Expression Of Α-tubulin Research Articles

Phase II Trial of Ixabepilone, an Epothilone B Analog, in Patients With Metastatic Breast Cancer Previously Untreated With Taxanes

Ixabepilone is an epothilone B analog that binds to microtubules and results in microtubule stabilization and mitotic arrest. Ixabepilone was evaluated for efficacy and safety in a phase II clinical trial for women with metastatic breast cancer. Patients were eligible if they had not previously received treatment with a taxane and had measurable metastatic breast cancer. Ixabepilone was administered at 6 mg/m(2)/d intravenously days 1 through 5 every 3 weeks until unacceptable toxicity or disease progression. Patients underwent pretreatment and post-treatment tumor biopsies, and tissues were analyzed for acetylated alpha-tubulin, tau-1, and p53 expression when possible. Twenty-three patients received 210 cycles with a median of eight cycles (range, two to 22 cycles) per patient. Thirteen patients (57%; exact 95% CI, 34.5% to 76.8%) had partial responses, six patients (26%) had stable disease, and four patients (17%) had progressive disease. Median time to progression and duration of response were 5.5 and 5.6 months, respectively. Four patients required dose reductions for neutropenia, neuropathy, or fatigue. Grade 3 or 4 toxicities included neutropenia (22%), fatigue (13%), anorexia (9%), and motor neuropathy (4%). Thirty-nine percent of patients experienced grade 1, 13% experienced grade 2, and none experienced grade 3/4 sensory neuropathy. The six patients with paired biopsies all had increases in tumor alpha-tubulin acetylation after treatment. Baseline or cycle 2 acetylated alpha-tubulin, tau-1, or p53 expression did not correlate with clinical response. Women with metastatic breast cancer previously untreated with taxanes have a meaningful durable response to single-agent ixabepilone therapy. Minimal hematologic toxicity and no grade 3 sensory neuropathy were noted.

Substrate-mediated Regulation of Cullin Neddylation

Cullin-based E3 ligases are a large family of multi-subunit ubiquitin ligases with diverse cellular functions, including the regulation of the cell cycle, of the DNA damage response, and of various transcription factors. These ligases are composed of one of six mammalian cullin homologs (Cul1, Cul2, Cul3, Cul4a, Cul4b, and Cul5), the Ring finger containing protein Roc1/Rbx1, and cullin homolog-specific adaptor and substrate recognition subunits. To be active, cullin-based ligases require the covalent modification of a conserved lysine residue in the cullin protein with the ubiquitin-like protein Nedd8. We show in this study that in intact cells Cul1 neddylation is dependent on binding to adaptor proteins and substrate recognition subunits. Mutant Cul1 that is unable to recruit adaptor and substrate recognition subunits exhibits markedly reduced neddylation, and inhibiting binding of adaptor and substrate recognition subunits to wild type Cul1 reduces Nedd8 modification. This regulatory mechanism also extends to other cullin-based E3 ligases, including Cul2, Cul3, and Cul4a. The regulation of cullin neddylation by adaptor proteins and substrate recognition subunits in cells was found to be independent of both CAND1 and the COP9 signalosome, two negative regulators of cullin Nedd8 modification. Using hypoxia-inducible factor-1alpha (HIF-1alpha), a substrate of the Elongin B/C-Cul2-VHL ligase, we demonstrate the critical role of substrate binding to promote Cul2 neddylation in a manner that does not require substrate ubiquitination but may involve a conformational change. These findings suggest a mechanism through which availability of substrate recognition subunits and substrates can regulate the ubiquitin ligase activity.

E2A-HLF Fusion Transcription Factor Mediating Aberrant Expression of LMO2 in B-Precursor ALL with t(17;19).

