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

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 1beta and CDKN2B genes were detected in 13 EACs (62%) and of CDKN2A exon 1alpha 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 approximately q33 and the CDKN2A locus) contribute to the development of EAC in rat, with the CDKN2A locus having a primary role.

Amplification studies of MET and Cdk6 in a rat endometrial tumor model and their correlation to human type I endometrial carcinoma tumors.

Cancer is known to be a genetic disease that is both polygenic and heterogeneous, in most cases involving changes in several genes in a stepwise fashion. The spectrum of individual genes involved in the initiation and progression of cancer is greatly influenced by genetic factors unique to each patient. A study of complex diseases such as cancer is complicated by the genetic heterogeneous background and environmental factors in the human population. Endometrial cancer (EC) is ranked fourth among invasive tumors in women. In Sweden, approximately 1300 women (27/100,000 women) are diagnosed annually. To be able to study the genetic alterations in cancer, the use of an animal model is very convenient. Females of the BDII strain are genetically predisposed to EC and 90% of female BDII rats develop EC during their lifetime. Thus, BDII rats have been used to model human EC with respect to the genetics of susceptibility and of tumor development. A set of rat EC tumors was analyzed using conventional cytogenetics and comparative genome hybridization (CGH). Chromosomal aberrations, i.e., gains, were found on rat chromosome 4 (RNO4). Using FISH analysis, we concluded that the Met oncogene and Cdk6 (cyclin-dependent kinase 6) were amplified in this set of EC tumors. The data from this investigation were used to analyze a set of human endometrial tumors for amplification of Cdk6 and Met. Our preliminary data are indicative for a good correlation between our findings in the BDII rat model for EAC and the situation in human EC. These data provide strong support for the use of animal model systems for better understanding and scrutinizing of human complex disease of cancer.

Recurrent gene amplifications in human type I endometrial adenocarcinoma detected by fluorescence in situ hybridization.

Determining what genes are actively involved in tumor development is important, because they may provide targets for directed therapy. Human tumors are greatly heterogeneous with respect to etiology and genetic background, which complicates the identification of common genetic aberrations. In contrast, genetic and environmental variation can be in part controlled in experimental animals, which facilitates identification of the important changes. In inbred BDII rats, which are genetically predisposed to endometrial adenocarcinomas (EAC), certain chromosome regions exhibit recurrent amplification in the tumors. Previous CGH analysis had shown that a subset of human EAC tumors exhibited increased copy numbers in the homologous chromosomal regions, located in human 2p21 approximately p25 and 7q21 approximately q31. Using fluorescence in situ hybridization analysis on imprints from 13 human EAC tumors, we determined the average copy numbers of each of 15 probes derived from cancer-related genes situated in these chromosome regions. Among the genes analyzed, those most often targeted by amplification were SDC1 and CYP1B1 in 2p21 approximately p25 and CDK6 and MET in 7q21 approximately q31, but all of the 15 genes tested were found to be amplified in at least two tumors.

Nonrandom pattern of chromosome aberrations in 17beta-estradiol-induced rat mammary tumors: indications of distinct pathways for tumor development.

Estrogens play an important role in breast cancer etiology and the ACI rat provides a novel animal model for defining the mechanisms through which estrogens contribute to mammary cancer development. In crossing experiments between the susceptible ACI strain and two resistant strains, COP (Copenhagen) and BN (Brown Norway), several quantitative trait loci (QTL) that affect development of 17beta-estradiol (E2)-induced mammary tumors have been defined. Using comparative genomic hybridization (CGH), we have analyzed cytogenetic aberrations in E2-induced mammary cancers and have found clear patterns of nonrandom chromosomal involvement. Approximately two thirds of the tumors exhibited copy number changes. Losses of rat chromosome 5 (RNO5) and RNO20 were particularly common, and it was found that these two aberrations often occurred together. A third recurrent aberration involving proximal gain and distal loss in RNO6 probably defined a distinct subgroup of tumors, since it never occurred in combination with RNO5 loss. Interestingly, QTL with powerful effects on mammary cancer development have been mapped to RNO5 and RNO6. These findings suggest that there were at least two genetic pathways to tumor formation in this rat model of E2-induced mammary cancer. By performing CGH on mammary tumors from ACI rats, F1 rats from crosses between the ACI and COP or BN strains and ACI.BN-Emca8 congenic rats, which carry the BN allele of the Emca8 QTL on RNO5 on the ACI genetic background, we were able to determine that the constitution of the germ line influences the pattern of chromosomal aberrations.

