DNA Flow Research Articles

The first case of natural spontaneous triploidy in the family Bombinatoridae

A triploid specimen of the diploid anuran species Bombina orientalis was identified by means of DNA flow cytometry. The finding is interpreted as a case of natural spontaneous autotriploidy. The incidence of triploidy was 1.8% per species (n = 55) and 11.1% per sample (n = 9).

On the southern distributional limit of the pool frog, Pelophylax lessonae (Camerano, 1882): the first record in Volgograd region, Russia

Distribution and types of population systems of green frogs (Pelophylax esculentus complex) in the northwest of Volgograd region were studied. The pool frog (P. lessonae) was reliably recorded in two localities of the area studied by use of the precise nuclear DNA flow cytometry method. That allowed to shift the southern boundary of this species area by 30 km. It is zoogeographically important that new records extend the range of the species distribution from the forest-steppe to the steppe zone. Two types of green frog population systems were revealed: a) R-type, ‘pure’ populations of P. ridibundus, and b) R-L-type, a mixed population system including P. ridibundus and P. lessonae. No hybrid P. esculentus was found. Refs 12. Figs 1. Tables 1.

Isoalantolactone Enhances the Radiosensitivity of UMSCC-10A Cells via Specific Inhibition of Erk1/2 Phosphorylation.

BackgroundAlthough radiotherapy is one of the mainstream approaches for the treatment of head and neck squamous cell carcinoma (HNSCC), this cancer is always associated with resistance to radiation. In this study, the mechanism of action of isoalantolactone as well as its radiosensitizing effect was investigated in UMSCC-10A cells.MethodsThe radiosensitization of UMSCC-10A cells treated with isoalantolactone was analyzed by colony formation assay. The radiosensitization effects of isoalantolactone on cell proliferation, cell cycle and apoptosis regulation were examined by BrdU incorporation assay, DNA content assay and flow cytometry, respectively. Western blotting was performed to determine the effects of isoalantolactone combined with radiation on the protein expression of Mek, extracellular signal-regulated kinase (Erk1/2) as well as phosphorylated Mek and Erk1/2. Erk1/2 knockdown by siRNA was used to confirm that isoalantolactone specifically inhibited the activation of Erk1/2 signaling pathway in UMSCC-10A cells.ResultsIsoalantolactone enhanced the radiosensitivity of UMSCC-10A cells; the sensitivity enhanced ratios (SERs) were 1.44 and 1.63, respectively, for 2.5 and 5 μM. Moreover, isoalantolactone enhanced radiation-induced cell proliferation and apoptosis and cell cycle arrested at G2/M phase. Furthermore, no marked changes were observed in the expression of total Erk1/2 and Mek protein after radiation treatment. However, isoalantolactone was significantly reduced radiation-induced the phosphorylation of Erk1/2, whereas it altered the phosphorylation of Mek to a lesser extent. In addition, the radiosensitivity of UMSCC-10A cells with Erk1/2 knockdown was increased. Isoalantolactone cannot further prevent the proliferation of UMSCC-10A cells with Erk1/2 knockdown which other mechanism regulated cell proliferation.ConclusionOur results suggested that isoalantolactone enhanced radiation-induced apoptosis, cell cycle arrested and reduced the cell proliferation of UMSCC-10A cells via specifically inhibited the phosphorylation of Erk1/2. Thus a low concentration of isoalantolactone may be used to overcome the resistance of UMSCC-10A cells to radiation and may be a promising radiosensitizer in cancer therapy.

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting.

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

Reproduction at the extremes: pseudovivipary, hybridization and genetic mosaicism inPosidonia australis(Posidoniaceae)

