DOCSLIB.ORG

Epidermal Growth Factor Modifies Cell Cycle Control in A431 Human Squamous Carcinoma Cells Damaged by Ionizing Radiation1

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Epidermal Growth Factor Modifies Cell Cycle Control in A431 Human Squamous Carcinoma Cells Damaged by Ionizing Radiation1 [CANCER RESEARCH 54, 1407-1411. March 15. 1W] Advances in Brief Epidermal Growth Factor Modifies Cell Cycle Control in A431 Human Squamous Carcinoma Cells Damaged by Ionizing Radiation1 Keith R. Laderoute, Walter A. Ausserer, A. Merrill Knapp, Tandora D. Grant, and Robert M. Sutherland Life Sciences Division, SRI /niernational, Menlo Park, California 94025 ¡K.R. L, A. M. K., T. D. G., R. M. S.¡,and Dionex Corporation, Sunnyvale, California 94088 ¡W.A. A.] Abstract characterized in this laboratory (8). The cells were grown without antibiotics in DMEM containing 10% FBS (Gibco BRL Life Technologies, Gaithersburg, Epidermal growth factor (EGF) has been shown to radiosensitize A431 MD) in a 5% CO2 atmosphere at 37°C.Cultures were used within 20 passages and other human squamous carcinoma cells with high numbers of surface after recovery from frozen stocks. Culture grade murine EGF (Collaborative EGF receptors. In this study of the mechanistic basis of EGF-induced Research, Bedford, MA) was used in all EGF experiments. radiosensitization, both EGF and ionizing radiation caused Gt phase ar Irradiation Procedure. Exponentially growing A431 cells were irradiated rests in cycling A431 cells, but only radiation caused a G2-M arrest. in 60- or 100-mm plastic Petri dishes in 4 or 10 ml of medium, respectively, However, EGF enhanced the magnitude of this G2-M arrest, suggesting an by using a 137Cs7-irradiator (Mark I Model 680A; J. Shepherd and Associates, interaction of signaling pathways involved in cellular responses to EGF San Fernando, CA) at a dose rate of 250 cGy/min. All of the experiments and radiation damage. EGF and radiation also uniquely perturbed cyclin described here included untreated control cells, cells treated with EGF only, A and B, mRNA levels during the time of maximum radiation-induced cells exposed to radiation only, and cells exposed to radiation followed by the G2-M arrest. The effects of EGF on G2-M events probably originated in addition of EGF. cells in G|. It is possible that aberrant EGF signal transduction in human Cell Cycle Distribution Studies. At various times after treatment, cells squamous carcinoma cells may be exploited as a novel strategy for were trypsinized, fixed in 70% ethanol/PBS, and stored at 4°C.Fixed cells radiotherapy. were analyzed by flow cytometry within 2 weeks of storage. Two h before analysis, 1 X 10'' cells were resuspended in PBS containing 1 mg/ml RNase for Introduction 30 min at room temperature and then pelleted and resuspended in 10 fig/ml The contribution of eukaryotic signal transduction pathways to the propidium iodide. Propidium iodide fluorescence was measured using a biological effects of ionizing radiation is attracting much interest. Coulter Epics Elite flow cytometer (Hialeah, FL) equipped with an air-cooled Several workers have shown that ionizing radiation can induce both argon laser delivering 15 mW of light at 488 nm. The red fluorescence from 1 X IO4 cells from each sample was collected through a 610 nm bandpass filter. immediate-early and late changes in gene expression in mammalian Multicycle software (Phoenix Flow Systems, Inc., San Diego, CA) was used to cells (1, 2). Research in our laboratory has concentrated on the inter action of EGF-R2 signal transduction with the response of human SC calculate the percentage of cells in the GÃŒ,S,and G2-M phases of the cell cycle. G2 Arrest Experiments. Cell cycle distributions of A431 cells exposed to cells to ionizing radiation. EGF-R signaling in these cells has aberrant different treatments were obtained by using both