Cyclic AMP specifically blocks proliferation of rat 3T3 cells transformed by polyomavirus.

0022-538X/87/051546-06$02.00/0

Copyright

© 1987, AmericanSocietyforMicrobiology

Cyclic AMP

Specifically

Blocks Proliferation of Rat 3T3

Cells

Transformed

by

Polyomavirus

NEDIAKAMECH,* ROLAND SEIF,ANDDOMINIQUE PANTALONI

Laboratoired'Enzymologie, Centre National de la Recherche

Scientifique,

91190

Gif-sur-Yvette,

France Received 28July 1986/Accepted 22January 1987

Elevated exogenous and intracellular levels ofcyclicAMP couldtotallyblockproliferationofpolyomavirus (PyV) transformants derived fromrat3T3cells withoutaffecting proliferationof normalcellsorsimianvirus 40 (SV40)-induced transformants. Concanavalin A (ConA) had the opposite effect; it could totally block proliferation of both normal cells and SV40 transformants but reducedproliferationofPyVtransformantsonly twofold. Adenylatecyclasewasthreefold less active in membranes ofPyV

transformants,

and the numberof ConAreceptors was similar tothatof normal cells.

Proliferating

PyVtransformants contained threefold less cyclic AMP than didproliferatingSV40 transformants. The

sensitivity

tocyclicAMP did not correlate withthe degree of transformation: cells transformed by Rous sarcoma virus and tumor cells derived from

SV40

transformants were not sensitivetocyclicAMP. The differential effect of

cyclic

AMP and ConAonproliferation wasprobably due to theactivityof anintact middletprotein.Thepresence of both

large

T and smallttogether withmiddle t was also required forcyclicAMP

sensitivity.

The cell membrane andelementsofthecytoskeletonatthe membrane level are targets for several viral and cellular oncogenes as well as tumor promoters(1, 2,8, 11, 13, 18,19, 21, 25). The cytoskeleton is postulated to associate at the membrane level withcertain glycoproteinstoforman assem-bly that regulates cell proliferation (3). Two agents associ-ated withthecell surface andatleastindirectlyconnected to thestructure andfunction of the cytoskeletonareknownto affect cell proliferation: cyclic AMP (cAMP) and thelectin concanavalin A(ConA).

cAMPis responsiblefor the regulation of diverse impor-tant cellularprocesses (14). Itisproduced atthemembrane level, and itsonly knownmoleculartarget is theactivation of cAMP-dependent kinases, an effect that provokes an en-hancement of protein phosphorylation (27). When added to certain cell cultures, cAMP inhibits proliferation, restores contact inhibition and the anchorage requirement, modifies cellmorphology, and increases adhesivenessto thesubstrate (9, 14-17, 23). Withothercelltypes,elevatedlevelsofcAMP haveanopposite effect; it is required for or evenstimulates cellproliferation(4, 5, 17).

The lectin ConAis capable of binding to various surface glycoproteins. ConA binding to the cell surface causes aggregation of itsownreceptors(patching and capping) and induces a redistribution ofcytoplasmic microtubules. Low doses ofConA stimulate mitogenesis oflymphocytes, and microtubule-disrupting agents inhibit this activity; on the otherhand, highdoses of ConA that induce capping inhibit mitogenesis. ConA can also inhibit proliferation of certain transformed cells(3, 12, 26).

We investigated the effects of cAMP and ConA on the proliferation of rat 3T3 cells and their transformed deriva-tives.

MATERIALS AND METHODS

Cells, medium, and drugs. Rat 3T3 cells and their trans-formed derivativesinduced either by simian virus 40

(SV40)

or polyomavirus (PyV) were previously described (18, 19,

*Corresponding author.

21, 22). Rous sarcoma virus transformants were

kindly

provided by G. Calothy, Institut Curie, Orsay, France. Bovine papillomavirus transformants were a gift from G. Meneguzzi, Centre de Biochimie, Nice, France. MTT4t14,

4MTLTltg,

and FRPBClt3 tranformants were kindly pro-vided by F. Cuzin, Centre de Biochimie. All cells were grown in Dulbecco modified Eagle medium (DMEM) sup-plemented with 10% newborn calf serum. 8-Bromo-cAMP sodium salt (molecular weight, 430) was purchased from SigmaChemical Co., St. Louis, Mo., and a stock solution 10-2 M was made in DMEM. ConA type IV (molecular weight of monomer, 25,000) was from Sigma, and a stock solution of 4 x 10-6 M was made in DMEM. ConAcoupled to rhodamine was purchased from IBF-France. Theophyl-line(molecularweight, 180) and prostaglandin E2(molecular weight, 352)werefromSigmaand weredissolved in DMEM or dimethyl sulfoxide, respectively. Stock solutions were 0-1 M for both theophylline andprostaglandin E2. cAMP, 1251-labeled cAMP, and antibodies against cAMP were from NewEngland Nuclear Corp., Boston, Mass.

