THE LARYNGOSCOPE, Issue 5 2002
Zhi Wang MD
Abstract Objectives Chemoprevention is a promising strategy to inhibit carcinoma before invasive tumors develop, but a new molecular target is desirable. Celecoxib is a newly developed cyclo-oxygenase (COX)-2 inhibitor with significantly less toxicity. The study was conducted to determine whether celecoxib is effective and safe in prevention of oral cancer. The antiangiogenic activity of celecoxib was studied to explore the potential mechanism involved. Study Design Randomized animal study. Methods The study consisted of two phases. In the phase 1, 10 mice were used to determine the efficacy and safety of celecoxib with intradermal inoculation with oral carcinoma cells. The 10 mice were equally divided into two groups 5 mice (30 inoculated sites) in each group to receive 1,500 parts per million (ppm) celecoxib mixed in with the diet or to eat a normal diet, respectively, for 21 days. In phase 2, 10 more mice were inoculated to determine the effect of celecoxib on angiogenesis. Five mice received 3,000 ppm celecoxib in the diet, with the other five mice as control animals. The antiangiogenic activity was evaluated by comparing the density of newly growing microvessels after the inoculation. Results The results indicated that celecoxib significantly delayed cell growth and reduced tumor volume. There was statistical significance in the quantity of new vasculature in the tumor sites between the two groups. No toxic effect was found by means of measurement of body weight loss and microscopic dissection of organs. Conclusions The study provided the first evidence to show the chemopreventive efficacy of celecoxib on oral cancer in a nude mouse model. Clinical trials are warranted to determine the efficacy in humans. [source]

Role of p38 MAPKs in Hypericin Photodynamic Therapy-induced Apoptosis of Nasopharyngeal Carcinoma Cells

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 5 2009
Pui S. Chan
The present study aims to determine the role of mitogen-activated protein kinases (MAPKs) in hypericin-mediated photodynamic therapy (HY-PDT)-induced apoptosis of the HK-1 nasopharyngeal carcinoma (NPC) cells. HY-PDT was found to induce proteolytic cleavage of procaspase-9 and -3 in HK-1 cells. Apoptotic nuclei were observed at 6 h after PDT whereas B-cell leukemia/lymphoma-2-associated-X-protein (Bax) translocation and formation of Bax channel is responsible for the cell death. Increase in phosphorylation of p38 MAPKs and c-Jun N-terminal kinase 1/2 (JNK1/2) was detected at 15,30 min after HY-PDT. The appearance of phosphorylated form of p38 MAPKs and JNK1/2 was inhibited by the singlet oxygen scavenger l -histidine. HY-PDT-induced cell death was enhanced by the chemical inhibitors for p38 MAPKs (SB202190 and SB203580), but not by the JNKs inhibitor SP600125. Knockdown of the p38, and p38, MAPK isoforms by small interfering RNA (siRNA) are more effective than the p38, in enhancing PDT-induced cell death. Augmentation of apoptosis by p38, or p38, knockdown is also correlated with the increased proteolytic cleavage of procaspase-9 after HY-PDT treatment. Our results suggested that HY-PDT activated p38 MAPKs through the production of singlet oxygen. Inhibition of p38 MAPKs with chemical inhibitors or siRNA enhances HY-PDT-induced apoptosis of the HK-1 NPC cells. [source]

Photosensitizing and Radiosensitizing Effects of Hypericin on Human Renal Carcinoma Cells in Vitro

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2008
Johannes Theodor Wessels
The renal cell carcinoma (RCC) is extremely resistant to chemotherapy and radiotherapy. The prognosis of patients with metastatic RCC still remains poor, the median survival is less than 12 months. Therefore, new therapeutic options are desirable. The aim of this study was to investigate the photosensitizing and radiosensitizing effects of hypericin on human RCC cells in vitro. First the RCC-derived cell lines A498 and ACHN were incubated with different concentrations of hypericin. In vitro uptake and intracellular distribution of hypericin were confirmed by fluorescence microscopy. Subsequently cells were illuminated and irradiated with a dose of 2,8 Gy, respectively. Finally, metabolic activity, apoptosis and clonogenic survival were investigated. Uptake of hypericin was observed for almost all cells. Hypericin treatment combined with illumination led to a 94,97% decrease in metabolic activity and caused apoptosis in nearly 100% of RCC cells. Hypericin enhanced the radiosensitivity of A498 cells in vitro. The clonogenic survival after irradiation was significantly reduced by hypericin treatment. Taken together, the photosensitizing and radiosensitizing effects of hypericin on human RCC cells we found in this investigation could be of clinical relevance, e.g. for radiotherapy and intraoperative photodynamic therapy, respectively. [source]

Alterations in Mitochondrial and Apoptosis-regulating Gene Expression in Photodynamic Therapy-resistant Variants of HT29 Colon Carcinoma Cells,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2005
Xiao Yun Shen
ABSTRACT Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragmentassociated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3,region Mbol complementary DNA, glutamate dehydrogenase, hepatomaderived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondria1 and apoptosisregulating genes contributes to PDT resistance. [source]

Immunohistochemical Absence of CD21 Membrane Receptor in Nasopharyngeal Carcinoma Cells Infected by Epstein-Barr Virus in Spanish Patients ,

