Significant Increase In Cells Research Articles

Constitutive Cyclin A1 Expression Impairs Retinoic Acid-Induced Growth Arrest and Differentiation of Myeloid Leukemia Cells

AbstractAbstract 2894Acute promyelocytic leukemia (APL) differentiation syndrome (DS) in all-trans retinoic acid (ATRA) therapy is often associated with increase of leukocyte count (hyperleukocytosis). It suggests deregulated cell proliferation of ATRA-induced APL cells is possibly involved in the development of DS. The molecular mechanism(s) of hyperleukocytosis are unknown. We previously found increased expression of cyclin A1 mRNA were associated with both increase of initial leukocyte count and development of DS in the therapy with ATRA in APL. To clarify role(s) of cyclin A1 in the proliferation and differentiation of myeloid leukemia cells, we generated U937.A1 which is stably transfected with cyclin A1 gene. U937.A1 showed increased expression of cyclin A1 protein compared with U937.C, which carries pCDNA3.1Hisc (2.5 fold, immunoblot). U937.A1 showed increased in vitro colony formation compared with U937.C (1.3 fold).Flow cytometry (FCM) analysis of cells stained with propidium iodide(PI) showed that U937.A1 was characterized with significant decrease in cells in G1 and increase in G2M (P<0.01)(U937.C=G1:70.1±1.1,S:26.5±1.1,G2M:13.8±1.0% and U937.A1=G1:55.5±0.9,S:30.1±2.9,G2M:21.1±0.8%). U937.A1 showed constitutive higher Cdk1 kinase activities compared with U937.C more than 100 times, as measured by ELISA which quantitate enzyme activities by detecting phospholyrated synthetic peptide using monoclonal antibody 4A4. It suggests that inappropriate activation of cyclin A1/Cdk1 complex is associated with altered cell cycle progression of U937.A1 cells. Immunoblot analysis showed that U937.A1 over-expressed checkpoint kinase 1(Chk1) protein compared with U937.C.(1.4 fold)suggesting deregulated G2M checkpoint in U937.A1. Anti-apoptotic protein, bcl2, was increased in U937.A1 cells(1.5 fold).Incubation with 1μ M ATRA for 4 days significantly inhibited cell growth in U937.C (U937.C=44.9±1% and U937.A1=70.8±3%, p<0.01, cell No. with ATRA/without ATRA).ATRA-induced growth inhibition in U937.C was accompanied with significant increase of cells in G1 (U937.C=88.9±0.9% and U937.A1=68.2±0.8%) and decreased in the G2M(U937.C=4.9±0.5% and U937.A1=19.7±1.2%). Expression of both cyclin A2 and cyclin B proteins were markedly down-regulated at days 3 and 4 in U937.C in the culture (U937.C=69.1±1.3%(cyclin A2) and 52.1±2.1%(cyclin B), U937.A1=90.2±1.7%(cyclin A2)and 94.5±2.4%(cyclin B),day4, ATRA/without ATRA) Cdk1 protein expression was also significantly down-regulated in U937.C(U937.C=53.5±2.5% and U937.A1=115±5%, p<0.01, ATRA/without ATRA). In both of U937.C and U937.A1 cells, Cdk1 kinase activities were significantly suppressed by 4 days' culture with 1μ M ATRA,however, the activities in U937.A1 still remained 10 times higher than those in U937.C (U937.C=0.06±0.01/0.25±0.02 and U937.A1=0.19±0.01/1.04±0.04,day4, O.D., ATRA/without ATRA). Incubated with ATRA, expression of Chk1 protein in U937.C cells time-dependently decreased (U937.C=44.3±0.1% and U937.A1=86.1±0.1%, p<0.01, day4. with ATRA/without ATRA) Checkpoint kinase activities, as measured by ELISA using anti-phospho-Cdc25C serine 216 specific antibody, was significantly suppressed by ATRA in U937.C (U937.C=1.25±0.02/2.69±0.09 and U937.A1=3.42±0.06/2.79±0.22,day4, O.D., ATRA/without ATRA). Incubation with Go6976 at 10 μ M, a specific chk1 inhibitor, produced significant cell growth inhibition in U937.A1 in combination with ATRA. The growth inhibition by ATRA plus Go6976 was accompanied with accumulation of cells in G1(79.2±0.9%) and decrease of cells in G2M (7.7%±0.3%)compared with those incubated with ATRA alone, suggesting deregulated chk1 activities are involved in impaired ATRA-induced growth inhibition in U937.A1 cells. ATRA-induced down-regulation of expression of bcl2 protein was significant in U937.C (U937.C=41.8±0.1% and U937.A1=77.8±0.2%, day4, with ATRA/without ATRA). Decreased bcl2 protein was inversely co-related with up-regulation of CD11b induced by ATRA (U937.C=4.3±0.2% and U937.A1=3.9±0.1%, FCM, day4).The present results showed that forced expression of cyclin A1 resulted in impaired ATRA-induced growth suppression and differentiation of U937 cells. It was related with inappropriately activated Chk1-Cdk1 pathway. Deregulated G2M cell cycle checkpoint(S)is a candidate for the therapeutic target in ATRA-induced hyperleukocytois in APL. Disclosures:No relevant conflicts of interest to declare.

