosomes 12 and 16. Oncogene, 16, 2747–2754.
22. Danielsen,H.E., Brogger,A. and Reith,A. (1991) Specific gain of
chromosome 19 in preneoplastic mouse liver cells after diethylnitrosamine
treatment. Carcinogenesis, 12, 1778–1780.
23. Manenti,G., De Gregorio,L., Gariboldi,M., Dragani,T.A. and Pierotti,M.A.
(1995) Analysis of loss of heterozygosity in murine hepatocellular tumors.
Mol. Carcinog., 13, 191–200.
24. Davis,L.M., Carpary,W.J., Slallah,A.S., Maronpot,R., Wiseman,R.,
Barrett,J.C., Elliott,R. and Hozier,J. (1994) Loss of heterozygosity in
spontaneous and chemically induced tumors of the B6C3F1 mouse.
Carcinogenesis, 15, 1637–1645.
25. Miyasaka,K., Ohtake,K., Nomura,K., Kanda,H., Kominami,R.,
Miyashita,N. and Kitagawa,T. (1995) Frequent loss of heterozygosity on
chromosome 4 in diethylnitrosamine-induced C3H/MSM mouse
hepatocellular carcinomas in culture. Mol. Carcinog., 13, 37–43.
26. Nishimori,H., Ogawa,K. and Tateno,H. (1994) Frequent deletion in
chromosome 4 and duplication of chromosme 15 in liver epithelial cells
derived from long-term culture of C3H mouse hepatocytes. Int. J. Cancer,
59, 108–113.
27. Lee,G.H., Ogawa,K., Nishimori,H. and Drinkwater,N.R. (1995) Most liver
epithelial cell lines from C3B6F1 mice exhibit paternally-biased loss of
heterozygosity at the Lci (liver cell immortalization) locus on chromosome
4. Oncogene, 11, 2281–2287.
28. Nesbitt,M.N. and Franke,U. (1973) A system of nomenclature for band
patterns of mouse chromosomes. Chromosoma, 41, 145–158.
29. Mitelman,F. (1995) Guidlines for Cancer Cytogenetics: Supplement to an
International System for Human Cytogenetic Nomenclature. S. Karger,
Basel, Switzerland.
30. Danielsen,H.E., Steen,H.B., Lindmo,T. and Reith,A. (1988) Ploidy
distinction in experimental liver carcinogenesis in mice. Carcinogenesis,
9, 59–63.
31. Herzog,C.R., Chen,B., Wang,Y., Schut,H.A.J. and You,M. (1996) Loss of
heterozygosity on chromosomes 1, 11, 12, and 14 in hybrid mouse lung
adenocarcinomas. Mol. Carcinog., 16, 83–90.
32. Otsuka,T., Kohno,T., Mori,M., Noguchi,M., Hirohashi,S. and Yokota,J.
(1996) Deletion mapping of chromosome 2 in human lung carcinoma.
Genes Chromosomes Cancer, 16, 113–119.
33. Suzuki,T., Yokota,J., Mugishima,H., Okabe,I., Ookuni,M., Sugimura,T.
and Terada,M. (1989) Frequent loss of heterozygosity on chromosome
14q in neuroblastoma. Cancer Res., 49, 1095–1098.
34. Chang,W.Y.H., Cairns,P., Schoenberg,M.P., Plascik,T.J. and Sidransky,D.
(1995) Novel suppressor loci on chromosome 14q in primary bladder
cancer. Cancer Res., 55, 3246–3249.
35. Cliby,W., Ritland,S., Hartmann,L., Dodson,M., Halling,K.C., Keeney,G.,
Podratz,K.C. and Jenkins,R.B. (1993) Human epithelial ovarian cancer
allelotype. Cancer Res., 53, 2393–2398.
36. Jacoby,R.F., Hohman,C., Marshall,D.J., Frick,T.J., Schlack,S., Broda,M.,
Smutko,J. and Elliot,R.W. (1994) Genetic analysis of colon cancer
susceptibility in mice. Genomics, 15, 381–387.
37. Gariboldi,M., Manenti,G., Canzian,F., Falvella,F.S., Pierotti,M.A., Della
Porta,G., Binelli,G. and Dragani,M.A. (1993) Chromosome mapping of
murine susceptibility loci to liver carcinogenesis. Cancer Res., 53, 209–211.
