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Xuan Huang

Publications and source records attributed to Xuan Huang.

At least 37 records · Page 2Linked to original sources
PubMed2005

Nanodroplet profiling of enzymatic activities in a microarray.

We describe a generic method for the large-scale functional characterization of enzymes in a microarray. Poly-l-lysine and amine reactive slides were coated with fluorogenic substrates sensitive to proteases and phosphatases. Patterning enzymes on the slides by robotic printing produced spatially addressable, segregated droplets that were simultaneously exposed to the on-chip sensors. Multiple enzymes were profiled using this system that provided fluorescence readouts across temporal and stoichiometric dimensions concurrently on a single microarray substrate. This integrated microarray platform is applicable not only for the functional annotation of proteins, but also for the rapid agonist and antagonist discovery and in performing on-chip kinetics.

Microarray Analysis↗
PubMed2005

Histone H2AX phosphorylation after cell irradiation with UV-B: relationship to cell cycle phase and induction of apoptosis.

Damage to DNA that engenders double-strand breaks (DSBs) triggers phosphorylation of histone H2AX on Ser-139. Expression of phosphorylated H2AX (gammaH2AX) can be revealed immunocytochemically; the intensity of gammaH2AX immunofluorescence (IF) measured by cytometry was reported to correlate with the frequency of DSBs induced by X-ray radiation or by DNA damaging antitumor drugs. The aim of the present study was to measure expression of gammaH2AX following exposure of HeLa and HL-60 cells to a wide range of doses of UV-B light (6.1 J/m(2)-3.45 kJ/m(2)) and using multiparameter flow and laser scanning cytometry (LSC) to correlate DNA damage with cell cycle phase and induction of apoptosis. In both cell lines, the highest degree of H2AX phosphorylation induced by UV was seen in S-phase cells, particularly during early portion of S. In cells that did not replicate DNA (G(1), G(2) and M) the degree of H2AX phosphorylation was markedly lower than that in S-phase cells, and was strongly UV dose-dependent. Furthermore, the level of UV-induced gammaH2AX in G(1), G(2) and M was much higher in HeLa- than in HL-60- cells. Apoptotic cells become apparent >2h after exposure to UV and exhibited nearly an order of magnitude higher intensity of gammaH2AX IF than that initially induced by UV; predominantly S-phase cells underwent apoptosis. While the suppression of DNA replication, by aphidicolin prevented the induction of H2AX phosphorylation by UV in most S phase cells, it had no effect on a small cohort of cells that appeared to be entering S-phase, that expressed very high levels of gammaH2AX. Furthermore, aphidicolin itself induced gammaH2AX in early-S phase cells. The induction of gammaH2AX by UV was inhibited, but the incidence of apoptosis increased, by 5 mM caffeine, a known inhibitor of PI-3-related kinases. The data are consistent with the notion that H2AX phosphorylation observed throughout S phase reflects formation of DSBs due to the collision of replication forks with the UV-induced primary DNA lesions. Induction of gammaH2AX in G(1), G(2) and M is likely a response to the primary DSBs generated during UV exposure and/or DNA repair. It is unclear why the latter process was more pronounced in HeLa than in HL-60 cells.

Aphidicolin↗
PubMed2005

[Clinical analysis of 39 cases of heterotopic gastric mucosa in the upper esophagus].