Cell proliferation. Actively proliferating cultures (1,500 cells percm2)were treated with thedrug. Proliferation was defined as thelogarithm of the ratioofthe number of cells after 3 days of incubation with the drug to the number of cells on the day the drug was added.

Adenylate cyclase activity. Adenylatecyclase activity was assayed essentially as previously described (24); 4 x 106 cells proliferating in 12 150-cm2 flasks were washed with phosphate-buffered saline and lysed in 1 mM sodium bicar-bonate (pH 9.2). The lysate was centrifuged for 30 min at '27,000 x g, and the pellet containing the membrane frag-ments wassuspended in 500 ,ul of 50 mM Tris (pH 7.6). The reaction mixture(final volume, 60

RI)

contained 50 mM Tris (pH 7.6), 2 mM MgCl2, 1 mM ATP, 2 x 106 cpm of [a-32P]ATP,3 mMcAMP, 2 x

104

cpm of[3H]cAMP,25 mM creatine phosphate, 1 mg of creatinephosphokinaseper ml, and50,ug ofmembraneproteins.Thereactionmixture was incubated at 33°C for 5 to 50 min, and the reaction was stopped by the addition of 200 ,ulof0.5 M HCl followed by incubation for 5 min at95°C. The solution was then neutral-izedwithimidazole(200,ul,1.5M) and passed over a neutral 1546

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0

0

o

Cyclc

AMP

z

2

50

I.-4 0*3T3

1L1 v *SV3T3

ILLPY3T3

0

2 6 10 14 1

-CONCENTRATION( 10 M)

FIG. 1. Effect of cAMP on proliferation of normal cells andSV40 orPyV transformants. Proliferation is expressed as a percentage of thatof untreated control cultures.

alumine (aluminum oxide 90 active, neutral for column chromatography [Merck]) column(1 g) thatretainsall nucle-otides. The cAMP was then eluted with 3 ml of 10 mM imidazolehydrochloride (pH 7.6) and counted. The recovery oftritiatedcAMPvariedatabout 30%.

Radioimmunoassay of intracellular cAMP. The radioim-munoassay of intracellular cAMP was done as described previously(7).Proliferating cellswerecollectedby scraping and

lysed

by sonication in 1.5ml of1 mM sodium bicarbon-ate (pH = 9.2). The proteinsin the lysate were precipitated withtrichloroacetic acid. Thetrichloroacetic acidwas then extracted with ether, and the supernatant was treated with acetic anhydride and triethylamine. Samples of the extract were incubated overnight at 4°C with antibodies against cAMP in the presence of

251I-labeled

cAMP (7). Immune complexes were collected on charcoal

particles,

and the associated radioactivity wasdeterminedin aliquid scintilla-tion counter. Standard curves were generated by

using

unlabeled cAMP.

Flow cytometry.

Living

cells attached to the substrate wereincubated for30minwith ConAcoupledtorhodamine (10-6 M), washed with

phosphate-buffered

saline,

sus-pended,and individuallyanalyzed

by

flowcytometrywithan Epics V system cytometer. Rhodamine wasexcited at556 nm, and the

emitted

fluorescence wasmeasuredat576nm.

RESULTS

Effect of cAMP onproliferationof normalandtransformed cells.Concentrations ofcAMP

ranging from

2 x 10-5to20x

l0-5

M were added to actively proliferating cultures of normal rat 3T3 cells and SV40 or

PyV

transformants. The effect of cAMP on cell

proliferation

was assessed 3 days (Fig. 1) and 10days

(Fig. 2)

later. A concentration of 6 x

10-5 M cAMP

totally

blocked

proliferation

of

PyV

trans-formants but did not affect

proliferation

of either normal

cells or SV40 transformants. At very high concentrations, cAMP slightly affected proliferation of rat 3T3 cells. The SV40 transformants in the presence of cAMP not only could proliferate well but also could accumulate beyond conflu-ence and reach a high saturation density (Fig. 2).

cAMPat a concentration of 6 x

10-5

Mor higher blocked proliferation (days 1 and 2) and then caused cell detachment and death by day 3. At 4 x

10-5

M, proliferation was seriously inhibited but no cell detachment occurred after 3 days. Prostaglandin E2 (10-4M) and theophylline (1.25 x

l0'

M),twoagentsknown to raise the intracellular concen-tration of cAMP (14), had a similar effect.

