Analysis of loss of heterozygosity in neoplastic nodules induced by diethylnitrosamine in the resistant BFF1 rat strain

Manuela Gariboldi, Rosa Pascale1, Giacomo Manenti, Maria R.De Miglio1, Diego Calvisi1, Angelo Carru1, Tommaso A. Dragani and Francesco Feo1,2

Department of Experimental Oncology A, Istituto Nazionale Tumori, Milan, Italy and
1 Department of Biomedical Sciences, Division of Experimental Pathology and Oncology, University of Sassari, Italy


    Abstract
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 Abstract
 Introduction
 References
 
Loss of heterozygosity (LOH) at specific chromosomal regions is a frequent event in poorly differentiated human hepatocellular carcinomas (HCCs), but rare in mouse HCCs. This behavior could depend on interspecies differences in mechanisms of hepatocarcinogenesis or in developmental stage of lesions. To verify if LOH is involved in rat hepatocarcinogenesis, we studied LOH frequency in slowly growing neoplastic nodules induced by Solt–Farber model in diethylnitrosamine-initiated BFF1 rats. We analyzed, with microsatellites, markers at 67 rat loci dispersed over all chromosomes, corresponding to regions homologous to those lost in human HCCs or containing hepatocellular susceptibility (Hcs) or resistance (Hcr) loci in rat and mouse. In agreement with previous findings with mouse HCCs, but at variance with human HCCs, no detectable LOH was found at any locus in rats, suggesting rare LOH involvement in neoplastic nodules, with low tendency to progress to full malignancy, of BFF1 rats.

Abbreviations: BFF1, (BNxF344)F1 hybrid; BN, Brown Norway; F344, Fisher 344; GST-P, placental glutathione S-transferase; HCC, hepatocellular carcinoma; LOH, loss of heterozygosity; SSLP, single sequence length polymorphism.


    Introduction
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 Abstract
 Introduction
 References
 
Human hepatocellular carcinoma (HCC) is one of the most common causes of cancer mortality worldwide (1). Molecular analysis has identified a high frequency of loss of heterozygosity (LOH) at specific chromosomal regions in human HCC suggesting the involvement of oncosuppressor genes (28). However, the occurrence of several LOHs at loci mapping on different chromosomes in the same tumor samples (28), makes it difficult to understand whether the lost loci are involved in the pathogenesis of human HCC. Mice and rats are good experimental models to study HCC. We and other investigators have previously reported rare or low frequency of LOH in mouse hepatocellular tumors (9,10). Following our report (9), Zenklusen et al. (11) described a high frequency of LOH on mouse chromosome 6 in B6C3F1 mice, in the regions surrounding the microsatellites D6Mit29 (27 cM) and D6Mit50 (4 cM), suggesting the presence of an oncosuppressor gene. We have tested these microsatellites in our collection of mouse HCCs (9), including B6C3F1, without observing any LOH at D6Mit29 and D6Mit50 loci (data not shown) and, therefore, we confirm our conclusions. These contrasting results may reflect interstrain and interspecies differences in developmental stages of liver tumors used by various investigators, or they may be due to technical approaches or material analyzed (primary tumors or tumor-derived cell lines). Indeed, frequent LOH has been documented in invasive and metastatic HCCs in liver of Mup-SV40 T antigen transgenic mice (12) as well as in surgery specimens of poorly differentiated human HCCs (28).

Rat and human hepatocellular carcinogenesis show a number of morphologic, molecular and biochemical commonalties (for instance, see refs 13,14). Genetic predisposition to hepatic carcinogenesis has been demonstrated in mice and rats and has been postulated in humans (15). LOH at chromosomal sites where oncosuppressor genes map, could be one of the mechanisms involved. This possibility was tested in the (BNxF344)F1 hybrid strain (BFF1), generated by crossing the resistant Brown Norway (BN), with the sensitive Fisher 344 (F344) strain, in which resistance to hepatocarcinogenesis is dominantly transmitted from BN to BFF1 rats (16).

