Agonist binding of herg K+ channels inhibits epithelial cell extrusion but not apoptosis in MDCK monolayers; providing possibilities to cancer metastasis regulation

Metastasis is commonly referred to as cancerous cell movement from an original site to one or more sites elsewhere in the body. When these moving cancerous cells are malignant, an individual's chance of survival is decreased. In order for epithelial cells to move they are detached from the epithelium in a process called extrusion. By regulating this extrusion pathway it may be possible to regulate metastasis as well. Unfortunately, compared to the amount of knowledge about apoptosis, or programmed cell death, very little is known about the signals that regulate extrusion. One possible target of extrusionsignaling regulation is HERG K+ Channels, It is hypothesized that HERG channels will prevent extrusion but not apoptosis, thus providing a means of extrusion regulation while still allowing for apoptotic death targeting of tumors. MDCK monolayers were treated with HERG antagonists, MK499 and E-4031, as well as an agonist, PD-118057 and exposed to UV irradiation to induce apoptosis and extrusion. For the first time, we show that increasing the conductivity of HERG inhibits extrusion but not apoptosis. This result provides possibilities of • future strategies for tumor metastasis regulation that have currently been unexplored. These possibilities are very promising because many tumors over express HERG K+ channels that contain a beta isoform, her gib, different from healthy tissues.

AGONIST BINDING OF HERG.K+'CHANNELS INHIBITS EPITHELIAL CELL EXTRUSION BUT NOT APOPTOSIS IN MDCK MONOLAYERS; PROVIDING POSSIBILITIES TO CANCER METASTASIS REGULATION by Patrick D. Loftus A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree of Bachelor of Science In Biomedical Engineering Approved: ' / \jr )dy Rosenblatt Supervisor Patrick Tresco Chair, Biomedical Engineering rtc Eddings Associate Dean, College of Engineering lomas Richmond Interim Dean, Honors College January 201^ ABSTRACT Metastasis is commonly referred to as cancerous cell movement from an original site to one or more sites elsewhere in the body. When these moving cancerous cells are malignant, an individual’s chance of survival is decreased. In order for epithelial cells to move they are detached from the epithelium in a process called extrusion. By regulating this extrusion pathway it may be possible to regulate metastasis as well. Unfortunately, compared to the amount of knowledge about apoptosis, or programmed cell death, very little is known about the signals that regulate extrusion. One possible target of extrusionsignaling regulation is HERG K+ Channels, It is hypothesized that HERG channels will prevent extrusion but not apoptosis, thus providing a means of extrusion regulation while still allowing for apoptotic death targeting of tumors. MDCK monolayers were treated with HERG antagonists, MK499 and E-4031, as well as an agonist, PD-118057 and exposed to UV irradiation to induce apoptosis and extrusion. For the first time, we show that increasing the conductivity of HERG inhibits extrusion but not apoptosis. This result provides possibilities of •future strategies for tumor metastasis regulation that have currently been unexplored. These possibilities are very promising because many tumors over express HERG K+ channels that contain a beta isoform, hergib, different from healthy tissues. TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iii INTRODUCTION 1 METHODS 3 CELL CULTURE 3 IIERG CHANNEL ASSAY 4 CELL STAINING 5 MICROSCOPY AND DATA ANALYSIS 5 RESULTS 6 DISCUSSION 9 ACKNOWLEDGEMENTS 13 REFERENCES 14 iii 1 INTRODUCTION One of the most vital parts of maintaining physical health is tissue and cell homeostasis. The number of epithelial cells that die must match the number of cells that divide in order to sustain the proper function of many adult organs. When this balance is lost and too many cells accumulate, epithelial cancers result. In order for the epithelium to maintain proper barrier function, each dying cell sends signals to its live neighbors, which form an actin and myosin ring to squeeze the dying cell out. This process is called