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(0522354) 09/02/2008 The Characterization and Transfection of Plasmid Vectors Expressing PMCA4b to Generate Stable Transfectant Cell Lines in Endothelial Cells ABSRACT: Tumour growth beyond 2-3 mm 3 depends on angiogenesis, since the tumour cells’ requirements cannot be satisfied by simple diffusion. Majority of the scientist agree that it’s better to eliminate the tumour than to correct it genetically. Therefore, targeting blood vessels that supply the tumour (antiangiogenesis) seems to be a potentially promising strategy in the treatment of cancer. Various attempts have been made to suppress angiogenesis in the tumour, including the blockade of the effects of vascular endothelial growth factor (VEGF) and subsequent influx of Ca 2+ that acts as a secondary messenger and activated intracellular pathways that are involved in angiogenesis. However, the diversity and distribution of Ca 2+ channels hinder the use of Ca2+ channel blockers and pose high risk of adverse effects. An alternative strategy is to over express the Ca2+ extrusion system, such as, plasma membrane calcium ATPase (PMCA), on tumour vascular endothelial cells; using endothelial cell specific vectors that express PMCA4b. Lowering [Ca 2+]i has been shown to be reducing tumour growth. Key words: angiogenesis: vascular endothelial growth factor (VEGF): Ca2+ signalling: plasma membrane calcium ATPase (PMCA). ………………………………………………………………………………………… …. Introduction: Sprouting of new blood vessel from a pre-existing blood vessel is called angiogenesis (Armesilla et al, 1999; Hernandes et al, 2001; Molina et al, 2005). There are two types of angiogenesis: physiological angiogenesis, which’s essential for life and occurs during embryogenesis, in endometrium during changes in menstrual cycle, during wound healing and post-ischaemic tissue repair (Felmeden et al, 2003; Cao et al, 1997). The second type is pathophysiological angiogenesis that’s implicated in various medical conditions such as rheumatoid arthritis, systemic lopus erythematosus, proliferative retinopathy, atherosclerosis and tumour (Felmeden at al, 2003). Here only angiogenesis that’s involved in tumour is considered. In order for the tumour to grow beyond 2-3 mm3, it requires angiogenesis (He et al, 2005; Churachambi et al, 2004; Xue at al, 2004) to receive adequate oxygen and nutrients and prevent tumour cells death. Apart from tumour growth it’s implicated in tumour metastasis and invasion (Jan et al, 2006;Davidoff et al, 2001). Angiogenesis is a multistep process that includes: activation of endothelial cells (ECs), fibrinolysis, degradation of basement membrane and extracellular matrix by matrix- metalloproteinases (MMPs), migration and proliferation of ECs and tube formation that sprouts into new branches of vasculature (Rafiee et al, 2004; Harold, 1999; Towson et al, 1996). Normally this process is regulated by the balance between angiogenic-inducer and angiogenic-inhibitor molecules (Molina et al, 2005). Therefore, the suppression of anti-angiogenic and
Kaker L. (0522354) 09/02/2008 activation of pro-angiogenic molecules are essential for angiogenesis (Cheng et al, 2005). Angiogenesis is induced by a plethora of angiogenic growth factors. They include; basic and acidic fibroblast growth factor (bFGF & aFGF), transforming growth factors alpha and beta (TGFα and TGFβ), tumour necrosis factor alpha (TNFα) interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF) (vannoorden at al, 2004). VEGF is highly selective endothelial cells mitogen in vitro and potent angiogenic factor in vivo (Lee et al, 2006; Liang et al, 1998). Members of VEGF family include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, sn-VEGF (sn= snake venom) and placenta growth factors. Their isoforms arise from the alternative splicing of the primary gene (Takashashi et al, 2005). They are multifunctional peptides, which activate tyrosine kinase receptors (flt/flk) on the endothelial cell membrane and trigger multiple intracellular signal transductional pathways (Felmeden at al, 2003; Kroll et al, 1997; Towson et al, 1996) fig 1. VEGF production is mainly caused by tumour-induced hypoxia (Soker et al, 1996; Ferrara et al, 2005). Carmeliet et al (1996) has shown that the VEGFA is the main inducer of angiogenesis that interact with flt-1 or VEGFR-1.