Organisms occupying the edges of natural geographical ranges usually survive at the extreme limits of their innate physiological tolerances. Extreme and prolonged fluctuations in environmental conditions, often associated with climate change and exacerbated at species' geographical range edges, are known to trigger alternative responses in reproduction. This study reports the first observations of adventitious inflorescence-derived plantlet formation in the marine angiosperm Posidonia australis, growing at the northern range edge (upper thermal and salinity tolerance) in Shark Bay, Western Australia. These novel plantlets are described and a combination of microsatellite DNA markers and flow cytometry is used to determine their origin. Polymorphic microsatellite DNA markers were used to generate multilocus genotypes to determine the origin of the adventitious inflorescence-derived plantlets. Ploidy and genome size were estimated using flow cytometry. All adventitious plantlets were genetically identical to the maternal plant and were therefore the product of a novel pseudoviviparous reproductive event. It was found that 87 % of the multilocus genotypes contained three alleles in at least one locus. Ploidy was identical in all sampled plants. The genome size (2 C value) for samples from Shark Bay and from a separate site much further south was not significantly different, implying they are the same ploidy level and ruling out a complete genome duplication (polyploidy). Survival at range edges often sees the development of novel responses in the struggle for survival and reproduction. This study documents a physiological response at the trailing edge, whereby reproductive strategy can adapt to fluctuating conditions and suggests that the lower-than-usual water temperature triggered unfertilized inflorescences to 'switch' to growing plantlets that were adventitious clones of their maternal parent. This may have important long-term implications as both genetic and ecological constraints may limit the ability to adapt or range-shift; this seagrass meadow in Shark Bay already has low genetic diversity, no sexual reproduction and no seedling recruitment.

Genomic DNA Copy Number Aberrations, Histological Diagnosis, Oral Subsite and Aneuploidy in OPMDs/OSCCs.

Oral potentially malignant disorders (OPMDs) characterized by the presence of dysplasia and DNA copy number aberrations (CNAs), may reflect chromosomal instability (CIN) and predispose to oral squamous cell carcinoma (OSCC). Early detection of OPMDs with such characteristics may play a crucial role in OSCC prevention. The aim of this study was to explore the relationship between CNAs, histological diagnosis, oral subsite and aneuploidy in OPMDs/OSCCs. Samples from OPMDs and OSCCs were processed by high-resolution DNA flow cytometry (hr DNA-FCM) to determine the relative nuclear DNA content. Additionally, CNAs were obtained for a subset of these samples by genome-wide array comparative genomic hybridization (aCGH) using DNA extracted from either diploid or aneuploid nuclei suspension sorted by FCM. Our study shows that: i) aneuploidy, global genomic imbalance (measured as the total number of CNAs) and specific focal CNAs occur early in the development of oral cancer and become more frequent at later stages; ii) OPMDs limited to tongue (TNG) mucosa display a higher frequency of aneuploidy compared to OPMDs confined to buccal mucosa (BM) as measured by DNA-FCM; iii) TNG OPMDs/OSCCs show peculiar features of CIN compared to BM OPMDs/OSCCs given the preferential association with total broad and specific focal CNA gains. Follow-up studies are warranted to establish whether the presence of DNA aneuploidy and specific focal or broad CNAs may predict cancer development in non-dysplastic OPMDs.

Flow Cytometric DNA Analysis and Histopathologic Re-Evaluation of Paraffin Embedded Samples from Hydatidiform Moles and Hydropic Abortions.

Distinction of hydatidiform moles (HMs) from non-molar abortions and sub-classification of HMs are important for clinical practice; yet, diagnosis based solely on morphology is affected by interobserver variability. The objective of this study was to determine the role of DNA flow cytometry in distinguishing molar from non-molar pregnancies. This retrospective study was conducted at the Department of Pathology, Women's Hospital, Tehran University of Medical Sciences, Tehran, Iran, between 2006 and 2010. DNA ploidy analysis and histopathologic re-evaluation were performed on paraffin-embedded tissue from 36 (17 complete and 19 partial) molar and 24 hydropic abortus (HA) cases which were previously diagnosed based on histomorphologic study. Of the 17 cases initially diagnosed as complete HM (CHM), 9 were diploid, 2 were triploid, 5 were tetraploid and 1 was aneuploid. Of the 19 initial partial HMs (PHMs), 2, 8, 1 and 8 cases were diploid, triploid, tetraploid and aneuploid, respectively. In the initial HA category (n=24), 14 diploid, 1 triploid, 5 tetraploid, and 4 aneuploid cases existed. Following flow cytometry and histopathologic reevaluation, 1 case with previous diagnosis of HA was reclassified as PHM, 2 initial PHMs were reclassified as CHM and 2 initial CHMs were categorized as PHM. The results show that correct diagnosis of PMH is the main challenge in histological diagnosis of gestational trophoblastic disease (GTD). DNA flow cytometric analysis could be an informative supplement to the histological interpretation of molar and hydropic placentas.