Evaluation of bacterial multiplication in cleaning cloths containing different quantities of organic matter

To determine a proper length of time for cleaning cloth usage, the present work aimed to evaluate bacterial multiplication in artificially contaminated cleaning cloths containing different amounts of organic matter. Cloths containing 1%, 5%, and 10% of bovine albumin were contaminated with Salmonella enteritidis 3091/05, Escherichia coli ATCC 25972, Staphylococcus aureus ATCC 25923, and Shigella sonnei CC07. They were incubated for different time periods at 30°C. Microbial multiplication was evaluated by bacterial counts and the ATP bioluminescence increase was monitored at sampling points. An ampicillin-resistant recombinant HSα E. coli strain was used as a pathogen surrogate to investigate the potential of microbial cloth dispersion. None of the strains showed expressive growth up to two hours of incubation. At three hours, the microorganisms demonstrated a slight increase, with E. coli ATCC 25972 showing a significant increase in cells (p < 0.05). The ATP bioluminescence did not increase during the incubation period and confirmed the microbial count results, demonstrating that the amounts of organic matter tested did not interfere with bacterial multiplication during the first three to four hours of incubation. The dispersion experiment indicated that a cleaning cloth contaminated with 10⁴ CFU/cm² was able to spread 10² CFU/cm² of recombinant E. coli onto a stainless steel surface. Based on these results, we suggest that an appropriate period of time for using disinfected cleaning cloths is around two hours, not exceeding three hours of usage.

Abstract 5645: Growth inhibitory activity of ethanol extracts of Chinese and Brazilian propolis in human colon carcinoma cell lines

Abstract Propolis is a natural product derived from plant resins collected by honeybees, and has been used globally as a folk medicine for centuries. More than 300 bio-active compounds have been identified from bee propolis. In the current study, we examined whether the ethanol extracts of Chinese and Brazilian propolis exert anticancer activities in four human colon carcinoma cell lines, CaCo2, HCT116, HT29 and SW480. Propolis samples were extracted with ethanol, and the crude extracts were dissolved in dimethylsulfoxide and used for the following experiments. We also examined the effects of the ethanol extract of Chinese propolis on cell proliferation, cell cycle progression, apoptosis, and the levels of expression of cell cycle control molecules in the HCT116 and HT29 cell lines. In the HCT116, HT29 and SW480 cell lines, the extracts of both Chinese and Brazilian propolis caused a marked dose-dependent growth inhibition, with IC50 values in the range of 4-41 μg/ml. In the FHC normal human colonic epithelial cell line, the extract of Chinese propolis failed to cause a marked growth inhibition, the growth inhibition rate was about 20%, whereas it was about 90% in the HCT116 and HT29 cell lines. In the HCT116 cell line, at concentrations of 10 and 20 μg/ml the extract of Chinese propolis caused a marked increase in sub-G1 fraction of cells in a dose-dependent fashion. The propolis extract (20 μg/ml) caused a significant increase of cells in sub-G1 and this increase was associated with a concomitant relative decrease of cells in the G1, S and G2-M phases of the cell cycle, indicating that Chinese propolis extract induced apoptosis in the HCT116 cell line. In addition, Chinese propolis extract caused a dose-dependent increase in the cellular mRNA levels of p21CIP1 and p53 in the HCT116 cell line. These findings indicate that the ethanol extracts of propolis contain components that may have anticancer activity. Thus, propolis and related products may provide a novel approach to the chemoprevention and/or chemotherapy of human colon carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5645.

Abstract 2884: Standardized mangosteen fruit extract promotes cell cycle arrest and apoptosis of PC3 cells and inhibits tumor formation in athymic nude mice