asynchronous and synchro features (3) which may be exploitable therapeutically. We reported nous cultures. Exponentially growing asynchronous cells were exposed to 10 that the addition of EGF to SC cells which overexpress the EGF-R can Gy of radiation, trypsinized, and plated at densities that ensured exponential sensitize the cells to killing by ionizing radiation. EGF-induced growth at various harvest times. Immediately after plating, cells (irradiated and radiosensitization occurred when EGF was present after irradiation nonirradiated) for EGF experiments were given EGF to a concentration of 50 during the colony formation period of a clonogenic assay (4—6).The ng/ml (7.8 HM).Cells were harvested and processed for flow cytometry at 24, sensitization was independent of the growth inhibition commonly 48, and 72 h after plating. To synchronize A431 cultures by the double thy- observed when cell lines expressing high levels of the EGF-R are midine block method, 6.8 X IO5 cells/100-mm Petri dish (8- and 24-h time points) or 3.4 X 10s cells/100-mm Petri dish (48-h time point) were plated in treated with EGF (6). In addition, the sensitization did not involve DMEM/10% FBS containing 3 ITIMthymidine. After incubation at 37°Cfor 16 enhancement of DNA single strand breaks or inhibition of potentially h, the medium was replaced with DMEM/10% FBS, and the cells were incu lethal damage repair or sublethal damage repair (4). Because cell bated for another 8 h at 37°C.The medium was then replaced with DMEM/ cycle analysis suggested that the cell cycle distribution of A431 cells 10% FBS containing 3 HIMthymidine, and the incubation was continued for 16 may be a factor in EGF-induced radiosensitization (7), we investi h at 37°C.At this time, the block was released and cells were exposed to 10 Gy gated this possibility further. The findings presented here show that of radiation. Immediately after irradiation, EGF-treated cells (irradiated and EGF stimulation of the EGF-R in irradiated A431 cells enhanced the nonirradiated) were given EGF to a concentration of 50 ng/ml. All the cells classical radiation-induced G2-M arrest or checkpoint and both EGF were incubated at 37°Cfor 8, 24, and 48 h. At each time, the cells were washed and radiation contributed to a G, arrest. EGF and radiation also caused once with ice-cold PBS and processed for flow cytometry. unique perturbations of the levels of cyclin A and cyclin B, mRNA in GI Arrest Experiments. Exponentially growing asynchronous A431 cells A431 cells. were exposed to 10 Gy of radiation and cells (irradiated and nonirradiated) for EGF experiments were given EGF to a concentration of 50 ng/ml. Vinblastine Materials and Methods was added to a concentration of 1 fig/ml at 0, 24, and 48 h after the radiation/ EGF protocol to prevent the repopulation of G, phase by cells exiting G2 Cell Culture. The A431 human squamous carcinoma cell line was origi phase. After the addition of vinblastine, the cells were allowed to exit G! for nally obtained from the American Type Culture Collection and has been 24 h and then processed for flow cytometry. Control cells were not treated with vinblastine and were harvested at the start of the vinblastine incubation period to determine the "percentage of cells in G, at the start of vinblastine treatment". Received 12/2/93; accepted 2/4/94. Data are presented as "percentage of G! arrest," defined as The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Ihis fact. % of cells in G, after 24 h of vinblastine treatment 1This work was supported by NIH/National Cancer Institute Grants CA 37618 and CA X 100 20329. % of cells in G, at the start of vinblastine treatment 2 The abbreviations used are: EGF-R. epidermal growth factor receptor; SC, squamous carcinoma; DMEM. Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, Mitotic Index Experiments. Exponentially growing A431 cells were irra phosphate-buffered saline. diated (2.5 or 10 Gy) followed immediately by the addition of EGF to a 1407 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1994 American Association for Cancer Research. CELL CYCLE EFFECTS OF EOF IN IRRADIATED A431 CELLS concentration of 50 ng/ml. At various times after treatment, the cells were trypsinized, swelled in 0.75 HIMKC1, fixed in methanol:acetic acid (3:1), and stained with Giemsa stain. The percentage of cells with condensed chromo somes was determined by scoring at least 3000 cells/time point. Northern Analysis. Exponentially growing A431 cells were exposed to 10 Gy of radiation followed immediately by the addition of EOF to a concentra tion of 50 ng/ml. At 8, 10, 12, and 14 h after treatment, total RNA was iso lated by using the guanidinium isothiocyanate method, purified by CsCl ul- tracentrifugation, resolved on 1% agarose/formaldehyde gels, and blotted onto nitrocellulose membranes. The blots were probed with approximately 1.6 kilobase complementary DNA fragments of human cyclins A and BI (ob tained from Dr. Tony Hunter, Salk Institute for Biological Studies, San Di ego, CA) labeled with [32P]dCTP (Dupont NEN, Boston, MA) by the random primer technique (Amersham, Arlington Heights, IL). To provide a normal ization standard for RNA loading, blots were stripped and reprobed with a DNA oligomer corresponding to a human 28S rRNA sequence (Clontech Laboratories, Palo Alto, CA) end-labeled with [32P]ATP (Amersham). 24 h 48 h 72 h 1 lUnt £2) EGF EGF+Rad Results EGF Enhanced the G2-M Arrest Caused by y-Irradiation of A431 Cells. A431 cells given high doses of ionizing radiation re sponded by arresting at a G2-M checkpoint, similar to the response of most eukaryotic cells subjected to substantial radiation damage. The results presented in Fig. L4 for asynchronous, exponential A431 cells exposed to 10 Gy of y-radiation show that this G2-M arrest persisted 100 for at least 72 h after irradiation.
Load more

Recommended publications
  • Isolation and Identification of a Distinct Side Population Cancer Cells in The
    Arch Dermatol Res (2011) 303:181–189 DOI 10.1007/s00403-010-1100-1 ORIGINAL PAPER Isolation and identification of a distinct side population cancer cells in the human epidermal squamous cancer cell line A431 Songmei Geng • Qianqian Wang • Jianli Wang • Zhishang Hu • Chunchun Liu • Junkang Qiu • Weihui Zeng Received: 22 July 2010 / Revised: 14 November 2010 / Accepted: 22 November 2010 / Published online: 15 January 2011 Ó Springer-Verlag 2011 Abstract Side population (SP) cells have been suggested Keywords Side population Á Squamous cell carcinoma Á to be multipotent cancer stem cells. To address whether SP A431 cells exist in epidermal squamous cancer cell line A431, A431 cells dyed with Hoechst 33342 were sorted through flow cytometry. The SP cells were then analyzed by colony- Introduction forming and cell proliferation assay. Further, tumorigenic- ity and microarray analysis were used to compare biological Solid tumors are characterized by the presence of poorly difference between SP and non-SP (NSP) cells. Our results differentiated cells that originate from the affected tissue showed that SP cells existed in the A431 cell line, showing and are presumably responsible for the long-term tumor higher proliferating and colony-forming ability than NSP maintenance, renewal, and drug resistance. Cells with these cells. Tumors generated from SP cells were larger than properties have been termed as cancer stem cells (CSC) or those from the NSP cells in NOD/SCID mice. The mRNA tumor-initiating cells (TIC) [11, 24]. The CSC hypothesis microarray profiling revealed that five cancer marker gene suggests that only the CSC within the tumor can self-renew expressions were up-regulated and one tumor suppressor and proliferate extensively to form new tumors, whereas gene expression was down-regulated.
