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Tumour metastasis suppressors are inhibitors of metastasis but their mechanisms of action are generally not understood. We previously showed that the suppressor Raf kinase inhibitory protein (RKIP) inhibits breast tumour metastasis in part via let-7. Here, we demonstrate an integrated approach combining statistical analysis of breast tumour gene expression data and experimental validation to extend the signalling pathway for RKIP. We show that RKIP inhibits let-7 targets (HMGA2, BACH1) that in turn upregulate bone metastasis genes (MMP1, OPN, CXCR4). Our results reveal BACH1 as a novel let-7-regulated transcription factor that induces matrix metalloproteinase1 (MMP1) expression and promotes metastasis. An RKIP pathway metastasis signature (designated RPMS) derived from the complete signalling cascade predicts high metastatic risk better than the individual genes. These results highlight a powerful approach for identifying signalling pathways downstream of a key metastasis suppressor and indicate that analysis of genes in the context of their signalling environment is critical for understanding their predictive and therapeutic potential.

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 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. 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 damage, 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.

We developed clonal cell lines of human bronchial smooth muscle origin by retroviral transduction of temperature-sensitive simian virus 40 large tumor (T) antigen. These cells show increased growth potential at 33 degrees C, but on shift to the nonpermissive temperature (39 degrees C), they show diminished or arrested growth. In addition to the expected reduction in the level of large T antigen, cells shifted to 39 degrees C show increased expression of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), characteristic of cells arrested in G1 of the cell cycle. Shifted cells undergo a process of cell hypertrophy, as demonstrated by increased time of flight and forward scatter, as well as increased expression of the contractile proteins alpha-smooth muscle actin, myosin light chain kinase, and SM22. Changes in contractile protein expression were regulated primarily in a posttranscriptional manner. Phosphatidylinositol 3-kinase activity was increased in shifted cells, and chemical inhibition of phosphatidylinositol 3-kinase attenuated alpha-actin and myosin light-chain kinase expression. We have developed clonal cell lines of human bronchial smooth muscle origin that may be useful for the study of airway smooth muscle biology. Furthermore, we demonstrate that arrest of airway smooth muscle cell cycle traversal can induce cellular hypertrophy, which parallels changes observed in the airways of patients with severe asthma.

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.

Basic fibroblast growth factor (FGF2) has many roles in neuronal development and maintenance including effects on mitogenesis, survival, fate determination, differentiation, and migration. Using a conditionally immortalized rat hippocampal cell line, H19-7, and primary hippocampal cultures, we now demonstrate that FGF2 treatment differentially regulates members of the tumor necrosis factor (TNF) superfamily of death domain receptors and their ligands. H19-7 cells transferred from serum to defined (N2) medium undergo apoptosis by a Fas-dependent mechanism similar to primary neurons. In contrast, H19-7 cells treated with FGF undergo apoptosis by a Fas-independent mechanism. FGF suppresses the Fas death pathway but also induces apoptosis by activation of a TNFα death pathway in both H19-7 and hippocampal progenitor cells. Expression of the TNF receptor 1 (TNFR1) or TNFR2 in H19-7 cells is sufficient to sensitize the cells to TNFα, similar to the effects of FGF. Because TNFα can be either proapoptotic or antiapoptotic, these results provide an explanation for the divergent trophic effects of FGF2 treatment and the observation that multiple trophic inputs are required for the survival of specific neurons.

