Analysis of multiple growth regulatory proteins using dissociable staining antibody arrays on solid tumor biopsy specimens.

Growth of tumor cells is often a function of deregulated growth factor receptors and their corresponding intracellular signalling molecules. The dissociable antibody staining arrays have the versatility to rapidly identify the expression, activation, and localization of such molecules and pathways in biopsy specimens. This report describes a protocol to quantify the activity of a panel of signalling molecules in Wilms tumor biopsy specimens and surrounding nonmalignant renal cells. We propose that this technique can be used to rapidly identify multiple markers and may aid in the study of aberrant growth regulatory mechanisms and potential targets for therapeutics from pathologic specimens.

Evidence of a role for the INK4 family of cyclin-dependent kinase inhibitors in ovarian granulosa cell tumors.

Granulosa cell tumors (GCTs) of the ovary are relatively rare and account for <5% of all ovarian cancers. The molecular pathogenesis of these tumors is not well understood. We tested the hypothesis that cyclin-dependent kinase inhibitors, specifically the inhibitors of the cyclin-dependent kinase 4 (INK4) family, are targets for altered gene expression in GCTs. The status of RB1, INK4A, INK4B, INK4C, INK4D, and ARF in 13 adult and 2 juvenile ovarian GCTs was determined by reverse transcription-polymerase chain reaction of total RNA and exon-specific sequencing of genomic DNA. Tumors showing loss of INK4A expression were assayed further by exon-deletion analysis and methylation-specific PCR. None of the juvenile tumors demonstrated altered expression, but 7/12 (58%) adult GCTs lacked expression of INK4A, INK4B, or both. In one of these cases, we noted a homozygous deletion of the INK4A locus, and in the remaining tumors we found hypermethylation of the promoter region, a mechanism that can lead to gene inactivation. These data support a role for the INK4 family of CDK inhibitors in the biology of GCTs.

Isolation and characterization of a novel, transforming allele of the c-Cbl proto-oncogene from a murine macrophage cell line.

The c-Cbl proto-oncogene acts as an E3 ubiquitin ligase via its RING finger domain to negatively regulate activated cellular signal transduction pathways. We have identified an aberrant Cbl-protein of approximately 95 kDa, which we have called p95Cbl, from the murine reticulum sarcoma cell-line, J-774. Cloning of the p95Cbl cDNA revealed that it contains a deletion resulting in the loss of 111 amino acids, eliminating two critical tyrosine residues in the linker region as well as the entire RING finger domain. p95Cbl displays a propensity for its interaction with the Src-family kinase Hck over cellular Cbl expressed in the same cells. Like its wildtype counterpart, p95Cbl is inducibly tyrosine phosphorylated in response to Fcgamma receptor engagement on hematopoietic cells, however this phosphorylation is sustained beyond that of cellular Cbl. NIH3T3 fibroblasts stably expressing p95Cbl acquire the typical refractile morphology associated with cellular transformation and form colonies in a focus-formation assay. The exogenously expressed mutant protein is constitutively phosphorylated in fibroblasts and partitions into the particulate fraction of cells, while cellular Cbl is exclusively cytoplasmic. p95Cbl is a novel, oncogenic mutant of the c-Cbl proto-oncogene, which might act in a dominant negative fashion to prolong normal cellular signaling responses by interfering with the down-regulation of activated signaling complexes through c-Cbl.