Characterization of cyclin E expression in multiple myeloma and its functional role in seliciclib-induced apoptotic cell death

Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator--cyclin dependent kinase (CDK). Genomic instability was reported to be affected by over expression of another CDK regulator--cyclin E (CCNE). This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs). Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion-mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy.     

Protocols for dry DNA storage and shipment at room temperature

The globalization of DNA barcoding will require core analytical facilities to develop cost‐effective, efficient protocols for the shipment and archival storage of DNA extracts and PCR products. We evaluated three dry‐state DNA stabilization systems: commercial Biomatrica^® DNAstable^® plates, home‐made trehalose and polyvinyl alcohol (PVA) plates on 96‐well panels of insect DNA stored at 56 °C and at room temperature. Controls included unprotected samples that were stored dry at room temperature and at 56 °C, and diluted samples held at 4 °C and at ?20 °C. PCR and selective sequencing were performed over a 4‐year interval to test the condition of DNA extracts. Biomatrica^® provided better protection of DNA at 56 °C and at room temperature than trehalose and PVA, especially for diluted samples. PVA was the second best protectant after Biomatrica^® at room temperature, whereas trehalose was the second best protectant at 56 °C. In spite of lower PCR success, the DNA stored at ?20 °C yielded longer sequence reads and stronger signal, indicating that temperature is a crucial factor for DNA quality which has to be considered especially for long‐term storage. Although it is premature to advocate a transition to DNA storage at room temperature, dry storage provides an additional layer of security for frozen samples, protecting them from degradation in the event of freezer failure. All three forms of DNA preservation enable shipment of dry DNA and PCR products between barcoding facilities.     

Rhodopsin: Evolution and comparative physiology

A review of physicochemical properties, photochemistry, functions, and evolution of retinal-containing proteins (microbial and of metazoan rhodopsins, mostly visual rhodopsins) is provided. Comparative physiology of visual rhodopsins is considered in detail, mainly the molecular mechanisms of their spectral tuning.     

Length matters: C-terminal tails regulate Mdm2–MdmX complexes

Comment on: Dolezelova P, et al. Cell Cycle 2012; 11:953–62Mechanisms controlling the p53 regulatory network remain the focus of numerous investigations in hopes of identifying more robust cancer therapies. Both Mdm2 and MdmX are found overexpressed in tumors with wild-type p53 and represent a key molecular device modulating p53 function. Thus, examining the interplay between these three proteins becomes highly relevant in the search for new pharmacological interventions in oncology.Mdm2 is a RING-type E3 ubiquitin ligase capable of forming homo-oligomers and hetero-oligomerization with MdmX via the extreme C termini of their RING domains. Since its discovery 15 years ago, MdmX has been assigned many roles in the regulation of p53, either on its own or in concert with Mdm2. While clearly an essential negative regulator or p53 in development, its lack of intrinsic ubiquitin ligase activity has made the mechanism of p53 regulation more elusive than in the case of Mdm2. The capacity of MdmX to stimulate Mdm2-mediated p53 ubiquitination was first reported in 2003.¹ Subsequent biochemical comparisons of the activity of Mdm2–MdmX complexes showed that not only does the presence of MdmX in the complex alter the substrate specificity of the holo-enzyme, it also allows for poly-ubiquitin chain formation on p53 (modification required for nuclear exclusion and degradation of p53).^2-4In vitro observations describing the importance of the MdmX RING domain in regulation of p53 turnover have now gained in vivo experimental support from the two knock-in animal models.^5,6 Consistent with the notion that MdmX is an essential component of p53 polyubiquitination/proteasomal degradation pathway, mice expressing either a point mutant in the MdmX RING domain or a RING domain deletion mutant succumbed to a p53-dependent embryonic lethality. These data implicate the RING domain of MdmX as the sole region of importance in the ability of MdmX to regulate p53 and, by extension, the Mdm2-MdmX complex (and not the Mdm2 homodimer), as the principle negative regulator of p53 activity during development.The growing body of evidence describing the presence of MdmX in the complex as crucial for target selectivity as well as the processivity of the holoezyme somewhat flies in the face of the existing structural data. Two published structures of the Mdm2 homodimer and Mdm2/MdmX heterodimer indicate virtually no difference in the complexes.^7,8 In the absence of structural differences, how then are such significant differences in function accomplished? A hypothesis unifying structural and functional data is brought forth by a very intriguing study from the Uldrijan group, which systematically looks at the differences between complex formation and activity of Mdm2 and MdmX.⁹ Phylogenetic analysis showed that the last cystein of the RING domain is followed by exactly 13 amino acids in all Mdm orthologs of vertebrate origin. Based on this, the authors hypothesized that not only the sequence of the C-terminal tails, but also their exact length are of central importance to the function of the complexes. Subsequent investigation of the ability of Mdm2 and MdmX proteins, which have been extended at the C terminus by 5, 14 or 18 amino acids, was designed to test the importance of the length of the C-terminal extensions. To the researchers surprise, when examined based on their ability to hetero-oligomerize and ubiquitinate p53, Mdm2 proteins behaved differently depending on whether the oligomeric partner was Mdm2 or MdmX. Dolezelova et al. present unexpected experimental evidence for the heterocomplex being structurally and functionally distinct from the Mdm2 homodimer, while providing a mechanism for the observed in vivo functional differences between the complexes. Although the work casts slight doubt on the complete accuracy of the existing structures, it nicely aligns with the above-mentioned results, showing the singular importance of the MdmX RING domain in the activity of the holoenzyme. In light of these results, additional structural studies that will take in to account reported differences between the complexes will undoubtedly be informative and contribute to our understanding of the biochemistry of RING-type ubiquitin ligases and the mechanisms regulating p53 in cells.     

Synthesis of a new organic pyrophosphate in large quantities is induced in some bacteria by oxidative stress.

Brevibacterium ammoniagenes and Micrococcus luteus were shown to synthesize up to 50 mM of a novel substance, 2-methylbutan-1,2,3,4-tetraol 2,4-cyclopyrophosphate, in response to oxidative stress created by benzyl viologen and other redox mediators under aerobic conditions. The substance, which represents greater than 50% of the extractable phosphorus, is suggested to play a role as a bacterial antistressor and is thought to be a product of condensation of two molecules of phosphoenolpyruvate whose accumulation is prompted by conversion of intracellular NADPH into an oxidized form.     

The mitogen-activated protein kinase cascade promotes myoblast cell survival by stabilizing the cyclin-dependent kinase inhibitor,p21WAF1 protein

During myogenesis, proliferating myoblasts withdraw from the cell cycle and are either eliminated by programmed cell death or differentiate into mature myotubes. Previous studies indicate that mitogen-activated protein kinase (MAPK) activity is significantly induced with the onset of terminal differentiation of C2 myoblasts. We have investigated the part played by the MAPK pathway in the differentiation of C2 myoblasts. Specific activation of MAPK by expression of an active Raf1-estrogen receptor chimera protein reduced significantly the number of myoblasts undergoing programmed cell death in the differentiation medium. Activation of Raf1 prevented the proteolytic activation of the proapoptotic caspase 9-protein during differentiation. The antiapoptotic function of Raf1 correlated with accumulation of the p21WAF1 protein resulting from its increased stability. Antisense expression of p21 was used to determine whether the p21WAF1 protein mediated the antiapoptotic activity of Raf1. Reduction of p21WAF1 protein in muscle cells abolished the antiapoptotic activity of the MAPK pathway. We conclude that MAPK contributes to muscle differentiation by preventing apoptotic cell death of differentiating myoblasts and that this activity is mediated by stabilization of the p21WAF1 protein.