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[Cancer Research 64, 7039-7044, October 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Pancreatic Insulinomas in Multiple Endocrine Neoplasia, Type I Knockout Mice Can Develop in the Absence of Chromosome Instability or Microsatellite Instability

Peter C. Scacheri1, Alyssa L. Kennedy1, Koei Chin4, Meghan T. Miller1, J. Graeme Hodgson4, Joe W. Gray4, Stephen J. Marx2, Allen M. Spiegel3 and Francis S. Collins1

1 National Human Genome Research Institute, 2 National Institute of Diabetes and Digestive and Kidney Diseases, and 3 National Institute of Deafness and Communication Disorders, National Institutes of Health, Bethesda, Maryland; and 4 Cancer Genetics and Breast Oncology, University of California San Francisco Comprehensive Cancer Center, San Francisco, California


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Multiple endocrine neoplasia, type I (MEN1) is an inherited cancer syndrome characterized by tumors arising primarily in endocrine tissues. The responsible gene acts as a tumor suppressor, and tumors in affected heterozygous individuals occur after inactivation of the wild-type allele. Previous studies have shown that Men1 knockout mice develop multiple pancreatic insulinomas, but this occurs many months after loss of both copies of the Men1 gene. These studies imply that loss of Men1 is not alone sufficient for tumor formation and that additional somatic genetic changes are most likely essential for tumorigenesis. The usual expectation is that such mutations would arise either by a chromosomal instability or microsatellite instability mechanism. In a study of more then a dozen such tumors, using the techniques of array-based comparative genomic hybridization, fluorescent in situ hybridization, loss of heterozygosity analysis using multiple microsatellite markers across the genome, and real time PCR to assess DNA copy number, it appears that many of these full-blown clonal adenomas remain remarkably euploid. Furthermore, the loss of the wild-type Men1 allele in heterozygous Men1 mice occurs by loss and reduplication of the entire mutant-bearing chromosome. Thus, the somatic genetic changes that are postulated to lead to tumorigenesis in a mouse model of MEN1 must be unusually subtle, occurring at either the nucleotide level or through epigenetic mechanisms.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Virtually all cancers are genetically unstable, and this instability has been used recently to classify solid tumors into three largely nonoverlapping groups (1) . Most frequently, instability is observed at the chromosomal level, recognized by deletions or additions of entire chromosomes or portions thereof. Other dramatic changes often seen in tumors that display chromosomal instability (CIN) include translocations and gene amplifications. A second type of instability involves mismatch repair. This is less common and is reflected by alterations in repetitive DNA sequences such as poly(CA) repeats or polyadenylic acid stretches. This type of instability is generally detected by noting instability of a standard panel of microsatellite markers and so is referred to as microsatellite instability (MIN). Microsatellite instability is often observed in tumors from patients with hereditary nonpolyposis colorectal cancer (2) where there are mutations in genes that encode mismatch repair proteins. Instability at the nucleotide level is also observed in xeroderma pigmentosum, Bloom syndrome, ataxia-telangiectasia, and Fanconia anemia. In these instances, defects in either nucleotide-excision or base-excision repair pathways are manifested by base substitutions, deletions, or insertions usually involving a few nucleotides. This is referred to as nucleotide-excision repair instability. The fact that the vast majority of tumors show one of these three modes of instability, but usually not more than one, suggests that genetic instability may be essential for a neoplasia to develop, although this hypothesis has been the subject of continued debate.

