Puromycin Treatment Research Articles

Heat shock proteins and cancer therapy: The trail grows hotter!

The physiological properties of the heat shock response were initially studied over three decades ago in Drosophila based on the observation that it had protective functions against teratogenic agents and heat induced animal death. While initially believed to be unique to flies, it was rapidly observed to be universal response common to all living organisms. Early mammalian studies (reviewed in ref. [1]) focused on the physiological role of heat shock proteins or HSPs in protecting the organism against heat, a phenomenon called thermotolerance. It was determined that HSPs were able to protect cellular proteins and therefore cells from proteotoxic stress such as heat shock. This general function of HSPs are not restricted to stress protection, but is an essential element in the natural operation of cellular machinery which can also lead to protein damage under non-stress conditions in a protein concentrated intracellular environment. Such interactions of the HSP with cellular proteins would protect the substrate protein from the damaging effects of heat and other proteotoxic conditions as well as to aid in the clearance of some damaged proteins via degradation. In cancer research, HSPs were initially perceived to play a role protecting tumors from hyperthermia when applied in multiple treatments, i.e. HSPs induced initially would protect the tumor from later treatments [2]. It took many more years to begin to appreciate that there are other and potentially more significant roles for HSPs in cancer. For example, it was recognized long ago that interference with HSP90, could revert tumor cells from a transformed to a normal phenotype due to the role of HSP90 in facilitating the functions of tyrosine kinases necessary in cell transformation. The ability to block HSP90 function has since been aggressively pursued as a cancer therapy using specific HSP90 inhibitors such as geldanamycin [3]. Later studies revealed the additional and more general role for HSPs in cancer. All proliferating cells require an increase in HSP chaperone concentrations due to an increase in macromolecular activities that occur compared to quiescent cells. This increase in activity can be expected to lead to an increase in various phenomena such as free radical formation and related protein damage in proliferating cells. In addition to this, the increased accumulation of mutated proteins expected in cancer also poses a further likelihood for cell damage compared to normal cells. Combined, these phenomena can result in the protein damage signals leading to the enhanced heat shock response and HSP levels, protecting the cancer cells from the accumulation of misfolded proteins. Thus, cancer cells can have an already activated heat shock response, at least in part, relative to normal cells. While a heat shock response can be robust, the pre-stress activated response in cancer suggests that cancer cells would be less capable of mounting a further strong response relative to normal cells. Therefore, additional proteotoxic stress such as hyperthermia may be more toxic to cancer cells relative to normal cells. A new paper by Neznanov et al [4] examines this hypothesis. It proposes that that proteotoxic stress such as hyperthermia might be more effective in killing cancer relative to normal tissues since a level of heat shock response is already partially occupied in cancer cells. Neznanov et al examined the combinations of three forms of proteotoxic stress, such as hyperthermia, puromycin treatment, and bortezomib treatment (which blocks protein degradation thereby increasing the accumulation of damaged proteins that would otherwise be eliminated). They show that these treatments caused substantial control of multiple myeloma cell growth, in vitro and in vivo. The increased cell death in cancer cells was found to be largely due to a p53-independent apoptotic process. This study provides tantalizing evidence that cancer cells are already addicted to the heat shock response for their own survival as hypothesized. The same group has previously used another approach to achieve a similar outcome. They have demonstrated that the inhibition of HSP expression or function through either inhibitors of the transcription factor heat shock factor 1or inhibitors of HSP70 function can also be toxic to cancer cells, again based on the same rationale, i.e. the enhanced need for HSPs in cancer cells [5]. Therefore the heat shock response has important survival functions in the development and progression of cancer. Importantly, studies from the Calderwood group also connect the heat shock response with carcinogenesis [6]. This information introduces new targets to exploit in cancer therapy. It is uplifting to see the very early and pioneering studies on the protective functions of HSPs in Drosophila being now applied to cancer therapy.

