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Carcinogenesis, Vol. 21, No. 9, 1753-1756, September 2000
© 2000 Oxford University Press

Carcinogenesis

Detection of somatic DNA alterations in azoxymethane-induced F344 rat colon tumors by random amplified polymorphic DNA analysis

Cristina Luceri2, Carlotta De Filippo, Giovanna Caderni, Lapo Gambacciani, Maddalena Salvadori, Augusto Giannini1 and Piero Dolara

Department of Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence and
1 USL, 10/H Antella, Florence, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Colon carcinogenesis induced in rats by azoxymethane (AOM) is a useful experimental model as it mimics the human adenoma–carcinoma sequence and allows the study of dietary variation and of the effects of chemopreventive substances. Alterations of specific oncogenes and tumor suppressor genes (APC and K-ras) play roles at different stages of this carcinogenesis process. Recently, it has been suggested that genomic instability is the necessary step for the generation of multiple mutations underlying the occurrence of cancer. We studied the frequency of K-ras and microsatellite instability (MSI) in 30 colorectal tumors induced by AOM (30 mg/kg) in F344 rats. We also used the random amplified polymorphic DNA (RAPD) method to identify genomic alterations in chemically induced aberrant crypt foci (ACF), adenomas and adenocarcinomas. K-ras mutations were identified in 16.7% of the cases (5/30; 9% in adenomas and 37.5% in adenocarcinomas) and MSI in 20% (6/30) of the tumors (only one sample exhibited instability at more than one locus). Of 21 primers used for the RAPD assay, six were very informative. All the analyzed tumors (16/16) showed at least one RAPD profile with lost or additional bands compared with the normal mucosa. A lower level of genomic alteration was present in the ACF analyzed (7/10). In conclusion, K-ras and MSI are not often involved in the AOM carcinogenesis in the rat, whereas extensive genomic instability is always present and can be detected using the RAPD analysis.

Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; MMR, mismatch repair; MSI, microsatellite instability; RAPD, random amplified polymorphic DNA.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Colon cancer is a well characterized model for understanding the genetic events that underlie the development of malignancy. Colorectal carcinogenesis is supposed to proceed through a series of genetic alterations (1) in which loss or inactivation of the tumor suppressor gene APC and activation of the oncogene K-ras are supposed to play key roles (1). In humans, mutations of the APC gene have been reported in ~80% of colon cancers (2), whereas mutations of K-ras occur with a frequency of 50% (1).

It was recently suggested that genomic instability is the essential prerequisite for the generation of multiple mutations underlying cancer (3). The spontaneous mutation rate is not sufficient to produce the number of mutations reported in human cancer cells. Genomic instability can be related to a defective mismatch repair (MMR), caused by recombination events or by defective mitosis.

Microsatellite instability (MSI) may monitor abnormalities in the MMR system. Microsatellites are simple repetitive sequences normally interspersed throughout the genome. These sequences are stably inherited, highly polymorphic and have a low mutation rate. Insertion or deletion errors of these short repeated sequences occur during DNA replication and can arise from defects in MMR mechanisms (4). While cases of MSI occur in only 12–15% of all colorectal tumors, it is found in virtually all hereditary non-polyposis colorectal cancer (5). Therefore, MSI may characterize a distinct mechanism of colorectal carcinogenesis, differing from the classical suppressor pathway (6).

Experimental models of colon cancers, in which tumors are induced by azoxymethane (AOM), are widely used to assess putative chemopreventive agents and to study the multistage development of colon cancer (7,8). In a previous paper we reported that in AOM-induced tumors, APC mutations are relatively rare (five out of 28 analyzed cancers) (9). Moreover, we did not find APC mutations in any of the aberrant crypt foci (ACF) analyzed. ACF are lesions of the colon epithelium, which can easily be visualized with methylene blue staining and can be scored in the whole colon (10). ACF are a heterogeneous group of lesions, frequently harboring mutations of K-ras (11). Genetic instability, tested by microsatellite analysis, has been also reported in ~10% of the human ACF analyzed (12).