Multiple endocrine neoplasia, type I (MEN1), is an inherited cancer syndrome characterized by multiple tumors, most striking for hormone secretion from the parathyroid gland, pancreatic islets, and pituitary gland. The gene responsible for MEN1 is located on chromosome 11q13 (3) and was identified in 1997 by positional cloning (4) . Germ-line mutations in MEN1 have been found in the vast majority of MEN1 kindreds (5, 6, 7) . Tumors in affected individuals occur after loss of the wild-type allele (the second hit), after the classic tumor suppressor model first proposed by Knudson (8) for retinoblastoma. Also consistent with the Knudson model, somatic MEN1 mutations have also been reported in sporadic parathyroid adenomas, pituitary tumors, insulinomas, gastrinomas, and lung carcinoids (9, 10, 11, 12, 13) . The protein product of MEN1, termed menin, is a ubiquitously expressed nuclear protein reported to interact with a multitude of proteins including JunD (14) , Smad3 (15) , Pem (16) , nm23 (17) , nuclear factor {kappa}B (18) , RPA2 (19) , NMMHC II-A (20) , FANCD2 (21) , and several members of a histone methyltransferase complex (22) . The identification of these interacting proteins have provided insights to the function of menin, but it remains unclear how the loss of menin leads to the development of tumors.

Our group and others have developed, "conventional" mouse models for MEN1 through homologous recombination and subsequent deletion of the mouse Men1 homologue (23 , 24) . Men1 homozygotes show lethality at day 11.5 to day 12.5 of embryonic development. Heterozygous Men1 mice develop a phenotype that closely resembles the human disorder, including multiple tumors involving pancreatic islets, parathyroids, adrenal cortex, pituitary, and occasionally other organs. Perhaps the most interesting finding is the presence of many pancreatic lesions. Over the course of 1 to 2 years, the pancreatic islet cells of Men1 knockout mice show a morphologic, neoplastic progression from normal islets to large islets, to large islets with dysplasia, to insulin-producing islet cell tumors. As in human MEN1 kindreds, tumors from Men1 knockout mice show loss of the remaining wild-type Men1 allele, but that loss is actually noted at the phase of dysplasia well before frank adenoma formation is apparent.

More recently, our group and others developed "conditional" mouse models using the cre-lox system (25, 26, 27) . By breeding homozygotes for the floxed Men1 allele to mice expressing cre-recombinase in the pancreatic islets, the homozygous loss of Men1 in the pancreatic ß cell was evaluated. Interestingly, even with early loss of both copies of Men1, there is a long delay in tumor appearance in these mice. The rather indolent course of neoplastic transformation of islets suggests that loss of function of Men1 alone is not sufficient for tumorigenesis. As is the case for virtually all forms of cancer, additional somatic mutations in genes that provide a selective advantage must be essential for tumor formation in MEN1. Identifying these mutations and the mechanism through which they are generated is a major challenge in MEN1 and other forms of cancer.

In humans, sporadic and MEN1-associated pancreatic endocrine tumors are reported to show signs of chromosomal instability (28, 29, 30, 31) . Microsatellite instability is seldom observed (32) . These observations have led to the hypotheses that either loss of MEN1 leads to an inability to maintain DNA integrity, or DNA integrity is compromised as a result of secondary somatic mutations. Mouse models of MEN1 are valuable resources to gain insights to the process of endocrine tumorigenesis and potentially to identify the secondary mutations that drive tumor development. We sought not only to determine whether chromosomal instability or microsatellite instability plays an essential role in the development of pancreatic endocrine tumors in Men1 knockout mice but also to compare, at the genetic level, how closely tumors from Men1-null mice resemble those from humans.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tumor Genomic DNA Isolation.
We isolated insulinomas from 40 to 50-week-old Men1 conditional knockout mice. The conditional mice were homozygous floxed for Men1 and expressing cre from the rat insulin promoter (Men1{Delta}N/{Delta}N; RIP-cre). Pancreatic insulinomas were also isolated from 17 to 21-month-old Men1 heterozygous conventional knockout mice containing a deletion of exons 3 to 8 on one of the two Men1 alleles (Men1{Delta}N3–8/+). The remaining allele was either wild-type (+) or contained a loxP site ({Delta}N). We have shown previously that in the absence of cre, the presence of the loxP site in Men1 does not affect the function of the Men1 allele (25) . All of the tumors ranged in size from 1 to 5 mm in diameter and histologically appeared similar in morphology to those described previously (23, 24, 25, 26, 27) . All of the Men1 knockout mice were maintained on a mixed B6;FVB:129Sv background. Table 1Citation lists the tumor samples used in this study.


