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Results for “Laser Scanning Cytometry”

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PubMed2000

Assessment of Tissue Estrogen and Progesterone Receptor Levels: A Survey of Current Practice, Techniques, and Quantitation Methods.

The assessment of steroid hormone receptors in resected breast carcinoma tissue is currently the standard of practice. The traditional method for assessment of receptor status is the ligand binding assay. More recently, immunohistochemistry (IHC) has become a popular method for such testing. Despite the widespread use of IHC and the availability of many antibodies, standardization of quantitative IHC for assessment of estrogen and progesterone receptors has not been achieved. While the College of American Pathologists (CAP) offers a Quality Assurance (QA) program for IHC quantitation of estrogen receptor (ER) and progesterone receptor (PgR), no universal standard is currently recognized in assessment of ER and PgR by IHC. We surveyed 300 laboratories within the United States for their current practices regarding the assessment of ER and PgR status in breast cancer tissue specimens. Eighty usable responses were received. Forty-nine (61%) laboratories performed the assay in-house, while the remainder sent the material out for assay. All responding laboratories performing their steroid receptor analysis in-house used the IHC technique. Forty-three (80%) laboratories answering the question on material accepted for analysis performed the assay only on paraffin-embedded material, three (6%) used either paraffin block or frozen material, and two (4%) used only frozen material. Eighty-eight percent of laboratories performing steroid receptor analysis in-house used a manual quantitation technique. Four (8%) used computer-assisted image analysis, and a single laboratory used laser scanning cytometry. Eight different antibodies were used among the 44 laboratories documenting the antibody supplier, and for any given commercially prepared antibody a wide variety of dilutions were used, with the exception of the standard solution used with the Ventana antibody. Of the laboratories using manual estimation techniques, 61% simply estimated the percentage of positive cells, 29% evaluated both the intensity of staining and percentage of nuclei staining, 6% used formal H-score analysis, 2% evaluated only intensity of nuclear staining, and 2% mainly counted the percentage of nuclei staining for ER but used a formal H score in the assessment of PgR. Cutoff points for the separation of positive and negative results varied widely, with some laboratories assessing any demonstrable positivity as a positive result, while others required as many as 19% of the nuclei to stain before a specimen was declared positive. Standardization techniques differed considerably among laboratories. Eighty-six percent used the CAP program for QA. While all laboratories utilized some form of intralaboratory control for assessment of ER and PgR, the nature of that control varied from laboratory to laboratory. Our survey indicates that a majority of laboratories perform their steroid hormone receptor analysis in-house using IHC. There is considerable variability in the antibodies utilized, the dilutions applied, and the quantitation method and level of expression used to dichotomize specimens into positive and negative groups. Finally, no universal control for interlaboratory standardization appears to exist.

Journal Article↗

PubMedDate not supplied

Curcumin enhances wound healing in streptozotocin induced diabetic rats and genetically diabetic mice.

Tissue repair and wound healing are complex processes that involve inflammation, granulation and tissue remodeling. Interactions of different cells, extracellular matrix proteins and their receptors are involved in wound healing, and are mediated by cytokines and growth factors. Previous studies from our laboratory have shown that curcumin (diferuloylmethane), a natural product obtained from the rhizomes of Curcuma longa, enhanced cutaneous wound healing in rats and guinea pigs. In this study, we have evaluated the efficacy of curcumin treatment by oral and topical applications on impaired wound healing in diabetic rats and genetically diabetic mice using a full thickness cutaneous punch wound model. Wounds of animals treated with curcumin showed earlier re-epithelialization, improved neovascularization, increased migration of various cells including dermal myofibroblasts, fibroblasts, and macrophages into the wound bed, and a higher collagen content. Immunohistochemical localization showed an increase in transforming growth factor-beta1 in curcumin-treated wounds compared to controls. Enhanced transforming growth factor-beta1 mRNA expression in treated wounds was confirmed by in situ hybridization, and laser scan cytometry. A delay in the apoptosis patterns was seen in diabetic wounds compared to curcumin treated wounds as shown by terminal deoxynucleotidyl transferase-mediated deoxyuridyl triphosphate nick end labeling analysis. Curcumin was effective both orally and topically. These results show that curcumin enhanced wound repair in diabetic impaired healing, and could be developed as a pharmacological agent in such clinical settings.

