Kornberg calls for scientists' association.
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Humans↗
Biomedical subjects
L Bonetta
Publications and source records attributed to L Bonetta.
Tumor-suppressor genes. Open questions on p16.
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Carrier Proteins↗
Absence of cyclin D/cdk complexes in cells lacking functional retinoblastoma protein.
Cyclins D1, D2 and D3 are thought to function in the G1 phase of the cell division cycle by regulating the activity of cyclin-dependent protein kinases. All three D-type cyclins can be shown to associate with two specific kinases, cdk4 and cdk6, providing at least six possible combinations. To establish whether different cell types require different subsets of these complexes and whether they are altered in tumours where D-cyclin expression is perturbed, we surveyed a series of tumour cell lines and compared them where possible to non-tumorigenic counterparts. Although complexes involving cdk4 or cdk6 were readily observed in many of the cell lines, no complexes were detectable in human cells harbouring DNA tumour virus oncoproteins or in which the retinblastoma gene product (pRb) is mutated or missing. These data suggest that as well as being a potential substrate for D-cyclin-kinases, functional pRb contributes to the formation or stability of the complexes, at least in human cells.
Breast Neoplasms↗
CDK6 (PLSTIRE) and CDK4 (PSK-J3) are a distinct subset of the cyclin-dependent kinases that associate with cyclin D1.
Deregulated expression of cyclin D1 is a feature of several neoplastic and proliferative disorders, but its normal role in the cell cycle remains unclear. Here we show that in a squamous carcinoma cell line with 11-fold amplification of the CCND1 gene, cyclin D1 associates specifically with p33cdk4 (PSK-J3) and p38cdk6 (PLSTIRE), two closely related members of the cyclin-dependent kinase (CDK) family. In these tumour cells, there is little evidence for an association between cyclin D1 and other CDKs, but in diploid human fibroblasts both CDK2 and CDK5 can be co-precipitated with cyclin D1, as well as CDK4. The data suggest that D-type cyclins participate in multiple interactions with CDKs but that the nature or stoichiometry of these associations may differ in different types of cell.
Amino Acid Sequence↗
Loss of heterozygosity mapping in Wilms tumor indicates the involvement of three distinct regions and a limited role for nondisjunction or mitotic recombination.
Loss of heterozygosity (LOH) for polymorphic markers is a frequently occurring event in some tumors, reflecting the role of allele loss in the development of these tumors. We have determined LOH in 38 cases of Wilms tumor for the 2 known loci on chromosome arm 11p and for a newly detected locus on chromosome arm 16q. Only 7 of the 38 tumors studied showed reduction to homozygosity of 11p13 markers. In 4 of these tumors, reduced expression of WT1 and WIT1, genes located at 11p13 and implicated in Wilms tumorigenesis, was noted. However, this was also found in 2 of 7 tumors showing LOH exclusively of 11p15 markers and in 15 of the remaining 24 tumors in which there was no LOH for 11p markers. This suggests that events not involving mitotic recombination or chromosome nondisjunction are the most common mechanisms for mutations at the 11p Wilms tumor locus. We also noted that mitotic recombination involving 11p15 loci occurred in addition to reduced expression of the 11p13 locus genes in 2 tumors, suggesting a possible interaction between these 2 loci. In addition, LOH for 16q markers was observed in 6 tumors. In one case this was coincident with reduction of WT1 and WIT1 gene expression, and in 3 other cases it occurred in addition to 11p LOH. This indicates that an additional locus on 16q is likely to be involved in Wilms tumorigenesis.
Adolescent↗
Coordinate expression of Wilms' tumor genes correlates with Wilms' tumor phenotypes.
The cloning and molecular characterization of two putative tumor genes, WT1 and WIT1, from the chromosome 11p13 region has provided a means of evaluating their role in the generation of Wilms' tumor heterogeneity. A series of 29 tumors were analyzed for WT1 and WIT1 expression by Northern blot or RNase protection analyses, and results were compared with tumor histopathology. Tumors were scored for the percentage of mesenchymal and epithelial derived tissue components. Homotypic tumors comprised blastema, tubular epithelium, and a fibroblast-like mesenchyme. In addition to these tissue components, the group of tumors designated as heterotypic also contained ectopic cell phenotypes such as muscle and squamous epithelium. The analyses suggest that heterotypic differentiation patterns occur when WT1 and WIT1 expression is low relative to normal fetal kidney. In situ hybridization using antisense RNA probes showed that WT1 and WIT1 were concordantly expressed in normal fetal kidney and in the blastema of tumors. The ratio of WT1:WIT1 expression remained relatively constant in homotypic tumors but deviated significantly in heterotypic tumors. These results suggest that expression patterns of the WT1 and WIT1 genes can be closely correlated to Wilms' tumor histopathology.