Proteotoxic stress targeted therapy (PSTT)

About a decade ago a multiple myeloma patient demonstrated a complete response to a proteasome inhibitor Bortezomib. This case generated a lot of hopes, and after successful clinical trials Bortezomib was approved by FDA and became the drug of choice for this disease. A high sensitivity of multiple myeloma to proteasome inhibition is probably related to extensive production immunoglobulins by the cancer cells. Bortezomib prevents degradation of these proteins, which accumulate and cause a strong ER stress, eventually triggering apoptosis [1, 2]. Unfortunately, many patients show only partial response to Bortezomib, in part due to the development of resistance [3]. Among multiple mechanism of resistance to Bortezomib, cells upregulate expression of molecular chaperones, e.g. Hsp27, which protect from the buildup of abnormal protein species [4-7]. The balance between toxicity of abnormal polypeptides and protective function of chaperones defines the outcome of the treatment with proteasome inhibitors. Bortezomib has also been tested in trials for treatment of solid tumors, either alone or in combination with other drugs, and results have been promising. The selective sensitivity of various cancer cells to proteasome inhibitors as compared to normal cells is probably because cancer cells have to handle higher levels of abnormal polypeptides, since they are exposed to adverse conditions of tumor microenvironments, oxidative stress and because of genetic instability and associated accumulation of mutant proteins [8]. Accordingly, cancer cells often have increased levels of molecular chaperones, like Hsp72 or Hsp27, as well as the heat shock transcription factor Hsf1 [9]. Furthermore, it was found that cancers have special requirements for these factors, since Hsf1 and hsp72 knockout mice demonstrate dramatic resistance to certain types of cancer, e.g. skin or breast cancer [10-12]. These data led to a concept of the “non-oncogene addiction” of cancers to Hsf1 and molecular chaperones, and predicted that inhibition of chaperones could be a potent novel approach towards development of therapeutics [8]. In fact, there have been number of publications that pharmacological inhibition or siRNA-mediated depletion of Hsf1, Hsp72 or Hsp27 can selectively cause apoptosis or growth inhibition in various types of cancer [9, 13]. Furthermore, anti-sense RNA against Hsp27 has been tested and demonstrated promising results with bladder cancer [14, 15]. Since downregulation of Hsf1 or chaperones increases the generation of abnormal proteins, while inhibition of proteasome blocks their degradation, the combination of these treatments could synergistically precipitate a selective demise of cancer cells. Indeed, it was demonstrated that downregulation or inhibition of Hsf1 can potently enhance sensitivity of cancer cells to Bortezomib [16, 17]. Accordingly, a lot of efforts and resources have been invested in academic labs and industry in development of inhibitors of Hsf1 and various chaperones, but so far there have been no breakthroughs. The paper by Neznanov et al. [18] offers an alternative approach towards sensitization of cancer cells to proteasome inhibitors. Instead of inhibition of chaperones, this group proposes to cause a buildup of abnormal polypeptides by heat shock. They demonstrated that a mild non-toxic heat shock strongly enhances apoptosis caused by sub-toxic concentrations of Bortezomib. Although there are no data on the effects of heat shock beyond cell culture models, an interesting possibility is that a combination of local hyperthermia and Bortezomib could be beneficial for treatment of localized tumors. Furthermore, since only mild heat treatment was necessary for the synergistic effect with Bortezomib, there is a possibility that the fever-range temperature, which could be triggered by pyrogenes can be sufficient for enhancement of Bortezomib-induced anti-cancer effects. This possibility opens up a range of approaches towards treatment. As an alternative way of generating abnormal proteins, the authors used puromycin. This inhibitor accepts growing polypeptide chains, and aborts further growth, leading to the release of incomplete chains, which of course cannot fold properly and are toxic. At low sub-toxic concentrations, puromycin could strongly potentiate killing of cancer cells by Bortezomib. In this set of experiments, the authors tested not only cell culture models, but also a mouse model of multiple myeloma. They demonstrated that Bortezomib alone only partially reduced growth of multiple myeloma tumors transplanted into syngenic animals. On the other hand, a combination of Bortezomib and low doses of puromycin led to almost complete inhibition. Overall, the combination of proteasome inhibitors with puromycin, heat shock or other treatments that generate a buildup of abnormal polypeptides represents a novel, and potentially powerful approach towards cancer treatment.

The ribosomal protein RACK1 is required for microRNA function in both C. elegans and humans.

Despite the importance of microRNAs (miRNAs) in gene regulation, it is unclear how the miRNA-Argonaute complex--or miRNA-induced silencing complex (miRISC)--can regulate the translation of their targets in such diverse ways. We demonstrate here a direct interaction between the miRISC and the ribosome by showing that a constituent of the eukaryotic 40S subunit, receptor for activated C-kinase (RACK1), is important for miRNA-mediated gene regulation in animals. In vivo studies demonstrate that RACK1 interacts with components of the miRISC in nematodes and mammals. In both systems, the alteration of RACK1 expression alters miRNA function and impairs the association of the miRNA complex with the translating ribosomes. Our data indicate that RACK1 can contribute to the recruitment of miRISC to the site of translation, and support a post-initiation mode of miRNA-mediated gene repression.