    [Show full text]
  • Influence of Gefitinib and Erlotinib on Apoptosis and C-MYC Expression in H23 Lung Cancer Cells
    ANTICANCER RESEARCH 33: 1547-1554 (2013) Influence of Gefitinib and Erlotinib on Apoptosis and c-MYC Expression in H23 Lung Cancer Cells MITSUHIRO SUENAGA1, MASATATSU YAMAMOTO2, SHO TABATA2, SUSUMU ITAKURA1, MASAAKI MIYATA1, SHUICHI HAMASAKI1 and TATSUHIKO FURUKAWA2 Departments of 1Cardiovascular, Respiratory and Metabolic Medicine, and 2Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan Abstract. Background: Gefitinib and erlotinib are inhibitors of mitogen-activated protein kinase (MAPK) cascade via of epidermal growth factor receptor tyrosine kinase. The RAS. Although theoretically a RAS mutation would attenuate effects of these tyrosine kinase inhibitors on RAS-mutated the therapeutic effect of EGFR inhibition, the effects of TKI cancer cells are unclear. Materials and Methods: Influence on the cells have been controversial. If these TKIs have any of gefitinib and erlotinib treatment was examined in H23 anticancer effects even at a high dose, this might provide a adenocarcinoma and A431 epidermoid carcinoma cells. The clue to evaluate for new anticancer targets. These TKIs have WST-1 assay was performed for evaluating cell growth. The advantages in that they have low adverse effects and simple phosphorylation status of extracellular-signal-regulated actions on kinases, making them of use as molecular tools to kinases (ERK) and AKT (protein kinase B) was examined by search for new molecular targets. western blot. Flow cytometry was used for analyzing cell- In addition to the oncogenic changes in cancer cells, the cycle status and apoptosis detection. Results: In H23 cells, status of TP53 tumor suppressor gene affects target therapy. 20 μM erlotinib suppressed growth, while gefitinib did not The genetic status of RAS and TP53 influences cell suppress proliferation after 48 h of treatment.
    [Show full text]
  • Establishment of a Cell Line from Conjunctival Squamous Cell Carcinoma: Peca-Ukhb-01
    Anatomy and Pathology/Oncology Establishment of a Cell Line From Conjunctival Squamous Cell Carcinoma: PeCa-UkHb-01 Henning Thomasen,1 Bettina Muller,¨ 1 Micaela Poetsch,2 Klaus-Peter Steuhl,1 and Daniel Meller1 1Department of Ophthalmology, University of Duisburg-Essen, Essen, Germany 2Institute of Legal Medicine, University of Duisburg-Essen, Essen, Germany Correspondence: Daniel Meller, PURPOSE. Until now, no epithelial cell line from conjunctival squamous cell carcinoma (SCC), Department of Ophthalmology, to our knowledge, has existed; therefore, the establishment of a model cell line would be a University of Duisburg-Essen, useful tool for further studies. In particular, the phenotypic and molecular characterization in Hufelandstraße 55, 45122 Essen, comparison to other SCC cells is of high interest because this would enable the development Germany; of new treatment options for clinical application in ophthalmic oncology. [email protected]. HT and BM are joint first authors. METHODS. Epithelial cells were isolated from a bulbar conjunctiva SCC obtained from a 74-year- old male, harvested by stepwise trypsinization and named PeCa-UkHb-01. Cell doubling and Submitted: October 16, 2014 the number of passages were determined. Short tandem repeats (STR) and karyotype analyses Accepted: May 20, 2015 were performed. Semiquantitative real-time PCR and immunocytochemical fluorescence Citation: Thomasen H, Muller¨ B, staining were carried out to detect tumor and epithelial cell markers. Poetsch M, Steuhl K-P, Meller D. Establishment of a cell line from RESULTS. The cells had an epithelial and conjunctival phenotype. They grew above passage conjunctival squamous cell carcino- number 50 in a doubling time at approximately 34.5 hours.
