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A correlation between membrane cholesterol level, cell adhesion and tumourigenicity of polyoma virus transformed cells

A correlation between membrane cholesterol level, cell adhesion and tumourigenicity of polyoma virus transformed cells
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   Molecular and Cellular Biochemistry  265:  85–95, 2004. c  2004  Kluwer Academic Publishers. Printed in the Netherlands. A correlation between membrane cholesterollevel, cell adhesion and tumourigenicity of polyoma virus transformed cells Tanvir Kaur, Praturi Gopalakrishna, Pulicat Subramanian Manogaranand Gopal Pande Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India Received 1 January 2004; accepted 24 February 2004 Abstract We have investigated the implications of the rise in membrane cholesterol levels on several  in vitro  and  in vivo  properties of polyoma virus transformed rat fibroblasts (PyF), with a special emphasis on  α 5 β 1  integrin functions. We show that increasedmembrane cholesterol causes the PyF cells to change their shape and become more bipolar in appearance. These cells alsoshow significantly higher adhesion to the cell-binding domain of fibronectin, increased localization of   α 5 β 1  integrin and talinmolecules in focal adhesions and a more robust actin cytoskeleton organization. PyF cells with increased membrane cholesterolshow reduced growth  in vitro  and tumours caused by these cells in nude mice are slow growing. These changes in the growthproperties of PyF cells are reversible when the cholesterol levels of PyF cells become normal. Our results suggest that changesin membrane cholesterol levels influence the growth and morphological properties of transformed cells, which can be exploitedin controlling the growth of tumours  in vivo . (Mol Cell Biochem  265:  85–95, 2004) Key words : tumours, cholesterol, cell membrane, integrins Introduction Most cells in multicellular organisms require attachment andspreading on the proteins of extracellular matrix (ECM) fortheir survival, growth and differentiation [1]. Cancer cells,however, can survive and proliferate in the absence of adhe-siontoECMproteins.Thispropertyofcancercells,termedasanchorage independent growth, correlates closely with theirtumourigenicity in animal models and it reflects their abilityto survive and grow in restrictive locations  in vivo  [2]. Inte-grinsareamongthemostsignificantmoleculesbywhichcellsattachtotheECM,although,someintegrinssupportcell–celladhesioneventsalso[3,4].TheinteractionbetweentheECMcomponents and integrins causes the integrin molecules tocluster at specific locations in the membrane, referred to as  Address for offprints : Gopal Pande, Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India (E-mail: focal contacts or focal adhesion points, and from there in-tegrins can influence the organization of cytoskeleton andother cytoplasmic functions [5]. Several types of proteins,present in focal adhesions, cooperate with integrins to per-form their function. These proteins can be classified as struc-turalproteins(e.g.talinandvinculin),signallingproteins(e.g.integrin-linked kinase, ilk, focal adhesion kinase, FAK andras-family of G-proteins) and docking proteins (e.g. paxillin, α -actinin, p131) [6, 7].Transformed cells are characterized by the disruption of the actin cytoskeleton, decreased adhesion to ECM proteinsand alterations in adhesion-dependent signalling [7, 8]. Pre-vious studies have shown that cell shape and organization of cytoskeleton are altered in cancer cells and the expressionof actin-associated proteins including  α -actinin is reduced  86[7]. It has been demonstrated further that cell morphology,adhesion and motility can be viewed as the cause ratherthan the effect of transformation because the restoration of  α -actinin level in tumour cells could result in the suppres-sion of tumourigenic ability of such cells [9]. Thus, by sim-ply modifying the cell shape, the cells could be switchedbetween different programs such as cell survival, differenti-ation or cell death [10]. These data indicate that restructur-ing of cell shape and cytoskeleton apparently tells the cells“ what to do next  ”.The lipid constitution of cell membrane, in particular thecholesterol content, can determine the architecture of thelipid bilayer and also influence the conformation and func-tion