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
1 Department of Developmental Biology and Oncology, Division of Molecular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chromosomal alterations were investigated in hepatocellular carcinoma cell lines, primary tumors and liver epithelial cell lines derived from normal livers of C57BL/6JxC3H/HeJ F1 and C3H/HeJxC57BL/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.

Abbreviations: B6, C57BL6/J; B6C3F1, C57BL6/JxC3H/HeJ F1; C3H, C3H/HeJ; C3B6F1, C3H/HeJxC57BL/6J F1; DEN, diethylnitrosamine; HCC, hepatocellular carcinoma; LE, liver epithelial; LOH, loss of heterozygosity.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 (821), 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 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 (2527). 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)xC3H/HeJ (C3H) F1 (B6C3F1) and C3HxB6 F1 (C3B6F1) mice.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
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 2x105 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'.

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 2x 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.

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 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 {chi}2 test. The significance level chosen was P < 0.05.


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 Materials and methods
 Results
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Cytogenetic changes
The general data on chromosomal changes are presented in Table IGo. 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 IGo and Figure 1aGo). Non-random structural alterations of chromosomes were generally rare in the primary HCCs, although telomere association of two chromosomes 15 was observed in one HCC (Figure 1bGo).


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Table I. Karyotypes of primary cultures of normal hepatocytes and HCC cells, and LE and HCC cell lines
 



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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.

 
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 1Go). 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 IGo). Of five HCC cell lines examined, three contained relatively abundant diploid range cells, while the other two mostly comprised hyperploid cells (Table IGo). 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 IGo and Figure 2aGo). In addition, centromere fusion between chromosomes 15 and 19 was detected in all cells in one case (Figure 2bGo). 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.




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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.

 
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 4 were detected in triploid and tetraploid range cells in two LE cell lines (Figure 3a and bGo and Table IIGo). In these cells, the length of chromosome 4 was reduced to ~50% in one or two copies (Figure 3aGo). 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 3bGo). Regarding chromosome 12, the copy numbers were two in most cells in the two LE cell lines and one of the two HCC cell lines (Figure 3cGo) and mainly one in the other HCC cell line (Figure 3dGo). 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.



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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.

 

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Table II. FISH analysis of chromosomes 4 and 12 on hypotriploid and hypotetraploid cells
 
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 was detected in six of eight LE lines and four of five HCC cell lines according to two to four markers (Figure 4Go). 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).



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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).

 

    Discussion
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 Materials and methods
 Results
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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 (1315) has been reported. The present study demonstrated that chromosome 12 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 (3235), 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 (4143). 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.


    Acknowledgments
 
This study was supported by a grant-in-aid from the Japanese Ministry of Education, Sciences, Sports and Culture.


    Notes
 
2 To whom correspondence should be addressedEmail: ogawak{at}asahikawa-med.ac.jp Back


    References
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 Abstract
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 Materials and methods
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 References
 

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Received April 12, 1999; revised July 13, 1999; accepted July 20, 1999.