Animals↗

PubMed2001

Malignant teratoma of the thyroid with primitive neuroepithelial and mesenchymal sarcomatous components.

A 15-year-old black girl had a near total resection of a malignant thyroid teratoma with bilateral nodal involvement and mediastinal extension. A predominant neuroepithelial pattern had ependymal rosettes and mitoses, stained for neuron-specific enolase, neuron-specific B tubulin, and synaptophysin. A malignant spindle cell component stained for smooth-muscle actin, muscle actin, and to a lesser extent S-100. Loose myxoid tissue resembled primitive cartilage. Epithelial membrane antigen and cytokeratin identified epithelial foci. Chromogranin A, MIC2, glial fibrillary acidic protein, and thyroid stimulating hormone receptor stains were negative. There was focal anaplasia. DNA ploidy by laser scanning cytometry was 1.2. The tumor from the left and right thyroid lobes exhibited trisomy 8, the right also had hyperdiploid cell lines. She was treated with aggressive combination chemotherapy and radiation. Presently there is no residual disease 16 months after diagnosis. Malignant thyroid teratoma is an aggressive tumor, with 15 of 27 reported patients dying 2 weeks to 3 years after diagnosis. Survivors have been treated with total or subtotal resection, combination chemotherapy with agents effective in the treatment of germ cell tumors as well as sarcomas, and radiation for either recurrent or residual disease. The heterologous elements, lacking MIC2 staining and t(11;22), support the diagnosis of malignant teratoma rather than a neuroepithelial tumor. Trisomy 8 is the first cytogenetic abnormality described in malignant thyroid teratoma. Therapy should be tailored to the management of all transformed histologies.

Adolescent↗

PubMed2000

Retinoblastoma protein-initiated cellular growth arrest overcomes the ability of cotransfected wild-type p53 to induce apoptosis.

The retinoblastoma gene, RB, participates in the regulation of the G1/S-phase transition and in p53-mediated apoptosis. We have previously reported that stably transfected RB functions as a growth and tumour suppressor in HTB9 human bladder carcinoma cells, which carry a mutation of the p53 gene at codon 280 and lack RB expression. To elucidate the potential role of RB in the regulation of p53-mediated apoptosis, we transfected a wt p53 expression plasmid under the control of the human cytomegalovirus promoter into parental and RB-transfected HTB9 cells. The p53(+)/RB(-)cells were susceptible to apoptosis under various experimental conditions: 1) incubation in serum-free culture for 72 h, 2) short-term (6 h) or long-term (48 h) exposure to etoposide, and 3) culturing in soft agar. In contrast, p53(+)/RB(+)cells were significantly resistant to apoptosis under similar conditions and exhibited efficient growth arrest, as measured by laser scanning cytometry. Tumorigenicity in nude mice of parental HTB9 cells was lost by exogenous expression of wt p53. Likewise, none of mice injected subcutaneously with either p53(-)/RB(+)or p53(+)/RB(+)cells developed tumours, indicating that RB allows suppression of tumorigenesis, regardless of p53 status. These results suggest that the growth-inhibitory function of RB may overcome the ability of wt p53 to induce apoptosis.

Animals↗

PubMed2000

Comparative methodologic study of NFkappaB activation in cultured endothelial cells.

