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Biomedical subjects

Wei Dai

Publications and source records attributed to Wei Dai.

16 recordsLinked to original sources
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

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↗
PubMed2003

VEGF receptors are differentially expressed by neuroblastoma cells in culture.

BACKGROUND/PURPOSE: Vascular endothelial growth factor (VEGF) is best known for its angiogenic properties, but its mitogenic capacity may be more important for tumorigenesis. The ability of VEGF to induce specific biologic activities may be dependent on the amount and type of VEGF receptors present. The authors hypothesize that neuroblastoma cells express specific VEGF receptors and that their expression may be altered when the cells are exposed to differing cytokines and culture environments. METHODS: Four groups of human neuroblastoma cells (IMR-32) are studied. (1) Control cells: cultured in standard media. (2) VEGF cells: VEGF added to the media. (3) Tumor necrosis factor alpha (TNF-alpha) cells: TNF-alpha added to the media. (4) Serum starved cells: cultured in serum-depleted media. Reverse transcriptase polymerase chain reaction (RT-PCR) is utilized to measure the VEGF receptors flt-1, KDR/flk-1, flt-4, neuropilin 1 (NRP-1), and neuropilin 2 (NRP-2). RESULTS: Flt-1 and KDR are not detected in any groups. Flt-4, NRP-1, and NRP-2 are present in the IMR-32 cells, and their expression is significantly increased by the administration of VEGF. Neuroblastoma cells cultured with TNF-alpha or in serum-depleted media have a significant decrease in the expression of these receptors. CONCLUSIONS: The authors show that neuroblastoma cells express specific VEGF receptors that may be altered by mitogenic or apoptotic stimuli. Specifically targeting VEGF and its receptors may be another therapeutic strategy for the treatment of neuroblastoma.

Culture Media, Serum-Free↗
PubMed2003

Neuroblastoma, apoptosis, and growth factors.

Neuroblastoma is the most common extracranial tumor of childhood. This tumor is associated with alterations in apoptosis and is affected by various growth factors and cytokines. In this short review we will discuss recent findings in our laboratory where we have been studying a cell culture model of neuroblastoma.

Apoptosis↗
PubMed2002

Ultraviolet-induced junD activation and apoptosis in myeloblastic leukemia ML-1 cells.

The exposure of mammalian cells to UV irradiation induces the expression of immediate early genes such as c-jun and c-fos and activates the transcription factors AP-1 and NF-kappaB. JunD is one of the three members of the Jun family and shares some functional characteristics with c-Jun. In the present study, we found that the exposure of myeloblastic leukemia ML-1 cells to UV light (UVC) caused a significant increase in junD mRNA expression within 5 min that persisted for a period of 3 h. The activation of protein kinase C (PKC) with 12-O-tetradecaoylphorbol-13-acetate (TPA) also induced increases in junD expression similar to those of UV irradiation. In addition, UV irradiation- and TPA-induced increases in junD expression were completely abolished by GF-109203X, a PKC-specific inhibitor. UV irradiation activated intracellular signaling pathways including extracellular regulated kinase-2 (Erk-2), c-Jun N-terminal kinases-1 (JNK-1), and p38. However, TPA-induced activation of PKC affected only Erk-2 activity, and GF-109203X (a PKC inhibitor) markedly suppressed UV-induced Erk-2 activation. To further investigate the effect of UV-induced Erk-2 activation on the expression of junD mRNA, cDNA encoding mitogen-activated protein kinase kinase (MEK1) was overexpressed in ML-1 cells. The overexpression of MEK1 enhanced substantially junD expression in response to UV or TPA. In contrast, the suppression of Erk activation with PD98059, a specific inhibitor of MEK1, inhibited UV- and TPA-induced junD mRNA expression, UV-induced increases in caspase-3 activities, and cell death. In addition, the overexpression of junD enhanced the UV irradiation-induced increases in caspase-3 activity and cell death. We conclude that UV irradiation-induced increases in junD expression in ML-1 cells are mediated through activation of the PKC-coupled Erk-2 signaling pathway and play an important role in ML-1 cell apoptosis.

Apoptosis↗
PubMed2002

Structure-activity relationships for inhibition of human 5alpha-reductases by polyphenols.

