Circadian Regulation of Temozolomide Sensitivity in Glioblastoma

The safety and efficacy of multiple cancer chemotherapeutics can vary as a function of when during the day they are delivered. This study aimed to improve the treatment of glioblastoma multiforme (GBM), the most common brain cancer, by testing the efficacy of the DNA alkylator Temozolomide (TMZ) on GBM in vitro and in vivo as a function of time of day. We found cell-intrinsic, daily rhythms in susceptibility of GBM tumor cells (mouse astrocytes deficient in NF1 and p53 signaling) to TMZ in vitro. The greatest TMZ-induced DNA damage response, activation of apoptosis and growth inhibition, occurred near the peak expression of the core clock gene Bmal1 in cultured GBM cells. Deletion of Bmal1 abolished rhythmic circadian clock gene expression and circadian rhythms in TMZ-induced activation of apoptosis and growth inhibition in GBM tumor cells in vitro. Taken together, these data suggest an important role for the core molecular clock in regulating the tumor cell-intrinsic response to TMZ-induced DNA damage. These results may be important broadly for how we design TMZ and other DNA damaging approaches to GBM treatment

Cloning and expression of a zebrafish SCN1B ortholog and identification of a species-specific splice variant

Abstract Background Voltage-gated Na+ channel β1 (Scn1b) subunits are multi-functional proteins that play roles in current modulation, channel cell surface expression, cell adhesion, cell migration, and neurite outgrowth. We have shown previously that β1 modulates electrical excitability in vivo using a mouse model. Scn1b null mice exhibit spontaneous seizures and ataxia, slowed action potential conduction, decreased numbers of nodes of Ranvier in myelinated axons, alterations in nodal architecture, and differences in Na+ channel α subunit localization. The early death of these mice at postnatal day 19, however, make them a challenging model system to study. As a first step toward development of an alternative model to investigate the physiological roles of β1 subunits in vivo we cloned two β1-like subunit cDNAs from D. rerio. Results Two β1-like subunit mRNAs from zebrafish, scn1ba_tv1 and scn1ba_tv2, arise from alternative splicing of scn1ba. The deduced amino acid sequences of Scn1ba_tv1 and Scn1ba_tv2 are identical except for their C-terminal domains. The C-terminus of Scn1ba_tv1 contains a tyrosine residue similar to that found to be critical for ankyrin association and Na+ channel modulation in mammalian β1. In contrast, Scn1ba_tv2 contains a unique, species-specific C-terminal domain that does not contain a tyrosine. Immunohistochemical analysis shows that, while the expression patterns of Scn1ba_tv1 and Scn1ba_tv2 overlap in some areas of the brain, retina, spinal cord, and skeletal muscle, only Scn1ba_tv1 is expressed in optic nerve where its staining pattern suggests nodal expression. Both scn1ba splice forms modulate Na+ currents expressed by zebrafish scn8aa, resulting in shifts in channel gating mode, increased current amplitude, negative shifts in the voltage dependence of current activation and inactivation, and increases in the rate of recovery from inactivation, similar to the function of mammalian β1 subunits. In contrast to mammalian β1, however, neither zebrafish subunit produces a complete shift to the fast gating mode and neither subunit produces complete channel inactivation or recovery from inactivation. Conclusion These data add to our understanding of structure-function relationships in Na+ channel β1 subunits and establish zebrafish as an ideal system in which to determine the contribution of scn1ba to electrical excitability in vivo.http://deepblue.lib.umich.edu/bitstream/2027.42/112585/1/12864_2007_Article_939.pd

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-7

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p>acetylated α-tubulin. : Anti Scn1ba_tv1 (green), anti-acetylated α-tubulin (red). : Anti-Scn1ba_tv2 (green), anti-acetylated α-tubulin (red). Images were viewed with an Olympus FluoView 500 confocal microscope at 100× magnification with 5× additional zoom. Scale bar: 50 μm

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-9

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p>ti-Scn1ba_tv1 produced two different staining patterns; staining at the t-tubules of striated muscle (A, D, and G), and punctate staining along the longitudinal edge of the muscle cells (arrowheads in B). Staining with anti-Scn1ba_tv2 labeled the t-tubule system and did not appear to label to muscle surface. Anti-Scn1ba_tv1 staining did not colocalize with α-BTX (D – F and H – I), suggesting that Scn1ba_tv1 is not expressed at neuromuscular junctions (arrows). Scale bar: 10 μm