LMO2 is a transcription factor critical for hematopoiesis, and its ectopic expression was reported in T-ALL having translocations such as t(11;14) in which the TCRα/δ locus is fused to LMO2. Moreover, aberrant activation of LMO2 via integration of the retroviral vector was reported in T-cell malignancies developed in the patients who received IL2Rγc gene therapy for X-SCID. Consistently, transgenic mice of LMO2 under the control of thymocyte-specific promoter developed T-cell lymphoma. Although these demonstrate that LMO2 is oncogenic in T-lymphoid cells, significance of LMO2 in B-precursor ALL remains totally unclarified. Gene expression of LMO2 is regulated by distal and proximal promoters: the former generates full-length transcript and the later generates transcript lacking exons 1–3, both of which encode the same open reading frame. Recently, the binding site of PAR transcription factors was identified as a critical region for distal promoter activity of LMO2 in hematopoietic cells. A family of PAR transcription factors belongs to the bZIP factor and includes HLF, TEF and DBP. Since E2A-HLF fusion derived from t(17;19) in B-precursor ALL has bZIP domain of HLF and transactivation domain of E2A, it is feasible to postulate that E2A-HLF might induce aberrant expression of LMO2 through its distal promoter. To confirm this, we first analyzed the expression of LMO2 transcripts derived from both distal and proximal promoters in t(17;19)-ALL cell lines by real time RT-PCR with the primers for exon 4/5. GAPDH expression was used as an internal control. Of note, all of 4 t(17;19)-ALL cell lines expressed LMO2 transcripts at an equivalent level to those in T-ALL cell lines with t(11;14). Moreover, the level of LMO2 transcripts in t(17;19)-ALL cell lines was significantly higher than that in 28 other B-precursor ALL cell lines including Ph1-ALL (n=6), t(1;19)-ALL (n=7), and MLL+-ALL (n=9). Similar results were obtained by real-time RT-PCR using primers for exon 1/2, which specifically detect the LMO2 transcripts derived from the distal promoter. We next analyzed LMO2 protein expression by Western blotting using α-tubulin expression as an internal control. LMO2 was expressed in all of 4 t(17;19)-ALL cell lines as high as that in T-ALL cell lines with t(11;14), whereas it was almost undetectable in 16 out of 28 other B-precursor ALL cell lines, indicating that overexpression of LMO2 in t(17;19)-ALL is a unique property among B-precursor ALLs. Next, to directly test the possibility that E2A-HLF induces LMO2 expression, we transfected E2A-HLF into the B-precursor ALL cell line 697, whose LMO2 expression level is very low, using Zn-inducible vector. When E2A-HLF expression was induced by Zn, LMO2 transcripts as well as LMO2 proteins were immediately upregulated. The induction of LMO2 expression by E2A-HLF was dependent on the DNA-binding and transactivation activity of E2A-HLF, since the mutants lacking either the DNA-binding or transactivation domains failed to induce LMO2 expression. Further, direct binding of E2A-HLF to the PAR binding site in the distal promoter was demonstrated in EMSA. These observations not only indicate that E2A-HLF directly induces aberrant expression of LMO2 in t(17;19)-ALL but also suggest that overexpression of LMO2 might involve in the leukemogenetic potential of E2A-HLF.

Raloxifene Increases Proliferation and Up-regulates Telomerase Activity in Human Umbilical Vein Endothelial Cells

Vascular endothelial senescence is involved in human atherosclerosis. Telomerase activity is known to be critical in cellular senescence and its level is modulated by regulation of telomerase catalytic subunit (telomerase reverse transcriptase (TERT)) at both the transcriptional and post-transcriptional levels. Since the cardioprotective effect of estrogen itself has not been ruled out, we examined that of raloxifene, which has been classified as a selective estrogen receptor modulator, on the proliferation and telomerase activity of human umbilical vein endothelial cells (HUVECs). Raloxifene, like estrogen, clearly induced the telomerase activity and human TERT (hTERT) expression via estrogen receptor (ER) alpha and ERbeta. Treatment with raloxifene for 5 days significantly induced cell growth, and either cotreatment with a telomerase inhibitor, 3'-azido-3'-deoxythymidine, or transfection with hTERT-specific small interfering RNA significantly attenuated the raloxifene-induced cell growth. Raloxifene also induced the phosphorylation of Akt, and pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002, significantly attenuated the raloxifene-induced telomerase activity. In addition, raloxifene induced both the phosphorylation of hTERT and IkappaB. Moreover, cotreatment with an IkappaBalpha phosphorylation inhibitor, BAY-11-7082, or a specific NFkappaB nuclear translocation inhibitor, SN50, significantly attenuated the raloxifene-induced telomerase activity and the association of NFkappaB with hTERT. These results show that raloxifene induced the up-regulation of telomerase activity not only by the transcriptional regulation of hTERT but also by post-translational regulation of the phosphorylation of Akt and hTERT and the association of hTERT with NFkappaB in HUVECs. Thus, the up-regulation of telomerase activity in vascular endothelial cells might be one mechanism contributing to the potential atheroprotective effect of raloxifene.

