The surviving fraction can be 20o/o of the population at input virus:cell ratios of 500 pfu/ cell

STUDIES ON MOUSE EMBRY9_CULTURES INFECTED WITH POLYOMA VIRUS

Thesis by

Robert Avrum Weisberg

In Partial Fulfillment of the F equirements For the Degree of

Doctor of Philosophy

California Institute of Technology Pasadena, California

1963

P..CKNOW LEDGEMENTS

The unique atmosphere of the Caltech Biology Division -- its close student-faculty relationships, its many opportunities for stimulating scientific discussion, its freedom from "red tape" -- have made my stay here both pleasurable and profitable. To my advisor, Professor F enato Dulbecco, are due especial thanks and gratitude for his invaluable advice during the course of my experimental work and the writing of this thesis. I would also like to thank Dr. Marguerite Vogt for her many perceptive suggestions and her unflagging interest in my work.

My stay here has been made possible by pre doctoral fellow- ships from the National Science Foundation, the United States Public Health Service, and the J\.rthur McCallum Fund. I gratefully

acknowledge their support.

The patient criticism, advice, and, above all, support of my wifeJJudyJhave been a major source of inspiration to me.

PBSTFP,CT

l·fter exposure of mouse embryo cultures to high concentrations of Py, a variable fraction of the cell population is converted to virus producers, but a fraction also survives and proliferates. The

surviving fraction can be 20o/o of the population at input virus:cell ratios of 500 pfu/ cell. F esistance to the cytocidal action of the virus in mouse embryo cultures is due neither to interferon nor to genetically resistant cells; it appears to be due to a transient physiological state of the cells.

No transformed cells have been found among the cells surviving a brief exposure to high concentrations of virus. Cultures derived from these cells by growth in antiviral medium resemble uninfected cultures in cell morphology, growth pattern, and sensitivity to reinfection. Transformed cells arise only in cultures which are 'exposed to Py over a period of two to five weeks. It has been shown that clonal cultures respond in the same way to Py infection as do un- cloned mouse embryo cultures; thus, transformation does not result from the infection of rare "transformable variants" preexisting in the cell population.

Changes similar to the transformation which takes place in infected mouse embryo cultures also occur , and rapidly, in uninfected cultures. The occurrence of these changes complicates the analysis of Py induced transformation. It has been shown that "spontaneous" and

virus -induced transformation are two different phenomena, since transplantable cells arising in infected cultures differ antigenically from those arising in uninfected cultures. The relationship between alterations of cell lines observable in vitro and the ability of these lines to produce tumors upon implantation have been studied; definite correlations have been demonstrated between these properties. These

facts have been discussed in the light of various theories of Py induced transformation.

TABLE OF CONTENTS

Part Title

GENEF_AL INTRC•DUC TION 1

A. Summary of the Findings with Rous Sarcoma Virus ·!!:

B. The Findings with Polyoma Vh•us 5

c .

^Plan of This The sis 17

MATERIALS AND J.'v!ETHC·.DS A. Mate ricu s

B. Methods C. Glossary

PART I. THE l.M!v1EDIATE EF·::..~ECTS OF POLY0?\1A VIRUS INFECTION CN M(:U'S£; .::;} .. i.::>l<..YC CULTURES

A. ",'lrus Multiplication a::d Cell Killing

B. The Properties of Cdls ;::.:~'l.l..·viving L"liection with J:>olymH.-1a

CULTURES OCGUii.R!l'!C, .f-UTT.::::t::. PROI./..)1-!GED

EXPOSURE TO 'VIR US

A. Prelimina:r.y Study

B. Further Studies on Transformation in Clonal Cultures

c.

^{Studies on ~he in \'}i"li.'O Ghal'ac·re:dctics of Selected Transformed Cell Liues

D. !_:.·resence oi the Polyoma-Specific A~~tigen in 'i:he

Transformed Linea DISCUSSION

A. Recapitulation of H.es"Ltlts

19

19

22 36

70

94

100

119

125 131 131

Pa1·t- Title

B. Discucsion of Recults

REFETI.ZNCES

Paee

133 150

-1-

GENERAL IN TROD UC TION

Th.-:: study of the induction of tumors by viru~es is a part of the n1uch broader area of research airr:eci. at under standing the initiation and maintenance of the neoplastic state of the cells. In both viral and non-viral tun1ors, the cells fail to respond to one or

more oi the growth regulating mechanisms which prevent unlimited and anarchic cell division in the nor mal organism. Tumors produced by viruses, a unique class among tumors, can be initiated and main- tained _.!l! ~· where they can be studied in a preferred way by using rr1oclern virological and tissue culture techniques. In the study of the neoplastic transformation of celle