OBJECTIVE: To determine accurately the incidence of heterotopic gastric mucosa in the upper esophagus (HGMUE) in China, and to study the macroscopic and microscopic aspects of the lesions and to evaluate the clinical importance of HGMUE. METHODS: A prospective study was made among a total of 15,228 consecutive patients, 8,573 male and 6,655 female, aged 54 (8-95), undergoing gastroscopy. Disease histories of all patients were carefully inquired, especially those regarding possible complaints including discomfort of throat and swallowing pain and so on. Special care was taken in the upper esophageal sphincter area to make sure whether the area was adequately inspected. Biopsy specimens from aberrant mucosa were obtained and the sections were stained with haematoxylin and eosin, and Giemsa stain for Helicobacter pylori. RESULTS: HGMUE was found in 39 patients (0.26%) with an average age of 50. Five patients with H. pylori infection in heterotopic gastric mucosa also presented the infection in the stomach. The gastric mucosa was gastric body type in 8 patients, transitional type in 11 patients, and antral pattern in 7 patients. Intestinal metaplasia was found in 5 patients, and mild atypical hyperplasia in 2 patients. An impressive finding was coexistent erosive gastritis in 14 patients (35.9%), Barrett's esophagus in one patient (2.6%), peptic ulcer in 8 patients (20.5%), and a patient had the complication of constriction in the upper esophagus. CONCLUSION: HGMUE is not rare in China. The presence of inlet patches is possibly correlated with specific symptoms. There are some severe complications in HGMUE, especially esophageal constriction. Close surveillance should be taken for rare cases with metaplasia or dysplasia in HGMUE.

Adolescent↗
PubMed2005

Novel assay utilizing fluorochrome-tagged physostigmine (Ph-F) to in situ detect active acetylcholinesterase (AChE) induced during apoptosis.

It was recently reported that acetylcholinesterase (AChE) is expressed in cells undergoing apoptosis and that its presence is essential for assembly of the apoptosome and subsequent caspase-9 activation. To obtain a marker of active AChE that could assay this enzyme in live intact cells and be applicable to fluorescence microscopy and cytometry, the fluorescein-tagged physostigmine (Ph-F), high affinity ligand (inhibitor) reactive with the active center of AChE, was constructed and tested for its ability to in situ label AChE and measure its induction during apoptosis. Ph-F inhibited cholinesterase activity in vitro (IC50 = 10(-6) and 5 x 10(-6) M for equine butyrylcholinesterase and human erythrocyte AChE, respectively) and was a selective marker of cells and structures that were AChE-positive. Thus, exposure of mouse bone marrow cells to Ph-F resulted in the exclusive labeling of megakaryocytes, and of the diaphragm muscle, preferential labeling of the nerve-muscle junctions (end-plates). During apoptosis of carcinoma HeLa cells and leukemic HL-60 or Jurkat cells triggered either by the DNA topoisomerase 1 inhibitor topotecan (TPT) or by oxidative stress (H2O2), the cells become reactive with Ph-F. Their Ph-F derived fluorescence was measured by flow and laser scanning cytometry. The appearance of Ph-F binding sites during apoptosis was preceded by the loss of mitochondrial potential, was concurrent with the presence of activated caspases, and was followed by loss of membrane integrity. At a very early stage of apoptosis, when nucleolar segregation was apparent, the Ph-F binding sites were distinctly localized within the nucleolus and at later stages of apoptosis in the cytoplasm. During apoptosis triggered by TPT, Ph-F binding was preferentially induced in S-phase cells. Our data on megakaryocytes and end-plates indicate that Ph-F reacts with active sites of AChE, and can be used to reveal the presence of this enzyme in live cells and possibly to study its expression in disorders of the neurological cholinergic system. The findings are also compatible with the reports that AChE may be induced during apoptosis. In fact, the simple and rapid Ph-F binding assay may serve as a convenient marker of apoptotic cells. However, the proposed role of active AChE as an essential factor for assembly of the apoptosome and caspase activation is in question because the AChE inhibitors Ph, Ph-F and BW284c51 did not protect the cells from apoptosis induced by TPT or H2O2. Further studies are thus needed to ascertain the induction and role of AChE in apoptosis.

Acetylcholinesterase↗
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

Comparative study of the effect of Shugan Shuru Granule on pathology and p53 gene expression in patients with hyperplastic disease of breast.