Although mitogenic and differentiating factors often activate a number of common signaling pathways, the mechanisms leading to their distinct cellular outcomes have not been elucidated. In a previous report, we demonstrated that mitogen-activated protein (MAP) kinase (ERK) activation by the neurogenic agents fibroblast growth factor (FGF) and nerve growth factor is dependent on protein kinase Cdelta (PKCdelta), whereas MAP kinase activation in response to the mitogen epidermal growth factor (EGF) is independent of PKCdelta in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells. We now show that EGF activates MAP kinase through a PKCzeta-dependent pathway involving phosphatidylinositol 3-kinase and PDK1 in H19-7 cells. PKCzeta, like PKCdelta, acts upstream of MEK, and PKCzeta can potentiate Raf-1 activation by EGF. Inhibition of PKCzeta also blocks EGF-induced DNA synthesis as monitored by bromodeoxyuridine incorporation in H19-7 cells. Finally, in embryonic rat brain hippocampal cell cultures, inhibitors of PKCzeta or PKCdelta suppress MAP kinase activation by EGF or FGF, respectively, indicating that these factors activate distinct signaling pathways in primary as well as immortalized neural cells. Taken together, these results implicate different PKC isoforms as determinants of growth factor signaling specificity within the same cell. Furthermore, these data provide a mechanism whereby different growth factors can differentially activate a common signaling intermediate and thereby generate biological diversity.

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. Interestingly, the initial DNA damage after RT plus BCNU was equivalent; whereas DNA repair was significantly less at 5 h after radiation in CHX-treated C6 glioma cells. Protecting non-dividing neuronal cells to avoid excesive functional deficit is an integral part of a successful brain tumor treatment regimen. Taking advantage of the differential effect of CHX on glioma and neuronal cells may improve tumor control without excessive neural toxicity.

A clonal cell line, AS583-8.E4.22, from the embryonic day 15 rat basal forebrain was established using retrovirus-mediated transduction of a temperature-sensitive mutant of the simian virus 40 (SV40) large tumour antigen. The cell line expresses cytoskeletal and neurotransmitter features indicative of neuronal commitment. In response to agents that increase intracellular cAMP, including forskolin and catecholamines, the cell line exhibits rapid process outgrowth and growth cone formation that does not require new gene expression or protein synthesis. The neurite outgrowth induced by catecholamines is mediated by beta 2-adrenergic receptors and is characterized by a rapid, reversible redistribution of filamentous actin. Neurons from primary cultures of embryonic day 15 basal forebrain were also found to respond to beta-adrenergic receptor agonists by enhancing growth cone formation. These results suggest that catecholamines provide cues that induce cytoskeletal rearrangements leading to neuronal process outgrowth and growth cone formation in the developing basal forebrain and possibly other neuronal progenitor cell populations. The neuronal basal forebrain cell line provides an ideal model to study the signalling mechanisms underlying the catecholamine-induced process outgrowth.

To elucidate signal transduction pathways leading to neuronal differentiation, we have investigated a conditionally immortalized cell line from rat hippocampal neurons (H19-7) that express a temperature sensitive simian virus 40 large T antigen. Treatment of H19-7 cells with the differentiating agent basic fibroblast growth factor at 39 degrees C, the nonpermissive temperature for T function, resulted in the activation of c-Raf-1, MEK, and mitogen-activated protein (MAP) kinases (ERK1 and -2). To evaluate the role of Raf-1 in neuronal cell differentiation, we stably transfected H19-7 cells with v-raf or an oncogenic human Raf-1-estrogen receptor fusion gene (deltaRaf-1:ER). deltaRaf-1:ER transfectants in the presence of estradiol for 1 to 2 days expressed a differentiation phenotype only at the nonpermissive temperature. However, extended exposure of the deltaRaf-1:ER transfectants to estradiol or stable expression of the v-raf construct yielded cells that extended processes at the permissive as well as the nonpermissive temperature, suggesting that cells expressing the large T antigen are capable of responding to the Raf differentiation signal. deltaRaf-1:ER, MEK, and MAP kinase activities in the deltaRaf-1:ER cells were elevated constitutively for up to 36 h of estradiol treatment at the permissive temperature. At the nonpermissive temperature, MEK and ERKs were activated to a significantly lesser extent, suggesting that prolonged MAP kinase activation may not be sufficient for differentiation. To test this possibility, H19-7 cells were transfected or microinjected with constitutively activated MEK. The results indicate that prolonged activation of MEK or MAP kinases (ERK1 and -2) is not sufficient for differentiation of H19-7 neuronal cells and raise the possibility that an alternative signaling pathway is required for differentiation of H19-7 cells by Raf.