12!

vitro b:,: tumor-producing viruses,

two main model systems were developed: one is based on the ribonucleic acid (RNA)*-containing Rous sarcoma virus, the other on \:he deox·i- ribonucleic (DNA}-containing polyoma viru::~. A variety of cell:> were u:;ed in both cases. This the.:ds is concerned primarily with the effect;:;

of the polyoma virus on cultures of mou.se embryo cell.:J.

Perhaps the first question of tumor virology is: do virus- induced tumors arise as a result of a genetic interaction. between the virus and a normal cell? The concept of virus-cell interactions as genetic interactions arises from the study of bacteriophage virology. Abundant evidence makes it clear that the process of phage r:1ultipli- cation can be regarded as the functioning

:f;l See Glossary (p. 36} for a li3t of the abbreviation:J used in this the3is.

-2.-

and replication of phage genes within e>.::: environment largely created and governed b) the activities of host cell genes. (l) In the case o; temperate bacteriophage, it is well-known that phage genes can interact with the bacterial genome in a rccombinational as well as in a func Uonal sense. H"or exaiYlple, genetic deterroinants oi teroperate phage can become linked to the bacterial chromosome in the form o~

1 (2., 3 ) db . . 1 ' . d i h

prop 1age, an ac·cer1a genes can oecoroe tn.corporate nto t e phage chromosome, thus giving rise to transducing phage. (4

, S) In fact, temperate phages have been placed in the class oi bacterial

. 1 k . ( 6) E . bl . th

genetic e ements nown as eptsornes. ptsoroes are a e e1 er to become integrated with the bacterial chromosome and replicate in

strict synchrony with it, or alternatively, to multiply autonomously in the cytoplasm. In addition, they may be completely absent fron•

the cell, in which ca:;e they can be acquired only from an external source. Temperate phages are epi~o:.nes which have the genetic inforrnation necessary to specify the elaborate mechanism of extra- cellular genetic transfer known as infection. \Vhen a temperate phage exists in the integrated state, a regulatory gene of the phage

synthesizes a substance, elaborated throughout the cell, which re- presses the functioning of the genes concerned with the production of the infectious virus particle.(?) It is this intracellular repressor which is held to be responsible both ior the maintenance of the lysogenic state and for the resistance of lysogenic bacteria to

ouperinfection with genetically related phage. Some phage-associated

- 3-

genes, which have no obvious relation either to the integrated state or to the state of autonomous replication of tee virus, function in both states. The changes in cells caused by thE. function of these gencf:l arc known as conversion; they include such phenomena aE alteration of the cell wall oi phage -infected Salmonella (B) and toxin l?roduction in the diptheria bacillus. (9

)

!n the light of theile conaiclerationr;, we may rephrase our quection: a1·e the cellt:i of virua-induced tumors descended from cells witl1.in which virual genes have functioned? The alternative hypo- thesic io tha~ tumor cells devcend from cello which were affected indirectly -- f.or inctance, by oubstanccs reieaeed fro;:n other infected cell::;. If t:l.1e firEit alternative io correct, we are led naturally to

further .qucstione, ~;uch ao these: Io the infecting virus genome lost from the neoplastic<.tlly ti·anoformed cell after its genes have functioned, or is there some fonTJ of intra.-cellula.:r transn1ission frorn n"lother to

da •.'.gh~er cell? Can. viral gcneG !:.ecorre integrated with the cell

cenome in a manner analoaous to the integration of temperate phage

·in lysogenic bacteria~· ~li!f.l the escape of the tumor cells from

r~gulatory mechanioms require the continued presence and functioning of vira.l g£;~nes within the affecteci cells and their descendants?

The answerc to some of these qucotions are probably different for different tun1or viru.ses. .I survey of the results obtained by using the two mociel oystemo of tumor virology will make this point clear.

"':: c shali begin with a brief review of the observations made wit..'l the

-4-

Rous sarcoma virus (RSV); polyoma vi ruG (Py), the principal object of this study, will then be considered in more detail.