Male and female germline specific expression of an EGFP reporter gene in a unique strain of transgenic rats.

A rat line was generated in which genomic integration of a ROSA-EGFP transgene resulted in exclusive expression of EGFP in the germ cells of both sexes. EGFP expression was uniform and robust in cleavage stage embryos beginning at the late 2-cell stage and continuing through blastocyst development where expression became restricted to cells of the inner cell mass. Subsequent analysis showed high EGFP expression exclusively in primordial, embryonic, and adult germ cells. This unique expression pattern makes this EGFP marked locus the first molecular marker of the germline lineage in both sexes in mammals. FISH was used to localize the transgene insertion to chromosome 11q11-q12, proximal to Grik1 and near Ncam2. Analysis of the region did not identify known germ cell-specific genes but did identify 19 ESTs or transcribed loci present in testes, ovary, or pre-implantation libraries from mice or rats. To assess the utility of the transgenic line for germ cell transplantation studies, non-selected, freshly isolated seminiferous tubule cells were transferred to the testis of recipient males. The donor cell population colonized the testis at a surprisingly high efficiency within 30 days following transfer. Since EGFP is a vital marker, the colonization process can be followed in vivo and the extent of colonization quantified. The unique germ cell specific expression of EGFP makes this line of transgenic rats an excellent novel tool to study germ cell origin, development, and differentiation, and to assess the plasticity of adult somatic stem cells to become male germ cells.

Oncogene amplification in the proximal part of chromosome 6 in rat endometrial adenocarcinoma as revealed by combined BAC/PAC FISH, chromosome painting, zoo-FISH, and allelotyping.

The inbred BDII rat is a valuable experimental model for the genetic analysis of endometrial adenocarcinoma (EAC). One common aberration detected by comparative genomic hybridization in rat EAC was gain/amplification affecting the proximal part of rat chromosome 6 (RNO6). We applied rat and mouse chromosome painting probes onto tumor cell metaphase preparations in order to detect and characterize gross RNO6 aberrations. In addition, the RNO6q11-q16 segment was analyzed by fluorescence in situ hybridization with probes representing 12 cancer-related genes in the region. The analysis revealed that seven tumors contained large RNO6-derived homogeneously staining regions (HSRs) in addition to several normal or near-normal RNO6 chromosomes. Five tumors (two of which also had HSRs) exhibited a selective increase of the RNO6q11-q16 segment, sometimes in conjunction with moderate amplification of one or a few genes. Most commonly, the amplification affected the region centered around band 6q16 and included the Mycn, Ddx1, and Rrm2 genes. A second region, centering around Slc8a1 and Xdh, also was affected by gene amplification but to a lesser extent. The aberrations in the proximal part of RNO6 were further analyzed using allelotyping of microsatellite markers in all tumors from animals that were heterozygous in the proximal RNO6 region. We could detect allelic imbalance (AI) in 12 of 20 informative tumors, 6 of which were in addition to those already analyzed by molecular cytogenetic methods as described. Our findings suggest that increase/amplification of genes in this chromosome region contribute to the development of this hormone-dependent tumor.

Cytogenetic aberrations in spontaneous endometrial adenocarcinomas in the BDII rat model as revealed by chromosome banding and comparative genome hybridization.