OBJECTIVE: To study the effect of Shugan Shuru Granule (SSG) on the p53 gene expression in patients with hyperplastic disease of breast (HDB) to indirectly explore the mechanism of SSG's effect on HDB on the molecular pathological level. METHODS: Sixty-six patients with HDB were allocated in the treated group and the control group, with the former treated with SSG and the latter not. All patients underwent breast operation and their diseased mammary tissues were cut out, sectioned, and observed under microscopy with HE staining and immunohistochemical staining, with ABC method adopted to estimate the degree of hyperplasia and p53 gene expression. The severity of HDB was classified into normal, mild, moderate and severe grades (marked as 0 to III), according to the degree of hyperplasia in the mammary gland. RESULTS: Hyperplasia in the control group mostly belonged to grade I-III before treatment, showing overgrowth of gland and proliferation of glandular epithelial cells, which were high columnar shaped, more stratified, with papillary or substantive dysplasia. While in the treated group, most belonged to grade 0-I after SSG treatment, with proliferated gland and dysplasia recovered to normal or disappeared. The positive rate of p53 gene expression in the treated group was 9.09%, and in the control group 39.39%, comparison between the two groups showing significant difference (P < 0.01), the intensity in the former was significantly weaker than that in the latter. CONCLUSION: SSG could not only inhibit the proliferation of mammary duct epithelia and lobuli, but also inhibit the over-expression of P53. Therefore, it could be regarded as an effective remedy for treatment of HDB and prevention of mammary cancer genesis.

Adult↗
PubMed2005

Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis.

Reviewed are the methods aimed to detect DNA damage in individual cells, estimate its extent and relate it to cell cycle phase and induction of apoptosis. They include the assays that reveal DNA fragmentation during apoptosis, as well as DNA damage induced by genotoxic agents. DNA fragmentation that occurs in the course of apoptosis is detected by selective extraction of degraded DNA. DNA in chromatin of apoptotic cells shows also increased propensity to undergo denaturation. The most common assay of DNA fragmentation relies on labelling DNA strand breaks with fluorochrome-tagged deoxynucleotides. The induction of double-strand DNA breaks (DSBs) by genotoxic agents provides a signal for histone H2AX phosphorylation on Ser139; the phosphorylated H2AX is named gammaH2AX. Also, ATM-kinase is activated through its autophosphorylation on Ser1981. Immunocytochemical detection of gammaH2AX and/or ATM-Ser1981(P) are sensitive probes to reveal induction of DSBs. When used concurrently with analysis of cellular DNA content and caspase-3 activation, they allow one to correlate the extent of DNA damage with the cell cycle phase and with activation of the apoptotic pathway. The presented data reveal cell cycle phase-specific patterns of H2AX phosphorylation and ATM autophosphorylation in response to induction of DSBs by ionizing radiation, topoisomerase I and II inhibitors and carcinogens. Detection of DNA damage in tumour cells during radio- or chemotherapy may provide an early marker predictive of response to treatment.

Animals↗
PubMed2005

The cytostatic and cytotoxic effects of oridonin (Rubescenin), a diterpenoid from Rabdosia rubescens, on tumor cells of different lineage.

