INTRODUCTION

Morphological change plays an important role in many cellularprocesses such as migration, differentiation, apoptosis,necrosis and senescence. Cell migration in mammalian cellsinvolves a series of morphological changes. Gaining functionin differentiated cells often requires morphological changes.Altered morphology is an important criterion for apoptosis ornecrosis. During cellular senescence, normal human diploidfibroblasts (HDFs) change their morphology from a spindleshape to an enlarged, flattened and irregular shape.

Normal animal cells from non-tumor origin undergosenescence as a result of serial passage in tissue culture.Senescent cells show a number of molecular changes that areobserved during the process of aging in vivo (Campisi, 1996;Campisi, 1997; Campisi et al., 1996; Chen, 2000). Usingsenescence-associated β-galactosidase as a biomarker,senescent cells are found in skin biopsy samples from elderlypersons (Dimri et al., 1995). Accelerated cellular senescenceis associated with the diseases that occur at high rates in elderlypersons such as atherosclerosis and osteoporosis (Bennett et

al., 1998; Kassem et al., 1997). Senescence has been used asa model for aging studies since Hayflick and Moorhead firstreported the limited replicative life span of HDFs in 1961(Hayflick and Moorhead, 1961).

A distinct feature of replicatively senescent cells is themorphological change including cell enlargement. The causeof this morphological change has not been well studied. Muchof the mechanistic studies on senescence have been focused onthe irreversible growth arrest. Since these cells are notreplicating but are enlarged, it is necessary to test whethergrowth arrest and cell enlargement share similar controlmechanisms. Traditionally, senescence results from cellreplication. Most strains of HDFs from fetal tissues canreplicate 50-60 population doublings before reachingsenescence. Several recent studies indicate that early passageHDFs can develop a phenotype resembling senescence inresponse to oxidants, inhibitors of histone deacetylase,hyperactivation of ras gene and overexpression of E2F1transcription factor (Chen and Ames, 1994; von Zglinicki etal., 1995; Ogryzko et al., 1996; Serrano et al., 1997; Lee et al.,1999; Dimri et al., 2000). In the model of induction of

4087Journal of Cell Science 113, 4087-4097 (2000)Printed in Great Britain © The Company of Biologists Limited 2000JCS1715

Early passage human diploid fibroblasts develop senescentmorphology prematurely within a week after a 2-hourpulse treatment with low or mild dose H2O2. We test herethe role of cell cycle checkpoints, cytoskeletal proteins andde novo protein synthesis in senescent morphogenesisfollowing H2O2 treatment. H2O2 treatment causes transientelevation of p53 protein and prolonged inhibition ofRb hyperphosphorylation. Expression of humanpapillomaviral E6 gene prevented elevation of p53 but didnot affect senescent morphogenesis. Expression of humanpapillomaviral E7 gene reduced the level of Rb protein andprevented induction of senescent morphology by H2O2. Themutants of the E7 gene, in which the Rb family proteinbinding site was destroyed, could not reduce Rb proteinor prevent H2O2 from inducing senescent morphology.Senescent-like cells showed enhanced actin stress fibers.In untreated cells, vinculin and paxillin preferentially

distributed along the edge of the cells. In contrast, vinculinand paxillin distributed randomly and sporadicallythroughout senescent-like cells. E7 expression preventedenhancement of actin filament formation andredistribution of vinculin or paxillin. Neither wild-type norE7 cells showed changes in the protein level of actin,vinculin or paxillin measured by western blot after H2O2treatment. Finally, depletion of methionine in the culturemedium after H2O2 treatment prevented senescentmorphogenesis without affecting dephosphorylation of Rbprotein. Our results suggest that senescent morphologylikely develops by a program involving activated Rb familyproteins, enhancement of actin stress fibers, redistributionof focal adhesion proteins and de novo protein synthesis.

Key words: Fibroblast, Senescence, Morphology, Cytoskeleton,Tumor suppressor gene

SUMMARY

Involvement of Rb family proteins, focal adhesion proteins and protein

synthesis in senescent morphogenesis induced by hydrogen peroxide

Qin M. Chen*, Victoria C. Tu, Jeffrey Catania, Maggi Burton ‡, Olivier Toussaint § and Tarrah Dilley

Department of Pharmacology, University of Arizona, Skaggs Pharmaceutical Science Building, 1703 E. Mabel St, Tucson, AZ85721, USA*Author for correspondence (e-mail: chen@pharmacy.arizona.edu)‡Present address: Unit of Animal Biology, University of Louvain, Batiment Carnoy, Croix du Sud 4/5, B-1348 Louvain-La-Neuve, Belgium§Present address: University of Namur (FUNDP), Unit of Cellular Biochemistry and Biology, Rue de Bruxelles 61, B-5000 Namur, Belgium

Accepted 7 September; published on WWW 31 October 2000

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premature senescence with oxidants a 2-hour pulse treatmentwith low or mild doses of H2O2 causes the cells to losereplicative potential immediately and to develop senescentmorphology one week later (Chen and Ames, 1994; Chen etal., 1998; Chen et al., 2000b). H2O2 treatment causes transientelevation of p53 protein and sustained inhibition of Rbhyperphosphorylation (Chen et al., 1998). Expressing humanpapillomaviral (HPV) protein E6 or E7 results in inactivationof p53 or Rb and abrogation of G1 cell cycle arrest (Chen etal., 1998). Since senescent morphogenesis is coupled to growtharrest, this model allows us to test whether or not cell cyclecheckpoint proteins are involved in the control of themorphological change. Nevertheless, the long time course (i.e.one week) of senescent morphogenesis following H2O2treatment indicates that the process may be a programmedevent involving new protein synthesis.

A large number of morphological studies in the literaturefocus on cytoskeletal proteins. Actin and focal adhesionproteins build the framework of a cell (Gumbiner, 1996).Expression levels or spatial arrangement of these cytoskeletalproteins determine the morphology of a cell. Actin is a highlyconserved protein with several isoforms existing in non-musclecells such as fibroblasts (Herman, 1993). Actin monomersalign to form a polymer string, which can bundle to form actinfilaments. Actin filaments assemble to form stress fibers whichtransverse a cell and are the major component of thecytoskeletal framework (Mitchison and Cramer, 1996). Theends of actin stress fibers meet at focal adhesion plaques, wherecells contact with the extracellular matrix (Hemmings et al.,1995; Mitchison and Cramer, 1996). Focal adhesion plaquesare composed of a number of proteins including vinculin andpaxillin (Craig and Johnson, 1996; Schaller and Parsons,1994). Since fibroblasts undergo drastic morphological changeafter treatment with low or mild doses of H2O2, we test herewhether senescent morphology is coupled to changes in actinand focal adhesion proteins and if so whether these changesare under the control of cell cycle checkpoints.

MATERIALS AND METHODS

ChemicalsAll chemicals were purchased from Sigma Chemical Inc. (St Louis,MO) unless otherwise indicated.