Clonal neuronal cell lines of rat hippocampal origin have been developed by two alternative strategies using retroviral transduction of a temperature-sensitive simian virus 40 large tumor antigen (Tts). Initially, Tts was introduced into dividing E17 rat embryonal hippocampal cells. In method 1, cell lines were generated directly from these progenitor cells by shifting the transductants to the permissive temperature under conditions promoting proliferation. Cell lines generated by this approach derived from neuronal, glial, or bipotential lineage. Alternatively, in method 2, the E17 rat hippocampal cells were infected with the Tts gene and then cultured at the nonpermissive temperature to enrich for postmitotic pyramidal neurons. The cells were shifted to the permissive temperature to allow the Tts gene product to promote cell cycle progression and were then cloned. Both approaches yielded cells that could differentiate in culture at the nonpermissive temperature and that expressed characteristics of a neuronal phenotype, ranging from expression of neuronal proteins such as neurofilament and induction of action potentials to synapse formation. These results indicate that it is possible to generate clonal neuronal cell lines from both precursor and differentiated hippocampal neurons and provide valuable model systems for investigating hippocampal neuronal survival, maturation, and function.

Breast cancer refers to a variety of types that each derive from distinct causes and merit different treatments. It is critical to apply a variety of treatment options that target a wide range of cellular mechanisms and targets, including those that are previously unexplored to enable new routes of therapy. Our proposal explores a new and previously under-appreciated avenue which is to use specifically engineered tRNAs that are always attached with an incorrect amino acid in a human cell. This mismatch of amino acid identity to the coding capacity of the tRNA results in increased production of mutated proteins which are misfolded, leading to over-simulation of an intrinsic cellular process that causes cell death. We are studying the effect of these engineered, mischarged tRNAs on breast cancer cells. Ultimately, we aim to demonstrate the suitability of these engineered RNAs as a new class of agents for the treatment of breast tumors.

Tumour metastasis suppressors are inhibitors of metastasis but their mechanisms of action are generally not understood. We previously showed that the suppressor Raf kinase inhibitory protein (RKIP) inhibits breast tumour metastasis in part via let-7. Here, we demonstrate an integrated approach combining statistical analysis of breast tumour gene expression data and experimental validation to extend the signalling pathway for RKIP. We show that RKIP inhibits let-7 targets (HMGA2, BACH1) that in turn upregulate bone metastasis genes (MMP1, OPN, CXCR4). Our results reveal BACH1 as a novel let-7-regulated transcription factor that induces matrix metalloproteinase1 (MMP1) expression and promotes metastasis. An RKIP pathway metastasis signature (designated RPMS) derived from the complete signalling cascade predicts high metastatic risk better than the individual genes. These results highlight a powerful approach for identifying signalling pathways downstream of a key metastasis suppressor and indicate that analysis of genes in the context of their signalling environment is critical for understanding their predictive and therapeutic potential.

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.

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.

Clonal cell lines of rat embryonic hippocampal origin have been developed by using retroviral transduction of temperature-sensitive simian virus 40 large tumor antigens. The cell lines undergo morphological differentiation at the nonpermissive temperature and in response to differentiating agents. Immunocytochemical analysis indicates that various lines are derived from progenitors of neuronal, glial, and bipotential lineages. Selected neuronal lines differentiate in response to diffusible factors released by primary glia, and one line of glial lineage supports the maturation of primary neurons in culture. Selected cell lines exhibit different patterns of neurotrophin gene expression that change after differentiation. In some lines, the relative levels of neurotrophin 3 and brain-derived neurotrophic factor message expression may reflect the developmental or regional differential expression seen for these genes in the hippocampus in situ. These hippocampal cell lines, which express markers indicative of commitment to neuronal or glial lineages, are valuable for studies of development and plasticity in these lineages, as well as for studies of the regulation of neural trophic interactions.

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.