Removal or Maintenance of Inositol-linked Acyl Chain in Glycosylphosphatidylinositol Is Critical in Trypanosome Life Cycle

The protozoan parasite Trypanosoma brucei is coated by glycosylphosphatidylinositol (GPI)-anchored proteins. During GPI biosynthesis, inositol in phosphatidylinositol becomes acylated. Inositol is deacylated prior to attachment to variant surface glycoproteins in the bloodstream form, whereas it remains acylated in procyclins in the procyclic form. We have cloned a T. brucei GPI inositol deacylase (GPIdeAc2). In accordance with the acylation/deacylation profile, the level of GPIdeAc2 mRNA was 6-fold higher in the bloodstream form than in the procyclic form. Knockdown of GPIdeAc2 in the bloodstream form caused accumulation of an inositol-acylated GPI, a decreased VSG expression on the cell surface and slower growth, indicating that inositol-deacylation is essential for the growth of the bloodstream form. Overexpression of GPIdeAc2 in the procyclic form caused an accumulation of GPI biosynthetic intermediates lacking inositol-linked acyl chain and decreased cell surface procyclins because of release into the culture medium, indicating that overexpression of GPIdeAc2 is deleterious to the surface coat of the procyclic form. Therefore, the GPI inositol deacylase activity must be tightly regulated in trypanosome life cycle.

Nitric Oxide-dependent Negative Feedback of PARP-1 trans-Activation of the Inducible Nitric-oxide Synthase Gene

Nitric oxide (NO) participates in a variety of physiologic and pathophysiologic processes in diverse tissues, including the kidney. Although mechanisms for cytokine induction of inducible nitric-oxide synthase (iNOS) have been increasingly clarified, the controls for termination of NO production remain unclear. Because excessive NO production can be cytotoxic to host cells, feedback inhibition of iNOS transcription would represent a means of cytoprotection. Many of the cGMP-independent functions of NO are mediated by S-nitrosylation of cysteine thiols of target proteins. We hypothesized that NO-mediated S-nitrosylation of transcription factors might serve to feedback inhibit their trans-activation potential and deactivate iNOS gene transcription. Transient transfection of murine mesangial cells with iNOS promoter deletion-luciferase constructs revealed the region -915 to -849 to be NO sensitive with respect to IL-1beta-induced promoter activity. In vitro DNase I footprinting identified a footprint at -865/-842 in the absence of NO, but not in the presence of endogenous or exogenously delivered NO. Southwestern blotting using this probe coupled with partial peptide sequencing of the protein bands revealed that poly(ADP-ribose) polymerase isoform 1 (PARP-1) bound the probe in a sequence-specific manner. Gel shift/supershift experiments and chromatin immunoprecipitation assay analysis confirmed this binding in vitro and in vivo. Functionally, mutation of the -859/-850 site to prevent PARP-1 binding or PARP-1 knockdown by RNA interference relieved the inhibitory effects of NO on iNOS promoter activity. Biotin-switch assays and co-immunoprecipitation with an anti-nitrocysteine antibody indicated that PARP-1 was S-nitrosylated. We conclude that NO feedback inhibits iNOS gene transcription by S-nitrosylating the trans-activator PARP-1 and decreasing its binding and/or action at the iNOS promoter.