The transcriptional regulatory protein nuclear factor kappaB (NFkappaB) participates in the control of gene expression of many modulators of the inflammatory and immune responses. Various activators trigger NFkappaB release and nuclear translocation after phosphorylation and proteolytic degradation of IkappaB. This study evaluated the abilities of fluorescence and confocal microscopies, laser scanning cytometry (LSC), electrophoretic mobility-shift assay (EMSA), and Western blotting to detect NFkappaB activation in endothelial cells (ECs) and to investigate the role of homocysteine (Hcy) in NFkappaB activation. ECs were treated with interleukin-1B (10 ng/mL) or Hcy thiolactone (1 and 5 mmol/L) as NFkappaB activators. Hcy, a thiol-containing amino acid, has been shown to directly damage ECs in vitro. Experimental evidence suggests that the atherogenic propensity associated with hyperhomocysteinemia results from EC dysfunction. When ECs were pretreated with an inhibitor (pyrrolidine dithiocarbamate, 100 micromol/L) or with staurosporine (5 microL/mL), no NFkappaB activation was observed. NFkappaB activation in ECs could be detected with all five techniques, clearly showing NFkappaB translocation from the cytoplasm to the nuclei. Confocal microscopy was more sensitive and less subjective than immunofluorescence microscopy. LSC was even more sensitive, specific, and reproducible. EMSA, the reference method, has the disadvantages of being radioactive, expensive, and time consuming. Western blot analysis detected the NFkappaB p50 subunit implicated in NFkappaB activation. The techniques usually used to detect NFkappaB activation in ECs are immunofluorescence microscopy and confocal microscopy, LSC, EMSA, and Western blot analysis, but none of them is ready for routine use.

Cells, Cultured↗

PubMed1999

Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum- and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. A novel convergence point of anti-proliferative and proliferative cell signaling pathways.

The serum- and glucocorticoid-inducible kinase (sgk) is a novel serine/threonine protein kinase that is transcriptionally regulated in rat mammary tumor cells by serum under proliferative conditions or by glucocorticoids that induce a G1 cell cycle arrest. Our results establish that the subcellular distribution of Sgk is under stringent cell cycle and hormonal control. Sgk is localized to the perinuclear or cytoplasmic compartment as a 50-kDa hypophosphorylated protein in cells arrested in G1 by treatment with the synthetic glucocorticoid dexamethasone. In serum-stimulated cells, Sgk was transiently hyperphosphorylated and resided in the nucleus. Laser scanning cytometry, which monitors Sgk localization and DNA content in individual mammary tumor cells of an asynchronously growing population, revealed that Sgk actively shuttles between the nucleus (in S and G2/M) and the cytoplasm (in G1) in synchrony with the cell cycle. In cells synchronously released from the G1/S boundary, Sgk localized to the nucleus during progression through S phase. The forced retention of exogenous Sgk in either the cytoplasmic compartment, using a wild type sgk gene, or the nucleus, using a nuclear localization signal-containing sgk gene (NLS-Sgk), suppressed the growth and DNA synthesis of serum-stimulated cells. Thus, our study implicates the nuclear-cytoplasmic shuttling of sgk as a requirement for cell cycle progression and represents a novel convergence point of anti-proliferative and proliferative signaling in mammary tumor cells.

Animals↗

PubMed2002

Fermented wheat germ extract inhibits glycolysis/pentose cycle enzymes and induces apoptosis through poly(ADP-ribose) polymerase activation in Jurkat T-cell leukemia tumor cells.