Neoplastic liver lesions were induced in 80 male BFF1 rats, by Solt–Farber protocol (17) that included initiation with 150 mg/kg of diethylnitrosamine, and selection by feeding a 0.02% 2-acetylaminofluorene diet with a partial hepatectomy at the midpoint of this treatment. Seventy weeks after initiation, the livers of 61 out of 69 surviving rats were perfused with 20 ml of ice-cold 0.9% NaCl, under ether anesthesia, before killing by bleeding through abdominal aorta. Perfused rats were used for LOH analysis and non-perfused rats for morphologic evaluation. Neoplastic nodules (5–15 mm in diameter) on liver surface or in 4 mm liver slices were isolated leaving a thin rim of nodular tissue, to avoid contamination by non-neoplastic cells. Tissues were frozen in liquid nitrogen and kept at –80°C until use for LOH analysis. Small pieces of surrounding liver and nodules were fixed in paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin (H&E). The percentage of non-parenchymal cells per lesion was evaluated on 20–30 slices, taken at regular intervals along the entire thickness of each of one to two nodules per rat, and stained with H&E. A surface area of 250–700 mm2 (sum of surface areas of single slices) was examined per nodule to obtain the mean percentage of non-parenchymal cells, with a 95% confidence interval. The presence of non-neoplastic hepatocytes and collagen fibers was evaluated in one to two nodules per rat by glutathione S-transferase (placental; GST-P) immunohistochemistry and Masson's trichrome stain, respectively. Liver lesions were classified according to the described criteria (18). Genomic DNAs from surrounding liver and nodules, and from normal liver of BN and F344 parental rats were extracted using the Genomix kit (Talent, Trieste, Italy). Single sequence length polymorphism (SSLP) analysis was performed to detect LOH and genetic instability by using primers (obtained from Research Genetics, Huntsville, AL) for microsatellite sequences which distinguished polymorphisms between parental strains (9). HCCs and the corresponding surrounding liver were screened for LOHs with 67 microsatellite marker loci dispersed long over the rat genome to cover: (i) genomic regions where susceptibility/resistance genes are located in mouse (1921) and rat (M.R.De Miglio et al., submitted for publication); (ii) genomic areas in which partial or total chromosomal deletions occur in neoplastic rat liver lesions (2224); and (iii) rat genomic areas syntenic with chromosomal sites where LOH was found in mice or humans (28,10,11). The PCR reaction was performed in 12 µl of a mixture containing 75–150 ng of DNA from surrounding liver and nodules of BFF1 rats and from the two parental strains, and normal F1 tissue. PCR reactions contained 1.5 mM of MgCl2, 2 pmol of primer mix, 70 µM of each dNTP, 0.5 µCi of [32P]dCTP (3000 Ci/mmol; Amersham, Branchburg, NJ) and 0.2 U Taq Polymerase (Perkin Elmer), and were subjected to 22 PCR cycles (annealing at 55°C for 30s) in a 9600 Thermal Cycler (Perkin Elmer). PCR products were loaded onto a 6% denaturating or non-denaturating polyacrylamide gel, depending on the marker analyzed. Gels were run at constant watts, dried and exposed to autoradiography.

No neoplastic nodules developed in BFF1 rats 1 year after initiation (16), while at 70 weeks non-infiltrating neoplastic nodules, consisting of atypical nodules/well differentiated carcinomas, some with glandular pattern, and moderately differentiated carcinomas (Figure 1AGo–D), developed in 34% of rats. Nodules showed a uniform pattern of GST-P immunostaining (Figure 1BGo) which excludes consistent contamination by normal hepatocytes, and Masson's trichrome stain of collagen fibers excluded large contamination by fibrous material (not shown). Non-neoplastic cells, including cells lining vases, ductular cells and inflammatory cells, represented 15–26% of nodular cell population (mean ± SD, 21.16 ± 2.4%, n = 30). No extensive fatty change, necrosis, fibrosis or bile duct hyperplasia occurred in surrounding liver, most of which exhibited an apparently normal pattern, except for the presence of rare clear/eosinophilic cell foci (Figure 1EGo).



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Fig. 1. Histologic pattern of liver lesions 70 weeks after initiation. (A) Well differentiated hepatic carcinoma with distortion of plate arrangement and hepatocytes in nests. (B) Uniform pattern of GST-P immunohistochemical staining of the same lesion. (C) Well differentiated carcinoma with glandular pattern. (D) Moderately differentiated carcinoma showing distortion of plate arrangement, thickened plates and atypical hepatocytes. (E) Surrounding liver showing a small clear cell focus (arrowheads) in a liver lobule. Magnification: x290 (A, C and D); x190 (B and E).