THE LARYNGOSCOPE, Issue 12 2000
Javier S. Burgos PhD
Abstract Objectives/Hypothesis The aim of this study was to analyze the relevance of the CD21 membrane receptor in nasopharyngeal carcinoma (NPC). CD21 is implicated in the introduction of the Epstein-Barr virus (EBV) genome into epithelial cells and B lymphocytes. Study Design Immunohistochemical analysis of CD21 in NPC. Methods Paraffin-embedded samples of NPC of different histological types with demonstrated presence of EBV were analyzed for CD21 expression using immunohistochemistry. Results We detected EBV by non-isotopic in situ hybridization (NISH) and nested polymerase chain reaction (PCR) in 100% of samples, regardless of histological type, supporting the previous view that all the types of NPC are variants of an EBV-associated malignancy. CD21 was not detected in NPC, and this absence was a typical feature in our data group. Conclusions The loss of the CD21 membrane receptor could be one of the immunophenotypical changes of the neoplastic cells that occur in the evolution of the NPC malignancy. [source]

Introduction of Clusterin Gene into Human Renal Cell Carcinoma Cells Enhances Their Resistance to Cytotoxic Chemotherapy through Inhibition of Apoptosis both in vitro and in vivo

CANCER SCIENCE, Issue 11 2001
Isao Hara
Recent studies have revealed the powerful antiapoptotic activity of clusterin in various malignant tumors; however, the significance of clusterin expression in the acquisition of a resistant phenotype against several kinds of treatment in human renal cell carcinoma (RCC) has not been well characterized. We, therefore, transfected the clusterin cDNA into RCC ACHN cells, that scarcely express clusterin protein, to examine whether overexpression of clusterin inhibits chemotherapy-induced apoptosis both in vitro and in vivo. Although no significant differences were observed in the in vitro growth rates between clusterin-transfected ACHN (ACHN/CL) and the vector only-transfected cell line (ACHN/Co), ACHN/CL exhibited high resistance to cisplatin treatment compared with ACHN/Co, with a greater than 5-fold higher IC50 through the inhibition of apoptotic cell death, which was demonstrated by DNA fragmentation analysis and western blotting of PARP protein. Moreover, intravenous administration of cisplatin into athymic nude mice bearing ACHN/CL tumors resulted in 2- to 3-times faster tumor growth compared with ACHN/Co tumors. These findings suggest that clusterin overexpression helps confer a chemoresistant phenotype through inhibition of apoptosis in human RCC cells. [source]

Reversing Effect of Agosterol A, a Spongean Sterol Acetate, on Multidrug Resistance in Human Carcinoma Cells

CANCER SCIENCE, Issue 8 2001
Shunji Aoki
The effect of agosterol A, a novel polyhydroxylated sterol acetate isolated from a marine sponge, on P-glycoprotein (P-gp)-mediated multidrug-resistant cells (KB-C2) and the multidrug resistance associated protein (MRPl)-mediated multidrug-resistant cells (KB-CV60) was examined. Agosterol A reversed the resistance to colchicine in KB-C2 cells and also the resistance to vincristine in KB-CV60 cells at 3 to 10 ,M concentration. Agosterol A at 3 ,M increased the vincristine concentration in both KB-C2 cells and KB-CV60 cells to the level in parental KB-3-1 cells. Agosterol A also decreased the efflux of vincristine from both KB-C2 cells and KB-CV60 cells to the level seen in KB-3-1 cells. Agosterol A inhibited the [3H]azidopine-photolabeling of P-gp and also inhibited the uptake of [3H]S-(2,4-dinitrophenyl)glutathione (DNP-SG) in inside-out membrane vesicles prepared from KB-CV60 cells. We conclude that agosterol A directly inhibited drug efflux through P-gp and/or MRP1. [source]

In vivo and in vitro Interactions between Human Colon Carcinoma Cells and Hepatic Stellate Cells

CANCER SCIENCE, Issue 12 2000
Sadatoshi Shimizu
Stromal reaction is important for the growth of cancer both in primary and metastatic sites. To demonstrate this reaction during the hepatic metastasis of human colon carcinoma, we histologically investigated alterations to the distribution and phenotype of hepatic stellate cells (HSCs), the only mesenchymal cells in the liver parenchyma, using a nude mouse model. Intrasplenically injected colon carcinoma LM-H3 cells migrated into the space of Disse and underwent proliferation, in close association with hepatocytes and HSCs, at 2 days. At 14 days, HSCs were accumulated around the tumor mass and expressed ,-smooth muscle actin, a marker for HSC activation. We next investigated in vitro the growth factors involved in the interactions between LM-H3 cells and HSCs. Conditioned medium of rat HSCs which underwent culture-induced activation contained platelet-derived growth factor (PDGF)-AB, hepatocyte growth factor (HGF) and transforming growth factor (TGF),, and could augment LM-H3-cell proliferation and migration. Neutralizing antibodies against PDGF-AA and PDGF-BB and those against PDGF-BB and HGF inhibited proliferation and migration, respectively, of LM-H3 cells, whereas antibody against TGF-, had no effect. LM-H3 cells expressed PDGF receptors-, and -, and c-met. Conditioned medium of LM-H3 cells contained PDGF-AB, and could enhance HSC proliferation and migration. This augmenting effect was suppressed by treatment with anti-PDGF-AB antibody. The present study has demonstrated that bidirectional interactions involving PDGF and HGF take place in vitro between colon carcinoma cells and HSCs, raising the possibility that similar interactions might be involved in the stromal reaction during hepatic metastasis. [source]