Six additional cell lines transformed by either SV40 or PyV (18) and all isolated in the soft-agar assay were tested for their ability to proliferate in the presence of

10-4

M cAMP(Table 1). All cell linestransformed by PyV failed to proliferate when the medium wassupplemented with cAMP. To further assess whether the block by cAMP was a general phenomenon in PyV-induced transformants, soft-agartransformation assays wereconducted in the presence of cAMP addedto the medium 7 days after viral infection. cAMP wasadded1weekafterviralinfection to determine its effect specifically on the maintenance of transformation without interference with the establishment step. Rat 3T3 cellswere infected with either PyV or SV40 and suspended in soft agar (Table 2). After 1 or 2 months, respectively, manyindependently induced transformants appeared in the cultures without cAMP infected with either SV40 or PyV. WithPyV no transformants weredetected when cAMP was added to the culture 1 week after viralinfection. With SV40 the numbers of induced transformants were similar in the presence and absence ofcAMP.

Elevated intracellular levelsofcAMPtherefore appear to be incompatible with proliferation of PyV transformants.

A

o

B

FIG. 2. Cultures of normaland transformed cellsincubatedwith 10- McAMPfor 10days. (A) Untreatedcells; (B)treated cells. Cultures: 1, normal cells; 2, SV40 transformants; 3, PyV transformants.

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TABLE 1. Proliferation ofvirus-induced transformants ofrat3T3 cells inthepresenceofcAMPorConA

Proliferation'inDMEM with: Cell linea No cAMP ConA (15 x

additions (10-4M) 10-7M)

WT-PyV-2 11.5 0.1c 7.5

WT-PyV-21 12.2 0.05C 8

WT-PyV-13 11.8 0.1C 8.3

WT-SV40-1 7 11.3 1

WT-SV40-11 7.5 10.8 1.2

WT-SV40-12 9.1 10.4 0.8

dl23-PyV-2310 7.8 10.2 1.2

dl23-PyV-2316 8.1 11.4 0.9

dl23-PyV-2319 7.2 11.8 1.1

aWT,Wild type.

bProliferation is the ratio of the celldensityafter 3daysofincubationat

33°C to the initialseedingdensity.

cNinety percent of the cells had detachedand couldnotproliferatewhen replated in the absence of cAMP.

This effectwas observed notonly under conditions restric-tivefornormal cell growth(suspensioninsoftagar)but also in sparse cultures seeded onplastic, conditionsunder which normalcells canactively proliferate.

Adenylate cyclase activity incell membranes and intracel-lularcAMP. TheintracellularlevelofcAMPisregulatedon the onehand by the synthetic activity ofadenylate cyclase and on theotherhand by thedegradation

activity

of phos-phodiesterase (14). We assayed the membrane-associated adenylate cyclase activity in actively

proliferating

normal and transformed cells. Adenylate cyclase in membranes of PyV-transformed cells was three times less active than in membranes of normal cells or SV40 tranformants (Fig. 3). Moreover, PyV transformants contained threefold less cAMP thandid SV40 transformants (Fig. 4).

EffectofConA onproliferation of normal and transformed cells.Various concentrations of ConAranging from2 x

10'

to20 x

10'

Mwereaddedtoproliferatingrat3T3cellsand SV40orPyVtransformants. Theeffectonproliferationwas examined3 (Fig. 5)and 10 days later(Fig. 6). A concentra-tion of15 x

10-7

MConA totally inhibited proliferation of normal and SV40-transformed cells but reduced the prolif-eration of PyV transformants only twofold (Fig. 5). Atthis concentration the PyVtransformants notonly were able to proliferate but could accumulate in multiple layers beyond confluence (Fig.6).

Six additional SV40 and PyV transformants were

exam-TABLE 2. Numberofcoloniesin soft-agar transformation assay inthe presenceofcAMP or ConAa

Time (wk) No. of colonies in DMEMwithc:

Transfomn ftermg

virusb after No cAMP ConA(15x

infection additions (10-4 M)

1o-7

M)

WT-SV40 8 78 85 0

WT-PyV 4 95 0 101

dl23-PyV 8 62 71 0

aProliferating3T3 cellswere infected while on plastic and then suspended

insoftagar 4 h after viralinfection.

bWT-SV40 was strain 776, WT-PyV was strain A2, and dl23 was isolated

byGriffin and Maddock (6). The multiplicity of infection was 100 PFU per

cell.WT, Wild type.

ccAMP orConA wasadded to the medium 1 week after infection and

maintaineduntil the colonies were counted.