    [Show full text]
  • Extracellular ATP Is a Mitogen for 3T3, 3T6, and A431 Cells and Acts
    Proc. Nati. Acad. Sci. USA Vol. 86, pp. 7904-7908, October 1989 Cell Biology Extracellular ATP is a mitogen for 3T3, 3T6, and A431 cells and acts synergistically with other growth factors (purinoceptor/competence factor/ectoenzymes) NINGNA HUANG, DINGJI WANG, AND LEON A. HEPPEL Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853 Contributed by Leon A. Heppel, July 3, 1989 ABSTRACT Extracellular ATP in concentrations of 5-50 increased formation of inositol phosphates (16). In addition, gLM displayed very little mitogenic activity by itself but it we observed a stimulation ofthe rate ofNa+, K+, and uridine caused synergistic stimulation of [3H]thymidine incorporation entry, and enhanced ornithine decarboxylase activity (un- in the presence of phorbol 12-tetradecanoate 13-acetate, epi- published observations). Other workers have reported that dermal growth factor, platelet-derived growth factor, insulin, extracellular ATP activates similar early signals in hepato- adenosine, or 5'-(N-ethyl)carboxamidoadenosine. Cultures of cytes (17-20), Ehrlich ascites tumor cells (21-23), mouse Swiss 3T3, Swiss 3T6, A431, DDT1-MF2, and HFF cells were macrophages (24), H35 hepatoma cells (25), endothelial cells used. The percent ofcell nuclei labeled with ['H]thymidine and (26-30), and turkey erythrocytes (31). cell number were also increased. ADP was equally mitogenic, These findings raise the possibility that exogenous ATP while UTP and ITP were much less active. The effect of ATP acts as a growth factor, and this was implied in two recent was not due to hydrolysis by ectoenzymes to form adenosine, a papers (23, 24), where ATP was compared to "more con- known growth factor.
    [Show full text]
  • Antitumor Effects of Dioscin in A431 Cells Via Adjusting ATM/P53‑Mediated Cell Apoptosis, DNA Damage and Migration
    ONCOLOGY LETTERS 21: 59, 2021 Antitumor effects of dioscin in A431 cells via adjusting ATM/p53‑mediated cell apoptosis, DNA damage and migration PENG WANG, CHUN WANG and CHUNYING LIU College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Shenyang, Liaoning 110847, P.R. China Received January 24, 2020; Accepted October 26, 2020 DOI: 10.3892/ol.2020.12321 Abstract. Skin cancer is the deadliest type of malignant Introduction disease and causes primary mortality worldwide. Dioscin, which exists in medicinal plants, has potent anticancer Skin cancer is one of the major causes of cancer‑associated effects. However, its effects on skin cancer remain unknown. mortality in humans, with 232,000 new cases worldwide in 2012, In the present study, the activity and mechanism of dioscin resulting in 55,000 deaths (1) There are two major types of on the human skin cancer A431 cell line were investigated, non‑melanoma skin cancer, named basal cell carcinoma and MTT, colony formation, Transwell, wound‑healing, TUNEL, squamous cell carcinoma (SCC) (1,2). A number of known chem‑ Comet, immunofluorescence and western blot assays were ical carcinogens, such as arsenide, and exposure to hazardous used to assess the effects of dioscin on A431 cells. The results UV rays can cause the development of SCC through mediation of of MTT, colony formation, Transwell and wound‑healing free radical production, resulting in a healthy epithelial cell being assays revealed that dioscin suppressed proliferation, colony transformed in a malignant one (3,4). As the prevalence of skin formation and invasion of the cancer cells. TUNEL and comet cancer is increasing, with skin cancer having the fastest‑growing assays demonstrated that dioscin exhibited significant effects cancer incidence among all malignancies in recent years with an on cell apoptosis and DNA damage.
