WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility.

We previously identified Waf1 Cip1 stabilizing protein 39 (WISp39) as a binding partner for heat shock protein 90 (Hsp90). We now report that WISp39 has an essential function in the control of directed cell migration, which requires WISp39 interaction with Hsp90. WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge. WISp39 binds Coronin 1B, known to regulate the Arp2/3 complex and Cofilin at the leading edge. WISp39 preferentially interacts with phosphorylated Coronin 1B, allowing it to complex with Slingshot phosphatase (SSH) to dephosphorylate and activate Cofilin. WISp39 also regulates Arp2/3 complex localization at the leading edge. WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant. We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge.

Maternal embryonic leucine zipper kinase regulates pancreatic ductal, but not ß-cell, regeneration.

The maternal embryonic leucine zipper kinase (MELK) is expressed in stem/progenitor cells in some adult tissues, where it has been implicated in diverse biological processes, including the control of cell proliferation. Here, we described studies on its role in adult pancreatic regeneration in response to injury induced by duct ligation and ß-cell ablation. MELK expression was studied using transgenic mice expressing GFP under the control of the MELK promoter, and the role of MELK was studied using transgenic mice deleted in the MELK kinase domain. Pancreatic damage was initiated using duct ligation and chemical beta-cell ablation. By tracing MELK expression using a MELK promoter-GFP transgene, we determined that expression was extremely low in the normal pancreas. However, following duct ligation and ß-cell ablation, it became highly expressed in pancreatic ductal cells while remaining weakly expressed in α-cells and ß- cells. In a mutant mouse in which the MELK kinase domain was deleted, there was no effect on pancreatic development. There was no apparent effect on islet regeneration, either. However, following duct ligation there was a dramatic increase in the number of small ducts, but no change in the total number of duct cells or duct cell proliferation. In vitro studies indicated that this was likely due to a defect in cell migration. These results implicate MELK in the control of the response of the pancreas to injury, specifically controlling cell migration in normal and transformed pancreatic duct cells.

Failure of cell cleavage induces senescence in tetraploid primary cells.

Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen-transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the γ-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-ß-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest.

TD-60 is required for interphase cell cycle progression.

We previously identified TD-60 (RCC2) as a mitotic centromere-associated protein that is necessary for proper completion of mitosis. We now report that TD-60 is an essential regulator of cell cycle progression during interphase. siRNA suppression blocks progression of mammalian G1/S phase cells and progression of G2 cells into mitosis. Prolonged arrest occurs both in non-transformed cells and in transformed cells lacking functional p53. TD-60 associates with Rac1 and Arf6 and has recently been demonstrated to be an element of α5ß1 integrin and cortactin interactomes. These associations with known elements of cell cycle control, together with our data, suggest that TD-60 is an essential component of one or more signaling pathways that drive cell cycle progression. During mitosis, TD-60 is required for correct assembly of the mitotic spindle and activation of key mitotic proteins. In contrast, in interphase TD-60 promotes cell cycle progression through what must be distinct mechanisms. TD-60 thus appears to be one of the growing categories of proteins that "moonlight," or have more than one distinct cellular function.

Glioblastoma motility occurs in the absence of actin polymer.

In fibroblasts and keratocytes, motility is actin dependent, while microtubules play a secondary role, providing directional guidance. We demonstrate here that the motility of glioblastoma cells is exceptional, in that it occurs in cells depleted of assembled actin. Cells display persistent motility in the presence of actin inhibitors at concentrations sufficient to fully disassemble actin. Such actin independent motility is characterized by the extension of cell protrusions containing abundant microtubule polymers. Strikingly, glioblastoma cells exhibit no motility in the presence of microtubule inhibitors, at concentrations that disassemble labile microtubule polymers. In accord with an unconventional mode of motility, glioblastoma cells have some unusual requirements for the Rho GTPases. While Rac1 is required for lamellipodial protrusions in fibroblasts, expression of dominant negative Rac1 does not suppress glioblastoma migration. Other GTPase mutants are largely without unique effect, except dominant positive Rac1-Q61L, and rapidly cycling Rac1-F28L, which substantially suppress glioblastoma motility. We conclude that glioblastoma cells display an unprecedented mode of intrinsic motility that can occur in the absence of actin polymer, and that appears to require polymerized microtubules.

Enzymes involved in the metabolism of gamma-hydroxybutyrate in SH-SY5Y cells: identification of an iron-dependent alcohol dehydrogenase ADHFe1.

