Tumor Suppressor Gene WT1 Research Articles

Different isoforms of the Wilms' tumour protein WT1 have distinct patterns of distribution and trafficking within the nucleus.

The Wilms' tumour suppressor gene WT1 encodes multiple isoforms of a transcription factor essential for correct mammalian urogenital development. Maintenance of the correct isoform ratio is critical. In humans, perturbation of this ratio causes Frasier syndrome, which is characterized by developmental defects of the kidney and urogenital tract. Different WT1 isoforms are thought to regulate transcription and participate in mRNA processing, functions reflected by a complex sub-nuclear distribution. However, the role of individual WT1 isoforms remains unclear and pathways leading to WT1 sub-nuclear localization are completely unknown. Here we use cells expressing green fluorescent protein-tagged WT1 to demonstrate that the two major WT1 isoforms occupy separate and dynamic intranuclear locations in which one isoform, WT1+KTS, preferentially associates with the nucleolus. The alternatively spliced zinc finger region is found to be critical for the initial sub-nuclear separation of WT1 isoforms, but interactions between different isoforms influence the sub-nuclear distribution of WT1. We illustrate how disruption of WT1 nuclear distribution might result in disease. This study contributes to the emerging picture of intranuclear protein trafficking.

The many facets of the Wilms' tumour gene, WT1

Over the years, many apparently contradictory findings and functions have been ascribed to the protein product of the WT1 tumour suppressor gene. These include being a transcriptional activator or repressor, a function in transcription versus RNA metabolism, and these days even a function as oncogene or tumour suppressor gene. To fully understand the role of WT1 in different diseases and normal development, we will need to understand these contradictions. In this review, we will discuss the present state of knowledge and suggest that a role for WT1 in influencing the mesenchymal-epithelial state of cells might be a common function that could explain many of the previously described findings.

The WT1 Wilms' tumor suppressor gene is a downstream target for insulin‐like growth factor‐I (IGF‐I) action in PC12 cells

The biological actions of the insulin-like growth factors, IGF-I and IGF-II, are mediated by the ligand-induced activation of the IGF-I receptor (IGF-IR), a transmembrane heterotetramer linked to the ras-raf-mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3 kinase (PI3K)-protein kinase B (PKB)/Akt signal transduction cascades. The Wilms' tumor suppressor gene (wt1) encodes a zinc finger transcription factor, WT1, which has been implicated in various cellular processes including proliferation, differentiation and apoptosis. In the present study we demonstrated that IGF-I modulates the WT1 gene expression in neurally derived PC12 cells in a dose- and time-dependent manner. This effect was mediated through both the MAPK and PI3-kinase signaling pathways, as shown by the ability of the specific inhibitors UO126 and LY294002 to abrogate IGF-I action. Moreover, using RT-PCR and transient transfection assays, we demonstrated that the IGF-I effect was associated with corresponding changes in WT1 mRNA levels and WT1 promoter activity. In addition, the results of the present study revealed that high WT1 levels were associated with the induction of apoptosis, whereas low WT1 levels were correlated with the inhibition of apoptosis, as demonstrated by poly ADP ribose polymerase (PARP) cleavage, Bax expression, Annexin V-FITC staining, and by the use of antisense oligonucleotides against WT1. In summary, our results show that the wt1 gene is a novel target for IGF-I action in neurally derived cells.

Phenotypic normalization of GN6TF rat liver tumor cells results from WT1 expression following transfection of human SYT13‐containing BACs

We identified Synaptotagmin 13 (SYT13) as a putative liver tumor suppressor gene using a microcell hybrid model system and transfer of human 11p11.2 into GN6TF rat liver tumor cells. In the current study, bacterial artificial chromosomes (BACs) corresponding to various regions of human 11p11.2 (retrofitted with a G418 cassette) were transferred into GN6TF tumor cells to facilitate characterization of SYT13 tumor suppressor function under the control of its natural proximal and long-range cis controlling elements. Selected BAC clones contained SYT13 and different overlapping portions of surrounding genomic DNA (CTD-2255G20 and RP11-10C6). The control BAC (CTD-3154D11) contained proximal genes (LOC219638 and PRDM11), but not SYT13. G418-resistant BAC-transfected clones were identified that exhibited a normalized, flattened, contact-inhibited morphology, suggesting that SYT13 transfection normalizes GN6TF cells. In addition, the WT1 tumor suppressor gene was induced following transfection of SYT13-containing BACs. Transfer of human 11p11.2 into GN6TF cells results in a normalized cell phenotype in vitro and induction of WT1 gene expression. Thus, the results of the current study support our previous supposition that human SYT13 complements a molecular defect in GN6TF liver tumor cells and gives rise to tumor suppression through induction of rat WT1. Support: NIH CA78343.