The fermented extract of wheat germ, trade name Avemar, is a complex mixture of biologically active molecules with potent anti-metastatic activities in various human malignancies. Here we report the effect of Avemar on Jurkat leukemia cell viability, proliferation, cell cycle distribution, apoptosis, and the activity of key glycolytic/pentose cycle enzymes that control carbon flow for nucleic acid synthesis. The cytotoxic IC(50) concentration of Avemar for Jurkat tumor cells is 0.2 mg/ml, and increasing doses of the crude powder inhibit Jurkat cell proliferation in a dose-dependent fashion. At concentrations higher than 0.2 mg/ml, Avemar inhibits cell growth by more than 50% (72 h of incubation), which is preceded by the appearance of a sub-G(1) peak on flow histograms at 48 h. Laser scanning cytometry of propidium iodide- and annexin V-stained cells indicated that the growth-inhibiting effect of Avemar was consistent with a strong induction of apoptosis. Inhibition by benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone of apoptosis but increased proteolysis of poly(ADP-ribose) indicate caspases mediate the cellular effects of Avemar. Activities of glucose-6-phosphate dehydrogenase and transketolase were inhibited in a dose-dependent fashion, which correlated with decreased (13)C incorporation and pentose cycle substrate flow into RNA ribose. This decrease in pentose cycle enzyme activities and carbon flow toward nucleic acid precursor synthesis provide the mechanistic understanding of the cell growth-controlling and apoptosis-inducing effects of fermented wheat germ. Avemar exhibits about a 50-fold higher IC(50) (10.02 mg/ml) for peripheral blood lymphocytes to induce a biological response, which provides the broad therapeutic window for this supplemental cancer treatment modality with no toxic effects.

Apoptosis↗

PubMed2003

Co-localization of apoptosis-regulating proteins in mouse mammary epithelial HC11 cells exposed to TGF-beta1.

TGF-beta1 is an apoptogenic agent for mammary epithelial cells (MEC). The molecular mechanism of the TGF-beta1-induced apoptosis remains, however, obscure. In the present study we used laser scanning cytometry, confocal microscopy and immunogold electron microscopy to analyze the expression, aggregation and co-localization of caspase-8, Bid, Bax and VDAC-1. These proteins are regarded as the most important factors involved in the regulatory phase of TGF-beta1-induced apoptosis. Apoptosis in HC11 mouse MEC manifested with a simultaneous increase in expression and subcellular aggregation of caspase-8, Bid, Bax and VDAC-1. Confocal microscopy revealed a strong pattern of co-localization of examined proteins during both early and late apoptosis. Experiments with double- and triple-staining immunoelectron microscopy showed a co-localization of Bax/Bid, caspase-8/Bax/Bid, and Bax/VDAC-1, on the membranes of mitochondria, Golgi apparatus, rough endoplasmic reticulum, nuclear envelope, nuclear pore, and within the nucleus. In conclusion, the observed pattern of changes in aggregation and subcellular localization of caspase-8, Bid, Bax and VDAC-1 during TGF-beta1-induced apoptosis in HC11 mouse MEC suggests an interaction between these proteins and formation of multimeric complexes on organellar membranes, thus controlling their permeability for intracellular mediators of apoptosis.

Animals↗

PubMed2003

Effects of epithelial growth factor receptor (EGFR) kinase inhibitors on genetically reconstituted mouse mammary glands.

UNLABELLED: The aim of the study was to determine the effects of a specific epithelial growth factor Receptor kinase inhibitor (EGFR-KI) and Taxol on tumor growth in a novel tumor model. MATERIAL & METHODS: A genetically engineered tumor model which uses "transgenic" organs in immune competent mice was used. NeuT-transfected immortalized HC11 epithelial cells and primary mouse mammary epithelial cells have been transplanted into the gland-free mammary fat pad of female BALB/c mice. Mammary tumors developed after a latency period of three to four weeks. The mice were thereafter daily orally treated over a 19 or 22-day period with 0, 38, 75, 2 x 75 mg/kg body weight (b.w.) EGFR-KI (n: 7-9 per group) or intravenously with 10 mg/kg b.w. Taxol. After necropsy the histopathological evaluation of the tumors was performed in a coded manner. The proliferation activity of tumor cells was analyzed by laser scanning cytometry (LSC) using anti-Ki67-antibodies. RESULTS: Oral Treatment with EGFR-KI in this transgenic organ model showed clear antitumor efficacy in a dose-dependent manner in the range between 38 and 75 mg/kg b.w. This antiproliferative effect appears to be minimally increased at 75 mg/kg/day twice per day. For all treatments a strong correlation between the biological behavior of the tumor, histopathology and cell proliferation could be established. In contrast, treatment with Taxol showed no significant reduction of tumor growth or cell proliferation in this model. This new transgenic organ model comprising histopathological evaluation and cell proliferation analysis appears to be a suitable test system for drug candidates that affect specific biochemical pathways. It may have greater predictive nature for clinical effects in humans as compared to conventional tumor models because of its c-erb B2 gene overexpression.