The enzyme steroid 5 alpha-reductase (EC 1.3.99.5) catalyzes the NADPH-dependent reduction of the double bond of a variety of 3-oxo-Delta(4) steroids including the conversion of testosterone to 5 alpha-dihydrotestosterone. In humans, 5 alpha-reductase activity is critical for certain aspects of male sexual differentiation, and may be involved in the development of benign prostatic hyperplasia, alopecia, hirsutism, and prostate cancer. Certain natural products contain components that are inhibitors of 5 alpha-reductase, such as the green tea catechin (-)-epigallocatechin gallate (EGCG). EGCG shows potent inhibition in cell-free but not in whole-cell assays of 5 alpha-reductase. Replacement of the gallate ester in EGCG with long-chain fatty acids produced potent 5 alpha-reductase inhibitors that were active in both cell-free and whole-cell assay systems. Other flavonoids that were potent inhibitors of the type 1 5alpha-reductase include myricetin, quercitin, baicalein, and fisetin. Biochanin A, daidzein, genistein, and kaempferol were much better inhibitors of the type 2 than the type 1 isozyme. Several other natural and synthetic polyphenolic compounds were more effective inhibitors of the type 1 than the type 2 isozyme, including alizarin, anthrarobin, gossypol, nordihydroguaiaretic acid, caffeic acid phenethyl ester, and octyl and dodecyl gallates. The presence of a catechol group was characteristic of almost all inhibitors that showed selectivity for the type 1 isozyme of 5 alpha-reductase. Since some of these compounds are consumed as part of the normal diet or in supplements, they have the potential to inhibit 5 alpha-reductase activity, which may be useful for the prevention or treatment of androgen-dependent disorders. However, these compounds also may adversely affect male sexual differentiation.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed2002

Inhibition of benzo(a)pyrene-induced lung tumorigenesis in A/J mice by dietary N-acetylcysteine conjugates of benzyl and phenethyl isothiocyanates during the postinitiation phase is associated with activation of mitogen-activated protein kinases and p53 activity and induction of apoptosis.

Recent studies in cell culture have shown that isothiocyanates (ITCs) induce apoptosis via activation of mitogen-activated protein (MAP) kinases and p53 pathways, suggesting a potential for ITCs or their conjugates to inhibit tumorigenesis during the postinitiation phase. To evaluate whether ITC compounds administered after carcinogen treatment inhibit lung tumorigenesis, we investigated in A/J mice the effects of the N-acetylcysteine (NAC) conjugates of benzyl (BITC-NAC) and phenethyl ITC (PEITC-NAC) in the diet (15 micromol/g) administered after a single dose of 20 micromol benzo(a)pyrene [B(a)P]. The formation of lung adenomas was examined 140 days after B(a)P dosing. Both the BITC-NAC and PEITC-NAC-treated groups showed a significant reduction in lung tumor multiplicity from 6.1 +/- 3.1 tumors/mouse in the B(a)P group fed the control diet to 3.7 +/- 2.9 and 3.4 +/- 2.7 tumors/mouse (P = 0.018 and 0.006, respectively). To investigate the mechanisms of tumor inhibition, lung tissues were obtained at 21, 84, and 140 days at interim sacrifices during the bioassay. These tissues showed a significant increase in apoptosis as determined by in situ end-labeling for both ITC-NAC-treated groups. The MAP kinase pathway was activated in the ITC-NAC-treated groups. The activation of c-Jun NH(2)-terminal kinase was higher in the BITC-NAC and PEITC-NAC groups when compared with B(a)P-treated control. The phosphorylation of p38 and extracellular signal-regulated kinases (ErKs) 1 and 2 was also induced by these treatments. To determine the downstream target of MAP kinases, activator protein-1 (AP-1) and nuclear factor-kappaB activities were evaluated by gel shift assay. The AP-1 binding activity was remarkably increased in lung tissue from both the BITC-NAC and PEITC-NAC groups. No change in nuclear factor-kappaB binding activity was found, however. Phosphorylation of p53 was also higher than the constitutive levels in both ITC-NAC-treated groups, but no induction of p53 expression was detected. This study demonstrates the chemopreventive efficacy of the NAC conjugates of PEITC and BITC administered in the diet after a single dose of B(a)P for lung tumorigenesis and provides the first in vivo evidence that activation of MAP kinases, AP-1 transcription factors, p53 phosphorylation, and the induction of apoptosis may be involved in the chemopreventive activity of these compounds.

Acetylcysteine↗
PubMed2002

Caspase 3 expression is altered in a coculture model of neuroblastoma.