Modulation of Biological Regulatory Networks During Nephrogenesis

Mesenchymal-to-epithelial transition is an important biological event during the course of renal cell differentiation as condensing mesenchyme gives rise to tubuloepithelial structures. Wilms' tumor suppressor gene (Wt1) has been identified as a master regulator of the complex genetic events that mediate mesenchymal transdifferentiation. Evidence is summarized here showing that the tightly regulated series of genetic and biochemical events during nephrogenesis is disrupted by superactivation of aryl hydrocarbon receptor (Ahr) by benzo(a)pyrene (BaP), a ubiquitous polycyclic aromatic hydrocarbon and renal carcinogen (). Nephron formation is inhibited by BaP, a response that involves inhibition of metanephric cell differentiation and shifts in the relative abundance of Wt1 splice variants. A systems biology paradigm that combined approaches from genomics, transcriptomics, and bioinformatics revealed that the global response of murine metanephric cultures to BaP involves downregulation of Ahr and disruption of downstream targets of Wt1. Discrete networks of genetic regulation were resolved using Boolean idealizations and included genes involved in renal cell differentiation and metabolic control. This work has established a role for Ahr in renal cell differentiation and kidney development and resolved putative molecular interactions between Ahr and Wt1.

Identification and comparative expression analysis of a second wt1 gene in zebrafish

The Wilms' tumor suppressor gene wt1 encodes a zinc-finger transcription factor that plays an important role in the development of the mammalian genitourinary system. Mutations in WT1 in humans lead to anomalies of kidney and gonad development and cause Wilms' tumor, a pediatric kidney cancer. The inactivation of both wt1 alleles in mice gives rise to multiple organ defects, among them agenesis of kidney, spleen, and gonads. In zebrafish, an ortholog of wt1 has been described that is expressed in the pronephric field and is later restricted to the podocytes. Here, we report the existence of a second wt1 gene in zebrafish, which we have named wt1b (we named the initial gene wt1a). The overall sequence identity of the two Wt1 proteins is 70% and 92% between the zinc-finger regions, respectively. In contrast to wt1a, wt1b is expressed from the earliest stages of development onward, albeit at low levels. Both wt1a and wt1b are expressed in the intermediate mesoderm, with wt1b being restricted to a smaller area lying at the caudal end of the wt1a expression domain. In adult fish, high expression levels for both genes can be found in gonads, kidney, heart, spleen, and muscle.

WT1 Induces Apoptosis through Transcriptional Regulation of the Proapoptotic Bcl-2 Family Member Bak

Wilms' tumor or nephroblastoma is believed to arise from embryonic nephrogenic rests of multipotent cells that fail to terminally differentiate into epithelium and continue to proliferate. The WT1 tumor suppressor gene, a transcription factor controlling the mesenchymal-epithelial transition in renal development, is mutated in 10% to 15% of Wilms' tumors. This potentially explains the disordered differentiation and proliferation program of a subset of Wilms' tumors. To elucidate the role of mutations of WT1 in the etiology of Wilms' tumor, we used an inducible cellular system for expressing wild-type and tumor-derived missense mutant WT1 proteins. Expression of wild-type WT1, but not mutant proteins, blocked cellular proliferation and DNA synthesis and rapidly induced apoptosis. We showed that wild-type WT1 induced transcription of one of the seven studied proapoptotic genes, Bak. Furthermore, WT1 protein bound to specific DNA-binding sites located in the Bak promoter and Bak was critical to WT1-mediated apoptosis, as overexpression of VDAC2, a specific Bak inhibitor, attenuated WT1-mediated cell death. These data support the hypothesis that Wilms' tumors arise, in part, because WT1 mutant proteins fail to promote programmed cell death during kidney development.

