alterations of enzymes associated with plasma - cancer research

9
ICANCER RESEARCH 35. 199 206. January 19751 SUMMARY The formation of cellular aggregates (foci) in CV-l cells following infection with Yaba tumor poxvirus is dependent upon cell passage level, temperature of incubation, and calcium concentration in the medium. Resistance of older cells can be reversed by maintaining calcium at 0. 1m@ior by adding cortisone acetate (I @zg/ml), hydrocortisone, or estradiol-l7f3 to the cultures. In susceptible cells, foci formation was inhibited slightly by methyltestosterone and inhibited completely by dexamethasone, aldosterone, and progesterone. Activities and patterns of enzymes associated with cytoplasmic membranes (alkaline phosphatase, mono nucleotidase, and Na@-K@-adenosine triphosphatase) and lysosomes (fl-glucuronidase and acid phosphatase)of the younger susceptible and the older resistant CV-l cells differed. These differences apparently occurred in concert with phenotypic changes in the membranes that reduced the mobility of older resistant cells. In susceptible cultures, uninfected cells migrated to the infected cell and partici pated in foci formation. Reduction ofthe calcium content to 0.1 m@i apparently removed some of the constraints on mobility of the resistant cells. Although the hormones may have had a similar effect, the changes in enzyme patterns indicated basic alterations in protein synthesis. The develop ment of resistance to foci formation occurred between the 45th and 50th passage level. Hormonal reversal of this resistance resulted in enzyme profiles that reflected the pattern of young susceptible cells. INTRODUCTION Yaba tumor poxvirus produces benign histiocytomas in primates and in man. The virus replicates in primary cell cultures of rhesus (1), cynomologus (16), Cercopithecus (18), and human origin (18). Foci develop in approximately 16 ±3 days after infection in stationary susceptible cul tures of the Cercopithecus monkey kidney cell line, CV-l. It has been proposed (13, 21) that these foci (areas of piled up cells), which have been called â€oemicrotumors―(13, 18), develop owing to migration of uninfected cells into the lo @ This work was supported in part by Grant CA-I 1613-04 from the USPHS and Grant 3192-Al from the Ohio Division of the American Cancer Society. Received December 20. 1973: accepted September 25, 974. calized infected area. Mitosis occurs in uninfected cells at the periphery of the foci. No evidence exists that produc tively infected cells proliferate (21). In view of the lack of evidence of neoplastic proliferation and/or transformation in the infected foci, it would seem appropriate to describe them as cellular â€oemicroaggregates― rather than micro tumors. If formation of microaggregates by Yaba tumor pox virus-infected cells involves chemical changes in cell mem branes, these events should be reflected in phenotypic changes in enzymes associated with the plasma membranes of the cells involved in their formation. Analyses of such changes are described in this report. MATERIALS AND METHODS Cell Culture. Passage of CV- I cells was performed as described previously (19). Cultures were classified as re sistant (R) or susceptible (S). Resistant cultures that were made susceptible by reducing calcium concentration were labeled (R-S). A 20% extension in the time of appearance of foci after infection was selected as the passage level when cells were no longer classifiable as susceptible. Passage levels up to Passage 35 were totally susceptible to virus in fections without manipulation. Cultures beyond Passage 35 were classifiable as R or R-S. The induction of susceptibil ity in the R-S cultures was accomplished by reducing the total calcium content of the complete media to 0. 1 m@i. In R cultures the total calcium content of the medium was maintained at 0.5 mM. When coverslip cultures were required, the cells were seeded at l0@ cells/sq cm and inoculated with virus at preconfluency, 48 hr after seeding the coverslips. The cultures were overlaid with Medium 199 in Hanks' balanced salts supplemented with 2% fetal calf serum. These cultures were incubated at 35°in a 5% carbon dioxide environment. Virus Preparation. Yaba tumor poxvirus used in these studies had been isolated by Yohn et a!. (19). The partial purification of this virus was described by Yohn and Gallagher (18). The virus was in its 17th passage in CV-l cells and contained 5.2 x l0@ FFU2/ml. For virus produc tion CV-l cells were infected at a multiplicity of 10 FFU /cell. 2 The abbreviations used are: FFU, focus-forming units: 5'-MNase, 5'-mononucleotidase. JANUARY 1975 199 Alterations of Enzymes Associated with Plasma Membranes and Cellular Organelles during Infection of C V-i Cells with Yaba Tumor Poxvirus' George E. Milo, Jr., and David S. Yohn Department of Veterinar@'Pathobiologv. College of Vezerinart Medicine. The Ohio Stale Universitt, Columbus, Ohio 43210 Research. on December 26, 2018. © 1975 American Association for Cancer cancerres.aacrjournals.org Downloaded from

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ICANCER RESEARCH 35. 199 206. January 19751