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  		Table 1 Summary of the assays performed on each tumor sample*

 
PCR Strategy to Assess Loss of the Wild-Type Men1Allele.
Tumor and corresponding normal tail DNA was subjected to a multiplex PCR using primers that flank the loxP site in intron 8 (8F 5'-GACAGGATTGGGAATTCTCTTTT-3', 8R 5'-AAGGTACAGCAGAGGTCACAGAG-3') in addition to a primer anchored in exon 2 (5'-CCCACATCCAGTCCCTCTTCAGCT-3'). Primers 8F and 8R can distinguish between the floxed Men1 allele and the smaller wild-type allele. When paired with 8R, the primer in exon 2 will only amplify an allele if excision of exons 3 to 8 has occurred.

Array Comparative Genomic Hybridization Analyses.
The array comparative genomic hybridization (CGH) was performed for genome-wide scanning of DNA copy number abnormalities as described (33, 34, 35) . Briefly, we digested 1 µg each of genomic DNA from 10 insulinomas in MEN1 knockout mice as test and 10 tail tissues of the same mice as reference. Four of these were isolated from three conditional Men1-null mice. The remaining 6 tumors were from 5 conventional Men1 heterozygous knockout mice. In all instances tumor DNA was compared with normal tail DNA from the same mouse to account for possible differences due to variations in genetic background. Digested test and reference DNA samples were labeled with Cy3 and Cy5, respectively, in random priming kit (Invitrogen, Carlsbad, CA). Test and reference DNA were combined with 50 µg of mouse Cot-1 DNA, EtOH precipitated, and then resuspended in 60 µg of hybridization buffer. Hybridization solutions after reannealing for 60 minutes were applied to arrays fitted with gaskets (Continental Lab Products, San Diego, CA). The array slides were placed in plastic slide holders with 100 µL of 2x SSC and hybridized on a rocking plate in 37°C incubator for 36 hours. After washing, slides were mounted with 4',6-diamidino-2-phenylindole (1 µmol/L in glycerol) buffer under coverslips and digitally imaged in 16-bit tiff format using a custom-built CCD imaging system in house. Cy3 and Cy5 intensities per spot and normalized log2 ratio of Cy3/Cy5 per bacterial artificial chromosome (BAC) clone were calculated using custom software for array CGH analysis in house (36) . The array consists of degenerate oligonucleotide-primed-PCR representations of 1,056 BAC clones across mouse genome, and their genomic positions are based on the February 2002 freeze through the University of California Santa Cruz genome browser (33) . DNA copy number profiles were plotted along genome so that we could easily identify copy number gains and losses in each tumor.

Quantitative PCR Analyses.
Real-time PCR was used to quantitate the number of copies of the Men1 gene in tumors and corresponding tail DNA. Tumor samples analyzed are listed in Table 1Citation . PCR was performed by using SYBR green PCR kits (Qiagen, Valencia, CA), and real-time PCR was performed on a 7900 real-time PCR machine (Applied Biosystems, Foster City, CA). Genes assayed include Men1, erythroid associated factor (Eraf), and p55, an erythrocyte membrane protein. The primers for the Men1 gene were located downstream of exon 8, in a region that was not deleted in the mutant Men1 allele (Men1{Delta}N3–8). Each gene was amplified separately, in triplicate, in both tumor and corresponding normal tail DNA samples. PCRs performed in triplicate generated highly reproducible results (SD <0.3). {Delta}Ct values were determined by subtracting the average cycle number at a given threshold (Ct) value of the tumor sample from the average Ct value from tail sample for each primer set. To determine whether there was a loss of the Men1 allele, the {Delta}Ct for ERAF and p55 were subtracted from the {Delta}Ct for Men1. This value was then converted to relative number of Men1 alleles compared with ERAF and p55. The experiment was duplicated, and the averages were graphed with the corresponding range of variability between the two experiments indicated with the error bars.

