Original article
Analysis of genetic changes in rat endometrial carcinomas by means of comparative genomic hybridization

https://doi.org/10.1016/S0165-4608(00)00435-0Get rights and content

Abstract

Animals of the BDII inbred rat strain are known to be genetically predisposed to endometrial adenocarcinoma (EAC). Using them as models of human EACs, we studied tumors arising in F1 and F2 progeny from BDII animals crossed to animals from two other inbred strains, in which EACs were quite rare. In order to identify chromosomal regions exhibiting DNA copy number changes, comparative genomic hybridization (CGH) was applied in a series corresponding to 27 different solid tumors, most of which were classified as EACs, from these animals. The main findings from the study were that, although many different chromosomes were involved in copy number variation, some of the changes detected were recurrent and quite specific. Among specific changes found were gains in rat chromosome (RNO) regions 4q12∼q22, 6q14∼q16, and whole chromosome arms in some of the small metacentric chromosomes (e.g., RNO14, 16, and 18). RNO10 was involved in gain in the terminal and proximal regions. Each of these regions contains previously identified cancer-related genes representing possible candidates to be involved in the development of EAC. Furthermore, it was observed that there were clear differences in the pattern of copy number changes between tumors occurring in the two different crosses, and also between solid tumors and cell cultures. Endometrial cancer is the most common human gynecological cancer, but not much is known about specific genetic changes influencing this disease. Two genetic alterations that have been reported from human endometrial cancer are amplification of the ERBB2 gene and mutations in the 12 codon of the KRAS gene. One case of Erbb2 amplification was found but there were no Kras mutations in the rat material studied. We conclude that molecular genetic analysis of the rat BDII model will provide important new information about EAC in mammals.

Introduction

Endometrial carcinoma is the most common gynecological cancer, ranking fourth in incidence among invasive tumors in women, following breast, lung, and colon cancers [1]. Consequently, it is important to try to determine the genetic and epigenetic events involved in the development of this disease. However, there have been relatively few reports on cytogenetic changes in endometrial carcinomas, probably due to the technical difficulties in applying chromosome banding techniques to the tumors. Furthermore, little is known about alterations on the molecular level that occur during the development and progression of the endometrial carcinomas, such as the involvement of specific oncogenes or tumor suppressor genes [2].

Like most other neoplastic diseases, endometrial carcinoma is a complex disease and its development is influenced by both genetic and environmental factors. It is well documented that, in cancer, the disease course usually depends on alterations in multiple cooperating genetic factors. Cancer is also, from a genetic viewpoint a highly heterogeneous disease, so that even if different patients present with identical tumors, the genetic alterations behind their conditions are likely to be entirely different. Taken together, these circumstances make the determination of the molecular genetic changes underlying the development of human endometrial carcinomas difficult.

One way to avoid some of the obstacles is to use an animal model system. Inbred rodent stains can be used under controlled conditions and the influence of many of the complicating factors, such as differences in genetic background and variations in environment and lifestyle, can be minimized. Information about cancer-related genes identified in the rodent model can easily be transferred to the human system via comparative mapping. Rats of the BDII inbred strain are known to be genetically predisposed to endometrial carcinoma 3, 4. In the present investigation, BDII rats were crossed to rats from two nonsusceptible inbred strains, and the occurrence of neoplasms was studied in both F1 and F2 animals. Comparative genome hybridization (CGH) has been used for detecting copy number changes in chromosomes and chromosome regions that occur during cancer development in a variety of tumors 5, 6, 7, 8. One major advantage of CGH is that information about cytogenetic changes can be obtained without performing cytogenetic analysis of the tumor cells themselves.

In order to identify chromosomal regions exhibiting DNA copy number changes, CGH was applied in a series corresponding to 27 different solid tumors from these animals. The main findings from the study were that, although many chromosomes were involved in copy number variation, some of the changes detected were quite specific. These included gains in RNO4q12∼q22, gains in RNO6q14∼q16, and gains of whole chromosome arms in some of the small metacentric chromosomes (e.g., RNO14, 16, 18). RNO10 was involved in gain terminally and loss of material proximally. Clear differences in the pattern of changes could be detected between tumors occurring in the two different crosses, and also between solid tumors and cell cultures. In human endometrial cancer, amplification of the ERBB2 gene and mutations in the codon of the KRAS gene have been reported. We found one case of Erbb2 amplification but no Kras mutations in the rat material studied.

Section snippets

Tumor material

Females of the inbred BDII rat strain are known to be predisposed to endometrial cancer 3, 4 and more than 90 percent of virgin females will contract this malignancy and succumb to it, most of them before 24 months of age. Two crossing experiments were set up, in which BDII/Han rats were crossed to rats from two inbred rat strains in which endometrial cancers are very rare, namely BN/Han and SPRD-Cu3/Han. The latter strain is known to have a high incidence of mammary cancers. In the BDII × BN

Calibrating the CGH analysis

First we set out to determine the normal variability in the ratio of test-to-reference DNA, and, also, in which rat chromosome regions observations of copy number imbalance cannot reliably be made. A control experiment was performed in which normal liver DNAs from a female rat (labeled with digoxigenin and used as the reference DNA) and from a male rat (labeled with biotin and used as the test DNA) were processed in the same CGH protocol as for the tumor DNAs. Metaphase chromosomes from 10

Discussion

In humans, the classification of endometrial cancer is based mainly on three histopathological criteria: myometrial invasion, tumor grading, and extra-uterine tumor spread [16]. There are two major categories of endometrial carcinoma: type I, which is estrogen-dependent, and the nonestrogen-dependent type II [17]. Family history of endometrial cancer in a first-degree relative may increase the risk of the development of endometrial cancer in that individual. In the BDII rat, endometrial cancer

Acknowledgements

This work was supported by the European Commission (contract ERBBio4CT960562) and by grants from the Swedish Cancer Society, the Swedish Medical Research Council, the Wallenberg Foundation, the IngaBritt and Arne Lundberg Research Foundation, the Royal Swedish Academy of Sciences, the Wilhelm and Martina Lundgrens Foundation, the Royal and Hvitfeldtska Foundation, and the Nilsson-Ehle-Foundation. We are grateful to Elisabet Magnusson and Gunilla Pettersson for technical assistance.

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