p63, a member of the p53 family of transcription factors, plays an important role in epithelial development, regulating both cell cycle and apoptosis. Even though p63 activity is regulated mainly at the posttranslational level, the control of p63 protein stability is far from being fully understood. Here, we show that the Hect (homologous to the E6-associated protein C terminus)-containing Nedd4-like ubiquitin protein ligase Itch binds, ubiquitylates, and promotes the degradation of p63. The physical interaction occurs at the border between the PY and the SAM (sterile ␣ motif) domains; a single Y504F mutation significantly affects p63 degradation. Itch and p63 are coexpressed in the epidermis and in primary keratinocytes where Itch controls the p63 protein steadystate level. Accordingly, p63 protein levels are significantly increased in Itch knockout keratinocytes. These data suggest that Itch has a fundamental role in the mechanism that controls endogenous p63 protein levels and therefore contributes to regulation of p63 in physiological conditions. keratinocytes ͉ ubiquitination ͉ p73 ͉ AIP4

Cajal bodies are small nuclear organelles with a number of nuclear functions. Here we show that FLICE-associated huge protein (FLASH), originally described as a component of the apoptosis signaling pathway, is mainly localized in Cajal bodies and is essential for their structure. Reduction in FLASH expression by short hairpin RNA results in disruption of the normal architecture of the Cajal body and relocalization of its components. Because the function of FLASH in the apoptosis receptor signaling pathway has been strongly questioned, we have now identified a clear function for this protein.coiled bodies ͉ nuclear organelles C ajal bodies (CBs) are small nuclear organelles described in vertebrate cells a century ago by Ramon y Cajal and which have since been observed in a variety of animal and plant nuclei. Many components of CBs are shared with the nucleolus, and CBs frequently localize to the nucleolar periphery or within the nucleoli (1-3). CBs disappear from prophase nuclei and reappear in late G 1 after resumption of transcription in the daughter nuclei (for review see refs. 1, 4, and 5). Although their function is still in part elusive, recent work suggests that they are involved in several nuclear functions, including modification of small nuclear RNAs and small nuclear ribonucleoproteins, important for spliceosome formation, and assembly of the three eukaryotic RNA polymerases (pol I, pol II, and pol III) with their respective transcription and processing factors that are then transported as multiprotein complexes to the sites of transcription (1). More recently CBs have been implicated in replication-dependent histone gene transcription and mRNA maturation (1, 6-10), and a subset of CBs is physically associated with histone gene clusters on chromosomes 1 and 6 (11). Here we identify FLASH (FLICEassociated huge protein) (12) as a new component of CBs and show that it is essential for their structure.FLASH was initially identified as a component of the apoptosis signaling complex known as the death-inducing signaling complex (12, 13), which is associated with caspase 8 in the death-inducing signaling complex and thus essential for caspase 8 activation. However, this role of FLASH has been questioned (14). More recently it has been shown that, in response to TNF␣, FLASH translocates to the nucleus and binds the glucocorticoid receptor-interacting protein (GRIP-1), inhibiting both its interaction with, and the transcriptional activity of, the glucocorticoid receptor (15, 16). Results FLASH Has a Nuclear Localization.Despite the presence of three nuclear localization signals FLASH was originally described as a cytoplasmic protein (12,17). Staining endogenous FLASH with four different anti-FLASH antibodies, however, showed that FLASH only localized to the nucleus with a clear punctate appearance (Fig. 5a, which is published as supporting information on the PNAS web site). The specificity of the antibodies used was confirmed by the disappearance of the staining after short hairpin RNA for FLASH (Fig. 4a). West...