Rabdosia rubescens is a herbal medicine used to treat esophageal cancer in China. In this study, the sesquiterpene oridonin, an isoprenoid, was isolated from Rabdosia rubescens. Mass spectroscopy and carbon 13 NMR spectroscopy were used to identify the structure of the purified compound. It was then evaluated for biological activity against human cell lines derived from prostate (DU-145, LNCaP), breast (MCF-7), and ovarian (A2780 and PTX10) cancers. Oridonin exhibited anti-proliferative activity toward all cancer cell lines tested, with an IC50 estimated by the MTT cell viability assay ranging from 5.8+/-2.3 to 11.72+/-4.8 microM. Flow cytometric analysis demonstrated that oridonin induced a G1 phase arrest in androgen receptor-positive LNCaP cells containing wt p53, while it blocked the cell cycle at G2 and M phases in androgen receptor-negative DU-145 cells with mutated p53; the arrest in M was verified by examination of cell morphology and by the increased frequency of cells with Ser-10 phosphorylated histone H3. The increased incidence of apoptosis, identified by characteristic changes in cell morphology, was seen in tumor lines treated with oridonin. Notably, at concentrations that induced apoptosis among tumor cells, oridonin failed to induce apoptosis in cultures of normal human fibroblasts. Western blot analysis was used to determine the protein expression of cancer suppressor genes, p53 (wt) and Bax, and the proto-oncogene, Bcl-2 in LNCaP cells following treatment with oridonin. Oridonin up-regulated p53 and Bax and down-regulated Bcl-2 expression in a dose-dependent manner. To further explore the possible interaction between oridonin and DNA, its absorption spectrum was measured in the presence and absence of double stranded (ds) DNA. Spectral shifts and an increase in absorption band intensity were observed indicating interaction of oridonin with DNA bases. The nature of the binding is not clear at present though no evidence of histone H2AX phosphorylation on Ser-139 was apparent in DU-145 cells treated with oridonin that would indicate the induction of ds DNA breaks. In conclusion, oridonin inhibits cancer cell growth in a cell cycle specific manner and shifts the balance between pro- and anti-apoptotic proteins in favor of apoptosis. The present data suggest that further studies are warranted to assess the potential of oridonin in cancer prevention and/or treatment.

Breast Neoplasms↗
PubMed2005

[Preparation and quality control of low molecular weight chitosan].

OBJECTIVE: To prepare the low molecular weight chitosan (LMWC) and establish the method for quality control. METHOD: Use enzymatic degradation to prepare LMWC with chitosan, and separate by ultrafiltration; the molecular weight and purity were determined by gel permeation chromatography (GPC) and colorimetry respectively. RESULT: LMWC was prepared by control the hours of enzymatic degradation and ultrafiltered through filter with cutoff molecular of 10K Dalton and 50 K dalton; the average molecular weight was 20 K dalton and the purity was (96.60 +/- 1.56)%. CONCLUSION: The condition of enzymatic degradation is geniality and easy to control, LMWCs with different molecular weight can separate by ultrafiltration efficiently; the quality of LMWC can control with gel permeation chromatography (GPC) and colorimetry.

Cellulase↗
PubMed2004

Developing photoactive affinity probes for proteomic profiling: hydroxamate-based probes for metalloproteases.

The denaturing aspect of current activity-based protein profiling strategies limits the classes of chemical probes to those which irreversibly and covalently modify their targeting enzymes. Herein, we present a complimentary, affinity-based labeling approach to profile enzymes which do not possess covalently bound substrate intermediates. Using a variety of enzymes belonging to the class of metalloproteases, the feasibility of the approach was successfully demonstrated in several proof-of-concept experiments. The design template of affinity-based probes targeting metalloproteases consists of a peptidyl hydroxamate zinc-binding group (ZBG), a fluorescent reporter tag, and a photolabile diazirine group. Photolysis of the photolabile unit in the probe effectively generates a covalent, irreversible linkage between the probe and the target enzyme, rendering the enzyme distinguishable from unlabeled proteins upon separation on a SDS-PAGE gel. A variety of labeling studies were carried out to confirm that the affinity-based approach selectively labeled metalloproteases in the presence of a large excess of other proteins and that the success of the labeling reaction depends intimately upon the catalytic activity of the enzyme. Addition of competitive inhibitors proportionally diminished the extent of enzyme labeling, making the approach useful for potential in situ screening of metalloprotease inhibitors. Using different probes with varying P(1) amino acids, we were able to generate unique "fingerprint" profiles of enzymes which may be used to determine their substrate specificities. Finally, by testing against a panel of yeast metalloproteases, we demonstrated that the affinity-based approach may be used for the large-scale profiling of metalloproteases in future proteomic experiments.

Affinity Labels↗
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

[Clinical application of mastoscopic axillary sentinel lymph node biopsy].