Administration, Oral↗

PubMed2001

Detection of viral RNA in CD4(-)CD8(-) and CD4(-)CD8(+) lymphocytes in vivo in rhesus monkeys infected with simian immunodeficiency virus of macaques.

A definition of the specific cell types that support HIV replication early in the course of infection will be important for understanding AIDS pathogenesis and designing strategies for preventing infection. Observations have indicated that the population of lymphocytes susceptible to productive infection extends beyond activated CD4(+) T cells. To explore this issue, we have employed laser scanning cytometry technology and the techniques of lymphocyte surface immunophenotyping followed by fluorescent in situ hybridization to detect simian immunodeficiency virus of macaques (SIVmac) RNA in phenotypically defined rhesus monkey lymphocytes. The immunophenotype of productively infected cells in either a rhesus monkey T cell line or in PBMCs infected in vitro with SIVmac was remarkably similar to that observed in productively infected PBMCs obtained from monkeys during primary infection. We observed low levels or no detectable expression of CD4 on cells infected in vitro or on PBMCs of infected monkeys. However, a substantial number of SIVmac-infected PBMCs both in cultured lymphocytes and sampled directly from infected monkeys expressed CD8 but not CD4. These observations are consistent with the possibility that the CD4 molecule may be modulated off the surface of CD4(+)CD8(-) or CD4(+)CD8(+) lymphocytes after infection or that infection occurred via a CD4-independent mechanism. Moreover, there was no preferential expression of CD25 on cells positive for SIVmac RNA, which might have been predicted if replication of the virus was occurring selectively in activated lymphocytes. These results broaden the range of lymphocytes that support productive SIVmac infection to include CD4(-)CD8(-) and CD4(-)CD8(+) subsets, and are consistent with virus replication occurring in nonactivated cells.

Animals↗

PubMed2004

Multiple pathways regulate MeCP2 expression in normal brain development and exhibit defects in autism-spectrum disorders.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in MECP2, encoding methyl-CpG-binding protein 2 (MeCP2). Although MECP2 is ubiquitously transcribed, MeCP2 expression is developmentally regulated and heterogeneous in neuronal subpopulations, defined as MeCP2(lo) and MeCP2(hi). To test the hypothesis that pathways affecting MeCP2 expression changes may be defective in RTT, autism and other neurodevelopmental disorders without MECP2 mutations, a high-throughput quantitation of MeCP2 expression was performed on a tissue microarray containing frontal cortex samples from 28 different patients with neurodevelopmental disorders and age-matched controls. Combined quantitative analyses of MeCP2 protein and alternatively polyadenylated transcript levels were performed by laser scanning cytometry and tested for significant differences from age-matched controls. Normal cerebral samples showed an increase in total MeCP2 expression and the percentage of MeCP2(hi) cells with age that could be explained by increased MECP2 transcription within the MeCP2(hi) population. A significant decrease in the relative usage of the long transcript in the MeCP2(lo) population was observed in postnatal compared to fetal brain, but alternate polyadenylation did not correlate with MeCP2 expression changes at the single cell level. Brain samples from several related neurodevelopmental disorders, including autism, pervasive developmental disorder, Prader-Willi and Angelman syndromes showed significant differences in MeCP2 expression from age-matched controls by apparently different transcriptional and post-transcriptional mechanisms. These results suggest that multiple pathways regulate the complex developmental expression of MeCP2 and are defective in autism-spectrum disorders in addition to RTT.