Impairment of FOS mRNA induction by a translation inhibitor puromycin in granulocytes from myelodysplastic syndrome patients

Impairment of <i>FOS</i> mRNA induction by a translation inhibitor puromycin in granulocytes from myelodysplastic syndrome patients

Melatonin promotes puromycin‐induced apoptosis with activation of caspase‐3 and 5′‐adenosine monophosphate‐activated kinase‐alpha in human leukemia HL‐60 cells

Melatonin, a naturally occurring molecule, is produced by the pineal gland in a circadian manner to regulate biologic rhythms in humans. Recent studies report that melatonin may be an attractive candidate as an anticancer agent or for combined therapy because of its antioxidant, oncostatic and immunoregulatory activities. In this study, the potentiating effect of melatonin was evaluated on the apoptosis induced by puromycin as an anticancer drug in acute promyelocytic leukemia HL-60 cells. Melatonin did not show significant cytotoxicity against HL-60 cells compared to puromycin. However, melatonin significantly augmented the cytotoxicity of puromycin. Consistently, combined treatment of melatonin and puromycin reduced the expression of anti-apoptotic proteins, such as bcl-2 and bcl-x(L) , and also induced caspase-3 activation and poly (ADP-ribose) polymerase (PARP) cleavage compared to puromycin treatment alone. Furthermore, cell cycle analysis revealed that melatonin promoted puromycin-induced apoptosis by increasing the sub-G1 population, but suppressing G2/M arrest in HL-60 cells. Interestingly, melatonin activated the phosphorylation of 5'-adenosine monophosphate-activated kinase (AMPK) in combination with puromycin. Taken together, our results suggest that melatonin potentiates puromycin-induced apoptosis with caspase-3 and AMPK activation in HL-60 cells, and thus, melatonin treatment can be effectively applied to leukemia treatment as a potential sensitizer for chemotherapeutic agents.

A New Method for Selection of Angular Aqueous Plexus Cells from Porcine Eyes: A Model for Schlemm's Canal Endothelium

The authors sought to develop a technique for isolating and culturing angular aqueous plexus (AAP) cells from more plentiful porcine eyes. AAP is an analogue of Schlemm's canal. Cells were differentially selected with puromycin, a toxin often used to select brain microvascular endothelial cells based on the expression of P-glycoprotein (P-gp), a multidrug resistance efflux pump. Trabecular meshwork containing AAP was dissected and pooled from fresh porcine eyes, digested in collagenase I, washed, filtered, and cultured for 8 days in a gelatin-coated plastic flask. Cells were then selected by exposure to 4 μg/mL puromycin for 2 days in the culture medium. Cells were fixed and immunostained for P-gp, ICAM II, von Willebrand factor (vWF), VE-cadherin, and α-smooth muscle actin (α-SMA). Histology of the limbus showed that the dissection was limited to the trabecular meshwork region, including the AAP. Before puromycin treatment, cells appeared heterogeneous and polygonal, suggestive of a mixed population. More than 90% of the cells were removed by puromycin, leaving a population that appeared uniformly cobblestone-like when grown to confluence and that was contact inhibited. Puromycin-selected cells stained positively for the endothelial markers ICAM II, vWF, and VE-cadherin but negatively for α-SMA, consistent with staining patterns in whole tissue. Based on marker expression, morphology, and behavior in culture, puromycin-selected cells from porcine outflow tissues are AAP endothelial cells. Thus, porcine eyes can provide a plentiful alternative cell source for studying Schlemm's canal biology related to ocular hypertension.