Abstract The purple mangosteen fruit (Garcinia mangostana) is native to southeast Asia and has been reported to contain a variety of health promoting properties that include antioxidant, anti-inflammatory, anit-microbial and anti-diabetic properties. The principle constituents isolated from the mangosteen fruit are the xanthones with well over 50 unique entities isolated to date. Here we report on the properties of a mangosteen fruit extract for cancer chemoprevention and/or chemotherapy using prostate cancer (PCa) as a model disease. Using a mangosteen fruit extract (MFE) a significant decrease in cell viability was observed against four different prostate cancer cell lines (LNCaP, CWR22, PC3, DU145). Several analytical standards were used to identify dietary xanthones by HPLC with alpha-mangostin found to be the most abundant xanthone of MFE representing approximately 59% of the extract. Next, we investigated the ability of MFE to promote cell cycle arrest by flow cytometry and observed a significant increase in cells in G1 phase when treated with 7.5 μg/mL of MFE after 24 hours. Further confirmatory studies were performed by western blot analysis of PC3 cells treated with MFE. To understand the possible interaction between xanthones and cyclins/cdks in a cell free system a biochemical based assay was performed and found to inhibit cyclinD1/CDK4 as well as other kinases. To determine the potential of MFE in an in vivo model we investigated the ability of MFE to inhibit colony formation of CWR22 and PC3 cells. MFE was found to completely inhibit colony formation of CWR22 cells at a concentration of 5 µg/mL. Next, using an athymic nude mouse model implanted with CWR22 cells mice were randomized to receive intraperitoneally the standardized mangosteen fruit extract (MFE) or placebo two times weekly with tumors measured twice weekly until tumors reached a volume of 1200 mm3. At the conclusion of the study the treatment group had a significantly smaller tumor with an average tumor volume of (143 mm3) compared to the placebo group (1135 mm3) representing an 87.5% smaller tumor in the treatment group. Based on these promising results we have evidence suggesting that MFE may be a promising agent for the prevention and/or treatment of prostate cancer. Further studies are needed to determine the potential of purified dietary xanthones from the mangosteen fruit for cancer chemoprevention and chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2884.

Mechanisms Underlying the Anti-Proliferative Actions of Adiponectin in Human Breast Cancer Cells, MCF7–Dependency on the cAMP/Protein Kinase-A Pathway

Obesity is a risk factor for breast cancer, and low blood concentrations of adiponectin are associated with high incidence and poor prognosis of breast cancer. However, the molecular mechanisms underlying such inhibitory effects of adiponectin remain to be defined. By using MCF7 cells, we investigated the mechanisms underlying the inhibitory effects of adiponectin on breast cancer cells, particularly in the context of opposing IGF-1-induced proliferation. We found that adiponectin, at 20 and 40 μg/ml, reduced MCF7 cell growth in the absence and presence of IGF-1. These inhibitory effects were primarily the result of the significant increase of cells at G1/G0 phase and concomitant decrease of cells at S phase. In addition, the percentage of apoptotic cells increased more than two-fold. Within 30-min of adiponectin addition, the phosphorylation of AMPKα was sharply elevated, and the level of IGF-1-activated Akt was decreased. Prolonged exposure to adiponectin resulted in reduction of cyclin D1 and cyclin E2 expression. Adiponectin also increased intracellular levels of cAMP and the activity of protein kinase-A (PKA). The inhibitors of PKA completely abolished the adiponectin's effects on cell growth. In conclusion, our studies revealed an important cellular mechanism underlying the relationship between reduced adiponectin concentrations and breast cancer development.

Abstract C192: Characterization of the target profile of XL228, a multi-targeted protein kinase inhibitor in phase 1 clinical development

Abstract Background: XL228 is a small molecule protein kinase inhibitor currently in Phase 1 clinical development targeting Ph+ leukemias, multiple myeloma, and solid tumors. Cellular and in vivo data have demonstrated activity of XL228 against IGF1R, ABL (including the T315I variant) and SRC family kinases. XL228 was tested in a panel of biochemical protein kinase assays, and additional putative targets were identified, including Aurora family kinases, FGFR1-3, and ALK. As these potential targets are known to enhance cell proliferation and survival in certain malignancies, the validation of their inhibition by XL228 through in vitro studies and in clinical pharmacodynamic samples was performed. Results: A panel of kinase inhibitors including XL228 was profiled against a series of cancer cell lines with known alterations in major signaling pathways. Approximately 30% of the lines demonstrated XL228 IC50 values of &amp;lt;100nM in viability assays, including many lines with characterized ALK or FGFR mutations or amplifications. Cell lines showing the highest level of response to XL228 were often sensitive to other Aurora, ALK or FGFR inhibitors as well. Sub-20nM activity was demonstrated against FGFR2 in a cellular kinase ELISA assay, and confirmed by immunoblot analysis. Potency against ALK in the cell kinase assay was lower (approximately 200 nM). The Aurora inhibitory activity of XL228 was examined through the use of immunoblots, immunofluorescence, and substrate phosphorylation assays. XL228 eliminated the phosphorylation of Aurora A and B at concentrations above 10 nM. Short-term treatment of HeLa cells led to disruption of mitotic spindle formation, with the majority of mitotic cells exhibiting a unipolar spindle and disorganized chromosomes. After exposure to XL228 for 24 hours, there was a significant increase in cells with &amp;gt;4N DNA content, suggesting a failure of cytokinesis and consequent endoreduplication. Activity of XL228 against Aurora kinases was further verified by dose-dependent inhibition of histone H3 phosphorylation, where it showed approximately 5-fold higher potency than the control compound VX-680. To confirm the relevance of the in vitro observations to the clinical activity of XL228, tumor biopsy specimens obtained from the solid tumor Phase 1 clinical study were evaluated for changes in phosphorylation of pathway markers by immunofluorescence. Inhibition of Aurora, FGFR, IGF1R, and SRC family kinases was observed, as measured by decreases in kinase substrate phosphorylation in these specimens. Conclusions: XL228 shows a broad pattern of protein kinase inhibition, including the tyrosine kinases IGF1R, SRC, ABL, FGFR1-3, and ALK and the serine/threonine kinases Aurora A and Aurora B. The clinical impact of this target profile is currently being assessed in two Phase 1 studies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C192.