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-0

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p> that are identical are indicted in red, strongly similar substitutions are indicated by (:), and weakly similar amino acids are indicated by (.). Identical resides in exon 5 of Scn1ba_tv1 and Scn1b are indicated in green. The two cysteine residues predicted to form the Ig loop are indicated in blue. The conserved regions that form the A/A' face of the Ig loop, sites of interaction with theα subunit [17], are underlined. Tyrosine-181 in Scn1b and the corresponding residues in Scn1ba_tv1 and Scn1ba_tv2 are highlighted in yellow. Predicted sites of N-linked glycosylation are indicated by ▼. These sites were determined using NetNGlyc 1.0 [61]. Transmembrane segments are indicated as boxes. Peptides used for antibody generation are underlined in blue. Predicted β-sheets in the Ig loop domain, based on the crystal structure of myelin P[62], are shown with labeled arrows and correspond to the ribbon diagram included in the lower panel. Lower panel: Proposed three-dimensional structure of the Ig domain of β1 using the crystal structure of myelin Po (PDB 1NEU) as a template. The figure was created with the KiNG Viewer program via the RCSB Protein Data Bank web site [63]. β strands corresponding to the arrows in the upper panel are labeled A through G. . Schematic showing the genomic organization of zebrafish . The positions of introns 1 through 5 (I1 – I5) are indicated. Positions of primers used for RT-PCR in panel D are indicated. The C-terminal alternate splice domains contained in and are encoded by exon 5. . Model of alternative splicing of . Exons 4 and 5 (boxes) and intron 4 (line) are illustrated. The splice acceptor sequence at the beginning of exon 5 is indicated by and the internal alternate splice acceptor site in exon 5 is indicated by a dashed line and by ▼. The location of stop codons in the resulting mRNAs are indicated. Drawings are not to scale. Consensus splice acceptor sequence [22] and the acceptor sequences found in exon 5 are indicated in the lower portion of the panel. P: pyrimidine. P: purine. Lower case: intronic sequence. Upper case: exonic sequence. The "T" indicated by the red arrow in the internal, alternate acceptor is rare and significantly weakens the site [22]. . RT-PCR from whole fish RNA demonstrating that both splice variants of are expressed in the mRNA pool. The upper band corresponds to and the lower band corresponds to . Translations of the resulting alternate C-terminal splice products are shown below. The sequence highlighted in green is found in Scn1ba_tv1 and corresponds to the green portion of exon 5 illustrated in panel C. The sequence highlighted in turquoise is found in Scn1ba_tv2 and corresponds to the turquoise portion of exon 5 illustrated in panel C

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-2

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p>ane 2: Chinese hamster lung 1610 cells transiently transfected with cDNA; Lane 3: Chinese hamster lung 1610 cells transiently transfected with cDNA; Lane 4: 5 μg rat brain membranes. Arrows indicate immunoreactive bands at ~30 kD in the transfected cells and at ~30 kD and ~38 kD in rat brain. Western blot probed with anti-Scn1ba_tv1. Lane 1: 5 μg rat brain membranes; Lane 2: 15 μg zebrafish brain membranes; Lane 3: 5 μg rat brain membranes probed with anti-Scn1ba_tv1 that had been preadsorbed to the immunizing peptide ("pre"); Lane 4: 15 μg zebrafish (zf) brain membranes probed with anti-Scn1ba_tv1 that had been preadsorbed to the immunizing peptide. Arrows indicate immunoreactive bands at ~30 kD and ~38 kD in both species. . Western blot probed with anti-Scn1ba_tv2. Lane 1: mock transfected Chinese hamster lung 1610 cells; Lane 2: Chinese hamster lung 1610 cells transiently transfected with cDNA; Lane 3: Chinese hamster lung 1610 cells transiently transfected with ; Lane 4: 5 μg rat brain membranes. Arrow indicates immunoreactive band at ~30 kD. Western blot probed with anti-Scn1ba_tv2. Lane 1: 15 μg zebrafish brain membranes; Lane 2: 15 μg zebrafish brain membranes probed with anti-Scn1ba_tv2 that had been preadsorbed to the immunizing peptide ("pre"). Arrow shows immunoreactive band at ~38 kD

