Genomic organization of the human p57KIP2 gene and its analysis in the G401 Wilms' tumor assay.

The p57KIP2 gene encodes an inhibitor of cyclin-dependent kinase activity, which negatively regulates cell cycle progression. The human p57 gene is located in 11p15.5, a region of DNA frequently altered in neoplasia. We have isolated a human genomic clone and mapped the p57 gene to a 2.2-kb region between D11S648 and D11S679. Sequence analysis revealed that the coding DNA of the human p57 gene is divided by 0.5-kb intron. A second intron was detected in the 3' untranslated region, indicating that the human p57 gene contains at least three exons. Our previous work with somatic cell hybrids mapped a tumor suppressor gene for the G401 Wilms' tumor cell line to a approximately 500-kb region of 11p15.5 that includes p57. Northern blot analysis detected a 0.8-kb p57 transcript in several of the G401 hybrid lines. However, p57 expression did not correlate with tumor suppression. These results suggest that p57 is not responsible for the tumor suppression observed in our somatic cell hybrid assay.

Cyclin-dependent kinase inhibitor p57KIP2 in soft tissue sarcomas and Wilms'tumors.

Mammalian cyclin-dependent kinase inhibitors fall into two families, the INK4 and the CIP/KIP. The CIP/KIP family comprises three structurally related members, including p21CiP1/WAF1, p27KIP1, and p57KIP2. These proteins are all capable of inhibiting the progression of the cell cycle by binding and inhibiting G(1) cyclin/cyclin-dependent kinase complexes. In humans, p57KIP2 is expressed specifically in skeletal muscle, heart, brain, kidney, and lung. Human KIP2 resides in 11p15.5, a chromosomal region that is a common site for loss of heterozygosity in certain sarcomas, Wilms' tumors, and tumors associated with the Beckwith-Wiedemann syndrome. Because of the function, selective expression, and chromosomal location of p57KIP2, we undertook the present study to search for potential mutations of KIP2 in a cohort of 126 tumors composed of 75 soft tissue sarcomas and 51 Wilms' tumors. The KIP2 gene was characterized by Southern blot, comparative multiplex PCR, PCR -single-strand conformational polymorphism, and DNA sequencing assays in these neoplasms. Deletions of the KIP2 gene or point mutations at the region encoding the cyclin-dependent kinase inhibitory domain were not found in the tumors analyzed. The absence of KIP2 mutations might indicate that these tumors arise due to defects at a closely linked but separate locus. Alternatively, similarly to the mouse homologue, inactivation of KIP2 could occur via genomic imprinting.

The transcription activation domains of v-Myc and VP16 interact with common factors required for cellular transformation and proliferation.

The amino terminus of the avian myelocytomatosis virus MC29 v-Myc oncoprotein contains sequences that are essential for cellular transformation (S. Farina, et al. J. Virol., 66: 2698-2708, 1992; S. Min and E. J. Taparowsky. Oncogene, 7:1531-1540, 1992) and for the ability to activate gene transcription (S. Min and E. J. Taparowsky. Oncogene, 7:1531-1540, 1992). To investigate the molecular interactions that mediate these v-Myc-associated activities, we performed competition assays in which various regions of the v-Myc amino terminal transcription activation domain (TAD) were examined for their ability to inhibit transcription activation by v-Myc, VP16, and the myogenic regulatory factor MyoD. Overexpression of these transcriptional activators also was used to investigate whether Myc-interacting proteins were required for cellular transformation and cell proliferation events. Our results demonstrate that at least two distinct cellular activities interact with the v-Myc TAD and that it is the synergism between these activities that is required for v-Myc to function fully as a transcriptional activator. In addition, v-Myc activators squelch VP16- and MyoD-dependent transcription activation, suggesting that the v-Myc TAD interacts with a component of the general transcription machinery. In support of this observation, we found that overexpression of the v-Myc TAD inhibits ras-mediated cellular transformation as well as cell proliferation, underscoring the critical role these amino terminal Myc-interacting factors play in regulating the physiology of both normal and transformed cells.

Novel transcribed sequences within the BWS/WT2 region in 11p15.5: tissue-specific expression correlates with cancer type.

Chromosome band 11p15.5 has proven to be an intriguing area of the human genome. Various studies have linked alterations in this region to growth-related disorders such as Beckwith-Wiedemann syndrome and a variety of human cancers. Furthermore, functional assays in G401 Wilms tumor cells and RD rhabdomyosarcoma cells support the existence of a tumor suppressor gene on 11p15.5, sometimes called WT2. In addition, several genes mapping to this region show imprinted expression, suggesting that 11p15.5 contains an imprinted domain. We have employed solution hybrid capture in combination with sequence analysis to identify 16 genes within the approximately 700-kb critical region of 11p15.5 between D11S601 and D11S1318. Two of these genes, NAP1L4 and KCNA9, had been previously reported. Ten novel transcripts were identified with partial cDNA sequences selected by solution hybrid capture. Sequence homology to known ESTs was used to identify the remaining gene transcripts. Interestingly, the tissue-specific mRNA expression of these genes correlates with the tumor types linked to this region. This work can be compiled into a transcript map, important in the elucidation of tumor suppressor activity on chromosome 11p15.5.

A 1-Mb physical map and PAC contig of the imprinted domain in 11p15.5 that contains TAPA1 and the BWSCR1/WT2 region.

We have constructed a 1-Mb contig in human chromosomal band 11p15.5, a region implicated in the etiology of several embryonal tumors, including Wilms tumor, and in Beckwith-Wiedemann syndrome. Cosmid, P1, PAC, and BAC clones were characterized by NotI/SalI digestion and hybridized to a variety of probes to generate a detailed physical map that extends from D11S517 to L23MRP. Included in the map are the CARS, NAP2, p57/KIP2, KVLQT1, ASCL2, TH, INS, IGF2, H19, and L23MRP genes as well as end probes isolated from PACs. The TAPA1 gene, whose protein product can transmit an antiproliferative signal, was also localized in the contig. However, Northern blot analysis demonstrated that its expression did not correlate with tumorigenicity in G401 Wilms tumor hybrids, suggesting that TAPA1 is not responsible for the tumor suppression associated with 11p15.5. Genomic clones were used as probes in FISH analysis to map the breakpoints from three Beckwith-Wiedemann syndrome patients and a rhabdoid tumor. Interestingly, each of the breakpoints disrupts the KVLQT1 gene, which is spread over a 400-kb region of the contig. Since 11p15.5 contains several genes with imprinted expression and one or more tumor suppressor genes, our contig and map provide a framework for characterizing this intriguing genetic environment.