The Cytotoxic Lymphocyte Protease, Granzyme B, Targets the Cytoskeleton and Perturbs Microtubule Polymerization Dynamics

Granzyme B, a serine protease derived from cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell granules, plays an important role in coordinating apoptosis of CTL and NK target cells. Here, we report that granzyme B targets the cytoskeleton by cleaving and removing the acidic C-terminal tail of alpha-tubulin. Consistent with this, Granzyme B markedly enhanced rates of microtubule polymerization in vitro, most likely by removal of an autoinhibitory domain within the tubulin C terminus. Moreover, delivery of Granzyme B into HeLa target cells promoted dramatic reorganization of the microtubule network in a caspase-independent manner. These data reveal that granzyme B directly attacks a major component of the cell cytoskeleton, which may contribute to the incapacitation of target cells during CTL/NK-mediated killing.

Immunoglobulin E Binding Reactivity of a Recombinant Allergen Homologous to α-Tubulin from Tyrophagus putrescentiae

Storage mites may cause allergic respiratory diseases in urban areas as well as pose an occupational hazard in rural areas. Characterization of storage mite allergens is important for the development of diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report on the cloning and expression of alpha-tubulin from the storage mite (Tyrophagus putrescentiae). The deduced amino acid sequence of the alpha-tubulin from the storage mite showed as much as 97.3% identity to the alpha-tubulin sequences from other organisms. The highly conserved amino acid sequences of alpha-tubulins across different species of mites may indicate that cross-reactivity for this potential allergen exists. The frequency of immunoglobulin E reactivity of this recombinant protein is 29.3% in sera from storage mite-allergic subjects.

Immunohistochemical detection of cdc2 is useful in predicting survival in patients with mantle cell lymphoma

Recent cDNA microarray studies have reported the prognostic value of several genes in mantle cell lymphoma patients. We aimed to validate the prognostic significance of three of these genes: α-tubulin, cdc2, and CENP-F. The protein expression of α-tubulin, cdc2, and CENP-F was assessed using immunohistochemistry. Their immunoreactivity in 48 formalin-fixed/paraffin-embedded mantle cell lymphoma tumors was determined by estimating the percentage of positive cells. These results were correlated with the expression of proliferation marker Ki67 and survival. Of these 48 mantle cell lymphoma patients, 41 were men and seven were women. The median age at time of diagnosis was 64.5 years, and the overall median survival was 40 months. In benign lymph nodes, the expression of cdc2 and α-tubulin was restricted to the germinal centers; mantle zones were negative. Expression of CENP-F was more uniformly distributed. In mantle cell lymphoma, Ki67 significantly correlated with all three markers (P<0.05, Spearman), but only Ki67 (>50%) and cdc2 (>25%) significantly correlated with shorter survival (P<0.0006, Spearman). Of several clinical parameters examined, international prognostic index of ≥2 correlated with worse clinical outcome, and high clinical stage (ie 4 vs ≤3) showed a trend for shorter survival. The prognostic significance of cdc2 and Ki67 was independent of international prognostic index and clinical stage. We have validated the prognostic value of cdc2, and confirmed that of Ki67, in a cohort of mantle cell lymphoma patients. Immunohistochemical detection of cdc2 and Ki67 may be a useful and simple method in evaluating the prognosis of mantle cell lymphoma patients.

Ras-ERK MAPK Cascade Regulates GATA3 Stability and Th2 Differentiation through Ubiquitin-Proteasome Pathway

Differentiation of naive CD4 T cells into Th2 cells requires protein expression of GATA3. Interleukin-4 induces STAT6 activation and subsequent GATA3 transcription. Little is known, however, on how T cell receptor-mediated signaling regulates GATA3 and Th2 cell differentiation. Here we demonstrated that T cell receptor-mediated activation of the Ras-ERK MAPK cascade stabilizes GATA3 protein in developing Th2 cells through the inhibition of the ubiquitin-proteasome pathway. Mdm2 was associated with GATA3 and induced ubiquitination on GATA3, suggesting its role as a ubiquitin-protein isopeptide ligase for GATA3 ubiquitination. Thus, the Ras-ERK MAPK cascade controls GATA3 protein stability by a post-transcriptional mechanism and facilitates GATA3-mediated chromatin remodeling at Th2 cytokine gene loci leading to successful Th2 cell differentiation.