 
LOH analysis was performed on 12 well differentiated and seven moderately differentiated carcinomas and from corresponding normal tissues, taken from perfused rats. For D3Mit7, D10Mgh14 and D15Mgh8 markers, only the allele belonging to B strain was amplified in F1 hybrid rats, although a length polymorphism was present in the PCR products from B and F parental strains. This preferential allele amplification may be due to a base change in the primer sequences. At least one microsatellite for each rat chromosome was analyzed (Table IGo). The choice of the microsatellite markers was based on rat–mouse, mouse–man and rat–man comparative maps (2527) (http://ratmap.gen.gu.se). Most of the SSLPs studied mapped on the rat chromosomal regions homologous to those frequently deleted in human HCCs. Regions containing known oncosuppressor genes (p53, Rb), homologous to those controlling genetics of liver tumor development in the mouse and rat (Hcs and Hcr loci) were also analyzed. Regions most frequently lost in human HCCs map on chromosomes 4 (4p15–q21), 5 (5q23–q32), 8 (8p22), 11 (11p13–p15), 13 (13q12–q32), 16 (16q22–q24) and 17 (17p13) (28). Human chromosome 4 shows homology with rat chromosome 14, where D14Mit1 (4p) and D14Mit7 (4q11–q13), which identifies the Alb gene, were tested. The region frequently lost on chromosome 8 corresponds to rat chromosome 19, where we analyzed D19Mit2 and D19Mit9. Hbb and Igf2 (human 11p15.4) map on rat chromosome 1 where we tested D1Mit3, close to Hbb, and D1Mit5 (Igf2). Genes mapped on human 11p13 were assigned to rat chromosome 3 where we tested D3Mit7 (Cat), D3Mit1, D3Mit8 (Scn2a1), and D3Mgh6. On rat chromosome 15 were positioned genes that map on human chromosome 13, homologous to mouse chromosome 17; by comparison of mouse maps we analyzed, D15Mgh8 and D15Mgh4, predicted to be positioned close to rat Rb1 gene (human 13q14.3). In the region lost on human chromosome 16, the locus Hp maps (16q22), which is identified by D19Mgh5, in the rat. On rat chromosome 10 (homologous to human chromosome 17p13), we tested D10Mit3, D10Mit8 (Syb2) and D10Mgh14. We found, however, that none of the regions affected by LOH in human HCC showed the same genetic alteration in the homologous rat chromosomal region.


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Table I. Marker loci analyzed for LOH in rat neoplastic nodules
 
HCC susceptibility (Hcs1–2) and resistance (Hcr1–3) loci map on rat chromosomes 1 and 7, and 4, 8 and 10, respectively, (M.R.De Miglio et al., submitted for publication). Hcs1–6 and Hcr1–2 loci map on mouse chromosomes 2, 5, 7, 8, 12 and 19 (19,20), and 4 and 10 (21), respectively. The rat regions containing Hcs and Hcr and rat regions homologous to the mouse ones containing Hcs and Hcr loci, were investigated. We also tested D4Mgh1, D4Mgh4 and D4Mgh22 markers that map in a region homologous to the mouse chromosome 6, where frequent LOH occurs (11). LOH was not found in any of these chromosomal regions in neoplastic nodules. A representative SSLP analysis with 75 ng of DNA and D7Rat27 and D18Mit1 markers is shown in Figure 2Go. Doubling DNA amounts did not result in evidence of complete or partial LOH with any microsatellite marker and tumor preparation (data not shown).



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Fig. 2. Polyacrylamide-gel analysis of microsatellites D7Rat27 and D18Mit1. The pairs of `normal' surrounding liver (N) and tumor(s) (T) from BFF1 rats, together with controls from parental strains (B, BN; F, F344) are indicated at the top of the figure.

 
A large portion of the rat chromosome 5 was investigated to include, based on mouse/rat homology, the region of Mom1 (modifier of Min) (28,29). This cancer modifier locus decreases multiplicity and size of intestinal tumors induced by Min (Apc) mutation (28). Mom1 lies in a region homologous to human chromosome 1p where frequent LOH occurs in various human tumors, including HCC (30). In the same region, Hcr1, lymphoma resistance (Lyr) and liver cell immortalization (Lci) loci map (21,31). However, none of 10 markers tested showed LOH, indicating that deletions in the regions homologous to these loci are not involved in BFF1 rat hepatocarcinogenesis.

Our data differ from results obtained in human HCCs but agree with those found in mouse. Neoplastic liver nodules exhibit morphologic and biochemical heterogeneity (13,14), to which contamination by non-tumoral cells may also contribute. The method used in this study to determine LOH was patterned on those used to detect LOH in surgery specimens of human HCC. These tumors generally develop in cirrhotic patients (27), and represent more advanced lesions, with higher cellular heterogeneity and contamination by non-neoplastic (mostly inflammatory and immune) cells and fibrous material, with respect to the liver nodules of BFF1 rats, in which mean contamination by non-tumoral cells was ~21%. This value does not affect LOH detection, as we demonstrated in pilot titration experiments (9 and data not shown). Indeed, an LOH event can be demonstrated easily (eventually with the help of Instant Imager analysis) even in the presence of a contamination of tumor cells by >40% of normal cells. At this contamination, the signal ratio of the two alleles changes from 0.5:0.5 (absence of LOH) to 0.8:0.2 (LOH in all tumor cells).

