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Wei Dai

Publications and source records attributed to Wei Dai.

At least 55 records · Page 3Linked to original sources
PubMed2005

Assessment of ATM phosphorylation on Ser-1981 induced by DNA topoisomerase I and II inhibitors in relation to Ser-139-histone H2AX phosphorylation, cell cycle phase, and apoptosis.

BACKGROUND: The ATM kinase regulates cell-cycle checkpoints by phosphorylating multiple proteins, including histone H2AX, CHK1, and CHK2 kinases and p53. ATM is activated through auto- or trans- phosphorylation of Ser-1981 in response to DNA damage, particularly induction of DNA double-strand breaks (DSBs). The aim of the present study was to reveal a possible correlation between activation of ATM vis-à-vis H2AX phosphorylation, cell cycle phase, and apoptosis in cells treated with DNA topoisomerase (topo) I (topotecan; Tpt) or topo2 (mitoxantrone; Mtx) inhibitor. MATERIALS AND METHODS: Cultures of HL-60 cells were treated with Tpt or Mtx for various time intervals. ATM or H2AX phosphorylation was detected immunocytochemically, using Ab specific for ATM phosphorylated on Ser-1981 (ATM-S1981(P)) or for H2AX (gammaH2AX) phosphorylated on Ser-139, respectively, concurrent with the analysis of cellular DNA content. Cellular fluorescence was measured by flow cytometry. RESULTS: Untreated cells showed a modest but variable level of labeling with ATM-S1981(P) Ab across the cell cycle, with exception of mitotic cells that were strongly labeled. Exposure of cells to 150 nM Tpt induced ATM phosphorylation concurrent with phosphorylation of H2AX within 10 min; phosphorylation of both proteins was essentially limited to S-phase and was suppressed by caffeine and wortmannin, inhibitors of PI-3-like kinases. Exposure of cells to Mtx also led to ATM and H2AX phosphorylation, which, compared to Tpt, occurred later and was not cell-cycle-phase specific. Apoptosis of HL-60 cells in Tpt or Mtx treated cultures was detected after 2 or 4 h, respectively, and was limited to S-phase cells. CONCLUSIONS: The data are consistent with the role of ATM as a mediator of H2AX phosphorylation in response to DNA damage by topo1 (Tpt) or topo 2 (Mtx) inhibitor. The observed cell-cycle-phase related differences in response to Tpt vs Mtx suggest that while the collisions of DNA replication forks with the "cleavable complexes" stabilized by topo1 inhibitor are the primary cause of DSBs induced by Tpt, the collisions of RNA polymerase molecules with the complexes stabilized by the topo2 inhibitor play a major role for induction of DSBs by Mtx. The present report is the first that (i) describes cytometric analysis of ATM activation and (ii) demonstrates activation of the enzyme (kinase) and its consequence (substrate phoshorylation), both in relation to cell-cycle phase and onset of apoptosis within the same cells.

Apoptosis↗
PubMed2005

[Effect and mechanism of rhubarb on fibrinolysis in secondary damaged central nerve system of rats with acute hemorrhagic stroke].

OBJECTIVE: To observe the effect of rhubarb in treating secondary damage of central nerve system (CNS) in rats with acute hemorrhagic stroke (AHS) and to explore the possible mechanism. METHODS: The rat's AHS model was established by autologous blood injection, the effect of rhubarb on the secondary damage of CNS, plasminogen (PLG) in brain and tissue type plasminogen activator (t-PA) were observed. RESULTS: (1) The nerve function deficit signs reappeared in about 70% model rats 4 - 6 days after modeling and reached the peak at day 6 - 8, scored as 1.63 +/- 0.72 on day 4 and as 2.32 +/- 1.12 on day 7; (2) Rhubarb could effectively improve the secondary nerve function damage, with the nerve deficit scores kept to 1.24 +/- 0.19 from day 4 on, and no sign of secondary CNS damage was shown. The nerve deficit score was 1.22 +/- 0.15 on day 7 in the rhubarb treated group, showing significant difference as compared with that in the model group (P<0.05); (3) The specific amplified products of t-PA mRNA on day 3 and that of PLG mRNA on day 7 in CNS of model group were significantly higher than those in the sham operated group and the rhubarb treated group. CONCLUSION: Rhubarb could effectively reduce the secondary CNS damage in rats with AHS, it might be related with the suppressive effect of rhubarb on tPA mRNA and PLG mRNA expression in CNS.