SUMMARY

The formation of cellular aggregates (foci) in CV-l cellsfollowing infection with Yaba tumor poxvirus is dependentupon cell passage level, temperature of incubation, andcalcium concentration in the medium. Resistance of oldercells can be reversed by maintaining calcium at 0. 1m@ior byadding cortisone acetate ( I @zg/ml), hydrocortisone, orestradiol-l7f3 to the cultures. In susceptible cells, fociformation was inhibited slightly by methyltestosterone andinhibited completely by dexamethasone,aldosterone, andprogesterone. Activities and patterns of enzymes associatedwith cytoplasmic membranes (alkaline phosphatase, mononucleotidase, and Na@-K@-adenosine triphosphatase) andlysosomes (fl-glucuronidase and acid phosphatase)of theyounger susceptible and the older resistant CV-l cellsdiffered. These differences apparently occurred in concertwith phenotypic changes in the membranes that reduced themobility of older resistant cells. In susceptible cultures,uninfected cells migrated to the infected cell and participated in foci formation. Reduction ofthe calcium content to0.1 m@i apparently removed some of the constraints onmobility of the resistant cells. Although the hormones mayhave had a similar effect, the changes in enzyme patternsindicated basic alterations in protein synthesis. The development of resistance to foci formation occurred between the45th and 50th passage level. Hormonal reversal of thisresistance resulted in enzyme profiles that reflected thepattern of young susceptible cells.

INTRODUCTION

Yaba tumor poxvirus produces benign histiocytomas inprimates and in man. The virus replicates in primary cellcultures of rhesus (1), cynomologus (16), Cercopithecus(18), and human origin (18). Foci develop in approximately16 ±3 days after infection in stationary susceptible cultures of the Cercopithecus monkey kidney cell line, CV-l.It has been proposed (13, 21) that these foci (areas of piledup cells), which have been called “microtumors―(13, 18),develop owing to migration of uninfected cells into the lo

@ This work was supported in part by Grant CA-I 1613-04 from theUSPHS and Grant 3192-Al from the Ohio Division of the AmericanCancer Society.

Received December 20. 1973: accepted September 25, 974.

calized infected area. Mitosis occurs in uninfected cells atthe periphery of the foci. No evidence exists that productively infected cells proliferate (21). In view of the lack ofevidence of neoplastic proliferation and/or transformationin the infected foci, it would seem appropriate to describethem as cellular “microaggregates― rather than microtumors.

If formation of microaggregates by Yaba tumor poxvirus-infected cells involves chemical changes in cell membranes, these events should be reflected in phenotypicchanges in enzymes associated with the plasma membranesof the cells involved in their formation. Analyses of suchchanges are described in this report.

MATERIALS AND METHODS

Cell Culture. Passage of CV- I cells was performed asdescribed previously (19). Cultures were classified as resistant (R) or susceptible (S). Resistant cultures that weremade susceptible by reducing calcium concentration werelabeled (R-S). A 20% extension in the time of appearanceof foci after infection was selected as the passage level whencells were no longer classifiable as susceptible. Passagelevels up to Passage 35 were totally susceptible to virus infections without manipulation. Cultures beyond Passage 35were classifiable as R or R-S. The induction of susceptibility in the R-S cultures was accomplished by reducing thetotal calcium content of the complete media to 0. 1 m@i. InR cultures the total calcium content of the medium wasmaintained at 0.5 mM.

When coverslip cultures were required, the cells wereseeded at l0@ cells/sq cm and inoculated with virus atpreconfluency, 48 hr after seeding the coverslips. Thecultures were overlaid with Medium 199 in Hanks' balancedsalts supplemented with 2% fetal calf serum. These cultureswere incubated at 35°in a 5% carbon dioxide environment.

Virus Preparation. Yaba tumor poxvirus used in thesestudies had been isolated by Yohn et a!. (19). The partialpurification of this virus was described by Yohn andGallagher (18). The virus was in its 17th passage in CV-lcells and contained 5.2 x l0@FFU2/ml. For virus production CV-l cells were infected at a multiplicity of 10FFU /cell.

2 The abbreviations used are: FFU, focus-forming units: 5'-MNase,

5'-mononucleotidase.

JANUARY 1975 199

Alterations of Enzymes Associated with Plasma Membranes andCellular Organelles during Infection of C V-i Cells withYaba Tumor Poxvirus'

George E. Milo, Jr., and David S. Yohn

Department of Veterinar@'Pathobiologv. College of Vezerinart Medicine. The Ohio Stale Universitt, Columbus, Ohio 43210

Research. on December 26, 2018. © 1975 American Association for Cancercancerres.aacrjournals.org Downloaded from

G. E. Milo, Jr., and D. S. Yohn

Fractionation of the Cellular Material on 8% AcrylamideGel. Infected and uninfected CV-l cultures, which remained untreated or were treated with hormones, werewashed with 100 ml of Medium 199 immediately beforetrypsinizing the cell sheet with I ml of 0.2% trypsin(Worthington lypholized trypsin with a specific activity of240 p-tosyl-L-arginine methyl ester units/mg of protein) for3 mm or 0.2% collagenase-0.Ol% trypsin solution for 30 mm(12). The tryptic action was inhibited by the addition of 0.1ml of fetal calf serum. The cell suspension was centrifugedat 600 x g for 25 mm and the pellet was washed twice. Thefinal pellet was resuspended in 2 ml of 0. 1 m@ Tris-HC Icontaining 0.1 mM calcium acetate at 4°. The cellularsuspension was homogenized in a Tri-R homogenizer with20 to 25 strokes of a pestle with a 0.006-inch clearance.After homogenization, the suspension was centrifuged at1000 x g for 10 mm to remove intact cells.

