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Vincent J Kidd

Publications and source records attributed to Vincent J Kidd.

15 recordsLinked to original sources
PubMed2006

Evaluation of IFN-gamma effects on apoptosis and gene expression in neuroblastoma--preclinical studies.

Loss of caspase-8 expression and resistance to cytotoxic agents occurs frequently in late stage neuroblastoma (NB). Interferon-gamma (IFN-gamma) induces caspase-8 in NB cells, sensitizing them to death receptor mediated apoptosis. This study characterizes the kinetics of this phenomenon and examines the effects of IFN-gamma on global gene expression to determine whether IFN-gamma responses are achievable at physiologically relevant doses and to define the biological effects of this cytokine. Here we examine the IFN-gamma responses of 16 NB cell lines. A single <5-min exposure to IFN-gamma (0.5 ng/ml) induced caspase-8 expression in all non-expressing cell lines and in 3/6 cell lines which already expressed high caspase-8. This increase in caspase-8 proteins was observed within 16 h and persisted for up to 9 days. Furthermore, IFN-gamma pretreatment of NB cells increased doxorubicin-induced apoptosis nearly 3-fold. Microarray analysis was used to identify additional genes involved in proliferation, signaling and apoptosis whose expression was modulated via IFN-gamma. Altered expression of these genes should further enhance the responsiveness of NB cells to chemotherapeutics. Thus, the use of IFN-gamma to sensitize NB cells to cytotoxic agents represents an attractive therapeutic strategy and warrants further investigation.

Apoptosis↗
PubMed2006

Potentiation of neuroblastoma metastasis by loss of caspase-8.

Neuroblastoma, the most common paediatric solid tumour, arises from defective neural crest cells. Genetic alterations occur frequently in the most aggressive neuroblastomas. In particular, deletion or suppression of the proapoptotic enzyme caspase-8 is common in malignant, disseminated disease, although the effect of this loss on disease progression is unclear. Here we show that suppression of caspase-8 expression occurs during the establishment of neuroblastoma metastases in vivo, and that reconstitution of caspase-8 expression in deficient neuroblastoma cells suppressed their metastases. Caspase-8 status was not a predictor of primary tumour growth; rather, caspase-8 selectively potentiated apoptosis in neuroblastoma cells invading the collagenous stroma at the tumour margin. Apoptosis was initiated by unligated integrins by means of a process known as integrin-mediated death. Loss of caspase-8 or integrin rendered these cells refractory to integrin-mediated death, allowed cellular survival in the stromal microenvironment, and promoted metastases. These findings define caspase-8 as a metastasis suppressor gene that, together with integrins, regulates the survival and invasive capacity of neuroblastoma cells.

Animals↗
PubMed2005

Phosphorylation of FADD at serine 194 by CKIalpha regulates its nonapoptotic activities.

FADD is essential for death receptor (DR)-induced apoptosis. However, it is also critical for cell cycle progression and proliferation, activities that are regulated by phosphorylation of its C-terminal Ser194, which has also been implicated in sensitizing cancer cells to chemotherapeutic drugs and in regulating FADD's intracellular localization. We now demonstrate that casein kinase Ialpha (CKIalpha) phosphorylates FADD at Ser194 both in vitro and in vivo. FADD-CKIalpha association regulates the subcellular localization of FADD, and phosphorylated FADD was found to colocalize with CKIalpha on the spindle poles in metaphase. Inhibition of CKIalpha diminished FADD phosphorylation, prevented the ability of Taxol to arrest cells in mitosis, and blocked mitogen-induced proliferation of mouse splenocytes. In contrast, a low level of cycling splenocytes from mice expressing FADD with a mutated phosphorylation site was insensitive to CKI inhibition. These data suggest that phosphorylation of FADD by CKI is a crucial event during mitosis.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Role of CDK/cyclin complexes in transcription and RNA splicing.

The production of mRNAs in all living organisms is an extremely complex process that includes multiple catalytic activities such as transcription, capping, splicing, polyadenylation, cleavage and export. All of these processes are controlled by a large group of proteins which form very dynamic complexes interacting with DNA and pre-mRNAs to coordinate these activities. Phosphorylations play a central role in regulating formation, activation and inactivation of these complexes. A growing number of protein kinases have been identified that are capable of phosphorylating proteins involved in mRNA production. Among them, Cyclin-dependent Kinases (CDKs) represent a family of serine/threonine protein kinases that become active upon binding to a cyclin regulatory partner. CDK/cyclin complexes were first identified as crucial regulators of cell cycle progression. More recently, CDK/cyclin complexes have also been implicated in transcription and mRNA processing leading to the concept of an intricate network of CDK/cyclin complexes regulating cell cycle, transcription and mRNA processing via cross-talk between multiple CDKs. In this review, we discuss the role of CDK/cyclin-dependent phosphorylation in the regulation of transcription and RNA splicing and highlight recent findings that indicate the involvement of CDK/cyclin complexes in connecting transcription and RNA splicing.