Endothelial Progenitor Populations in Differentiating Embryonic Stem Cells. II. Drug Selection and Functional Characterization

Despite the enormous potential of embryonic stem cells (ESCs) for use in tissue engineering and cell therapies, clinical use of ESCs has been deterred by difficulties in obtaining desired cell populations without undifferentiated or other undesired cell phenotypes. Previously we demonstrated the time-dependent variation of endothelial specific promoter activities in differentiating mouse ESCs. Here, we evaluated the functional behavior of endothelial cells (ECs) selected using a drug resistance gene (Puro(R)) under the control of different EC promoters (Flk1, Tie1, and platelet endothelial cell adhesion molecule (PECAM), as well as vascular endothelial (VE)-cadherin). All four promoters tested yielded cells with EC-specific protein expression and acetylated low density lipoprotein, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL) uptake after puromycin treatment. However, the percentage of puromycin-surviving cells varied depending on EC promoters, indicating different levels of promoter activity. Continuous selection of ECs in puromycin reduced the presence of smooth muscle cells or undifferentiated cells. Puro-selected cells under all the EC promoters were positive for capillary-like network formation in Matrigel. Prostacyclin secretion by ESC-derived ECs showed improvement with orbital shear stress exposure corresponding to 0-16 dynes/cm(2); however, prostacyclin levels were lower than those seen in immortalized mouse EC lines. Tissue plasminogen activator (tPA) secretion by Puro-selected cells was comparable to that seen in cell lines, but tPA secretion after shear stress exposure showed mixed results, with tPA increasing upon shear under PECAM and Tie1 promoters and tPA decreasing upon shear under Flk1 and VE-cadherin promoters.

APC germ-line allele-specific expression in familial adenomatous polyposis

e22037 Background: About 13% of Familial Adenomatous Polyposis (FAP) families and 70% of Attenuated FAP families remain with unknown molecular pathogenic cause after APC and MYH mutational analyses. Also, mutations can affect specific allele expression (ASE) at the germline level. The aim of the study was to determine the presence of germline ASE in the APC gene in FAP and AFP with and without detectable APC or MYH mutations. Methods: Germline RNA from fresh frozen and/or cultured lymphocytes of 17 APC/MYH-negative Polyposis (7 FAP, 10 AFAP) families (21 individuals) and 35 APC-mutated Polyposis (30 FAP, 5 AFAP) families (60 individuals) was analyzed. Fourteen controls were also studied. ASE was investigated by single nucleotide primer extension (SNuPE) of rs2229992 APC coding SNP. Results: In controls ASE was 1.04± 0.3. We found that 17% (3 of 17) APC/MYH(-) FAP and AFAP families showed ASE (range=1.17–1.39) and ASE co-segregated with disease. ASE was more intense in short-cultured lymphocytes except for two cases and completely reversed by puromycin treatment. Eleven of 35 (31%) APC-FAP/AFAP harbored ASE (range=1.20–7.76), and the mutant allele was underexpressed in each case. ASE was restricted to splicing (4 families), nonsense (3 families) and frameshift (3 families) mutations outside of exon 15. Puromycin reversed ASE in all cases analyzed. Conclusions: APC ASE is present in a significant proportion (17%) of APC/MYH(-) FAP or AFAP. ASE, due to nonsense-mediated decay (NMD), is present in APC-FAP and is associated with specific mutation location, similar to reports for other hereditary syndromes. No significant financial relationships to disclose.

Cotranslational Targeting of XBP1 Protein to the Membrane Promotes Cytoplasmic Splicing of Its Own mRNA

Endoplasmic reticulum (ER) stress triggers the cytoplasmic splicing of XBP1 mRNA by the transmembrane endoribonuclease IRE1alpha, resulting in activation of the unfolded protein response, which maintains ER homeostasis. We show that the unspliced XBP1 (XBP1u) mRNA is localized to the membrane, although its product is neither a secretory nor a membrane protein and is released to the cytosol after splicing. Biochemical and mutagenic analyses demonstrated that membrane localization of XBP1u mRNA required its in-frame translation. An insertional frame-shift mutation greatly diminished both membrane localization and splicing of the XBP1u mRNA. Furthermore, membrane localization was compromised by puromycin treatment and required a hydrophobic region within XBP1u. These data demonstrate that the nascent XBP1u polypeptide recruits its own mRNA to the membrane. This system serves to enhance cytoplasmic splicing and could facilitate a more rapid response to ER stress, and represents a unique way of cotranslational protein targeting coupled to mRNA maturation.