BACKGROUND: Previously, we demonstrated that neuroblastoma cells cocultured with hepatocytes are protected from apoptosis, while apoptosis is upregulated in the hepatocytes. The mechanisms responsible for these findings are unknown. We hypothesize that caspase 3, a cysteine protease central to the apoptotic pathway, will be altered in this coculture model that simulates metastatic neuroblastoma. METHODS: Control human neuroblastoma cells and liver cells are plated in standard media. For the study group, a noncontact, coculture system is used. Hepatocytes are plated on cell culture inserts, placed above a growing layer of neuroblastoma cells, and incubated. Activated caspase 3 is measured after 1, 2, 3, or 4 days. RESULTS: Activated caspase 3 levels are significantly decreased in the cocultured neuroblastoma cells on days 2, 3, and 4. Conversely, cocultured hepatocytes have a significant increase in caspase 3 activation at all time periods, with the largest difference seen after 1 day in coculture. CONCLUSIONS: The capacity for neuroblastoma to differentially alter caspase 3 activation may provide a significant survival advantage for the neuroblastoma cells in metastatic environments. Understanding the mechanisms for this altered regulation may lead to improved and better targeted therapy for this malignancy.

Caspase 3↗
PubMed2002

Proteasome-dependent downregulation of p21(Waf1/Cip1) induced by reactive oxygen species.

After hydrogen peroxide (H(2)O(2)) treatment, the p21 (p21(Waf1/Cip1)) protein level in GM00637 fibroblast cells was rapidly decreased, reaching its nadir around 3 h. However, it rebounded within 5 hours to a level higher than that before treatment. Fluorescence microscopic analyses revealed that nuclear p21 was downregulated during the initial oxidative stress. H(2)O(2)-induced downregulation of p21 protein was accompanied by a gradual increase in p21 mRNA levels. Other inducers of genotoxic stress, such as treatment with adriamycin, a DNA damage compound, did not cause a significant decrease in p21 protein levels. Pretreatment of GM00637 cells with the proteasome inhibitors, lactacystin or MG132, completely blocked H(2)O(2)-induced p21 downregulation, suggesting that H(2)O(2) treatment accelerated p21 degradation. Conversely, cotreatment of cells with a protein synthesis inhibitor, cycloheximide, and H(2)O(2) drastically shortened the half-life of p21. Moreover, p21 mRNA levels were not downregulated by treatment with proteasome or protein synthesis inhibitors. Taken together, our studies indicate that oxidative stress induces rapid, but reversible, downregulation of functional p21 by accelerating its protein turnover.

Acetylcysteine↗
PubMed2002

Cell cycle arrest and apoptosis induced by human Polo-like kinase 3 is mediated through perturbation of microtubule integrity.

Human Polo-like kinase 3 (Plk3, previously termed Prk or Fnk) is involved in regulation of cell cycle progression through the M phase (B. Ouyang, H. Pan, L. Lu, J. Li, P. Stambrook, B. Li, and W. Dai, J. Biol. Chem. 272:28646-28651, 1997). Here we report that in most interphase cells endogenous Plk3 was predominantly localized around the nuclear membrane. Double labeling with Plk3 and gamma-tubulin, the latter a major component of pericentriole materials, revealed that Plk3 was closely associated with centrosomes and that its localization to centrosomes was dependent on the integrity of microtubules. Throughout mitosis, Plk3 appeared to be localized to mitotic apparatus such as spindle poles and mitotic spindles. During telophase, a significant amount of Plk3 was also detected in the midbody. Ectopic expression of Plk3 mutants dramatically changed cell morphology primarily due to their effects on microtubule dynamics. Expression of a constitutively active Plk3 (Plk3-A) resulted in rapid cell shrinkage, which led to formation of cells with an elongated, unsevered, and taxol-sensitive midbody. In contrast, cells expressing a kinase-defective Plk3 (Plk3(K52R)) mutant exhibited extended, deformed cytoplasmic structures, the phenotype of which was somewhat refractory to taxol treatment. Expression of both Plk3-A and Plk3(K52R) induced apparent G(2)/M arrest followed by apoptosis, although the kinase-defective mutant was less effective. Taken together, our studies strongly suggest that Plk3 plays an important role in the regulation of microtubule dynamics and centrosomal function in the cell and that deregulated expression of Plk3 results in cell cycle arrest and apoptosis.

