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Background: p27(BBP) is a regulator of ribosome assembly and an essential nuclear and cytoplasmic component of eukaryotes., Methods: We investigated the immunochemical distribution of p27(BBP) in head and neck carcinomas, in the associated normal mucosa, and in regional lymph nodes., Results: p27(BBP) is detectable in mucosal cells but is overexpressed in carcinomas, highly concentrated in large polymorphous nucleoli, and even larger and more evident in lymph node metastatic foci. Western blotting confirms increased p27(BBP) in carcinomas versus normal mucosa and also in metastatic versus normal lymph nodes. The overexpression of p27(BBP) corresponds to mRNA upregulation in carcinomas. Unexpectedly, a 52-kDa band specifically reacting with antibodies to p27(BBP) was observed in several carcinomas., Conclusions: p27(BBP) alterations are common events in the transition to malignancy and are probably involved in squamous carcinoma progression. Immune reagents raised to p27(BBP) may provide additional diagnostic tools for surgical pathology of tumor boundaries and lymph nodes. The 52-kDa band may represent an abnormal form of p27(BBP) expressed by transformed airway epithelia., (Copyright 2004 Wiley Periodicals, Inc. Head Neck 26: 408-417, 2004)

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

The ITGB4BP gene encodes for a highly conserved protein, named p27BBP (also known as eIF6), originally identified in mammals as a cytoplasmic interactor of beta4 integrin. In vitro and in vivo studies demonstrated that p27BBP is essential for cell viability and has a primary function in the biogenesis of the 60S ribosomal subunit. Here we report the genomic organization of the human ITGB4BP gene and show that its gene product is expressed with features of a housekeeping element in vitro, but is regulated in a cell specific fashion in vivo. The human gene spans 10 kb and comprises seven exons and six introns. The 5' flanking region shows a TATA-less promoter, canonical CpG islands, and binding sites for serum responsive elements. In cultured cells, p27BBP mRNA and protein are constitutively expressed and stable. A gradual loss of p27BBP mRNA can be observed only after prolonged serum starvation, and heat shock treatment. In contrast, p27BBP mRNA and protein levels in vivo are variable among different organs. More strikingly, immunohistochemical analysis shows that the p27BBP protein is present in a cell specific fashion, even within the same tissue. Taken together, these data show that ITGB4BP gene expression is highly regulated in vivo, possibly by the combination of tissue specific factors and protein synthesis pathways.

The highly conserved protein p27BBP is a cytoplasmic interactor of integrin beta4 expressed in epithelia. p27BBP is found in two pools: one nuclear pool enriched in the perinucleolar region, and one cytoplasmic pool. Deletion of p27BBP in yeast is lethal as a result of loss of the ribosomal 60S subunit. The aim of this study was to investigate the distribution of p27BBP in gut epithelium and its behavior during progression of human colorectal carcinomas. Results indicated that p27BBP is high in rapidly cycling cells and decreased in villous cells committed to apoptotic cell death. In dysplastic adenomas and carcinomas, p27BBP displayed a large increase of its nucleolar component that was superimposable to argyrophylic nucleolar organizing region-associated proteins and was associated with the nuclear matrix. Western blotting confirmed increased p27BBP in dysplastic adenomas and in carcinomas. In particular, p27BBP increased progressively from adenomas to carcinomas and, in the latter, was related to the tumor stage. The overexpression of p27BBP corresponded to mRNA up-regulation in carcinomas, supporting the idea of transcriptional or post-transcriptional regulation of its expression. Results suggested that p27BBP alterations are an early event in the transition from benign to malignant colorectal phenotypes and provide a novel tool in surgical pathology.

p27(BBP/eIF6) is an evolutionarily conserved protein that was originally identified as p27(BBP), an interactor of the cytoplasmic domain of integrin beta4 and, independently, as the putative translation initiation factor eIF6. To establish the in vivo function of p27(BBP/eIF6), its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing beta4 integrin, p27(BBP/eIF6) is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of beta4 integrin. Surprisingly, in the absence and in the presence of the beta4 integrin subunit, p27(BBP/eIF6) is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27(BBP/eIF6) homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit. Furthermore, human p27(BBP/eIF6) can rescue the lethal effect of the iihDelta yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27(BBP/eIF6) in ribosome biogenesis or assembly rather than in translation. A further function related to the beta4 integrin subunit may have evolved specifically in higher eukaryotic cells.

The integrin beta4 has a long cytodomain necessary for hemidesmosome formation. A yeast two-hybrid screen using beta4 cytodomain uncovered a protein called p27(BBP) that represents a beta4 interactor. Both in yeast and in vitro, p27(BBP) binds the two NH2-terminal fibronectin type III modules of beta4, a region required for signaling and hemidesmosome formation. Sequence analysis of p27(BBP) revealed that p27(BBP) was not previously known and has no homology with any isolated mammalian protein, but 85% identical to a yeast gene product of unknown function. Expression studies by Northern analysis and in situ hybridization showed that, in vivo, p27(BBP) mRNA is highly expressed in epithelia and proliferating embryonic epithelial cells. An antibody raised against p27(BBP) COOH-terminal domain showed that all beta4-containing epithelial cell lines expressed p27(BBP). The p27(BBP) protein is insoluble and present in the intermediate filament pool. Furthermore, subcellular fractionation indicated the presence of p27(BBP) both in the cytoplasm and in the nucleus. Confocal analysis of cultured cells showed that part of p27(BBP) immunoreactivity was both nuclear and in the membrane closely apposed to beta4. These results suggest that the p27(BBP) is an in vivo interactor of beta4, possibly linking beta4 to the intermediate filament cytoskeleton.