Amino Acid Sequence↗
PubMed2005

Activation of pre-mRNA splicing by human RNPS1 is regulated by CK2 phosphorylation.

Human RNPS1 was originally characterized as a pre-mRNA splicing activator in vitro and was shown to regulate alternative splicing in vivo. RNPS1 was also identified as a protein component of the splicing-dependent mRNP complex, or exon-exon junction complex (EJC), and a role for RNPS1 in postsplicing processes has been proposed. Here we demonstrate that RNPS1 incorporates into active spliceosomes, enhances the formation of the ATP-dependent A complex, and promotes the generation of both intermediate and final spliced products. RNPS1 is phosphorylated in vivo and interacts with the CK2 (casein kinase II) protein kinase. Serine 53 (Ser-53) of RNPS1 was identified as the major phosphorylation site for CK2 in vitro, and the same site is also phosphorylated in vivo. The phosphorylation status of Ser-53 significantly affects splicing activation in vitro, but it does not perturb the nuclear localization of RNPS1. In vivo experiments indicated that the phosphorylation of RNPS1 at Ser-53 influences the efficiencies of both splicing and translation. We propose that RNPS1 is a splicing regulator whose activator function is controlled in part by CK2 phosphorylation.

Casein Kinase II↗
PubMed2004

Failure to proliferate and mitotic arrest of CDK11(p110/p58)-null mutant mice at the blastocyst stage of embryonic cell development.

The CDK11(p110) protein kinases are part of large-molecular-weight complexes that also contain RNA polymerase II, transcriptional elongation factors, and general pre-mRNA splicing factors. CDK11(p110) isoforms may therefore couple transcription and pre-mRNA splicing by their effect(s) on certain proteins required for these processes. The CDK11(p58) kinase isoform is generated from the CDK11(p110) mRNA through the use of an internal ribosome entry site in a mitosis-specific manner, suggesting that this kinase may regulate the cell cycle during mitosis. The in vivo role and necessity of CDK11(p110/p58) kinase function during mammalian development were examined by generating CDK11(p110/p58)-null mice through targeted disruption of the corresponding gene using homologous recombination. While heterozygous mice develop normally, disruption of both CDK11(p110/p58) alleles results in early embryonic lethality due to apoptosis of the blastocyst cells between 3.5 and 4 days postcoitus. Cells within these embryos exhibit both proliferative defect(s) and a mitotic arrest. These results are consistent with the proposed cellular functions of the CDK11(p110/p58) kinases and confirm that the CDK11(p110/p58) kinases are essential for cellular viability as well as normal early embryonic development.

Animals↗
PubMed2003

The murine G+C-rich promoter binding protein mGPBP is required for promoter-specific transcription.

The archetypal TATA-box deficient G+C-rich promoter of the murine adenosine deaminase gene (Ada) requires a 48-bp minimal self-sufficient promoter element (MSPE) for function. This MSPE was used to isolate a novel full-length cDNA clone that encodes a 66-kDa murine G+C-rich promoter binding protein (mGPBP). The mGPBP mRNAs are ubiquitously expressed as either 3.0- or 3.5-kb forms differing in 3' polyadenylation site usage. Purified recombinant mGPBP, in the absence of any other mammalian cofactors, binds specifically to both the murine Ada gene promoter's MSPE and the nonhomologous human Topo IIalpha gene's G+C-rich promoter. In situ binding assays, immunoprecipitation, and Western blot analyses demonstrated that mGPBP is a nuclear factor that can form complexes with TATA-binding protein, TFIIB, TFIIF, RNA polymerase II, and P300/CBP both in vitro and in intact cells. In cotransfection assays, increased mGPBP expression transactivated the murine Ada gene's promoter. Sequestering of GPBP present in HeLa cell nuclear extract by immunoabsorption completely and reversibly suppressed extract-dependent in vitro transcription from the murine Ada gene's G+C-rich promoter. However, transcription from the human Topo IIalpha gene's TATA box-containing G+C-rich promoter was only partially suppressed and the adenovirus major late gene's classical TATA box-dependent promoter is totally unaffected under identical assay conditions. These results implicate GPBP as a requisite G+C-rich promoter-specific transcription factor and provide a mechanistic basis for distinguishing transcription initiated at a TATA box-deficient G+C-rich promoter from that initiated at a TATA box-dependent promoter.

Adenosine Deaminase↗
PubMed2003

ASAP, a novel protein complex involved in RNA processing and apoptosis.