    [Show full text]
  • Effects of Anti-EGFR Antibody Cetuximab on Androgen-Independent Prostate Cancer Cells
    ANTICANCER RESEARCH 30: 1905-1910 (2010) Effects of Anti-EGFR Antibody Cetuximab on Androgen-independent Prostate Cancer Cells POOJA DHUPKAR1, MELISSA DOWLING2, KEITH CENGEL2 and BIN CHEN1,2 1Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences in Philadelphia, Philadelphia, PA 19104, U.S.A.; 2Department of Radiation Oncology, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, U.S.A. Abstract. Aim: Epidermal growth factor receptor (EGFR) is often used for patients with androgen-independent prostate is a novel molecular target for anticancer therapy. This study cancer. However, the median patient survival time with examined the effects of anti-EGFR antibody cetuximab on current standard docetaxel-based combination chemotherapy two human androgen-independent prostate carcinoma cell is only about 18 months (3), which necessitates new lines, Du145 and PC-3. Materials and Methods: Cell therapeutic approaches for this disease. proliferation was monitored with a trypan blue viability Activation of receptor tyrosine kinase (RTK)-mediated assay. Cell apoptosis and cell cycle profile was evaluated by downstream pathways is known to partially contribute to flow cytometry. The expression of various signaling prostate cancer androgen independence by stimulating tumor molecules was examined by Western immunoblotting. cell proliferation at low androgen levels (2). Epidermal Results: Cetuximab (100 μg/ml) caused a significant growth growth factor receptor (EGFR), the first and most studied inhibition by inducing cell apoptosis in Du145 cells, but not RTK, is overexpressed in about one third of epithelial in PC-3 cells. It caused EGFR down-regulation and inhibited cancers including head and neck, colorectal, breast, ovarian, EGFR Tyr-845 autophosphorylation in both Du145 and PC- prostate, bladder and lung cancer (4).
    [Show full text]
  • Autocrine IL-1Β-TRAF6 Signalling Promotes Squamous Cell
    Oncogene (2013) 32, 747 --758 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Autocrine IL-1b-TRAF6 signalling promotes squamous cell carcinoma invasion through paracrine TNFa signalling to carcinoma-associated fibroblasts SI Chaudhry1,2, S Hooper1, E Nye3, P Williamson4, K Harrington5 and E Sahai1 The invasion of squamous cell carcinoma (SCC) is a significant cause of morbidity and mortality. Here, we identify an E3 ligase, Traf6 and a de-ubiquitinating enzyme, Cezanne/ZA20D1, as important regulators of this process in organotypic models. Traf6 can promote the formation of Cdc42-dependent F-actin microspikes. Furthermore, Traf6 has a key role in autocrine interleukin- 1b signalling in SCC cells, which in turn is required to drive the expression of tumour necrosis factor a (TNFa). TNFa acts in a paracrine manner to increase the invasion-promoting potential of carcinoma-associated fibroblasts (CAFs). Exogenous TNFa signalling can restore invasion in cells depleted of Traf6. In conclusion, Traf6 has two important roles in SCC invasion: it promotes cell intrinsic Cdc42-dependent regulation of the actin cytoskeleton and enables production of the paracrine signal, TNFa, that enhances the activity of CAFs. Oncogene (2013) 32, 747--758; doi:10.1038/onc.2012.91; published online 26 March 2012 Keywords: Traf6; Interleukin-1; TNFa; invasion; carcinoma-associated fibroblast INTRODUCTION cytokine signalling. Both TNFa and IL-1a/b bind to membrane Squamous cell carcinoma (SCC) is an epithelial-derived cancer spanning receptors that then trigger a range of intracellular of variable clinical presentation and histological appearance. signalling pathways. Canonical NFkB signalling is activated by Important tumour-related factors that govern patient prognosis both cytokines.
    [Show full text]
  • Growth Stimulation of A431 Cells Byepidermal Growth Factor
    Proc. Natt Acad. Sci. USA Vol. 80, pp. 1337-1341, March 1983 Cell Biology Growth stimulation of A431 cells by epidermal growth factor: Identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody (hormone receptors/mitogenesis) TOMOYUKI KAWAMOTO, J. DENRY SATO*, ANH LE, JONATHAN POLIKOFF, GORDON H. SATO, AND JOHN MENDELSOHN Cancer Center, Q-058, University of California at San Diego, La Jolla, California 92093 Communicated by Morris Friedkin, November 15, 1982 ABSTRACT Epidermal growth factor (EGF) at 3 nM maxi- for more than 8 hr when occupied by EGF (15). King and Cua- mally inhibits the proliferation of A431 epidermoid carcinoma cells. trecasas also have suggested that the accumulation of stable in- We show that at lower concentrations, in the range of 3-100 pM, tracellular complexes between high-affinity receptors and EGF EGF has a mitogenic effect on A431 cells. In the presence of 100 are involved in growth stimulation, but the role of these high- nM anti-EGF-receptor monoclonal IgG (designated 528), which affinity receptors in mitogenesis remains unclear (16). inhibits A431 cell proliferation and blocks >95% of EGF binding, A431 cells lend themselves to the study of EGF interactions EGF becomes mitogenic for A431 cells at concentrations up to 3 with receptors because of their extremely high number of EGF nM. These results suggest that a minor population of high-affinity receptors (1-3 x 10' per cell) (1, 17, 18). They are atypical in EGF receptors may be involved in stimulation of A431 cell pro- that doses of EGF that are mitogenic in many other cell lines liferation.
