Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma.

The MTS1 gene on chromosome 9p21 encodes the p16 inhibitor of cyclinD/Cdk-4 complexes, and is deleted or mutated in a variety of tumour types. We found allelic deletions of 9p21-p22 in 85% of pancreatic adenocarcinomas. Analysis of MTS1 in pancreatic carcinomas (27 xenografts and 10 cell lines) showed homozygous deletions in 15 (41%) and sequence changes in 14 (38%). These included eight point mutations (four nonsense, two missense and two splice site mutations) and six deletions/insertions, all accompanied by loss of the wild-type allele. Sequencing of MTS1 from primary tumours confirmed the mutations. Coexistent inactivations of both MTS1 and p53 was common and suggests that abnormal regulation of cyclin-dependent kinases may play an important role in the biology of pancreatic carcinoma.

DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1.

About 90 percent of human pancreatic carcinomas show allelic loss at chromosome 18q. To identify candidate tumor suppressor genes on 18q, a panel of pancreatic carcinomas were analyzed for convergent sites of homozygous deletion. Twenty-five of 84 tumors had homozygous deletions at 18q21.1, a site that excludes DCC (a candidate suppressor gene for colorectal cancer) and includes DPC4, a gene similar in sequence to a Drosophila melanogaster gene (Mad) implicated in a transforming growth factor-beta (TGF-beta)-like signaling pathway. Potentially inactivating mutations in DPC4 were identified in six of 27 pancreatic carcinomas that did not have homozygous deletions at 18q21.1. These results identify DPC4 as a candidate tumor suppressor gene whose inactivation may play a role in pancreatic and possibly other human cancers.

Identification of c-MYC as a target of the APC pathway.

The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c-MYC oncogene is identified as a target gene in this signaling pathway. Expression of c-MYC was shown to be repressed by wild-type APC and activated by beta-catenin, and these effects were mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.

An integrated high-resolution physical map of the DPC/BRCA2 region at chromosome 13q12.

We identified a homozygous deletion in a pancreatic carcinoma (DPC) that localized to a 1-cM region at chromosome 13q12.3, which lay within the 6-cM locus of familial breast cancer susceptibility (BRCA-2). Here we present a physical map of the region, consisting of YAC, PAC, and cosmid contigs. The YAC contig comprises 16 clones that together span the entire BRCA2 region. The PAC contig comprises 22 clones that together span the DPC region. Seventy cosmid clones were localized within and near the DPC region. Thirty-five sequence-tagged sites were defined and localized within the map. The map indicates the size of the DPC region to be near 250 kb, and provides mapped and cloned resources for the search for the putative tumor suppressor gene(s) in the region.

Allelotype of pancreatic adenocarcinoma using xenograft enrichment.

p53 and MTS1 are known to be mutationally inactivated in pancreatic adenocarcinoma. Other tumor suppressor genes are likely also to play a role. To define chromosomal arms which may harbor additional tumor suppressor genes, we performed an extensive allelotype on pancreatic cancer utilizing a xenograft enrichment technique. Eighty-eight percent (28/32) of primary tumors gave rise to xenografts. Eighteen cases were used in a PCR-based allelotype using 283 polymorphic markers, over 2800 informative assays, and an average coverage of 4.1 informative markers per chromosomal arm per case. Highly frequent allelic loss (> 60%) was seen at chromosomes 1p, 9p, 17p, and 18q. Moderately frequent allelic loss (40-60%) was seen at 3p, 6p, 6q, 8p, 10q, 12q, 13q, 18p, 21q, and 22q. The average fractional allelic loss was 0.36. Allelic and sequence stability was demonstrated among 64 parallel and second-passage xenografts derived from 12 cases of pancreatic adenocarcinoma with the ascertainment of over 3000 single alleles. The findings were confirmed in primary tumors. In only two instances were discrepancies revealed between the allelic loss data obtained from corresponding parallel xenografts, probably due to the xenografting of minor subpopulations, reflecting genetic heterogeneity of the primary tumor.

Homozygous deletion map at 18q21.1 in pancreatic cancer.

Absolute genetic differences between neoplastic and nonneoplastic cells can be discerned at sites of homozygous deletions. These deletions are of critical interest because they might be useful in the identification of defective biochemical pathways in neoplastic cells, and subsequently for the development of new treatment strategies in human cancer. We identified an area at 18q21.1 involved by homozygous deletions in 30% of pancreatic carcinomas. To characterize the homozygous deletions, we constructed a detailed physical map of nearly 2 Mb, containing yeast artificial chromosomes, P1-derived artificial chromosomes, cosmids and 24 sequence-tagged sites. The homozygously deleted are contained a new candidate tumor-suppressor gene (DPC4). To date, 23 (64%) of 35 pancreatic carcinomas carry at least one homozygous deletion at a published locus. The study of the total gene content of these loci, facilitated by the sequence-tagged site markers and maps of these regions, should help to reveal the absolute biochemical differences between neoplastic and nonneoplastic cells for a common human tumor.