Microsatellite Analysis for Assessment of Loss of Heterozygosity and Microsatellite Instability.
Short tandem repeat markers on mouse chromosomes 1 to 19 were chosen for PCR-loss of heterozygosity (LOH) analysis (37) . Samples tested are listed in Table 1Citation . PCR was used to amplify genomic DNA from matched pairs of tail and tumor DNA using primers that were fluorescently tagged with either FAM or HEX (Applied Biosystems). All of the PCR products were run on a PE3100 Genetic Analyzer, and allele sizes were calculated using Genescan software (Applied Biosystems). LOH and microsatellite instability were assessed visually by comparing allele peaks between the tumor and normal DNA samples. LOH was confirmed if the reduction of >90% of an allele peak was observed in the tumor DNA for one of the constitutive alleles. Microsatellite instability was confirmed if the allele sizes between the tumor and tail DNA were different or if multiple alleles were observed in the tumor but not the tail DNA sample.

Fluorescent In situ Hybridization.
Three insulinomas from 2 Men1 heterozygous knockout mice (Men1{Delta}N3–8/+) were excised from surrounding pancreas. Fluorescent in situ hybridization (FISH) was performed on interphase cells using fluorescently labeled (Spectrum Orange, Vysis, Downers Grove, IL) BAC DNA containing the entire Men1 gene (BAC clone 7 D 23).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tumors from Men1 Knockout Mice Show Loss of the Wild-Type Allele.
A total of 17 pancreatic insulinomas, 1 prolactinoma, and 1 adrenal tumor excised from Men1 knockout mice were analyzed for loss of the wild-type Men1 allele (Table 1Citation and Fig. 1Citation ). Controls included tail DNA from wild-type mice. Samples were subjected to LOH analyses using a PCR assay capable of distinguishing the floxed and wild-type Men1 alleles from the mutant Men1 {Delta}N3–8 allele. PCR products corresponding to the Men1 wild-type and mutant alleles were coamplified in normal tail DNA samples. In the tumor samples, in comparison to the mutant Men1 allele ({Delta}N3–8), we detected a dramatic reduction in the intensity of the band corresponding to the wild-type or floxed Men1 allele. The relatively minor amount of wild-type or floxed PCR product remaining in some tumor samples is most likely due to stromal cell contamination. These data are consistent with previous findings that tumors from Men1 heterozygotes lose the remaining wild-type Men1 allele (23 , 24) .



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  		Fig. 1. PCR to detect loss of the wild-type Men1 allele. A—K, individual mice (see Table 1Citation ). Top band arises from the mutant Men1 allele ({Delta}N3–8). Bottom band is either from the wild-type (+) or floxed Men1 allele ({Delta}N). In comparison to normal DNA samples (N) that show relatively similar amounts of the wild-type and mutant alleles, tumors show absence or dramatic reduction of either the wild-type or floxed allele. P, pancreatic insulinoma DNA; n, normal tail DNA; A, adrenal tumor DNA; Pt, pituitary tumor DNA.

 
CGH Analyses Fail to Detect Copy Number Changes in Men1-Null Insulinomas.
We previously performed CGH analyses on 13 pancreatic insulinomas from conventional and conditional Men1 knockout mice (ref. 25 ; data not shown). No evidence of chromosomal loss or duplication was detected in 1 of 5 insulinomas from conditional knockout animals and 6 of 8 insulinomas from conventional knockout mice. The apparent lack of aneuploidy in the majority of insulinomas from conventional Men1-null mice suggested that these tumors may contain small chromosomal aberrations that are below the resolution of CGH (~20 Mb). Array-CGH provides a sensitivity of detection and a mapping resolution that is potentially several orders of magnitude greater than traditional CGH, thereby allowing for the detection of much shorter segments of DNA gain or loss. We used array-CGH to analyze 6 pancreatic insulinomas from Men1 conventional mice and 4 insulinomas from conditional knockout mice. Of the 4 insulinomas from conditionally null mice, one showed a gain of entire chromosome 11 (Fig. 2A)Citation , and one showed losses of chromosomes 4 and 9 (Fig. 2B)Citation . Interestingly, the decrease in CGH ratio for chromosome 4 was less that that for chromosome 9. We speculate that most of the cells in this tumor had lost chromosome 9, whereas only a fraction had lost chromosome 4. No whole chromosomal gains or losses were detected in the 2 remaining tumors from the conditional knockout mice or in any of the 6 tumors from the conventional knockout mice (data not shown). Some differences in CGH ratios were detected for single BAC elements (Fig. 2Citation ; data not shown), but elements that lay nearby showed CGH ratios that were well within the normal range. We attribute these variants to experimental noise and, therefore, discount them when interpreting CGH profiles. To date, we have analyzed a combined total of 22 insulinomas from Men1-knockout animals by either CGH or array-CGH. Eleven of 13 insulinomas from conventional knockout mice and 3 of 9 tumors from conditionally null mice showed no changes in DNA copy number by either CGH or array CGH. The lack of detectable aneuploidy suggests that chromosomal instability may not be the predominant mode of genetic instability in these insulinomas.