Cell culture and H 2O2 treatmentIMR-90 cells (obtained from the Coriell Institute for MedicalResearch at the population doubling level (PDL)10.85, Camden, NJ)were subcultured weekly in 10 ml of Dulbecco’s modified Eaglemedium (DMEM; Life Science Technologies, Grand Island, NY)containing 10% (v/v) fetal bovine serum (FBS, Life ScienceTechnologies, Grand Island, NY) at a seeding density of 0.5×106 cellsper 100 mm Corning dish. The cells reach confluence at 6-7 days aftersubculture. For treatment with H2O2, cells were seeded at the densityof 2×106 per 100 mm dish 20-24 hours before the treatment. Afterincubating for 2 hours in the presence of H2O2, the cells were placedin fresh DMEM containing 10% (v/v) FBS.

Infection with recombinant HPV E6 or E7 retroviralconstructsThe retroviral producing cells of human papillomavirus (HPV) type16 E6, E7 and E6E7 genes were obtained from the American TypeCulture Collection (Rockville, MD) (Demers et al., 1994). The cells

producing E7 mutant d21-24, C24G or E26G virus were provided byDr Denise Galloway (Demers et al., 1996). Exponentially growingIMR-90 cells (PDL ≤17) were infected with retroviruses carrying E6,E7, E6E7 or a mutant E7 gene and were selected by culturing in 500µg/ml of G418 as described (Chen et al., 1998).

Western blot analysisCells in a 100 mm dish were lysed by scraping in Laemmli buffer (0.5M Tris, pH 6.8, 2.4% (w/v) SDS, 50% (v/v) glycerol with proteaseinhibitors). Protein concentration was determined by thebicinchoninic acid (BCA) method (Pierce Inc, Rockford, IL). Proteinswere separated by SDS-polyacrylamide gel electrophoresis using amini-Protean II electrophoresis apparatus (Bio-Rad, Richmond, CA)run at 100 volts. The separating gel contained 8% acrylamide fordetecting p53, actin or paxillin and 6% acrylamide for detecting Rbor vinculin. The separated proteins were transferred to immobilon-Pmembranes (Millipore, Bedford, MA) by electrophoresis aspreviously described (Chen et al., 1998). The membrane wasincubated with anti-p53 (Ab-6, 1:100, Oncogene Science, NY), anti-Rb (polyclonal, 1:100, Santa Cruz Biotechnology, CA), anti-actin(clone AC-40, 1:1,000, Sigma, St Louis, MO), anti-vinculin (1:1,000,Sigma, St Louis, MO), or anti-paxillin (1:1,000, Zymed, South SanFrancisco, CA) antibody as described (Chen et al., 1998). The boundantibodies were detected by secondary antibodies conjugated withhorseradish peroxidase and enhanced chemiluminescence reaction.The densities of the bands were quantified using an Eagle Eye IIImage System (Stratagene, La Jolla, CA).

Measurement of cell volume and percentage of enlargedcellsAt 10 days after H2O2 treatment, adherent cells were detached bytrypsinization. Rounded cells were loaded onto a microslide fieldfinder (Fisher Scientific, Pittsburgh, PA). The diameters of the cellsthat randomly landed on the grids of the microslide field finder wereviewed with a microscope and recorded. For each group, at least 99cells were measured for diameters within one experiment. The cellvolume was calculated based on the diameter using the equation fora sphere 1.33 × π × radius3. The number of cells showing diametersgreater than 0.04 millimeter (mm) was counted for calculating thepercentage of enlarged cells within a group of 33 cells.

Flow cytometry analysis for cell enlargementAdherent cells were detached by trypsinization and collected bycentrifugation at 3,300 rpm before fixation with 25% (v/v) ethanolcontaining 15 mM MgCl2. After RNase digestion (0.1 µg RNase Aand 2 units RNase T1/ml at 37°C for 1 hour), the cells were stainedwith 50 µg/ml propidium iodide for at least 30 minutes before analysisby flow cytometry (Becton Dickinson FACSorter) using CELLQuestsoftware. The machine was set to collect 20,000 events. Forwardlight scatter, side light scatter, and DNA content were recordedsimultaneously. During data analysis, the cells distributed in G1, S, orG2/M phase were gated for light scatters. Since the cells distributedin the S or G2/M phase showed a plateau in the parameters of forwardand side light scatters before H2O2 treatment, the data are presentedfrom the cells distributed in G1 phase of the cell cycle.

Phalloidin staining for actin filaments andimmunocytochemical staining for cytoskeletal proteinsControl or H2O2 treated cells were seeded 7 days after H2O2 treatmentonto 12 mm round coverglass (Carolina Biological Supply, NC) in 24-well plates at a density of 1.25×104 cells per well. After culturing foran additional 48 hours, cells that were attached and spread on thecoverglass were fixed by a 15-minute incubation in 5% formalin. After70% (v/v) ethanol and PBS washes, the cells were incubated with 1µg/ml tetramethylrhodamine B isothiocyanate (TRITC)-conjugatedphalloidin for 1 hour with gentle shaking.

For indirect immunocytochemical staining, the cells grown on

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coverglass were fixed with 100% methanol for actin staining or 5%formalin for vinculin or paxillin staining. The coverglass was washedin sequence with PBS twice, PBS containing 0.5% NP-40 once, PBSthree times, and PBS containing 1% bovine serum albumin (BSA)once. The coverglass was incubated for 2 hours with an antibodyspecific to actin (clone AC-40, 1:200, Sigma, St Louis, MO), vinculin(1:500, Sigma, St Louis, MO) or paxillin (1:500, Zymed, South SanFrancisco, CA). A secondary antibody conjugated with fluoresceinisothiocyanate (FITC) was added to bind to the primary antibody for1 hour. The cells on the coverglass were washed with PBS andmounted with 50% glycerol/PBS for fluorescent microscopy.Fluorescence images were obtained with a digital camera attached toa fluorescent microscope using IPlab spectrum software.

Measurement of protein synthesisCells were seeded at a density of 5×104 per well in 24-well plates andtreated with 150 µM H2O2 for 2 hours at 20-24 hours after seeding.Untreated cells or treated cells were incubated in complete mediumcontaining 0.5 µCi/well [3H]leucine (ICN Pharmaceuticals, CostaMesa, CA) for additional 24 hours before fixation with 10%trichloroacetic acid for determination of incorporated [3H]leucine asdescribed (Chen and Ames, 1994).