Expression of Class III β-Tubulin Is Predictive of Patient Outcome in Patients with Non–Small Cell Lung Cancer Receiving Vinorelbine-Based Chemotherapy

To determine the prevalence and the prognostic value of microtubule component expression in tumors of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC). Expression of microtubular components was immunohistochemically examined in 93 tumor samples from untreated patients with stage III and IV NSCLC. All patients received vinorelbine-based chemotherapy. Response to chemotherapy, progression-free survival, and overall survival were correlated with the expression of microtubule proteins. The response rate was 27.3% (21 partial responses among 77 valuable patients). Although expression of microtubule components was not associated with the response rate, high class III beta-tubulin expression was correlated with resistance to vinorelbine, defined as disease progression under treatment. Patients whose tumors expressed high levels of class III beta-tubulin isotype had shorter progression-free survival and overall survival (P = 0.002 and 0.001, respectively). High Delta2 alpha-tubulin expression was associated with a shorter overall survival (P = 0.018). Tubulin II levels were not found to be correlated with patient outcome. A multivariate analysis, taking into account sex, age, histology, stage, weight loss, and class II beta-tubulin, class III beta-tubulin, and Delta2 alpha-tubulin levels, confirmed that class III beta-tubulin expression was independently correlated with progression-free survival (P = 0.04) and overall survival (P = 0.012). These findings suggest that a high level of expression of class III beta-tubulin in tumor cells is associated with resistance to vinorelbine and a poor prognosis in patients with NSCLC receiving vinorelbine-based chemotherapy.

Woc gene mutation causes 20E-dependent α-tubulin detyrosination inDrosophila melanogaster

The mutation without children(rgl) (woc(rgl)) is a newly described ecdysone-deficient Drosophila mutant. The woc(rgl) mutant larvae show developmental arrestment at the late larval stage and fail to form a puparium due to the failure of the ring gland to secret normal levels of ecdysone. Although a 6.8-kb woc gene transcript encoding a 187-kDa potential transcription factor has been cloned and lack of a specific cholesterol 7,8-dehydrogenease that mediates the first step in the ecdysteroidogenic pathway is likely the cause for ecdysteroid deficiency, the cellular events controlled by the woc gene remain unclear. In the present study, we investigated the effect of the woc gene mutation on the expression and tyrosination of alpha-tubulin in the woc(rgl) mutant. Our results demonstrated that the mutation in the woc gene caused 20E-dependent alpha-tubulin detyrosination, but had no significant effect on the expression of total alpha- and beta-tubulin in the homozygous woc(rgl) mutant larvae. Immunocytochemical study revealed that 20E-induced alpha-tubulin detyrosination led to the diminishing of tyrosinated alpha-tubulin signals from microtubules, resulting in the disruption of microtubule structure. The composite data suggest that the woc gene may regulate 20E-dependent alpha-tubulin detyrosination and that microtubules may be involved in sterol transport and sterol utilization in insect.

Varicella-Zoster Virus Infection Influences Expression and Organization of Actin and α-Tubulin but Does Not Affect Lamin A and Vimentin

Objective: The aim of this study was to examine the effects of varicella-zoster virus (VZV) infection on the cytoskeletal components actin, lamin A, α-tubulin and vimentin. Methods: The expression patterns of these four proteins during VZV infection were studied by Northern and Western blotting. The filaments were also studied in their cellular environment by immunofluorescence using confocal microscopy. Treatment with nocodazole and cytochalasin B was performed to examine the effects of the destruction of actin or tubulin networks on the VZV replicative cycle. Results: The amounts of the mRNAs of actin, lamin A, α-tubulin and vimentin decreased slightly at 48 h post infection (p.i.) with VZV. The cellular content of the lamin A protein appeared to remain stable during the time period analyzed, whereas the amounts of actin, α-tubulin and vimentin decreased slightly at 24 h p.i. until the end of the viral cycle. Rearrangement of microfilaments and microtubules was observed at 24 h p.i. The addition of nocodazole or cytochalasin B decreased viral replication. Conclusions: During the VZV replicative cycle, tubulin and actin networks undergo significant changes including fiber elongation. If destroyed intentionally, viral replication is diminished, suggesting that these systems are vital for an efficient infection and viral replication.