Upregulation of the MCL-1S protein variant following dihydroartemisinin treatment induces apoptosis in cholangiocarcinoma cells.

The aim of the present study was to determine whether dihydroartemisinin (DHA) induces apoptosis in the human cholangiocarcinoma QBC939 cell line through the regulation of myeloid cell leukemia-1 (MCL-1) expression. The inhibitory rates of DHA on QBC939 cell proliferation and the effects of DHA on the cell death rates at various DHA concentrations and following various treatment times were examined. The rate of apoptosis and cell cycle changes following DHA treatment were examined and the changes in the expression of MCL-1 mRNAs and MCL-1 proteins following DHA treatment were also examined. The MTT assay and trypan blue staining demonstrated that DHA significantly inhibited the proliferation (P<0.05) and promoted the death of QBC939 cells (P<0.05). The DNA ladder assay and flow cytometry (FCM) analysis demonstrated that the rate of apoptosis in the experimental group was significantly increased following DHA treatment (P<0.01). FCM analysis also demonstrated that DHA treatment led to a reduction in the percentage of QBC939 cells in the G0/G1 and G2/M phases, and the majority of the DNA-treated cells were arrested in the S phase of the cell cycle (P<0.01). Western blot analysis demonstrated that DHA treatment significantly upregulated the expression of the pro-apoptotic MCL-1S protein. In contrast, no significant difference in the expression of the anti-apoptotic MCL-1L protein was observed following DHA treatment. DHA affected the expression of the apoptosis-associated protein MCL-1 through multiple mechanisms. DHA treatment increased the ratio of MCL-1S/MCL-1L protein, thus inducing apoptosis in cholangiocarcinoma cells.

E. adenophorum Induces Cell Cycle and Apoptosis of Renal Cells through Mitochondrial Pathway and Caspase Activation in Saanen Goat.

The cytotoxicity effects of E. adenophorum on cell cycle and apoptosis of renal cells in Saanen goat was evaluated by TUNEL, DAPI, AO/EB staining, DNA fragmentation assay, Caspase activity, Western-blot, qRT-PCR and flow cytometry analysis. 16 saanen goats randomly divided into four groups were fed on 0%, 40%, 60% and 80% E. adenophorum diets. The Results showed that E. adenophorum induced typical apoptotic features of renal cells. E. adenophorum significantly suppressed renal cells viability, caused cell cycle activity arrest and induced typical apoptotic features in a dose-dependent manner. However, the protein levels of Fas/FasL, Bid and caspase-8 did not appear significant changes in the process of E. adenophorum-induced apoptosis. Moreover, E. adenophorum administration slightly decreased Bcl-2 expression, promoted Bax translocation to mitochondria, triggered the release of Cyt c from mitochondria into cytosol and activated caspase-9, -3, and cleaved PARP. The mitochondrial p53 translocation was significantly activated, accompanied by a significant increase in the loss of ΔΨm, Cyt c release and caspase-9 activation. Above all, these data suggest that E. adenophorum induces renal cells apoptosis via the activation of mitochondria-mediated apoptosis pathway in renal cells. These findings may provide new insights to understand the mechanisms involved in E. adenophorum-caused cytotoxicity of renal cells.

Anti-proliferative Activities of Metallic Nanoparticles in an in Vitro Breast Cancer Model.