We chose to study K-ras mutations and MSI in AOM-induced rat ACF and colon cancers previously characterized for APC gene mutations, to define the involvement of the two different carcinogenesis pathways in experimental rat carcinogenesis.

We also decided to study the genomic alterations induced by AOM using a DNA fingerprinting method. Random amplified polymorphic DNA (RAPD) is a PCR-based technique that amplifies random DNA fragments with single short primers of arbitrary nucleotide sequence under low annealing conditions. This technique is used extensively for species classification and microorganism strain determination. RAPD analysis detects rearrangements, additions or deletions of DNA and ploidy changes in cells by visualizing banding shifts, missing bands or the appearance of new bands in a DNA gel electrophoresis (13).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Tissue samples
Male F344 rats (Nossan, Correzzana, Italy) were treated twice s.c. with 15 mg/kg of AOM and killed after 230–245 days. The colon was opened longitudinally. The distal part of the colon was washed with saline and stained in ice with methylene blue (0.5% in saline) and observed under a stereomicroscope (40x). ACF were identified, harvested and stored at –80°C. Macroscopic tumors were dissected and divided into two parts. Half were stored at –80°C, as well as samples of apparently normal mucosa for comparative analysis; half were fixed in buffered formalin and embedded in paraffin blocks. Blocks were then sectioned and stained with hematoxylin and eosin to confirm the presence and type of tumors by histopathological examination, which was performed by a pathologist. Cancer histological types were evaluated on the basis of the histotype, grading and pattern of growth (14). Adenomas were classified on the basis of their microscopic architecture as tubular, tubulovillous and villous according to Morson et al. (15).

DNA extraction
Total DNA was extracted from frozen tumors and normal tissue using the QIAamp tissue kit (Qiagen, Hilden, Germany), according to the manufacturer's specifications.

Microsatellite analysis
We screened 30 colon tumors harvested from 24 rats (eight adenocarcinomas and 22 adenomas) with microsatellite analysis using a PCR-based approach. Dinucleotide loci were selected from published rat sequences (ADRB2, APOC3, PPY, PRLR, IGHE, FGG) (16). One of two primers from each marker was first end-labeled with [{gamma}-33P]ATP (2000 Ci/mmol; NEN, Germany) and T4 DNA polynucleotide kinase [Pharmacia Biotech Italia, Cologno Monzese (Mi), Italy]. PCR reactions were carried out in a 15 µl vol containing ~100 ng of genomic DNA, 1x PCR buffer, 1.5 mM MgCl2, 0.25 mM dNTPs, 0.2 µM primers, 0.2 µl of the end-labeled primer and 1.25 U Taq polymerase (Advanced Biotechnologies, UK). The six marker PCR conditions were the same: 30 cycles at 94°C for 1 min, 60°C for 1 min and 72°C for 1 min and a final extension at 72°C for 5 min. PCR products were separated on a 7% polyacrylamide–urea–formamide gel and visualized by autoradiography.

K-ras analysis (codons 12 and 13)
The K-ras mutational analysis was performed on the same 30 rat tumors samples by PCR–RFLP according to Singh et al. (17). Briefly, K-ras exon 1, spanning codons 12 and 13, was amplified using a mismatch 5'-end primer. The PCR amplification creates a BstNI restriction enzyme recognition site (CCTGG) overlapping the first two nucleotides of codon 12. Digestion with this enzyme originates in the wild-type two fragments of 29 and 87 bp, respectively, while in case of a mutation at either of the first two positions, this BstNI site was abolished in the PCR-amplified product, and in polyacrylamide gel electrophoresis results in one band of 116 bp. Similarly, the PCR amplification of the wild-type K-ras codon 13 using the mismatch 5'-end primer as described above also created a BglI restriction enzyme recognition site (GCC4NGGC) overlapping the entire codon 13. The digestion with this enzyme originates in the wild-type two fragments of 32 and 87 bp, respectively, whereas a point mutation at either of the three positions, destroys this BglI site in the PCR amplified product. Gel electrophoresis results, as in the case of codon 12, in one band of 116 bp. Mutated samples were sequenced using the dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Foster City, CA) and a DNA sequencer (ABI Prism 310 Genetic Analyzer; Perkin Elmer), following the manufacturer's protocol.