Apoptosis↗
PubMed2002

Down-regulation of PLK3 gene expression by types and amount of dietary fat in rat colon tumors.

Epidemiological studies suggest that high intake of dietary fat rich in saturated fatty acids increases the colon cancer risk whereas dietary fish oil high in omega-3 fatty acids reduces the colon cancer risk. Previously, we reported that consumption of omega-6 fatty acid rich diets such as corn oil strongly promotes azoxymethane (AOM)-induced colon carcinogenesis in rats as compared to ingestion of a diet with equivalent amount of fat containing fish oil (HFFO) or low-fat diet (LFCO). Expression of PLK3 (Polo-like kinase-3, previously named Prk) is negatively correlated with the development of certain cancers. Ectopic expression of human PLK3 results in cell cycle arrest or induces apoptosis. To understand the role of PLK3 in colon carcinogenesis and to study the effect of types and amount of dietary fat on the expression levels of PLK3 in colon tumors, we analyzed the colon tumors and mucosa of rats administered the diets containing fish oil and corn oil for PLK mRNA expression. Here we report that expression of PLK3 was down-regulated in rat colon tumors. Quantitative polymerase chain reaction demonstrated that PLK3 mRNA levels were significantly lower in carcinogen (azoxymethane)-induced rat colon tumors than their uninvolved normal colonic mucosa. Among the normal mucosa isolated from rats fed on diets with various levels of fat (LFCO, or high fat diet with corn oil, HFCO, or supplemented with fish oil, HFFO), no significant changes in PLK3 mRNA expression was detected. Tumors isolated from rats fed with HFCO diet contained a very low level of PLK3 mRNA expression. Interestingly, tumors from rats fed the HFFO diet did not exhibit as dramatic down-regulation of PLK3 as the tumors of animals fed the HFCO diet. Furthermore, our results also indicate that the ectopic expression of a kinase active PLK3 construct induced apoptosis in HT-29 colon carcinoma cells. These observations suggest for the first time that a decreased activity of PLK3 may play a key role in colon tumor development as well as in HFCO-induced colon tumorigenesis.

Animals↗
PubMedDate not supplied

Genotoxic stress-induced activation of Plk3 is partly mediated by Chk2.

Polo-like kinase 3 (Plk3, alternatively termed Prk) is involved in the regulation of DNA damage checkpoint as well as in M-phase function. Plk3 physically interacts with p53 and phosphorylates this tumor suppressor protein on serine-20, suggesting that the role of Plk3 in cell cycle progression is mediated, at least in part, through direct regulation of p53. Here we show that Plk3 is rapidly activated by reactive oxygen species in normal diploid fibroblast cells (WI-38), correlating with a subsequent increase in p53 protein level. Plk3 physically interacts with Chk2 and the interaction is enhanced upon DNA damage. In addition, Chk2 immunoprecipitated from cell lysates of Daudi (which expressed little Plk3) is capable of stimulating the kinase activity of purified recombinant Plk3 in vitro, and this stimulation is more pronounced when Plk3 is supplemented with Chk2 immunoprecipitated from Daudi after DNA damage. Furthermore, ectopic expression Chk2 activates cellular Plk3. Together, our studies suggest Chk2 may mediate direct activation of Plk3 in response to genotoxic stresses.

Cell Cycle↗
PubMedDate not supplied

Arsenic trioxide arrests cells early in mitosis leading to apoptosis.

Arsenic trioxide (As(2)O(3)) is highly effective in the treatment of acute promyelocytic leukemias that express the promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARalpha) fusion protein. However, evidence has accumulated that As(2)O(3) induces apoptosis regardless of PML-RARalpha status. Here we show that, at clinically relevant concentrations, As(2)O(3) causes S and G(2)M phase arrest of both PML-RARalpha-positive and -negative leukemia cell lines, thus inhibiting their growth. Apoptotic cells are generated predominately from the G(2)M fraction. Using several independent methods, we demonstrate that the cells accumulated in the G(2)M peak consist primarily of cells arrested in the early stages of mitosis, prophase, prometaphase and metaphase. In mitotic cells, there was significant activation of caspases, PARP cleavage, and morphological changes characteristic of apoptosis. Unlike microtubule-active drugs that arrest cells in metaphase, arsenic trioxide did not affect the architecture of microtubules. Our data suggest that the antileukemic activities of arsenic may be a result of mitotic arrest which culminates in apoptosis.

Antineoplastic Agents↗