p73 is a recently described member of the p53 family, and, like p53, it undergoes a number of posttranslational modifications. Here we show, by yeast two-hybrid screening, pull-down assays, and coimmunoprecipitation, that p73␣, -␤, and -␥ bind to the protein inhibitor of activated STAT-1 (PIAS-1) and that this binding stabilizes p73. PIAS-1 also sumoylates p73␣, although not the C-terminally truncated isoforms p73␤ and -␥, and this requires the RING finger domain of PIAS-1. The ⌬Np73␣ isoform can also bind, and be sumoylated by, PIAS-1. PIAS-1-mediated sumoylation decreases p73 transcriptional activity on several target promoters, such as Bax. p73 is colocalized in the nucleus with PIAS-1, and sumoylated p73 is located exclusively in the nuclear matrix. PIAS-1 is expressed predominantly during S phase, and PIAS-1 overexpression reduces p73-mediated transcription of p21, with a reduction of cells in G 1 and cell cycle reentry. Inhibition of endogenous PIAS-1 by RNA interference reduces the proportion of cells in S phase and induces G 2 arrest. These data suggest that PIAS-1, acting partly through binding and sumoylation of p73, is an important component of the cell cycle machinery.Signal transducer and activator of transcription (STAT) proteins are cytoplasmic transcription factors which, after phosphorylation, translocate to the nucleus, where they regulate expression of STAT-responsive genes (reviewed in references 4 and 14). STAT activity is partly regulated by a family of at least six related proteins, the protein inhibitors of activated STATs (PIAS). Thus, PIAS-1 inhibits DNA binding and gene activation by activated, tyrosine-phosphorylated STAT-1 (24). Another protein, PIASy, also inhibits the transcriptional activity of activated STAT-1, without, however, affecting STAT-1 DNA binding (25). A region located at the carboxy terminus of PIAS-1 directly interacts with STAT-1 dimers, though its interaction with phosphorylated and nonphosphorylated monomers is inhibited by the N-terminal domain (23). Recently, it has become clear that PIAS proteins can also regulate the activity of other transcription factors such as Smads (25), the mineralocorticoid and glucocorticoid receptors (40), and p53 (38).At least part of the mechanism of transcriptional regulation by PIAS proteins depends on their E3 SUMO ligase activity, and PIAS-1 and SUMO-1 colocalize in nuclear granules (19). Thus, PIAS proteins sumoylate activated STAT-1, resulting in reduced STAT-1-mediated transcription (42). Moreover, the RING finger domain of PIAS-1 binds to the C terminus of the tumor suppressor p53 (33) and catalyzes its sumoylation (17), a modification which represses p53 activity on a reporter plasmid containing consensus p53 DNA binding sites (38). However, other studies using the p21 promoter have shown enhanced p53 transcriptional activity after interaction with PIAS-1, although this is independent of the RING finger domain and therefore of p53 sumoylation (26). Two homologues of p53, p63 and p73, have been described. The three proteins ...

Background: We wished to evaluate the clinical response following ATP-Tumor Chemosensitivity Assay (ATP-TCA) directed salvage chemotherapy in a series of UK patients with advanced ovarian cancer. The results are compared with that of a similar assay used in a different country in terms of evaluability and clinical endpoints.

Aims-Overexpression of c-myc protein has independent prognostic significance in a variety of primary and metastatic cutaneous melanomas which suggests a possible role for this gene in melanomagenesis. We have therefore examined the importance of this oncogene in uveal melanoma and studied the coexpression of two other gene products, Bcl-2 and p53, which might contribute to its eVect. Methods-The percentage of cells positive for nuclear c-myc expression was estimated by flow cytometric analysis of nuclei extracted from paraYn blocks. The expression of Bcl-2 and p53 protein was assessed by immunohistochemistry. A total of 71 tumours were studied and the results compared with survival with a mean follow up period of 6 years. Results-c-myc was expressed in >50% of the cells by 70% of the tumours, and was independently associated with improved survival in a Cox multiple regression model. Although Bcl-2 was expressed by the majority of the cells in 67% tumours, it was without eVect on prognosis. None of the cases studied showed convincing positivity for p53. Analysis of coexpression showed that the best survival was seen in c-myc+/Bcl-2+ tumours and the worst in c-myc−/Bcl-2− tumours. Conclusion-The finding of improved rather than reduced survival in c-myc positive tumours is at variance with skin melanoma. There was no evidence to suggest that c-myc was modulated by upregulation of Bcl-2 or p53 inactivation/ mutation. Although Bcl-2 is unlikely to have any eVect on tumour growth or metastasis, it could contribute to the general lack of susceptibility to apoptosis in these tumours. (Br J Ophthalmol 1999;83:110-114) Although uveal melanoma is the commonest tumour of the eye, it represents less than 1% of cancer registrations and is a good example of a "rare" tumour. However, it accounts for 13% of deaths from melanoma. Death is invariably due to metastatic disease.