Stochasticity of Manganese Superoxide Dismutase mRNA Expression in Breast Carcinoma Cells by Molecular Beacon Imaging

CHEMBIOCHEM, Issue 11 2005
Timothy J. Drake
Abstract Visual and quantitative monitoring of cell-to-cell variation in the expression of manganese superoxide dismutase (MnSOD) mRNA by using novel ratiometric imaging with molecular beacons (MB) reveals a distinct change in patterns following induction of human breast-carcinoma cells with lipopolysaccharide. Interestingly, the pattern of cell-to-cell variation in a cell line stably transfected with a plasmid bearing a cDNA clone of MnSOD and overproducing the enzyme is significantly different from the pattern associated with MnSOD induction. The levels and the patterns of cell-population heterogeneity for ,-actin mRNA expression do not show distinct changes either following induction or in stably transfected cells. These results are significant in light of the reported relationship between this enzyme and malignant phenotype of breast-carcinoma cells. Use of MBs in ratiometric image analyses for cytoplasmic mRNAs represents a novel means of directly examining the stochasticity of transcription of MnSOD and other genes implicated in cellular phenotype regulation. [source]

Small Interfering RNA-Mediated Silencing of Glutathione- S -Transferase,A1 Sensitizes Hepatic Carcinoma Cells to Photodynamic Therapy with Pentaphyrins

CHEMMEDCHEM, Issue 4 2008
Valentina Rapozzi Dr.
Photodynamic therapy (PDT) uses nontoxic photosensitizers and visible light to produce reactive oxygen species that kill malignant cells by apoptosis or necrosis. Silencing the antioxidant GSTA1-1 gene by siRNA sensitizes hepatic HepG2 cells to PDT with pentaphyrins. The study is a proof-of-concept for combining PDT with antigene molecules that decrease cellular response to oxidative stress. [source]

Use of magnetic enrichment for detection of carcinoma cells in fluid specimens

CANCER, Issue 1 2002
Eric Kielhorn B.S.
BACKGROUND Ascites fluid or a pleural effusion are common events in metastatic carcinoma, but they also can be associated with several other medical conditions. The standard for determination of malignancy in these situations is cytologic evaluation of these fluids. Although this method is frequently successful, there are times when it fails, even when the patient has a malignancy, either because of insufficient cells in the fluid or for other reasons. This study addresses this problem taking advantage of the recent advances in technology for detection of rare epithelial cells in liquid specimens. METHODS The authors examined fluid specimens from 59 patients to determine the frequency of recovery of epithelial cells compared with that achieved by conventional cytopathology. The Dynal CELLection Epithelial Enrich (Dynal AS, Oslo, Norway) method was used. This method is based on immunomagnetic selection of cells binding to EpCAM antibodies. Carcinoma cells were confirmed by morphology and, when there was sufficient material, by E-cadherin staining. RESULTS Grouping the cases by cytologic diagnosis, the authors found malignant cells using the cell enrichment assay in 11 of 12 malignant cases, 2 of 5 atypical cases, and 3 of 42 negative cases. Further investigations were conducted on the five cases that were cytologically negative or atypical but yielded epithelial cells after immunomagnetic enrichment. Four cases ultimately were proven malignant by other methods and one had incomplete follow-up. CONCLUSIONS The new methods available for epithelial cell enrichment in liquids may be used successfully on cytologic fluid specimens and may lead to increased sensitivity for detection of malignancy, and consequently more accurate staging. Cancer 2002;94:205,11. © 2002 American Cancer Society. [source]

Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell lines

INTERNATIONAL JOURNAL OF CANCER, Issue 4 2004
Byong Chul Yoo
Abstract Resistance to anticancer drugs is a major obstacle preventing effective treatment of disseminated cancers. Understanding the molecular basis to chemoresistance is likely to provide better treatment. Cell lines resistant to cisplatin or 5-fluorouracil (5-FU) were established from human gastric carcinoma cell lines SNU-638 and SNU-620. Comparative proteomics involving 2-dimensional gel electrophoresis (2-DE) and matrix-associated laser desorption ionization-mass spectroscopy (MALDI-MS) was performed on protein extracts from these parental and drug-resistant derivative lines to screen drug resistance-related proteins. Pyruvate kinase M2 (PK-M2) was identified as a protein showing lower expression in cisplatin-resistant cells compared to parental cells. Consistent with this finding, PK-M2 activity was also lower in cisplatin-resistant cells. Suppression of PK-M2 expression by antisense oligonucleotide resulted in acquired cisplatin resistance in SNU-638 cells. Furthermore, PK-M2 activity in 11 individual human gastric carcinoma cell lines positively correlated with cisplatin sensitivity. Taken together, PK-M2 protein and activity levels were lower in cisplatin-resistant human gastric carcinoma cell lines compared to their parental cell lines. Furthermore, suppression of PK-M2 expression using antisense oligonucleotides increased cisplatin resistance. These data clearly link PK-M2 and cisplatin resistance mechanisms. © 2003 Wiley-Liss, Inc. [source]

