The genes coding for adenosine kinase (ADK; ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) and esterase- 10 (ES-10; carboxylesterase, carboxylic-ester hydrolase, EC 3.1.1.1) are both located on chromosome 14 in the mouse. The near-diploid mouse cell line CAK is heterozygous for two electro- phoretic variants of ES-10. Recessive Adk- mutants of CAK have een isolated and analyzed for Es-10 phenotype and karyotypic abnormalities. Two classes of mutants were found with approxi- mately equal frequencies: those that remained heterozygous in the expression of Es- 10 and those that expressed only one Es- 10 allele. Of the mutants that lacked one form of ES-10, approximately half were missing most or all of one copy of chromosome 14; the others contained two copies of 14, frequently in the form of an isochro- mosome. There were no abnormalities of this chromosome found among the mutants that were Es-10 heterozygotes. These results suggest that the expression of an autosomal recessive mutation in near-diploid mouse cells is frequently associated with events that result in the segregation of a physically linked marker and part or all of a chromosome.

Stem-like “Tumor Initiating Cells” (TICs) have been isolated from a number of tumors including breast, and they are likely involved in all aspects of tumor biology including metastasis. Recent studies have established a link between epithelial stem-like properties and the epithelial-mesenchymal transition (EMT), a step that leads to invasion and metastasis. Identifying key signaling pathways that regulate both tumor initiating and metastatic events could enable more accurate prognosis and reveal important mediators of the process that could serve as novel therapeutic targets. Previous studies from our laboratory and others have shown that let-7 inhibits breast tumor initiation and metastasis. Previously, we have shown that BACH1, a transcription factor that is a direct target of let-7, promotes metastasis. Using gene expression arrays to quantify transcription in BACH1 depleted cells, we show that BACH1 regulates a number of downstream targets including Bone Metastasis Signature (BMS) genes. Utilizing an approach based on experimental and clinical validation, we identify genes implicated in both tumor initiation and metastasis. Gene set analysis confirmed that the BACH1 target genes significantly correlate to the metastatic pathway including BACH1, let-7 and BMS gene expression across a set of more than 1200 primary human tumors, and, in tumors selected for a basal phenotype, the expression levels of individual BACH1 target genes can significantly stratify metastasis free survival in patients. Using FACS to sort metastatic breast cancer cell lines, we see a significant increase in the transcription levels of BACH1 regulated genes when comparing TICs to non-tumor initiating cells. Additionally, in metastatic cell lines known to be enriched in TICs, BACH1 depletion reduces the population of cells expressing cell surface markers (CD44+CD24-/lowESA+) specific to a tumor initiating phenotype. And finally, gene set enrichment analysis indicates that BACH1 and the BACH1 target genes are enriched in human and mouse primary breast TICs relative to non-initiating tumor cells. Taken together, these results suggest that let-7 regulates a signaling cascade involving BACH1 and BACH1 target genes that promotes both tumor initiation and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3329. doi:1538-7445.AM2012-3329

Cells have a multitude of controls to maintain their integrity and prevent random switching from one biological state to another. Raf Kinase Inhibitory protein (RKIP or PEBP1), a member of the phosphatidylethanolamine-binding protein family, is representative of a new class of modulators of signaling cascades that functions to maintain the balance of biological systems. RKIP inhibits MAP kinase (Raf-MEK-ERK), G protein–coupled receptor (GPCR), and NF-κB-signaling cascades. RKIP targets different kinases dependent on its phosphorylation state, and integrates cross talk initiated by multiple environmental stimuli. We have shown that RKIP is a unique kinase inhibitor and substrate that uses a flexible pocket to integrate ligand-binding and phosphorylation-dependent interactions to modulate the MAPK signaling pathway. This mechanism is likely conserved among RKIP homologues in eukaryotes. RKIP also functions as a suppressor of metastasis. We have identified two mechanisms leading to altered cellular signaling and potentiation of tumorigenesis and metastasis. First, loss or depletion of RKIP results in chromosomal abnormalities and genomic instability via disregulation of the spindle checkpoint. Second, RKIP inhibits a signaling cascade involving MAPK, Myc, LIN28, let-7, and downstream let-7 targets that promotes invasion and metastasis of breast cancer cells. Our results highlight the importance of RKIP as a key metastasis suppressor and potential therapeutic agent.