A Role for NBR1 in Autophagosomal Degradation of Ubiquitinated Substrates

Autophagy is a catabolic process where cytosolic cellular components are delivered to the lysosome for degradation. Recent studies have indicated the existence of specific receptors, such as p62, which link ubiquitinated targets to autophagosomal degradation pathways. Here we show that NBR1 (neighbor of BRCA1 gene 1) is an autophagy receptor containing LC3- and ubiquitin (Ub)-binding domains. NBR1 is recruited to Ub-positive protein aggregates and degraded by autophagy depending on an LC3-interacting region (LIR) and LC3 family modifiers. Although NBR1 and p62 interact and form oligomers, they can function independently, as shown by autophagosomal clearance of NBR1 in p62-deficient cells. NBR1 was localized to Ub-positive inclusions in patients with liver dysfunction, and depletion of NBR1 abolished the formation of Ub-positive p62 bodies upon puromycin treatment of cells. We propose that NBR1 and p62 act as receptors for selective autophagosomal degradation of ubiquitinated targets.

Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability (Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is approximately 3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane.

A single Sec61-complex functions as a protein-conducting channel

During cotranslational translocation of proteins into the endoplasmic reticulum (ER) translating ribosomes bind to Sec61-complexes. Presently two models exist how these membrane protein complexes might form protein-conducting channels. While electron microscopic data suggest that a ring-like structure consisting of four Sec61-complexes build the channel, the recently solved crystal structure of a homologous bacterial protein complex led to the speculation that the actual tunnel is formed by just one individual Sec61-complex. Using protease protection assays together with quantitative immunoblotting we directly examined the structure of mammalian protein-conducting channels. We found that in native ER-membranes one single Sec61α-molecule is preferentially protected by a membrane bound ribosome, both, in the presence and absence of nascent polypeptides. In addition we present evidence that the nascent polypeptide destabilizes the ring-like translocation apparatus formed by four Sec61-complexes. Moreover, we found that after solubilization of ER-membranes a single Sec61-complex is sufficient to protect the nascent polypeptide chain against added proteases. Finally, we could show that this single Sec61-complex allows the movement of the nascent chain, when it has been released from the ribosome by puromycin treatment. Collectively, our data suggest that the active protein-conducting channel in the ER is formed by a single Sec61-complex.

Identification of a mutation in exon 27 of the RB1 gene associated with incomplete penetrance retinoblastoma

Retinoblastoma (Rb) is initiated by germline mutations in the RB1 gene. Up to date, no mutation was identified in exons 26 and 27. We have identified a 2 bp frameshift insertion in exon 27 of the RB1 gene (RBg.177008_177009dup) in a boy with unilateral Rb and his healthy father that has occurred de novo on the allele transmitted by the father's father. RT-PCR showed that the mutant +2 bp transcript is present in RNA from peripheral leukocytes after short-term culture. The level of the mutant transcript was low compared to the normal transcript indicating abnormal expression of the variant allele. The mutant transcript was further reduced after puromycin treatment suggesting that NMD is not involved. Although oncogenic mutations in the terminal exons of the RB1 gene are rare molecular testing is important as those terminal mutations can be associated with incomplete penetrance and cause high recurrence risk in family members.

Differential Effect of the Protein Synthesis Inhibitors Puromycin and Cycloheximide on Vascular Smooth Muscle Cell Viability

Recent evidence indicates that the protein synthesis inhibitor cycloheximide triggers selective macrophage death in rabbit atheroma-like lesions without affecting smooth muscle cells (SMCs) or the endothelium, thereby favoring a stable plaque phenotype. In this study, we report that puromycin, a protein synthesis inhibitor with a different mode of action but with similar ability to inhibit de novo protein synthesis, did not reveal plaque-stabilizing effects. The macrophage and the SMC content readily decreased in puromycin-treated atheroma-like lesions in rabbit carotid arteries. Moreover, puromycin induced apoptosis in macrophages and SMCs in vitro. Puromycin-treated SMCs showed signs of endoplasmic reticulum (ER) stress, as demonstrated by CCAAT/enhancer-binding protein homologous protein (CHOP) protein expression, splicing of X-box-binding protein 1 mRNA, and phosphorylation of eukaryotic translation initiation factor 2alpha. The ER stress inducer thapsigargin up-regulated CHOP protein expression in SMCs without affecting their viability, indicating that ER stress not necessarily results in cell death. Puromycin, but not thapsigargin, activated the ER stress-related caspase-12. Treatment of SMCs with a combination of cycloheximide and puromycin inhibited ER stress and partially improved SMC viability. In addition, puromycin, but not cycloheximide or thapsigargin, induced intracellular accumulation of polyubiquitinated proteins in SMCs, whereas the proteasome function was not affected. Taken together, puromycin, in contrast to cycloheximide, induces SMC apoptosis, thereby favoring an unstable plaque phenotype. SMC death upon puromycin treatment could only be partially prevented by cycloheximide, which completely blocked ER stress. However, other or additional mechanisms, such as increased polyubiquitination of proteins, might be involved in puromycin-induced SMC death.