EKLF/KLF1 Controls Cell Cycle Entry via Direct Regulation of E2f2

Differentiation of erythroid cells requires precise control over the cell cycle to regulate the balance between cell proliferation and differentiation. The zinc finger transcription factor, erythroid Krüppel-like factor (EKLF/KLF1), is essential for proper erythroid cell differentiation and regulates many erythroid genes. Here we show that loss of EKLF leads to aberrant entry into S-phase of the cell cycle during both primitive and definitive erythropoiesis. This cell cycle defect was associated with a significant reduction in the expression levels of E2f2 and E2f4, key factors necessary for the induction of S-phase gene expression and erythropoiesis. We found and validated novel intronic enhancers in both the E2f2 and E2f4 genes, which contain conserved CACC, GATA, and E-BOX elements. The E2f2 enhancer was occupied by EKLF in vivo. Furthermore, we were able to partially restore cell cycle dynamics in EKLF(-/-) fetal liver upon additional genetic depletion of Rb, establishing a genetic causal link between reduced E2f2 and the EKLF cell cycle defect. Finally, we propose direct regulation of the E2f2 enhancer is a generic mechanism by which many KLFs regulate proliferation and differentiation.

Up-regulation of ROS by mitochondria-dependent bystander signaling contributes to genotoxicity of bystander effects

Genomic instability can be observed in bystander cells. However, the underlying mechanism(s) is still relatively unclear. In a previous study, we found that irradiated cells released mitochondria-dependent intracellular factor(s) which could lead to bystander γ-H2AX induction. In this paper, we used normal (ρ +) and mtDNA-depleted (ρ 0) human–hamster hybrid cells to investigate mitochondrial effects on the genotoxicity in bystander effect through medium transfer experiments. Through the detection of DNA double-strand breaks with γ-H2AX, we found that the fraction of γ-H2AX positive cells changed with time when irradiation conditioned cell medium (ICCM) were harvested. ICCM harvested from irradiated ρ + cells at 10 min post-irradiation (ρ + ICCM 10 min ) caused larger increases of bystander γ-H2AX induction comparing to ρ 0 ICCM 10 min , which only caused a slight increase of bystander γ-H2AX induction. The ρ + ICCM 10 min could also result in the up-regulation of ROS production (increased by 35% at 10 min), while there was no significant increase in cells treated with ρ 0 ICCM 10 min . We treated cells with dimethyl sulfoxide (DMSO), the scavenger of ROS, and quenched γ-H2AX induction by ρ + ICCM. Furthermore, after the medium had been transferred and the cells were continuously cultured for 7 days, we found significantly increased CD59 − gene loci mutation (increased by 45.9%) and delayed cell death in the progeny of ρ + ICCM-treated bystander cells. In conclusion, the work presented here suggested that up-regulation of the mitochondria-dependent ROS might be very important in mediating genotoxicity of bystander effects.

CCR6 Regulation of the Actin Cytoskeleton Orchestrates Human Beta Defensin-2- and CCL20-mediated Restitution of Colonic Epithelial Cells

Intestinal inflammation is exacerbated by defects in the epithelial barrier and subsequent infiltration of microbes and toxins into the underlying mucosa. Production of chemokines and antimicrobial peptides by an intact epithelium provide the first line of defense against invading organisms. In addition to its antimicrobial actions, human beta defensin-2 (HBD2) may also stimulate the migration of dendritic cells through binding the chemokine receptor CCR6. As human colonic epithelium expresses CCR6, we investigated the potential of HBD2 to stimulate intestinal epithelial migration. Using polarized human intestinal Caco2 and T84 cells and non-transformed IEC6 cells, HBD2 was equipotent to CCL20 in stimulating migration. Neutralizing antibodies confirmed HBD2 and CCL20 engagement to CCR6 were sufficient to induce epithelial cell migration. Consistent with restitution, motogenic concentrations of HBD2 and CCL20 did not induce proliferation. Stimulation with those CCR6 ligands leads to calcium mobilization and elevated active RhoA, phosphorylated myosin light chain, and F-actin accumulation. HBD2 and CCL20 were unable to stimulate migration in the presence of either Rho-kinase or phosphoinositide 3-kinase inhibitors or an intracellular calcium chelator. Together, these data indicate that the canonical wound healing regulatory pathway, along with calcium mobilization, regulates CCR6-directed epithelial cell migration. These findings expand the mechanistic role for chemokines and HBD2 in mucosal inflammation to include immunocyte trafficking and killing of microbes with the concomitant activation of restitutive migration and barrier repair.