Thank you for submitting your manuscript for consideration by the EMBO Journal. It has now been seen by two referees whose comments are enclosed. As you will see, both referees express interest in your work and are broadly in favour of publication, pending satisfactory revision. Both referees request a number of additional experiments, as well as various changes and clarifications to the text. I would just draw your attention to two points. Firstly, referee 1 argues that it would be important to measure let7 levels directly in breast tumour samples, and I agree that this would be very valuable; currently the use of predicted targets as a readout of let7 is rather indirect. Secondly, referee 2 questions whether the identified pathway is also relevant upon orthotopic transplantation of breast cancer cells: it would be important to address this issue experimentally.

Maintaining the integrity of the cell cycle is critical for ensuring that cells only undergo DNA replication and proliferation under controlled conditions in response to discrete stimuli. One mechanism by which the fidelity of this process is guaranteed is through the activation of cell cycle checkpoints. The mitotic spindle checkpoint, which is regulated by Aurora B kinase, ensures proper kinetochore attachment to chromosomes leading to equal distribution of chromosomes to daughter cells. We demonstrated that the mitogen-activated protein kinase (MAPK) cascade regulates mitotic progression and the spindle checkpoint. As demonstrated by immunofluorescence at kinetochores, depletion of Raf Kinase Inhibitory Protein (RKIP), an inhibitor of Raf/MEK/ERK signaling, causes an increase in MAPK activity that inhibits Aurora B kinase activity. By monitoring mitotic index and transit time from nuclear envelope breakdown to anaphase, we demonstrated that RKIP depletion leads to a defective spindle checkpoint and genomic instability, particularly in response to drugs that disrupt microtubule function.

For cells to function properly the process of translating RNA messengers into proteins needs to be accurate, on the whole. Yet work in HeLa cells now shows that about 1% of methionine residues used in protein synthesis are aminoacylated to 'textbook-incorrect' tRNAs. Surprisingly, the proportion of Met-misacylated tRNAs increases significantly when cells are under stress through viral infection or treatment with viral or bacterial Toll-like receptor ligands. Tests with other amino acids indicate that the phenomenon is limited to Met, and as Met residues are known to protect proteins against damage from reactive oxygen species, one possibility is that Met-misacylation is a natural protective response to cellular stress. Accurate transfer RNA (tRNA) aminoacylation is necessary for translational fidelity; however, the accuracy of tRNA aminoacylation in vivo is uncertain. In mammalian cells, approximately 1% of methionine residues used in protein synthesis are now shown to be aminoacylated to non-methionyl-tRNAs. Furthermore, misacylation of methionine increases up to tenfold upon exposing cells to viruses, toll-like receptor ligands or oxidative stress. Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings1,2. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower3,4,5,6. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage7, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.

Raf kinase inhibitory protein (RKIP) negatively regulates the MAP kinase (MAPK), G protein-coupled receptor kinase-2, and NF-kappaB signalling cascades. RKIP has been implicated as a metastasis suppressor for prostate cancer, but the mechanism is not known. Here, we show that RKIP inhibits invasion by metastatic breast cancer cells and represses breast tumour cell intravasation and bone metastasis in an orthotopic murine model. The mechanism involves inhibition of MAPK, leading to decreased transcription of LIN28 by Myc. Suppression of LIN28 enables enhanced let-7 processing in breast cancer cells. Elevated let-7 expression inhibits HMGA2, a chromatin remodelling protein that activates pro-invasive and pro-metastatic genes, including Snail. LIN28 depletion and let-7 expression suppress bone metastasis, and LIN28 restores bone metastasis in mice bearing RKIP-expressing breast tumour cells. These results indicate that RKIP suppresses invasion and metastasis in part through a signalling cascade involving MAPK, Myc, LIN28, let-7, and downstream let-7 targets. RKIP regulation of two pluripotent stem cell genes, Myc and LIN28, highlights the importance of RKIP as a key metastasis suppressor and potential therapeutic agent.