Human Cdt1 Lacking the Evolutionarily Conserved Region That Interacts with MCM2–7 Is Capable of Inducing Re-replication

Replication initiation must be a carefully regulated process to avoid genomic instability caused by aberrant replication. In eukaryotic cells, distinct steps of protein loading (origin licensing) and replication activation are choreographed such that a cell can replicate only once per cell cycle. The first proteins recruited to the origins form the pre-replication complex. Of these proteins, Cdt1 is of interest, as it is the focus of several pathways to control replication initiation. It is degraded by two different pathways, mediated by the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) or with cyclin-Cdk2 and inhibited by geminin once cells are in S-phase, presumably to prevent reloading of pre-replication complexes once S-phase has begun. Although the requirement of Cdt1 in loading MCM2-7 is known, the mechanism by which overexpressed Cdt1 stimulates re-replication is unclear. In this study we have designed various mutations in Cdt1 to determine which portion of Cdt1 is important for re-replication, providing insight into possible mechanisms. Surprisingly, we found that mutants of Cdt1 that do not interact with MCM2-7 are able to induce re-replication when overexpressed. The re-replication is not due to titration of geminin from endogenous Cdt1 and is not accompanied by stabilization of endogenous Cdt1. Additionally, the N-terminal one-third of Cdt1 is sufficient to induce re-replication. The N terminus contains the PCNA- and cyclin-interacting motifs, and deletion of both motifs simultaneously in the overexpressed Cdt1 prevents re-replication. These findings suggest that exogenous Cdt1 induces re-replication by de-repressing endogenous Cdt1 through the titration of PCNA and cyclin.

Intracellular localization of human Ins(1,3,4,5,6)P5 2-kinase.

InsP6 is an intracellular signal with several proposed functions that is synthesized by IP5K [Ins(1,3,4,5,6)P5 2-kinase]. In the present study, we overexpressed EGFP (enhanced green fluorescent protein)-IP5K fusion proteins in NRK (normal rat kidney), COS7 and H1299 cells. The results indicate that there is spatial microheterogeneity in the intracellular localization of IP5K that could also be confirmed for the endogenous enzyme. This may facilitate changes in InsP6 levels at its sites of action. For example, overexpressed IP5K showed a structured organization within the nucleus. The kinase was preferentially localized in euchromatin and nucleoli, and co-localized with mRNA. In the cytoplasm, the overexpressed IP5K showed locally high concentrations in discrete foci. The latter were attributed to stress granules by using mRNA, PABP [poly(A)-binding protein] and TIAR (TIA-1-related protein) as markers. The incidence of stress granules, in which IP5K remained highly concentrated, was further increased by puromycin treatment. Using FRAP (fluorescence recovery after photobleaching) we established that IP5K was actively transported into the nucleus. By site-directed mutagenesis we identified a nuclear import signal and a peptide segment mediating the nuclear export of IP5K.

A High-Throughput Arrayed Lentiviral RNAi Screen To Identify Novel Oncogenes in Multiple Myeloma.

Karyotypic complexity is one of the hallmarks of multiple myeloma, complicating efforts to identify the specific genetic alterations involved in the development of multiple myeloma. To identify genes essential for myeloma proliferation, we used a high-throughput loss-of-function approach using lentivirally delivered short hairpin RNA (shRNA) library targeting human kinases as part of a Multiple Myeloma Research Foundation sponsored pilot project. Three myeloma cell lines, MM.1S, RPMI8226 and INA6, were infected in an arrayed 384-well format. All infections were performed in quadruplicate; two replicates were treated with puromycin to select for infected cells and two were left untreated. Wells in which more than 25% of the cells survived puromycin treatment, as determined by a cell viability assay, were included in future analysis. Genes in which 2 or more hairpins scored below a defined threshold were considered as ‘hits'. To identify physiologically relevant hits, we have integrated these findings with whole genome analysis of copy number and gene expression of multiple myeloma. We have identified a number of genes that are both amplified in and required for multiple myeloma cell viability. These observations suggest potential new targets for therapeutic targeting in myeloma.

