Original article
Rearrangement and allelic imbalance on chromosome 5 leads to homozygous deletions in the CDKN2A/2B tumor suppressor gene region in rat endometrial cancer

https://doi.org/10.1016/j.cancergencyto.2008.02.016Get rights and content

Abstract

The inbred BDII rat is a valuable experimental model for the genetic analysis of hormone-dependent endometrial adenocarcinoma (EAC). One common aberration detected previously by comparative genomic hybridization in rat EAC is loss affecting mostly the middle part of rat chromosome 5 (RNO5). First, we applied an RNO5-specific painting probe and four region-specific gene probes onto tumor cell metaphases from 21 EACs, and found that rearrangements involving RNO5 were common. The copy numbers of loci situated on RNO5 were found to be reduced, particularly for the CDKN2A/2B locus. Second, polymerase chain reaction analysis was performed with 22 genes and markers and homozygous deletions of the CDKN2A exon 1β and CDKN2B genes were detected in 13 EACs (62%) and of CDKN2A exon 1α in 12 EACs (57%) Third, the occurrence of allelic imbalance in RNO5 was analyzed using 39 microsatellite markers covering the entire chromosome and frequent loss of heterozygosity was detected. Even more intriguing was the repeated finding of allele switching in a narrow region of 7 Mb across the CDKN2A/2B locus. We conclude that genetic events affecting the middle part of RNO5 (including bands 5q31∼q33 and the CDKN2A locus) contribute to the development of EAC in rat, with the CDKN2A locus having a primary role.

Introduction

Endometrial adenocarcinoma (EAC) is the fourth most common type of malignant tumor among women [1]. As with most cancers, EAC is a complex disease, and its development is influenced by multiple genetic and environmental factors [2], but not many genetic changes typical for EAC have been identified and characterized—in part because genetic changes important for cancer development can be very difficult to define against the heterogeneous genetic background inherent in the human population. This difficulty may be reduced if an animal model is employed. Using inbred rodent strains, the background genetic heterogeneity can be minimized, and, furthermore, variations in the influence of environmental factors can be reasonably controlled.

Females from the BDII inbred rat strain are predisposed to endometrial carcinoma, and >90% of virgin females will develop EAC before the age of 24 months [3], [4]. Thus, the BDII inbred strain can be used as a model for studies of human EAC. In the present investigation, BDII females were crossed to males from two other rat strains that rarely develop EAC, to produce intercross (F1, F2) and backcross (N1) offspring. EAC tumors developed spontaneously in a subset of all three types of offspring. From cytogenetic and comparative genome hybridization (CGH) analysis of the tumors, we found that certain chromosomes or chromosomal regions were selectively involved in copy number changes [5], [6]. One of those regions involved rat chromosome 5 (RNO5). Here we report application of molecular cytogenetic techniques to characterizing the genetic events that have taken place during tumor development. RNO5 was often affected by complex rearrangements in EAC tumors, frequently leading to homozygous deletion of sequences in or near the CDKN2A/2B loci.

A variety of human tumors manifest a homozygous deletion in this region (9p21 in humans) encompassing the CDKN2A and CDKN2B tumor suppressor genes [7], [8]. The p16/p14ARF protein (encoded by the CDKN2A gene) and the p15 protein (encoded by CDKN2B) play important roles in human tumorigenesis. Little is known, however, about the potential role of these genes in reproductive tract biology or, specifically, in uterine tumors.

Section snippets

Tumor material

Breeding pairs were set up in which inbred BDII/Han rats, prone to develop EAC, were crossed to animals from two other inbred rat strains, BN/Han and SPRD-Cu3/Han, both of which exhibit very low incidence of EAC. Animals from the F1 progeny were intercrossed to produce F2 offspring or were backcrossed to BDII females to generate N1 offspring. All animals were examined weekly for uterine tumors. In case of a suspected tumor, the animal was humanely killed and subjected to necropsy. Tumor

Results

Our previous CGH analysis had indicated that a subset of EAC tumors exhibit aberrations (copy number changes) in RNO5 [4], [5]. In fact, aberrant CGH patterns in RNO5 were seen in 15 of 56 tumors (27%) in animals from F1, F2, and backcrosses involving the BDII strain [5]. Some examples of aberrant RNO5 patterns are shown in Figure 1. In most cases, there was loss in the middle region of the chromosome, with losses centering around bands 5q31∼q33. In a few cases, loss in the middle part was

Discussion

The present study was prompted by the finding from our previous CGH analysis that copy number changes affecting RNO5 were common in rat EAC tumors. Our cytogenetic analysis confirmed that rearrangements involving RNO5 were common in these tumors, but that the majority of RNO5 paint-positive chromosomes looked normal and seemed to be unaffected by rearrangement as far as could be determined by traditional cytogenetic methods. Subsequent analysis involving locus-specific probes generally

Acknowledgments

The RNO5 paint probe was generously provided by Dr. Malcolm Ferguson-Smith and Dr. Fengtang Yang (University of Cambridge, Clinical Veterinary Medicine, Cambridge Resource Centre for Comparative Genomics, Cambridge, UK). We are grateful to Elisabeth Jansson and Brita Bjönness for excellent technical assistance. This work was supported by the Swedish Cancer Society, Swedish Natural Science Research Council, SWEGENE, Erik Philip-Sörensen Foundation, Nilsson-Ehle Foundation, and Inga Britt and

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