Adolescent↗

PubMed2004

X-Chromosome inactivation ratios affect wild-type MeCP2 expression within mosaic Rett syndrome and Mecp2-/+ mouse brain.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in MECP2, encoding methyl-CpG-binding protein 2 (MeCP2). The onset of symptoms in RTT is delayed until 6-18 months and 4-6 months in the Mecp2(-/+) mouse model, corresponding to a dynamic and gradual accumulation of MeCP2 expression in individual neurons of the postnatal brain. Because of X chromosome inactivation (XCI), cells within RTT females are mosaic for expression of the heterozygous MECP2 mutation. Using the targeted Mecp2 mouse model, we investigated the effect of Mecp2 mutation on XCI and developmental MeCP2 expression in wild-type (wt)-expressing neurons by quantitative laser scanning cytometry. Mecp2(-/+) female mice exhibited uniform regional distribution of Mecp2 mutant-expressing cells in brain, but unbalanced XCI in the population, favoring expression of the Mecp2 wt allele. Interestingly, MeCP2 expression in Mecp2 wt-expressing cells from Mecp2(-/+) mice was significantly lower than those from Mecp2(+/+) age-matched controls. The negative effect of Mecp2 mutation on wt Mecp2 expression correlated with the percentage of Mecp2 mutant-expressing cells in the cortex. Similar results were observed in two RTT females with identical MECP2 mutations but different XCI ratios. These results demonstrate that Mecp2-mutant neurons affect the development of surrounding neurons in a non-cell-autonomous manner and suggest that environmental influences affect the level of MeCP2 expression in wt neurons. These results help in explaining the role of XCI in the pathogenesis of RTT and have important implications in designing therapies for female RTT patients.

Animals↗

PubMed2001

Facile induction of apoptosis into plant cells associated with temperature-sensitive lethality shown on interspecific hybrid from the cross Nicotiana suaveolens x N. tabacum.

Two lines of suspension culture cells were obtained from a hybrid seedling of Nicotiana suaveolens Lehm. x N. tabacum L. cv. Hicks-2 expressing temperature-sensitive lethality. One of them (LH line) was inducible cell death in accordance with the lethality at 28 degrees C but not under high-temperature conditions (36 degrees C). Another one (SH line) lost the lethality and survived at 28 degrees C. The cells of LH line showed apoptotic changes when they were cultured at 28 degrees C. Fragmentation of nuclei was correlated with the lethality in the cells, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from the cells expressing the lethality revealed a specific ladder pattern suggesting nucleosomal fragmentation that is one of the biochemical characteristics of apoptosis. From these facts, we confirmed that the process of cell death leading to hybrid lethality in the cells is certainly apoptosis. Hybrid cells were used in the experiments to estimate the point of no return in temperature-sensitive lethality and to examine the influence of cation in DNA fragmentation during apoptosis. The utility of hybrid cells as an experimental system for studies of hybrid lethality and apoptosis in plants was confirmed.

Apoptosis↗

PubMed2000

Distribution and activation of uterine mononuclear phagocytes in peripartum endometrium and myometrium of the mouse.

The present study tested the hypothesis that macrophage distribution and activation are enhanced in the uterus before term. Mid-uterine horn tissue strips from mice on Days 15 and 18 of pregnancy, the day of birth (= Day 19), and one day postpartum were paraffin-embedded and then sectioned, stained with a monoclonal pan-macrophage marker (BM8), and processed for visualization and quantification of resident macrophages per nuclear area. Macrophages were dispersed throughout the endometrium and subluminal epithelium; cell numbers declined on the day before term, then increased postpartum. Within myometrium, macrophages congregated in stroma surrounding muscle bundles, and staining was enhanced near term. Macrophage numbers were similar in pregnant and postpartum uteri, enhanced more than 2-fold over those in nonpregnant controls. Uterine sections were also analyzed by laser-scanning cytometry to enumerate activated macrophages (i.e., those that express the intercellular adhesion molecule marker CD54+) and to determine cell cycle (propidium iodide fluorescence). Activated macrophages were directly proportional to cell numbers and, by cell cycle analysis, were not terminally differentiated. Highest cell numbers occurred on Day 15: 4-fold greater than those in nonpregnant controls and 2-fold higher than those at Day 18 or in postpartum groups. These findings indicate a decline in endometrial macrophage numbers at least one day before the onset of parturition and raise the possibility that trafficking of this immune cell may contribute to onset of labor.