1H and hyperpolarized 3He MR imaging of mouse with LPS‐induced inflammation

To evaluate the lipopolysaccharide (LPS) model of chronic obstructive pulmonary disease (COPD) in mouse with (1)H and hyperpolarized (HP) (3)He MR imaging. Axial slices of the lung volume were acquired with HP (3)He and (1)H MRI at 4, 24, and 48 h after LPS exposure. A quantitative ventilation index was calculated from two HP (3)He acquisitions. A bronchoalveolar lavage (BAL) for a cell count was performed following magnetic resonance imaging (MRI). The LPS exposure resulted in a significant increase of cells in BAL, with maximum at 48 h. Lesions on (3)He images were characterized by ventilation defects, whereas lesions on (1)H images were hyperintense and were attributed to edema. The number of lesions was at maximum at 48 h. At this time point, and for both (3)He and (1)H MRI, the volume of the lesions was significantly higher for LPS-exposed mice compared to controls. At 4, 24, and 48 h the ventilation index from the (3)He data was significantly smaller for the LPS-exposed animals compared to controls. The time point 48 h after LPS exposure was advantageous for MRI evaluation. Functional read-out with (3)He MRI seems to be more sensitive than conventional (1)H MRI.

Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells

BackgroundPseudomonas aeruginosa is the major pathogen associated with chronic and ultimately fatal lung infections in patients with cystic fibrosis (CF). To investigate how P. aeruginosa-derived vesicles may contribute to lung disease, we explored their ability to associate with human lung cells.ResultsPurified vesicles associated with lung cells and were internalized in a time- and dose-dependent manner. Vesicles from a CF isolate exhibited a 3- to 4-fold greater association with lung cells than vesicles from the lab strain PAO1. Vesicle internalization was temperature-dependent and was inhibited by hypertonic sucrose and cyclodextrins. Surface-bound vesicles rarely colocalized with clathrin. Internalized vesicles colocalized with the endoplasmic reticulum (ER) marker, TRAPα, as well as with ER-localized pools of cholera toxin and transferrin. CF isolates of P. aeruginosa abundantly secrete PaAP (PA2939), an aminopeptidase that associates with the surface of vesicles. Vesicles from a PaAP knockout strain exhibited a 40% decrease in cell association. Likewise, vesicles from PAO1 overexpressing PaAP displayed a significant increase in cell association.ConclusionThese data reveal that PaAP promotes the association of vesicles with lung cells. Taken together, these results suggest that P. aeruginosa vesicles can interact with and be internalized by lung epithelial cells and contribute to the inflammatory response during infection.

Comments on the paper by E. M. Minicucci et al. “DNA damage in lymphocytes and buccal mucosa cells of children with malignant tumours undergoing chemotherapy”

I read the paper by Minicucci et al. ‘‘DNA damage in lymphocytes and buccal mucosa cells of children with malignant tumours undergoing chemotherapy’’ [1] with great interest because recently I analyzed all available literature sources concerning the influence of chemoand radiotherapies on exfoliated mucosal cells [2]. The problem of possible micronuclei (MN)-inducing activity of antiblastics on epithelial oral mucosa cells has not been studied well. Only four papers were published concerning cancer patients (for review see [2]) and two concerning patients with other diseases treated with cytostatics [3, 4]. It has been shown that in these papers positive effect was registered in some cases. But no firm conclusion could be drawn if chemotherapy induces MN in oral epithelial cells. I think it is because all investigators applied various time points to collect exfoliated epithelial cells after the application of cytostatics. For example, it is practically not clear when the samples of buccal cells were collected by Minicucci et al. [1]. The time factor could play a key role in finding of MN because of kinetics (or the life cycle) of these types of cells. According to Szete et al. [5], the half-life of buccal cells is about 15 days and Kassie et al. [6] showed that MN level is highest 28 days after the effect of mutagen. After that there is an elimination of cells with MN. Hence, collection of oral cells ought to be done not later than 30 days after the last application of cytostatic drug. In my opinion, success of such kind of investigations depends mainly on the study of primary patients (without any treatment which can induce damage of cytogenetic structures) after one cycle of chemotherapy because it is mostly toxic for organism, and it could induce changes in basal cell kinetics. Also, due to this the so-called nuclear anomalies (karyolysis, karyorrhexis, pycnosis) can appear [7]. The evaluation of the so-called nuclear anomalies along with MN will improve the quality of the studies. Minicucci et al. [1] found significantly increased levels of MN in lymphocytes which were collected at the same time as exfoliated cells. It is because, unlike epithelial cells, half-life of lymphocytes is substantially longer (some years) that it is possible to find an increased number of MN in lymphocytes of children under therapy of much longer time than 30 days (the critical time point to register MN in epithelial cells) [8]. It is noteworthy that Torres-Bugarin et al. [7] investigated about 40 patients under antiblastic therapy, and found significantly increased levels of MN in some of them. But among them were subjects after two or even three cycles of therapy. Like Minicucci et al. [1], no firm information was presented as to when the buccal cells were collected. Based on the data presented by the authors, the number of cells with MN only in primary cancer patients treated with three chemotherapeutic schedules was calculated. Totally among them 19 were primary, and MN frequencies were 1.6 ± 0.4% before and 2.6 ± 0.7% after treatment (p [ 0.05, Mann–Whitney test). In ten patients treated with carboplatin ?5-fluorouracil MN numbers were 0.95 ± 0.12% before and 1.35 ± 0.42% after treatment, and in nine patients treated with isophosphamide ? epirubicin the frequencies were 2.9 ± 1.0% (before) and 4.9 ± 1.6% (after treatment) (p [ 0.05 in both cases, Mann–Whitney U test). As can be noted, no significant increase of cells with MN was found also in primary patients after one course of therapy. But the time of cell collection was not clearly indicated by the authors [1]. Ramos-Remus studied and A. Nersesyan (&) Institute of Cancer Research, Vienna, Austria e-mail: armen.nersesyan@meduniwien.ac.at