extrusion [1]. The orientation of extruding cells within a monolayer, as well as the location of different membrane proteins, is extremely important in tumors where apoptosis, programmed cell death, is blocked. When a cell extrudes basally, compared to apically, metastasis can be initiated, thus decreasing a cancer patient’s chance of survival [2J. The SEER Cancer Statistics Review published annually by the Cancer Statistics Branch of the National Cancer Institute estimates that in 2009 1,479,350 men and women would be diagnosed with cancer with an estimated 562,340 people dying from the consequences of the disease [3]. The National Institutes of Health estimated a total cost of $228.1 billion in 2008 due to cancer health expenditures and individual loss of productivity [4]. A greater understanding of the cell signaling pathway that leads to extrusion and metastasis may greatly decrease the number of individuals dying each year and reduce the costs of cancer health care. The goal of this study is to further deduce the role of potassium channels, specifically HERG K+ channels, in extrusion and apoptosis as a possible means of targeting for tumor metastasis regulation. Many studies have focused on cell death pathways triggered by a variety of apoptotic stimuli; however, relatively little is known about the signals that induce extrusion [5]~[10], Despite these different pathways of apoptosis, all cells seem to have a common cell surge-shrinkage event [11]. This surge-shrinkage event is thought to be caused by a potassium efflux, which is a prerequisite to and not a consequence of cell death [12]. It has been shown that adding 4-aminopyridine (4-AP), a type 1 delayed rectifying voltage gated K+ channel (Kvl) inhibitor, to Madin-Darbey Canine Kidney (MDCIC) cells, prior to UV radiation, prevents apoptosis and extrusion [1]. By inhibiting extrusion, this K+ channel antagonist prevents the possibility of metastasis by basal extrusion. However, it is not feasible to use 4-AP as a potential metastasis regulator because it also inhibits apoptosis. Tumors are already anti-apoptotic and it is this anti­ death feature which leads to their formation. Finding a tumor specific drug which prevents metastasis but does not inhibit apoptosis or cause undesired side effects would be a novel innovation to epithelial tumor eradication. It is therefore hypothesized that inhibition of inwardly rectifying K+ channels, K vll, brought about by the human-ethera-go-go related gene (HERG), or HERG K+ channels, will prevent extrusion but not apoptosis by UV light. Since HERG K+ Channels are inward rectifying it is assumed they will not be involved in the potassium efflux and will therefore not inhibit apoptosis [13]. The drugs MK499 and E-4031, HERG channel antagonists, as well as PD-118057, a HERG channel agonist, were used to further delineate the effects of the inwardly rectifying Kvl 1 channels on cell contraction prior to apoptosis and extrusion. Our study shows that when inhibited and exposed to UV light, inwardly rectifying HERG K+ channels do not prevent apoptosis, but do prevent extrusion. MDCK cells were grown to confluence treated with each drug at different concentrations for thirty minutes, exposed to UV irradiation, and examined at different time points. Our results show that when treating HERG K+ channels of MDCK cells with antagonists, MK499 and E-4031, extrusion and apoptosis were not inhibited or affected. However, treatment with agonist PD-118057 increased apoptosis and inhibited extrusion. These significant findings could be used as a possible means of preventing metastasis while promoting apoptosis. Many studies have shown that HERG K+ channels are over expressed in gastric carcinomas, leukemia, glioblastoma multiforme, neoplastic hematopoietic cells, colorectal cancers, and endometrial adenocarcinomas [14]-[19]. By drug targeting a gene over expressed in tumors but not in normal tissues with PD-118057, the prevention of further metastasis may be possible, as well as inhibition of proliferation. METHODS Cell Culture MDCK II cells (gift from K. Matlin, University of Chicago, Chicago, IL) were cultured with Dulbecco's minimum essential medium (DMEM) high glucose, sodium