RESULTS

Inhibition of senescent morphogenesis by HPV E7A pulse treatment with low or mild doses of oxidants causesHDFs to develop features of senescence including growtharrest governed by cell cycle checkpoint proteins anddevelopment of senescent morphology. Since growth arrest iscoupled to senescent morphogenesis, we test whether cell cyclecheckpoint proteins play a role in senescent morphogenesis byexpressing HPV E6 and E7 genes. These genes wereintroduced into HDFs by retrovirus-mediated gene transfer(Crystal, 1995; Sokol and Gewirtz, 1996), which gives nearly100% transduction efficiency. The infection does not interferewith cell proliferation or produce cytotoxicity. To ensure thatthe E6, E7 or E6E7 genes were indeed expressed, we selectedthe cells for at least two weeks by G418 resistance conferredfrom a neo resistant gene under the control of the samepromoter that drives the expression of E6, E7 or E6E7 genesin the pLXSN plasmid. Since almost all cells were resistant toG418, this method allowed us to study the consequence of E6,E7 or E6E7 gene expression in a population of cells withoutclonal selection.

Subconfluent cultures were used in this study because cellsrequire space to develop senescent morphology after H2O2treatment. This condition differs from our previous work inwhich cells were treated with H2O2 after reaching confluenceand becoming quiescent (Chen and Ames, 1994; Chen et al.,1998). Since the response to H2O2 is determined by pmol percell concentration rather than absolute micromolar (µM)concentration, addition of 150 µM (or 0.85 pmol/cell) H2O2into the culture medium is sufficient for inducing senescentmorphology in subconfluent cultures. Wild-type cells, cellsexpressing HPV E6 gene (E6 cells), cells expressing HPV E7gene (E7 cells), or cells expressing both HPV E6 and E7 genes(E6E7 cells) at an equal density (2×106 cells per 100 mm dish)were treated with 150 µM H2O2 for 2 hours. The cells werecollected 20 hours later for measurement of p53 and Rbproteins using western blot analysis. An equal amount ofproteins from different cell samples was loaded to each lane.

Equal loading was verified by staining the gel with Coomassieblue after electrophoresis or by blotting the membrane withan antibody against actin. The results showed that E6expression reduced the level of p53 in untreated and H2O2treated cells (Fig. 1A). HPV E7 reduced levels ofunderphosphorylated Rb and phosphorylated Rb in control orH2O2 treated cells (Fig. 1A,B). In addition to enhancing theproteolytic degradation of Rb, HPV E7 presumably bound toRb family proteins and prevented them from interactingwith their cellular partners (Farthing and Vousden, 1994;Tommasino and Crawford, 1995). E6E7 cells failed to elevatep53 in response to H2O2 and showed some reduction of overallRb protein levels (Fig. 1A,B). Rb was mainly presented in ahyperphosphorylated state after H2O2 treatment in E6E7 cells(Fig. 1A,B). An absence of underphosphorylated (activated)

Fig. 1. Expression of HPV E6 or E7 gene causes reduction of p53 orRb protein in IMR-90 cells. Early passage IMR-90 cells (PDL ≤30)were seeded at a density of 2×106 cells per 100 mm dish. At 20-24hours after seeding, cells were treated with 150 µM H2O2 for 2 hoursand were harvested 20 hours later for determining p53, Rb or actinlevel by western blot with 20 µg protein loaded into each lane (A).The densities of phosphorylated Rb (B, top panel) andunderphosphorylated Rb (B, bottom panel) were quantified by anEagle Eye II Image System.

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Rb was observed in E6E7 cells regardless of H2O2 treatment(Fig. 1A,B).

The cells expressing E6, E7 or E6E7 do not appear to bedifferent from wild-type cells morphologically (Fig. 2A). E6,E7 or E6E7 cells showed similar proliferation doubling timewith wild-type cells. E7 and E6E7 cells can achieve a highersaturation density at confluence than wild-type or E6 cells.Wild-type, E6, E7 or E6E7 cells at similar PDLs were seededat an equal density for H2O2 treatment (Fig. 2A for themorphology of wild-type cells before H2O2 treatment. Themorphology of E6, E7, or E6E7 cells before H2O2 treatmentwas not different from that of wild-type cells). After H2O2treatment, wild-type cells or E6 cells developed senescentmorphology while E7 cells could not do so (Fig. 2A). E6E7cells were also reluctant to develop senescent morphology (Fig.2A). Flow cytometry allows semiquantitative analysis of cellvolume (reflected by X-axis forward light scatter) and cellsurface irregularity (reflected by Y-axis side light scatter) froma large population of cells. The analysis confirmed thereluctance of E7 and E6E7 cells to increase cell volume or cellsurface irregularity in response to H2O2 treatment (Fig. 2B).Since senescent morphology is coupled to cell volumeincrease, we quantified the average cell volume and scored thepercentage of cells showing increased diameters after roundingup the cells by trypsin treatment. At 10 days after H2O2

treatment, wild-type or E6 cells showed about 10-fold increasein cell volume (Fig. 2C, top panel). Similar increase in cellvolume was observed in replicatively senescent cells (Fig. 2C,top panel). In contrast, E7 cells and E6E7 cells failed toincrease cell volume following H2O2 treatment (Fig. 2C, toppanel). Most early passage cells showed diameters of 0.02 to0.03 millimeter (mm). Replicatively senescent cells or H2O2treated early passage wild-type cells show diameters varyingfrom 0.02 to 0.07 mm. We scored the percentage of cellsshowing diameters greater than 0.04 mm as a way to quantifycell enlargement. Using this method, cell enlargement was notobserved in E7 cells (Fig. 2C, bottom panel). E6E7 cellsshowed only a small proportion of enlarged cells in responseto H2O2 treatment (Fig. 2C, bottom panel). The data point tothe possibility that E7 interacting proteins namely Rb familyproteins, but not E6 interacting protein such as p53, play a rolein senescent morphogenesis induced by H2O2.

Since E7 interacts with Rb family proteins as well as a fewother cellular proteins, the E7 mutants that are defective inbinding to Rb family proteins allow us to further test the roleof Rb family proteins in senescent morphogenesis. E7 proteininteracts with Rb family proteins through LxCxE consensussequence at the amino acid position 21-25 (Demers et al., 1996;Farthing and Vousden, 1994; Tommasino and Crawford, 1995).Deletion of amino acids 21 to 24 or mutation at the position24 or 26 reduces the Rb binding activity of E7 (Demers et al.,1996). The d21-24 (deletion of amino acids at the position 21to 24), C24G (replacing cysteine at the position 24 to glycine),and E26G (replacing glutamate at the position 26 to glycine)mutants of E7 gene were introduced into IMR-90 cells by theretroviral technique described above. Expression of d21-24,C24G or E26G did not result in changes in growth rate or cellmorphology. Western blot analysis showed that cellsexpressing d21-24, C24G or E26G mutant E7 gene containedRb protein at the level similar to that of wild-type cells (Fig.3A,B). Like wild-type cells, cells expressing d21-24, C24G orE26G mutant E7 gene contained only underphosphorylated Rbprotein 20 hours after H2O2 treatment (Fig. 3A,B). Unlike E7cells, the cells expressing mutant E7 genes appeared to developsenescent morphology and showed a degree of cell volumeincrease or percentage of enlarged cells similar to that of wild-type cells in response to H2O2 (Fig. 3C,D). These data furtherpoint to the possibility that HPV E7 prohibits senescentmorphogenesis through interaction with Rb family proteins.