269 POSTER Recombinant human epidermal growth factor (rhEGF) inhibits proliferation of cancer cell lines with high level of EGFR expression

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L), a member of the TNF gene superfamily, induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells. Nevertheless, some human hepatoma cells are particularly resistant to the effects of TRAIL. In this study, we show that J7, a novel methyl jasmonate analogue, sensitizes TRAIL-resistant HepG2 human hepatocarcinoma cells to TRAIL-mediated apoptosis. Our results indicate that J7 substantially enhances TRAIL-induced apoptosis, compared with treatment with either agent alone. Combined treatment with J7 and TRAIL effectively induced Bid cleavage, down-regulation of XIAP, cIAP-1 and Bcl-xL, activation of caspases, and cleavage of poly(ADP-ribose) polymerase and phopholipase γ-1. In addition, generation of reactive oxygen species (ROS) showed a significant increase in cells following exposure to J7 in a time-dependent manner. However, the cytotoxic effects induced by co-treatment with J7 and TRAIL were markedly attenuated by caspase inhibitors, indicating an important role for caspases. Administration of N-acetyl cysteine, a scavenger of ROS, also resulted in significant inhibition of apoptosis induced by combinatory treatment with J7 and TRAIL. These results support a mechanism whereby J7 plus TRAIL induces apoptosis of HepG2 human hepatoma cells through a signaling cascade involving a ROS-mediated caspase pathway.

Induction of cyclooxygenase-2 expression and prostaglandin E2 secretion by macrophage migration inhibitory factor in ectopic endometrial cells: role in the pathophysiology of endometriosis

OBJECTIVE: To assess whether macrophage migration inhibitory factor (MIF), a potent proinflammatory and angiogenic factor found at elevated levels in the peritoneal fluid and active endometriotic lesions of women with endometriosis, may influence the expression of cyclooxygenases (COX) 1 and 2 and the secretion of prostaglandin E2 (PGE2) in ectopic endometrial cells. DESIGN: Primary cultures of ectopic endometrial cells exposed to different concentrations of MIF and assessment of COX-1 and COX-2 expression and PGE2 secretion. MATERIALS AND METHODS: Ectopic endometrial cells were isolated and cultured from endometriotic lesions (n=9) according to routinely used procedures. At confluence, cells were starved 16h in DMEM F12 and then stimulated with various concentrations of MIF (0-50 ng/mL) for several periods of time (0-24 h). In some cultures, cells were co-incubated with a specific inhibitor of MIF, ISO-1 or with NS-398, a specific inhibitor of COX-2 activity. Expression of COX-1 and COX-2 was analysed by quantitative real-time PCR at mRNA level and by Western Blot and immunocytofluorescence at protein level. PGE2 concentration in supernatants was measured using a PGE2 Enzyme Immunoassay (EIA) kit. Statistical analysis was performed using ANOVA followed by the Dunnet's or the Bonferonni's multiple comparison test. RESULTS: MIF stimulates the synthesis of COX-2 in endometriotic cells in vitro, whereas COX-1 is not affected. Cell exposure to MIF led to a dose- and time-dependent upregulation of COX-2 mRNA expression, as shown by quantitative real-time PCR (p<0.01), and ISO-1 significantly inhibited such a MIF-induced effect. Results from Western blot and immunocytofluorescence further demonstrated an increase in COX-2 protein synthesis in response to MIF. Analysis of PGE2 secretion revealed a significant increase in cells exposed to MIF (p<0.01), whereas addition of NS-398 resulted in a significant inhibition of the MIF-induced PGE2 secretion (p<0.01). CONCLUSIONS: MIF up-regulates COX-2 expression and PGE2 secretion in ectopic endometrial cells. Considering the wide spectrum of PGE2 inflammatory, growth-promoting and angiogenic properties and implication in various female reproductive functions, these data unveil a new mechanism by which MIF may favour the development of endometriotic lesions and the manifestation of the disease's clinical symptoms.