pyruvate, L-glutamine, and other non essential amino acids (Invitrogen). The DMEM was supplemented with 5% fetal bovine serum (FBS) and 100 |ig/ml penicillin/streptomycin (all from Invitrogen) at 5% CO2 and 37°C. MDCK cells were chosen because of their natural ability to form an epithelial monolayer. All cells were detached from their original container with 0.05% Trypsin EDTA in DMEM at 37°C, centrifuged for 1 min 30 sec, and re-suspended onto coverslips with DMEM media. Cells were allowed to proliferate until full confluency in order to create an epithelial monolayer similar to many epithelial layers in the human body. HERG Channel Assay 10 mM stock solutions of PD-118057, MK499 (both gifts from Dr. Michael Sanguinetti, University of Utah, Salt Lake City, UT), and E-4031 (Biocris) were made in DMSO. A 10 |liM concentration of PD-118057 was used as a standard for each trial. E4031 and MK499 were both tested at 10 jliM and 50 \xM in each trial. A 0.5 M stock solution of 4-AP (Sigma-Aldrich) was made with water. 4-AP was used at a 2 mM concentration. 24 cover slips were seeded with MDCK cells at a density of 755,000 cells/mL of media and grown to confluency. Each cover slip was placed inside a well of a 4-well dish, covered with media, and treated with each corresponding amount of drug independently for 15 - 60 min depending on the trial. This incubation time was to ensure the MDCK. cells had begun to take up each drug. To induce apoptosis the drug treated cells and control cells were exposed to 1,200 |uJ/cm2 UV254 irradiation in a UV series II (Spectroline) and allowed to incubate for 1-4 hrs before fixation depending on the trial. This assay was repeated to test drug treated MDCK cells with No UV, UV radiated cells with post drug treatment, and drug pre-treated cells with UV at different incubation times. Cell Staining All cell fixations were done with 4% formaldehyde in lxPBS for 20 minutes at 37°C. lxPBS containing 0.5% Triton X-100 was used to permeabilize the cells for 10 min. The cells were blocked in Abdil (PBS with 0.1% Triton X-100 and 2% BSA) for 20 min, and incubated in Rabbit Anti-Active Caspase 3 Antibody (Cell Signaling Technology) for 1 hr 30 min, followed by secondary antibody for 45 min. The following secondary antibodies were used: actin was monitored using 0.1 [ig/ml Alexa Flour 568phalloidin (Invitrogen), DNA was monitored using 1 |ig/ml Hoechst 33342 (SigmaAldrich), and caspase 3 was monitored using Alexa Flour 488 goat anti-rabbit (Invitrogen). Microscopy and Data Analysis Image sections were taken using a CTR 6000 microscope (Leica) with a 63x oil lens and Qlmaging Retiga 200R camera. IPLab 4.0.8 s and Image-J software were used to view places of apoptosis and extrusion. Montage pictures were made from projections of 8-10 consecutive 1 \xm z slices. For quantification of extrusion, 100 extruding cells in each control and drug treated trial were manually counted and classified as basal if the actin extrusion ring appeared in the top 4-8 jum, apical if the ring appeared in the bottom 0-4 |im, or inhibited if a ring never formed when a cell was caspase positive. Statistical analysis was done on three different extrusion assays for the control, MK499, E-4031 and PD-118057. 4-AP was not statistically analyzed for extrusion because it was not possible to count 100 extruding or apoptotic cells. The error was calculated by taking the standard deviation of the different trials. The standard error of the mean (SEM) was then taken for this standard deviation. This SEM of the standard deviation is the final represented error. 