RAPD analysis
Twenty-one random primers (decanucleotide GC-rich) were used to score alterations. These primers were newly designed or have been reported previously (18). The sequences, the percentage of CG bases and the polymorphism status are shown in Table IGo.


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  		Table I. Primers utilized for the RAPD analysis to score alterations in AOM-induced tumors
 
We analyzed 16 colorectal tumors, 10 ACF and the respective normal tissue of eight rats. Normal tissue, ACF and tumor DNA were amplified with each primer (20 ng/µl), 3 mM MgCl2, 0.8 mM dNTPs and 1.25 U Taq polymerase (Advanced Biotechnologies, UK) in 25 µl of 1x PCR reaction buffer for 45 cycles. PCR conditions were: denaturing at 94°C for 0.5 min, annealing at 36°C for 1 min and extension at 72°C for 2 min.

PCR products were resolved by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. We defined all tumors that presented an RAPD profile with loss or addition of bands, if compared with their normal mucosa, as `genomic unstable' and for each tumor we calculated the frequency of altered RAPD profile. An example of such analysis is shown in Figure 1Go.



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  		Fig. 1. Examples of RAPD profiles using primer 1281. The numbers at the top represent codes for specific rats; M, marker; N, normal mucosa DNA; 1 and 2 are different tumors from the same rat, localized in the proximal colon (a), distal colon (b), rectum (r) or ileum (d). The arrow indicates a tumor-specific band.

 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
We found microsatellite instability in six of 30 (20%) AOM-induced colon tumors [4/22 (18.2%) adenomas and 2/8 (25%) adenocarcinomas]; five tumors exhibited instability at one locus and one showed instability at three loci (Table IIGo).


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  		Table II. Microsatellite instability in AOM-induced tumors
 
The K-ras analysis (Table IIIGo) was performed on the same 30 tumors (eight adenocarcinomas and 22 adenomas). Three tumors were mutated in codon 12 (one adenoma and two adenocarcinomas) and in codon 13 (one adenoma and one adenocarcinoma). The overall mutation frequency in the K-ras gene was 16.7% in all tumors; 9% in adenomas and 37.5% in adenocarcinomas. Most of the mutations (60%) were located in codon 12.


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  		Table III. Mutation analysis of codons 12 and 13 of K-ras exon 1
 
For the RAPD analysis, 21 random primers (decanucleotide GC-rich) were used to amplify 16 colorectal tumors, 10 ACF and the respective normal tissue. PCR products were resolved by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. Tumors that presented a RAPD profile with loss or addition of bands, if compared with their normal mucosa were defined as `genomic unstable'. Of all the primers used, six were very informative (Table IGo).

All the analyzed tumors (adenomas + carcinomas) (16/16) showed at least one RAPD profile with lost or additional bands, and some tumors (9/16, 56.25%) exhibited a high frequency of genomic alterations. It is apparent that the use of the RAPD analysis allows the detection of genetic instability also in ACF (Figure 2Go). The level of instability was lower in ACF than in adenomas and carcinomas and no difference was observed in the level of instability between adenomas and carcinomas. We did not find any correlation between the degree of genomic alteration of the tumors and their level of dysplasia, cancer invasiveness and dimensions (data not shown).



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  		Fig. 2. Percentage of altered RAPD profiles in ACF, hyperplastic polyp (light gray), adenomas (gray) and carcinomas (black) from rats administered AOM.