Cyclophilin C-associated Protein Is a Mediator for Fibronectin Fragment-induced Matrix Metalloproteinase-13 Expression

The function of cyclophilin C-associated protein (CyC-AP) on expression of extracellular matrix and matrix metalloproteinases (MMPs) was studied in CyC-AP-null mice. Fibronectin showed increased expression of the 53- and 29-kDa fragments in skin and wounds from CyC-AP-null mice. Type I collagen had an initial degraded pattern in the skin of CyC-AP-null mice, which did not occur in wild-type mice. MMP-3, MMP-13, MMP-14, and tumor necrosis factor-alpha (TNFalpha) had a higher expression in CyC-AP-null skin. During wound healing, MMP-13 and TNFalpha were stimulated to an even higher level, suggesting they are regulated by multiple factors. To understand the regulatory mechanisms of the up-regulated MMPs, the direct effects of TNFalpha, IL-1beta, 45-kDa fibronectin fragment (FN-45), and the 70-kDa fibronectin fragments (FN-70) on the expression of MMPs were studied. MMP-13 expression increased significantly in both CyC-AP-null and wild-type dermal fibroblasts after treatment with IL-1beta or with TNFalpha. However, MMP-13 expression did not increase in CyC-AP-null fibroblasts but did increase only in wild-type fibroblasts after FN-45 and FN-70 treatment. MMP-3 activation was induced by FN-45 and did not show a difference between CyC-AP-null and wild-type fibroblasts, suggesting different regulatory pathways for FN-45 on MMP-13 and MMP-3 expression. Our data are the first to demonstrate that deletion of CyC-AP can abolish fibronectin fragment-induced MMP-13 expression through an unknown mechanism. CyC-AP is an important factor for the regulation of MMP-13 expression.

The effects of hypoxia on growth cones in the ovine fetal brain

Objective: This study was designed to investigate the effects of hypoxia on neural process proliferation by studying its effects on growth cone tubulin and insulin-like growth factor (IGF)-I receptor content.Methods: Six fetal lambs were catheterized in the brachial artery and vein. Maternal oxygenation was reduced in steps from a fractional inspired oxygen concentration (FiO2) of 20% to 6% by addition of nitrogen to the inhaled gas mixture for a period of 4 h of reduced oxygen intake. Fetal arterial blood was sampled after the maternal FiO2 and oxygen were stable for > 5 min at maternal FiO2 of 20% to 6%. Controls were obtained from normoxic fetuses whose ewes had similar surgery and were kept at an FiO2 of 20% throughout the experiment. Growth cones were isolated from the fetal cerebrum and cerebellum. α-Tubulin and IGF-I receptors were quantified by immunoblotting. Tubulin and IGF-I receptor mRNA expressions were quantified by real-time polymerase chain reaction.Results: Maternal nitrogen breathing reduced fetal arterial pH from 7.32 ± 0.06 to 6.99 ± 0.02 (p < 0.001). Hypoxia increased IGF-I receptors from 143 ± 10 to 327 ± 14 (p < 0.001) and from 272 ± 26 to 396 ± 34 (p < 0.001) fluorescence units/μg protein in the cerebrum and cerebellum, respectively. It also increased α-tubulin from 713 ± 30 to 1873 ± 126 (p < 0.001) and from 780 ± 34 to 2362 ± 79 (p < 0.001) fluorescence units/μg protein in the cerebrum and cerebellum, respectively. Expression of IGF-I receptor mRNA increased significantly in the hypoxic animals both in the cerebrum and the cerebellum, but there was no change in expression of α-tubulin mRNA.Conclusions: This increase in IGF-I receptor expression and growth cone content may be an adaptive response to hypoxia to maintain neurite growth by facilitating binding of IGF-I. Hypoxia also increased the growth cone level of α-tubulin but did not increase its mRNA expression, which may indicate an inability to polymerize tubulin and build microtubules.