7 RESULTS After analyzing the data from 1-4 hours, it was found that two hours was the best time to view changes in the monolayer due to UV and drug treatment. At one hour, the cells did not have enough time to fully respond to UV induced extrusion and death. At three hours, many of the monolayers were too far damaged to properly quantify the number of extruding cells. Therefore, the data presented here is shown at two hours. Figure 1 shows images of the control, 2 mM 4-AP, 10 jiM MK499, 10 jiM E-4031, and less than 10 jaM PD-118057 two hours after UV treatment. When treating the cells with 10 |aM PD-118057 there was too much death at two hours to properly quantify the number of non-extruding cells. For this reason a concentration less than 10 jaM PD118057 was used for imaging and quantification. Approximately 5 juM PD-118057 was used, however due to small pipeting limitations this amount can only be approximated and properly stated as less than 10 juM PD-118057. When viewing from left to right in Figure 1, each picture represents an image captured 1 jam lower in the z-direction. When DNA was in the top z~plane and a thick actin ring was in the bottom plane, we assumed the cell extruded apically. When a cell was extruded basally the actin ring was in the top plane and DNA was in the bottom plane. Condensed DNA, not separated by mitosis, as well as active caspase 3 was a sign of apoptosis. If apoptosis occurred without ring formation, extrusion was inhibited. 8 Control 4-AP No Extrusion MK499 Basal Extrusion Apical E-4031 PD-118057 TOP Z-PLANE BOTTOM 9 Fig. 1 Images of the control, 2 mM 4-AP, 10 joM MK499, 10 [iM E-4031, and < 10 |aM PD-118057 are shown two hours after UV treatment. When viewing from left to right each picture is 1 |im lower in the zdirection. When DNA was in the top z-plane and a thick actin ring was in the bottom plane, a cell was extruded apically. When a cell extruded basally the actin ring was in the top plane and DNA was in the bottom plane. Condensed DNA and activated caspase was a sign of apoptosis. After UV treatment the control began to show induced extrusion and apoptosis. 4-AP inhibited both of these processes from happening. MK499 and E-4031 showed a similar response as the control and PD-118057 showed extrusion was inhibited. Bars, 10 jum. Figure 2 shows the quantification of extruding cells for each drug treatment. After UV treatment the control began to show induced extrusion and apoptosis. Each of the extruding cells was found to be apoptotic due to caspase 3 activation and DNA condensing/fragmentation. In the control without UV, 100 extruding cells were not counted, but rather the same area of cells in the monolayer was covered as the control with UV treatment to illustrate the increase in apoptosis occurring due to UV. The control no-UV ratio of apical to basal extrusions is approximately the same ratio as MK499 no-UV and E-4031 no-UV, and would have likely reached the same quantified numbers had 100 control no-UV cells been counted. The only significant change from the control occurred at two hours after drug treatment with agonist 10 PD-118057. Both UV and No-UV < 10 jxM PD-118057 treatment showed an increase in blocked extrusions. With concentrations >10 |iM, we assumed PD-118057 blocked every extrusion. This assumption was made because once too many cells die in close proximity to each other, it is longer possible to carry out the extrusion process due to lack of living cells. 4-AP inhibited both extrusion and apoptosis from happening, and therefore made it impossible to count 100 extruding or apoptotic cells in this assay. Overall, the antagonists, MK499 and E-4031, showed no difference from the control with both UV and non-UV treatment. Apoptosis and extrusion were not inhibited. 10 Increasing HERG K+ Channel Conductivity Inhibits Extrusion i _____ w O 13% <oo pH c ______ 5 1 SS^ & Jp & j f — 1 1 tSS^ ^ V* — E1 * njV (S' # V1 # Not Extruded — c\ ,0 ,/ Fig. 2. Out of 100 extruding cells, the results of apical, basal, and extrusion inhibited are shown for each drug treatment. MK499 and E-4031 showed a non-significant difference from the control. However, < 10 HM PD-118057 showed a quantifiable amount of inhibited extrusions. In the control without UV, 100 extruding cells were not counted, but rather the same number of cells in the monolayer was covered as the control with UV treatment to illustrate the increase in apoptosis occurring due to UV. The non-UV control ratio of apical/basal/not extruded is approximately the same non-UV ratio for MK499 and E-4031, 4-AP inhibited both extrusion and apoptosis, and therefore made it not possible to count 100 extruding or apoptotic cells in this assay. 