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  		Fig. 2. Array-CGH analyses of genome copy number in Men1-null tumors. The CGH ratio for each BAC array element is plotted as a function of its genome location. Arrows, gain of chromosome 11 in sample I-P1 (A), and losses of chromsomes 4 and 9 in sample I-P2 (B). C, results from sample F-P1 and is representative of other tumor that showed a normal copy number across all chromosomes.

 
Quantitative PCR Analyses Reveal Two Copies of the Men1 Gene in Insulinomas.
In many forms of inherited cancer, elimination of the remaining wild-type allele occurs by complete or partial chromosome deletion. Considering that Men1-null tumors from mice and humans invariably show loss of the wild-type Men1 allele, we were puzzled that our CGH results showed neither loss of chromosome 19 nor deletion of the region containing the Men1 locus. BAC RP23–391B20, which maps ~200 kb distal to the Men1 gene, was not deleted in any of the samples tested. Whereas this data strongly suggested that deletion of the Men1 gene was not occurring in these tumors, we investigated in detail the copy number status of the Men1 gene using quantitative (real-time) PCR. By doing so, we could test whether microdeletions of Men1 were present (undetectable by the arrays used in this study) or whether mechanisms that eliminate the wild-type copy of a tumor suppressor gene (38) without changing the gene copy number were involved. Examples of such mechanisms include mitotic nondisjunction with reduplication of the mutant chromosome or mitotic recombination between the mutant tumor suppressor gene and the centromere. Independent mutation or gene conversion could also cripple the remaining wild-type allele.

We tested this hypothesis using real-time PCR to quantitate the number of Men1 alleles in each tumor sample. Fig. 3Citation shows that pancreatic insulinomas contained approximately the same relative amounts of Men1 DNA as compared with randomly chosen genes Eraf and p55. These genes were chosen because they map to regions of the genome not observed to be altered in copy number by array CGH. These data suggest the existence of two Men1 copies in the insulinomas, possibly arising through mitotic nondisjunction and reduplication of the mutant chromosome. In contrast, adrenal and pituitary tumors showed half as much Men1 DNA relative to Eraf and p55, suggesting the presence of only one Men1 copy in these tumor samples. The data from the pituitary tumor is consistent with previous data showing loss of entire chromosome 19 in pituitary tumors from Men1 knockout mice (25) .



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  		Fig. 3. Quantitative PCR analyses used to measure DNA copy number in Men1-null tumors. Pancreatic insulinomas show approximately the same relative amounts of Men1 as compared with randomly chosen genes, Eraf (light bars) and p55 (dark bars). Tumors arising from the pituitary (H-Pt1) and adrenal (E-A1) glands show approximately one-half the level of Men1 when compared with ERAF and p55. Note that tumors from sample H arose in a female, whereas control DNA was male. This results in the p55 copy number being affected by a factor of two, because p55 is on the X chromosome. Therefore, when Men1 is compared with p55, located on chromosome X, the Men1 copy number appears to be reduced by one-half.