CDX2 is mutated in a colorectal cancer with normal APC/beta-catenin signaling.

The majority of human colorectal cancers have elevated beta-catenin/TCF regulated transcription due to either inactivating mutations of the APC tumor suppressor gene or activating mutations of beta-catenin. Surprisingly, one commonly used colorectal cancer cell line was found to have intact APC and beta-catenin and no demonstrable beta-catenin/TCF regulated transcription. However, this line did possess a truncating mutation in one allele of CDX2, a gene whose inactivation has recently been shown to cause colon tumorigenesis in mice. Expression of CDX2 was found to be induced by restoring expression of wild type APC in a colorectal cancer cell line. These findings raise the intriguing possibility that CDX2 contributes to APC's tumor suppressive effects.

Homeosis and polyposis: a tale from the mouse.

Homeobox genes play essential roles in specifying the fates of different cell types during embryogenesis. In Drosophila, the homeotic gene caudal is important for the generation of posterior structures. In the mouse, the caudal homologue Cdx2 has been implicated in directing early processes in intestinal morphogenesis and in the maintenance of the differentiated phenotype. A recent study showed that Cdx2 null mutation was embryonically lethal, whereas Cdx2+/- mice developed multiple intestinal polyps in the proximal colon in addition to developmental defects. There are striking phenotypic similarities and differences between Cdx2+/- and other mice predisposed to intestinal neoplasia. The possible role of Cdx2 in human colorectal tumorigenesis is discussed.

A simplified system for generating recombinant adenoviruses.

Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We report herein a strategy that simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system should expedite the process of generating and testing recombinant adenoviruses for a variety of purposes.

Converting cancer genes into killer genes.

Over the past decade, it has become clear that tumorigenesis is driven by alterations in genes that control cell growth or cell death. Theoretically, the proteins encoded by these genes provide excellent targets for new therapeutic agents. Here, we describe a gene therapy approach to specifically kill tumor cells expressing such oncoproteins. In outline, the target oncoprotein binds to exogenously introduced gene products, resulting in transcriptional activation of a toxic gene. As an example, we show that this approach can be used to specifically kill cells overexpressing a mutant p53 gene in cell culture. The strategy may be generally applicable to neoplastic diseases in which the underlying patterns of genetic alterations or abnormal gene expression are known.

Characterization of MAD2B and other mitotic spindle checkpoint genes.

Aneuploidy is a characteristic of the majority of human cancers, and recent work has suggested that mitotic checkpoint defects play a role in its development. To further explore this issue, we isolated a novel human gene, MAD2B (MAD2L2), which is homologous to the spindle checkpoint gene MAD2 (MAD2L1). We determined the chromosomal localization of it and other spindle checkpoint genes, including MAD1L1, MAD2, BUB3, TTK (MPS1L1), and CDC20. In addition, we resolved the genomic intron-exon structure of the human BUB1 gene. We then searched for mutations in these genes in a panel of 19 aneuploid colorectal tumors. No new mutations were identified, suggesting that genes yet to be discovered are responsible for most of the checkpoint defects observed in aneuploid cancers.

Identification by representational difference analysis of a homozygous deletion in pancreatic carcinoma that lies within the BRCA2 region.

Homozygous deletions have been central to the discovery of several tumor-suppressor genes, but their finding has often been either serendipitous or the result of a directed search. A recently described technique [Lisitsyn, N., Lisitsyn, N. & Wigler, M. (1993) Science 259, 946-951] held out the potential to efficiently discover such events in an unbiased manner. Here we present the application of the representational difference analysis (RDA) to the study of cancer. We cloned two DNA fragments that identified a homozygous deletion in a human pancreatic adenocarcinoma, mapping to a 1-centimorgan region at chromosome 13q12.3 flanked by the markers D13S171 and D13S260. Interestingly, this lies within the 6-centimorgan region recently identified as the BRCA2 locus of heritable breast cancer susceptibility. This suggests that the same gene may be involved in multiple tumor types and that its function is that of a tumor suppressor rather than that of a dominant oncogene.

Isolation of YAC insert sequences by representational difference analysis.

We present a method for the isolation of YAC insert sequences by representational difference analysis (RDA). To achieve maximal representation of the sequences, the amplicons were generated from a Mbol digestion product. RDA was performed using a 970 kb insert YAC clone. After two rounds of re-association and selective amplification 92% of the difference product represented sequences derived from the YAC insert. Twenty insert-specific sequence-tagged sites were readily defined. The difference product was also successfully used to isolate microsatellite markers, to identify clones from a human PAC library and as a chromosome painting probe in fluorescence in situ hybridization.