To investigate effect of metallic nanoparticles, silver (AgNPs) and gold nanoparticles (AuNPs) as antitumor treatment in vitro against human breast cancer cells (MCF-7) and their associated mechanisms. This could provide new class of engineered nanoparticles with desired physicochemical properties and may present newer approaches for therapeutic modalities to breast cancer in women. A human breast cancer cell line (MCF-7) was used as a model of cells. Metallic nanoparticles were characterized using UV-visible spectra and transmission electron microscopy (TEM). Cytotoxic effects of metallic nanoparticles on MCF-7 cells were followed by colorimetric SRB cell viability assays, microscopy, and cellular uptake. Nature of cell death was further investigated by DNA analysis and flow cytometry. Treatment of MCF-7 with different concentrations of 5-10nm diameter of AgNPs inhibited cell viability in a dose-dependent manner, with IC50 value of 6.28μM, whereas treatment of MCF-7 with different concentrations of 13-15nm diameter of AuNPs inhibited cell viability in a dose-dependent manner, with IC50 value of 14.48μM. Treatment of cells with a IC50 concentration of AgNPs generated progressive accumulation of cells in the S phase of the cell cycle and prevented entry into the M phase. The treatment of cells with IC50 concentrations of AuNPs similarly generated progressive accumulation of cells in sub-G1 and S phase, and inhibited the entrance of cells into the M phase of the cell cycle. DNA fragmentation, as demonstrated by electrophoresis, indicated induction of apoptosis. Our engineered silver nanoparticles effectively inhibit the proliferation of human breast carcinoma cell line MCF-7 in vitro at high concentration (1000 μM) through apoptotic mechanisms, and may be a beneficial agent against human carcinoma but further detailed study is still needed.

Effects of Physalis peruviana L on Toxicity and Lung Cancer Induction by Nicotine Derived Nitrosamine Ketone in Rats.

Nicotine-derived nitrosamine ketone (NNK) is considered a key tobacco smoke carcinogen inducing lung tumors. Physalis peruviana L (harankash) is considered one plant with marked health benefits. This study aimed to evaluate Physalis peruviana L effect on the toxic effect of NNK induced lung cancer in the rats by using pulmonary histopathological, immunohistochemical and DNA flow cytometric analyses. Sixty adult male rats were divided into four groups, each consisting of fifteen animals. The first group received saline, the second received two successive toxic doses of NNK only while the third received two successive toxic doses of NNK with a single daily dose of Physalis peruviana L. The fourth group received a single daily dose of Physalis peruviana L only. Toxic doses of NNK induced hyperplasia and adenocarcinoma in the lung and positive immunoreactivity for Ki-67 and p53 staining with disturbance of the lung DNA content. Administration of Physalis peruviana L with NNK led to a mild pulmonary hyperplasia and weak expression of Ki-67 and p53 with an improvement in the lung DNA content. Physalis peruviana L may protect against NNK induced lung carcinogenesis due to its antioxidant and anti-proliferative effects.

Mitochondrial OGG1 protects against PM2.5-induced oxidative DNA damage in BEAS-2B cells

The enzyme 8-oxoguanine glycosylase 1 (OGG1) has been shown to be involved in the repair of oxidative DNA damage. However, the effect of OGG1 on oxidative DNA damage caused by particulate matter 2.5 (PM2.5) is unknown. Herein, we demonstrated that OGG1 could inhibit the generation of ROS and alleviate mitochondrial dysfunction and increased the expression of IL-1β caused by PM2.5. The dichlorodihydrofluorescein diacetate (DCFH-DA) staining and 5,5′,6,6′-tetrachloro-1,1′,3,3′-.tetraethylbenzi-midazolylcarbocyanine iodide (JC-1) staining using flow cytometry showed that PM2.5 induces the generation of ROS and leads to a reduction in mitochondrial membrane potential (MMP) in BEAS-2B cells. Overexpression of OGG1 inhibited the generation of ROS and the decline in MMP. Knockdown of OGG1 by RNA interference (RNAi) increased the generation of ROS and reduced the MMP. Real-time quantitative PCR (RT-qPCR) for the mitochondrial DNA copy number (mtDNAcn) and flow cytometry for apoptosis revealed that OGG1 inhibits the apoptosis and decreases mtDNAcn induced by PM2.5. Additionally, the results of the comet assay showed that OGG1 had a significant repair effect on DNA strand breaks caused by PM2.5. Overexpression of OGG1 also significantly suppressed the expression of IL-1β caused by PM2.5. Together, these data suggest that PM2.5 leads to mitochondrial dysfunction and the up-regulation of IL-1β could be reversed by overexpression of OGG1. The mitochondrial dysfunction caused by PM2.5 could be relieved by OGG1. Thus, the base excision repair enzyme OGG1 may alleviate mitochondrial dysfunction caused by PM2.5 involved in the expression of IL-1β.