of some important proteins [11]. In this context, it hasbeen shown that modification of the cholesterol content of cell membranes could significantly alter membrane fluidityand also influences the distribution and the function of someproteins [12]. In this report, we have investigated the effectof elevating membrane cholesterol content on cell morphol-ogy, ECM adhesion properties and the tumourigenic poten-tial of a polyoma virus transformed cell line. Our resultsshow that elevation of membrane cholesterol caused sig-nificant alteration in the morphology of cells and their ad-hesion to fibronectin through  α 5 β 1  integrin was increased.These cells also showed increased clustering of   α 5 β 1  inte-grin and talin molecules in the membrane and more orga-nized actin cytoskeleton. Concomitantly, a significant reduc-tion in  in vitro  cell proliferation as well as  in vivo  tumourgrowth in nude mice was observed. Our data indicate thatmembrane cholesterol modulates many aspects of the distri-bution and function of  α 5 β 1  integrins. Based on these results,we propose that changes in the lipid constitution of plasmamembraneaffecttheshapeandadhesionoftransformedcellsto fibronectin and thus in turn affects surface membraneproteins. Material and methods  Animals Nude mice (NIH (S) (Sex: male/female; age: 30 days) wereobtainedfromtheNationalInstituteofNutrition,Hyderabad,India. Chemicals All cell culture media and sera were obtained fromGibco-BRL (Gaithersburg, MD, USA). The bufferswere prepared from analytical grade chemicals avail-able locally. Sources for other chemicals are mentionedsubsequently. Cell lines Alltheexperimentsweredonewitharatfibroblastlinetrans-formed by polyoma virus; PyF or its non-transformed coun-terpart the F111 cells. The cell lines were kindly providedby Prof. T.L. Benjamin of Harvard Medical School, USAPyF cells were maintained in Dulbecco’s modified Eagle’smedium (DMEM) containing 5% foetal calf serum (FCS)(Sigma, USA) (or 10% FCS for F111), and streptomycin(50 µ g/ml)andpenicillin(50 µ g/ml)(Sigma,USA)in75cm 2 plastic culture flasks (Costar, USA). Cells were incubated at37  ◦ C in an atmosphere of 5% CO 2  and 95% relative humid-ity. Cells were subcultured before they became confluent andexperiments were done within 10 passages after revival of cells from liquid nitrogen storage.Cholesterol treatment of cells was done as described byArbogast et al. [13]. The three groups of the cells used forour study, were as follows: (a) normal untreated PyF cells –cells grown through out in DMEM with 5% FCS; (b) starvedcells – cells grown in serum free DMEM for 24–30 h be-fore harvesting; (c) cholesterol treated cells – cells treatedwith different concentrations of cholesterol – 1.25, 2.5 and5.0  µ g/ml in serum free DMEM for 12–16 h, after 12 h of incubation in serum free DMEM without any cholesterol.These cholesterol treated cells were referred to as 1.25 , 2.5and 5.0  µ g (CHO) cells, respectively. Cell morphology studies Themorphologyofuntreated,serum-starvedandcholesterol-treated PyF cells, was studied using phase contrast mi-croscopy. Cells were photographed on Kodak TMAX 100film using a Nikon inverted microscope at 200X magnifica-tion through a 20X-phase contrast objective lens.  Membrane cholesterol measurement  The cholesterol content of plasma membrane fractions wasestimated by the cholesterol oxidase method as described[14]. This method ensures that only plasma membranecholesterol was measured without accounting for cytosolic-free cholesterol because cholesterol oxidase (the enzymeusedinthisassay)actsononlyfreecholesterol(whichisseenin plasma membrane) and not on cholesterol esters (whichareonlypresentinthecytoplasm–mostoftheinternalmem-braneshaveeithernoorverylittlecholesterol).Thecellswereharvested by scraping with a policeman and lysed by soni-cation. Membrane fractions from the lysates were collectedafter centrifugation. The oxidation of cholesterol to cholest-4-ene-3-one was monitored by measuring fluorescence in aHitachi F-4000 steady state spectrofluorimeter ( λ ex  325 nm; λ em  415 nm).  