 
We observed also that different lesions from the same rat were not all positive for the same assay (Figure 1Go) and that a common pattern of instability among the samples from the same animal was not detectable. We also noted that some primers allowed the detection of tumor-specific bands (Figure 1Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In humans, colon cancer results from the progressive accumulation of mutations in genes involved in the control of the cell cycle (1). The K-ras and APC genes are two of the earliest and most frequently mutated genes.

Previously, we reported that APC gene mutations are relatively rare in AOM-induced rat carcinogenesis (9). In this study we analyzed the same tumors at codons 12 and 13 of the K-ras gene. AOM causes O6-methylguanine adduct formation which leads to G->A transitions and O6-methylguanine adducts can cause activating mutations in the K-ras genes (19). The presence of K-ras point mutations has been reported in AOM-induced rat colonic tumors with variable frequency (17,20). We found K-ras mutations in two of 22 and three of eight adenomas and adenocarcinomas, respectively; these frequencies are very low and have been reported by others in the same model (20).

We detected microsatellite alterations in 20% of the tumor samples (four of 22 adenomas and two of eight adenocarcinomas) but only one tumor exhibited instability at more than one locus; in most cases, this instability was characterized by a single microsatellite change (insertion or deletion). In humans, widespread microsatellite instability is highly suggestive of underlying genetic instability caused by mutations of DNA mismatched repair genes (6). In contrast, in AOM-induced tumors of the rat, the number and type of microsatellite alterations are so limited that they probably result from spontaneous mutations and play a limited role in the generation of cancer.

Finally, we analyzed our samples by the RAPD method. Recently the RAPD method has been used to identify genomic instability in human cancers, revealing that genomic alterations occur frequently in lung and brain (13,18). Moreover, a significant association between the frequency of genomic instability and the degree of tumor differentiation has been reported in head and neck squamous cell carcinomas (21).

Twenty-one decamers were screened to identify the primers generating the most distinctive fingerprinting. We observed that the polymorphism status as defined by RAPD was not correlated with the GC content. Six of the 21 primers were very informative (MP13, OPA11, 1281, MP41 GP85 and MP77). Using these primers, 100% of the tumors (adenomas and carcinomas) exhibited a different pattern of bands compared with the normal mucosa. In our study, DNA genomic alterations were identified as band loss or gain. In some cases it was possible to observe an increase or a decrease of the band intensity as well; however, variations of band intensity were not considered as a criterion for classifying instability. These alterations seemed to be early events in colon carcinogenesis, appearing also in ACF. Using the RAPD assay with different primers, we observed a widespread and very high instability also in human colon cancers (preliminary observations).

It is not possible to say whether the genetic defects observed in tumors with the RAPD technique are due to translocations or deletions causing inactivation of tumor suppressor genes or to point mutations causing activation of oncogenes. Therefore, some of these alterations observed could be inconsequential in terms of the control of cell function.

It is clear from this and other studies that the genetic progression in the rat differs from that found in humans. We found that mutations in APC and K-ras genes and MSI are not particularly involved in the development of colon cancer induced by AOM in the rat. It is possible that chemically induced rat tumors are different from human lesions since they are harvested at an early stage in which invasion and metastasis have not progressed. However, AOM-induced tumors present a widespread genomic instability as revealed by the RAPD assay. Likely, the apparently normal mucosa might have same degree of instability caused by the AOM treatment, which was not detectable with our comparative analysis. Theoretically at least it should be possible to select specific primers to reveal different patterns between carcinogen-treated and control rodents performing RAPD on normal mucosa samples.

In conclusion, determination of the RAPD profile allows the detection of genomic instability in tumor cells, which appears to be an early event in colon carcinogenesis.


   Notes
 
2 To whom correspondence should be addressed Email: dipfar4{at}ds.unifi.it Back


   Acknowledgments
 
This work was supported by grants from the European Community FAIR program (grant no. PL95/0653), the European Community QLRT programs (grant nos 1999/00346 and 1999/00505), the Ministero della Università e della Ricerca Scientifica e Tecnologica and the University of Florence (Florence, Italy).


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 13, 2000; revised June 8, 2000; accepted June 15, 2000.


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