One-tenth ml of the homogenate or cellular membranepreparation was adjusted to 200 @tgof protein in 20%sucrose containing 0.1 M Tris-HCI at pH 7.0, 0.1% TritonX-lOO, and 0.01 ml 2 m@imercaptoethanol: this was layeredover the 8% small-pore acrylamide gel under buffer. Thecurrent was maintained at 2 ma/tube until the proteinentered the gel. At that time the current was raised to 4ma/tube for 4.5 hr. The gels were then prepared for enzymelocalization or fixed in 20% sulfosalicylic acid overnight andstained with 0.25% Coomassie blue (7).

Enzyme Assay on the 8% Gels. The gels selected to beassayed for the presence of specific enzymes were usedwithin 4 hr after completion of the electrophoretic run. Gelsto be assayed for alkaline phosphatase or acid phosphatasewere run in a phosphate-free buffer. The gels were polymerized and preelectrophoretically extracted in the phosphatefree buffer before the addition of the sample.

Gels were assayed for esterase (EC 3. 1.3) activity (4) witha-naphthyl acetate as a substrate: for fl-glucuronidase (EC3.2. I .3 1) activity (4) with 6-bromo-2-naphthyl-j3-D-glucuronide as a substrate; for 5'-MNase (EC 3.1.3.5) (4) in thepresence of 0.1 mM magnesium and AMP: for membraneassociated potassi um-sodi um-dependent AT Pase ( EC3.6. 1.3) (4) with ATP present: for alkaline phosphatase (EC3. 1.3. I ) ( I I ) with @-naphthyl phosphate sodium salt presentat pH 7.8: for acid phosphatase (EC 3.1.3.2) (4) witha-naphthyl phosphate (sodium salt) at pH 5.0. The P1released in the gels from the ATP, AMP, and a-naphthylphosphate sodium salt after the action of the enzymes on thesubstrates was captured by lead (Pb@@ ) and the lead wasprecipitated with ammoniacal sulfide (2, 4). Proteins werelocated on the gels after fixation of the gels in 10%sulfosalicylic acid with Coomassie blue (4).

Quantitation of Enzyme Activities. After localization ofthe enzymes on the gels, other gels were cut into 2-mm sliceswith a razor blade and the protein was extracted in 3.0 ml of0.1 @iNa2PO4-(Tris-HCI) pH 6.8, or as indicated in theassay. The 3.0-mI extraction medium was recovered bycentrifugation at 6000 x g in a Seprafuge tube (CitationOccomy Association, Chicago, Ill.). To assay the enzymesin theseextracts, the appropriate substrateswereaddedandthe change in absorbance units was measured on a Zeiss

PMQ-II spectrophotometer. Three readings for each assaywere performed on each extract. We routinely recovered85% of the protein from the gels in these areas.

In the region on the gel where acid phosphatase waslocated, the acid phosphatase band was extracted with 3.0ml of 0. 15 M sodium acetate, pH 5.0, and assayed with 3.65x l0@ M o-carboxyphenyl phosphate (3); alkaline phosphate band(s) were extracted (5) with 3.0 ml of0.2 Mglycinebuffer, pH 8.8, and assayed with 3.65 x l0@ @io-carboxyphenyl phosphate as the substrate: 5'-MNase bands wereextracted into 3 ml of 0. 1 Mglycine-NaOH buffer at pH 9.0and assayed in the presence of AMP (2): Na@-K@-ATPaseband(s) were extracted into 3 ml of 0. I @iTris-HCI, pH 7.5,and assayed with 0.005 m@i disodium-ATP (2) in thepresence of 5 mM MgCl2. The Na@-K@-ATPase is defined asthe difference between total ATPase activity and Mg@ATPase activity. The P1 was determined using the methodof Fiske and Subbarow (9). The reaction velocities of eachof these enzyme substrates was measured at various substrate concentrations and the Vmax was determined experimentally. Concentrations were well over the Vmax concentration for each enzyme. The amount of nonspecific phosphatase in each of the above assays was determined usingp-nitrophenyl phosphate in 0. 1 M glycine-NaOH at pH 9.5in the presence of Mg@ . Units of activity were expressed asunits of activity per ..@A280nm of enzyme solution per mg ofprotein.

Protein content was measured by the method of Lowry eta!. (10) using bovine serum albumin as the standard. Thereactions were stopped by adding a 10% (v/v) volume ofcold (4°)trichloroacetic acid to the reaction vessels, and theP1 was assayed as described above.

The esterase activity was assayed by a potentiometricprocedure (6). f3-Glucuronidase activity was assayed in thepresence of0.0l @iphenolphthalein glucuronate (8).