11 DISCUSSION We hypothesized that inhibition of inwardly rectifying K+ channels, K vll, brought about by the humcm-ether-a-go-go related gene (HERG), or HERG K+ channels, would prevent extrusion but not apoptosis by UV light. Since HERG K+ Channels are inward rectifying we assumed they would not be involved in the potassium efflux necessary for apoptosis to occur [13]. This assumption is important because our overall goal was to identify a molecular target over expressed in epithelial cancers that would prevent extrusion but not apoptosis. The results in this paper show that HERG K+ channels are an important part of cell signaling in the extrusion pathway and demonstrate that the proposed hypothesis was correct. This result proves to be very valuable because it opens a new pathway for possibly preventing metastasis while still allowing current techniques of targeting tumors for death to occur. Further tests are necessary to determine what, if any, role HERG channels may play in the potassium efflux necessary for apoptosis. These tests will help determine any unforeseen limitations with our previous assumption that HERG does not play a role in this K+ efflux. The result of HERG not inhibiting apoptosis in this experiment, however, suggests our assumption was not proven wrong and until proven wrong remains valid. Other types of outward rectifying K+ channels most likely play the strongest role in the potassium efflux required for apoptosis. It has been shown previously that HERG expression is cell cycle dependent with the greatest expression of HERG proteins in the G1 phase [21]. Evidence exists that IO channels are involved in the regulation of cell proliferation [22]-[24]. Although these mechanisms are not fully understood, it is possible that activation of K+ channels is required to pass through different phases of the mitotic cycle [24]-[25]. Based on this hypothesis, it is possible that as a cell ages through the G1 phase, when HERG expression is the highest, it may be prime for extrusion from an epithelial layer. This is also evidence why proliferating cells are not seen extruding. During S or M phases, signals for extrusion would be working against DNA replication and mitosis. Through future examinations of extrusion during the mitotic phases it will be possible to further distinguish the role of HERG in extrusion and the validity of our hypothesis. An alternative transcript of the hergl gene, hergib, was demonstrated to be mainly expressed during the S phase of the mitotic cycle [21]. It has also been demonstrated that tumor cells preferentially express hergib over herg leading to an increase in survival [21]. Currently, no research on extrusion and herglb has been explored. This study, along with future studies of hergl, herg2, and herg3 on extrusion, will lay a foundation for clearer understanding of the role of herglb in tumor cells and the extrusion pathway. In future work western blot analysis will be used to determine which of the herg proteins may or may not be present in MDCK cells. Testing this extrusion inhibition assay with the herglb transcript in tumor cells to examine if extrusion is inhibited and apoptosis is increased is the next goal. One of the biggest limitations of this study, that will also be addressed next, is the surety that both the antagonists, E-4031 and MK499, and agonist, PD-118057, were acting specifically on HERG K+ Channels, and only on HERG K+ channels. Using shRNAs and gene knockdown of HERG K+ channels this limitation will be addressed. 13 One of the most important questions concerning HERG K+ channels that is still unanswered is how they directly or indirectly fit into the cell signaling pathway of extrusion. It has been demonstrated that HERG channels are physically linked to integrins, which are adhesion receptors mainly involved in migration, proliferation, survival, and differentiation [26]-[27], A few integrin-HERG activated pathways have been identified that may play a key role in extrusion signaling [28]-[29]. One pathway leads to activation of the focal adhesion kinase (FAK), and ultimately the activation of mitogen-activated protein kinases [28]. Another pathway is a signal cascade involving caveolin-1, the Src family kinase Fyn, and the adapter protein She [28], A link between integrins and small GTPases, RhoA, Racl, and Cdc42 has been found [29], This last