 
Microsatellite Analysis of Chromosomes 1 to 19 for Loss of Heterozygosity Assessment.
If loss of the chromosome carrying the wild-type allele in a tumor is accompanied by a duplication of the remaining chromosome (1 , 38) , one would observe LOH at all of the loci on the chromosome but no loss of copy number. Such events would be undetectable by CGH analyses. We reasoned that the detection of LOH on a chromosome that was shown to have a normal copy number by CGH analysis would be considered strong evidence for a duplication event. To test for this, we performed LOH analyses on microsatellites located on chromosomes 1 to 19 in matched pairs of tumor and normal tissues. Microsatellites on chromosomes 1 to 19 were analyzed in the same 10 tumor samples from Men1 heterozygotes that were analyzed by array CGH. No evidence for LOH was detected for chromosomes 1 to 18 in any of the tumors tested, although in some cases uninformative markers prevented assessment of certain chromosomes (Table 1)Citation . In tumor samples from conventional knockout mice, we detected evidence for LOH on chromosome 19, which harbors the Men1 gene. When combined with the LOH, CGH, and quantitative PCR results described above, these data imply that chromosome 19, or a portion thereof, is duplicated in Men1-null insulinomas.

The Men1 gene is located ~4.5 Mb from the centromere of acrocentric chromosome 19. To map the extent of the duplication, we analyzed 10 microsatellites that were distributed at a 6 cM resolution on chromosome 19 in 5 insulinomas. PCR was successfully performed for all 10 of the markers with 6 yielding informative allelotypes in the normal DNA (Table 2)Citation . PCR of 5 tumors revealed loss of one of the two peaks detected in the corresponding tail DNA, and this pattern was seen for all 6 of the informative markers. We then tested for LOH in 8 additional insulinomas using short tandem repeat markers located at the distal portion of chromosome 19, ~40 cM distal to the Men1 gene. LOH was detected in all but one tumor, which was uninformative. These data suggest a duplication event involving the entire mutant chromosome 19, but we cannot rule out a mitotic recombination event between the centromere and the Men1.


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  		Table 2 Microsatellite analysis for detection of LOH on chromosome 19*

 
Men1-Null Tumors Do Not Display Microsatellite Instability.
Tumors that display a microsatellite instability phenotype as a result of defective mismatch repair typically show widespread alterations in repetitive sequences including microsatellites. Therefore, in addition to detecting LOH, microsatellite analysis can be simultaneously used to detect a microsatellite instability phenotype. We analyzed >60 microsatellites in 6 tumors from conventional knockout mice (Table 2Citation ; data not shown). In all of the instances, allele sizes between matched tumor and tail samples were identical, without any evidence for "extra" alleles in the tumor samples. These results are inconsistent with tumors that show microsatellite instability as a result of functional deficiencies in DNA mismatch repair (2 , 39) and, therefore, argue against a microsatellite instability phenotype in Men1-null tumors.

Fluorescent In situ Hybridization Analyses of Insulinomas Reveal Two Men1 Alleles.
Men1-null insulinomas fail to grow in culture, and therefore metaphase spreads were not available for chromosome analyses. Instead, we performed FISH analysis of interphase tumor cells using a fluorescently labeled BAC clone containing the entire Men1 gene. FISH was performed on 3 tumors from conventional Men1 heterozygous mice that were validated by PCR to have lost the wild-type Men1 allele (data not shown). All 3 of the insulinomas revealed two normal positive signals (Fig. 4)Citation , suggesting that the Men1 gene was present in two copies. These data, combined with the CGH, quantitative PCR, and LOH results described above, additionally support the occurrence of a duplication event involving the Men1 locus.