Preparation of Cytoplasmic Membranes. Con11uentmonolayers were treated with 0.5% collagenase (140 units/mg)/0.05% trypsin (250 p-tosyl-t.-arginine methyl esterunits/mg) (Worthington Biochemical Corp., Freehold, N.J.), pH 6.8, dissolved in basal medium Hanks' salts. Thecells were washed, as desceibed above, before they weresuspended in calcium acetate and centrifuged at 600 x g.The pellet was suspended in 1.2 ml of0.Ol M EDTA in 0.02M Tris-HCI, pH 7.0, at 4° and homogenized until 90% of

the cells were disrupted. The suspension was centrifuged for10 mm at 400 x g over 2 ml of 2 M sucrose. The upper layerwas withdrawn with a Pasteur pipet and examined microscopically. If any nuclei were present, the centrifugationprocess was repeated. The interphase was washed twice andrecentrifuged over a 2 @isucrose cushion. The supernatantsolutions were combined and adjusted to a 45% (w/w)sucrose solution. Three ml were layered on the bottom ofeach I3-ml tube and 2.5-mI volumes of 35, 30, 25, and 20%sucrose were layered over the sample. The gradient was thencentrifuged in a L-2-(65) B Beckman ultracentrifuge at70,000 x g for 16 hr at 3°.The gradient was fractionatedinto 1.6- to 1.8-mi volumes. Each fraction was diluted 1:4with 0. 1 M Tris-HC I, pH 7.0, and centrifuged at 70,000 x gfor I hr at 4°(2). The pellets were suspended in 0.2 ml of

200 CANCER RESEARCH VOL. 35

Research. on December 26, 2018. © 1975 American Association for Cancercancerres.aacrjournals.org Downloaded from

ConcentrationCation(mM)FFU/mIpaMagnesium0.01

0.10.50

00<0.001

<0.001<0.001Calcium0.01―

0.10.50.74.6

x2.3x0

10' ±108102±25

0<0.001

NS'<0.01

<0.001Manganese0.01

0.10.50

00K0.00l

<0.001<0.001

Effects of Yaba Tumor Poxvirus on P!asma Membrane Enzymes

Table I

Influence ofdivaleni caiion.s onfoci formation bt Yaha turnorpoxvirus inR-S culture of CV.! cells

Infected cultures were incubated at 33°until the primary foci formed.The cultures were infected at a multiplicity of 10 FFU/celI from a virusstock of 5.0 x 10' FFU/ml. The values represented here are the meanvalues from 4 Leighton tubes for each concentration of cation for 2experiments.

0.05 M Tris-HCI, pH 7.0. lfthere was any acid phosphataseactivity presentin the preparations, thesepreparations werererun. When no acid phosphatase activity was detected inthe membrane preparations they were prepared for chromatography. Fractions from above the pellet were stored at— 19°.

The membranes were dissolved in the Triton X-lOOphosphate buffer or Triton X-lOO phosphate-free buffer.The membrane solution was added to the tops of 8.0%acrylam ide gel columns and electrophoretically fractionated as described above.

Addition of Hormones to Resistant Cell Systems. Cultures were infected at a multiplicity of I or 10 in mediumcontaining 0.1 mM calcium as described above, and hormones were added to the infected cells. The hormones usedin this study were cortisone acetate, progesterone, hydrocortisone, methyltestosterone,aldosterone,estrone, and dexamethasone. The hormones were solubilized in ethanol oracetone and incorporated into the medium at I @.tg/ml(14).The medium was changed every 3 or 4 days until focideveloped in the control dishes.

Addition of Divalent Cations to Cultures. Magnesiumchloride and manganese chloride (Fisher Scientific Co.,Pittsburgh, Pa.) were dissolvedin the overlay media at I m@iand added to the media deficient in these ions at theappropriate concentrations.

RESULTS

Influence of Cations on Foci Formation. CV- 1 cell passages listed as resistant to virus infection were completelyresistant after 45 passages in vitro. Once the cells wereresistant to virus infection at 0.5 m@icalcium, lowering thecalcium (Ca@@ ) concentration to 0. 1 m@ calcium increasedsusceptibility. Manganese (Mn@) or magnesium (Mg@)(Table I) would not substitute for calcium. Both cationstotally inhibited foci formation from 0.01 to 0.5 [email protected]@@ and/or Mg@@ were, combined with Ca@@ at 0.01 mMCa@@ and 0.01 to 0.5 mM Mn4@ or Mg@4, both cationstotally inhibited foci formation.

Influence of Steroids on Foci Formation in Resistant(R-S)CV-I Cells. Steroids were selected for their glucocorticoidor mineralocorticoid activity or as a representative hormonewith estrogenic or androgenic activity. Results obtainedwith cultures infected at a multiplicity of I werecomparableto those results obtained at a multiplicity of 10. The culturestreated with cortisone acetate ( I @zg/ml)exhibited nearly a5-fold increase in the number of foci. The morphologicalappearance of foci in 5- and in R-S-infected cultures isshown in Fig. 1. The cells comprising the foci in R-Scultures appeared more rounded than did those in Scultures. Foci were surrounded by fibroblast cells orientedtowards the focus.