A . Summary of the Findings with RSV

RSV is a mernber oi a class of viruses--tl1.e Aviar1leuk.osis group--whose members ehow Berologic cro01s reactions. (IO) RSV

ta. RN" . . d 1' 'd ( ll'

l~)

^{1 . d' . d ·}

con 1ns - 1 ,.. ... , proi:eln an 1p1 . t 1s a rne tUnl s1ze vtrus,

·with a diameter of approxirna tely 70 m~. Neither RSV, nor any other oi the Avian leukosis viruses is known to undergo a 'lytic ¹ cycle of multiplication on cultured fowl cells with rapid and large production

f . d ll d • ( 13 J 14)

v. .

^{b . d .}

o v1rus an consequent ce eatn. true 1s synt .es1ze 1n the cytoplasm of RSV -infected cells and assembled at their surface; ( l S) it is released from the infected cells continuously rather than in a

bur st. ( 13

) An infected cell retains the capacity to divide u.nd produce 11 11 f h. h . ^{1.. •} ~· 1 . . ( 13) progeny ce 9 , a o w 1c. reta1n t11e capac1ty or re eastng v1rus.

Recent evidence shows, however, that infectious RSV is released on...ly when the cells are supel"infected ^bj' another virus of the Avian

I 1 ( 16 • 17) Th' - h h

:.euk.osis comp ex. 1s pnenomenon as suggeeted t at RSV

. d f t' d . h 1 . t d -.• . ( l?)

1s e ec tve an requ1res a e per v1rus o pro uce ^ac~.1ve progeny.

Colonies iormed by RSV infected cells can be· distinguished

in a background of uninfected cells by virtue of their altered morphology and growth characteristics. ( 18

) These altered cells are known as

"tran:Jformed¹¹ cells. Transformation occurs in the absence of

-5-

helper virus. ( l?) The fraction of transformed cells in a population of susceptible cells i a directly proportional to the virul'il input. This observation ha::> led to a convenient in ~assay for the biological ac'i:ivity of the vit·ut3. The study of transfor mation by this method has had two important conclusions: ( 1) the majority, if not all, of the celli3 o£ cultures infected by high concentrations of virus can.become trans- formed; ( 19

) and (2.) different virus mutants induce recognizably different

morphological a.lteration0 oi the infected cells. (.::!O) All o£ these finding:::;

strongly indicate that RSV has a direct and contirming role in the trans- formation of cultured cells.

B . The Findings with Polyoma Virua

! . Experiments in the anir.oal

Py is a member of the Papova tumor virus group which includes the simian virus 40 (SV

40), the rabbit papilloma virus and the hur~•an warts virus. (ll) These viruses are similar in size--about 45

m~-~o

ⁱⁿ

diameter--, in cornpo8ition--DNA and pr otein--, and in the s·:rmmetry of the protein shell or capsid--icosahedral with a sirnilar number of rnorphological subunits. All of these viruses produce turnor:il in ou:; -

ceptible hosts.

Other interesting properties of some of these virusea will now be summarized. The relative DNA content of Py is 13o/o (corresponding to a

!~: olecular

^{weight of 5 x 106).}

{.:!~)

^Infectious DNA can be extracted

~

^{· f ,- d ·11 (2.3 ,}

~

⁴

)

^{,.,..,_ · ·d •}

r--

xrorn preparations o '~'i an pap1 on1a. .\.K!e v1rus caps1 ot ~y

-6-

can adsorb to and agglutinate euspenGions of the erythrocytes of . . ( 2 5) '""'' . k . 1 . . cert:nn epec1es. .Ull3 property, ·nown as ner..-.>agg utlnatlon, affords an extremely useful method of assaying virus concentration.

1'\pparently the other virusec of the Papova group do not rJhare thic property. The site of replica~ion oi f'y, SV

40 and papilloma in cytocidal infections. is the cell nucleus, as showrt by fluorescent

... b d t . . l 1 ^{t .} {26. 27)

an~.1 o y s ^~um.ng cnc\ e ec ron m1croscopy.