EGFR Regulates the Side Population in Head and Neck Squamous Cell Carcinoma

THE LARYNGOSCOPE, Issue 3 2006
Jocelyn S. Chen BS
Abstract Objective: To identify the presence of side population (SP) cells in established head and neck squamous carcinoma cell (HNSCC) lines and to determine the role of EGFR in the regulation of the side population of these cells. Methods: SP cells were identified using flow cytometry analysis by the ability of these cells to extrude the Hoechst 33342 dye via the drug transporter BCRP1/ABCG2. Effect of EGFR on the side population was determined also by difference in Hoechst extrusion and by immunofluorescence. Immunohistochemical staining was performed to show the presence of the BCRP1/ABCG2 transporter and the phosphorylated form of EGFR in HNSCC tissue. Results: SP cells are present in HNSCC cell lines. With the Hoechst 33342 extrusion assay, SP cells were found to comprise an average of 0.69% of the UMSCC10B cells and 0.91% of HN12 cells. Addition of the EGF ligand increased the SP population while inactivation of the EGFR kinase by Iressa significantly decreased SP. Conclusion: In established head and neck squamous cell carcinoma cell lines, SP cells were found using methods that determine expression and function of the drug transporter BCRP1/ABCG2. Activation of EGFR, a gene implicated in tumorigenesis in HNSCC leads to increased SP, and conversely, inhibition of EGFR leads to decrease in SP. This finding could help explain the role of EGFR in regulating cancer stem cells and thus tumorigenesis in HNSCC. [source]

Membrane type-1 matrix metalloproteinase stimulates tumour cell-induced platelet aggregation: role of receptor glycoproteins

BRITISH JOURNAL OF PHARMACOLOGY, Issue 2 2004
David Alonso-Escolano
Matrix metalloproteinase-2 (MMP-2) plays a role in agonist- and tumour cell-induced platelet aggregation (TCIPA). MMP-2 is synthesized as a proenzyme and is activated at the cell surface by membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14). The significance of tumour cell-associated MT1-MMP for TCIPA was investigated using human breast carcinoma MCF7 cells stably coexpressing the integrin ,v,3 with MT1-MMP, cells expressing ,v,3 alone and mock-transfected cells. Western blot and zymography confirmed that ,v,3/MT1-MMP cells expressed MT1-MMP and efficiently processed proMMP-2 to MMP-2. Aggregometry, phase-contrast and transmission electron microscopy and flow cytometry were used to characterize TCIPA induced by MCF7 cell lines. The aggregating potency of cells was: ,v,3/MT1-MMP >,v,3=mock cells, as shown by aggregometry and phase-contrast microscopy. Electron microscopy revealed close, membrane,membrane interactions between activated platelets and ,v,3/MT1-MMP cells during TCIPA. Inhibition of MMP-2 with the neutralizing anti-MMP-2 antibody (5 ,g ml,1) and o -phenanthroline (100 ,M) reduced aggregation induced by ,v,3/MT1-MMP cells. TCIPA induced by ,v,3/MT1-MMP cells was also reduced by inhibiting the generation and actions of ADP with apyrase (250 ,g ml,1) and 2-methylthio-AMP (2-MeSAMP) (30 ,M), but not N6 -methyl-2,-deoxyadenosine-3,,5,-bisphosphate (MRS2179) (30 ,M). Flow cytometry demonstrated that TCIPA enhanced expression of glycoprotein (GP) Ib and IIb/IIIa receptors not only on platelets but also on breast cancer cells. Thus, (a) human breast carcinoma cell surface-associated MT1-MMP, via activating proMMP-2, stimulates TCIPA; (b) ADP amplifies the effects of MMPs via stimulation of P2Y12 receptors and (c) both tumour- and platelet-derived GPIb and GPIIb/IIIa are involved in the aggregatory effects of MT1-MMP. British Journal of Pharmacology (2004) 141, 241,252. doi:10.1038/sj.bjp.0705606 [source]

Protein kinase C involvement in aloe-emodin- and emodin-induced apoptosis in lung carcinoma cell

BRITISH JOURNAL OF PHARMACOLOGY, Issue 5 2001
Hong-Zin Lee
This study demonstrated aloe-emodin- and emodin-induced apoptosis in lung carcinoma cell lines CH27 (human lung squamous carcinoma cell) and H460 (human lung non-small cell carcinoma cell). Aloe-emodin- and emodin-induced apoptosis was characterized by nuclear morphological changes and DNA fragmentation. During apoptosis, an increase in cytochrome c of cytosolic fraction and activation of caspase-3, identified by the cleavage of its proform, were observed. To elucidate whether the expression of protein kinase C (PKC) isozymes are involved in aloe-emodin- and emodin-induced apoptosis, this study examined the changes of PKC isozymes by Western blotting techniques during aloe-emodin- and emodin-induced apoptosis. The expression of PKC isozymes involved in aloe-emodin- and emodin-induced apoptosis of CH27 and H460 cells. In this study, aloe-emodin and emodin induced the changes of each of PKC isozymes in CH27 and H460 cells. The decrease in the expression of PKC, and , may play a critical role in aloe-emodin- and emodin-induced apoptosis in CH27 and H460 cells. The present study also demonstrated that PKC stimulation occurs at a site downstream of caspase-3 in the emodin-mediated apoptotic pathway. British Journal of Pharmacology (2001) 134, 1093,1103; doi:10.1038/sj.bjp.0704342 [source]