Blotting, Northern↗
Tissue, developmental, and tumor-specific expression of divergent transcripts in Wilms tumor.
The Wilms tumor locus on chromosome 11p13 has been mapped to a region defined by overlapping, tumor-specific deletions. Complementary DNA clones representing transcripts of 2.5 (WIT-1) and 3.5 kb (WIT-2) mapping to this region were isolated from a kidney complementary DNA library. Expression of WIT-1 and WIT-2 was restricted to kidney and spleen. RNase protection revealed divergent transcription of WIT-1 and WIT-2, originating from a DNA region of less than 600 bp. Both transcripts were present at high concentrations in fetal kidney and at much reduced amounts in 5-year-old and adult kidneys. Eleven of 12 Wilms tumors classified as histopathologically heterogeneous exhibited absent or reduced expression of WIT-2, whereas only 4 of 14 histopathologically homogeneous tumors showed reduced expression. These data demonstrate a molecular basis for the pathogenetic heterogeneity in Wilms tumorigenesis.
Base Sequence↗
Wilms tumor locus on 11p13 defined by multiple CpG island-associated transcripts.
Wilms tumor is an embryonal kidney tumor involving complex pathology and genetics. The Wilms tumor locus on chromosome 11p13 is defined by the region of overlap of constitutional and tumor-associated deletions. Chromosome walking and yeast artificial chromosome (YAC) cloning were used to clone and map 850 kilobases of DNA. Nine CpG islands, constituting a "CpG island archipelago," were identified, including three islands that were not apparent by conventional pulsed-field mapping, and thus were at least partially methylated. Three distinct transcriptional units were found closely associated with a CpG island within the boundaries of a homozygous DNA deletion in a Wilms tumor.
Chromosome Mapping↗
Complete physical map of the WAGR region of 11p13 localizes a candidate Wilms' tumor gene.
A complete physical map of the 11p13 region containing the Wilms' tumor locus has been developed and used to localize a candidate Wilms' tumor gene. Our strategy to construct the map combined the use of pulsed-field gel electrophoresis and irradiation-reduced somatic cell hybrids. These hybrids, which contain limited segments of human chromosome 11 segregated from the remainder of the human genome, permit direct visualization of restriction fragments located in 11p13 using human interspersed repeated DNA sequences as hybridization probes. The physical map has provided a framework to identify the sites of genes responsible for the complex of disorders associated with hemizygous 11p13 deletion: Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation. The Wilms' tumor locus has been limited to a region of less than 345 kb, and a transcript with many of the characteristics expected for the Wilms' tumor gene has been localized to this region.
Cell Line↗
Definition of the limits of the Wilms tumor locus on human chromosome 11p13.
In a previous report, we described a contiguous restriction map of chromosome band 11p13 that localized the Wilms tumor locus to a small group of NotI fragments. In an effort to identify and isolate the 11p13-associated sporadic Wilms tumor locus, we developed a panel of NotI fragment-specific DNA probes. These probes were selected from genomic libraries constructed using the Chinese hamster ovary-human somatic cell hybrid carrying only human 11p. The libraries were prepared from NotI-digested DNA after size selection by pulsed-field gel electrophoresis. The selected NotI fragments had been previously targeted on the basis of deletion mapping as having a high probability of containing the Wilms tumor locus. We used these newly identified 11p13-specific probes to improve the resolution of the restriction map spanning the Wilms tumor locus. The locus has been defined by a homozygous deletion in a sporadic Wilms tumor. Using these probes, the region of homozygous deletion in this tumor and presumably all or part of the Wilms tumor gene have been confined to two small SfiI fragments spanning less than 350 kb.
Animals↗
Homozygous deletion of a DNA marker from chromosome 11p13 in sporadic Wilms tumor.
A random DNA fragment, probe p2.3 (locus D11S87), was cloned from the 11p13 region between a translocation breakpoint associated with familial aniridia and another translocation breakpoint associated with childhood T-cell leukemia. The D11S87 locus maps between the catalase (CAT) locus and the beta subunit of follicle stimulating hormone (FSHB). The D11S87 locus is deleted in a Wilms tumor patient with a constitutional deletion of 11p and in a case of sporadic Wilms tumor (WiT-13) apparently with normal karyotype. In the WiT-13 tumor both maternal and paternal chromosomes 11 are retained; D11S87 is deleted homozygously and FSHB hemizygously. These results suggest two mutational events resulting in homozygous deletion in this patient. The D11S87 homozygous deletion was also demonstrated in WiT-13 nude mouse heterotransplants and in fibroblast-like cell line derived from the primary tumor. The minimum size of the deletion was estimated to be 30 kb as determined by cosmid screening and hybridization. As homozygous deletions in the 11p13 region have not been previously reported for sporadic Wilms tumors, these findings place the D11S87 locus within or approximate to the Wilms tumor gene.
Blotting, Southern↗