Py waz originally isolc.ted from leukemic tir;suee of mice. (28 ) .Apparently, the virus exist:ed as a contaminant in thes~: tissue::;,

becaus~ it was suboequently shown to h&ve no leukemogenic . . <29 ) .... h . . t d. t t'bl . l acttvtty. bow ever, vL en 1n;ec e 1n o Bu£cep 1 e c::.nur;a s

(rodent~), the virus is able to induce a wide variety of solid tumor e ..

Theae tumors are frequently locali2>ed and non-invaoive, ali::hough occasional invasive atld metastaoi:z.ing growths are produced. Th(o:

types of tumor o found depend upon the dose of virus and the opeciee . . f . 1 . . d ( 3 0) --- b bl . . . d ana stra1n o an:;.I:na lnJecte • t-'ro a y no ^tH:~sue 1n nuce a.n hamsters. at lea.et, is completely immune, although the parotid gland in some atraino of mice, (3

l) and th.e kidney in harnBtero, are eapccially . bl t . d . ( 3 2 )

susceptl e o tumor 1n uction.

Py multiplies extensively and rapidly with accompanying de- gl:!nerative changec when it is injected into baby mice and baby h<>.msters. (33

• 34

) The kidneys of infected hamsters illur;trate especially clet>.rly the cffcctc of !:he viruc: profound degenerative

-7-

changes and a rapid neoplastic response both occur . (3

S) In fact, discrete microscopic foci of neoplastic growth can be found in the

. . . . ,. . (36)

.1.udney s as early a s one week a;:ter tnJection or vtrus. The number of these foci is appro:x:irnate~y proportional to the virus dose, a fact which suggests that one particle is sufficient to induce a neoplastic change. This, in turn, suggests that the virus has a direct and

hn::~ediate role in tumor induction.

1\fiic~. and to a lesser extent, harnstet·s, d8vclop, with age,

. . . h . . "f. " .,. ( 3 ?) I ·

a very strong 1rnmun1ty to 1: e carcu'logentc e1 ects ot -"'Y. t ts known that the rapid immunological response of adult n1ice to .Py

reduces virus proliferation in the tissues of.older a.nirnals, and,

in. addition, that X -irradiation allows the production o( tumors in adult

. (38)

~l ~-

^{f 'h . d" . . . 1} •! 'h

tm.ce. l. 1e e:ttect o t e 1rra 1auon 1s pro .;ao y to .suppress t e i:...~.wnune response. These f.acts ouggest th3.t the i!:.•~·ilUrtity of adult mice Co l""y carcinogeneais may b0 due to th0ir greater h:..1munological

In spite of the increased reaction.,

r-y

does multiply to a certain extent in adult mice: minute quantities oi virus, when injected, can p1·oliiex-ate and i:hereuy induce the 3yn·i:he;:;is oZ la1:~e q:..m.ntiti.es of anti- body. Cn this .Zact io based aver·~ D13:·1sitivo e•:1d-point :.:.nethod of assay

~o1· the biological activity oi t:he viruo-·-the !DOU6e antibody production te-·. (39)

roo~ t..•

To GU~::1 up, the expe:rL:..>ents ^h! the anir-.'1al show that the virus can uiiect cell3 in two ways--by causing either cell proliferation or cell de3truction. The dose response of the production

-8-

of discrete foci of neoplagtic growth in the kidneys of infected han.'lsters suggests that the virus h..as a direct and immediate role in the induction of neoplasia. Hm.vever, the inability of the virus to induce turnor s in adults suggests either that Py may be very

inefficient in promoting neoplasia and/or that mice may have efficient rnea.ns for suppressing such change once it has occurred; imrnuno- logical mechanisms appear to participate in this suppression.

It seertls clear, however, thai conclusions about the nature of. the cell-virus interaction leading to the neoplastic state can only be suggestive when drawn froL"l experiments with intact animals. The irnmunological defenses of infected animals, their wide variety of target cell::!, and the uPl<I'lO'JI;n rnultiplicity of infection of each variety create difficulties for quantitative virology. Therefore, we shall turn to experhnent.:;; with ti!~sue culture s:,ri:Jte:ms which offer better opportunitieG for thene studies.

,~. E;xperin1ents in tLssue culture

The irnrnediate consequ•~nce of .Py virus in cultures of mouse cells

L~

^{to cnuse c.:dl}

cle •3tru:.cti o~"!.

