The cytokine midkine and its receptor RPTPζ regulate B cell survival in a pathway induced by CD74

Lasting B cell persistence depends on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism essential for survival and homeostasis of normal peripheral mature B cells and chronic lymphocytic leukemia cells, regulated by the heparin-binding cytokine, midkine (MK), and its proteoglycan receptor, the receptor-type tyrosine phosphatase ζ (RPTPζ). We demonstrate that MK initiates a signaling cascade leading to B cell survival by binding to RPTPζ. In mice lacking PTPRZ, the proportion and number of the mature B cell population are reduced. Our results emphasize a unique and critical function for MK signaling in the previously described MIF/CD74-induced survival pathway. Stimulation of CD74 with MIF leads to c-Met activation, resulting in elevation of MK expression in both normal mouse splenic B and chronic lymphocytic leukemia cells. Our results indicate that MK and RPTPζ are important regulators of the B cell repertoire. These findings could pave the way toward understanding the mechanisms shaping B cell survival and suggest novel therapeutic strategies based on the blockade of the MK/RPTPζ-dependent survival pathway.     

Identification of plant cytoskeleton-interacting proteins by screening for actin stress fiber association in mammalian fibroblasts

Taking advantage of the high conservation of the cytoskeleton building blocks actin and tubulin between plant and animal kingdoms, we developed a functional genomic screen for the isolation of new plant cytoskeleton-binding proteins that uses a mammalian cell expression system. A yellow fluorescent protein (YFP)-fusion cDNA library from Arabidopsis was inserted into rat fibroblasts and screened for fluorescent chimeras localizing to cytoskeletal structures. The high-throughput screen was performed by an automated microscope. An initial set of candidate genes identified in the screen was isolated, sequenced, the full-length cDNAs were synthesized by RT-PCR and tested by biochemical approaches to verify the ability of the genes to bind actin directly. Alternatively, indirect binding via interaction with other actin-binding proteins was studied. The full-length cDNAs were transferred back to plants as YFP chimeras behind the CAMV-35S promoter. We give here two examples of new plant cytoskeletal proteins identified in the pilot screen. ERD10, a member of the dehydrin family of proteins, was localized to actin stress fibers in rat fibroblasts. Its direct binding to actin filaments was confirmed by several biochemical approaches. Touch-induced calmodulin-like protein, TCH2, was also localized to actin stress fibers in fibroblasts, but was unable to bind actin filaments directly in vitro. Nevertheless, it did bind to the IQ domains of Arabidopsis myosin VIII in a calcium-dependent manner. Further evidence for a cytoskeletal function of ERD10 was obtained in planta; GFP-ERD10 was able to protect the actin cytoskeleton from latrunculin-mediated disruption in Nicotiana benthamiana leaves.     

A Comparative Study of the Involvement of 17 Arabidopsis Myosin Family Members on the Motility of Golgi and Other Organelles