Small interfering double-stranded RNAs as therapeutic molecules to restore chemosensitivity to thymidylate synthase inhibitor compounds.

RNA interference is a post-transcriptional mechanism by which double-stranded RNA specifically silence expression of a corresponding gene. Small interfering double-stranded RNA (siRNA) of 21-23 nucleotides can induce the process of RNA interference. Studies from our laboratory have shown that translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this process gives rise to increased synthesis of TS and leads to the development of cellular drug resistance to TS-targeted compounds. As a strategy to inhibit TS expression at the mRNA level, siRNAs were designed to target nucleotides 1058-1077 on human TS mRNA. Transfection of TS1058 siRNA into human colon cancer RKO cells resulted in a dose-dependent inhibition of TS expression with an IC(50) value of 10 pM but had no effect on the expression of alpha-tubulin or topoisomerase I. Inhibition of TS expression by TS1058 was maximal at 48 h and remained suppressed for up to 5 days. Pretreatment of RKO cells with TS1058 siRNA suppressed TS protein induction following exposure to raltitrexed. In addition, TS1058 restored chemosensitivity of the resistant RKO-HTStet cell line to various TS inhibitor compounds. On treatment with TS1058, IC(50) values for raltitrexed, 1843U89, and 5-fluoro-2'-deoxyuridine decreased by approximately 15-16-fold. These studies suggest that TS-targeted siRNAs are effective inhibitors of TS expression and may have therapeutic potential by themselves or as chemosensitizers in combination with TS inhibitor compounds.

Removal of a single alpha-tubulin gene intron suppresses cell cycle arrest phenotypes of splicing factor mutations in Saccharomyces cerevisiae.

Genetic and biochemical studies of Schizosaccharomyces pombe and Saccharomyces cerevisiae have identified gene products that play essential functions in both pre-mRNA splicing and cell cycle control. Among these are the conserved, Myb-related CDC5 (also known as Cef1p in S. cerevisiae) proteins. The mechanism by which loss of CDC5/Cef1p function causes both splicing and cell cycle defects has been unclear. Here we provide evidence that cell cycle arrest in a new temperature-sensitive CEF1 mutant, cef1-13, is an indirect consequence of defects in pre-mRNA splicing. Although cef1-13 cells harbor global defects in pre-mRNA splicing discovered through intron microarray analysis, inefficient splicing of the alpha-tubulin-encoding TUB1 mRNA was considered as a potential cause of the cef1-13 cell cycle arrest because cef1-13 cells arrest uniformly at G(2)/M with many hallmarks of a defective microtubule cytoskeleton. Consistent with this possibility, cef1-13 cells possess reduced levels of total TUB1 mRNA and alpha-tubulin protein. Removing the intron from TUB1 in cef1-13 cells boosts TUB1 mRNA and alpha-tubulin expression to near wild-type levels and restores microtubule stability in the cef1-13 mutant. As a result, cef1-13 tub1Deltai cells progress through mitosis and their cell cycle arrest phenotype is alleviated. Removing the TUB1 intron from two other splicing mutants that arrest at G(2)/M, prp17Delta and prp22-1 strains, permits nuclear division, but suppression of the cell cycle block is less efficient. Our data raise the possibility that although cell cycle arrest phenotypes in prp mutants can be explained by defects in pre-mRNA splicing, the transcript(s) whose inefficient splicing contributes to cell cycle arrest is likely to be prp mutant dependent.