Animals↗

PubMed2001

Subcellular redistribution of BAX during apoptosis induced by anticancer drugs.

BAX is the 192-amino acid, 21-kDa protein which is ubiquitously distributed in normal tissues and is regarded as a tumor suppressor sensitizing malignant cells to anticancer drugs. In spite of many studies, the molecular mechanism of BAX action is still obscure. In the present study subcellular BAX translocations in human colon adenocarcinoma COLO 205 cells exposed to various anticancer drugs [camptothecin (CPT), etoposide (ETO), staurosporine (STP), 2-chloro-2'-deoxyadenosine (2CdA) and nimesulide (NIM)] was examined. Cells were grown on coverslips under optimal conditions (10% FCS/DMEM) or were stimulated to apoptosis with the drugs examined. Laser scanning cytometry was applied for the quantitative analysis of BAX expression, and distribution in the cytoplasmic (BAX Cf) and nuclear (BAX Nf) area. BAX maximal pixel (BAX MP), the parameter corresponding to aggregation of BAX in the cell, was also measured. All examined drugs increased the number of cells with high BAX MP, reaching the peak at 60 min after drug administration. The most pronounced effect was in the case of 2CdA, CPT and STP. The increase in BAX MP was observed only when antibody recognizing the 43-61 amino acid sequence was used. When antibody binding the N-terminal epitope (11-30 amino acid sequence) was applied, the number of cells expressing high BAX MP significantly decreased. These results indicate that apoptotic stimuli delivered by anticancer drugs led to aggregation of BAX in cancer cells, which is dependent on BAX activation by its cleavage at the N-terminal epitope and exposure of the BH3 domain. It was shown that BAX Nf increased in cells treated with CPT, STP, ETO, 2CdA and NIM, whereas BAX Cf rose after STP and NIM. The increase in BAX Nf and, occurring in most treatments, the increase in the BAX Nf:Cf ratio indicates a BAX shift from the cytoplasm to the nucleus. Furthermore, staining with different antibodies showed that only the activated form of BAX was translocated to the nucleus. Immunoelectron microscopy revealed that CPT-induced apoptosis was associated with translocation of BAX from the cytosol to organellar membranes (mitochondrial, Golgi apparatus and endoplasmic reticulum) and via nuclear envelope pores to the nucleus, occurring within 60-180 min of cell exposure to the drug. The subcellular translocations of BAX preceded in time the appearance of morphological symptoms of apoptosis. In conclusion, (i) in spite of different molecular mechanisms of apoptosis induction by the anticancer drugs examined, BAX remains a common link in the chain of reactions leading to cell death, and (ii) BAX activation and subcellular translocations from the cytosol to organellar membranes and nucleus are key cellular responses to drugs bearing proapoptotic properties.

Adenocarcinoma↗

PubMed2002

Effects of WR-2721 and cyclophosphamide on the cell cycle phase specificity of apoptosis in mouse bone marrow.