    [Show full text]
  • Internalization of NK Cells Into Tumor Cells Requires Ezrin and Leads to Programmed Cell-In-Cell Death
    npg Ezrin functions in NK cell internalization Cell Research (2009) 19:1350-1362. 1350 © 2009 IBCB, SIBS, CAS All rights reserved 1001-0602/09 $ 32.00 npg ORIGINAL ARTICLE www.nature.com/cr Internalization of NK cells into tumor cells requires ezrin and leads to programmed cell-in-cell death Shan Wang1, 2, *, Zhen Guo3, *, Peng Xia3, Tingting Liu1, Jufang Wang1, Shan Li1, Lihua Sun1, Jianxin Lu2, Qian Wen4, Mingqian Zhou4, Li Ma4, Xia Ding5, Xiaoning Wang1, 2, 4, Xuebiao Yao3 1School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510641, China; 2Wenzhou Medical College, Wenzhou 325035, China; 3Anhui Key Laboratory for Cellular Dynamics and Chemical Biology, University of Science and Technology of China, Hefei 230027, China; 4Nanfang Medical University, Guangzhou 510515, China; 5Beijing University of Chi- nese Medicine, Beijing 100086, China Cytotoxic lymphocytes are key players in the orchestration of immune response and elimination of defective cells. We have previously reported that natural killer (NK) cells enter target tumor cells, leading to either target cell death or self-destruction within tumor cells. However, it has remained elusive as to the fate of NK cells after internaliza- tion and whether the heterotypic cell-in-cell process is different from that of the homotypic cell-in-cell event recently named entosis. Here, we show that NK cells undergo a cell-in-cell process with the ultimate fate of apoptosis within tumor cells and reveal that the internalization process requires the actin cytoskeletal regulator, ezrin. To visualize how NK cells enter into tumor cells, we carried out real-time dual color imaging analyses of NK cell internalization into tumor cells.
    [Show full text]
  • 5 Radiosensitivity of A431, CHO, and SK-BR-3 Cell Lines to Low-Intensity
    RADIOSENSITIVITY OF A431, CHO, AND SK-BR-3 CELL LINES TO LOW-INTENSITY BETA RADIATION FROM A SR-90+Y-90 MIXED SOURCE S.S. Soroko*, L.N. Shestakova, A.А. Brilkina, E.K. Akinchits, V.A. Vodeneev, N.Yu. Shilyagina Lobachevsky State University, Gagarin Ave., 23, Nizhny Novgorod, 603950, Russia. * Corresponding author: [email protected] Abstract. Currently, radionuclide therapy of tumors using sources of alpha and beta radiation is actively developing. However, the radiosensitivity of tumor cells has been studied mainly using acute gamma radiation. In this regard, studies aimed at determining the ranges of radiosensitivity of cells of various origins in relation to beta-emission radionuclides are gaining relevance. The study was carried out on A431, CHO and SK-BR-3 cell lines using beta-emission sealed sources Sr-90+Y-90. Cell viability was assessed via MTT-assay. Dose dependences were obtained for irradiating cells with a beta source: the LD50 range was from 17 to 19 Gy, and LD37 was from 24 to 36 Gy. It was shown that at the same dose of radiation, the percentage of viable cells relative to the control of 72 hours after irradiation is significantly less than after 24 hours. The revealed LD50 values for tumor cells under chronic beta-irradiation are higher than with acute gamma-irradiation, which should be considered when selecting doses during the development of potential radiopharma- ceutical treatment. Decreased cell viability in response to beta radiation is due to both cytotoxic and cytostatic manifes- tations. Keywords: radionuclide therapy, Sr-90+Y-90, cell culture, radiosensitivity, cancer, beta radiation.