Animals↗
PubMed2005

[Investigation on activities of hammerhead ribozyme embedded in genomic RNA of hepatitis delta virus].

OBJECTIVE: To develop HDV as a vehicle to deliver hammerhead ribozyme into hepatocytes, the effects of modified HDV was assessed on the activity of embedded hammerhead ribozyme in vitro and in vivo. METHODS: In vitro activity of ribozyme or HDV-driven ribozyme was assessed by incubating with the [alpha-32 P]-ATP labeled HBV RNA substrates at different temperature. Huh-7 cells were cotransfected with ribozyme or HDV-ribozyme chimera and HBV genome, by which inhibition of ribozymes on HBV transcription in vivo were examined. RESULTS: The results indicated that both temperature and secondary structure influenced the cleavage activity of HDV-driven ribozyme significantly. When the factors were eliminated, the HDV-driven ribozyme could act as well as its counterpart naked ribozyme. While in cultured cells the HDV-driven ribozyme had higher inhibition to HBV gene expression than that of ribozyme alone. CONCLUSION: The results demonstrated that HDV may weaken the activity of embedded ribozyme in vitro, but make it enhanced in cultured cells. Thus, this study could provide a useful evidence to develop HDV as vector for liver-special delivery of ribozyme to against chronic HBV infection.

Base Sequence↗
PubMed2004

K(+) channel activity and redox status are differentially required for JNK activation by UV and reactive oxygen species.

Upon exposure to ultraviolet (UV) radiation, osmotic changes or the presence of reactive oxygen species (ROS) c-Jun N-terminal kinases (JNKs) are rapidly activated. Extensive studies have elucidated molecular components that mediate the activation of JNKs. However, it remains unclear whether activation of JNKs by various stress signals involves different pathways. Here we show that K(+) channel activity is involved in mediating apoptosis induced by UV but not by H(2)O(2) in myelocytic leukemic ML-1 cells. Specifically, JNKs were rapidly phosphorylated upon treatment of ML-1 cells with UV and H(2)O(2). UV-induced, but not H(2)O(2)-induced, JNK-1 phosphorylation was inhibited by pretreatment with 4-aminopyridine (4-AP), a K(+) channel blocker. 4-AP also blocked UV-induced increase in JNK activity as well as p38 phosphorylation. Immunofluorescent microscopy revealed that phosphorylated JNKs were concentrated at centrosomes in ML-1 cells and that these proteins underwent rapid subcellular translocation upon UV treatment. Consistently, the subcellular translocation of JNKs induced by UV was largely blocked by 4-AP. Furthermore, UV-induced JNK activation was blocked by NEM, a sulfhydryl alkylating agent also affecting K(+) current. Both UV- and H(2)O(2)-induced JNK activities were inhibited by glutathione, suggesting that the redox status does play an important role in the activation of JNKs. Taken together, our findings suggest that JNK activation by UV and H(2)O(2) is mediated by distinct yet overlapping pathways and that K(+) channel activity and redox status are differentially required for UV- and H(2)O(2)-induced activation of JNKs.

4-Aminopyridine↗
PubMed2004

Physical and functional interactions between mitotic kinases during polyploidization and megakaryocytic differentiation.