Raf Kinase Inhibitory Protein (RKIP), a member of the Phosphatidylethanolamine Binding Protein family, is a negative regulator of the Raf‐1/MAPK pathway. Recently, locostatin was reported to bind RKIP and inhibit cell migration by a mechanism involving Raf kinase and RKIP. We have analyzed the interaction of RKIP with locostatin and examined the biological consequences of locostatin binding to RKIP. NMR studies [SK1]showed that a locostatin precursor binds the conserved PE binding pocket of RKIP with no effect on its association with Raf‐1, nor phosphorylation by PKC at a site that prevents RKIP interaction with Raf‐1. Moreover, exposure of wild type, RKIP‐depleted HeLa cells or RKIP‐deficient MEFs[SK2] to locostatin had no effect on MAP kinase activation. However, locostatin induced cytoskeletal defects and spindle abnormalities and resulted in inhibition of cell migration following wounding which was exacerbated in RKIP deficient MEFs. These results show that locostatin binds to the RKIP ligand‐binding pocket but alters cytoskeletal structure and inhibits migration independently of RKIP and Raf/MAPK signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.[SK3][SK1]should we use the past tense for our results?[SK2]is "MEFs" a common term?[SK3]I like this conclusion!

Raf kinase inhibitory protein (RKIP or PEBP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. We now show that RKIP associates with centrosomes and kinetochores and regulates the spindle checkpoint in mammalian cells. RKIP depletion causes decreases in the mitotic index, the number of metaphase cells, and traversal times from nuclear envelope breakdown to anaphase, and an override of mitotic checkpoints induced by spindle poisons. Raf-1 depletion or MEK inhibition reverses the reduction in the mitotic index, whereas hyperactivation of Raf mimics the RKIP-depletion phenotype. Finally, RKIP depletion or Raf hyperactivation reduces kinetochore localization and kinase activity of Aurora B, a regulator of the spindle checkpoint. These results indicate that RKIP regulates Aurora B kinase and the spindle checkpoint via the Raf-1/MEK/ERK cascade and demonstrate that small changes in the MAP kinase (MAPK) pathway can profoundly impact the fidelity of the cell cycle.

Laboratory Investigations Mitogenesis in glioblastoma multiforme cell lines: a role for NGF and its TrkA receptors Harvey S. Singer, Bendt Hansen, Daniel Martinie and Christopher L. Karp 1–8 ACNU, MTX and 5-FU penetration of rat brain tissue and tumors Tzuu-Yuan Huang, Norio Arita, Toru Hayakawa and Yukitaka Ushio 9–17 Differential effect of cycloheximide on neuronal and glioma cells treated with chemotherapy and radiation K.S. Clifford Chao, J.S. Joy Hsu, Jan Xu, Uthayashanker R. Ezekiel, Eva Eves, Marsha Rosner and Chung Y. Hsu 19–26

Generation of oxidative stress/reactive oxygen species (ROS) is one of the causes of neuronal apoptosis. We have examined the effects of ROS at the transcriptional level in an immortalized hippocampal neuronal cell line (H19-7) and in rat primary hippocampal neurons. Treatment of H19-7 cells with hydrogen peroxide (150 micro m) resulted in a 40% decrease in Bcl-2 protein and a parallel decrease in bcl-2 mRNA levels. H19-7 cells overexpressing bcl-2 were found to be resistant to ROS-induced apoptosis. We had previously shown that bcl-2 promoter activity is positively regulated by the transcription factor cyclic AMP response element binding protein (CREB) in neurons. In the present study, we demonstrate that ROS decreases the activity of luciferase reporter gene driven by a cyclic AMP response element site containing bcl-2 promoter. Exposure of neurons to ROS for 6 h resulted in basal and fibroblast growth factor-2-stimulated phosphorylation/activation of CREB. Chronic 24 h treatment with ROS led to a significant (p < 0.01) decrease in CREB protein and CREB mRNA levels. Adenoviral overexpression of wild type CREB in H19-7 cells resulted in significant (p < 0.01) protection against ROS-induced apoptosis through up-regulation of Bcl-2 expression whereas dominant negative CREB exaggerated the injury. These findings demonstrate that loss of CREB function contributes to oxidative stress-induced neuronal dysfunction.