Differential Expression of WT1 Gene Product in Non-Hodgkin Lymphomas

The tumor suppressor gene wt1 (Wilms tumor 1) encodes a zinc finger transcription factor reported to be expressed in many tumors, including mesotheliomas, carcinomas, and acute leukemias. However, WT1 expression in non-Hodgkin lymphomas (NHLs) has not been studied. The authors assessed for WT1 expression in six lymphoma/leukemia cell lines using Western blot methods after subcellular fractionation. We also assessed for WT1 expression in 167 NHLs using immunohistochemical methods. The B-cell NHLs analyzed were 18 diffuse large B-cell lymphomas, 13 marginal zone B-cell lymphomas, 9 small lymphocytic lymphomas, (DLBCLs), 8 follicular lymphomas, 6 mantle cell lymphomas, 5 Burkitt lymphomas, 3 lymphoplasmacytic lymphomas, and 2 B-cell lymphoblastic lymphomas. The T-cell NHLs analyzed were 43 anaplastic large cell lymphomas (ALCLs), 26 peripheral T-cell lymphomas unspecified, 13 angioimmunoblastic T-cell lymphomas, 6 cutaneous ALCLs, 6 cases of mycosis fungoides, 5 extranodal NK/T-cell lymphomas of nasal type, and 4 T-cell lymphoblastic lymphomas. WT1 levels were higher in cytoplasmic extracts than in nuclear extracts of the Karpas 299 and SU-DHL-1 lymphoma cell lines but were higher in nuclear extracts than in the cytoplasmic extracts of the Jurkat, HH, U-937, and K562 leukemia cell lines. In NHLs, WT1 was positive in 4 of 5 (80%) Burkitt lymphomas, 9 of 12 (75%) ALK-positive ALCLs, 3 of 6 (50%) lymphoblastic lymphomas (2 of 4 T-cell, 1 of 2 B-cell), 14 of 31 (45%) ALK-negative ALCLs, 6 of 18 (33%) DLBCLs, and 1 of 6 (17%) cutaneous ALCLs. WT1 was negative in all other NHLs tested. WT1 immunoreactivity was primarily cytoplasmic in all positive NHLs except T-cell lymphoblastic lymphoma. In conclusion, WT1 protein is frequently detected in the cytoplasm of a subset of high-grade NHLs.

Prevalence of WT1 mutations in a large cohort of patients with steroid-resistant and steroid-sensitive nephrotic syndrome

Nephrotic syndrome (NS) represents the association of proteinuria, hypoalbuminemia, edema, and hyperlipidemia. Steroid-resistant nephrotic syndrome (SRNS) is defined by primary resistance to standard steroid therapy. It remains one of the most intractable causes for end-stage renal disease (ESRD) in the first two decades of life. Sporadic mutations in the Wilms' tumor suppressor gene WT1 have been found to be present in patients with SRNS in association with Wilms' tumor (WT) and urinary or genital malformations, as well as in patients with isolated SRNS. To further evaluate the incidence of WT1 mutations in patients with NS we performed mutational analysis in 115 sporadic cases of SRNS and in 110 sporadic cases of steroid-sensitive nephrotic syndrome (SSNS) as a control group. Sixty out of 115 (52%) patients with sporadic SRNS were male, 55/115 (48%) were female. Sex genotype was verified by haplotype analysis. Mutational analysis was performed by direct sequencing and by denaturing high-performance liquid chromatography (DHPLC). Mutations in WT1 were found in 3/60 (5%) male (sex genotype) cases and 5/55 (9%) female (sex genotype) cases of sporadic SRNS, and 0/110 (0%) sporadic cases of SSNS. One out of five female patients with mutations in WT1 developed a WT, 2/3 male patients presented with the association of urinary and genital malformations, 1/3 male patients presented with sexual reversal (female phenotype) and bilateral gonadoblastoma, and 4/5 female patients presented with isolated SRNS. According to the data acquired in this study, patients presenting with a female phenotype and SRNS and male patients presenting with genital abnormalities should especially be screened to take advantage of the important genetic information on potential Wilms' tumor risk and differential therapy. This will also help to provide more data on the phenotype/genotype correlation in this patient population.

Activation of the WT1 tumor suppressor gene promoter by Pea3

Gene array profiling of RNA from cells engineered to express a dominant-negative version of the ETS family member transcription factor Pea3 (polyomavirus enhancer activator 3) identified WT1 as a candidate downstream gene. Given the co-expression of WT1 and Pea3 in developing kidney and breast tissue undergoing mesenchymal to epithelial transitions, we further characterized this potential gene hierarchy. Analysis of the human WT1 promoter revealed several potential binding sites for Pea3. Pea3 transactivated the WT1 promoter in transient transfection assays and bound to specific sites within the WT1 promoter in vitro. Our results position Pea3 upstream of WT1 and define a gene hierarchy important for mesenchymal–epithelial transitions.