Human Polo-like kinase 3 (Plk3), a protein serine/threonine kinase, is involved in the regulation of cell cycle progression at multiple stages. Our previous studies revealed that Plk3 is closely associated with centrosomes and plays an important role in the regulation of microtubule dynamics. Here we describe the physical interaction of Plk3 with Aurora A and BubR1 kinases, and the significance of this interaction during terminal differentiation and polyploidization of megakaryocytes. Specifically, double immunofluorescence staining confirms that Plk3 and Aurora A colocalize to centrosomes or spindle poles during essentially all phases of the cell cycle and that BubR1 also exhibits spindle pole localization during metaphase. Pull-down assays show that Plk3 physically interacts with Aurora A as well as BubR1. Upon treatment with phorbol 12-myristate 13-acetate (PMA), human erythroleukemic cells (K562) underwent megakaryocytic differentiation characterized by polyploidization and expression of mature megakaryocyte surface markers such as CD41. Plk3 protein levels were seen to be increased during PMA-induced megakaryocytic differentiation of K562 cells, correlating well with the ploidy level in these cells. Similarly, Aurora A and its phosphorylated form also increased after PMA treatment. In contrast, BubR1 levels were markedly reduced. Taken together, our study suggests that Plk3 and Aurora A kinases may lie in the same regulatory pathway and that Plk3 and Aurora A as well as BubR1 may play an important role in polyploidization and megakaryocytic differentiation.

Aurora Kinases↗
PubMed2004

B23/nucleophosmin serine 4 phosphorylation mediates mitotic functions of polo-like kinase 1.

Phosphoprotein profiling by Kinetworks trade mark analysis of M-phase-arrested HeLa cells by nocodazole treatment revealed that a novel mitosis-specific phosphorylation event occurred in the nucleolar protein B23/nucleophosmin at a conserved Ser-4 residue. Consistent with the resemblance of the Ser-4 phosphorylation site to the Polo-like kinase 1 (Plk1) consensus recognition sequence, inhibition of Plk1 by a kinase-defective mutation (K82M) abrogated B23 Ser-4 phosphorylation, whereas activation of Plk1 by a constitutively active mutation (T210D) enhanced its phosphorylation following in vivo transfection and in vitro phosphorylation assays. Depletion of endogenous Plk1 by RNA interference abolished B23 Ser-4 phosphorylation. The physical interaction of Plk1 and B23 was further demonstrated by their co-immunoprecipitation and glutathione S-transferase fusion protein pull-down assays. Interference of Ser-4 phosphorylation of B23 induced multiple mitotic defects in HeLa cells, including aberrant numbers of centrosomes, elongation and fragmentation of nuclei, and incomplete cytokinesis. The phenotypes of B23 mutants are reminiscent of a subset of those described previously in Plk1 mutants. Our findings provide insights into the biochemical mechanism underlying the role of Plk1 in mitosis regulation through the identification of Ser-4 in B23 as a major physiological substrate of Plk1.

Cell Cycle Proteins↗
PubMed2004

Artificial rearing of mouse pups: development of a mouse pup in a cup model.

Artificial rearing of rat pups has been used in the investigation of the neonatal gut. We propose to adapt the model of artificially rearing rat pups for use in mouse pups, thereby allowing the use of transgenic animals for our research. We hypothesized that gastrostomy catheters may be placed successfully into neonatal mouse pups and that the pups may be artificially reared without significant alterations in their growth or intestinal development. Gastrostomy tubes are placed into 5-d-old mouse pups [artificially reared (AR); n = 32], and the mice are fed rodent milk substitute. Littermate pups [maternally reared (MR); n = 22] are used as controls. After 5 d, pups are killed and their organs are harvested. Intestinal villus measurements, protein content, and DNA content are determined. Data are reported as mean +/- SEM, compared with appropriate statistical methods, and significance is determined at P < 0.05. Initial weights and lengths are not different between the two groups, but after 5 d, MR pups weigh more than their AR counterparts (5.0 +/- 0.13 versus 4.1 +/- 0.14 g, MR versus AR; P < 0.01). However, the pups' length and the intestinal villus height-to-width ratios, protein, and DNA content are not different between the MR and AR pups. To our knowledge, this is the first report of artificially rearing mouse pups. Development of this technique will permit nutritional manipulation in neonatal mice, a mammalian model wherein the genome is sequenced and transgenic mutants are available.

Animals↗
PubMed2004

Expression of VEGF receptors in cocultured neuroblastoma cells.