Short Communication: Characterization of Madin-Darby Bovine Kidney Cell Line for Peroxisome Proliferator-Activated Receptors: Temporal Response and Sensitivity to Fatty Acids

The peroxisome proliferator-activated receptors (PPAR) are critical for lipid metabolism, and many fatty acids are PPAR agonists. Madin-Darby bovine kidney (MDBK) cells were tested as an in vitro bovine model for PPAR activation, and preliminary evaluation of the effect of fatty acids on bovine PPAR was performed. Cells were treated with Wy-14643 (WY, specific PPARα agonist) and rosiglitazone (ROSI, specific PPARγ agonist). The gene expression of specific PPARα-responsive genes such as carnitine palmitoyl transferase-1 (CPT1A) and acetyl coenzyme A oxidase (ACOX1) and of PPARγ-responsive gene lipoprotein lipase (LPL) were analyzed using real-time reverse transcription PCR. It was found that CPT1A exhibited a significant increase in cells treated with WY, whereas the ACOX1 gene expression was not altered. The LPL gene expression showed an increase in response to ROSI. Interestingly, LPL was almost undetectable in MDBK cells not treated with ROSI. The potency of different fatty acids in activating PPARα as assessed by CPT1A mRNA abundance in MDBK cells was also tested. The mRNA of CPT1A (2.5- to 1.4-fold) was significantly increased by fatty acids in the order of palmitate>linolenate>linoleate>conjugated linoleate, and oleate. The results demonstrated MDBK cells to be responsive to PPAR agonists and thus a promising model to evaluate the role of PPAR in bovine cells. In addition, fatty acids were proven to have a different potency in modulating expression of CPT1A through PPARα.

Potential role of mast cells in hamster cheek pouch carcinogenesis

During the process of activation, mast cells release products stored in their granules. Tryptase, a protease released from mast cell granules after activation, induces tumor cell proliferation through the activation of PAR-2 (protease activated receptor 2) on the plasma membrane of carcinoma cells. Chemical cancerization (DMBA) of the hamster cheek pouch is the most accepted model of oral cancer. However, there are no reports on the activation of mast cells during experimental carcinogenesis or on the correlation between mast cell activation and cell proliferation. The aim of the present study was to evaluate the potential effect of mast cells on the proliferation of epithelial cells at different times during the cancerization process. Paraffin serial sections of cancerized, tumor-bearing pouches were stained with Alcian Blue-Safranin to identify the different degrees of mast cell activation. Immunohistochemistry was performed to identify BrdU-positive cells to study tumor cell proliferation. Mast cells were counted and grouped into two categories: inactive mast cells AB-S+++ (red) and active mast cells AB+++S- (blue). Mast cell counts were performed in tumor stroma, base of the tumor (connective tissue immediately below the exophytic tumor), connective and muscle tissue underlying the cancerized epithelium (pouch wall) and adventitious tissue underlying the pouch wall. There was a significant increase in the number of mast cells at the base of tumors (p<0.001) compared to the number of mast cells in the wall of the pouch and in tumor stroma. In normal non-cancerized pouches, inactive mast cells were prevalent both in the wall (AB:S=1:2.15; p<0.001) and in the adventitious tissue (AB:S=1:1.6; p<0.004) of the hamster cheek pouch. At most of the experimental times examined, the ratio of active/inactive mast cells (AB/S) in the wall approximated unity and even reverted. The ratio of mast cells was AB:S 1:1.05 at the base of the tumor and 1:0.24 in tumor stroma (p<0.001). The evaluation of epithelial nuclei labeled for BrdU revealed a statistically significant increase in cells undergoing DNA synthesis in the epithelium of the wall of the cancerized pouch compared to control (p<0.017). Tumor parenchyma exhibited a highly statistically significant increase in DNA synthesis compared to control (p<0.001) and compared to the epithelium of the wall of the cancerized pouch (p<0.036). We conclude that mast cell activation in this model is associated to the increase in tumor cell proliferation, conceivably mediated by the release of tryptase.

Mitotic, but not meiotic, oriented cell divisions in rat spermatogenesis

The process of mammalian spermatogenesis involves both mitosis and meiosis at the same developmental age. Most previous studies have focused on mitotic spindle orientation during development, but not during meiotic division. Therefore, we asked whether there is a difference between mitotic and meiotic germ cell spindle orientation during rat spermatogenesis. Our results showed that mitotic spindles of spermatogonia were mainly oriented with angles ranging from 60 to 90 degrees, perpendicular in relation to the basement membrane of the seminiferous tubules. On the other hand, meiotic spindles showed a random orientation. Nocodazole treatment (at a concentration that depolymerizes only astral microtubules) induced a significant increase in cells with an angle between 0 and 30 degrees (parallel) in relation to the basement membrane. Meiotic spindles did not show a significant change in their orientation after the Nocodazole treatment. Therefore, our results suggest differences between the mechanisms controlling positioning and orientation of mitotic and meiotic spindles during rat spermatogenesis. It seems that a phylogenetically conserved programme controls the mitotic spindle orientation in organisms ranging from worms to mammals.