Estrogen Effects on Neurite Outgrowth and Cytoskeletal Gene Expression in ERα-Transfected PC12 Cell Lines

The potential of gonadal steroids like estrogen (E) to promote neurite sprouting is of interest in development and aging, as well as after neural trauma. The specific roles of the two main estrogen receptors, ERα and ERβ, in neuronal sprouting are not yet well understood. We examined the hypothesis that E can enhance nerve growth factor (NGF)-stimulated neurite sprouting in an ERα-dependent manner. PC12 cells that were stably transfected with the full-length rat ERα gene (PCER) and a control line of cells transfected with vector DNA alone (PCCON) were compared. Both cell lines vigorously differentiate neurites when treated with NGF. We determined that both lines show basal expression of ERβ mRNA, but only the PCER cells express ERα mRNA. Estrogen treatment markedly enhanced NGF-stimulated neurite outgrowth from PCER but not from PCCON cells. Significantly larger proportions of PCER cells (34 and 53% at 24 and 48 h, respectively) had neurites than did the PCCON cells (17 and 26% at 24 and 48 h) after E plus NGF treatment. We also examined the effects of E and NGF treatment of PCER and PCCON cells on peripherin, α-tubulin, and tau mRNA expression. In undifferentiated PCER cells, E treatment increased peripherin, reduced α-tubulin, and did not alter tau mRNA levels. No changes in these mRNAs were observed in the controls (undifferentiated PCCON cells) after E treatment. NGF treatment markedly stimulated expression of peripherin, α-tubulin, and tau mRNAs in both PCER and PCCON cells. From these observations we conclude that E synergizes with NGF and stimulates neurite sprouting and also modulates expression of several cytoskeletal mRNAs through ERα.

Akt Stimulates the Transactivation Potential of the RelA/p65 Subunit of NF-κB through Utilization of the IκB Kinase and Activation of the Mitogen-activated Protein Kinase p38

The serine/threonine kinase Akt/PKB is a potent regulator of cell survival and has oncogenic transformation potential. Previously, it has been shown that Akt can activate the transcription factor NF-kappaB and that this functions to block apoptosis induced by certain stimuli. The mechanism whereby Akt activates NF-kappaB has been controversial, with evidence supporting induction of nuclear translocation of NF-kappaB via activation of IkappaB kinase activity and/or the stimulation of the transcription function of NF-kappaB. Here we demonstrate that Akt targets the transactivation function of NF-kappaB by stimulating the transactivation domain of RelA/p65 in a manner that is dependent on IkappaB kinase beta activity and on the mitogen-activated protein kinase p38 (p38). Activation of RelA/p65 transactivation function requires serines 529 and 536, sites shown previously to be inducibly phosphorylated. Consistent with the requirement of p38 in the activation of NF-kappaB transcriptional function, expression of activated Akt induces p38 activity. Furthermore, the ability of IL-1beta to activate NF-kappaB is known to involve Akt, and we show here that IL-1beta induces p38 activity in manner dependent on Akt and IkappaB kinase activation. Interestingly, activated Akt and the transcriptional co-activators CBP/p300 synergize in the activation of the RelA/p65 transactivation domain, and this synergy is blocked by p38 inhibitors. These studies demonstrate that Akt, functioning through IkappaB kinase and p38, induces the transcription function of NF-kappaB by stimulating the RelA/p65 transactivation subunit of NF-kappaB.

Sugar-regulated control of α-tubulin in maize cell suspension culture

The data reported here shows that α-tubulin, the subunit of microtubulin in the cytoskeleton, is regulated by sugars. A 48-h sugar-depletion treatment in BMS maize cell suspension culture medium led to a marked reduction in the levels of α-tubulin mRNA and protein. This response was reversible; the addition of metabolizable sugars led to a rapid increase in the levels of the α-tubulin transcript. Levels of α-tubulin protein also increased, albeit gradually. Surprisingly, there was little or no effect of phosphate depletion and phosphate addition on α-tubulin expression. Furthermore, the data show that sugars modulate a co-regulation of α-tubulin and cell-wall invertase. We propose that microtubules might be a major component of a sugar-signaling pathway that is regulated by, among other factors, cellular carbon metabolism.