OBJECTIVE: To evaluate the technique and significance of mastoscopic axillary sentinel lymph node biopsy. METHODS: Sixty-two patients with breast cancer use methylene blue to test axillary sentinel lymph node. Sentinel lymph node was moved with endoscopy, and endoscopic axillary lymph nod dissection was performed. Pathological examination of sentinel lymph node and axillary lymph node was made with HE. To evaluate detection rate and false negative rate in sentinel lymph node. RESULTS: Among the 62 patients, 61 were confirmed by endoscopic axillary sentinel lymph nod biopsy. Detection rate was 98.4%. Thirty-five cases were no metastasis, 27 cases were metastasis, false negative rate was 0. CONCLUSIONS: Mastoscopic axillary sentinel lymph node biopsy has a high detection rate, good efficiency of cosmetic and lower complications. It has higher sensitivity than traditional axillary lymph nod dissection and provide accurate lymph node stages.

Adult↗
PubMed2004

Significance of vascular endothelial growth factor expression and its correlation with inducible nitric oxide synthase in gastric cancer.

AIM: To investigate the clinical significance of the expression of VEGF165mRNA and the correlation with vascular endothelial growth factor (VEGF) protein and inducible nitric oxide synthase (iNO) in human gastric cancer. METHODS: We tested VEGF165mRNA expression in 31 cases of resected gastric cancer specimens and normal paired gastric mucosae by RT-PCR. Total RNA was extracted with TRIzol reagents, transcribed into cDNA with oligo (dT15) priming, inner controlled with beta-actin expression and agarose gel isolated after PCR. VEGF expression was quantitated with IS1000 imaging system. Meanwhile we also examined expression levels of VEGF protein and iNOS in 85 cases of gastric cancer. All paraffin-embedded samples were immunohistochemically stained by streptavidin -peroxidase method (SP). RESULTS: The mean expression of VEGF165mRNA in gastric cancer was 1.125+/-0.356, significantly higher than that of normal paired mucosae, which was 0.760+/-0.278. The data indicated that the expression level of VEGF165mRNA was well related to lymph node metastasis and TNM stages of UICC. The expression levels in patients with lymph node metastasis and without lymph node metastasis were 1.219+/-0.377 and 0.927+/-0.205 respectively (P<0.05). The expression in stages I, II, III, IV was 0.934+/-0.194, 1.262+/-0.386 respectively (P<0.01). Further analysis showed the lymph node metastasis rate in the group with over-expression of VEGF was higher than that in the group with low expression of VEGF (83.3% vs 46.2%), and the ratio of stage III+IV in the group with over-expression of VEGF was also higher than that in the group with low expression with VEGF (77.8% vs 33.8%) (P<0.05). The positive rates of expression of VEGF protein and iNOS in 85 cases of gastric cancer were 75.4% and 58.8% respectively, and 50.1% of the patients showed positive staining both for iNOS and VEGF, the correlation with the two factors was significant (P=0.018). But more intensive analysis showed the immunoreactive grades of VEGF were not associated with that of iNOS. CONCLUSIONS: The expression of VEGF165mRNA is well related with lymph node metastasis and TNM stages of UICC in gastric cancer, and is concerned with the invasiveness and metastasis of gastric cancer. The relationship can be observed between the expression of VEGF and iNOS in gastric cancer.

Adult↗
PubMed2004

Simple, semiautomatic assay of cytostatic and cytotoxic effects of antitumor drugs by laser scanning cytometry: effects of the bis-intercalator WP631 on growth and cell cycle of T-24 cells.