Elucidation of the mechanisms of action of the thiol and alkylating agents on normal cells requires the knowledge of their cell cycle phase specificity in terms of their ability to induce apoptosis. The effects of S-2-/3-aminopropylamino/ethyl phosphorothioic acid (WR-2721, Amifostine) and cyclophosphamide (CP) on apoptosis and cell cycle progression were assessed in the mouse bone marrow. Adult male Swiss mice were treated with WR-2721, at a dose of 400 mg/kg body weight, and/or CP, at a dose of 200 mg/kg body weight. Application of the laser scanning cytometry APO-BRDU assay, a two-color staining method for labeling of DNA breaks and cellular DNA, allowed an identification of apoptotic and non-apoptotic cells, and their position with respect to their cell cycle phase. Temporary alterations in the number of apoptotic cells and also all bone marrow cells, including apoptotic and non-apoptotic ones, were determined throughout the 240-h period after treatment of mice with WR-2721 and/or CP. These drugs, given alone, affected apoptotic cell death and caused deregulation of the cell cycle in the bone marrow. WR-2721, applied 30 min prior to CP administration, resulted in a suppressing effect on apoptosis and the cell cycle perturbation triggered in normal bone marrow cells by the alkylating drug. The patterns of changes in the frequency of apoptotic cells and the number of apoptotic and non-apoptotic bone marrow cells, observed in all phases of the cell cycle, were dependent on the agent(s) given and the time interval after WR-2721 and/or CP administration.

Amifostine↗

PubMed2002

Colocalization of BAX with BID and VDAC-1 in nimesulide-induced apoptosis of human colon adenocarcinoma COLO 205 cells.

Cyclooxygenase (COX)-2 inhibitors that belong to non-steroid anti-inflammatory drug family have been shown to have an apoptosis-inducing effect on neoplastic cells. In the present study the effect of nimesulide (NIM), a specific COX-2 inhibitor, on apoptosis and interactions between BCL-2 family death promoters BAX and BID and BAX and VDAC-1 were examined in human colon adenocarcinoma COLO 205 cells. Laser scanning cytometry was applied for the measurement of expression and aggregation of apoptosis-related proteins and quantitative analysis of NIM-induced apoptosis. Double-staining immunoconfocal and immunoelectron microscopy were used for subcellular colocalization of examined proteins. NIM induced apoptosis of COLO 205 cells in a dose-dependent manner. This was accompanied by: (1) a decrease in intracellular prostaglandin (PG) E content; (2) subcellular redistribution and aggregation of BAX and BID on organellar membranes and within the nucleus; (3) colocalization of BAX with BID and BAX with VDAC-1 on organelles; and (4) survival of cells with the highest BCL-2 aggregation. A similar pattern of subcellular redistribution and colocalization of BAX with BID and BAX with VDAC-1 suggests that BAX (in association with BID) controls the function of VDAC-1 and its permeability for apoptogenic factors released from mitochondria of COLO 205 cells stimulated to apoptosis with NIM.

Apoptosis↗

PubMed2005

Autophagy is the dominant type of programmed cell death in breast cancer MCF-7 cells exposed to AGS 115 and EFDAC, new sesquiterpene analogs of paclitaxel.

The molecular mechanism of cell death induced by AGS 115 and EFDAC, sesquiterpene analogs of paclitaxel, was investigated in human breast cancer MCF-7 cells. The study was carried out using laser scanning cytometry, homeostatic confocal microscopy, atomic force microscopy and electron microscopy. AGS 115 and EFDAC exhibited a microtubule-stabilizing effect as confirmed by a significant increase in alpha-tubulin aggregation. Both paclitaxel analogs also induced death in MCF-7 cells. Evaluation of biochemical and morphological features suggested that the major form of programmed cell death induced by AGS 115 and EFDAC was autophagy. This was confirmed by MAP I LC3 expression and the ultrastructural pattern revealed by electron microscopy. Surface images of cells undergoing autophagy showed that, unlike during apoptosis, the dimensions remained unchanged, but the surface of the cell was deformed. The occurrence of apoptosis was confirmed by the efflux of Smac/DIABLO from mitochondria, caspase-7 activation and DNA loss, and did not exceed 9.7%. Therefore, AGS 115 and EFDAC appear to be promising candidates for further investigation in anti-cancer therapy.

Animals↗