Female rats of the inbred strain BDII are genetically predisposed to endometrial estrogen-dependent adenocarcinomas (EAC). More than 90% of them spontaneously develop this tumor type before the age of 24 months. In order to dissect out the genetic components behind these tumors we have made crosses between BDII females and rats from 2 other strains that are nonsusceptible to EAC. It was found that EAC tumors developed in a subset of intercross and backcross animals from both interstrain crosses. The chromosomal changes in the developing tumors were studied using cytogenetic and molecular cytogenetic methods. From these studies, we conclude that certain chromosome regions were recurrently engaged in chromosomal changes such as increases in copy number (e.g., trisomy, amplification) or decreases (e.g., deletion). Based on the analysis of 56 tumors, 8 regions were found to be particularly often involved: RNO4prx, gain=34 (61%) (amplification 12 cases); RNO5mid, loss=15 (27%); RNO6prx, gain=25 (45%) (amplification 8 cases); RNO10 loss, prx-mid/gain dst=25 (45%) (amplification 1 case); RNO12q, gain=23 (41%); RNO15p loss/RNO15q gain=29 (52%) (amplification 1 case) [RNO, rat chromosome; prx, proximal; mid, middle; dst, distal; p, short arm; q, long arm]. We begun to analyze these regions in detail using various molecular methods and within them there are certain possible target genes, such as MET (RNO4q21), CDKN2A/2B (RNO5q32), MYCN (RNO6q15 approximately q16), and TP53 (RNO10q24 approximately q25), but it is clear that several other genes, still unidentified, must also be involved.

Assessment of allele dosage at polymorphic microsatellite loci displaying allelic imbalance in tumors by means of quantitative competitive-polymerase chain reaction.

Analysis of allelic imbalance at polymorphic marker loci is usually employed to identify chromosomal regions affected by recurrent aberrations in tumor genomes. Such regions are likely to harbor genes involved in the onset and/or progression of cancer. Although often used to identify regions of loss of heterozygosity caused by deletions/rearrangements near tumor suppressor gene loci, allelic imbalance can also reflect regional amplification, indicating the presence of oncogenes. It is difficult to tell these two situations apart after ordinary polymerase chain reaction (PCR), but here we describe a method that distinguishes allelic loss from allelic gain. The level of allelic imbalance was determined by quantitative PCR (QPCR) in the presence of an internal control DNA that displayed a third allele at the locus studied. To validate the efficiency of allele quantitation, we analyzed an amplified region in a set of rat fibrosarcomas. In four tumor samples with amplification of the Met oncogene, we could show with QPCR that there was amplification of one of the alleles at a microsatellite marker located close to Met. QPCR may be useful for cancer studies because experiments may be predesigned for using either suitable microsatellite markers or the abundant and polymorphic poly-A tails of rodent identifier sequences.

Allelic imbalance on chromosome 10 in rat endometrial adenocarcinomas.

Earlier work using comparative genome hybridization (CGH) has shown that rat chromosome 10 (RNO10) is frequently involved in cytogenetic aberrations in BDII rat endometrial adenocarcinomas (EAC). Relative reduction in copy number (chromosomal deletions) was seen in the proximal to middle part of the chromosome, whereas there were increases in copy number in the distal part. The occurrence of RNO10 aberrations was further analyzed in DNA from primary tumor material from 42 EACs and 3 benign endometrial tumors using allelotyping of microsatellite markers. We found frequently that there were 4 quite distinct RNO10 regions that exhibited allelic imbalance. Based on these findings we believe that genes with relevance to EAC tumor development are situated in each of these chromosome regions. Extrapolation of our microsatellite marker data to the rat draft DNA sequence will facilitate the definition of the regions at the level of the DNA and to select and characterize candidate genes within each of the affected chromosome regions.

Genomically complex lymphomas undergo sustained tumor regression upon MYC inactivation unless they acquire novel chromosomal translocations.