BACKGROUND: Common assays of drug-induced cytotoxicity on adherent cells rely on cell trypsinization followed by count of live and dead cells. To estimate the cell cycle effects, cellular DNA content is analyzed by flow cytometry. This procedure is laborious and time consuming. The alternative viability assays, e.g., based on reduction of 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide, although rapid and convenient, do not provide information about individual cells or cell cycle effects and may be biased by growth imbalance. METHODS: The bladder carcinoma T-24 cells were seeded onto eight-chamber microscope slide-based tissue culture vessels. The novel antitumor drug, the bis-intercalating anthracycline WP631, was administered at various concentrations to different chamber cultures on the same slide; the control cultures were left untreated. After 24, 48, and 72 h, the cultures were fixed, and cellular DNA was stained with 4,6-diamidino-2-phenyl indole (DAPI). The slides were scanned by laser scanning cytometry (LSC) to obtain the number of attached cells per culture chamber and reveal their cell cycle distribution. RESULTS: The cell growth and viability plots in the absence and presence of WP621 were constructed from the frequency of the attached cells per chamber. A 50% reduction in cell number was observed at the 75 nM concentration of WP321. Mitotic and postmitotic cells were identified based on high intensity of maximal pixel of DAPI fluorescence. An increase in proportion of cells in G2 was seen at 75-300 nM of WP631. Relatively few (<12%) apoptotic cells, identified by the presence of DNA strand breaks, remained attached in the WP631-treated cultures. CONCLUSIONS: Because late apoptotic cells detach during culturing, the cells that remain attached in the multi-chamber cultures represent predominantly live cells; the deficit in their number compared with the untreated cultures, recorded by LSC during scanning, provides information about the degree of cytostatic and cytotoxic effects of the studied drug. The possibility to demonstrate the cell cycle distribution, including a distinction between G2 and M cells, provides an additional advantage of this assay. Other parameters that may be associated with the cell cycle perturbation or with induction of apoptosis also can be measured in the same cultures by using the multiparameter capabilities of LSC. Each measured cell can be relocated for imaging or measurement after subsequent staining with other probes.

Antineoplastic Agents↗
PubMed2004

Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin.

BACKGROUND: DNA double-strand breaks (DSBs) in chromatin, whether induced by radiation, antitumor drugs, or by apoptosis-associated (AA) DNA fragmentation, provide a signal for histone H2AX phosphorylation on Ser-139; the phosphorylated H2AX is denoted gammaH2AX. The intensity of immunofluorescence (IF) of gammaH2AX was reported to reveal the frequency of DSBs in chromatin induced by radiation or by DNA topoisomerase I (topo 1) and II (topo 2) inhibitors. The purpose of this study was to further characterize the drug-induced (DI) IF of gammaH2AX, and in particular to distinguish it from AA gammaH2AX IF triggered by DNA breaks that occur in the course of AA DNA fragmentation. METHODS: HL-60 cells in cultures were treated with topotecan (TPT), mitoxantrone (MTX), or with DNA cross-linking drug cisplatin (CP); using multiparameter flow and laser-scanning cytometry, induction of gammaH2AX was correlated with: 1) caspase-3 activation; 2) chromatin condensation, 3) cell cycle phase, and 4) AA DNA fragmentation. The intensity of gammaH2AX IF was compensated for by an increase in histone/DNA content, which doubles during the cell cycle, and for the "programmed" H2AX phosphorylation, which occurs in untreated cells. RESULTS: In cells treated with TPT or MTX, the increase in DI-gammaH2AX IF peaked at 1.5 or 2 h, and was maximal in S- or G(1)-phase cells, respectively, for each drug. In cells treated with CP, compared with TPT, the gammaH2AX IF was less intense, peaked later (3 h) and showed no cell cycle-phase specificity. In the presence of phosphatase inhibitor calyculin A, a continuous increase in the TPT-induced gammaH2AX IF was still seen past 1.5 h, and after 3 h gammaH2AX IF was 2.7- to 3.4-fold higher than in the absence of the inhibitor. The AA gammaH2AX IF was distinguished from the DI-gammaH2AX IF by: 1) its greater intensity; 2) its prevention by caspase inhibitor zVAD-FMK; and 3) the concurrent activation of caspase-3 in the same cells. A decrease in AA gammaH2AX IF coinciding with AA chromatin condensation was seen in the late stages of apoptosis. CONCLUSIONS: Multiparameter analysis of gammaH2AX IF, caspase-3 activation, cellular DNA content, and chromatin condensation allowed us to distinguish the DI from AA H2AX phosphorylation and relate them to the cell cycle phase and stage of apoptosis. With a comparable degree of ds DNA breaks, the cells arrested at the G1 or G2/M checkpoint were less prone to undergo apoptosis than the cells replicating DNA. H2AX phosphorylation seen in CP-treated cells may be associated with DNA repair that involves nucleotide excision repair (NER) and nonhomologous end joining (NHEJ). When the primary drug-induced lesions do not involve ds DNA breaks, but ds DNA breaks are formed during DNA repair, as in the case of CP, analysis of H2AX phosphorylation may reflect extent of the repair process.