87 Cell attachment assay Cell attachment assays were carried out as described [15].Cholesterol-treated and untreated cells were suspended inplainDMEMataconcentrationof5 × 10 5 cells/ml(viability > 90%) and 100  µ l cell suspension containing 5  ×  10 4 cells were added to each fibronectin coated well. The cellswere incubated at 37  ◦ C for 1 h in a 5% CO 2  atmosphereunattached cells were removed by gently flicking the plate;the number of attached cells were estimated by MTT assay. Colorimetric estimation of cell number – MTT assay MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-biphenyl-tetrazolium bromide; Sigma, USA) was used for estimation of cell number in cell attachment assays (as above) and cellproliferative assays [16]. One hundred micro litres of DMEM followed by 25  µ l MTT solution (2 mg/ml inPBS, freshly prepared) was added to each well. The cellswere incubated at 37  ◦ C and 5% CO 2  for 4 h after which150  µ l acidified isopropanol mixed with 4 ml of IN HClwas added to each well and contents were mixed thoroughlyusing a micropipette. The optical density of the solution ineach well was determined using a  V  max  ELISA plate reader(Molecular Devices, USA) using a 490 nm detection filter. Cell proliferation assay Twothousandandfivehundredviablecellswereplatedin96-well plates. Serum starvation was done for 12 h as describedearlier. After which, serum-starved cells were treated with5.0  µ g/ml cholesterol. Cell number was measured at 48 h of plating the cells.To study the reversal of effect of cholesterol treatment oncell growth, cholesterol containing medium was replaced byDMEM containing 5% FCS after 12 h of cholesterol treat-ment.Cellnumberwasmeasuredat12and24hafterreplace-mentofserumcontainingmedium,anditwascomparedwiththe cell number in PyF cultures growing through out the pe-riod of the experiment in 5% FCS. Tumourigenicity assay One million viable cells, with or without treatment withcholesterol or after serum starvation, were suspended in200  µ l PBS and injected subcutaneously at one site on theback of nude mice using a 24G hypodermic needle. Eachgroup included 10–12 animals/cell type. The tumour sizewas measured by calculating diameters on two orthogonalaxes after five days of cell inoculation. Growth of tumours,or death of animals, was monitored at intervals of every twodays for upto 30 days post-cell inoculation.  Integrin, talin and actin localization The cells were grown on cover slips (24  ×  24 mm 2 ) andwere treated with cholesterol as described above. After 12 hcholesterol treatment, the cells were fixed in 3% formalde-hydeatroomtemperaturefor30min.Fixedcellsweretreatedwith 1: 500 dilution of polyclonal anti-rabbit-anti- α 5 β 1  anti-body (Gibco, BRL) followed by staining with TRITC taggedanti-rabbitIgG(SigmaInc;USA)indarkfor1hatroomtem-perature.CoverslipswerewashedwithPBSandmountedona slide with 10  µ l Antifade solution (Molecular Probes, Inc;USA).For F-actin visualization, formaldehyde fixed cells werepermeabilised with chilled acetone at − 20  ◦ C for 4 min andwashedwithPBSatroomtemperature.Thecellswerestainedwith1:50dilutionofrhodaminetaggedphalloidin(MolecularProbesInc;USA)for30–40minatroomtemperature.Allthestained cells were mounted on a slide in 50% glycerol andvisualized under a confocal microscope (Meridian, UltimaInc; USA) using suitable filters and 40X Plan-Apochromaticobjective lens. Optical sectioning of cells was done as perstandard procedure and analyzed for localization of integrin,talin and actin molecules.  Membrane fluidity measurementsFluorescence recovery after photobleaching (FRAP) Cells were grown on cover slips (in subconfluent con-ditions) and were treated with cholesterol as describedabove. Subsequently they were incubated in 100  µ l DMEMcontaining 1  µ g of N-(-7-)-nitrobenz-2-oxa-1,3-diazol-4-yl)-dipalmitoyl-1- α -phosphatidyl ethanolamine (NBD-PE[Molecular Probes] stock-solution was made at 1 mg/ml inmethanol) in 5% CO 2  atmosphere for 10 min at 37  ◦ C. Thecells were washed in plain DMEM and subjected to FRAPanalysis within 15 min after the staining was over. Fluores-cence recovery of NBD-PE was monitored at 530 nm usinga confocal microscope (Meridian, Ultima, USA) at 22  ◦ C.Eight to ten recordings per cell, of each cell type were taken.Florescence recovery of NBD-PE was monitored for 20 secand the diffusion coefficient ( µ  =  cm 2 sec − 1 ) values werecalculated using Meridian System software V4.15. Fluorescence polarization of DPH  Changes in the membrane fluidity were monitored by thefluorescence anisotropy of 1,6 diphenyl-1, 3,5 hexatriene(DPH) according to the method described [17]. Cells werelabelled with DPH (1  µ M final concentration) at 37  ◦ C for20 mins, while