0~~~~~~~~~~~~~ 6

E

CL

4

0 0

ind(al 1) The thre 304trnsomat coldno

proliferate

when ConA

(15

x

10-7

M)

was added to the medium. A transformation

soft-agar

assay was also con-ducted with either

PyV

orSV40 in the presence of ConA added 1 week after infection

(Table

2).

Cultures infected with SV40 gave rise to many

independent

transformants in theabsenceof ConAbut tonone in the presenceofConA. On theother

hand,

culturesinfected with

PyV

gave riseto transformants both in the presenceand absenceofConA.

PyV

transformants were therefore less sensitive than SV40 transformants to the

inhibitory

effect of ConA on

proliferation.

ConA sites on the cell surface. The number of ConA receptorsonthesurface ofnormalcells and

PyV

transform-ants was determined

by

flow cytometry. ConA

coupled

to rhodamine

(10-1

M)

was added to the culture medium of

0.

0

0 0 0

0.

.06

.04

0 50 100 150

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number of cells(

103)

FIG. 4. Titrationof cAMPinactivelyproliferating PyVorSM40

transformants.

,8 mw SV3T3

I

11~~~~~

Py3T3

'

to I

o

I I

.06

.04.

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200

h.~~~~~~o.

0 0

z - 50

m 0~~3T3

WU *Sv3T3

U. ~VPy3T3

0

cc

0

2 6 10 14 18

CONCENTRATION (

167M

) FIG. 5. Effect of ConA on proliferation of normal cells and SV40 orPyVtransformants. Proliferation is expressed as a percentage of thatof untreated control cultures.

proliferating cellsand incubated for30 min. The cells were then detached and individually analyzed for fluorescence. Actively proliferatingnormal cellsdifferedfrom oneanother in the numberof ConA receptors ontheir surface (Fig. 7). Thiswasalsotruefor thePyVtransformants. When normal cells were compared with transformed

cells,

the overall

distribution

of cells relativetotheirabilitytobind ConAwas similar. The higher resistance of PyV transformants to the inhibitory effect of ConA did not result from a reduced numberof ConAreceptors ontheir surface.

EffectsofConA and cAMP oncell morphology. A concen-tration of15 x

1i-7

MConAnot only prevented prolifera-tion of normal cells but also induced adramatic

change

in cell morphology. The cells became extensively elongated. Thesameconcentration

had

noeffectonthe

morphology

of PyV transformants. A concentration of

10-4

M cAMP

in-FIG. 7. ConA sites on the surface of normal cells and PyV transformants. Cell distribution is shown as a function of the total amount of fluorescence per cell. Upper and lower histograms: normal and transformed cells,respectively. Abscissa and ordinate: fluorescence and cell number,respectively. BU and BL: upper and lower gates, respectively.

duced slight elongation of normal rat 3T3 cells but did not interfere with their proliferation.

PyV middle t protein wasrequired for thedifferentialeffects of cAMP and ConA on cellproliferation.Cellstransformedby a mutant ofPyV (d123) that does not encode a full-sized middle t protein display a less intense transformed pheno-type andappear similar toSV40transformants(6, 18). They proliferate more slowly andhave mildermembrane-related modifications than do wild-type-PyV transformants. The effects of cAMP and ConA on the proliferation of three transformed cell lines induced by d123 were analyzed. The three cell lines could proliferate well in the presence of cAMP

(10-4

M)but not inthe presenceof ConA(15 x 10-7 M) (Table 1). Normalcells infected with d123 andscreened for transformedcoloniesintheagar assay

yielded

transform-TABLE 3. Proliferation of various transformed cells,derived fromrat3T3cells, in the presence ofcAMP

Proliferationa in:

Cell line DMEM + cAMP

DMEM (10-4M)

SV40-A30-Tulb 9.8 10.3

SV40-A30-Tu9b 13 12

SV40-A30-Tullb 11.2 13.4

RSV-3T3-cllc 10 11

RSV-3T3-c12c 12 10.8

RSV-3T3-c13c 10.4 12.5

BPV-3T3d 7.9 7.4

MNNG-3T3-clle 6.8 7.2

MNNG-3T3-cl5e 7.4 6.9

aDefined in Table1,footnoteb.

bTumorcellsderived from in vitro-inducedSV40transformants(22).

cIndependentcelllineinducedbyatemperature-sensitive Roussarcoma

virus.

dCells transformedbybovinepapillomavirus.

eCells transformedbymethylnitronitrosoguanidine.