Loss of heterozygosity of chromosome 8p and 11p in the dysplastic nodule and hepatocellular carcinoma.

In hepatocarcinogenesis, both de novo and multistep pathways have been suggested, and in the latter a dysplastic nodule is the proposed precancerous lesion. But genetic changes involved in the dysplastic nodule are not well understood. In this study, we tried to determine whether allelic loss of the chromosome 8p and/or 11p could be involved in the development of the dysplastic nodule and/or hepatocellular carcinoma. Platelet-derived growth factor-receptor beta-like tumor suppressor gene (PRLTS) and deletion in liver cancer-1 tumor suppressor gene are located at 8p21.3-p22. The hepatitis B virus integration site and WT1 tumor suppressor gene are located at 11p13. We therefore studied loss of heterozygosity (LOH) of chromosome 8p21.3-p22 and 11p13 in 22 dysplastic nodules and 21 hepatocellular carcinomas. The samples, microdissected from paraffin-embedded tissues, were examined using a polymerase chain reaction-based LOH assay using microsatellite markers. Loss of heterozygosity was detected for chromosome 8p21.3-p22 in nine (40.9%) of 22 dysplastic nodules and in eight (42.1%) of 19 hepatocellular carcinomas. D8S261, located adjacent to PRLTS, showed most frequent LOH: 28.6% in dysplastic nodule and 40.0% in hepatocellular carcinoma. Loss of heterozygosity on chromosome 11p13 was found in three (15.8%) of 19 dysplastic nodules and in six (31.6%) of 19 hepatocellular carcinomas. Loss of heterozygosity of D11S995 and D11S907 was found in 33.3% and 7.1% of dysplastic nodules, and 8.3% and 44.4% of hepatocellular carcinomas, respectively. These results suggest that at least one putative tumor suppressor gene involved in the development and progression of hepatocellular carcinoma may be located on 8p21.3-p22 and 11p13. Particularly, PRLTS might be related to an early genetic event of hepatocarcinogenesis.

Role of the WT1 tumor suppressor in murine hematopoiesis

The WT1 tumor-suppressor gene is expressed by many forms of acute myeloid leukemia. Inhibition of this expression can lead to the differentiation and reduced growth of leukemia cells and cell lines, suggesting that WT1 participates in regulating the proliferation of leukemic cells. However, the role of WT1 in normal hematopoiesis is not well understood. To investigate this question, we have used murine cells in which the WT1 gene has been inactivated by homologous recombination. We have found that cells lacking WT1 show deficits in hematopoietic stem cell function. Embryonic stem cells lacking WT1, although contributing efficiently to other organ systems, make only a minimal contribution to the hematopoietic system in chimeras, indicating that hematopoietic stem cells lacking WT1 compete poorly with healthy stem cells. In addition, fetal liver cells lacking WT1 have an approximately 75% reduction in erythroid blast-forming unit (BFU-E), erythroid colony-forming unit (CFU-E), and colony-forming unit-granulocyte macrophage-erythroid-megakaryocyte (CFU-GEMM). However, transplantation of fetal liver hematopoietic cells lacking WT1 will repopulate the hematopoietic system of an irradiated adult recipient in the absence of competition. We conclude that the absence of WT1 in hematopoietic cells leads to functional defects in growth potential that may be of consequence to leukemic cells that have alterations in the expression of WT1.

Exclusion of WTAP and HOXA13 as candidate genes for isolated hypospadias.

Different molecular factors have been identified as being associated with isolated or syndrome-associated forms of hypospadias. Nevertheless, the etiology of hypospadias is unknown in 70% of cases. As mutations in the homeobox gene A13 (HOXA13) were all found to be associated with hypospadias in affected males, some types of mutation may solely lead to the isolated form. Moreover, mutations in the Wilms' tumor suppressor gene WT1 have been found in patients with hypospadias without evidence of a Wilms' tumor and, therefore, its recently identified associated protein (WTAP) may be a further candidate gene for the genesis of hypospadias. A total of 37 patients affected with different forms of isolated hypospadias were analyzed for mutations in their HOXA13 and WTAP genes. With the exception of two novel WTAP polymorphisms, no mutations could be observed. There seems to be no evidence that isolated hypospadias is commonly caused by mutations in HOXA13 or WTAP genes.