Organometallic Ruthenium Inhibitors of Glutathione- S -Transferase P1-1 as Anticancer Drugs

CHEMMEDCHEM, Issue 12 2007
Han Ang Dr.
Abstract Ruthenium,arene complexes conjugated to ethacrynic acid were prepared as part of a strategy to develop novel glutathione- S -transferase (GST) inhibitors with alternate modes of activity through the organometallic fragment, ultimately to provide targeted ruthenium-based anticancer drugs. Enzyme kinetics and electrospray mass spectrometry experiments using GST P1-1 and its cysteine-modified mutant forms revealed that the complexes are effective enzyme inhibitors, but they also rapidly inactivate the enzyme by covalent binding at Cys,47 and, to a lesser extent, Cys,101. They are highly effective against the GST Pi-positive A2780 and A2780cisR ovarian carcinoma cell lines, are among the most effective ruthenium complexes reported so far, and target ubiquitous GST Pi overexpressed in many cancers. [source]

A European interlaboratory testing of three well-known procedures for immunocytochemical detection of epithelial cells in bone marrow.

CYTOMETRY, Issue 6 2006
Results from analysis of normal bone marrow
Abstract Background: This investigation intended to study the unspecific background to be expected in normal bone marrow (BM), comparing three well recognized protocols for immunocytochemical detection of disseminated carcinoma cells. The interlaboratory variation in screening and evaluation of stained cells was analyzed and different screening methods were compared. Methods: BM mononuclear cells (BM MNC) from 48 normal BMs were immunostained in parallel by three participating laboratories. The protocols, based on three different anti-cytokeratin antibodies, have all been in common use for detection of disseminated carcinoma cells: the A45-B/B3 protocol (Hamburg), the CK2 protocol (Augsburg) and the AE1AE3 protocol (Oslo). For all protocols, the immunostained cells were visualized by the same alkaline-phosphatase (AP) detection system (APAAP) followed by detection of the cells by manual screening and by two different automated screening systems (ACIS from Chromavision and MDS1 from Applied Imaging). Detected AP-visualized cells were morphologically classified into unambiguous hematopoietic (Uhc) and questionable cells (Qc, potentially interpreted as tumor cells). Results: Seven of 48 BMs (15%) harbored ,1 AP-visualized cell(s) among 1 × 106 BM MNC, both for the A45-B/B3- and for the AE1AE3 protocol, while for CK2 a higher proportion of BMs (21 BMs; 44%) harbored AP-visualized cells (P < 0.01, McNemar's test). The number of Qc was, for all protocols, 1 log lower than the total number of AP-visualized cells. On average, the frequency of Qc was 0.04, 0.08, and 0.02 per 106 BM MNC with A45-B/B3, CK2 and AE1AE3, respectively, and the number of Qc-positive BMs 1, 4, and 1. The MDS1 screening sensitivity was similar to manual screening, while ACIS detected fewer cells (P < 0.001, McNemar's test). Conclusions: All protocols resulted in AP-visualization of occasional hematopoietic cells. However, morphological classification brings the specificity to a satisfactory high level. Approximately 10% of AP-visualized cells were categorized "questionable". The CK2 protocol turned out less specific than the A45-B/B3 and AE1AE3 protocols. © 2006 International Society for Analytical Cytology. [source]

Radiation-induced changes in oral carcinoma cells , a multiparametric evaluation

CYTOPATHOLOGY, Issue 5 2003
L. Bindu
The aim of this study was to see whether serial cytological evaluation of various cellular abnormalities in tumours from patients receiving fractionated radiotherapy can predict radio-response in oral carcinoma. Cytological assessment was carried out in scrape smears collected prior to and during the course of radiotherapy in 68 patients with squamous cell carcinoma of the oral cavity planned for radical radiotherapy with accelerated fraction schedule. Smears were evaluated for a set of 15 radiation-induced cellular abnormalities. The relationship between the cellular alterations and the cumulative radiation dose was analysed by Kruskal,Wallis one-way anova. The results showed that among the various quantifiable changes that occur in irradiated cancer cells, karyolysis, karyorrhexis, pyknosis, cytolysis, multinucleation, micronucleation and nuclear budding show significant increase depending on the dose of radiation. The radio-resistant group of patients exhibited a lesser degree of change compared with the radio-sensitive group. This suggests that radio-resistance may be due to the defective induction of cell damage and that these cytological features may have potential use as predictive markers of radio-sensitivity in oral carcinoma. [source]

,3-Tubulin is induced by estradiol in human breast carcinoma cells through an estrogen-receptor dependent pathway

CYTOSKELETON, Issue 7 2009
Jennifer Saussede-Aim
Abstract Microtubules are involved in a variety of essential cell functions. Their role during mitosis has made them a target for anti-cancer drugs. However development of resistance has limited their use. It has been established that enhanced ,3-tubulin expression is correlated with reduced response to antimicrotubule agent-based chemotherapy or worse outcome in a variety of tumor settings. However little is known regarding the regulation of ,3-tubulin expression. We investigated the regulatory mechanisms of expression of ,3-tubulin in the MCF-7 cell line, a model of hormone-dependent breast cancer. Exposure of MCF-7 cells to estradiol was found to induce ,3-tubulin mRNA as well as ,3-tubulin protein expression. Conversely, we did not observe induction of ,3-tubulin mRNA by estradiol in MDA-MB-231 cells which are negative for the estrogen receptor (ER). In order to determine whether ,3-tubulin up-regulation is mediated through the ER pathway, MCF-7 cells were exposed to two ER modulators. Exposure to tamoxifen, a selective estrogen receptor modulator, completely abolished the ,3-tubulin mRNA induction due to estradiol in MCF-7 cells. This result was confirmed with fulvestrant, a pure antagonist of ER. These results demonstrate that the effect of estradiol on ,3-tubulin transcription is mediated through an ER dependent pathway. Cell Motil. Cytoskeleton 66:378,388, 2009. © 2009 Wiley-Liss, Inc. [source]