Fig 1.The VEGF pathway. Interactions between members of VEGF family with their receptors (VEGFRs). Activation of VEGFr1 and VEGFR2 results in angiogenesis and vasculogenesis, while the activation of VEGFR3 leads to lymphangiogenesis.
Calcium (Ca2+) messenger:
Cancer is characterised by uncontrolled cell proliferation and cell cycle that arise from disordered cellular signalling pathways (Rasado et al, 2004). Calcium plays a critical role in cell proliferation, thus, in angiogenesis and subsequent tumour growth (Munaron, 2006). Phospholiphase C (PLCγ) is one of the intracellular pathway, which is triggered as a consequence of tyrosine kinase receptor activation on the EC membrane
by VEGF (Zachary, 2001). PLCγ elevates the intracellular inositolphosphate (InsP3) level by converting phosphoinositol-bisphosphate (PIP2) into diacylglycerole (DAG) and inositol1,4,5-triphosphate (InsP3). Elevated level of InsP3 causes the release of Ca2+ from intracellular stores. High level of intracellular Ca2+ open voltageindependent calcium channels on the cell surface and cause the influx of extracellular Ca2+. (Cunningham et al, 1999; Patton et al, 2003; Jho et al, 2005).
Kaker L. (0522354) 09/02/2008 High intracellular Ca concentration activates Ca2+-calmodulin dependent phosphatase, calcineurin. Calcineurin dephosphorylates cytosolic nuclear factor of activated T-cells (NFAT) and translocates it into the nucleus (Hogan et al, 2003; Guerini et al, 2000). In the nucleus NFAT regulates the transcription of a number of cytokines’ gene (IL-1, IL-6 & IL-8) that are roduced by endothelial cells (Ruff et al, 1995; Loh et al, 1996; Rafiee et al, 2004). A threshold of [Ca2+]i is required to activate VEGF-mediated pathways (Jho et al, 2005). This means that the prevention of this threshold will inhibit VEGFmediated signalling pathways. Normally cytosolic Ca2+ concentration is maintained at about 1x10-7 M (Munaron, 2006). After transient efflux of Ca2+ into the cell, to activate intracellular signalling pathways, 2+ intracellular Ca homeostasis is achieved by plasma membrane calcium ATPases (PMCAs) on the
cell surface (Pang et al, 2005). They remove cytosolic Ca2+ into extracellular space until its concentration drop below 10-7M (Brand et al, 1992; Strehler et al, 2007). PMCAs are P-type ATPases (Armesilla et al, 2004) and they are activated by Ca2+calmodulin complex (Du et al, 1995). PMCAs are protein molecules, composed of ten membrane spanning segment with NH2 and COOH ends on the cytosolic side of the plasma membrane (fig 2). There are four types of PMCAs: PMCA1, PMCA2, PMCA3 and PMCA4. Their isoforms results from the mRNA alternative splicing (Strehler et al, 2001) and they differ mainly in the amino acid sequence of carboxyl (–COOH) terminal (Kip at al, 2003). Isoform 1&4 are ubiquitous and are expressed in most tissues, while isoform 2&3 are exclusive to excitable tissues such as brain and striated muscles (Strehler et al, 2001).
Fig 2. Simple schematic diagram of plasma membrane calcium ATPase (PMCA). Membranespanning domains are numbered as shown. Amino (NH2) and carboxyl (–COOH) groups are on the cytosolic side. The region between transmembrane domains 4 and 5 is the catalytic domain, which is responsible for transporting the Ca2+ across the membrane.