they were still attached to their substrata. Theunincorporated dye was removed by repeated washings withplain DMEM, and the cells were finally suspended in PBSat a concentration of 2  ×  10 6 cells/ml. DPH fluorescence  88 Fig. 1 . The relationship between membrane cholesterol levels and the  invitro c holesterol treatment of PyF cells and F111 cells. PyF cells weretreated with different concentrations of cholesterol, and plasma membranecholesterolcontentwasestimatedasdescribed.F111cellswerealsostudied,without cholesterol treatment. Cholesterol levels in different cell types areshown. Each value in the bar charts represents the average of eight indepen-dent estimations. was monitored at room temperature in a Hitachi F4000spectrofluorimeter equipped with a polariser,  λ ex  at 358 nmand  λ em  at 430 nm. Fluorescence polarisation values werecalculated as per the following equation:P = I VV − GI VH / I VV + GI VH Where I VV  is the fluorescence intensity when the excitationandemissionpolariserswerebothinverticalposition,andI VH isthefluorescenceintensitywhentheexcitationpolariserwasin vertical, and emission polariser in horizontal position withrespect to the to the beam path.  G  is the grating correctionfactor and is equal to I HV  /I HH . Results  Measurement of membrane cholesterol content  Membrane cholesterol content increased significantly uponincubation of PyF cells in different concentrations of solu-ble cholesterol in DMEM (Fig. 1). All values for membranecholesterol content have been equated for 1 × 10 5 cells andeach level in the bar chart represents the average of eight in-dependent experiments. 1.25  µ g CHO cells showed almostthe same level of membrane cholesterol (1.52 ± 0.14  µ g) asuntreatedPyFcells(1.31 ± 0.14 µ g)  p  value > µ gCHO cells showed a marginal increase in membrane choles-terol (1.65  ±  0.22  µ g;  p  >  0 . 05) and 5.0  µ g CHO cellsshowed almost twice as much membrane cholesterol (2.8 ± 0.34  µ g;  p  <  0 . 001) as untreated PyF. We also comparedthe membrane cholesterol level of the un-transformed coun- Fig. 2 . Cell attachment to fibronectin in untreated, starved and cholesteroltreated PyF cells. Cell attachment assays to FN-CBD were carried out asdescribed in Material and Methods. The percentage of attached cells, asa fraction of total cells added to each well (5  ×  10 4 ), was calculated andplotted on the  y -axis against the FN-CBD concentration shown on the  x  -axis. Control (  ), Starved ( ◦ ), 2.5  µ g/ml Cholesterol (  ), and 5.0  µ g/mlCholesterol ( • ). Each data point represents the mean of three independentexperiments keeping triplicate wells for each FN-CBD concentration in ev-ery experiment. Cholesterol treated cells showed the maximum attachmentto fibronectin at a concentration 2.5  µ g ml − 1 . terpart of PyF cells (F111) and interestingly the membranecholesterol of the F111 cells was 2.18 ± 0.25  µ g. Compar-ison of this membrane cholesterol content with that of PyFcells suggests (Fig. 1) that transformation of the F111 fi-broblasts by polyoma virus significantly reduced the levelof membrane cholesterol and restoration of the cholesterollevels in the membranes of the PyF cells reversed manyof the transformed properties of PyF cells as shown in oursubsequent. Cell attachment to fibronectin (FN) Adhesion of PyF cells to FN was assayed on a range of FNconcentrations (0.5 –5.0 µ g/ml). Figure 2 shows that all cellsshowed a concentration dependent increase in attachment toFN and the maximum attachment of cells to FN was seen atthe concentration of 2.5 µ g/ml. At this concentration, 2.5 µ gCHO cells showed almost 2.5 times, and 5.0  µ g CHO cellsshowed four times more attachment to FN, as compared withuntreated PyF cells.  In vitro cell proliferation and in vivo tumourigenicity Invitro cellproliferationwasmeasuredasdescribedinMate-rials and Methods. The results are shown in Fig. 3 where 2.5and 5.0  µ g CHO cells showed 40 and 25% lesser increase incell number respectively; as compared with the control PyF  89 Fig. 3 .  In vitro  cell proliferation in untreated and cholesterol treated PyFcells. Cell proliferation assays, with or without cholesterol treatment andreaddition of serum containing medium were done as described in Materialand Methods. The description of the bar charts is as follows: (1) untreatedcells, (2) serum starved cells, (3) 2.5  µ g CHO cells, (4) 5.0  µ g CHO cells,(5) cells at 24 h after addition of serum containing medium and (6) cellsat 48 h after addition of serum containing medium. 