The targeted inactivation of oncogenes may be a specific and effective treatment for cancer. However, because human cancers are the consequence of multiple genetic changes, the inactivation of one oncogene may not be sufficient to cause sustained tumor regression. Moreover, cancers are genomically unstable and may readily compensate for the inactivation of a single oncogene. Here we confirm by spectral karyotypic analysis that MYC-induced hematopoietic tumors are highly genetically complex and genomically unstable. Nevertheless, the inactivation of MYC alone was found to be sufficient to induce sustained tumor regression. After prolonged MYC inactivation, some tumors exhibited a distinct propensity to relapse. When tumors relapsed, they no longer required the overexpression of MYC but instead acquired novel chromosomal translocations. We conclude that even highly genetically complex cancers are reversible on the inactivation of MYC, unless they acquire novel genetic alterations that can sustain a neoplastic phenotype.

Recurrent allelic imbalance at the rat Pten locus in DMBA-induced fibrosarcomas.

The tumor-suppressor gene PTEN (phosphatase and tensin homolog) is frequently inactivated in different types of human tumors. Less is known about the involvement of the homologous gene Pten in animal model systems of cancer. By sequencing one of the introns of rat Pten, we found an informative intragenic PCR marker suitable for genetic studies. Through use of this marker, the position of Pten in the genetic linkage map was localized to the distal part of rat chromosome 1 (RNO1) by analysis of F2 progeny from an intercross between inbred strains BN and LE. Subsequently, 22 markers from this region (including the intragenic Pten marker) were used to study the occurrence of allelic imbalance in distal RNO1 in fibrosarcomas that had been induced by DMBA in F1(BNxLE) rats. The analysis revealed that allelic imbalance was common in the vicinity of Pten, and there was loss or reduction of one of the Pten alleles in more than 60% of the fibrosarcomas. DNA sequencing was preformed to investigate whether the Pten allele remaining in the tumors was inactivated by mutation. However, no mutations were detected in the genomic sequence of Pten exons 5 to 9 in any of the fibrosarcomas, and normal mRNA transcripts were expressed in all tumors. Thus, based on the targeted selection for loss of Pten observed in some of these tumors and the absence of inactivation of the remaining allele, we suggest that haploinsufficiency of Pten may be an important factor in rat DMBA-induced fibrosarcomas.

Identification, characterization and cytogenetic mapping of a yeast Vps54 homolog in rat and mouse.

A novel gene, VPS54-like (Vps54l), is described in the rat that is homologous to the yeast Vps54 gene which is known to be involved in intracellular protein sorting. Furthermore, Vps54-related sequences of human, mouse, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana could be identified in the EMBL/GenBank/DDBJ database. Each of the deduced amino acid sequences of the Vps54 genes in these species contain a coiled-coil region and eight to 13 dileucine motifs. The rat Vps54l gene could be mapped to the end of chromosome 14 by radiation hybrid analysis 7 cR(3000) from the D14Rat22 marker and to 14q22 by fluorescence in situ hybridization. Using a rat Vps54l-containing P1-derived artificial chromosome (PAC) clone the respective ortholog was mapped to chromosome 11A3 in the mouse. In addition, the rat genome contains a processed pseudogene of Vps54l on chromosome 7q22. PAC clone analysis shows that the rat Vps54l gene maps close to the UDP-glucose-pyrophosphorylase 2 gene. The two genes are in tail to tail orientation with their polyadenylation sites 497 bp apart. Rat Vps54l appears to be expressed ubiquitously, but at a relatively low level. Alternatively spliced transcripts could be isolated which lack the sequence coding for the coiled-coil region.

The functional significance of absence: the chromosomal segment harboring Tp53 is absent from the T55 rat radiation hybrid mapping panel.

The T55 rat radiation hybrid (RH) mapping panel has been reported to retain the entire rat genome at retention frequencies between 22% and 37%. However, we found that a small segment of rat chromosome 10 harboring at least four different genes, including Tp53, was completely absent from the panel (retention frequency = 0%). Two other markers located in the vicinity exhibited much reduced retention (2-6%). RH clones are generated by transferring highly fragmented DNA into a recipient cell. There might be a strong selection against the transfer and retention of chromosome segments harboring an intact Tp53, as the action of this gene might prevent proliferation and establishment of the RH clone. Our finding further suggests that unexpected low retention or absence of chromosome segments in an RH panel may represent indications that the segments harbor genes with important functions in cell proliferation control.