Role of cytoskeletal proteins in H 2O2 inducedsenescent morphologyThe drastic change in morphology following H2O2 treatmentsuggests the involvement of cytoskeletal proteins, since actin,focal adhesion proteins and other cytoskeletal proteins buildthe framework of mammalian cells. Phalloidin, a toxinoriginally isolated from the poisonous fungus Amanitaphalloides, binds to polymeric actin. Fluorescence labeledphalloidin allows the determination of intensity andarrangement of actin filaments. Fluorescent phalloidin wasadded to formalin-fixed cells collected at 9-10 days after H2O2treatment. The results indicated that most untreated wild-typecells contained limited actin stress fibers visible at themagnification of 66 times (Fig. 4A). In contrast, H2O2 treatedwild-type cells showing senescent morphology containedextensive actin stress fibers transversing the entire cells at the

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Fig. 2. Inhibition of senescent morphogenesis by HPV E7. Cells atsimilar PDLs (≤35) were seeded at an equal density (2×106 cells/100mm dish). At 20-24 hours after seeding, cells were treated with 150µM H2O2 for 2 hours. The morphology was recorded 7 days laterusing a digital camera attached to a phase contrast microscope andIPlab spectrum software (A). Cells were collected 24 hours or 4 daysafter H2O2 treatment for flow cytometry analysis (B). Cell volume isexpressed as means ± standard deviation from 99 cells whosediameters were measured randomly 10 days after H2O2 treatment or7 days after plating (senescent cells, PDL52) as described inMaterials and Methods (C, top panel). The number of cells withdiameters greater than 0.04 mm was recorded within a group of 33cells for calculating the percentage of enlarged cells from triplicatesamples of one representative experiment (C, bottom panel).

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same magnification (Fig. 4A). Indirect immunocytochemicalstaining with the antibody developed against the conserved C-terminal peptide sequence (Ser-Gly-Pro-Ser-Ile-Val-His-Arg-Lys-Cys-Phe) of all actin isoforms generated the same results(data not shown). Enhanced actin filaments were also observedin replicatively senescent cells (Fig. 4B).

Since H2O2 treatment results in an enhanced actin filamentformation and HPV E7 can prevent H2O2 from inducingsenescent morphology, we test whether E7 expression can

abolish changes in actin following H2O2 treatment. Phalloidinstaining of formalin-fixed cells revealed that E7 cells did notcontain detectable changes in actin filament density, strengthor length after H2O2 treatment (Fig. 4A).

The enhancement of actin filaments in wild-type cellsfollowing H2O2 treatment suggests a possible increase in thelevel of actin protein. Using an antibody that recognizesvarious actin isoforms (e.g. α, β and γ actin), we determinedthe level of actin protein 7-10 days after H2O2 treatment by

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Fig. 3. Rb binding defective E7 cannot inhibit senescentmorphogenesis. Cells at similar PDLs (≤35) were seeded at an equaldensity (2×106 cells/100 mm dish). At 20-24 hours after seeding,cells were treated with 150 µM H2O2 for 2 hours. The cells wereharvested 20 hours later for western blot (A,B), 7 days later forrecording the morphology (C), or 10 days later for measuring cellvolume or percentage enlarged cells (D). The densities ofphosphorylated Rb (B, top panel) and underphosphorylated Rb (B, bottom panel) were quantified by an Eagle Eye II Image System.Cell volume is expressed as means ± standard deviation from 99 cellswhose diameters were measured randomly (D, top panel). Thenumber of cells with diameters greater than 0.04 mm was recordedwithin a group of 33 cells for calculating the percentage of enlargedcells from triplicate samples of one representative experiment (D, bottom panel).

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western blot. The results indicate no changes in the level ofactin protein in wild-type, pLXSN or E7 cells treated withvarious concentrations of H2O2 (Fig. 5).

In addition to actin filaments, focal adhesion plaques are alsoimportant components of cell morphology. The distribution offocal adhesion protein vinculin or paxillin was determined

using an immunocytochemical technique with cells fixed at 9-10 days after H2O2 treatment. The results showed that vinculinand paxillin preferentially distributed at the edge of untreatedcells (Fig. 4C,D), while many H2O2 treated wild-type cellsshowed a sporadic and random distribution of vinculin andpaxillin (Fig. 4C,D). Changes in vinculin distribution were also

Fig. 4. Enhanced actin stress fibers and redistribution of vinculin or paxillin in H2O2 induced senescent-like cells and replicatively senescentcells. Early passage wild-type (wt) IMR-90 cells or IMR-90 cells expressing HPV E7 (A,C,D) were seeded at an equal density (2×106 cells/100mm dish). At 20-24 hours after seeding, cells were treated with 150 µM H2O2 for 2 hours. The cells were seeded onto coverglass in 24-well (2cm2/well) plates at the density of 1.25×104 cells per well 7 days later (A,C,D). A similar density was used for seeding early passage andreplicatively senescent F65 human skin fibroblasts onto coverglass (B). After 48-hour incubation, the cells grown onto coverglass were fixed forstaining actin stress fibers (A,B), vinculin (B,C) or paxillin (D) as described in Materials and Methods. The pictures were acquired by an imagesystem attached to an Olympus fluorescent microscope at the magnification of 66 times (×20 lens).

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Table 1. Distribution of focal adhesion plaques in wild-type or E7 cells treated with H2O2

Vinculin Paxillin

Cells Edge Non-edge Total Edge Non-edge Total

wt ctr 19.8±7.3 2.8±2.3 22.6±9.3 16.8±8.6 2.7±2.1 19.5±9.9wt HP 10.5±9.9 145.2±22.8 155.7±23.9 10.2±8.3 130.7±25.8 140.9±25.7E7 ctr 16.9±8.9 2.1±2.2 18.9±10.3 14.7±6.2 3.3±3.0 17.9±8.3E7 HP 22.7±8.3 3.9±2.9 26.4±9.8 21.5±7.3 5.8±5.3 27.7±10.1

Early passage wild-type (wt) IMR-90 cells or cells expressing HPV E7 were seeded at an equal density (2×106 cells/100 mm dish) and treated with 150 µMH2O2 for 2 hours. The cells were seeded onto coverglass in 24-well dish at 5×104 cells per well 9 days after the treatment. After 48-hour incubation, the cellswere fixed for vinculin or paxillin staining. Each fluorescent spot, rod or stick was counted as one unit under an Olympus fluorescent microscope with ×66magnification (×20 lens). The data are means ± standard deviation from 20 cells in each sample from one representative experiment.