pathway, with activation of Rho GTPases, is the most promising connection because it has already been shown that inhibition of Rho GTPases inhibits extrusion [1]. By understanding the coprecipitation of HERG proteins and integrins it may be possible to further deduce the extrusion signaling pathway and thereby increase the current understanding of cancer metastasis. In summary, our study outlines, for the first time, the role of HERG K+ channels in the extrusion pathway. It has been shown that increasing the conductivity of HERG K+ channels inhibits extrusion but not apoptosis. Since many tumors are already antiapoptotic, a protein which allows for regulation of extrusion independent of apoptosis is of the utmost importance in broadening the foundation of metastasis knowledge. The HERG K+ channel opens this new doorway of study and has the possibility of leading to complete metastasis regulation. 14 ACKNOWLEDGEMENTS A tremendous thank you is in order for Dr. Jody Rosenblatt and Dr. George T. Eisenhoffer, both from the Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah. Thank you to Dr. Michael Sanguinetti, University of Utah, for his donations of MK499 and PD-118057, as well as the Undergraduate Research Opportunities Program for funding. 15 REFERENCES [1] J. Rosenblatt J, M.C. Raff, and L.P. Cramer, “An epithelial cell destined for apoptosis signals its neighbors to extrude by an actin- and myosin-dependent mechanism,” Current Biology vol. 11, pp. 1847-1857,2001. [2] G. Slattum, K.M. McGee, J. Rosenblatt, "PI 15 RhoGEF and microtubules decide the direction apoptotic cells extrude from an epithelium, " Journal of Cell Biology, pp. 186(5):693~702, Sep 2009. [3] M.J. Horner, L.A.G. Ries, M. Krapcho, N. Neyman, R. Aminou, N. Howlader, S.F. Altekruse, E.J. Feuer, L. Huang, A. Mariotto, B.A. Miller, D.R. Lewis, M.P. Eisner, D.G. Stinchcomb, B.K. Edwards (eds), "SEER Cancer Statistics Review, 1975-2006, National Cancer Institute," Bethesda, MD, http://seer.cancer.gov/csr/1975_2006/, based on November 2008 SEER data submission, posted to the SEER web site, 2009. [4] American Cancer Society. Cancer Facts & Figures 2009. Atlanta, GA. 2009 [5] B. Xu, Z. Xu, T. Xia, P. Gao, W. He, M. Zhang, L. Guo, Q. Miu, A. Wang, "Effects of the Fas/Fas~L pathway on fluoride-induced apoptosis," Environmental Toxicology, Oct 22, 2009. [Epub ahead of print] [6] S. Cagnol, J.C. Chambard, "ERK and cell death: Mechanisms of ERK-induced cell death - apoptosis, autophagy, and senescence," FEBS J, Oct 16, 2009. [Epub ahead of print] [7] J. Gao, M. Senthil, B. Ren, J. Yan, Q. Xing, J. Yu, L. Zhang, J.H. Yim, "IRF-1 transcriptionally upregulates PUMA which mediates the mitochondrial apoptotic pathway in IRF-1-induced apoptosis in cancer cells," Cell Death Differentiation Journal, Oct 23, 2009. [8] A.F. Renert, P. Leprince, M. Dieu, J. Renaut, M. Raes, V. Bours, J.P. Chapelle, J. Piette, M.P. Merville, M. Fillet, "The proapoptotic C16-ceramide-dependent pathway requires the death-promoting factor Btf in colon adenocarcinoma cells," Journal of Proteome Research, pp. 8(10):4810-22, Oct 2009. [9] M. Li, H. Li, C. Li, S. Zhou, L. Guo, H. Liu, W. Jiang, X. Liu, P. Li, M.A. McNutt, G. Li, "Alpha fetoprotein is a novel protein-binding partner for caspase-3 and blocks the apoptotic signaling pathway in human hepatoma cells," International Journal of Cancer, pp. 124(12):2845-54, Jun 2009. [10] G. Ouyang, L. Yao, K. Ruan, G. Song, Y. Mao, S. Bao, "Genistein Induces G2/M cell cycle arrest and apoptosis of human ovarian cancer cells via activation of DNA damage checkpoint," Cell Biology International, Sep 2, 2009. [Epub ahead of print] [11] L. Wangdanger, D. XuDagger, W. Dai, and L. LuDagger, “An ultra-violetactivated K+ channel mediates apoptosis of myeloblastic leukemia cells,” J Biol Chem, vol 274, Issue 6, pp. 3678-3685, Feb 1999. [12] A. Grishin, H. Ford, J. Wang, H. Li, V. Salvador-Recatala, E.S. Levitan, and E. Zaks-Makhina, “Attenuation of apoptosis in enterocytes by blockade of potassium channels,” Am J Physiol Gastrointest Liver Physiol, pp. 289(5):G815-21, Nov 2005. [13] G.A, Gutman, K.G. Chandy, S. Grissmer, M. Lazdunski, D. McKinnon, L.A. Pardo, G.A. Robertson, B. Rudy, M.C. Sannguinetti, W. Stuhmer, X. Wang, "International Union of Pharmacology. LIII. Nomenclature and molecular relationships 17 of voltage-gated potassium channels," Pharmacoogical Reviews, pp. 57 (4): 473-508. 