Abstract 5539: Antiproliferative and apoptotic effects of some African natural products in solid and hematopoietic malignant cell lines

Abstract Background: Natural products have played a very important role in health care from time immemorial in curation and prevention of diseases. Antiproliferative and apoptotic effects of extracts of some African natural products like Hymelocardia acida, Moringa oleifera leaves as well as Pleurotus pulmonarius mushroom metabolites were studied in a panel of solid and hematopoietic malignant derived cell lines such as SUIT2-007, MDA-MB 231, SW480, K562 and HL60 cells. Methods: MTT assay was used to determine the growth inhibition of respective cells in vitro. Apoptosis of the cells was demonstrated by fluorescence microscopy and Hoechst 33342 staining, DNA ladder formation assay and flow cytometry with propidium iodide staining. Results: MTT results showed that extracts of Hymelocardia acida stem, Moringa oleifera leaves as well as Pleurotus pulmonarius metabolites could inhibit the proliferation of SUIT2-007, MDA-MB 231, SW480, K562 and HL60 cells in a time and dose dependent manner. Our results show that the ethanol extract of Hymelocardia acida stem had a positive effect and induced apoptosis as shown by fluorescence microscopy and flow cytometry assay. Apoptotic effects were demonstrated by nuclear staining with Hoechst showing clear signs for nuclear fragmentation in cells treated with Hymelocardia acida stem and Moringa oleifera leaves extracts. FACS analysis with propidium iodide showed that the sub G1 fraction increased in treated cells. DNA ladder formation, however, showed no clear DNA fragmentation, which does not exclude a clearer picture with isolated compounds. Conclusion: The present study indicates that the extracts studied can effectively inhibit proliferation and induce apoptosis in cells of panel of tumor cell lines investigated. Citation Format: Olufemi E. Akanni, Martin R. Berger. Antiproliferative and apoptotic effects of some African natural products in solid and hematopoietic malignant cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5539. doi:10.1158/1538-7445.AM2015-5539

Abstract 4505: Activity and avascular penetration of FTY720 (fingolimod) and its non-immune suppressant analogue (OSU2S) within three dimensional tissue culture model of colorectal cancer

Abstract Fingolimod (FTY-720) and its non-immunosuppressive analogue (OSU2S) possess anticancer activity against hepatocellular carcinoma attributed to interference with protein kinase Cδ (PKCδ) signaling. Avascular penetration represents a major limitation for the treatment of solid tumors nonetheless colorectal cancer. Herein, we are assessing the differential antiproliferative activity of OSU2S compared to FTY720 against multicellular spheroids of HCT-116 and HT-29 colorectal cancer cell lines. In addition, we are comparing the avascular penetration properties of OSU2S to FTY720 within the avascular multicellular layers model. The IC50's of FTY720 and OSU2S were calculated in monolayer and spheroid culture models of HCT-116 and HT-29 cells using Emax model. Generally, OSU2S was found to be 1.5 to 3 folds more potent than FTY720 in both cell lines using monolayer and spheroid culture systems. The ratios between the IC50's of FTY720 in spheroid compared to monolayer cultures of HCT-116 and HT-29 cells were found to be to 5.7 and 3.4 folds, respectively. On the other hand, the ratios between the IC50's of OSU2S in spheroid compared to monolayer cultures of HCT-116 and HT-29 cells were found to be 4.5 and 3.4 folds, respectively. This might be attributed to avascular penetration problem for OSU2S and FTY720 in colorectal cancer. The pre-calculated IC50's of FTY720 and OSU2S induced significant increase in the cleaved fraction of PKCδ in both monolayer and spheroid model in HCT-116 and HT-29 cells. The influence of FTY720 and OSU2S on cell cycle analysis was studied using DNA flow cytometry analysis. Both FTY720 and OSU2S changed the cell cycle distribution of both HCT-116 and HT-29 cell lines in the monolayer culture and to a lesser extent in the spheroid culture model. The avascular penetration of OSU2S through multicellular layers of HCT-116 was 1.9 and 1.2 fold faster than FTY720 after 12 and 24 h of penetration, respectively. Also, OSU2S was faster than FTY720 in penetrating multicellular layers of HT-29 by 2.1 to 1.7 folds after 12 and 24 h of penetration, respectively. In conclusion, OSU2S possess better antiproliferative and avascular penetration profile within colorectal cancer cells compared to FTY720. Citation Format: Samir A. Zaahkouk, Serag Eldin I. Elbehairi, Aly F. El-Sayed, Sayed Bakry, Alaa Khedr, Hany A. Omar, Ahmed M. Al-Abd. Activity and avascular penetration of FTY720 (fingolimod) and its non-immune suppressant analogue (OSU2S) within three dimensional tissue culture model of colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4505. doi:10.1158/1538-7445.AM2015-4505