Description and characterization of a chamber for viewing and quantifying cancer cell chemotaxis

CYTOSKELETON, Issue 1 2005
Lilian Soon
Abstract Direct observations of cancer cell invasion underscore the importance of chemotaxis in invasion and metastasis. Yet, there is to date, no established method for real-time imaging of cancer chemotaxis towards factors clinically correlated with metastasis. A chamber has been designed and tested, called the Soon chamber, which allows the direct observation and quantification of cancer cell chemotaxis. The premise for the design of the Soon chamber is the incorporation of a dam, which creates a steep gradient while retaining stability associated with a pressure-driven system. The design is based on the characteristics of cancer cell motility such as relatively low speeds, and slower motility responses to stimuli compared to classical amoeboid cells like neutrophils and Dictyostelium. We tested MTLn3 breast carcinoma cells in the Soon chamber in the presence of an EGF gradient, obtaining hour-long time-lapses of chemotaxis. MTLn3 cells migrated further, more linearly, and at greater speeds within an EGF gradient compared to buffer controls. Computation of the degree of orientation towards the EGF/buffer source showed that MTLn3 cells were significantly more directional toward the EGF gradient compared to buffer controls. Analysis of the time-lapse data obtained during chemotaxis demonstrated that two populations of cancer cells were present. One population exhibited oscillations in directionality occurring at average intervals of 12 min while the second population exhibited sustained high levels of directionality toward the source of EGF. This result suggests that polarized cancer cells can avoid the need for oscillatory path corrections during chemotaxis. Cell Motil. Cytoskeleton 62:27,34, 2005. © 2005 Wiley-Liss, Inc. [source]

Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation,

CYTOSKELETON, Issue 4 2004
Mike Lorenz
Abstract Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-,-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured. Cell Motil. Cytoskeleton 57:207,217, 2004. © 2004 Wiley-Liss, Inc. [source]

TSC-box is essential for the nuclear localization and antiproliferative effect of XTSC-22

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2007
Akiko Hashiguchi
Transforming growth factor- ,1-stimulated clone 22 (TSC-22) encodes a leucine zipper-containing protein that is highly conserved among various species. Mammalian TSC-22 is a potential tumor suppressor gene. It translocates into nuclei and suppresses cell division upon antiproliferative stimuli. In human colon carcinoma cells, TSC-22 inhibits cell growth by upregulating expression of the p21 gene, a cyclin-dependent kinase (Cdk) inhibitor. We previously showed that the Xenopus laevis homologue of the TSC-22 gene (XTSC-22) is required for cell movement during gastrulation through cell cycle regulation. In this report, we investigated the molecular mechanism of the antiproliferative effect of XTSC-22. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis suggested that XTSC-22 did not affect the expression levels of the p21 family of Cdk inhibitors or other cell cycle regulators. Analysis of deletion mutants of XTSC-22 revealed that nuclear localization of the N-terminal TSC-box is necessary for cell cycle inhibition by XTSC-22. Further experiments suggested that p27Xic1, a key Cdk inhibitor in Xenopus, interacts with XTSC-22. Because p27Xic1 is a cell cycle inhibitor with a nuclear localization signal, it is possible that XTSC-22 suppresses cell division by translocating into the nucleus with p27Xic1, where it may potentiate the intranuclear action of p27Xic1. [source]

The basic helix-loop-helix factor Hand2 regulates autonomic nervous system development

DEVELOPMENTAL DYNAMICS, Issue 3 2005
Yuka Morikawa
Abstract Mammalian autonomic nervous system (ANS) development requires the combinatorial action of a number of transcription factors, which include Mash1, Phox2b, and GATA3. Here we show that the bHLH transcription factor, Hand2 (dHAND), is expressed concurrently with Mash1 during sympathetic nervous system (SNS) development and that the expression of Hand2 is not dependent on Mash1. This suggests that these two bHLH factors work in parallel during SNS development. We also show that ectopic expression of Hand2 activates the neuronal program and promotes the acquisition of a phenotype corresponding to peripheral neurons including neurons of the SNS lineage in P19 embryonic carcinoma cells. We propose that Hand2 works in parallel with other members of the transcriptional network to regulate ANS developmental but can ectopically activate the program by a cross-regulatory mechanism that includes the activation of Mash1. We show that this function is dependent on its interaction with the histone acetyltransferase p300/CBP, indicating that Hand2 functions to promote ANS development as part of a larger transcriptional complex. Developmental Dynamics 234:613,621, 2005. © 2005 Wiley-Liss, Inc. [source]

Evidence-based guidelines to optimize the selection of antibody panels in cytopathology: Pleural effusions with malignant epithelioid cells