Gene families with multiple members are predicted to have individuals with overlapping functions. We examined all of the Arabidopsis (Arabidopsis thaliana) myosin family members for their involvement in Golgi and other organelle motility. Truncated fragments of all 17 annotated Arabidopsis myosins containing either the IQ tail or tail domains only were fused to fluorescent markers and coexpressed with a Golgi marker in two different plants. We tracked and calculated Golgi body displacement rate in the presence of all myosin truncations and found that tail fragments of myosins MYA1, MYA2, XI-C, XI-E, XI-I, and XI-K were the best inhibitors of Golgi body movement in the two plants. Tail fragments of myosins XI-B, XI-F, XI-H, and ATM1 had an inhibitory effect on Golgi bodies only in Nicotiana tabacum, while tail fragments of myosins XI-G and ATM2 had a slight effect on Golgi body motility only in Nicotiana benthamiana. The best myosin inhibitors of Golgi body motility were able to arrest mitochondrial movement too. No exclusive colocalization was found between these myosins and Golgi bodies in our system, although the excess of cytosolic signal observed could mask myosin molecules bound to the surface of the organelle. From the preserved actin filaments found in the presence of enhanced green fluorescent protein fusions of truncated myosins and the motility of myosin punctae, we conclude that global arrest of actomyosin-derived cytoplasmic streaming had not occurred. Taken together, our data suggest that the above myosins are involved, directly or indirectly, in the movement of Golgi and mitochondria in plant cells.The Arabidopsis (Arabidopsis thaliana) myosin gene family contains 17 members: myosin group XI, which includes 13 members (myosins XI-A, -B, -C, -D, -E, -F, -G, -H, -I, -J, and -K, MYA1, and MYA2), and myosin group VIII, which includes four members (ATM1, ATM2, myosin VIIIA, and myosin VIIIB). Both groups are related to unconventional myosin V (Berg et al., 2001; Foth et al., 2006). The Arabidopsis myosins contain a conserved motor domain with ATPase and actin-binding activities, a number of IQ domains that bind myosin light chains, a coiled-coil domain for dimerization, and a specific tail that binds different cargo (Kinkema and Schiefelbein, 1994; Tominaga et al., 2003). Using these functional domains, myosins convert chemical energy from ATP hydrolysis into physical movement along actin fibers, carrying with their tails membrane-bound organelles or RNA/protein complexes (Li and Nebenführ, 2008b).Plant myosins have been implicated in various cellular activities, such as cytoplasmic streaming (Shimmen and Yokota, 2004; Esseling-Ozdoba et al., 2008), plasmodesmata function (Baluska et al., 2001; Volkmann et al., 2003), organelle movement (Nebenführ et al., 1999; Jedd and Chua, 2002), cytokinesis (Molchan et al., 2002; Collings et al., 2003; Volkmann et al., 2003), endocytosis (Volkmann et al., 2003; Baluska et al., 2004; Samaj et al., 2005), and targeted RNA transport (Hamada et al., 2003). Actomyosin mediated cytoplasmic streaming found in various algae cells reach velocities of up to 100 μm s⁻¹, which is the fastest known myosin-mediated movement (Shimmen and Yokota, 1994).The information that exists regarding specific roles of each plant myosin is rather limited. Immunolocalization studies indicated that myosin XIs are associated with various particles in lily (Lilium longiflorum) and tobacco (Nicotiana tabacum) pollen tubes (Yokota et al., 1995), with mitochondria, plastids, and low-density membranes in maize (Zea mays) root cells (Liu et al., 2001; Wang and Pesacreta, 2004), and with endoplasmic reticulum (ER) in tobacco BY2 cells (Yokota et al., 2008). Specific antibodies against MYA2 showed that it is associated with peroxisomes in epidermal and guard cells of Arabidopsis leaves (Hashimoto et al., 2005). More recent studies using recombinant DNA fusions to fluorescent proteins showed localization of the tails of MYA2, MYA1, XI-K, and XI-I to peroxisomes (Li and Nebenführ, 2007; Reisen and Hanson, 2007) and MYA1 partially localized to Golgi (Li and Nebenführ, 2007). Furthermore, it was shown that peroxisomes, Golgi, and mitochondrial motility were arrested by dominant negative mutants of myosin XI-K and myosin XI-E (Avisar et al., 2008b; Sparkes et al., 2008). Arrest of organelle motility was also found in Arabidopsis knockout plants xi-k and mya2 (Peremyslov et al., 2008) and double mutants xi-k/mya1, xi-k/mya2, and mya2/xi-b (Prokhnevsky et al., 2008). In contrast, the association of the single globular tail domain of MYA1 or MYA2 with peroxisomes did not arrest their motility (Li and Nebenführ, 2007). Knockout plants for myosin xi-k and mya2 had root hair phenotypes (Ojangu et al., 2007; Peremyslov et al., 2008); however, all other 11 myosin XI single knockouts looked normal under regular growth conditions (Peremyslov et al., 2008). Reciprocal stimulation between dimerization via the coiled-coil domains of MYA1 and organelle binding was suggested (Li and Nebenführ, 2008a). As for myosin VIII, immunostaining studies showed that it localized to the cell periphery at plant-specific structures such as plasmodesmata and cytokinetic cell plates (Reichelt et al., 1999; Baluska et al., 2001). Recent data from our laboratory and from others confirmed the presence of myosin VIII in plasmodesmata (Golomb et al., 2008) and the cell plate (Van Damme et al., 2004) and further provided evidence for its involvement with endocytosis (Golomb et al., 2008; Sattarzadeh et al., 2008) and its colocalization with the ER (Golomb et al., 2008). In addition, it was shown that myosin VIII is involved in the plasmodesmata targeting of the beet yellows virus protein Hsp70h (Avisar et al., 2008a).We have determined the role of all 17 genes through transient overexpression of dominant negative forms in leaf epidermal cells. Fluorescent dominant negative fusions not only provide data on the subcellular location but also provide a relatively easy way of determining expression. Additionally, overexpression of dominant negative forms can expose a role of an individual member, which might be masked by redundant activity, if it was silenced. In order to undertake such a large-scale study, we needed to choose an efficient, fast, and reproducible expression system. Therefore, Agrobacterium tumefaciens-mediated transient expression in Nicotiana leaves was suitable.     