Anti-angiogenesis as treatment for tumour: The consensus of scientists is that it’s better to eliminate the tumour rather than to correct it genetically. Therefore, most of the anti-cancer therapies, such as,
immunopotentiation, chemotherapy, oncolytic agents etc, are aimed at killing the target tumour cells (Majhen et al, 2006). Alternatively, targeting blood vessels that nourish the tumour cells, by suppressing angiogenesis, seems to be a promising approach (Jiao et al, 2006). 3
Kaker L. (0522354) 09/02/2008 One of the antiangiogenic strategies is to increase the concentration of endogenous angiogenic inhibitor molecules (Cheng et al, 2005), using gene therapy that target normal and genetically stable endothelial cells (Majhen et al, 2006). These molecules include: endostatin, angiostatin, vasohimbin, vasostatin and tamstatin (Ferrara et al, 2005). Endostatin and angiostatin can be detected in the blood of tumour-bearing individuals (Jiang et al, 2001) Endostatin is a 29Kda peptide and strongly inhibits angiogenesis (Chang et al, 2005; Wu et al, 2005) by inhibiting the migration and proliferation of ECs and angiostatin inhibits ECs migration only. They both induce ECs apoptosis (Jiang et al, 2001). Angiogenex is an exogenous synthetic peptide that blocks angiogenesis by preventing the adhesion and migration of activated ECs (Branwijk et al, 2005). Another strategy involves the blockade of VEGF and its receptors (VEGFRs), using monoclonal antibodies or VEGF antagonists, which significantly reduce tumour growth. Bevcacizumab, an antiVEGF monoclonal antibody, has been approved by food and drug administration (FDA) in USA, in combination with cytotoxic 5fluorouracil for cancer treatment. The combination of antiangiogenic gene therapy with chemotherapy lowers the rate of tumour progression and increases patient survival (Ferrara et al, 2005). However, the effectiveness of gene therapy is limited by inadequate transgene expression and its maintenance for a long time (Fukumor et al, 2002). Ca2+ as an anti-angiogenic target: Numerous studies have elucidated the role of Ca2+ in angiogenesis. Kohn et al (1995) showed that the blocking of receptor operated Ca2+ influx; using immunosuppressive agent carboxyamidotriazole (CAI), inhibits ECs proliferation and proteolysis both in vivo and in vitro. Faehling et al (2002) reported that lower intracellular concentration inhibits VEGF-A induced ECs proliferation. Smith et al (1986) explained that in order for preneoplastic mouse JB6 epidermal cell to transform into neoplastic cell, influx of extracellular Ca2+ is necessary. Furthermore, intracellular calcium affects the rate of cellular protein synthesis and lowers cell proliferation in experimental cancer (Palakurthi et al, 2000). According to Haverstic et al (2000) the blockade of Ca2+ with TH1177 increased the mean life span of mice by 38%, who were inoculated with human prostate cancer cells. Similar observation has been noted by Yoshida et al (2004) that the blocking of Ca2+ with amlodipine, reduces tumour growth and increases the survival of mice who were inoculated with human epidermoid carcinona A43 cells. From these results it can be deduced that reduction in cytosolic Ca2+ level can reduce tumour progression. Therefore, it might be one of the potential targets for cancer therapy. However, the selectivity of calcium antagonists and the ubiquitous nature of calcium channels is a major problem with this approach. Alternative approach is to over-express PMCAs in tumour vascular endothelial cells, using endothelial cell specific expression vectors. Kip et al (2003) showed that over expression of PMCA4b in the madin-darby canine kidney (MDCK) cells increases 2+ transcellular Ca transport by 140%. Buch et al (2005) describes PMCA as negative regulator of calcineurin/NFAT signalling pathway, since it has inhibitory effects on the pathway. For the reason that PMCAs are indigenous Ca2+ systems, they will not pose the risk of adverse effects that are associated with calcium antagonists.