BACKGROUND: VEGF is best known for its angiogenic properties. We have found that VEGF expression is increased in neuroblastoma cells cocultured with hepatocytes. In addition, we have previously shown that neuroblastoma cells cultured with exogenous VEGF have an increase in the expression of VEGF receptors. Therefore, we hypothesized that the expression of VEGF receptors would be up-regulated in neuroblastoma cells grown in the coculture environment. MATERIALS AND METHODS: Two neuroblastoma cell lines (IMR-32 or SK-N-DZ) are used. These cells are cultured alone and in a coculture system with hepatocytes. Message for VEGF and the VEGF receptors KDR, flt-1, flt-4, neuropilin 1 (NRP-1), and neuropilin 2 (NRP-2) are measured with RT-PCR. Flt-4, NRP-1, and NRP-2 protein expression is measured with Western blot. RESULTS: The receptors KDR and flt-1 are not detected in either cell line in either control or coculture conditions. Message for VEGF and flt-4 is significantly increased in the cocultured IMR-32 cells, while that for NRP-1 and NRP-2 is unchanged in these cells. VEGF and its receptors are unchanged in cocultured SK-N-DZ cells. CONCLUSIONS: Neuroblastoma cells express specific VEGF receptors that are differentially regulated in the different cell lines. These findings suggest that the heterogeneity of neuroblastomas may limit the utility of targeting VEGF and its receptors as sole treatments for the tumor, and that successful therapies will be dependent upon the specific biology of the tumor.

Blotting, Western↗
PubMed2004

MEK1-induced Golgi dynamics during cell cycle progression is partly mediated by Polo-like kinase-3.

MEK1, a gene product that regulates cell growth and differentiation, also plays an important role in Golgi breakdown during the cell cycle. We have recently shown that polo-like kinase (Plk3) is Golgi localized and involved in Golgi dynamics during the cell cycle. To study the mode of action of Plk3 in the Golgi fragmentation cascade, we examined functional as well as physical interactions between Plk3 and MEK1/ERKs. In HeLa cells, although a significant amount of Plk3 signals dispersed in a manner similar to those of Golgi during mitosis concentrated Plk3 was detected at spindle poles, which colocalized with phospho-MEKs and phospho-ERKs. Pull-down assays showed that Plk3 physically interacted with MEK1 and ERK2. Nocodazole activated Plk3 and its activation was blocked by MEK-specific inhibitors (PD98059 or U0126). Moreover, transfection of activated MEK1 resulted in an enhanced kinase activity of Plk3; Plk3-induced fragmentation of Golgi stacks was significantly reduced after treatment with MEK inhibitors. Consistently, ectopic expression of activated MEK1, but not kinase-dead MEK1(K97R), stimulated Plk3 to induce Golgi breakdown and the stimulation was not observed in cells expressing Plk3(K52R). Furthermore, PLK3(-/-) murine embryonic fibroblast cells exhibited a significantly less fragmentation of the Golgi complex than that in wild-type cells after exposed to nocodazole. Thus, our studies strongly suggest that Plk3 may be a key protein kinase mediating MEK1 function in the Golgi fragmentation pathway during cell division.

Cell Cycle↗
PubMed2004

Polo-like kinase 3 is Golgi localized and involved in regulating Golgi fragmentation during the cell cycle.

The Golgi apparatus undergoes extensive fragmentation during mitosis in animal cells. Protein kinases play a critical role during fragmentation of the Golgi apparatus. We reported here that Polo-like kinase 3 (Plk3) may be an important mediator during Golgi breakdown. Specifically, Plk3 was concentrated at the Golgi apparatus in HeLa and A549 cells during interphase. At the onset of mitosis, Plk3 signals disintegrated and redistributed in a manner similar to those of Golgi stacks. Nocodazole activated Plk3 kinase activity, correlating with redistribution of Plk3 signals and Golgi fragmentation. In addition, treatment with brefeldin A (BFA), a Golgi-specific poison, also resulted in disappearance of concentrated Plk3 signals. Plk3 interacted with giantin, a Golgi-specific protein. Expression of Plk3, but not the kinase-defective Plk3 (Plk3(K52R)), resulted in significant Golgi breakdown. Given its role in cell cycle progression, Plk3 may be a protein kinase involved in regulation of Golgi fragmentation during the cell cycle.