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observed in replicatively senescent cells (Fig. 4B). The numberof vinculin or paxillin foci was quantified to further documentchanges in the distribution of focal adhesion plaques. Table 1shows an overall 7-fold increase in the number of vinculin orpaxillin foci per senescent-like cell compared to untreatedcontrol. While 87% vinculin foci were distributed along theedge of the untreated cells, 93% vinculin foci were notassociated with edge distribution in senescent-like cells (Table1). Similar results were obtained with paxillin (Table 1). Incomparison, E7 cells did not change vinculin or paxillindistribution significantly after H2O2 treatment (Fig. 4C,D;Table 1).

The apparent increase in the number of vinculin or paxillinfoci per H2O2 treated wild-type cell indicated a possibleincrease in the level of vinculin or paxillin protein. When thelevel of vinculin or paxillin was measured by western blot, wedid not observe any significant increase resulting from H2O2treatment in wild-type, pLXSN or E7 cells (Fig. 5).

Senescent morphogenesis requires de novo proteinsynthesisH2O2 is a toxic agent. If senescent morphogenesis is a resultof degeneration after oxidative injury, the process may notrequire de novo protein synthesis. The long time courserequired for the development of senescent morphology and thecorrelation of senescent morphogenesis with the presence ofunderphosphorylated (activated) Rb protein plus changes inthe cytoskeletal proteins indicate the process may be aprogrammed event involving de novo protein synthesis. H2O2at the dose range capable of inducing premature senescencedoes not appear to abolish protein synthesis (Chen and Ames,1994). An increase in the number of vinculin or paxillin fociper H2O2-treated wild-type cell in the absence of an increasein the protein level indicates a possible increase in the overallprotein content per H2O2 treated wild-type cell. We testedwhether de novo protein synthesis was required for themorphological change by placing the cells in methionine-freemedium following a 2-hour treatment with 150 µM H2O2.

[3H]Leucine incorporation was reduced to 40% in untreated orH2O2 treated cells by methionine depletion when measuredduring a 24-hour time period. When morphology was observed7 days after H2O2 treatment, the cells incubated in methionine-free medium did not appear to enlarge (Fig. 6A,B). These datasuggest a role of de novo protein synthesis in the induction ofsenescent morphology by H2O2 treatment.

To determine the relationship between de novo proteinsynthesis and underphosphorylated (activated) Rb in senescentmorphogenesis, we measured protein synthesis rate after

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Fig. 5. Levels of actin, vinculin and paxillin proteins in H2O2 treatedcells. Cells at similar PDLs (≤35) were seeded at an equal density(2×106 cells/100 mm dish). At 20-24 hours after seeding, cells weretreated with 0, 125, 150, or 180 µM H2O2 for 2 hours. The cells wereplaced in fresh medium and cultured for 7 days before harvesting. Anequal amount of protein (10 µg/lane) was loaded into each lane forelectrophoresis and western blot as described in Materials andMethods.

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Fig. 6. Inhibition of senescent morphogenesis by methionine (Met)depletion. Early passage IMR-90 cells (2×106 cells /100 mm dish,PDL ≤30) were treated with 150 µM H2O2 for 2 hours. The cellswere placed in fresh complete medium or Met free medium for 7days following the treatment for determining the morphology (A), cell volume (B, top panel) and percentage of enlarged cells (B, bottom panel). Cell volume is expressed as means ± standarddeviation from 99 cells whose diameters were measured randomly asdescribed in Materials and Methods (B, top panel). The number ofcells with diameters greater than 0.04 mm was recorded within agroup of 33 cells for calculating the percentage of enlarged cellsfrom triplicate samples of one representative experiment (B, bottompanel).

4095Mechanism of senescent morphogenesis

introducing the E7 gene or the status of Rb phosphorylation bymethionine depletion. Introducing the E7 gene into IMR-90cells did not affect the rate of protein synthesis (data notshown). In contrast, H2O2 treated cells showed mainlyunderphosphorylated Rb when kept in complete medium ormethionine-free medium for 20 hours (Fig. 7). The dataindicate that H2O2 treated cells were capable ofdephosphorylating Rb in methionine-free medium.

DISCUSSION

The present study supports that senescent morphogenesis iscontrolled by specific molecular changes. Senescentmorphology develops when H2O2 treated cells are kept inculture for 7 or more days. Among a few early molecularchanges induced by H2O2 treatment are transient elevation ofp53 and prolonged inhibition of Rb phosphorylation (Chen etal., 1998). HPV E6 reduces p53 protein level while HPV E7reduces Rb protein level in HDFs. We found that HPV E7 butnot E6 can abolish senescent morphogenesis.

Rb protein can be inactivated by three mechanisms:hyperphosphorylation, proteolytic degradation and E7 binding.E7 expression in IMR-90 cells results in decreases of Rbprotein levels. This observation is consistent with the report byBoyer et al. (Boyer et al., 1996) showing that E7 expressionenhances proteolytic degradation of Rb protein. In addition toreducing the level of Rb protein, E7 binds to Rb family proteinssuch as Rb, p107 and p130 (Farthing and Vousden, 1994;Tommasino and Crawford, 1995). Upon binding to Rb, E7prevents Rb from interacting with its cellular partnersincluding E2F (Farthing and Vousden, 1994; Tommasino andCrawford, 1995). Although the decrease of Rb protein in E6E7cells is not as dramatic as in E7 cells, the observedhyperphosphorylation and likely E7 binding can inactivate Rbin E6E7 cells. Both E7 cells and E6E7 cells show a shift in thedose response of DNA synthesis inhibition towards higherconcentration compared to wild-type cells (Chen et al., 2000a).Other similarity between E7 and E6E7 cells include failures toundergo G1 arrest immediately, to replicate and to developsenescent morphology after H2O2 treatment (Chen et al., 1998;Chen et al., 2000a). Deletion or mutation at LxCxE consensussequence abolishes the interaction of E7 with Rb familyproteins (Demers et al., 1996). These mutants failed to reducethe level of Rb protein (Fig. 3A,B) or prevent senescentmorphogenesis, indicating a role of Rb family proteins insenescent morphogenesis. The argument that Rb familyproteins play a critical role in senescent morphogenesis issupported by a recent report showing that overexpression of theRb gene can induce senescent morphology in an immortalizedcell line (Xu et al., 1997). However, HPV E7 has been reportedto interact with a number of cellular proteins other than Rbfamily proteins, for example p27KIP1 (Zerfass-Thome et al.,1996), AP1 family transcription factors (Antinore et al., 1996),cyclin E (McIntyre et al., 1996), cyclin A and p33CDK2(Tommasino et al., 1993). HPV E7 expression has been linkedto elevated expression of cyclin A, cyclin E and p21 (Jian etal., 1999; Martin et al., 1998; Schulze et al., 1998; Vogt et al.,1999). Although some of these E7 functions appear to bemediated through its interaction with Rb family proteins(Martin et al., 1998), the mechanisms of how E7 interacting

with these cellular proteins or how E7 affecting the expressionof these cellular proteins are unknown. Because we have notexamined the effect of the E7 mutants on each of these cellularproteins, we cannot exclude the possibility that some of thesecellular proteins may participate in the control of senescentmorphogenesis.