The type I insulin-like growth factor receptor (IGF-IR) is known to send two seemingly contradictory signals inducing either cell proliferation or cell differentiation, depending on cell type and/or conditions. H19-7 cells are rat hippocampal neuronal cells immortalized by a temperature-sensitive SV40 large T antigen that grow at 34 degrees C in epidermal growth factor or serum but differentiate at 39 degrees C when induced by basic fibroblast growth factor. At 39 degrees C, expression of the human IGF-IR in H19-7 cells induces an insulin-like growth factor (IGF) I-dependent differentiation. We have investigated the domains of the IGF-IR required for differentiation of H19-7 cells. The tyrosine 950 residue and serines 1280-1283 in the COOH terminus of the receptor are required for IGF-I-induced differentiation at 39 degrees C, although they are dispensable for IGF-I-mediated growth at 34 degrees C. Both domains have to be mutated to inactivate the differentiating function. The inability of these mutant receptors to induce differentiation correlates with mitogen-activated protein kinase activation. In contrast, inhibitors of phosphatidylinositol 3'-kinase have no effect on IGF-I-mediated differentiation of H19-7 cells, although they do inhibit the mitogenic response.

Dividing cells and non-dividing cells are distinct in their cell cycle kinetics, and react differently when facing cytotoxic stimuli. A protein synthesis inhibitor, cycloheximide (CHX), has recently been found to protect neuronal cells from oxidative stress. We investigated whether CHX exerts differential effects on dividing and non-dividing cells in the brain under cytotoxic stimuli. Mitotic C6 rat glioma cells and postmitotic neuronal cells were studied with a cytotoxic regimen combining γ-irradiation (RT) and 1,3-bis,2-chloroethyl-1-nitrosurea (BCNU). Cells were exposed to BCNU (1 µg/ml) for 15 h before γ-irradiation and incubated with CHX (1 µg/ml) from 30 min before and until 5 h after irradiation. Clonogenic assay was used to assess cytotoxic effects on C6 glioma cells. LDH assay was used for the viability of H19-7 postmitotic neuronal cells. A 2.27–3.75 fold enhancement of cytotoxicity was noticed with the addition of CHX to BCNU and 2–10 Gy of radiation. Our data demonstrated that CHX enhanced cytotoxocity of RT plus BCNU, while no additional toxicity was incurred to the postmitotic neuronal cells when CHX was added. We further studied whether the inhibition of DNA repair, assayed by single-cell DNA electrophoresis (comet assay), is a contributing factor for the enhanced cytotoxicity on C6 glioma cells.

Employing reverse transcription–polymerase chain reaction and clonal cell lines derived by retroviral transduction of the temperature sensitive simian virus 40 large T-antigen into dispersed rat embryonic hippocampal cells, we detected the ancestral gene-insulin II mRNA in three progenitor subcloned cell lines. These cell lines upon differentiation are known to express markers indicative of commitment to either neuronal (H19-7; NF+, GFAP−), glial (H19-5; GFAP+, NF−), or bipotential (H583-5; NF+, GFAP+) lineages. No duplicated, i.e., insulin I gene expression, was observed in any of the three cell lines. Induction of differentiation was associated with the persistence of insulin II mRNA and in the cells expressing a neuronal phenotype (H19-7; NF+, GFAP−) a relative doubling in insulin II mRNA level was present (P<0.05). Minimal cellular insulin immunoreactivity was detected only in a subpopulation of cells with a differentiated neuronal phenotype. Radioimmunoassayable insulin peptide in the H19-7 cellular conditioned medium revealed a 5-fold increase in the differentiated state. In contrast, peripheral sympathetic PC-12 neuronal cells both in the undifferentiated and nerve growth factor-driven differentiated states, failed to express both insulin I and insulin II genes. We conclude that insulin II is expressed by cultured rat hippocampal clonal cell lines, and not by the peripheral sympathetic PC-12 neuronal cell line.

A clonal cell line of rat embryonic hippocampal origin (H19-7) has been examined for the expression of glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). H19-7 cells grown at 33 degrees C continue to divide, however when grown at 39 degrees C in reduced levels of serum the cells undergo morphological differentiation and express neuronal properties. Immunocytochemistry demonstrated that H19-7 cells express both MR and GR when grown at either 33 degrees C or 39 degrees C. GR mRNA is readily detected in H19-7 cells by RNase protection assay. MR mRNA levels in H19-7 cells are too low to detect by RNase protection, but can be detected by RT-PCR. RT-PCR also demonstrated that H19-7 cells express more GR mRNA than primary hippocampal neurons. Since previous studies have shown that the level of MR mRNA is higher than that of GR mRNA in hippocampal neurons, these studies suggest that H19-7 cells represent hippocampal neurons immortalized at an early stage when the MR system is not yet fully differentiated.