Brefeldin A↗
PubMed2004

Slippage of mitotic arrest and enhanced tumor development in mice with BubR1 haploinsufficiency.

A compromised spindle checkpoint is thought to play a key role in genetic instability that predisposes cells to malignant transformation. Loss of function mutations of BubR1, an important component of the spindle checkpoint, have been detected in human cancers. Here we show that BubR1(+/-) mouse embryonic fibroblasts are defective in spindle checkpoint activation, contain a significantly reduced amount of securin and Cdc20, and exhibit a greater level of micronuclei than do wild-type cells. RNA interference-mediated down-regulation of BubR1 also greatly reduced securin level. Moreover, compared with wild-type littermates, BubR1(+/-) mice rapidly develop lung as well as intestinal adenocarcinomas in response to challenge with carcinogen. BubR1 is thus essential for spindle checkpoint activation and tumor suppression.

Adenoma↗
PubMed2004

The putative recombination of hepatitis B virus genotype B with pre-C/C region of genotype C.

Among hepatitis B virus (HBV) genotypes the B and C are most prevalent in China. To further study on the inside story of the intertypes, the genotype of 136 sequences from Chinese patients were analyzed either by restriction fragment length polymorphism on fragments or by phylogenetic analysis and bootscanning on full genome. The 22 complete sequences of genotype B clustered with different genotypes depending on gene fragments analyzed, which indicated that recombinant events occurred during HBV evolutionary history. To locate the recombinant regions, the sequences of HBV entire genome were analyzed by SimPlot program. The recombinant regions of B genotype with recombination were mapped in the pre-C/C region with relatively less varied size. Besides, three sequences of genotype C have recombination with genotype B or D in different regions. However, among all of the 136 sequences, none of authentic genotype B was identified. To investigate the possible mechanism responsible for intertype recombination, the selection pressure on the recombinant region was estimated by using CODEML program. All models allow for positively selected sites suggest existence of positive selection pressure. In conclusion, the genotype B with recombination was exclusive subgroup of genotype B in China. The mosaic genotype B might result from immune pressure on the pre-C/C gene.

Carrier State↗
PubMed2003

BUBR1 deficiency results in abnormal megakaryopoiesis.

The physiologic function of BUBR1, a key component of the spindle checkpoint, was examined by generating BUBR1-mutant mice. BUBR1(-/-) embryos failed to survive beyond day 8.5 in utero as a result of extensive apoptosis. Whereas BUBR1(+/-) blastocysts grew relatively normally in vitro, BUBR1(-/-) blastocysts exhibited impaired proliferation and atrophied. Adult BUBR1(+/-) mice manifested splenomegaly and abnormal megakaryopoiesis. BUBR1 haploinsufficiency resulted in an increase in the number of splenic megakaryocytes, which was correlated with an increase in megakaryocytic, but a decrease in erythroid, progenitors in bone marrow cells. RNA interference-mediated down-regulation of BUBR1 also caused an increase in polyploidy formation in murine embryonic fibroblast cells and enhanced megakaryopoiesis in bone marrow progenitor cells. However, enhanced megakaryopoiesis in BUBR1(+/-) mice was not correlated with a significant increase in platelets in peripheral blood, which was at least partly due to a defect in the formation of proplatelet-producing megakaryocytes. Together, these results indicate that BUBR1 is essential for early embryonic development and normal hematopoiesis.

Animals↗
PubMed2003

Polo-like kinases in cell cycle checkpoint control.

Recent studies from various eukaryotic model systems indicate that polo-like kinases (Plks) play an ever-increasing role in the regulation of cell cycle progression. Early genetic studies have demonstrated that Cdc5, a budding yeast counterpart of vertebrate Plks, is essential for mitosis. Mammalian Plks primarily localize to the microtubule organization center during interphase and undergo dramatic subcellular relocation during mitotic progression. Many key cell cycle regulators such as p53, Cdc25C, cyclin B, and components of the anaphase promoting complex are directly targeted by Plks. Although the exact mechanisms of action of these protein kinases in vivo remain to be elucidated, Plks appear to orchestrate various cell cycle checkpoints (intra-S phase, G2/M transition, spindle assembly, and cytokinesis checkpoints) that protect cells against genetic instability during cell division.

