The presence of two tightly bound zn<SUP>2+</SUP> ions is essential for the structural and functional integrity of yeast RNA polymerase II

DNA-dependent RNA polymerases (RNApol) are Zn2+ metalloproteins where the Zn2+ ion plays both catalytic and structural roles. Although the ubiquitous presence of Zn2+ with the RNApol from eukaryotes had already been established, the exact stoichiometry of Zn2+ ion(s) per mole enzyme is not well documented, and its role in enzymatic function remains elusive. We show here that RNApolII from Saccharomyces cerevisiae has two Zn2+ ions tightly associated with it which are necessary for its transcriptional activity. Upon prolonged dialysis against 10 mM EDTA for 4-5 h, the enzyme loses one Zn2+, as well as partial activity. However, Zn2+ can be added back to the enzyme, but without recovering its total activity. 5 mM orthophenanthroline (OP) removes one Zn2+ within 2 h; the enzyme, however, cannot be reconstituted back with Zn2+. Circular dichroism (CD) studies showed that the conformation of the native enzyme is unique and cannot be reproduced with Zn2+-reconstituted RNApolII. Similarly, the rate of abortive synthesis of a dinucleotide product over a non-specific template is faster when catalyzed by two Zn2+-native enzymes. Zn2+-reconstituted RNApolII or one Zn2+-RNApolII showed a slower abortive synthesis rate. 65Zn2+-blotting experiments indicated that the removal of one Zn2+ from the enzyme destroys the Zn2+-binding ability of the larger subunits of yeast RNApolII. In order to check whether the presence of Zn2+ ions has any effect on substrate recognition, we followed the binding of (&#947; -AmNS)UTP, a fluorescent substrate analog to RNApol||. It was observed that OP-treated enzyme showed non-specific substrate recognition, whereas two Zn2+-native RNApol binds substrate at a single site

ΔNp63 induces β-catenin nuclear accumulation and signaling

AbstractThe P53 homolog p63 encodes multiple proteins with transactivating, apoptosis-inducing, and oncogenic activities. We showed that p63 is amplified and that ΔNp63 isotypes are overexpressed in squamous cell carcinoma (SCC) and enhance oncogenic growth in vitro and in vivo. Moreover, p53 associated with ΔNp63α and mediated its degradation. Here, we report that ΔNp63 associates with the B56α regulatory subunit of protein phosphatase 2A (PP2A) and glycogen synthase kinase 3β (GSK3β), leading to a dramatic inhibition of PP2A-mediated GSK3β reactivation. The inhibitory effect of ΔNp63 on GSK3β mediates a decrease in phosphorylation levels of β-catenin, which induces intranuclear accumulation of β-catenin and activates β-catenin-dependent transcription. Our results suggest that ΔNp63 isotypes act as positive regulators of the β-catenin signaling pathway, providing a basis for their oncogenic properties

DeltaNp63 induces beta-catenin nuclear accumulation and signaling.

The P53 homolog p63 encodes multiple proteins with transactivating, apoptosis-inducing, and oncogenic activities. We showed that p63 is amplified and that DeltaNp63 isotypes are overexpressed in squamous cell carcinoma (SCC) and enhance oncogenic growth in vitro and in vivo. Moreover, p53 associated with DeltaNp63alpha and mediated its degradation. Here, we report that DeltaNp63 associates with the B56alpha regulatory subunit of protein phosphatase 2A (PP2A) and glycogen synthase kinase 3beta (GSK3beta), leading to a dramatic inhibition of PP2A-mediated GSK3beta reactivation. The inhibitory effect of DeltaNp63 on GSK3beta mediates a decrease in phosphorylation levels of beta-catenin, which induces intranuclear accumulation of beta-catenin and activates beta-catenin-dependent transcription. Our results suggest that DeltaNp63 isotypes act as positive regulators of the beta-catenin signaling pathway, providing a basis for their oncogenic properties

Prospective subgroup analyses of the randomized MCL-002 (SPRINT) study : lenalidomide versus investigator's choice in relapsed or refractory mantle cell lymphoma

International audienceIn the mantle cell lymphoma (MCL)-002 study, lenalidomide demonstrated significantly improved median progression-free survival (PFS) compared with investigator's choice (IC) in patients with relapsed/refractory MCL. Here we present the long-term follow-up data and results of preplanned subgroup exploratory analyses from MCL-002 to evaluate the potential impact of demographic factors, baseline clinical characteristics and prior therapies on PFS. In MCL-002, patients with relapsed/refractory MCL were randomized 2:1 to receive lenalidomide (25 mg/day orally on days 1-21; 28-day cycles) or single-agent IC therapy (rituximab, gemcitabine, fludarabine, chlorambucil or cytarabine). The intent-to-treat population comprised 254 patients (lenalidomide, n = 170; IC, n = 84). Subgroup analyses of PFS favoured lenalidomide over IC across most characteristics, including risk factors, such as high MCL International Prognostic Index score, age ≥65 years, high lactate dehydrogenase (LDH), stage III/IV disease, high tumour burden, and refractoriness to last prior therapy. By multivariate Cox regression analysis, factors associated with significantly longer PFS (other than lenalidomide treatment) included normal LDH levels (P < 0·001), nonbulky disease (P = 0·045), <3 prior antilymphoma treatments (P = 0·005), and ≥6 months since last prior treatment (P = 0·032). Overall, lenalidomide improved PFS versus single-agent IC therapy in patients with relapsed/refractory MCL, irrespective of many demographic factors, disease characteristics and prior treatment history

Efficacy and safety of azacitidine in pediatric patients with newly diagnosed advanced myelodysplastic syndromes before hematopoietic stem cell transplantation in the AZA-JMML-001 trial

Here we report efficacy, pharmacokinetics, and safety data obtained in treatment-naive, pediatric patients with newly diagnosed advanced MDS receiving azacitidine in the AZA-JMML-001 study. The primary endpoint was response rate (proportion of patients with complete response [CR], partial response [PR], or marrow CR, sustained for &gt;= 4 weeks). Of the 10 patients enrolled, one had an unconfirmed marrow CR and none had confirmed responses after three cycles; the study was therefore closed after stage 1. Azacitidine was well tolerated. The lack of efficacy of azacitidine in pediatric patients with newly diagnosed advanced MDS highlights the need for effective new treatments in these patients

Efficacy and safety of azacitidine in pediatric patients with newly diagnosed advanced myelodysplastic syndromes before hematopoietic stem cell transplantation in the AZA-JMML-001 trial

Here we report efficacy, pharmacokinetics, and safety data obtained in treatment-naive, pediatric patients with newly diagnosed advanced MDS receiving azacitidine in the AZA-JMML-001 study. The primary endpoint was response rate (proportion of patients with complete response [CR], partial response [PR], or marrow CR, sustained for &gt;= 4 weeks). Of the 10 patients enrolled, one had an unconfirmed marrow CR and none had confirmed responses after three cycles; the study was therefore closed after stage 1. Azacitidine was well tolerated. The lack of efficacy of azacitidine in pediatric patients with newly diagnosed advanced MDS highlights the need for effective new treatments in these patients