Activity-dependent Neuroprotective Protein Constitutes a Novel Element in the SWI/SNF Chromatin Remodeling Complex

Complete deficiency in activity-dependent neuroprotective protein (ADNP), a heterochromatin 1-binding protein, results in dramatic changes in gene expression, neural tube closure defects, and death at gestation day 9 in mice. To further understand the cellular roles played by ADNP, the HEK293 human embryonic kidney cell line that allows efficient transfection with recombinant DNA was used as a model for the identification of ADNP-interacting proteins. Recombinant green fluorescent protein (GFP)-ADNP was localized to cell nuclei. When nuclear extracts were subjected to immunoprecipitation with specific GFP antibodies followed by polyacrylamide gel electrophoresis, several minor protein bands were observed in addition to GFP-ADNP. In-gel protein digests followed by mass spectrometry identified BRG1, BAF250a, and BAF170, all components of the SWI/SNF (mating type switching/sucrose nonfermenting) chromatin remodeling complex, as proteins that co-immunoprecipitate with ADNP. These results were verified utilizing BRG1 antibodies. ADNP short hairpin RNA down-regulation resulted in microtubule reorganization and changes in cell morphology including reduction in cell process formation and cell number. These morphological changes are closely associated with the SWI/SNF complex multifunctionality. Taken together, the current study uncovers a molecular basis for the essential function of the ADNP gene and protein.

Podocyte protection by darbepoetin: preservation of the cytoskeleton and nephrin expression

Podocyte injury is a significant contributor to proteinuria and glomerulosclerosis. Recent studies have shown a renoprotective effect of erythropoietin (EPO) during ischemic kidney disease. In this study, we examine mechanisms by which a long acting recombinant EPO analog, darbepoetin, may confer renoprotection in the puromycin aminonucleoside-induced model of nephrotic syndrome. Darbepoetin decreased the proteinuria of rats treated with puromycin. This protective effect was correlated with the immunohistochemical disappearance of the podocyte injury markers desmin and the immune costimulator molecule B7.1 with the reappearance of nephrin expression in the slit diaphragm. Podocyte foot process retraction and effacement along with actin filament rearrangement, determined by electron microscopy, were all reversed by darbepoetin treatment. The protective effects were confirmed in puromycin-induced nephrotic rats that had been hemodiluted to normal hematocrit levels. Furthermore, puromycin treatment of rat podocytes in culture caused actin cytoskeletal reorganization along with deranged nephrin distribution. All these effects in vitro were reversed by darbepoetin. Our study demonstrates that darbepoetin treatment ameliorates podocyte injury and decreases proteinuria by a direct effect on podocytes. This may be accomplished by maintenance of the actin cytoskeleton and nephrin expression.

Translocon pores in the endoplasmic reticulum are permeable to small anions

Contribution of translocon peptide channels to the permeation of low molecular mass anions was investigated in rat liver microsomes. Puromycin, which purges translocon pores of nascent polypeptides, creating additional empty pores, raised the microsomal uptake of radiolabeled UDP-glucuronic acid, while it did not increase the uptake of glucose-6-phosphate or glutathione. The role of translocon pores in the transport of small anions was also investigated by measuring the effect of puromycin on the activity of microsomal enzymes with intraluminal active sites. The mannose-6-phosphatase activity of glucose-6-phosphatase and the activity of UDP-glucuronosyltransferase were elevated upon addition of puromycin, but glucose-6-phosphatase and beta-glucuronidase activities were not changed. The increase in enzyme activities was due to a better access of the substrates to the luminal compartment rather than to activation of the enzymes. Antibody against Sec61 translocon component decreased the activity of UDP-glucuronosyltransferase and antagonized the effect of puromycin. Similarly, the addition of the puromycin antagonist anisomycin or treatments of microsomes, resulting in the release of attached ribosomes, prevented the puromycin-dependent increase in the activity. Mannose-6-phosphatase and UDP-glucuronosyltransferase activities of smooth microsomal vesicles showed higher basal latencies that were not affected by puromycin. In conclusion, translationally inactive, ribosome-bound translocons allow small anions to cross the endoplasmic reticulum membrane. This pathway can contribute to the nonspecific substrate supply of enzymes with intraluminal active centers.