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adenoviral vector. Life Sciences 77: 1331–1340. 7. Chura-Chambi RM,Tornieri PH, Spencer PJ, Nascimento PA,Mathor MB Morganti L (2004) High-level synthesis of recombinant murine Endostatin in Chinese hamster ovary cells. Protein Expression and Purification 35: 11–16. 8. Xue Y, Bi F,Zhang X, Pan Y, Liu N, Zheng Y and Fan D (2004) Inhibition of endothelial cell proliferation by targeting Rac1 GTPase with small interference RNA in tumor cells.Biochemical and Biophysical Research Communications 320 1309–1315. 9. Jan SL, Meur NL, Cazes A, Philippea J, Cunff ML, Le´ger J, Corvol P and Germain S (2006) Characterization of the expression of the hypoxia-induced genes neuritin, TXNIP and IGFBP3 in cancer. FEBS 580: 3395–3400. 10. Davidoff AM, Leary MA, Ng CYC and Vanin EF (2001) Gene Therapy-Mediated Expression by Tumor Cells of the Angiogenesis Inhibitor flk-I Results in Inhibition of Neuroblastoma Growth In Vivo. Journal of Pediatric Surgery 36 (1): 30-36. 11. Rafiee P, Heidemann J, Ogawa H, Johnson NA, Fisher PJ, Li MS, Otterson MF, Johnson CP and Binion DG (2004) Cyclosporin A differentially inhibits multiple steps in VEGF induced angiogenesis in human microvascular endothelial cells through altered intracellular signaling. Cell Communication and Signaling 2: 12. Townson S, Lagercrantz J, Grimmond S, Silins G, Nordenskjöld M, Weber G and Hayward N (1996) Characterization of the Murine VEGF-Related Factor Gene. Biochemical And Biophysical Research Communications 220: 922–928. 13. Van Noorden CJF, Jansen M, Hamer PCW, Witmer AN and Troost D (2004) Current Perspective on
References: 1. Armesilla AL, Lorenzo E, Arco PG,
Martinez A, Alfranca A and Redondo JM (1999) Vascular Endothelial Growth Factor Activates Nuclear Factor Of Activated T Cells in Human Endothelial Cells: A Role For Tissue Factor Gene Expression. Molecular and Cellular Biology 19: 2032-2043. Hernández GL, Volpert OV, Íñiguez MA, Lorenzo E, Martínez S, Grau R, Fresno M and Redondo JM (2001) Selective Inhibition of Vascular Endothelial Growth Factor–mediated Angiogenesis by Cyclosporin A: Roles of the Nuclear Factor of Activated T Cells and Cyclooxygenase 2. J. Exp. Med 193 (5): 607–620. Molina MC, Ferreira V, Valck C, Aguilar L, Orellana J, Rojas A, Ramirez G, Billetta R, Schwaeble W, Lemus D and Ferreira A (2005) An in vivo role for Trypanosoma cruzi calreticulin in antiangiogenesis. Molecular & Biochemical Parasitology 140: 133-140. Felmeden DC, Blann AD and Lip GYH (2003) Angiogenesis: Basic Pathophysiology and Implications for Disease. European Heart Journal 24: 586-603. Cao Y, Ji WR, Qi P, Rosin A and Cao Y (1997) Placenta Growth Factor: Identification and Characterization of a Novel Isoform Generated by RNA Alternative Splicing. Biochemical and Biophysical Research Communications 234: 493-498. He GA, Xue G, Xiao L, Wu JX, Xu BL, Huang JL, Liang ZH, Xiao X, Huang BJ, Liu RY and Huang W (2005) Dynamic distribution and expression in vivo of human Endostatin gene delivered by
Kaker L. (0522354) 09/02/2008
Antiangiogenesis Strategies in the Treatment of Malignant Gliomas. Brain Research Review 45: 143163.
20. Ferrara N and Kerbel RS (2005)
Angiogenesis as a therapeutic target. Nature 438: 967-974. 21. Majhen D and Ambriovi´cRistov A (2006) Adenoviral vectors—How to use them in cancer gene therapy? Virus Research 119:121–133. 22. Jiao JG, Li YN, Wang H, Liu Q, Cao JX, Bai RZ and Huang FY (2006) A plasmid DNA vaccine encoding the extracellular domain of porcine endoglin induces anti-tumour immune response against selfendoglin-related angiogenesis in two liver cancer models. Digestive and Liver Disease 38: 578– 587. 23. Chang JH, Javier JAD, Chang GY, Oliveira HB and Azar DT (2005) Functional characterization of neostatins, the MMP-derived, enzymatic cleavage products of type XVIII collagen. FEBS Letters 579: 3601–3606. 24. Wu J, Fu W, Luo J and Zhang T (2005) Expression and purification of human Endostatin from Hansenula polymorpha A16. Protein Expression and PuriWcation 42: 12–19. 25. Branwijk RJMGE, Nesmelova I, Dings RPM, Mayo KH, Thijssen VLJL and riffioen AW (2005) Cloning an artificial gene encoding angiostatic anginex: From designed peptide to functional recombinant protein. Biochemical and Biophysical Research Communications 333: 1261–1268. 26. Fukumori T, Nishitani MA, Naroda T, Onishi T, Oka N, Kanayama HO and Kagawa S (2002) Expression Of Angiostatin cDNA In A Murine Renal Cell Carcinoma Suppresses Tumor Growth In Vivo. Urology 59 (6): 973-977. 27. Rosado JA, Redondo PC, Pariente JA and Salido JM (2004) Calcium Signalling and tumorigenesis. Cancer Therapy 2, 263-270.