Abstract 1734: Preclinical study of HSP-90 inhibitor drug, AUY922 showed good efficacy in treatment of nasopharyngeal cancer

Abstract Introduction: Prognosis of patients with metastatic or recurrent nasopharyngeal cancer (NPC) is usually poor. Despite some good response to chemotherapy by cisplatin-containing regimen, long term remission in NPC patients is not common. Response rate to second line drug after failing cisplatin is poor. Heat shock protein-90 (HSP-90) is a molecular chaperone essential to the folding of a wide variety of oncogenic client proteins. Inhibition of HSP-90 can lead to a combinatorial blockage of signal transduction leading to cancer cell death. Here, we reported a successful preclinical study showing good efficacy of an anti-HSP-90 drug, AUY922 in treating NPC in cell culture in vitro and xenograft model in vivo in nude mice. Experimental Design: WST assay was used to determine the growth inhibitory dose of AUY922 in 3 NPC cell lines (C666, HONE1 &amp; CNE2) and 2 cisplatin resistant NPC cell lines (HONE1-EBV-Cisp-Res &amp; HK1-LMP1-Cisp-Res). Subcutaneous NPC xenografts was treated by AUY922 i.p. to examine tumor growth inhibition. DNA flow cytometric analysis (DNA-FCM) and Yo-Pro staining were carried out to analyze the effect of AUY922 in cell cycle progression and apoptosis. RT-PCR and Western blotting were performed to examine the effects of AUY922 in regulating Epstein-Barr virus (EBV) gene transcripts and kinase pathway phosphorylation respectively. Results: AUY922 (at GI-50 of 22 nM for C666) could effectively induce tumor growth inhibition in all the 3 NPC cell lines with 300-900 and 180-2700 folds of higher sensitivity than cisplatin or 5-FU. NPC xenograft regression was accomplished by AUY922 treatment at 35-65 mg/kg alone with additive effect when combined with cisplatin. Importantly, AUY922 was also growth inhibitory in the 2 cisplatin resistant NPC cell lines in vitro or in vivo hinting its potential use in the clinical setting in patients refractory to cisplatin. By DNA-FCM, AUY922 could induce prominent G2 cell cycle phase arrest and sub-G1 apoptotic peak at 60 nM (with low level of early apoptosis by YoPro staining). &amp;gt;95% NPC patients in Hong Kong are EBV positive. AUY922 can dramatically induce the expression of 2 EBV transcripts - BZLF1 (for viral lytic cycle) and LMP2A (associated with cytotoxic T cell lysis) indicating its potential in facilitating tumor lysis through these processes. Western blot delineated substantial regulation of phosphorylation in AKT, mTOR, GSK-3-beta, NF-KappaB, IKK-alpha/beta and IKB-alpha pathways but not in CREB, p38 and PARP pathways. Conclusion: The findings of this preclinical study clearly showed the good potential of this HSP90 inhibitor drug, AUY922 in NPC treatment. Ethics approval is pending to initial clinical trials of AUY922 in patients with metastatic/locally recurrent NPC in Hong Kong. Citation Format: Timothy T.C. Yip, Roger K.C. Ngan, Wai-Tong Ng, Lewis T.C. Chan, William C.S. Tai, Wai-Wai Cheng, Victor W.S. Ma, Kwok-Wai Lo, Ya-Ping Li, Michael B.H. Yang, Eric C.H. Wong, Brigette B.Y. Ma, Nai-Ki Mak, George S.W. Tsao, Maria Li-Lung. Preclinical study of HSP-90 inhibitor drug, AUY922 showed good efficacy in treatment of nasopharyngeal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1734. doi:10.1158/1538-7445.AM2015-1734

Guggulsterone inhibits human cholangiocarcinoma Sk-ChA-1 and Mz-ChA-1 cell growth by inducing caspase-dependent apoptosis and downregulation of survivin and Bcl-2 expression.