Developmental regulation of gamma-globin gene expression probably occurs through developmental-stage-specific trans-acting factors able to promote the interaction of enhancer elements located in the far upstream locus control region with regulatory elements in the gamma gene promoters and 3' A gamma enhancer located in close proximity to the genes. We have detected a nuclear protein in K562 and baboon fetal bone marrow nuclear extracts capable of binding to A+T-rich sequences in the locus control region, gamma gene promoter, and 3' A gamma enhancer. SDS/polyacrylamide gel analysis of the purified K562 binding activity revealed a single protein of 87 kDa. A K562 cDNA clone was isolated encoding a beta-galactosidase fusion protein with a DNA binding specificity identical to that of the K562/fetal bone marrow nuclear protein. The cDNA clone encodes a homeodomain homologous to the Drosophila antennapedia protein.

Store-operated calcium entry (SOCE) and TRPC protein expression were investigated in the rat-derived hippocampal H19-7 cell line. Thapsigargin-stimulated Ba2+ entry and the expression of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 mRNA and protein were observed in proliferating H19-7 cells. When cells were placed under differentiating conditions, a change in TRPC homolog expression profile occurred. The expression of TRPC1 and TRPC3 mRNA and protein dramatically increased, while the expression of TRPC4 and TRPC7 mRNA and protein dramatically decreased; in parallel a 3.4-fold increase in the level of thapsigargin-stimulated Ba2+ entry was observed and found to be inhibited by 2-aminoethoxydiphenylborane. The selective suppression of TRPC protein levels by small interfering RNA (siRNA) approaches indicated that TRPC1 and TRPC3 are involved in mediating SOCE in proliferating H19-7 cells. Although TRPC4 and TRPC7 are expressed at much higher levels than TRPC1 and TRPC3 in proliferating cells, they do not appear to mediate SOCE. The co-expression of siRNA specific for TRPC1 and TRPC3 in proliferating cells inhibited approximately the same amount of SOCE as observed with expression of either siRNA alone, suggesting that TRPC1 and TRPC3 work in tandem to mediate SOCE. Under differentiating conditions, co-expression of siRNA for TRPC1 and TRPC3 blocked the normal 3.4-fold increase in SOCE and in turn blocked the differentiation of H19-7 cells. This study suggests that placing H19-7 cells under differentiating conditions significantly alters TRPC gene expression and increases the level of SOCE and that this increase in SOCE is necessary for cell differentiation.

Phosphatidylinositol (PI) 3-kinase has been suggested to mediate cell survival. Consistent with this possibility, apoptosis of conditionally (simian virus 40 Tts) immortalized rat hippocampal H19-7 neuronal cells was increased in response to wortmannin, an inhibitor of PI 3-kinase. Downstream effectors of PI 3-kinase include Rac1, protein kinase C, and the serine-threonine kinase Akt (protein kinase B). Here, we show that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation. While ectopic expression of wild-type Akt (c-Akt) does not significantly enhance survival in H19-7 cells, expression of activated forms of Akt (v-Akt or myristoylated Akt) results in enhanced survival which can be comparable to that conferred by Bcl-2. Conversely, expression of a dominant-negative mutant of Akt accelerates cell death upon serum deprivation or differentiation. Finally, the results indicate that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or Bcl-xL or inhibition of Jun kinase activity.

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 (P0.05). 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.