Different isoforms of a protein complex termed the apoptosis- and splicing-associated protein (ASAP) were isolated from HeLa cell extract. ASAP complexes are composed of the polypeptides SAP18 and RNPS1 and different isoforms of the Acinus protein. While Acinus had previously been implicated in apoptosis and was recently identified as a component of the spliceosome, RNPS1 has been described as a general activator of RNA processing. Addition of ASAP isoforms to in vitro splicing reactions inhibits RNA processing mediated by ASF/SF2, by SC35, or by RNPS1. Additionally, microinjection of ASAP complexes into mammalian cells resulted in acceleration of cell death. Importantly, after induction of apoptosis the ASAP complex disassembles. Taken together, our results suggest an important role for the ASAP complexes in linking RNA processing and apoptosis.

Apoptosis↗
PubMed2002

CDK11 complexes promote pre-mRNA splicing.

The PITSLRE protein kinases, hereafter referred to as CDK11 because of their association with the cyclin L regulatory partner, belong to large molecular weight protein complexes that contain RNA polymerase II. These CDK11(p110) complexes have been reported to influence transcription as well as interact with the general pre-mRNA-splicing factor RNPS1. Some of these complexes may also play a role in pre-mRNA splicing. Using a two-hybrid interactive screen, the splicing protein 9G8 was identified as an in vivo partner for CDK11(p110). The identification of several splicing-related factors as CDK11(p110) interactors along with the close relationship between transcription and splicing indicated that CDK11(p110) might influence splicing activity directly. Immunodepletion of CDK11(p110) from splicing extracts greatly reduced the appearance of spliced products using an in vitro assay system. Moreover, the re-addition of these CDK11(p110) immune complexes to the CDK11(p110)-immunodepleted splicing reactions completely restored splicing activity. Similarly, the addition of purified CDK11(p110) amino-terminal domain protein was sufficient to inhibit the splicing reaction. Finally, 9G8 is a phosphoprotein in vivo and is a substrate for CDK11(p110) phosphorylation in vitro. These data are among the first demonstrations showing that a CDK activity is functionally coupled to the regulation of pre-mRNA-splicing events and further support the hypothesis that CDK11(p110) is in a signaling pathway that may help to coordinate transcription and RNA-processing events.

Base Sequence↗
PubMed2002

Casein kinase 2 interacts with cyclin-dependent kinase 11 (CDK11) in vivo and phosphorylates both the RNA polymerase II carboxyl-terminal domain and CDK11 in vitro.

The PITSLRE protein kinases, hereafter referred to as cyclin-dependent kinase 11 (CDK11) due to their association with cyclin L, are part of large molecular weight protein complexes that contain RNA polymerase II (RNAP II) as well as numerous transcription and RNA processing factors. Data presented here demonstrate that the influence of CDK11(p110) on transcription and splicing does not involve phosphorylation of the RNAP II carboxyl-terminal domain by CDK11(p110). We have isolated a DRB- and heparin-sensitive protein kinase activity that co-purifies with CDK11(p110) after ion exchange and affinity purification chromatography. This protein kinase was identified as casein kinase 2 (CK2) by immunoblot and mass spectrometry analyses. In addition to the RNAP II carboxyl-terminal domain, CK2 phosphorylates the CDK11(p110) amino-terminal domain. These data suggest that CDK11(p110) isoforms participate in signaling pathways that include CK2 and that its function may help to coordinate the regulation of RNA transcription and processing events. Future experiments will determine how phosphorylation of CDK11(p110) by CK2 specifically affects RNA transcription and/or processing events.

Casein Kinase II↗
PubMed2002

Caspase-9 and Apaf-1 are expressed and functionally active in human neuroblastoma tumor cell lines with 1p36 LOH and amplified MYCN.

Important roles have been suggested for caspase-8, caspase-9 and Apaf-1 in controlling tumor development and their sensitivity to chemotherapeutic agents. Methylation and deletion of Apaf-1 and CASP8 results in the loss of their expression in melanoma and neuroblastoma, respectively, while CASP9 localization to 1p36.1 suggests it is a good candidate tumor suppressor. The status of CASP9 and Apaf-1 expression in numerous neuroblastoma cell lines with/without amplified MYCN and chromosome 1p36 loss-of-heterozygosity (LOH) was therefore examined to test the hypothesis that one or both of these genes are tumor suppressors in neuroblastoma. Although CASP9 is included in the region encompassing 1p36 LOH in all neuroblastoma cell lines examined, the remaining CASP9 allele(s) express a functional caspase-9 enzyme. Apaf-1 is also expressed in all neuroblastoma tumor cell lines examined. Thus, the CASP9 or Apaf-1 genes do not appear to function as tumor suppressors in MYCN amplified neuroblastomas. However, approximately 20% of the neuroblastoma cell lines with methylated CASP8 alleles are also highly resistant to staurosporine (STS)- and radiation-induced cell death, presumably because cytochrome c is not released from mitochondria. This suggests that a second, smaller sub-group of MYCN amplified neuroblastoma tumors exists with defect(s) in apoptotic signaling components upstream of caspase-9 and Apaf-1. Since no consistent differences in Bcl-2, Bcl-x(L) or Bax expression were seen in the STS- and radiation-resistant neuroblastomas, it suggests that a unique mitochondrial signaling factor(s) is responsible for the defect in cytochrome c release in this sub-group of tumors.