DIAGNOSTIC CYTOPATHOLOGY, Issue 1 2010
Danielle E. Westfall M.D.
Abstract There is no established methodology to help select cost effective antibody panels. We used Bayesian statistics and an evidence-based pathology (EBP) approach to retrospectively review the use of immunohistochemistry (IHC) in 153 consecutive pleural effusions evaluated in our laboratory from 2005,2007 for the differential diagnosis of malignant mesothelial cells versus carcinoma cells and to estimate the likely site of origin of a carcinoma. The results in this "training" set were used to design antibody panels and test their clinical applicability on a "test set" of 44 pleural effusions collected in early 2008. Cytopathologists had used 6 ± 4.5 IHC tests per case for the diagnosis of malignant mesothelioma (n = 9) and carcinomas of lung (n = 60), breast (n = 47), Müllerian (n = 25), and other origins in the "training set". The sensitivity and specificity of pleural cytology using all these IHC tests were 32% and 95%, respectively. Sensitivity, specificity and post-test odds (PTO) of a positive IHC result were calculated for each antibody and by the following classes: malignant mesothelial cells and carcinoma cells by primary site of origin. The antibodies that provided the best PTO to diagnose the most prevalent tumors in our population were included in diagnostic panels for male (calretinin, TTF-1, PSA and CDX-2) and female (calretinin, TTF-1, ER and CA125) patients. These panels provided 100% specificity and 77% and 50% sensitivity, respectively, for the pleural effusions from female and male patients in the "test set." The use of an EBP approach for test selection in cytopathology is discussed. Diagn. Cytopathol. 2010. © 2009 Wiley-Liss, Inc. [source]

Diagnostic utility of mammaglobin and GCDFP-15 in the identification of metastatic breast carcinoma in fluid specimens

DIAGNOSTIC CYTOPATHOLOGY, Issue 7 2009
Z. Yan M.D.
Abstract Morphologic differentiation of breast carcinoma from nonmammary malignancies in fluid specimens can be a diagnostic challenge. Immunocytochemistry is often employed in the differential diagnosis. In this study, we evaluated the expression of mammoglobin (MGB1) in body-cavity fluid specimens and compared its efficacy as a marker for metastatic breast carcinomas with that of gross cystic disease fluid protein-15 (GCDFP-15). Cell blocks from 40 fluid specimens were immunostained with monoclonal antibodies against MGB1 and GCDFP-15. They included 15 breast carcinomas and 25 nonmammary carcinomas (10 lungs, 10 ovaries, 3 gastrointestinal tracts, 1 kidney, and 1 urinary bladder). Positivity was defined as the presence of cytoplasmic staining in 10% or more carcinoma cells. Thirteen (87%) and seven (47%) breast carcinomas showed positive staining with MGB1 and GCDFP-15, respectively. Three (12%) nonmammary carcinomas (2 ovarian and 1 colonic) showed positive MGB1 staining; one (3%) nonmammary carcinoma demonstrated positive GCDFP-15 staining. The differences of MGB1 and GCDFP-15 staining between breast and nonmammary carcinomas were statistically significant (P < 0.05). Both MGB1 and GCDFP-15 are specific markers for metastatic breast carcinomas in cell block fluid specimens (88 vs. 96%). However, MGB1 is more sensitive than GCDFP-15 as a marker for metastatic breast carcinoma (87 vs. 46%). Diagn. Cytopathol. 2009. © 2009 Wiley-Liss, Inc. [source]

MUC4 is upregulated in ovarian carcinoma effusions and differentiates carcinoma cells from mesothelial cells

DIAGNOSTIC CYTOPATHOLOGY, Issue 12 2007
Ben Davidson M.D., Ph.D.
Abstract Using gene expression arrays, we recently showed that MUC4 expression is significantly higher in ovarian/primary peritoneal serous carcinoma (OC/PPC) compared to diffuse peritoneal malignant mesothelioma (DMPM). In the present study, we analyzed the anatomic site-related expression of MUC4 in OC/PPC and studied its prognostic role. We additionally studied the ability of MUC4 to differentiate between OC/PPC and reactive mesothelial cells (RMC). OC/PPC effusions (n = 142) and benign reactive effusions (n = 10) were immunostained for MUC4 expression. Immunoreactivity was scored in carcinoma cells and RMC and was compared with tumor cell expression in 60 previously studied primary carcinomas and solid metastases and analyzed for association with clinicopathologic parameters, including survival. MUC4 was detected in carcinoma cells in 141/142 (99%) effusions, with comparable expression in peritoneal and pleural effusions. RMC were present in 72 malignant effusions and were MUC4-negative in all specimens, as well as in the 10 reactive effusions. MUC4 expression in carcinoma cells in effusions was significantly higher than in primary carcinomas and solid metastases (P < 0.001). Higher MUC4 expression was seen in tumors from older (>60 year) patients (P = 0.049). No association was found between MUC4 expression and other clinicopathologic parameters, including survival. MUC4 is universally expressed in OC/PPC effusions and is upregulated at this anatomic site compared to primary carcinomas and solid metastases. The data in the present study, together with our earlier report, show that MUC4 is an excellent marker for differentiating OC/PPC from both benign and malignant mesothelial cells. Diagn. Cytopathol. 2007;35:756,760. © 2007 Wiley-Liss, Inc. [source]