To characterize the nature of programmed cell death (PCD) induced in neuronal cells during development, three regulators of apoptosis were investigated : one, the bcl-2-related genes, modulate cell survival, and the other two, the interleukin-1β converting enzyme ICE)-related enzymes and the tumor suppressor protein p53, have been implicated as mediators of apoptosis. These regulators were studied in H19-7 cells, an SV40 T ts -immortalized rat hippocampal neuronal cell line that can be differentiated with basic fibroblast growth factor at the nonpermissive temperature, resulting in a rapid attrition of cells by apoptosis. PCD occurred by two mechanisms in H19-7 cells : The first was initiated by removal of serum from undifferentiated cells, and the second was a consequence of neuronal differentiation. In differentiated H19-7 cells, the survival time was increased by both human bcl-2 and bcl-x L , and this could be reversed by bcl-x S . Addition of a peptide inhibitor of the ICE enzyme family to H19-7 cells resulted in a transient protection against differentiation-associated apoptosis, whereas no further protection was observed in the BCL-2- or BCL-X L -expressing cells. Shifting the differentiated cells to 33°C to inactivate p53 did not significantly affect the apoptotic process, indicating that apoptosis induced by neuronal differentiation is not dependent on the continued presence of p53. By contrast, in undifferentiated cells, cell loss induced by transfer to serum-free media occurred more rapidly on inactivation of large T, consistent with p53 involvement. This medium-induced decrease in cell survival could not be rescued by the ICE inhibitor but was partially rescued by BCL-2 or BCL-X L . Furthermore, studies involving expression of BCL-2 and BCL-X L alone or together revealed differences in the survival dependent on the cellular environment. These results suggest that apoptosis of neuronal cells occurs by at least two processes : one in undifferentiated cells initiated by removal of serum and one linked to differentiation. The data implicate the ICE enzyme family but not p53 in apoptosis induced by differentiation and demonstrate that either BCL-2 or BCL-X L can prolong the survival of differentiated neuronal cells.

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

To ddetermine whether postmitotic neurons can be immortalized by oncogenic transduction, we used two approaches involving conditional expression of a temperature-sensitive SV40 large T antigen (Tts). Initially, Tts was introduced into E17 rat embryonal hippocampal cells that were then cultured at the non-permissive temperature to enrich for postmitotic pyramidal neurons, and subsequently cloned at the permissive temperature. One clonal line (HMR10-3) expressed neuron-specific proteins upon differentiation, was capable of generating action potentials, and formed synapses with primary rat neurons in co-culture. Replating of these postmitotic cells at the permissive temperature resulted in reversible loss of neurofilament expression. Conditionally immortalized cell lines were also generated from the brain of an adult mouse carrying an inducible Tts transgene. These lines proliferated in a T antigen-dependent manner and expressed neuron-specific proteins upon differentiation at the non-permissive temperature. These results suggest that postmitotic neurons can be induced to enter the cell cycle without losing their commitment to a neuronal lineage.

Epidermal growth factor (EGF) functions in a bimodal capacity in the nervous system, acting as a mitogen in neuronal stem cells and a neurotrophic factor in differentiated adult neurons. Thus, it is likely that EGF signal transduction, as well as receptor expression, differs among various cell types and possibly in the same cell type at different stages of development. We used hippocampal neuronal cell lines capable of terminal differentiation to investigate changes in EGF receptor expression, DNA synthesis, and stimulation of mitogen-activated protein (MAP) kinase by EGF before and after differentiation. H19-7, the line that was most representative of hippocampal neurons, was mitogenically responsive to EGF only before differentiation and increased in EGF binding after differentiation. MAP kinase was stimulated by EGF in both undifferentiated and differentiated cells, as well as in primary hippocampal cultures treated with either EGF or glutamate. These results indicate that the activation of MAP kinase by EGF is an early signaling event in both mitotic and postmitotic neuronal cells. Furthermore, these studies demonstrate the usefulness of hippocampal cell lines as a homogeneous neuronal system for studies of EGF signaling or other receptor signaling mechanisms in the brain.

Karyotypes of recessive mutants at the autosomal adenine phosphoribosyltransferase (Aprt) locus in a clone of the neardiploid mouse CAK cell line have been analyzed. The Aprt gene is located on chromosome 8. One copy of chromosome 8 was morphologically abnormal in the parental clone (CAK-B3-Toyr13) from whichAprt − mutants were isolated. Among 22 mutants, there were ten in which one copy of chromosome 8 had been lost. Four of these were monosomic, and in the others duplication of the remaining homolog had occurred. These findings indicate that newly induced recessive mutations in cultured mammalian cells can be expressed as the result of loss of one chromosome carrying a wild-type allele, with or without duplication of the homolog carrying the mutant allele. Loss and duplication would not be detcted in cell lines lacking morphologically marked chromosomes.