Guggulsterone has recently been reported to demonstrate anti-tumor effects in a variety of cancers. The present study aims to investigate the biological roles and underlying mechanism of the action of guggulsterone in cholangiocarcinoma. The immortalized human cholangiocarcinoma Sk-ChA-1 and Mz-ChA-1 cell lines were treated with various concentrations of the trans isomer of guggulsterone, Z-guggulsterone. Cellular proliferation was determined using the XTT assay. The apoptotic status of cholangiocarcinoma cells was assessed by Hoechst 33258 staining, DNA fragmentation assay and flow cytometry. Specific caspase inhibitor was used to explore the role of caspase in guggulsterone-induced apoptosis. A colorimetric assay was performed to measure the alterations of the activities of caspase-3, -8 and -9. Western blot analysis was used to detect the protein expression of survivin, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein and cleaved poly (adenosine diphosphate-ribose) polymerase (PARP). As revealed by the present data, guggulsterone significantly inhibited the growth of the two human cholangiocarcinoma cell lines by inducing cellular apoptosis. In addition, guggulsterone-induced apoptosis of cholangiocarcinoma cells was demonstrated to be partially inhibited by the caspase inhibitors z-VAD-fmk, z-LEHD-fmk and z-IETD-fmk, accompanied by the activation of caspases-3, -8 and -9, accumulation of cleaved PARP and decreased expression of survivin and Bcl-2. In conclusion, the present study demonstrated that guggulsterone was able to suppress the proliferation of cholangiocarcinoma by inducing caspase-dependent apoptosis and downregulating survivin and Bcl-2.

POLYPLOID INDUCTION OF DENDROBIUM DRACONIS RCHB.F

This study was carried out to find the optimal conditions for in vitro polyploid induction of Dendrobium draconis Rchb.f. Seed-derived protocorms of D. draconis were transferred to Murashige and Skoog (MS) medium containing different concentrations of colchicine (0-0.1%, in v/v) and maintained in the dark for 1, 2, 3 or 4 days at 25±2°C. The results showed that prolonged exposure (4 days) of the protocorms to high concentrations of colchicine (0.1%) produced a substantial reduction in their survival percentage and shoot regeneration capacity. Three-day exposure of the protocorms to 0.05% colchicine was most effective in polyploid induction. Only tetraploids were achieved, as suggested by squash technique. Levels of polyploidy induced in D. draconis were confirmed by DNA content and flow cytometry. The obtained polyploid plants had thicker, dark green leaves with larger guard cells when compared to diploid plants. The number of guard cells per unit area observed for the polyploid plants produced is however lower than that found in diploid plants, which might be due to the increase in the size of the guard cells.

Aneuploidy identifies subsets of patients with poor clinical outcome in grade 1 and grade 2 breast cancer

ObjectiveHistological grade is a well-established prognostic/predictive factor in breast cancer. However, mainly within intermediate categories, patients may have unpredictable outcome. We hypothesised whether ploidy status can distinguish different prognostic groups among breast cancer patients with similar tumour grade. Material and methodsThe study involved 684 patients with invasive breast carcinoma, and median follow-up of 134.5 months. Pathological staging was evaluated according to WHO classification. Tumour differentiation was assessed using the Nottingham grading system. Ploidy was determined prospectively by DNA flow cytometry. Disease-free survival (DFS) and overall survival (OS) were estimated by the Kaplan–Meier method. ResultsThere were 179 (26.2%) deaths and 239 (33.3%) disease recurrences. For grading, tumours were classified as follows: 163 (23.8%) G1, 356 (52.1%) G2 and 165 (24.1%) G3, while 389 (56.9%) tumours presented aneuploidy. Ploidy and grading are strongly associated (P < 0.001). Patients with aneuploid G2 tumours showed worse DFS (P = 0.001) and OS (P < 0.001), as well as those with aneuploid G1 tumours in relation to OS (P = 0.013). When a subset analysis was performed in early breast cancer patients (n = 451) with Stage I/IIA of disease, it remained the same significant associations of aneuploid G1 (to OS) and G2 tumours (to DFS and OS) with unfavourable prognosis. ConclusionsAneuploidy identifies subsets of breast cancer patients with G1 and G2 tumours who showed poor clinical outcome. The finding has therapeutic implications, as these patients are potential candidates to risk-adapted adjuvant therapy.