Apoptosis↗
PubMed2002

ERK activation mediates cell cycle arrest and apoptosis after DNA damage independently of p53.

In response to DNA damage, ataxia-telangiectasia mutant and ataxia-telangiectasia and Rad-3 activate p53, resulting in either cell cycle arrest or apoptosis. We report here that DNA damage stimuli, including etoposide (ETOP), adriamycin (ADR), ionizing irradiation (IR), and ultraviolet irradiation (UV) activate ERK1/2 (ERK) mitogen-activated protein kinase in primary (MEF and IMR90), immortalized (NIH3T3) and transformed (MCF-7) cells. ERK activation in response to ETOP was abolished in ATM-/- fibroblasts (GM05823) and was independent of p53. The MEK1 inhibitor PD98059 prevented ERK activation but not p53 stabilization. Maximal ERK activation in response to DNA damage was not attenuated in MEF(p53-/-). However, ERK activation contributes to either cell cycle arrest or apoptosis in response to low or high intensity DNA insults, respectively. Inhibition of ERK activation by PD98059 or U0126 attenuated p21(CIP1) induction, resulting in partial release of the G(2)/M cell cycle arrest induced by ETOP. Furthermore, PD98059 or U0126 also strongly attenuated apoptosis induced by high dose ETOP, ADR, or UV. Conversely, enforced activation of ERK by overexpression of MEK-1/Q56P sensitized cells to DNA damage-induced apoptosis. Taken together, these results indicate that DNA damage activates parallel ERK and p53 pathways in an ATM-dependent manner. These pathways might function cooperatively in cell cycle arrest and apoptosis.

3T3 Cells↗
PubMed2002

Apoptotic release of histones from nucleosomes.

Chromatin structure is influenced by histone modification, and this may help direct chromatin behavior to facilitate transcription, DNA replication, and DNA repair. Chromatin condensation and DNA fragmentation are the classic nuclear features but remain poorly characterized. It is highly probable that nucleosomal structure must be altered to allow these features to become apparent, but data to support this construct are lacking. We report here that in response to apoptotic signals from a death receptor (CD95 and tumor necrosis factor-alpha) or mitochondrial (staurosporine) apoptotic stimulus, the core nucleosomal histones H2A, H2B, H3, and H4 become separated from DNA during apoptosis in Jurkat and HeLa cells and are consequently detectable in the cell lysate prepared using a non-ionic detergent. The timing of this histone release from DNA correlates well with the progression of apoptosis. We also show expression of a caspase cleavage-resistant form of ICAD (ICAD-DM) in Jurkat and HeLa cells abolished DNA fragmentation and also dramatically reduced histone release in apoptotic cells. However, we demonstrate that apoptotic histone release is not an inevitable consequence of CAD/DFF-40-mediated DNA destruction as DNA fragmentation but not histone release occurs efficiently in tumor necrosis factor-alpha- and etoposide-treated NIH3T3 cells. Furthermore, in an in vitro apoptotic assay, incubation of apoptotic Jurkat cellular extract with non-apoptotic Jurkat nuclei led to nuclear DNA fragmentation without obvious histone release. Taken together, these data demonstrate that CAD/DFF-40 functions indirectly in mediating nucleosomal destruction during apoptosis.

3T3 Cells↗
PubMed2001

PITSLRE p110 protein kinases associate with transcription complexes and affect their activity.

Although the PITSLRE protein kinases are members of the cyclin-dependent kinase superfamily, their cellular function is unclear. Previously we demonstrated that the general RNA splicing factor RNPS1 is a specific PITSLRE p110 kinase interactor in vivo. This suggests that the PITSLRE family of protein kinases is involved in some aspect of RNA processing or transcription. Here we identify multiple transcriptional elongation factors, including ELL2, TFIIF(1), TFIIS, and FACT, as PITSLRE kinase-associated proteins. We demonstrate that PITSLRE p110 protein kinases co-immunoprecipitate and/or co-purify with these elongation factors as well as with RNA polymerase II. Antibody-mediated inhibition of PITSLRE kinase specifically suppressed RNA polymerase II-dependent in vitro transcription initiated at a GC-rich (adenosine deaminase) or TATA box-dependent (Ad2ML) promoter, and this suppression was rescued by readdition of purified PITSLRE p110 kinase. Together, these data strongly suggest that PITSLRE protein kinases participate in a signaling pathway that potentially regulates or links transcription and RNA processing events.

Animals↗