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-10

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p> shows the mean current elicited by depolarization to 0 mV from a holding potential of -80 mV in oocytes injected with the indicated combinations of α and β subunits. . Current Density. Coexpression of , , or cRNA with cRNA results in increased current amplitude compared to α alone. Individual peak current amplitudes for each condition were measured and normalized to the mean current amplitude of for each experiment to account for variability between different oocyte preparations. . Representative Nacurrent traces for alone (upper left), plus (upper right), plus (lower left), and plus (lower right) . Current-voltage relationships for the families of Nacurrents shown in panel C. . Voltage dependence of activation. Coexpression of with (●), (△), or (▽) results in hyperpolarizing shifts in the voltage dependence of activation compared to the expression of alone (■). Coexpression of with results in a significantly greater hyperpolarizing shift than coexpression with . . Voltage dependence of inactivation. Coexpression of with , , or resulted in hyperpolarizing shifts in the voltage dependence of inactivation compared to alone. The effects of and on the voltage-dependence of inactivation are indistinguishable from each other. . Zebrafish β subunits speed recovery from inactivation. Coexpression of with (●), (△), or (▽) results in a dramatic increase in the rate of recovery from inactivation compared with alone (■). Zebrafish (■) expressed alone has a very slow rate of recovery, and a full recovery was never achieved during the duration of the experiment

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-8

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p> α-tubulin (red). SC: spinal cord. Scale bar: 50 μm

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-11

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p>that are identical are indicted in red, strongly similar substitutions are indicated by (:), and weakly similar amino acids are indicated by (.). Identical resides in exon 5 of Scn1ba_tv1 and Scn1b are indicated in green. The two cysteine residues predicted to form the Ig loop are indicated in blue. The conserved regions that form the A/A' face of the Ig loop, sites of interaction with theα subunit [17], are underlined. Tyrosine-181 in Scn1b and the corresponding residues in Scn1ba_tv1 and Scn1ba_tv2 are highlighted in yellow. Predicted sites of N-linked glycosylation are indicated by ▼. These sites were determined using NetNGlyc 1.0 [61]. Transmembrane segments are indicated as boxes. Peptides used for antibody generation are underlined in blue. Predicted β-sheets in the Ig loop domain, based on the crystal structure of myelin P[62], are shown with labeled arrows and correspond to the ribbon diagram included in the lower panel. Lower panel: Proposed three-dimensional structure of the Ig domain of β1 using the crystal structure of myelin Po (PDB 1NEU) as a template. The figure was created with the KiNG Viewer program via the RCSB Protein Data Bank web site [63]. β strands corresponding to the arrows in the upper panel are labeled A through G. . Schematic showing the genomic organization of zebrafish . The positions of introns 1 through 5 (I1 – I5) are indicated. Positions of primers used for RT-PCR in panel D are indicated. The C-terminal alternate splice domains contained in and are encoded by exon 5. . Model of alternative splicing of . Exons 4 and 5 (boxes) and intron 4 (line) are illustrated. The splice acceptor sequence at the beginning of exon 5 is indicated by and the internal alternate splice acceptor site in exon 5 is indicated by a dashed line and by ▼. The location of stop codons in the resulting mRNAs are indicated. Drawings are not to scale. Consensus splice acceptor sequence [22] and the acceptor sequences found in exon 5 are indicated in the lower portion of the panel. P: pyrimidine. P: purine. Lower case: intronic sequence. Upper case: exonic sequence. The "T" indicated by the red arrow in the internal, alternate acceptor is rare and significantly weakens the site [22]. . RT-PCR from whole fish RNA demonstrating that both splice variants of are expressed in the mRNA pool. The upper band corresponds to and the lower band corresponds to . Translations of the resulting alternate C-terminal splice products are shown below. The sequence highlighted in green is found in Scn1ba_tv1 and corresponds to the green portion of exon 5 illustrated in panel C. The sequence highlighted in turquoise is found in Scn1ba_tv2 and corresponds to the turquoise portion of exon 5 illustrated in panel C

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-4

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p> α-tubulin (red). OP: olfactory pit. Scale bar: 50 μm