    [Show full text]
  • Adenovirus-Mediated Wild-Type P53 Radiosensitizes Human Tumor Cells by Suppressing DNA Repair Capacity
    Molecular Cancer Therapeutics 1223 Adenovirus-mediated wild-type p53 radiosensitizes human tumor cells by suppressing DNA repair capacity Nand K. Sah,1 Anupama Munshi,1 Enhancing the antitumor effects by combining radiation Takashi Nishikawa,1 Tapas Mukhopadhyay,2 with other agents often allows lower doses to be used, Jack A. Roth,2 and Raymond E. Meyn1 thereby minimizing side effects. Gene therapy in combi- 1 2 nation with radiation is one such promising strategy (1–6). Departments of Experimental Radiation Oncology and Thoracic It has been estimated that about 50% of all tumors have and Cardiovascular Surgery, University of Texas MD Anderson Cancer Center, Houston, TX mutations in p53, and the p53 pathway may be nonfunc- tional for other reasons in many more. A number of investigators have shown that exogenous expression of Abstract wild-type (wt) p53 sensitizes human tumor cells to Functional inactivation of the p53 gene and robust DNA radiation in vitro and in vivo (7–16). repair capacity may be among the salient causes of radio- p53 is well known for its role in monitoring genomic resistance in tumor cells. We expressed the wild-type (wt) stability. The mechanisms underlying this function of p53 p53 gene in a p53-mutant human epidermoid carcinoma cell are not fully understood (17). Nevertheless, it is known that line, A431, using an adenoviral vector [adenovirus-p53 (Ad- genetic insults activate p53, which in turn induce down- p53), INGN 201], examined its radiosensitivity, and corre- stream repair genes including GADD45, p48XPE, and XPC lated p53 status and radiosensitivity with cellular repair (17–19) that are involved in the nucleotide excision repair functions.
    [Show full text]
  • Tumor Promoter Phorbol 12-Myristate 13-Acetate Inhibits Mitogen-Stimulated Na+/H ÷ Exchange in Human Epidermoid Carcinoma A431 Cells
    Tumor Promoter Phorbol 12-Myristate 13-Acetate Inhibits Mitogen-stimulated Na+/H ÷ Exchange in Human Epidermoid Carcinoma A431 Cells BRIAN WHITELFY, DAN CASSEL, YING-XIN ZHUANG, and LUIS GLASER Department of Biological Chemistry, Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, Missouri 63110. Dr. Cassel's present address is Department of Biology, The Technion, Haifa 32000, Israel. ABSTRACT Addition of polypeptide growth factors to cultured cells results in a rapid stimulation of Na+/H ÷ exchange, which leads to cytoplasmic alkalinization. We studied the effects of the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) on the Na+/H ÷ exchange system of A431 cells. Stimulation of Na+/H + exchange by epidermal growth factor (EGF) and serum as well as by vanadate ions is strongly inhibited after treatment of cells with nanomolar concentrations of PMA. Phorbol esters that have no activity as tumor promotors also do not modulate the activation of Na+/ H ÷ exchange. By contrast, the stimulation of Na+/H ÷ exchange that is produced upon exposure of cells to hypertonic solution is only slightly inhibited by PMA treatment, indicating that PMA treatment does not directly block the activity of the Na+/H + antiporter. Furthermore, incubation of cells with PMA causes a weak stimulation of Na+/H + exchange, although this effect is mostly observed at relatively high PMA concentrations and appears to require external Ca 2+. The inhibition BY PMA of EGF-promoted Na+/H + exchange is not due to inhibition of EGF-binding to the EGF receptor. Since PMA activates protein kinase C, our observations are consistent with the hypothesis that protein kinase C functions to attenuate the stimulation of Na+/H + exchange by polypeptide growth factors.
    [Show full text]