Similar morphological types of foci were seen in R-Sinfected cultures treated with hydrocortisone, methyltestosterone, or estradiol- I7fl. The frequency of foci formation inR-S cultures was significantly enhanced by cortisone acetate(I j@g/ml), estradiol- I 7fl, and to a lesser degree by hydrocortisone (Table 2). While methyltestosterone ( I @zg/ml)

(, Expected FFU value was 5.2 x 10' based on simultaneous titration insusceptible CV-l cells (t test).

h The medium, containing 0.01 msi calcium, contained sufficient

dialyzed fetal calf serum, with a total calcium content of 0.6 msi, to bringthe culture to 0.01 mM calcium. This medium was fortified with I xvitamins, I x essential amino acids, and 200 m@ glutamine.

@ Mean ±S.D.(I NS, not significant.

slightly inhibited formation of foci, complete inhibitionoccurred with progesterone, aldosterone, dexamethasone,and estrone; estrone was also completely inhibitory at 0.1and 0.01 @g/ml.In experiments not reported here, aldosterone, dexamethasone, progesterone, and estrone completelyinhibited focus formation in susceptible(S) cells.

Changes in Enzyme Activities Associated with Cytoplas-.mic Membranes and Lysosomesduring Foci Formation at35°. The influence of Yaba tumor poxvirus infection onactivities of the membrane-associated enzymes 5'-MNase,alkaline phosphatase, and sodium-potassium-ATPase areshown in Table 3, while effects on the lysosomal enzymes,acid phosphatase and fl-glucuronidase, and of microsomalenzyme esterase are shown in Table 4. The influence of 0.1mM calcium with and without cortisone acetate (I @zg/ml)also are shown. Statistical comparisons are shown in Table5. The 5'-MNase, alkaline phosphatase, and Na@-K@ATPase activities were determined for cytoplasmic membrane preparations. @-Glucuronidase activity and acid phosphatase activity were assayed on total cellular extracts.Esterase activity was assayed on both membrane preparations and total cellular extracts; the values presented inTable 4 were obtained with the total cellular extracts.

Activities of membrane-associated enzymes 5'-M Naseand ATPase from uninfected R and S cells did not varystatistically as measured by analysis of variances (Table 5, Ftest) between the 2 cell types (Table 3). Furthermore, theisoenzyme patterns of 5'-MNase and ATPase (Chart 1, Aand C) did not differ. In addition, these same enzymeactivities from the uninfected R-S cells cultured in low-calcium media and infected R-S cells cultured in the same

JANUARY 1975 201

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FoldSteroid@zg/mlFFU/ml

(6 samples)increase(+

decrease ()or—)p'Noneb2.0

x l0@:1:131'CortisoneacetateI5.7x l0@±240+2.9<0.01HydrocortisoneI3.2x 10' ±171+1.60.05ProgesteroneI0<0.001MethyltestosteroneI1.2

x l0@±176—0.6>0.5AldosteroneI0<0.001Estrone1.0.1,0.010<0.001DexamethasoneI0<0.001Estradiol-l7fl14.3

x l0@±176+2.20.01

G. E. Mb, Jr., and D. S. Yohn

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Effects ofselected corticoid and steroid hormones on formation of Yaha tumor poxvirus-inducedfoci in R-S CV-! cells

aExpected FFU value was 5.2 x l0@based on simultaneous titration in susceptible CV-l cells(t test).

h Resistant cells rendered susceptible by reducing Ca' ‘ to 0.1 msi were used in this experiment.

In a similar experiment with young susceptible cells similar results were obtained.â€M̃ean ±S.D.

low-calcium media were not statistically different (F test).The isoenzyme patterns of 5'-MNase and ATPase fromboth cultures were the same (Chart lE). (The pattern forR-S uninfected cells is not shown.)

The addition of cortisone acetate to uninfected R-Scultures markedly enhanced 5'-MNase and ATPase activities. However, infection of these treated cells with virus

reduced the differences to near base line (uninfected) levels(Table 3). These enzyme activities in the S cultures wereelevated following infection but only the increase in ATPaseactivity was significant (Table 5). The levels of ATPase and5'-MNase in infected S cells were essentially identical tothose of cortisone acetate-treated infected R-S cells.

The extracts from uninfected R-S cells that were treated

CANCER RESEARCH VOL. 35202

Fig. I. A, foci formation in susceptible cells at Passage 25 (5) inoculated at a multiplicity of 0 FFU/cell. Time offormation ofthese foci was I I ±2days. B, formation of foci in R-S cultures grown in Medium 199, Hanks' salts, 2% fetal calf serum, and cortisone acetate ( I @zg/ml)at 0. I msi total calcium required 17 days. x 400.