2088
38. Pataer,A., Nishimura,M., Kamoto,T., Ichioka,K., Sato,M. and Hiai,H.
(1997) Genetic resistance to urethan-induced pulmonary adenomas in
SM3A recombinant inbred mouse strains. Cancer Res., 57, 2904–2908.
39. Lee,G.H., Sawada,N., Mochizuki,Y., Nomura,K. and Kitagawa,T. (1989)
Immortal epithelial cells of normal C3H mouse liver in culture: possible
precursor populations for spontaneous hepatocellular carcinoma. Cancer
Res., 49, 403–409.
40. Yoshie,M., Nishimori,H., Lee,G.H. and Ogawa,K. (1998) High colony
forming capacity of cultured hepatocytes as a dominant trait in C3H/HeJ,
C57BL/6J and DBA/2J mice. Carcinogenesis, 19, 1103–1107.
41. Romach,E.H., Goldswarthy,T.L., Maronpot,R.R. and Fox,T.R. (1997)
Altered gene expression in spontaneous hepatocellular carcinomas from
male B6C3F1 mice. Mol. Carcinog., 19, 31–38.
42. Giri,R.K. and Das,B.R. (1996) Differential expression of c-jun and c-myc
in N-nitrosodiethylamine-induced hepatic oncogenesis in AKR mice.
Cancer Lett., 109, 121–127.
43. Dragani,T.A., Manenti,G., Della Porta,G., Gattoni-Celli,S. and
Weinstein,I.B. (1989) Expression of retroviral sequences and oncogenes
in murine hepatocellular tumors. Cancer Res., 46, 1915–1919.
44. Pascale,R.M., De Miglio,M.R., Muroni,M.R., Simile,M.M., Daino,L.,
Seddaiu,M.A., Nufris,A., Gaspa,L., Deiana,L. and Feo,F. (1996) c-myc
amplification in premalignant and malignant lesions induced in rat liver
by the resistant hepatocyte model. Int. J. Cancer, 68, 136–142.
45. Sargent,L., Dragan,Y.P., Xu,Y.-H., Sattler,G., Wiley,J. and Pitot,H.C. (1996)
Karyotypic changes in a multistage model of chemical
hepatocarcinogenesis in the rat. Cancer Res., 56, 2985–2991.
46. Manenti,G., Binelli,G., Gariboldi,M., Canzian,F., Gregoria,D.L.,
Falvella,F.S., Dragani,T.A. and Pierotti,M.A. (1994) Multiple loci affect
genetic predisposition to hepatocarcinogenesis in mice. Genomics, 23,
118–124.
47. Devereux,T.R., Wiseman,R.W., Kaplan,N. et al. (1994) Assignment of a
locus for mouse lung tumor susceptibility to proximal chromosome 19.
Mamm. Genome, 5, 749–755.
48. Richardson,K.K. and Helvering,L.M. (1992) Genetic alterations in the 61st
codon of the H-ras oncogene isolated from archival sections of hepatic
hyperplasias, adenomas and carcinomas in control groups of B6C3F1
mouse bioassay studies conducted from 1979 to 1986. Carcinogenesis,
13, 935–941.
49. Maronpot,R.R., Fox,T.R., Malarkey,D.E. and Goldworthy,T.L. (1995)
Mutations in the ras proto-oncogene: clues to etiology and molecular
pathogenesis of mouse liver tumors. Toxicology, 101, 125–156.
50. Schirmacher,P., Held,W.A., Yang,D., Chisari,F.V., Rustum,Y. and
Rogler,C.E. (1992) Reactivation of insulin-like growth factor II during
hepatocarcinogenesis in transgenic mice suggests a role in malignant
growth. Cancer Res., 52, 2549–2556.
51. Ooasa,T., Karasaki,H., Kanda,H., Nomura,K., Kitagawa,T. and Ogawa,K.
(1998) Loss of imprinting of the insulin-like growth factor II (Igf2) gene
in mouse hepatocellular carcinoma cell lines. Mol. Carcinog., 22, 248–253.
52. Haddad,R. and Held,W.A. (1997) Genomic imprinting and IGF2 influence
liver tumorigenesis and loss of heterozygosity in SV40 T antigen transgenic
mice. Cancer Res., 57, 4615–4623.
Received April 12, 1999; revised July 13, 1999; accepted July 20, 1999