Underphosphorylated (activated) Rb is known to interactwith a large number of cellular proteins (Taya, 1997). Inductionof senescent morphology correlates with the presence ofunderphosphorylated (activated) Rb, indicating a possible roleof Rb-interacting proteins in senescent morphogenesis. Onecandidate is the nuclear tyrosine kinase c-abl (Welch andWang, 1993; Wen et al., 1996). c-Abl tyrosine kinase can beactivated by DNA-damaging agents (Kharbanda et al., 1995;Liu et al., 1996; Welch and Wang, 1993). The activated c-ablcan translocate to the cytoplasm and phosphorylate the FocalAdhesion Kinase and a number of focal adhesion proteins(Salgia et al., 1995; Welch and Wang, 1993). Phosphorylationof focal adhesion proteins is thought to regulate focal adhesionassembly and cell-matrix contact (Hanks and Polte, 1997;Schaller and Parsons, 1994). In addition, c-abl is found capableof binding to actin filaments and cooperating the actin filamentbundling (Van et al., 1994). The presence of Rb appears to becritical for c-abl function (Wen et al., 1996). In parallel withthis c-abl hypothesis, Rb may control senescent morphogenesisthrough interaction with the chromatin modulating factorhBRG1/hBRM (Strober et al., 1996). These proteins are themammalian homologues of the yeast SNF2/SWI2 orDrosophila brmgene and form a large complex that serves toremodel chromatin and facilitates the function of specifictranscription factors (Phelan et al., 1999; Wang et al., 1996).The interaction of hBRG1/hBRM and Rb is known to result incell spreading and enlargement (Strober et al., 1996).Therefore c-abl and hBRG1/hBRM are two possible mediatorsbetween Rb and senescent morphogenesis.

Our data from methionine depletion experiments indicatethat the presence of underphosphorylated (activated) Rb maybe necessary but not sufficient for senescent morphogenesis.Methionine is an essential amino acid for human cells anddepletion of methionine ultimately results in inhibition of cellproliferation. Depletion of methionine alone cannot lead to

Fig. 7. Methionine (Met) depletion does not affect Rbdephosphorylation. Early passage IMR-90 cells (2×106 cells/100 mmdish, PDL ≤30) were treated with 150 µM H2O2 for 2 hours. Thecells were placed in complete medium or Met free medium for 20hours. An equal amount of protein (30 µg/lane) was loaded forelectrophoresis and western blot. The Rb antibody reacts with anonspecific protein, which has a molecular mass lower than that ofRb and serves as a loading control.

4096

senescent morphology but can prevent H2O2 from inducingsenescent morphology (Fig. 6), supporting that senescentmorphogenesis is not simply a consequence of inhibition ofcell proliferation. Overall protein synthesis is likely importantin driving the complex process of morphological change. Thedramatic increases in cell surface area and cell volume indicatean increase in organic cellular content. Since H2O2 treated cellscannot synthesize DNA or replicate but remain capable ofsynthesizing protein, the protein content likely accumulatesand contributes to cell enlargement. Nevertheless, H2O2 hasbeen shown to activate mitogen activated protein (MAP)kinases (Aikawa et al., 1997; Guyton et al., 1996) and proteinkinase C (Konishi et al., 1997), which can lead to changes ingene expression. A number of genes increase their expressionas a result of oxidative stress (Sen and Packer, 1996). Thesesuggest the possibility that synthesis of a particular factor isequally possible as the overall protein synthesis for senescentmorphogenesis following H2O2 treatment.

Senescent morphology correlates with enhanced actin stressfibers and redistribution of focal adhesion plaques in senescent-like cells. Similar changes of actin have been reported (Wangand Gundersen, 1984) and observed here with replicativelysenescent cells (Fig. 4B). Our data further indicate thehomology between H2O2 treated cells and replicativelysenescent cells. Since the protein level of actin does not appearto change, a likely explanation of observed enhancement ofactin stress fibers is an increased rate of actin polymerizationor a decreased rate of actin filament depolymerization in H2O2treated cells. On the other hand, the distribution pattern of focaladhesion plaques explains the observed increases in adhesionand spreading of senescent-like cells or replicatively senescentcells. Rodriguez et al. (Rodriguez et al., 1992; Rodriguez et al.,1993) report that the level of vinculin protein influences thenumber of focal adhesion plaques and cell surface area.Although we did not observe an increase in the overall proteinlevel of vinculin or paxillin by western blot analysis, theimmunocytochemical studies indicated an increase in thenumber of focal adhesion plaques per senescent-like cell. Asdiscussed in the above section, H2O2 treated cells appear to bebigger and may contain more protein per cell than untreatedearly passage cells. This phenomenon may explain thedifference between western blot versus immunocytochemistry,since western blot measures the concentration of a particularprotein in the pool of total cellular proteins whileimmunocytochemistry measures a particular protein in eachindividual cell. Functionally while enhanced actin stress fibersmay construct a frame structure necessary to support theenlarged cells, the increased number of focal adhesion plaquesper cell and their random distribution may be necessary for theenlarged cells to adhere and to spread.

Senescence is thought to be controlled by mechanismsoverlapping with tumor suppression (Campisi et al., 1996;Smith and Pereira, 1996). Changes in cell morphology andadhesion are important parameters of cancer metastasis andinvasion (Button et al., 1995; Gumbiner, 1996). Tumor cells ingeneral are often smaller and less adhesive than their parentalcells. Many tumor cells appear to have less actin stress fibersand often reduce the level of vinculin or the number of focaladhesion plaques compared to their normal counterparts(Button et al., 1995; Otto, 1990; Zigmond, 1996). In contrast,senescent or senescent-like cells are enlarged and contain

enhanced actin stress fibers and an increased number of focaladhesion plaques. From the morphological point of view,senescent or senescent-like cells may contribute to themechanism of tumor suppression simply by cell enlargementand changes in cytoskeletal proteins.

This work was supported by the Burroughs Wellcome NewInvestigator Award (Q.M.C.), the start-up fund from the Departmentof Pharmacology and the Dean’s Research Award from College ofMedicine, University of Arizona (Q.M.C.), Flinn predoctoralfellowship (V.C.T.), Fulbright, CIES, and NATO fellowships (M.B.and O.T.). We are greatly indebted to Dr Denise Galloway for mutantE7 producing cells, Dr John Regan for access to the microscopicimaging system and Juping Liu for technical assistance.

REFERENCES

Aikawa, R., Komuro, I., Yamazaki, T., Zou, Y., Kudoh, S., Tanaka, M.,Shiojima, I., Hiroi, Y. and Yazaki, Y. (1997). Oxidative stress activatesextracellular signal-regulated kinases through Src and Ras in culturedcardiac myocytes of neonatal rats. J. Clin. Invest. 100, 1813-1821.