Cell (A549)–Particle (Jasada Bhasma) interactions using Raman spectroscopy

Current methods for the evaluation of cell interactions with particles are nonspecific, slow, and invasive to the cells. Raman spectroscopy is a noninvasive technique, and is used in the present study to investigate particle-cell interactions. The main focus of the present study is to employ Raman spectroscopy for investigating the interaction of human lung adenocarcinoma cell line (A549) with the particulate system Jasada Bhasma, a traditional Indian medicine. Jasada Bhasma is a unique preparation of zinc and is traditionally used for the treatment of various diseases like diabetes, age-related eye diseases, and as a health promotional tonic. The Raman spectral analysis is executed by identifying the difference in intracellular DNA/RNA, and proteins and lipids concentration between particles--treated and untreated cells. Comparison between Bhasma-treated and -untreated cells indicates that vibrational peaks corresponding to the DNA/RNA molecule show a significant increase in cells treated with the Jasada Bhasma. Apart from the DNA molecule, several other vibrational peaks related to the protein molecules also show a significant increase in A549 cells after treatment with Bhasma. These results indicate that Bhasma treatment of A549 possibly delays DNA degradation and enables retention of higher amount of protein molecules in the cells.

The buccal cytome and micronucleus frequency is substantially altered in Down's syndrome and normal ageing compared to young healthy controls

The buccal micronucleus cytome assay was used to investigate biomarkers for DNA damage, cell death and basal cell frequency in buccal cells of healthy young, healthy old and young Down's syndrome cohorts. With normal ageing a significant increase in cells with micronuclei ( P < 0.05, average increase +366%), karyorrhectic cells ( P < 0.001, average increase +439%), condensed chromatin cells ( P < 0.01, average increase +45.8%) and basal cells ( P < 0.001, average increase +233%) is reported relative to young controls. In Down's syndrome we report a significant increase in cells with micronuclei ( P < 0.001, average increase +733%) and binucleated cells ( P < 0.001, average increase +84.5%) and a significant decrease in condensed chromatin cells ( P < 0.01, average decrease −52%), karyolytic cells ( P < 0.001, average decrease −51.8%) and pyknotic cells ( P < 0.001, average decrease −75.0%) relative to young controls. These changes show distinct differences between the cytome profile of normal ageing relative to that for a premature ageing syndrome, and highlight the diagnostic value of the cytome approach for measuring the profile of cells with DNA damage, cell death and proportion of cells with proliferative potential (i.e., basal cells). Significant correlations amongst cell death biomarkers observed in this study were used to propose a new model of the inter-relationship of cell types scored within the buccal micronucleus cytome assay. This study validates the use of a cytome approach to investigate DNA damage, cell death and cell proliferation in buccal cells with ageing.

INTESTINAL ISCHEMIA-REPERFUSION-INDUCED ACUTE LUNG INJURY AND ONCOTIC CELL DEATH IN MULTIPLE ORGANS

Most acute respiratory distress syndrome studies have been focused on the lung injury. Little is known about other organs during the development of acute respiratory distress syndrome. Herein, we investigated the injury and cell death in multiple organs after intestinal ischemia-reperfusion (IIR) in C57BL/6 mice. Terminal transferase dUTP nick end labeling staining was used as a marker of cell death. Caspase 3 and cathepsin B activation as markers of caspase-dependent and caspase-independent apoptosis, respectively, and electron microscopy for ultimate characterization of cell death were used. In comparison with control and sham-operated mice, the IIR group showed interstitial inflammatory infiltrates in the lung and significant increases of lung injury parameters and plasma lactate dehydrogenase and aspartate aminotransferase levels. Terminal transferase dUTP nick end labeling-positive cells and immunostaining for hemeoxygenase 1, an enzyme induced by inflammatory stimuli, were increased in the lung, heart, and kidney, but not in the liver. The number of hemeoxygenase 1-positive cells positively and significantly correlated to the number of terminal transferase dUTP nick end labeling-positive cells. Cell death was not associated with caspase 3 or cathepsin B activation. Electron microscopy showed morphological features compatible with oncotic rather than apoptotic cell death or necrosis, including mitochondrial swelling and cytoplasm disorganization in pulmonary and renal epithelial cells, lung and cardiac endothelial cells, and myocytes. These results indicate that, although lung injury is the most significant manifestation after IIR, oncotic cell death occurs in the lung, heart, and kidney, which may be related to ischemia and inflammation.