Animals↗
PubMed2003

Identification of an electron densification of the glomerular basement membrane in renal biopsy specimens.

Ultrastructural examination of renal biopsy specimens is often necessary to establish a diagnosis. We have noted, in some renal biopsies, that normal-appearing glomerular basement membrane (GBM) shows an electron densification. The aim of this study was to describe this phenomenon and assess the degree of its occurrence. We reviewed ultrastructural pathology records of 153 renal biopsy patients with special reference to the careful examination of the electron micrographs. Of these, 28 cases (18%) showed a definite and homogeneous electron densification of normal-appearing GBM in the glomeruli. The pathological diagnosis in these cases was IgA nephropathy in 15, minimal change nephrotic syndrome in 6, interstitial nephritis in 3, membranoproliferative glomerulonephritis in 2, and nephritis of Henoch-Schonlein purpura and membranous nephropathy in 1 case each. In addition, frequent deposition of IgG without C3 in the GBM (86%) and glomerular endothelial cell edema (96%) were observed. No apparent correlation could be ascertained between the presence of the electron densification and pathological diagnosis. It is suggested that electron densification of the GBM may be caused by the penetration of some electron-dense materials into the GBM as a result of an abnormality in the glomerular filtration barrier and that this could be considered as a sign of altered properties of the lamina densa.

Adolescent↗
PubMed2003

Use of oolong tea extract (OTE) for elastin staining and enhancement in ultrathin sections.

To assess the usefulness of oolong tea extract (OTE) staining for connective tissue observation, we examined the visceral pleural mesothelium of rat lung by transmission electron microscope. Four kinds of electron microscopic staining methods (routine, tannic acid, OTE in distilled water, and OTE in 0.1 M phosphate buffer) were compared to determine the most suitable method for electron microscopic observation of ultrathin sections. Elastin (elastic laminae) was selectively stained by tannic acid and both water and buffered OTE. Except for elastin, connective tissue and cell ultrastructures were also electron enhanced by tannic acid and both water and buffered OTE staining. However, using water OTE, the electron-dense filaments (10-12 nm in diameter) were obscured. In tannic acid staining, the unit membranes of the visceral pleural mesothelial cells were weaker as compared with routine and buffered OTE stains. Thus, the buffered OTE staining method is a highly useful technique for connective tissue observation and electron-enhanced staining in transmission electron microscopic preparations.

Animals↗
PubMed2003

Differential expression of Bcl-2 and Bax may enhance neuroblastoma survival.

BACKGROUND/PURPOSE: Aggressive tumors may alter their expression of Bcl-2 proteins to decrease apoptosis and increase survival. The authors reported previously that neuroblastoma cells have diminished apoptosis when placed in coculture with hepatocytes to stimulate a metastatic environment. It was hypothesized that the expression of proapoptotic (Bax) and prosurvival (Bcl-2 and Mcl-1) proteins would be altered in neuroblastoma cells grown in a cell culture model of metastatic neuroblastoma. METHODS: Human neuroblastoma cells (IMR-32) were grown alone or in coculture with human hepatocytes for 2, 3, or 4 days. Bcl-2, Mcl-1, and Bax mRNA were measured with reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Bcl-2, an antiapoptotic protein, was significantly increased in cocultured neuroblastoma cells by day 4. Bax, a proapoptotic protein, was significantly diminished by day 3. No significant change in Mcl-1 occurred in this study. CONCLUSIONS: When neuroblastoma cells placed in coculture, the prosurvival protein, Bcl-2, is upregulated whereas the proapoptotic protein, Bax, is downregulated. The combination of these changes can maximally enhance the survival rate of neuroblastoma cells in coculture. The propensity for neuroblastoma to either metastasize or regress may be associated with its ability to differentially regulate the expression of different members of the Bcl-2 protein family.

Apoptosis↗