Antinore, M. J., Birrer, M. J., Patel, D., Nader, L. and McCance, D. J.(1996). The human papillomavirus type 16 E7 gene product interacts withand trans-activates the AP1 family of transcription factors. EMBO J. 15,1950-1960.

Bennett, M. R., Macdonald, K., Chan, S. W., Boyle, J. J. and Weissberg,P. L. (1998). Cooperative interactions between RB and p53 regulate cellproliferation, cell senescence, and apoptosis in human vascular smoothmuscle cells from atherosclerotic plaques. Circ. Res.82, 704-712.

Boyer, S. N., Wazer, D. E. and Band, V. (1996). E7 protein of humanpapilloma virus-16 induces degradation of retinoblastoma protein throughthe ubiquitin-proteasome pathway. Cancer Res.56, 4620-4624.

Button, E., Shapland, C. and Lawson, D. (1995). Actin, its associatedproteins and metastasis Cell Motil. Cytoskel.30, 247-251.

Campisi, J. (1996). Replicative senescence: An old lives’ tale? Cell 84, 497-500.

Campisi, J., Dimri, G. and Hara, E. (1996). In Control of ReplicativeSenescence (ed. E. Schneider and J. Rowe), pp. 121-149. New York:Academic press.

Campisi, J. (1997). Aging and cancer: the double-edged sword of replicativesenescence. J. Am. Geriatrics Soc.45, 482-488.

Chen, Q. and Ames, B. N. (1994). Senescence-like growth arrest induced byhydrogen peroxide in human diploid fibroblast F65 cells. Proc. Nat. Acad.Sci. USA91, 4130-4134.

Chen, Q. M., Bartholomew, J. C., Campisi, J., Acosta, M., Reagan, J. D.and Ames, B. N. (1998). Molecular analysis of H2O2-induced senescent-like growth arrest in normal human fibroblasts: p53 and Rb control G1 arrestbut not cell replication. Biochem. J.332, 43-50.

Chen, Q. M. (2000). Replicative senescence and oxidant induced prematuresenescence: beyond the control of cell cycle checkpoints. Ann. NY Acad.Sci.908, 111-125.

Chen, Q. M., Liu, J. P. and Tu, V. C. (2000a). Measurements of hydrogenperoxide induced premature senescence: senescence-associated β-galactosidase and DNA synthesis index in human diploid fibroblasts withdown-regulated p53 or Rb. Biogerontology(in press).

Chen, Q., Liu, J. P. and Merrett, J. (2000b). Apoptosis or senescence-likegrowth arrest: influence of cell cycle position, p53, p21 and bax in H2O2response of normal human fibroblasts. Biochem. J.347, 543-551.

Craig, S. W. and Johnson, R. P. (1996). Assembly of focal adhesions:progress, paradigms, and portents. Curr. Opin. Cell Biol. 8, 74-85.

Crystal, R. G. (1995). Transfer of genes to humans: early lessons andobstacles to success. Science270, 404-410.

Demers, G. W., Foster, S. A., Halbert, C. L. and Galloway, D. A. (1994).Growth arrest by induction of p53 in DNA damaged keratinocytes isbypassed by human papillomavirus 16 E7. Proc. Nat. Acad. Sci. USA 91,4382-4386.

Demers, G. W., Espling, E., Harry, J. B., Etscheid, B. G. and Galloway, D.A. (1996). Abrogation of growth arrest signals by human papillomavirustype 16 E7 is mediated by sequences required for transformation. J. Virol.70, 6862-6869.

Dimri, G., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C.,

Q. M. Chen and others

4097Mechanism of senescent morphogenesis

Medrano, E., Linskens, M., Rubelj, I., Pereira-Smith, O., Peacocke, M.and Campisi, J. (1995). A biomarker that identifies senescent human cellsin culture and in aging skin in vivo. Proc. Nat. Acad. Sci. USA92, 9363-9367.

Dimri, G. P., Itahana, K., Acosta, M. and Campisi, J. (2000). Regulationof a senescence checkpoint response by the E2F1 transcription factor andp14(ARF) tumor suppressor. Mol. Cell. Biol.20, 273-285.

Farthing, A. J. and Vousden, K. H. (1994). Functions of humanpapillomavirus E6 and E7 oncoproteins. Trends Microbiol. 2, 170-174.

Gumbiner, B. M. (1996). Cell adhesion: the molecular basis of tissuearchitecture and morphogenesis. Cell 84, 345-357.

Guyton, K. Z., Liu, Y., Gorospe, M., Xu, Q. and Holbrook, N. J. (1996).Activation of mitogen-activated protein kinase by H2O2 – role in cell survivalfollowing oxidant injury. J. Biol. Chem.271, 4138-4142.

Hanks, S. K. and Polte, T. R. (1997). Signaling through focal adhesion kinase.BioEssays19, 137-145.

Hayflick, L. and Moorhead, P. S. (1961). The serial cultivation of humandiploid cell strains. Exp. Cell Res.25, 585-621.

Hemmings, L., Barry, S. T. and Critchley, D. R. (1995). Cell-matrixadhesion: structure and regulation. Biochem. Soc. Trans23, 619-626.

Herman, I. M. (1993). Actin isoforms. Curr. Opin. Cell Biol.5, 48-55. Jian, Y., Van Tine, B. A., Chien, W. M., Shaw, G. M., Broker, T. R. and

Chow, L. T. (1999). Concordant induction of cyclin E and p21cip1 indifferentiated keratinocytes by the human papillomavirus E7 protein inhibitscellular and viral DNA synthesis. Cell Growth Differ.10, 101-111.

Kassem, M., Ankersen, L., Eriksen, E. F., Clark, B. F. and Rattan, S. I.(1997). Demonstration of cellular aging and senescence in serially passagedlong-term cultures of human trabecular osteoblasts. Osteoporosis Int.7, 514-524.

Kharbanda, S., Ren, R., Pandey, P., Shafman, T. D., Feller, S. M.,Weichselbaum, R. R. and Kufe, D. W. (1995). Activation of the c-Abltyrosine kinase in the stress response to DNA-damaging agents. Nature376,785-788.

Konishi, H., Tanaka, M., Takemura, Y., Matsuzaki, H., Ono, Y., Kikkawa,U. and Nishizuka, Y. (1997). Activation of protein kinase C by tyrosinephosphorylation in response to H2O2. Proc. Nat. Acad. Sci. USA94, 11233-11237.

Lee, A. C., Fenster, B. E., Ito, H., Takeda, K., Bae, N. S., Hirai, T., Yu, Z.X., Ferrans, V. J., Howard, B. H. and Finkel, T. (1999). Ras proteinsinduce senescence by altering the intracellular levels of reactive oxygenspecies. J. Biol. Chem.274, 7936-7940.