Table 2

Research. on December 26, 2018. © 1975 American Association for Cancercancerres.aacrjournals.org Downloaded from

Activity/mgprotein5'-MNaseAlkaline

phosphataseATPaseCulture―(pgP/hr)(pmoles/hr)(@.tgP/hr)Resistant

(P-45)UninfectedR

—0.5 [email protected] ±4.2k15.0 ±6.26.2 ±2.3R-S'—0.1 msiCa -36.2 ±4.60.07.3 ±1.5R-S

+ cortisone58.2 ± 6.214.0 ±3.117.5 ±4.6IInfectedR-S2

0.1 msiCa -40.1 ± 3.70.09.8 ±3.2R-S+ cortisone44.2 ± 3.88.0 ±1.812.2 ±2.6acetate2Susceptible

(P- I9)Uninfected5'35.0 ±4.623.0 ±4.78.3 ±1.6Infected

[email protected] ±5.64.0 ±1.114.6 ±2.4

Activity/mgproteinfl-GlucuronidaseAcid

phosphataseEsteraseCulture―(nsi/hr)(psi/hr)(@z@i—H/min)Resistant

(P-45)UninfectedR

—[email protected]±2.lh19.3±4.167.0±13.1R-S'—0.1 msi Ca- -19.1 ±1.727.2 ±4.372.1±8.2R-S

+ cortisone14.1 ±3.225.1 ±3.264.0±6.4acetate'InfectedR-S2

0.1 m@iCa -19.8 ±4.128.7 ±3.860.0±3.7R-S—cortisone24.3 ±3.133.1 ±5.650.1±8.2acetate2Susceptible

(P-l9)UninfectedS'12.0±2.817.0±2.785.1±7.8Infected

S222.1 ±2.834.3 ±4.136.0 ±4.1

Effects of Yaba Tumor Poxvirus on Plasma Membrane Enzymes

with cortisone acetate contained 5 and 4 isoenzymes of5'-MNase and ATPase activity, respectively, while only 3and 2 isoenzymes were detected in the extracts from lowpassage cells (Chart I, B and C). Patterns of5'-MNase andATPase in infected S and infected RS cortisone acetatetreated cultures suggested that both calcium and cortisoneacetate, in addition to the virus, effected changes in theisoenzyme patterns (Chart I, D to F). The reproducibility of

these values for membrane-associated enzymes wasachieved when total cell numbers from all cultures werewithin 90% and fractionation of the cells followed theregimen described in “Materials and Methods.― Thesecultures did not exhibit cell number variation due toincrease in cell size with age in cell culture or an increase inthe nondividing population until Passage 92.

Alkaline phoaphatase activities differed significantly be

Table 3

En:Lrne activities in membrane preparations of uninfected and infected CV-l cells at low passage(5) and high passage levels (R) or made susceptible(R-S) to virus infection

See text for details of assays from membrane preparations. The values reported here are means of3 assays for 3 extracts from 3 gels for a total of 12 gels per tissue from one 733-sq cm roller bottlerepeated 3 times.

‘SR, R-S, S represent Passages P-45 R, R-S, and P-19 S. The R cultures were cultured in Medium

199 containing 0.5 m@icalcium. R-S cultures were cultured in Medium 199 containing 0.1 msicalcium, and S cultures were cultured in Medium 199 in 0.5 mM calcium. ‘,uninfected cultures forusein Table 5; @,infectedcultures for usein Table 5.

h Mean ± S.D.

Table 4

En:vme activities ofen.:@vmesassociated with intracellular organelles after treatment with cortisoneacetate and/or Yaba tumor poxvirus infection

See text for details of enzyme assay for cellular homogenates.

“R, R-S, S represent Passages P-45 R. R-S, and P-l9 S. The R cultures were cultured in Medium199 containing 0.5 msi calcium, R-S cultures were cultured in Medium 199 containing 0.1 msicalcium, and S cultures were cultured in Medium 199 in 0.5 m@calcium. ‘,uninfected cultures forusein Table 5; @,infectedcultures for usein Table 5.

h Mean ± S.D.

JANUARY 1975 203

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Cultureap5'-MNaseAlkaline phosphataseATPase$-GlucuronidaseAcidphosphataseEsteraseI

testResistant

(P-45)U ninfected

(R)vs.(R-S)'(R) vs. (R-S) + CA'(R-S)'vs.(R-S)+CA'

Infected(R-S)2vs.(R-S)+CA2(R-S) + CA' vs. (R-S) +(5)' vs. (5)2CA2NS5

<0.001<0.001

NSNSNS<0.001

NS<0.001

<0.0010.05

<0.001NS

<0.01<0.001

NSNS

<0.001NS

0.05NS

NS<0.001<0.001NS

NSNS

NSNS

<0.001NS

NSNS

NSNS

0.05F

test(R)vs.(S)'

(R-S) + CA2 vs. (5)2NS NSNS 0.05NS NSNS NSNS NSNS 0.05

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CANCER RESEARCH VOL. 35204

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115.MP4A5@][IIIIESTERASE]111ACEIPHASE1I

III!ATP.ATL7

G. E. Mio, Jr., and D. S. Yohn

Table 5

Statistical significance of mean activity values of enzyme activitie.s from normal and infected cultures

See text for details of enzyme assays.