Intracytoplasmic lumina in invasive micropapillary carcinoma of the lung

DIAGNOSTIC CYTOPATHOLOGY, Issue 3 2006
Naoto Kuroda M.D.
Abstract Micropapillary carcinoma of the lung is a rare neoplasm, and several reports on micropapillary carcinoma of the lung have been presented to date. We present a case of micropapillary carcinoma of the lung here. A 75-yr-old Japanese man received the medical checkup and his chest X-ray disclosed the abnormal shadow of the lower lobe of the left lung. The histological examination of resected lung and extirpated lymph node showed the finding of micropapillary carcinoma. Some neoplastic cells of primary site contained intracytoplasmic lumina positive for Alcian blue and PAS stains. Pleural effusion appeared 9-mo after the operation. The cytology of pleural effusion showed cohesive clusters of neoplastic cells consisting of 3,20 cells without fibrovascular core. Additionally, intracytoplasmic lumina were observed in some neoplastic cells. Finally, carcinoma cells with micropapillary morphology may possess the intracytoplasmic lumina in the cytoplasm of metastatic site as well as primary site. Diagn. Cytopathol. 2006;34:224,226. © 2006 Wiley-Liss, Inc. [source]

Urinary bladder biopsy with denuded mucosa: Denuding cystitis,Cytopathologic correlates

DIAGNOSTIC CYTOPATHOLOGY, Issue 5 2004
Anil V. Parwani M.D., Ph.D.
Abstract Denuding cystitis is often encountered in tissue biopsies of bladder mucosa performed by either cold-cup forceps or wire loop electrocautery to evaluate hematuria or to rule out recurrent urothelial carcinoma. Lack of urothelium in these biopsies is often a frustrating experience, leading to a nonspecific interpretation. In this study, 151 cases of denuding cystitis were retrieved from the surgical pathology files of The Johns Hopkins Hospital over a 4-year period (1996,1999). Patients under the age of 40 years and outside consultation material were excluded. Of the 151 cases of denuding cystitis, 48 patients were identified who had concurrent urinary cytologic studies. Of these patients, 35 were male (73%) and 13 were female (27%). Patient ages ranged from 43 to 85 years (mean, 67). Twenty-six of these 48 patients (54%) had at least one concurrently positive urinary cytology, which was histologically confirmed. All except three cases were high-grade urothelial carcinoma with the following histologic subtypes: flat carcinoma in situ (n = 11), noninvasive papillary (n = 9), and invasive urothelial carcinoma (n = 3). We conclude that urinary cytology is a sensitive modality that detects exfoliated carcinoma cells in patients with a histologic diagnosis of denuding cystitis. An inconclusive diagnosis of denuding cystitis on tissue might be related to biopsy method and technique, small sample size, or biopsy of cystoscopically abnormal urothelium that is denuded. A cytologic diagnosis of high-grade urothelial carcinoma in these cases leads to a timely clinical intervention for optimal patient management. Diagn. Cytopathol. 2004;30:297,300. © 2004 Wiley-Liss, Inc. [source]

True carcinosarcoma of the esophagus

DISEASES OF THE ESOPHAGUS, Issue 1 2006
T. Iwaya
SUMMARY., Most esophageal carcinosarcomas are diagnosed as so-called carcinosarcoma, in which individual elements may be derived from a single common ancestor cell, and there have been a few reports describing true carcinosarcoma originating from two individual stem cells. We describe a case of esophageal carcinosarcoma exhibiting neoplastic osteoid formation. Immunoreactivity for vimentin and p53 was limited to only the sarcomatous component and was absent in the carcinomatous component. Furthermore, a point mutation in exon 7 of the p53 gene was observed only in the sarcomatous component. Both sarcoma and carcinoma cells distinctively metastasized to different lymph nodes. These observations led us to diagnose the esophageal tumor as a true carcinosarcoma. [source]

Tumour cell,dendritic cell fusion for cancer immunotherapy: comparison of therapeutic efficiency of polyethylen-glycol versus electro-fusion protocols

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 3 2002
M. Lindner
Abstract Background ,Fusion of tumour cells with dendritic cells (DC) is a powerful new technology to increase tumour vaccine immunogenicity. The aim of this study was to compare fusion protocols with syngenic DCs with respect to the efficiency of polyethylen-glycol-(PEG) and electric pulse-mediated fusions for induction of protective anti-tumour immune responses. As a model we chose a low immunogenic and metastatic murine mammary carcinoma cell line, which mimics clinically relevant tumour features. Methods FACS-staining, chromium release assay, therapeutic immunization, adoptive transfer. Results ,We show that the parental line with low cell surface expression of MHC molecules as well as a lacZ transfectant becomes highly immunogenic upon fusion with DCs. This was true for PEG- as well as for electro-fused cells. Immunization with products of DCs and tumour cells cocultivated for 16 h without the fusing agent PEG also caused induction of profound anti-tumour immunity, while this was not the case when using parental tumour cells or their lacZ transfectants as vaccines. Immune protection against the parental tumour cells after vaccination with fused cells was long-lasting and could be transferred via immune spleen cells into immuno-incompetent nude (nu/nu) mice. Conclusion ,Fusion products of DA3hi mammary carcinoma cells and DCs produced by an electric pulse were similar to those produced by PEG fusion with regard to vaccine potency in prophylactic antitumour immunization assays in vivo. Therefore, both techniques seem to be promising for clinical application. [source]