Liu, Z. G., Baskaran, R., Lea-Chou, E. T., Wood, L. D., Chen, Y., Karin,M. and Wang, J. Y. (1996). Three distinct signalling responses by murinefibroblasts to genotoxic stress. Nature384, 273-276.

Martin, L. G., Demers, G. W. and Galloway, D. A. (1998). Disruption ofthe G1/S transition in human papillomavirus type 16 E7-expressing humancells is associated with altered regulation of cyclin E. J. Virol. 72, 975-985.

McIntyre, M. C., Ruesch, M. N. and Laimins, L. A. (1996). Humanpapillomavirus E7 oncoproteins bind a single form of cyclin E in a complexwith cdk2 and p107. Virology 215, 73-82.

Mitchison, T. J. and Cramer, L. P. (1996). Actin-based cell motility and celllocomotion. Cell 84, 371-379.

Ogryzko, V. V., Hirai, T. H., Russanova, V. R., Barbie, D. A. and Howard,B. H. (1996). Human fibroblast commitment to a senescence-like state inresponse to histone deacetylase inhibitors is cell cycle dependent. Mol. Cell.Biol. 16, 5210-5218.

Otto, J. J. (1990). Vinculin. Cell Motil. Cytoskel. 16, 1-6. Phelan, M. L., Sif, S., Narlikar, G. J. and Kingston, R. E. (1999).

Reconstitution of a core chromatin remodeling complex from SWI/SNFsubunits. Mol. Cell 3, 247-253.

Rodriguez, F. J. I., Geiger, B., Salomon, D. and Ben, Z. A.(1992).Overexpression of vinculin suppresses cell motility in BALB/c 3T3 cells.Cell Motil. Cytoskel. 22, 127-134.

Rodriguez, F. J. I., Geiger, B., Salomon, D. and Ben, Z. A.(1993).Suppression of vinculin expression by antisense transfection confers

changes in cell morphology, motility, and anchorage-dependent growth of3T3 cells. J. Cell Biol. 122, 1285-1294.

Salgia, R., Brunkhorst, B., Pisick, E., Li, J. L., Lo, S. H., Chen, L. B. andGriffin, J. D. (1995). Increased tyrosine phosphorylation of focal adhesionproteins in myeloid cell lines expressing p210BCR/ABL. Oncogene11,1149-1155.

Schaller, M. D. and Parsons, J. T. (1994). Focal adhesion kinase andassociated proteins. Curr. Opin. Cell Biol.6, 705-710.

Schulze, A., Mannhardt, B., Zerfass-Thome, K., Zwerschke, W. andJansen-Durr, P. (1998). Anchorage-independent transcription of the cyclinA gene induced by the E7 oncoprotein of human papillomavirus type 16. J.Virol. 72, 2323-2334.

Sen, C. K. and Packer, L. (1996). Antioxidant and redox regulation of genetranscription. FASEB J.10, 709-720.

Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. and Lowe, S. W.(1997). Oncogenic ras provokes premature cell senescence associated withaccumulation of p53 and p16INK4a. Cell 88, 593-602.

Smith, J. R. and Pereira, S. O. (1996). Replicative senescence: implicationsfor in vivo aging and tumor suppression. Science273, 63-67.

Sokol, D. L. and Gewirtz, A. M. (1996). Gene therapy: basic concepts andrecent advances. Crit. Rev. Euk. Gene Exp.6, 29-57.

Strober, B. E., Dunaief, J. L., Guha and Goff, S. P. (1996). Functionalinteractions between the hBRM/hBRG1 transcriptional activators and thepRB family of proteins. Mol. Cell. Biol.16, 1576-1583.

Taya, Y. (1997). RB kinases and RB-binding proteins: new points of view.Trends Biochem. Sci. 22, 14-17.

Tommasino, M., Adamczewski, J. P., Carlotti, F., Barth, C. F., Manetti, R.,Contorni, M., Cavalieri, F., Hunt, T. and Crawford, L. (1993). HPV16E7 protein associates with the protein kinase p33CDK2 and cyclin A.Oncogene8, 195-202.

Tommasino, M. and Crawford, L. (1995). Human papillomavirus E6 and E7:proteins which deregulate the cell cycle. BioEssays17, 509-518.

Van, E. R., Jackson, P. K., Baltimore, D., Sanders, M. C., Matsudaira, P.T. and Janmey, P. A. (1994). The COOH terminus of the c-Abl tyrosinekinase contains distinct F- and G-actin binding domains with bundlingactivity. J. Cell Biol.124, 325-340.

Vogt, B., Zerfass-Thome, K., Schulze, A., Botz, J. W., Zwerschke, W. andJansen-Durr, P. (1999). Regulation of cyclin E gene expression by thehuman papillomavirus type 16 E7 oncoprotein. J. Gen. Virol.80, 2103-2113.

von Zglinicki, T., Saretzki, G., Docke, W. and Lotze, C. (1995). Mildhyperoxia shortens telomeres and inhibits proliferation of fibroblasts: amodel for senescence? Exp. Cell Res.220, 186-193.

Wang, E. and Gundersen, D. (1984). Increased organization of cytoskeletonaccompanying the aging of human fibroblasts in vitro. Exp. Cell Res. 154,191-202.

Wang, W., Cote, J., Xue, Y., Zhou, S., Khavari, P. A., Biggar, S. R.,Muchardt, C., Kalpana, G. V., Goff, S. P., Yaniv, M., Workman, J. L.and Crabtree, G. R. (1996). Purification and biochemical heterogeneity ofthe mammalian SWI-SNF complex. EMBO J.15, 5370-5382.

Welch, P. J. and Wang, J. Y. (1993). A C-terminal protein-binding domainin the retinoblastoma protein regulates nuclear c-Abl tyrosine kinase in thecell cycle. Cell 75, 779-790.

Wen, S. T., Jackson, P. K. and Van, E. R. (1996). The cytostatic function ofc-Abl is controlled by multiple nuclear localization signals and requires thep53 and Rb tumor suppressor gene products. EMBO J.15, 1583-1595.

Xu, H. J., Zhou, Y., Ji, W., Perng, G. S., Kruzelock, R., Kong, C. T., Bast,R. C., Mills, G. B., Li, J. and Hu, S. X. (1997). Reexpression of theretinoblastoma protein in tumor cells induces senescence and telomeraseinhibition. Oncogene15, 2589-2596.

Zerfass-Thome, K., Zwerschke, W., Mannhardt, B., Tindle, R., Botz, J. W.and Jansen-Durr, P. (1996). Inactivation of the cdk inhibitor p27KIP1 bythe human papillomarivus type 16 E7 oncoprotein. Oncogene 13, 2323-2330.

Zigmond, S. H. (1996). Signal transduction and actin filament organization.Curr. Opin. Cell Biol.8, 66-73.