The metabolism of the endogenous metabolite gamma-hydroxybutyrate (GHB) has been studied in a human neuroblastoma cell line SH-SY5Y as a model for examining neuronal metabolism. We show that GHB can be synthesized and released from these cells, indicating that pathways for GHB synthesis and secretion are present. Activities for the major enzymes that are involved in GHB metabolism are reported, and transcripts for AKR1A1, AKR7A2, ALDH5A1 and GABA-T can be detected by RT-PCR. We also demonstrate the presence of the ADHFe1 transcript, a gene that has been reported to encode a hydroxyacid-oxoacid transhydrogenase (HOT). We show that the ADHFe1 gene is related to bacterial GHB dehydrogenases and has a conserved NAD-binding site. The potential for using the SH-SY5Y cell line for investigating GHB catabolism is discussed.

Mitosis persists in the absence of Cdk1 activity when proteolysis or protein phosphatase activity is suppressed.

Cellular transition to anaphase and mitotic exit has been linked to the loss of cyclin-dependent kinase 1 (Cdk1) kinase activity as a result of anaphase-promoting complex/cyclosome (APC/C)-dependent specific degradation of its cyclin B1 subunit. Cdk1 inhibition by roscovitine is known to induce premature mitotic exit, whereas inhibition of the APC/C-dependent degradation of cyclin B1 by MG132 induces mitotic arrest. In this study, we find that combining both drugs causes prolonged mitotic arrest in the absence of Cdk1 activity. Different Cdk1 and proteasome inhibitors produce similar results, indicating that the effect is not drug specific. We verify mitotic status by the retention of mitosis-specific markers and Cdk1 phosphorylation substrates, although cells can undergo late mitotic furrowing while still in mitosis. Overall, we conclude that continuous Cdk1 activity is not essential to maintain the mitotic state and that phosphatase activity directed at Cdk1 substrates is largely quiescent during mitosis. Furthermore, the degradation of a protein other than cyclin B1 is essential to activate a phosphatase that, in turn, enables mitotic exit.

TNFalpha induces chromosomal abnormalities independent of ROS through IKK, JNK, p38 and caspase pathways.

A role for pro-inflammatory cytokines in inflammation-related cancers has been suggested, but mechanisms are not defined. Here, we demonstrate that treatment of HeLa cells with TNFalpha increases chromosomal aberration. In contrast, IL-1beta did not increase, but rather decreased chromosomal aberration. TNFalpha and IL-1beta increased the production of H2O2 to similar levels in cells, suggesting that increased production of reactive oxygen species might not be the premier factor involved. Reducing H2O2 through overexpression of catalase or treatment of cells with NAC or BHA did not have an effect on TNF-induced chromosomal aberration. TNFalpha-induced NO production has been implicated in DNA damage. Inhibiting NO did not reduce TNF-induced chromosomal aberration. Inhibiting IKK, JNK, and p38 kinase as well as caspases decreased TNF-induced chromosomal aberration, and a correlation between TNF-induced apoptosis and CA generation was not found. Single-strand DNA breaks give rise to double-strand breaks, which then results in chromosomal breaks, when replication forks reach the single-strand breaks during S-phase. In cells progressing through S-phase, TNFalpha activation of IKK, JNK, and p38 is significantly reduced. However, these kinases were activated by IL-1beta in S-phase. The possibility that these pathways, in a TNF-specific manner, may regulate either the generation of single- and double-strand breaks or their repair, thereby resulting in increased chromosomal aberration, is discussed.

Iron-mediated H2O2 production as a mechanism for cell type-specific inhibition of tumor necrosis factor alpha-induced but not interleukin-1beta-induced IkappaB kinase complex/nuclear factor-kappaB activation.

Coordinated and specific regulation of tumor necrosis factor (TNF) and interleukin (IL)-1 signaling pathways and how and whether they are modified by different agents are key events for proper immune responses. The IkappaB kinase complex (IKK)/NF-kappaB and JNK/AP-1 pathways are central mediators of TNF and IL-1 during inflammatory responses. Here we show that l-mimosine, a toxic non-protein amino acid that has been shown to reduce serum TNFalpha levels and affect inflammatory responses, specifically inhibits TNF-induced IKK but not JNK in a cell type-specific manner. l-Mimosine did not affect IKK and NF-kappaB activation by IL-1beta. l-Mimosine caused cell cycle arrest at G(1)-S phase, but inhibition of IKK was found to be independent of cell cycle arrest. Treatment of cells with l-mimosine resulted in production of H(2)O(2). Addition of FeSO(4) restored IKK activation by TNFalpha as did ectopic expression of catalase or pretreatment of cells with N-aceltyl-l-cysteine, indicating a role for intracellular H(2)O(2) as a mediator of inhibition. Cleavage and degradation of TNF pathway components TNFR1, RIP, and Hsp90 were observed in l-mimosine and H(2)O(2) treated cells indicating a putative mechanism for selective inhibition of TNF but not IL-1beta-induced IKK activation.