2.5 and 5.0  µ g CHOcells showed 40 and 25% lesser cell growth respectively as compared withuntreated cells (  p = <  0 . 001). Cell proliferation recovered to normal levelsat 24 h after readdition of serum containing medium. cells,at48hafterplatingthecells.Interestinglythecellnum-ber in serum free DMEM without cholesterol was similar tothe control PyF cells (  p  >  0 . 01), indicating that these cellsare capable growing for a few generations in serum free con-ditions. The reduction in the growth of cholesterol-treatedPyF cells was restored to same levels as untreated PyF cellsafter they are allowed to grow in DMEM containing 5% FCSfor 24 h.  In vivo  proliferation of these cells was estimated by mea-suring the size of tumours in nude mice at the subcutaneoussite as described. The tumour sizes were observed for over30 days post-cell inoculation as shown in Fig. 4. The animalsinjected with untreated and serum-starved PyF cells devel-oped tumours (first visible nodules) within 7 days post-cellinoculation, whereas animals injected with 1.25, 2.5 and 5.0 µ g CHO first palpable visible nodules appeared after 9, 11and 15 days respectively, post-cell inoculation. The differ-ence among the size of tumours of the various groups wasconsiderably reduced after 15–20 days post-cell inoculationand after 30 days, tumours from all cell types were of thesame size; animals died after 26–35 days of cell inocula-tion. It appears that the initial tumourigenicity of PyF cellsis related inversely to the membrane cholesterol content andtheir ability to attach to FN, i.e. cells with higher membranecholesterol show more adhesion to FN  in vitro  but lesser tu-mourigenicity  in vivo .  Effect of increased membrane cholesterol on cell shape The incubation of PyF cells in different concentrations of cholesterol in DMEM showed a significant effect on cellshape. Cells treated with 5  µ g/ml cholesterol showed thechanges most clearly and reproducibly. The 5  µ g CHO cellsappearedflatandbipolarinshapeascomparedwithuntreatedand serum-starved PyF cells (Fig. 5c). Serum starvation for24 h did not show any change in cell shape as compared withcontrol cells (compare 5a with b) however, the cell viabil-ity was decreased by 5–10%. Cholesterol concentration of  > 5  µ g/ml in medium was toxic to the cells. Organization of focal adhesion plaques, talinand actin cytoskeleton In order to correlate the increase of cell adhesion andthe distribution of   α 5 β 1  integrins and the cytoskeletalorganization of cholesterol treated cells, we performedthe following immunocytochemical staining and confocalmicroscopy: α 5 β 1  integrin and talin distribution The staining of untreated PyF cells with anti- α 5 β 1  antibodyshowed the diffused localization of integrins (Fig. 6a). Thedistribution was diffused in serum-starved cells (Fig. 6b)but the integrins were prominently localized in focal adhe-sion points in cholesterol treated cells (Fig. 6c). Put togetherthese results indicate that increased cholesterol in PyF cellssupports formation of more focal contacts and thus the betteradhesion of these cells to the FN. It also possibly assists inthe reorganization of the cytoskeleton which leads to a moreflattened morphology of these cells (compare with the mor-phology of cholesterol treated cells inbreak Fig. 5).  Actin cytoskeleton The staining of cells was done with rhodamine phalloidinand anti-talin antibody simultaneously. The results show theabsence of F-actin in most of the cytoplasm of untreatedand starved cells except in the sub-membranous lamellipo-dialprojections(Figs.7aand7brespectively).Thisisindica-tive of high membrane ruffling in untreated PyF cells. Theactin distribution also did not coincide with the distributionof talin in these cells indicating that actin does not associatewith the focal adhesions in these cells. In cholesterol treatedcells, however, the distribution of F-actin is remarkably dif-ferent(Fig.7c).Aconsiderableamountofactinfilamentscanbe seen in the cytoplasm, although they are not organized asstressfibers,andalmostalltheactinisco-localizedwithtalin,i.e. is in association with the focal adhesion points. Interest-ingly even in these cells a marginal staining of lamellipodiacould be observed.
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