‘,R, R-S, S represent Passages P-45 R, R-S, and P-l9 S. The R cultures were cultured in Medium 199 containing 0.5 mM calcium,R-S cultures were cultured in Medium 199 containing 0.1 mM calcium, and S cultures were cultured in Medium 199 in 0.5 msicalcium. ‘,uninfected cultures: 2, infected cultures.

h NS. not significant: CA. cortisone acetate.

Chart I . Representation of electrophoreticpatterns of cellular extracts on 8% acrylamidegel run in a phosphate or phosphate-freebuffer as described in “Materialsand Methods.―P.30 or P-70, passagelevel or doublingnumber based upon serial subcultivation of the _______________________cells. A , an electrophoretic pattern of a cellfree extract of normal tissue at P.70 separatedon an 8% acrylamide gel as described in“Materialsand Methods―(R). B, an eletro -_________________________phoretic pattern of a cell-free extract of normal tissue at P-70, grown in the presence of0. 1 msi calcium and I @gcortisone acetate per

ml of complete medium (R-S). C, an electro -_________________________phoretic pattern of a cell-free extract of normal tissue at P-30 (5). D, an electrophoreticpattern of a cell-free extract of Yaba tumorvirus-infected cultures at P-70. The infected _________________________ _________________________cultures were incubated in the presence of 0. ImM Ca@@ and I @gcortisone acetate per ml ofcomplete medium (R-S). E. an electropho -_________________________retic pattern of a cell-free extract of virusinfected cultures at P-70. The infected cultureswere incubated at 0.1 mM Ca@@ (R-S). F, anelectrophoretic pattern of a cell-free extract ofvirus-infected cultures at P-30 (5). The migration of the protein was from the anode ( —) tothe cathode ( + ). The top diagram in each caseis a protein profile of a cellular extract stained

with Coomassie blue. These diagrams areextract replicas of results obtained from65-mm gels. Each bar on the enzyme profilesrepresents a specific chromophore location ofenzyme activities (“Materialsand Metheds'@).Each bar represents I extraction. ALKPHASE, alkaline phosphatase: ACIDPHASE, acid phosphatase.

A. P.70(R) B. P-70(R-S) C. P-30 (S)

D. P-70 (R-S) Infected

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Effects of Yaba Tumor Poxvirus on Plasma Membrane Enzymes

Mg@@ was added to 0. 1 mM Ca@@ at concentrations from0.01 to 0.5 mM, foci formation was inhibited. These resultsare similar to those noted in transformation by adenoviruses(14), where calcium concentration is an important majorfactor in the expression of viral-induced transformation.

The addition of cortisone acetate to R-S cultures stimulated formation of foci. Substitution of dialyzed fetal calfserum for undialyzed serum did not alter the effects. Otherhormones that stimulated foci formation included estradiol- 17$ and hydrocortisone. Methyltestosterone reducedfoci formation by nearly 50%, while progesterone, estrone,dexamethasone, and aldosterone completely inhibited formation of foci. Estrone killed S cells at I zg/ml but not at0. 1 zg/ml. Cells tolerated all other hormones at 1 @zg/ml.Estrone completely inhibited foci formation in R-S infectedcultures at concentrations as low as 0.01 zg/ml. Thehormonal effects obtained here are similar to those obtainedwith hormone treatment during adenovirus-12 transformation of hamster cells in vitro (14). The glucocorticoid orestrogen hormones may act antagonistically or synergistically with the divalent calcium cation in this system.Cortisone acetate, hydrocortisone, or estradiol-l7@3 maypromote foci formation by augmenting calcium transport orutilization, although many other alternative mechanismsare conceivable.

The glucocorticoids that stimulated formation of foci ininfected R-S cultures have been reported to inhibit hydrolase activity in lytically infected cells. Examination ofchanges in enzyme activities of membrane-associatedenzymes and protein patterns by acrylamide gel electrophoresis in infected S and R-S cultures indicated that membraneprotein composition changed during foci formation. Forexample, alkaline phosphatase activity in infected R-Scultures virtually becameundetectable.The rearrangementand/or formation of additional membrane-associated isoenzymes in infected cultures strongly indicated alterations infunctional capacity associated with these cells. The formation of additional isoenzymes of Na@-K@-ATPase couldhave a differential effect on the cell transport system.

Enhancement of foci formation with cortisone acetate,hydrocortisone, or estradiol-l7f3 at pharmacological levelsmay be associated with alterations in cell membranes (17).The nucleus of the steroid may become packed in themembrane so that its physiological activity is correlatedwith terminal groups of the molecules. This packing occursin a definite order. With a change in the structure of thecytoplasmic membrane, additional receptor sites for thevirus may be produced, resulting in an increasein susceptible cells. Changes in the composition of membraneassociated enzymes following steroid treatment were notedsupporting the feasibility ofthis rearrangement hypothesis.