2005. [14] L. Qing, L. Huiyu, L. Xiaoming, G. Wang, “HERG K+ channels expression in gastric cancers and analysis of its regulation in tumor cell proliferation and apoptosis,” Journal o f Nanjing Medical University, pp. 23 (3): 157-162. 2009. [15] S. Pillozzi, M.F. Brizzi, M. Balzi, O. Crociani, A. Cherubini, L. Guasti, B. Bartolozzi, A. Becchetti, E. Wanke, P.A. Bernabei, M. Olivotto, L. Pegoraro, A. Arcangeli, "HERG potassium channels are constitutively expressed in primary human acute myeloid leukemias and regulater cell proliferation of normal and leukemic hemopoietic progenitors,” Leukemia, Nature Publishing Group, pp. (16): 1791-1798. 2002. [16] A. Masi, A. Becchetti, R. Restano-Cassulini, S. Polvani, G. Hoffmann, A.M. Buccoliero, M. Paglierani, B. Polio, G.L. Taddei, P. Gallina, N. Di Lorenzo, S. Franceshetti, E. Wanke, A. Arcangeli, "HERG1 channels are overexpressed in glioblastoma multiforme and modulate VEGF secretion in glioblastoma cell lines," British Journal o f Cancer, pp (93): 781-792. 2005. [17] G. Smith, H.W. Tsui, E.W. Newell, X. Jiang, X.P. Zhu, F.W.L. Tsui, L.C. Schlichter, "Functional Up-regulation of HERG K+ Channels in Neoplastic Hematopoietic Cells,” The Journal o f Biological Chemistry, pp 277 (21): 18528-18534. 2002 [18] . E. Lastraioli, L. Guasti, O. Crociani, S. Polvani, G. Hofmann, H. Witchel, L. Bencini, M. Calistri, L. Messerini, M. Scatizzi, R. Moretti, E. Wanke, M. Olivotto, G. Mugnai, A. Arcangeli, "hergl Gene and HERG1 protein are overexpressed in colorectal 18 cancers and regulate cell invasion of tumor cells," Cancer Research, pp 64: 606-611. 2004. [19] A. Cherubini, G.L. Taddei, O. Crociani, M. Paglierani, A.M. Buccoliero, L. Fontana, I. Noci, P. Borri, E. Borrani, M. Giachi, A. Becchetti, B. Rosati, E. Wanke, M. Olivotto, A. Arcangeli, "HERG potassium channels are more frequently expressed in human endometrial cancer as compared to non cancerous endometrium," British Journal o f Cancer, pp. 83:1722-1729.2000. [20] J. Monks, C. Smith-Steinheart, E. Kruk, V. Fadok, P. Henson, "Epithelial cells remove apoptotic epithelial cells during post-lactation involution of the mouse mammary gland," Biology o f Reproduction, pp. 78:586-594. 2008. [21] O. Crociani, L. Guasti, M. Balzi, A, Becchetti, E. Wanke, M. Olivotto, R. Wymore, A. Arcangeli, "Cell cycle-dependent expression of HERG1 and HERG1B isoforms in tumor cells," The Journal of Biological Chemistry, pp. 278 (5):2947-2955. 2003. [22] R.S. Lewis, M.D. Cahalan," Molecular Properties and Physiological Roles of Ion Channels in the Immune System Annu. Rev. Immunology, pp. 13:623-653. 1995. [23] S.A. Kotecha, L.C. Schlichter, "A Kvl.5 to Kvl.3 Switch in Endogenous Hippocampal Microglia and a Role in Proliferation," Journal o f Neuroscience, pp 19: 10680-10693. 1999. [24] W.F. Wonderlin, J.S. Strobl, "Potassium Channels, Proliferation, and G1 Progression," Journal Membrane Bioliogy, pp. 154:91-107. 1996. [25] S. Wang, Z. Mellcoumian, K. Woodfork, C. Gather, A. Davidson, W.F. Wonderlin, J.S. Strobl, Journal o f Cell Physiology, pp. 176:456-464. 1998. [26] A. Cherubini, G. Hofmann, S. Pillozzi, L. Guasti, O. Crociani, E. Cilia, P. Di Stefano, S. Degani, M. Balzi, M. Olivotoo, E. Wanke, A. Becchetti, P. Defilippi, R. Wymore, A. Arcangeli, "Human ether-a-go-go-related gene 1 channels are physically linked to B1 integrins and modulate adhesion-dependent signaling," Molecular Biology of the Cell, pp. 16:2972-2983. 2005. [27] A. Arcangeli, A. Becchetti, A. Mannini, G. Mugnai, P. Defilippi, G. Tarone, M.R. Del Bene, E. Barletta, E. Wanke, M. Olivotto, "Integrin mediated neurite outgrowth in neuroblastoma cells depends on the activation of potassium channel," Journal of Cell Biology, pp 122:1131-1143. 1993. [28] J.W. Lee, R. Juliano, "Mitogenic signal transduction by integrin and growth factor receptor-mediated pathways,"Molecules and Cell, pp. 17:188-202. 2004. [29] L.V. Parise, J. Weon Lee, R.L. Juliano, "New aspects of integrin signaling in cancer," Seminars in Cancer Biology, pp. 10(6):407-414. 2000.