14. Cheng WF, Hung CF, Chen CA, Lee CN, Su YN, Chai CY, Boyd DAK, Hsieh CY and Wu TC (2005) Characterization of DNA vaccines encoding the domains of calreticulin for their ability to elicit tumor-specific immunity and antiangiogenesis. Vaccine 23: 3864–3874. 15. Lee GY, Jung WW, Kang CS and
Bang IS (2006) Expression and characterization of human vascular endothelial growth factor (VEGF165) in insect cells. Protein Expression and Purification 46:503–509. 16. Liang D, Xu X, Chin AJ, Balasubramaniyan NV, Teo MAL, Lam TJ, Weinberg ES and Ge R (1998) Cloning and characterization of vascular endothelial growth factor / VEGF from zebrafish, Danio rerio. Biochimica et Biophysica Acta 1397: 14–20. 17. Takashashi H and Shibuya M (2005) The Vascular Endothelial Growth Factor (VEGF)/VEGF Receptor System and its Role Under Physiological and Pathological Conditions. Clinical Science 109: 227-241. 18. Kroll J and Waltenberger J (1997) The Vascular Endothelial Growth Factor Receptor KDR Activates Multiple Signal Transduction Pathways in Porcine Aortic Endothelial Cells. The Journal Of Biological Chemistry 272 (51): 32521–32527. 19. Soker S, Fidder H, Neufeld G and Klagsbrun M (1996) Characterization of Novel Vascular Endothelial Growth Factor (VEGF) Receptors on Tumour Cells That Bind VEGF165 Via Its Exon 7Encoded Domain. The Journal of Biological Chemistry 271: 57615767.
Kaker L. (0522354) 09/02/2008 28. Munaron L (2006) Intracellular
Calcium, Endothelial Cells and Angiogenesis. Recent Patents on Anti-Cancer Drug Discovery 1: 105-119. 29. Zachary I (2001) Actions of Vascular Endothelial Growth Factor Signaling Mechanisms Mediating Vascular Protective. Am J Physiol Cell Physiol 280:1375-1386. 30. Cunningham SA, Tran TM, Arrate MP, Bjercke R, and Brock TA (1999) KDR activation is crucial for VEGF165-mediated Ca2+ mobilization in human umbilical vein endothelial cells. Am J Physiol Cell Physiol 276:176-181. 31. Patton AM, Kassis J, Doong H, and Kohn EC (2003) Calcium as a Molecular Target in Angiogenesis. Current Pharmaceutical Design 9: 543-551. 32. Hogan PG, Chen l, Nardone J and Rao A (2007) Transcriptional regulation by calcium, calcineurin, and NFAT. Genes & Dev 17: 22052232. 33. Guerini D, Wang X, Li L, Genazzani A, and Carafoli E (2000) Calcineurin Controls the Expression of Isoform 4CII of the Plasma Membrane Ca21 Pump in Neurons. The Journal Of Biological Chemistry 275: 3706–3712. 34. Ruff VA and Leach KL (1995) Direct Demonstration of NFATp Dephosphorylation and Nuclear Localization in Activated HT-2 Cells Using a Specific NFATp Polyclonal Antibody. The Journal Of Biological Chemistry 270: 22602–22607. 35. Loh C, Shaw KTY, Carew J, Viola JPB, Luo C, Perrino BA, and Rao A (1996) Calcineurin Binds the Transcription Factor NFAT1 and Reversibly Regulates Its ctivity. The Journal Of Biological Chemistry 271:10884–10891. 36. Pang Y, Zhu H, Wu P, Chen J (2005) The characterization of plasma membrane Ca2+-ATPase in rich sphingomyelin–cholesterol domains. FEBS Letters 579: 2397– 2403. 37. Jho D, Mehta D, Ahmmed G, Gao X, Tiruppathi C, Broman M and Malik AB (2005) Angiopoietin-1 Opposes VEGF-Induced Increase in Endothelial Permeability by Inhibiting TRPC1-Dependent Ca2_ Influx. Circ. Res. 96; 12821290; [online] available at http://circres.ahajournals.or g. DOI: 10.1161/01.RES.000017189 4.03801.03 [cited on; June 7, 2007] 38. Brand P, Neve RL, Kammesheidt A, Rhoadsll RE, and Vanamann TC (1992) Analysis of the Tissuespecific Distribution of mRNAs Encoding the Plasma Membrane Calcium-pumping ATPases and Characterization of an Alternately Spliced Form of PMCA4 at the cDNA and Genomic Levels. The Journal Of Biological Chemistry 267: 4376-4385. 39. Strehler EE, Caride AJ, Filoteo AG, Xiong Y, Penniston JT and Enyedi A (2007) Plasma Membrane Ca2+ ATPases as Dynamic Regulators of Cellular Calcium Handling. Ann. N.Y. Acad. Sci 1099: 226–236.