A piscine birnavirus induces inhibition of protein synthesis in CHSE-214 cells primarily through the induction of eIF2α phosphorylation.

Inhibition of protein synthesis represents one of the antiviral mechanisms employed by cells and it is also used by viruses for their own propagation. To what extent members of the Birnaviridae family employ such strategies is not well understood. Here we use a type-strain of the Aquabirnavirus, infectious pancreatic necrosis virus (IPNV), to investigate this phenomenon in vitro. CHSE-214 cells were infected with IPNV and at 3, 12, 24, and 48 hours post infection (hpi) before the cells were harvested and labeled with S35 methionine to assess protein synthesis. eIF2α phosphorylation was examined by Western blot while RT-qPCR was used to assess virus replication and the expression levels of IFN-α, Mx1 and PKR. Cellular responses to IPNV infection were assessed by DNA laddering, Caspase-3 assays and flow cytometry. The results show that the onset and kinetics of eIF2α phosphorylation was similar to that of protein synthesis inhibition as shown by metabolic labeling. Increased virus replication and virus protein formation was observed by 12 hpi, peaking at 24 hpi. Apoptosis was induced in a small fraction (1−2%) of IPNV-infected CHSE cells from 24 hpi while necrotic/late apoptotic cells increased from 10% by 24 hpi to 59% at 48 hpi, as shown by flow cytometry. These results were in accordance with a small decline in cell viability by 24hpi, dropping below 50% by 48 hpi. IPNV induced IFN-α mRNA upregulation by 24 hpi while no change was observed in the expression of Mx1 and PKR mRNA. Collectively, these findings show that IPNV induces inhibition of protein synthesis in CHSE cells through phosphorylation of eIF2α with minimal involvement of apoptosis. The anticipation is that protein inhibition is used by the virus to evade the host innate antiviral responses.

Outcome of "indefinite for dysplasia" in inflammatory bowel disease: correlation with DNA flow cytometry and other risk factors of colorectal cancer.

Dysplasia that develops in the setting of inflammatory bowel disease precedes colorectal cancer (CRC). The category of "indefinite for dysplasia (IND)" is used often in equivocal cases, but its clinical significance remains unclear. Flow cytometric analysis of DNA content (aneuploidy) has shown some promise in stratifying patients into low or high risk of CRC, but there are few reports that have specifically evaluated the outcome of IND. As such, we analyzed a series of 84 IND inflammatory bowel disease patients seen at the University of Washington and Harborview Medical Centers from 2003 to 2013 to determine the outcome of IND. Hospital electronic medical records were further reviewed to correlate outcome with the type of lesion (flat versus polypoid), primary sclerosing cholangitis, active inflammation in the area of IND, and DNA flow cytometric data. The data show that 13% of IND cases were found to have low-grade dysplasia, whereas only 2% of IND cases showed advanced neoplasia (high-grade dysplasia or CRC) after a mean follow-up of 28 months. The risk of neoplasia was not significantly associated with the type of lesion (P = .94 from log-rank test), primary sclerosing cholangitis (P = .94), or active inflammation (P = .41) in this cohort. However, the finding of DNA aneuploidy at baseline IND was predictive of subsequent detection of neoplasia (P = .037). IND patients with abnormal DNA flow cytometric results may warrant more careful follow-up, but conversely, IND in the setting of normal DNA content may require less frequent surveillance colonoscopy.