PIG. 6. Cultures of normal and transformedcells incubatedwith 15t IO-- MConA for10days. Cultures: 1, normalcells;2,SV40

traasfoimants;

3,PyV transformants.

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TABLE 4. Proliferation ofrat3T3cells,transformedbyDNA transfection and notexpressingone or moreof three viral

proteins, in the presence of cAMP Proliferationb:

Cell linea(protein(s) expressed) DMEM + cAMP

DMEM (10-4M)

MTI4tl4(middle t) 6.2 5.3

FRPBClt3 (middletand smallt) 10 6.29 4MTLTltg(middletandlarge T) 6 4.52

aThe cell lines were generously provided by F. Cuzin, Centre de

Biochimie.

bProliferationis the ratio ofthecelldensityafter2daysofincubationat

33°Ctothe initialseedingdensity.

ants in the presence of cAMP but not in the presence of ConA(Table2).

Cells that displayed an intense transformed

phenotype

comparable tothat ofPyV transformants(transformed cells induced byRous sarcomavirusor tumorcellsderivedfrom SV40 transformants) were not sensitive to cAMP. More-over, cells transformed by bovine papillomavirus or the chemicalcarcinogen methyl nitro

nitrosoguanidine

werealso insensitiveto cAMP (Table 3).

Rat 3T3 cells transformed by DNAtransfection and ex-pressing either middletandlarge T, middletand smallt,or middletalone were notfully sensitiveto cAMP,

indicating

that both large T and small t are also

required

for cAMP sensitivity (Table 4).

DISCUSSION

Rat 3T3

cells,

an immortal cell line derived from a rat

embryo, have the

capacity

to

limit

their

proliferation

under certain conditions invitro(20). They can

permanently

lose this property after transformation by SV40 or PyV. PyV-induced transformed derivatives of rat 3T3 cells

display

a more intense transformed phenotype than do their SV40-induced counterparts (18). They proliferate faster under conditions restrictive for normal cell

proliferation,

and

they

have more profound membrane alterations. This enhance-ment of the transformed phenotype is due to an active middletprotein codedby PyV butnotby SV40(18). Middle tprotein canbe found insertedatthe inner face ofthe cell membraneandisassociatedwiththecellularsrcproteinthat possesses akinaseactivity (2, 10).

Inthispaper we report thatcAMP, or agents that increase the intracellular concentrations ofcAMP, like theophylline and prostaglandin E2, when added to the culture medium couldtotally block proliferation of PyV-induced transform-antsbut did not have asignificant effect on the proliferation oftheir parental normal cells or transformants induced by SV40. The membrane-associated adenylate cyclase was threefold less active in PyV transformants than in normal cells or SV40 transformants, and PyV transformants con-tained threefoldless cAMP. Transformation byPyV appears to impose a low level of intracellular cAMP which, when increasedby

external

agents, leads to a block incell prolif-eration.

The lectinConA, when added to the culture medium, had the opposite effect. It totally blocked proliferation of both normalcells and SV40transformants but reduced prolifera-tion ofPyV transformants only twofold. Normal and trans-formed cells analyzed by flow cytometry hadsimilar num-bers of ConAreceptors on their surface.

The sensitivityto cAMP did not correlate withthe degree of cell transformation: cells which displayed an intense

transformed phenotype

(Rous

sarcomavirus transformants andtumorcells derived fromSV40transformants)werenot sensitive to cAMP. The physical association between the middlet

protein

of

PyV

and thesrc

product

of Roussarcoma virus isthoughttoplayanimportantrole in transformation (2). However, based on the results of experiments with cAMP, it is clear that the transformed

phenotypes

induced by PyV and Roussarcomavirusare notidentical.

The differential effects of cAMP and ConA on

prolifera-tionofSV40and

PyV

transformants appeartobe relatedto the activity of an intact middle t protein. However, the presence ofmiddle t, although requiredfor cAMP

sensitiv-ity,

isnot

sufficient,

since middletin the absence of either largeT orsmall tdidnotconferanincreased

sensitivity

on thetransformedcell. cAMP and ConAcanbe usedto

rapidly

distinguish

between SV40 and

PyV

transformants derived from rat 3T3 cells. This may be of

help

in

elucidating

the molecular basis for the

transforming activity

of middletand in

shedding

some

light

on themechanismoftumor progres-sion

(18).

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