We have used the eukaryotic-prokaryotic shuttle vector pSV2Neo to demonstrate that cultured mammalian somatic cells have the enzymatic machinery to mediate homologous recombination and that the frequency of this recombination can be enhanced by pretreatment of the input DNA. Two nonoverlapping deletion mutants of pSV2Neo were constructed, each affecting the bacterial aminoglycoside 3'-phosphorylase gene (the neo gene), which confers resistance to aminoglycoside antibiotics on bacteria and resistance to the antibiotic G418 on mammalian cells. Mammalian cells transfected with either deletion plasmid alone yield no G418 -resistant colonies. Cells cotransfected with both deletion plasmids yield G418 -resistant colonies with high frequency. We show that these resistant colonies result from recombination involving homologous crossing-over or gene conversion between the deletion plasmids by rescuing from the resistant cells both types of reciprocal recombinant, full-length plasmids, and doubly deleted plasmids. Cutting one of the input plasmids to generate a double-stranded gap in the neo gene considerably enhances the frequency of homologous recombination within the gene. This suggests that targeting exogenous DNA to specific sites in mammalian chromosomes could be facilitated by suitable pretreatment of the DNA.

The transmission of two non-Mendelian drug resistance markers has been studied in crosses of Chlamydomonas reinhardtii involving diploids and aneuploids with different mating type genotypes. Under normal laboratory conditions for gametogenesis, mating and zygote maturation, the transmission pattern of the non-Mendelian markers sr-u-1 (resistance to streptomycin) and spr-u-1-27-3 (resistance to spectinomycin) is primarily determined by the mating type genotypes of the parental cells. Our results confirm and expand an earlier observation suggesting that an apparent codominant function of the female (mt +) allele in regulating chloroplast gene transmission in meiosis appears to be distinct and separate from its recessive function in regulating mating behavior. The chloroplast DNA complement (as indexed by the number of extranuclear DNA-containing bodies) may exert a secondary effect on the transmission of these markers. Within a mating type group (mt+/mt- or mt-/mt-) a cell line with more chloroplast DNA tended to transmit its non-Mendelian markers more frequently than a cell line with less chloroplast DNA.

A strain of Chlamydomonas reinhardtii has been investigated which, when mated with known wild-types, produces very few viable germination products and transmits its Mendelian markers to more than half of those products. Cytogenetic observations, fluorometric measurements of DNA and genetic data all suggest that the strain, d mt-ery-M3a sr-u-1 is a stable homozygous diploid. This strain has twice as many nuclear chromatin bodies at metaphase and twice as much DNA as its haploid progenitor, and the phenotypes of its meiotic progeny are consistent with predictions based on triploid meiosis. Data from crosses involving d mt-ery-M3a sr-u-1 and from crosses involving hybrid diploids indicate that the frequency of second division segregation increases in triploid zygotes and that mitotic segregation following triploid meiosis is a frequent event which may more often result from mitotic recombination than from chromosome loss.

The cellular distribution of thymidine kinase activities in Chlamydomonas reinhardtii, as manifested by the in vivo incorporation of exogenous thymidine and 5-bromodeoxyuridine into different DNA species, appeared to be organelle specific and varied with different developmental stages in the life cycle of this organism. During vegetative growth and gametogenic differentiation, thymidine and 5-bromodeoxyuridine were shown to be selectively incorporated into chloroplast but not nuclear DNA. On the other hand, during zygotic germination in which meiosis occurs and the ensuing vegetative divisions of meiotic products, thymidine as well as 5-bromodeoxyuridine were incorporated into both nuclear and chloroplast DNA. These results suggest that, in addition to the thymidine kinase activity that is constantly present in the chloroplast, a cytoplasmic thymidine kinase is derepressed only during the sexual reproductive cycle of C. reinhardtii.

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 electrophoretic variants of ES-10. Recessive Adk- mutants of CAK have been isolated and analyzed for Es-10 phenotype and karyotypic abnormalities. Two classes of mutants were found with approximatley 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 other contained two copies of 14, frequently in the form of an isochromosome. 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.