AL, Williams JC, BuchMH, Pickard A, Emerson M, Cartwright EJ, Oceandy D, Vos MD, Gillies S, Clark JG and Neyses L (2004) Novel Functional Interaction between the Plasma Membrane Ca2+ Pump 4b and the Proapoptotic Tumor Suppressor Ras-associated Factor 1 (RASSF1). The Journal Of Biological Chemistry 279 (30): 31318–31328. 41. Strehler EE and Zacharias DA (2001) Role Of Alternative Splicing In Generation Isoform Diversity Among Plasma Membrane Calcium Pumps. Physiology Review 81: 2150.
Kaker L. (0522354) 09/02/2008 42. Kip NS and Strehler EE (2003)
Characterization of PMCA isoforms and their Contribution to transcellular Ca2+ influx in MDCK cells. Am J Physiol Renal Physiol 284: 122–132. 43. Kohn EC, Alessandro R, Spoonster J, Wersto RP and Liotta LA (1995) Angiogenesis: Role of calcium-mediated signal transduction. Cell Biology 92: 1307-1311. 44. Faehling M, Kroll J, Föhr KJ, Fellbrich G, Mayr U, Trischler G and Waltenberger J (2002) Essential role of calcium in vascular endothelial growth factor A-induced signaling: mechanism of the antiangiogenic effect of carboxyamidotriazole. The FASEB Journal express article 10.1096/fj.01-0938fje. Published online September 19, 2002. 45. Du Y, Carlock L and Kuo TH (1995) The mouse plasma membrane Ca2+ pump isoform 1 promoter: Cloning and Characterization. Archive of biochemistry and biophysics 316 (1): 302-310. 46. Smith BM, Gindhart TD and Colburn NH (1986) Extracellular calcium requirement for promotion of transformation in JB6 cells. Cancer Res 46: 701-706. Harneit S, Kilic E and Halperin JA. (2000) Inhibition of translation initiation mediates the anticancer effect of the n-3 polyunsaturated fatty acid eicosapentaenoic acid. Cancer Res 60: 2919-2925. 48. Haverstick DM, Heady TN, Macdonald TL and Gray LS. (2000) Inhibition of human prostate cancer proliferation in vitro and in a mouse model by a compound synthesized to block Ca2+ entry. Cancer Res 60: 1002-1008. 49. Yoshida J, Ishibashi T and Nishio M. (2004) Antitumor effects of amlodipine, a Ca2+ channel blocker, on human epidermoid carcinoma A431 cells in vitro and in vivo. Eur J Pharmacol 492: 103-112. 50. Buch MH, Pickard A, Rodriguez A, Gillies S, Maass HA, Emerson M, Cartwright EJ, Williams J, Oceandy D, Redondo JM, Neyses L and Armesilla al (2005) The Sarcolemmal Calcium Pump Inhibits the Calcineurin/Nuclear Factor of Activated T-cell Pathway via Interaction with the Calcineurin a Catalytic Subunit. The Journal Of Biological Chemistry 280 (33): 29479–29487.
47. Palakurthi SS, Fluckiger R, Aktas
H, Changolkar AK, Shahsafaei A,
51. Research VEGF (2005) The VEGF Pathway [online] < http://www.researchvegf.com/res earchvegf/vegf-overview/vegfpathway/index.m> [Cited on 19.0607].
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