Telomere Shortening Sensitizes Cancer Cells to Selected Cytotoxic Agents: In Vitro and In Vivo Studies and Putative Mechanisms

Background

Telomere/telomerase system has been recently recognized as an attractive target for anticancer therapy. Telomerase inhibition results in tumor regression and increased sensitivity to various cytotoxic drugs. However, it has not been fully established yet whether the mediator of these effects is telomerase inhibition per se or telomere shortening resulting from inhibition of telomerase activity. In addition, the characteristics and mechanisms of sensitization to cytotoxic drugs caused by telomerase inhibition has not been elucidated in a systematic manner.

Methodology/Principal Findings

In this study we characterized the relative importance of telomerase inhibition versus telomere shortening in cancer cells. Sensitization of cancer cells to cytotoxic drugs was achieved by telomere shortening in a length dependent manner and not by telomerase inhibition per se. In our system this sensitization was related to the mechanism of action of the cytotoxic drug. In addition, telomere shortening affected also other cancer cell functions such as migration. Telomere shortening induced DNA damage whose repair was impaired after administration of cisplatinum while doxorubicin or vincristine did not affect the DNA repair. These findings were verified also in in vivo mouse model. The putative explanation underlying the phenotype induced by telomere shortening may be related to changes in expression of various microRNAs triggered by telomere shortening.

Conclusions/Significance

To our best knowledge this is the first study characterizing the relative impact of telomerase inhibition and telomere shortening on several aspects of cancer cell phenotype, especially related to sensitivity to cytotoxic drugs and its putative mechanisms. The microRNA changes in cancer cells upon telomere shortening are novel information. These findings may facilitate the development of telomere based approaches in treatment of cancer.     

A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death

The tumor suppressor functions of p19(ARF) have been attributed to its ability to induce cell cycle arrest or apoptosis by activating p53 and regulating ribosome biogenesis. Here we describe another cellular function of p19(ARF), involving a short isoform (smARF, short mitochondrial ARF) that localizes to a Proteinase K-resistant compartment of the mitochondria. smARF is a product of internal initiation of translation at Met45, which lacks the nucleolar functional domains. The human p14(ARF) mRNA likewise produces a shorter isoform. smARF is maintained at low levels via proteasome-mediated degradation, but it increases in response to viral and cellular oncogenes. Ectopic expression of smARF reduces mitochondrial membrane potential (DeltaPsim) without causing cytochrome c release or caspase activation. The dissipation of DeltaPsim does not depend on p53 or Bcl-2 family members. smARF induces massive autophagy and caspase-independent cell death that can be partially rescued by knocking down ATG5 or Beclin-1, suggesting a different prodeath function for this short isoform.     

Mutational analysis of the hMSH6 gene in familial and early-onset colorectal and endometrial cancer in Israeli patients

Familial colorectal cancer (CRC) is noted in about 15% of incident CRC cases, and at times is hallmarked by an age at diagnosis less than 50 years. Familial adenomatous polyposis (FAP) and hereditary non-polyposis colon cancer (HNPCC) account for about 40% of familial cases. Thus, the majority of familial and early-onset CRC remain genetically elusive. Similarly, the majority of familial and early onset endometrial cancer (EC), the most prevalent extracolonic tumor in HNPCC, are genetically undefined. An attractive candidate is the hMSH6 gene. Israeli patients with early onset (age under 50 years) (n = 44) and familial nonsyndromic (n = 23) CRC, and women with familial clustering of EC or CRC (n = 12), and those diagnosed with EC at, or under, the age of 50 years (n = 5) were genotyped for germ-line mutations within the hMSH6 gene. Exon-specific PCR was followed by denaturing gradient gel electrophoresis (DGGE) analysis, complemented by DNA sequencing of abnormally migrating fragments. No patients displayed a truncating mutation, and 1 CRC patient harbored a novel missense mutation (V878A). In addition, 6 previously described polymorphisms were detected. In conclusion, mutations in the hMSH6 gene occur uncommonly in Israeli patients with familial and early-onset CRC and EC.