Resistance of BN rats to tumor development, dominantly transmitted to BFF1 (16), and identification of Hcr loci in backcrosses BFF1xF344 (M.R.De Miglio et al., submitted for publication), suggest that the resistance of these rat strains at least in part depends on active oncosuppressor genes transmitted by parental BN rats. This could contribute to slow progression of these lesions to poorly differentiated neoplasms. Our results do not exclude that allelic loss occurs at chromosome sites not yet explored for LOH in rat genome. Our analysis was addressed to those areas that could be reasonably thought to be `at risk' of LOH, on the basis of the presence of susceptible/resistance loci in rat or mouse, or of the synteny with human chromosomal sites where LOH, affecting putative oncosuppressor genes (3).

Cytogenetic studies on in vitro cultures of rapidly progressing preneoplastic hepatocytes from rats treated with the initiation–promotion–progression protocol (23) and on WB cell lines developed from normal rat liver (32), showed duplications of chromosome 1q or a trisomy of rat chromosome 1 and deletions of chromosomes 3p and 6q. Frequent duplication of chromosome 1 and an ~20% aneuploidy rate of chromosomes 3, 6 and 15 occur in primary cultures of cells derived from HCCs of alb-SV40 T antigen transgenic rats (24). All these changes involve genomic portions that have their counterparts in regions containing Hcs loci in mice or rat, or in chromosomal segments affected by LOHs in humans. Rat chromosomes 1q, 3p and 6p are syntenic with mouse chromosomes 7, 2 and 12, respectively, and with human chromosomes 11p15, 11p13 and 14q32. In man, 11p13, 11p15 and 14q32 are associated with LOH in HCCs. Hcs1 is located on rat chromosome 1q, and Hcs loci are positioned on mouse chromosomes 2 and 12. However, analysis of several microsatellites on chromosomes 1, 3, 6, 10 and 15 did not show any allelic loss. Therefore, the primary rat liver neoplastic nodules that we have analyzed, behave in the same way as primary mouse HCCs (9), while in vitro cultured rat liver tumor cells show LOHs like primary human HCCs. These results are consistent with data obtained on mouse cell lines established from HCCs, where frequent LOHs were found on chromosome 4 (33,34), whereas none of the primary tumors, from which cell lines derived, presented LOH. These data and the present results indicate that LOH alterations may be more easily detected after in vitro passage of precancerous and cancerous cells, which may result in the selection of cells carrying chromosomal aberrations, or it may induce or accelerate breakage of chromosome fragile sites, not recognizable in primary tumors in vivo. Recent data have shown the presence of fragile sites on various chromosomes of c-myc/TGF{alpha} transgenic mice (35), predisposing to genetic aberrations, including LOH. Interestingly, genomic instability evidenced by amplification and overexpression of c-myc (36) and TGF{alpha} (37) genes, occurs in nodules and HCCs induced, in susceptible F344 rats, by RH protocol. In contrast, c-myc is not overexpressed in liver nodules with low propensity to progress to full malignancy, in rats resistant to hepatocarcinogenesis (38), as those used in the present study.

In conclusion, LOH at chromosomal sites at which are located genes involved in susceptibility/resistance to hepatic carcinogenesis or syntenic with human chromosomal sites where putative oncosuppressor genes are present, does not occur in >60% of nodular cells, not prone to evolve to undifferentiated HCCs, in rats resistant to hepatic carcinogenesis. A corollary of this is that LOHs may represent late events, occurring in unstable genomes, of fast growing and poorly differentiated neoplastic liver lesions. This conclusion is consistent with recent observations of LOH of several oncosuppressor genes, much more frequent in undifferentiated than in well differentiated human HCCs (39).


    Acknowledgments
 
This work was partially funded by grants from Finalized Project CNR `ACRO' and from Associazione and Fondazione Italiana Ricerca Cancro (AIRC and FIRC) of Italy.


    Notes
 
2 To whom correspondence should be addressed Email: feo{at}ssmain.uniss.it Back


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Received June 3, 1998; revised June 3, 1998; accepted March 22, 1999.