Antineoplastic Agents↗
PubMed2004

Histone H2AX phosphorylation induced by selective photolysis of BrdU-labeled DNA with UV light: relation to cell cycle phase.

BACKGROUND: The induction of DNA double-strand breaks (DSBs) in chromatin triggers histone H2AX phosphorylation (on Ser-139) by ATM-, ATR-, or DNA-dependent protein kinases (DNA-PK). Phosphorylated H2AX, denoted as gammaH2AX, can be detected immunocytochemically using an antibody that is specific to the Ser-139-phosphorylated epitope. We previously reported that the induction DSBs by DNA topoisomerase I or II inhibitors can be monitored in individual cells by measuring gammaH2AX immunofluorescence (IF) by cytometry. The present study explored whether the detection of gammaH2AX IF can serve as a marker of the presence of the DNA precursor bromodeoxyuridine (BrdU) that is incorporated into DNA. METHODS: HeLa cells growing on microscope slides were incubated with BrdU for 1 h, rinsed free of the precursor, and incubated for different periods for up to 12 h. The cells were then briefly incubated with Hoechst 33342 (to sensitize BrdU-labeled DNA to ultraviolet [UV] light), irradiated with 300 nm UV light to photolyze BrdU-labeled DNA, transferred back into culture for an additional hour, and fixed. Cells were concurrently immunostained for gammaH2AX (Alexa Fluor 633) and cyclin A (fluorescein isothiocyanate); their DNA was counterstained with 4,6-diamidino-2-phenylindole. The intensities of cellular far red (gammaH2AX), green (cyclin A), and blue (DNA) fluorescences were measured by laser scanning cytometry. RESULTS: After a 1-h pulse of BrdU followed by exposure to UV, nearly all cells with S-phase DNA content had many-fold higher gammaH2AX IF than G(1) or G(2)/M cells. The nonirradiated cells had minimal ("programmed") expression of gammaH2AX, whereas the irradiated cells incubated without BrdU had uniformly elevated levels of gammaH2AX IF independent of the cell cycle phase. Pulse-chase experiments showed that the cohort of BrdU-labeled (gammaH2AX-positive) cells progressed through G(2)/M and into G(1) phase after 8 and 12 h of growth in BrdU-free medium, respectively. Bivariate analysis of gammaH2AX versus cyclin A expression for the gated S-phase cells showed a correlation between these variables, suggesting that the rate of BrdU incorporation (DNA replication) correlates with expression of cyclin A. CONCLUSIONS: Photolysis of BrdU-labeled DNA induces DSBs and leads to H2AX phosphorylation. Detection of gammaH2AX IF indicates the presence of the incorporated BrdU, is compatible with concurrent detection of other intracellular antigens, and can be used to demonstrate cell cycle kinetics.

Benzimidazoles↗
PubMed2004

Recent advances in gel-based proteome profiling techniques.

This review focuses on recent developments in gel-based proteomics techniques. By combining traditional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoretic techniques with recent advances in protein labeling using different classes of molecules (i.e., fluorescent dyes, chemical probes, radioisotopes), new technologies have been developed that allow for high-throughput studies of proteins at the whole-proteome scale.

Electrophoresis, Gel, Two-Dimensional↗