If the temperature of incubation of Yaba tumor poxvirusinfected cultures was raised to 37°,lysis ofthe infected cellsin the focus occurred. However, examination of the changesin @3-glucuronidaseactivities of infected cultures at thesetemperatures suggested that the cells should have lysedduring foci formation at the lower temperature of 33°.Examination of the acid phosphatase isoenzyme patterns ofinfected cultures, whether of the R-S class or S class,revealed that acid phosphataseactivity increasedas a result

tween S uninfected, S infected, and R-S infected cultures.The S uninfected cultures contained 53% more activity thandid R uninfected cultures, but no detectable change wasobserved in the enzyme patterns (Chart I, A and C).Infection of S cultures resulted in a marked decrease inalkaline phosphataseactivity. Infected cortisone acetatetreated R-S cultures showed less activity than did R-Suninfected cultures (Table 3) but, in contrast, showed adefinite increase in activity when compared to the R-Suntreated infected cultures. Again, the RS cultures exhibited differential effects due to the low calcium and/orcortisone acetate in the medium in the presenceor absenceof the virus. The addition of cortisone acetate to infectedcultures, while increasing the activity of the enzyme, did notalter isoenzyme patterns.

0 HThe enzyme esterase (R—II—O—C—R')generally found

C Hassociated with the microsomal fraction was identified inour membrane preparations. Repeated centrifugation ofthe membrane pellet over the discontinuous gradient didnot decrease the activity. The esterase activity in S infectedcells decreasedin activity (Table 4), while in the R-S infected cultures there were no apparent significant changesin activity. Esterase patterns in the gels revealed 2 isoenzymes in the uninfected cultures and 4 bands in the infectedcultures. The intensity of the bands varied with treatmentwith calcium or cortisone acetate; this occurred in uninfected and infected cultures. This suggests that these 2agents affected various responses within the cell as well asin the cell membrane as noted above.

Activities of enzymes associated with lysosomes. of Sinfected cells increased significantly in the infected cultures(Table 4). The increase in acid phosphatase activity of Sinfected cultures could be due to the formation of isoenzymes (Chart I , C and F). While the total acid phosphataseactivity of the infected treated R-S cultures was notsignificantly greater than the activity of the controls (R-Sand cortisone acetate uninfected: Table 4), the isoenzymesformed only when the cells were infected. The isoenzymepatterns in R-S infected cells was similar to the isoenzymepatterns of S infected cells (Chart 1, D to F). Anotherlysosomal hydrolase enzyme, fl-glucuronidase, increasedsignificantly in the infected S cultures but not significantlyin R-S infected cultures. There appearedto be no markedchanges in the migration pattern ofthe enzyme (Chart I, Dto F).

DISCUSSION

The formation of foci in CV-l cells following infectionwith Yaba tumor poxvirus is temperature dependent (19,

20), requiring incubation at 35°or less. Normally resistantCV-I cells, even at 33°,can be made susceptible, R-S, byproper adjustment of Ca@ concentration (13) and, asshown herein, by the addition of steroid hormones.

Study of the effect of the addition of Mg@, Mn@ @,orCa , ions individually and together on foci formation inR-S cultures indicated that neither Mn@ nor Mg@@wouldsubstitute for Ca* + in the overlay medium. When Mn@@ or

JANUARY 1975 205

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G. E. Milo, Jr., and D. S. Yohn

of the formation of isoenzymes. Infected cultures that weretreated with cortisone acetate also formed similar isoenzymes. If treatment with the hormone did not prevent theformation of the isoenzymes in the lysosomes of infectedcultures, it may have prevented the distribution of theisoenzymes throughout the cell during infection and therebyprevented lysis of the cells. These cells do not divide oncethey form the focus, and when removed from culture bytrypsin they do not reattach and grow.

The type of cellular migration by uninfected cells into afocus is probably analogous to the migration of histiocytesinto a site of Yaba virus infection in vivo (15). It appearsthat the migration of adjacent cells into a focus to formmicroaggregates is stimulated by a chemotactic factor. Thischemotactic factor can be isolated from the membranes ofYaba-infected cells and has been tentatively identified as aglycoprotein (G. E. Milo, Jr., and D. S. Yohn, in preparation). The marked disturbances in cellular proteins andmembrane-associated enzyme patterns of infected cellsindicate that structural alterations in membranes probablyoccurred. These results are interpreted as evidence that thephenotypic result, i.e., formation of a focus, can occur afteralterations in the structure of the cytoplasmic membranes.Apparently, these alterations can be augmented or controlled by administration of selected hormones to infectedcells. In the case of Yaba poxvirus, these effects may bemore relevant to inflammation than to viral-induced malignant transformation. However, the same hormones thatenhance Yaba poxvirus foci formation also enhance transformation of hamster cells by adenovirus 12 ( 14), and thosethat inhibit the formation inhibit in both systems. Similarobservations have been obtained during transformation ofvarious cells by SV4O and by feline sarcoma virus (D. S.Yohn el a!., unpublished data). Further studies are warranted to distinguish whether 2 separable processes invirus-host cell interactions are influenced by steroid hormones in similar ways.

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CANCER RESEARCH VOL. 35206

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1975;35:199-206. Cancer Res   George E. Milo, Jr. and David S. Yohn  Tumor PoxvirusCellular Organelles during Infection of CV-1 Cells with Yaba Alterations of Enzymes Associated with Plasma Membranes and

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