A mutant form of the Wilms' tumor suppressor gene WT1 observed in Denys-Drash syndrome interferes with glomerular capillary development.

The Wilms' tumor suppressor gene WT1 encodes a zinc finger protein that is required for urogenital development. In the kidney, WT1 is most highly expressed in glomerular epithelial cells or podocytes, which are an essential component of the filtering system. Human subjects heterozygous for point mutations in the WT1 gene develop renal failure because of the formation of scar tissue within glomeruli. The relationship between WT1 expression in podocytes during development and glomerular scarring is not well understood. In this study, transgenic mice that expressed a mutant form of WT1 in podocytes were derived. The capillaries within transgenic glomeruli were dilated, indicating that WT1 might regulate the expression of growth factors that affect capillary development. Platelet endothelial cell adhesion molecule-1 expression was greatly reduced on glomerular endothelial cells of transgenic kidneys. These results suggest that WT1 controls the expression of growth factors that regulate glomerular capillary development and that abnormal capillary development might lead to glomerular disease.

The Wilms tumor suppressor WT1 regulates early gonad development by activation of Sf1.

In mammals, several genes including the Wilms tumor suppressor gene Wt1, the Lim homeobox gene Lhx9, and the gene encoding steroidogenic factor 1 (Sf1) have been implicated in the development of the indifferent gonad prior to sexual differentiation. Interactions among these genes have not yet been elucidated. Using biochemical and genetic experiments, we demonstrate here that WT1 and LHX9 function as direct activators of the Sf1 gene. Interestingly, only the -KTS form of WT1 is able to bind to and transactivate the Sf1 promoter. This observation is consistent with differential roles for the -KTS and +KTS variants of WT1 which have been postulated on the basis of human disorders such as the Frasier syndrome. Our data suggest a pathway in which the products of the Wt1 and Lhx9 genes activate expression of Sf1 and thus mediate early gonadogenesis.

A mammal-specific exon of WT1 is not required for development or fertility.

The WT1 tumor suppressor gene is a zinc finger-containing transcription factor which is required for development of the kidney and gonads. A mammal-specific alternative splicing event within this gene results in the presence or absence of a 17-amino-acid sequence within the WT1 protein. To determine the function of this sequence in vivo, gene targeting was utilized to specifically eliminate the exon encoding this sequence in mice. Mice lacking WT1 exon 5 develop normally. Adult mice lacking this exon are viable and fertile, and females are capable of lactation.

WT1 regulates the expression of the major glomerular podocyte membrane protein Podocalyxin

The WT1 tumor suppressor gene encodes a zinc finger transcription factor expressed in differentiating glomerular podocytes. Complete inactivation of WT1 in the mouse leads to failure of mesenchymal induction and renal agenesis, an early developmental phenotype that prevents analysis of subsequent stages in glomerular differentiation [1]. In humans with Denys-Drash Syndrome, a heterozygous germline mutation in WT1 is associated with specific defects in glomeruli and an increased risk for developing Wilms Tumor [2, 3]. WT1 target genes implicated in cell cycle regulation and cellular proliferation have been proposed [4], but the link between WT1 function and glomerular differentiation is unexplained. Here, we show that inducible expression of WT1 in rat embryonic kidney cell precursors leads to the induction of endogenous Podocalyxin, the major structural membrane protein of glomerular podocytes, which is implicated in the maintenance of filtration slits. Binding of WT1 to conserved elements within the Podocalyxin gene promoter results in potent transcriptional activation, and the specific expression pattern of Podocalyxin in the developing kidney mirrors that of WT1 itself. These observations support a role for WT1 in the specific activation of a glomerular differentiation program in renal precursors and provide a molecular basis for the glomerulonephropathy that is characteristic of Denys-Drash Syndrome.