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  		Fig. 4. FISH analysis of an insulinoma reveals two alleles at the Men1 locus. Two other insulinomas from Men1 heterozygotes showed a similar result (data not shown).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The Men1 knockout mice described here develop a tumor spectrum that closely resembles that found in the corresponding human condition. Previous research on these Men1 knockout mice have shown that, as in humans with MEN1, loss of both copies of the Men1 tumor suppressor gene is not sufficient for adenoma formation, but additional somatic mutations are required. The usual expectation is that such additional mutations occur at the chromosomal level (chromosomal instability), recognized by deletions or additions of entire chromosomes or portions thereof. In fact, it has been proposed that genomic instability is the driving force for development of a variety of tumor types (1) , including pancreatic endocrine neoplasms in human MEN1 (40 , 41) . Although a number of studies have reported widespread chromosomal abnormalities in sporadic and MEN1-associated pancreatic endocrine tumors (28, 29, 30, 31, 32) , it is not clear whether menin is involved in maintaining DNA integrity. To gain insights to this issue, and to determine how closely Men1-null insulinomas from mice resemble those in humans, we used a number of approaches to identify abnormalities in DNA copy number in full-blown clonal adenomas. Remarkably and unexpectedly, our data indicate that insulinomas in these Men1 knockout mice can remain euploid. Men1-null tumors also had stable microsatellites. These data suggest that chromosomal instability and microsatellite instability, although reported in some human MEN1 insulinomas, is apparently not the only pathway to insulinoma formation.

So why do tumors in Men1 knockout mice take so long to develop after loss of Men1? And what mode of genetic instability, if any, is driving the process? We hypothesize that either tumors arise after loss of menin due to accumulation of base substitutions and/or small deletions or insertions that have escaped our detection, or epigenetic mechanisms lead to gene silencing or activation. Whether loss of menin directly contributes to these additional somatic steps or whether background rate of mutation or epigenetic change is simply unmasked by its creation of a neoplastic phenotype in a menin null cell cannot be determined at present. However, there are possibly reasons to propose that menin loss might be a direct contributor. Menin was reported recently to be associated with FANCD2 (21) , a protein involved in DNA repair and mutated in an inherited cancer syndrome, Fanconia anemia. Interestingly, menin-null fibroblasts were demonstrated to have an increased sensitivity to {gamma}-irradiation, suggesting that menin and FANCD2 function together to repair damaged DNA. These findings invite speculation that loss of menin compromises the DNA damage response machinery, leading to the accumulation of subtle nucleotide aberrations that contribute to tumorigenesis. Large-scale sequencing of Men1-null insulinomas could help test this hypothesis. The second "epigenetic" hypothesis is supported by a recent study in which menin was reported to associate with several members of a methlytransferase complex (22) . It was proposed that this complex functions to activate transcription through histone methylation. The loss of menin may deregulate the transcription of target genes, and over time this could lead to tumor development. Expression arrays might be helpful to additionally explore this situation, but technical difficulties related to the isolation of wild-type islet ß cells make this approach very challenging.

Our findings that tumors can be chromosomal instability(–) and microsatellite instability(–) are strikingly reminiscent of a previous study of tumors from a mouse model of inherited intestinal cancer (42) . Whereas human colorectal tumors nearly always display either chromosomal instability or microsatellite instability phenotypes, intestinal adenomas from mice heterozygous for the adenomatous polyposis coli (Apc) tumor suppressor gene apparently can develop with a stable karyotype and stable microsatellites. Similar to the Men1-null insulinomas containing two mutant copies of Men1, adenomas from heterozygous microsatellite instability+ mice were shown to contain two mutant copies of the Apc locus on chromosome 18, presumably due to homologous somatic recombination. In conclusion, whereas additional somatic changes are clearly needed to convert menin-null endocrine cells into tumors, that process appears in general not to involve either chromosomal or microsatellite instability. Other methods for identifying these somatic genetic or epigenetic changes will be required to identify these collaborators in the process of neoplastic transformation of pancreatic islets.


   ACKNOWLEDGMENTS
 
We thank Amalia Dutra for FISH analyses and Ken Myambo for technical assistance with array-CGH.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Francis S. Collins, National Human Genome Research Institute, NIH, Building 31, Room 4B09, 31 Center Drive, Bethesda, MD 20892-2152. Phone: 301-496-0844; Fax: 301-402-0837; E-mail: fc23a{at}nih.gov

Received 5/12/04. Revised 7/ 2/04. Accepted 7/22/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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