The murine Wilms tumor suppressor gene ( wt1) locus

The Wilms tumor suppressor gene WT1 plays a crucial role in the etiology of various human diseases as well as in the development of specific organs including the kidneys, gonads and the spleen. At present the human as well as the Fugu wt1 locus have been characterized. We have used a PAC clone to analyze the murine wt1 locus and report here the structure of the wt1 gene as well as a characterization of the nine wt1 introns regarding their size and sequence at the exon/intron and intron/exon boundaries. In addition we provide a restriction map of the murine wt1 locus which should prove useful for the cloning of various constructs designed for the generation of mouse models. Prompted by the existence of a WT1 antisense transcript in humans we also examined strand-specific transcription at the murine wt1 locus. Our analysis suggests that there is no detectable antisense transcription of sequences within or immediately downstream of wt1 exon 1. We find, however, evidence for a divergent transcript which encompasses sequences at and around minor transcriptional initiation sites of wt1 and which is transcribed in the opposite direction. Despite the very high degree of similarity between the human and the murine wt1 sequence and expression as well as the presence of divergent transcripts in both cases, the existence of antisense transcription does not seem to be conserved between the two species.

Pre-pattern in the pronephric kidney field of zebrafish.

Vertebrate embryos use a series of transient kidneys to regulate fluid balance, osmolarity and metabolic waste during development. The first kidney to form in the embryo is the pronephros. This kidney is composed of several cell types with very different functions and is organized into discrete segments: glomerulus, tubules and nephric duct. The site of origin of these cells is poorly understood, as are their lineage relationships. We have defined regions of the intermediate mesoderm as candidates for the pronephric field by expression patterns of the Wilms' Tumor suppressor gene (wt1), single-minded 1 (sim1) and pax2.1. All of these potential kidney markers are expressed in a stripe of intermediate mesoderm, with distinct, overlapping antero-posterior borders. We labeled small groups of cells in this area by laser uncaging of a fluorescent dextran, and then tracked their fates. We found that there was a bounded contiguous region of the intermediate mesoderm that provides pronephric progenitors. As is true for other organ fields, the pronephric field regulates after focal destruction, such that a normal pronephros forms after laser-mediated removal of the wt1 domain. The progenitors for podocytes, tubular cells and duct are restricted to subdomains within the pronephric field. The most anterior cells in the pronephric field give rise to podocytes. This corresponds to the wt1-expressing region. The next more posterior cells contribute to the tubule, and express both wt1 and pax2.1. The most posterior cells contribute to the nephric duct, and these express pax2.1 and sim1, but not wt1. Thus, there is a field for the pronephric kidney with classical attributes of defined border, pre-pattern and regulation. The pattern of the fate map reflects particular combinations of transcription factors.

Defining a common region of deletion at 13q21 in human cancers

Previous molecular genetic analyses identified a region of deletion at 13q21 in a variety of human cancers, suggesting the existence of a tumor suppressor gene(s) at this locus. In our earlier study on prostate cancer, the region of deletion was confined to a 3.1 cM interval between D13S152 and D13S162. At present, however, no known gene located in this interval has been firmly implicated in cancer, and the region remains too large for gene identification. To fine-map the area of interest, we established a contig of bacterial artificial chromosome (BAC) clones, narrowed the region of deletion by loss of heterozygosity (LOH) and homozygosity-mapping-of-deletion (HOMOD) analyses in different types of cancers, and tested a candidate gene from the region for mutation and alteration of expression in prostate cancers. The contig consisted of 75 overlapping BAC clones. In addition to the generation of 47 new sequence-tagged-site (STS) markers from the ends of BAC inserts, 76 known STS and expressed sequence tag markers were mapped to the contig (25 kb per marker on average). The minimal region of deletion was further defined to be about 700 kb between markers D13S791 and D13S166 by LOH analysis of 42 cases of prostate cancer, and by HOMOD analysis of eight prostate cancer cell lines/xenografts and 49 cell lines from cancers of the breast, ovary, endometrium, and cervix, using 18 microsatellite markers encompassing the deletion region. A gene that is homologous to the WT1 tumor suppressor gene, AP-2rep (KLF12), was mapped in this region and was analyzed for its expression and genetic mutation. In addition to low levels of expression in both normal and neoplastic cells of the prostate, this gene did not have any mutations in a group of aggressive prostate cancers and cell lines/xenografts, as assessed by the methods of polymerase chain reaction-single strand conformational polymorphism analysis and direct sequencing. These studies suggest that a 700 kb interval at 13q21 harbors a tumor suppressor gene(s) that seems to be involved in multiple types of cancer, and that the AP-2rep gene is unlikely to be an important tumor suppressor gene in prostate cancer. The BAC contig and high-resolution physical map of the defined region of deletion should facilitate the cloning of a tumor suppressor gene(s) at 13q21.