^{(called cytopathic effect rcPE] ). (40)}

The virus multiplies extensiV"ely in these cultures; their fluids have hieh hemagglutinating titer o ar::d high iniectious titers, whether

deterrninecl b;.· production of tumors in the anirna.ls or of CPE in tissue

-9-

1 (40) ~-h ^{r···· - .} '1' 1 . ' f h . (41, 42)

cu tures. 1 e .__lJ_-:~ 18 utl 1zec.. 1n a p.aque a.soay o ^t e v1rus,

a method of bioassay which ia as sensitive ao, but more accurate than,.

' 'b d c1 • (43)

tne 1-r,ouS(.: antl o y p:::-o uctlon test.

Seve1·al workers hav'~ deocribed the transformation of culture3 f . ' 11 . . ~ . . h """' ( 44 • 4 5 • 46 )

o mouse, .-12.m cter ane rat cc £ oy uuectlon w1t ^.~.-y.

L'efore thiu phenmY.lenon iG diacussed in more detail, however, the n:ore general probler;.--, of ~1ow neoplneia can be defined and detected in vitro will be considered.

a) Definition and detection of neoplasia in vitro. The only direct test for neoplae;ia in cultured cells ie the ability of the cells in question to give rise to a tumor upon ir:1plantation into an intact

animal of the same h.ietocompatibiliiy genotype. For various reasons, some of which wiil be discuosed ;xore fully in a later section, this test is often inconcluzive, and it is always inconvenient. Therefore, other indirect tests will aloo be used in t~1.is work to determine the state of the cells.

!twill be recalled that cells respond to I\SV infection by a transform_ation of cell morphology and of growth pattern: Both are qualities which can oe determined visually in living cultures with the microscope. Cell n"'wrphology r eferc to the shape o:f the cell and to itf'J refractility. The growth pattern of the cells refers to their

. (47)

tendency to gro'\7 e1ther as a monolayer (regulated growth ) or as a multilayered- mat (non-regulated growth} on the Bu:dace of a petri dish.

*

^For a more complete definition of these terms see Glossary (p. 36 ).

t/1

-10-

Cells showing regulated nrowt7.l grow on the substrate rather than on top of one another and, moat important, stop dividing once they have exhausted the our face area of the substrate. Cells showing non-rer;ulated growth, on the ot:lex hand, continue to divid~~ actively when they reach confluency. It ic this uncontrolled cell diviDion v;·hich ce.tH:~ee the forr~>ation of a rDultilayered mat.

In general, freshly explan'l:ed embryo cella show regulated growth. \',hen. these celis, a~ •;.;ill be seen below, are infected with

?y, they develop cha~1ges in cell morphology and a non-regulated growth pattern. ':'Chis process will be called transformation and the reoultant cells transfor1:'l1cd cells.

lt should be pointed out that trandorr:r,ed cell2, aa defined above, are op_rationally dic:.tinct from neoplaeth. cells, which are clefined by their ability to produce tumoro upon implantation. In fa.ct, Py transformed

harn~ter

cells axe neoplastic in the animal, (44

) wherear3 transfor~:;·;cd mouse ceils are frequently unaole to produce

. ' . . d (48,~19, 50) ,.~, 1 t• f t' . 1

tumors w .. 1cn lmpiantc . .1.1e co:r:re a 1on o ne morpho ogy and gro·.r.;th pattern oi. mouse cells with their ability to produce tumors 'a hen inlplunted is one o£ the subjects of ti::ds thesie.

b) The transformation of cell cultures. ^A fraction of the cellc: in mouse ernbryo cul'i:ures do not degenerate after infection with a large dos•:: of virus. i' period of about !our to eight weeks ensues, in which cell killing is approximately balanced by cell division, so that

-11 -

there is very little or 110 n.ef: increase in cell number. This period is known as the stendy state period. Virua production is abundant through the entire steady state period. Finally, a new type of cell appears and overgrows the culture, the transformed cell., with changed morphology and growth pattern. Coincident with this, the amount of

C? ,- ~

and virus p:-oduction decreaoes. <⁴⁴)

In comparison with mouse embryo cultures, Py~nfected

cultures of hamster and rat ceils show much less cell degeneration

d . 1''' . (44,45,~16) . f 1 h b

an Vlrtts J?ro ueratlon -- 1n e.ct, rat ce le ave een

reported to be incor."'lpetent to support the nmltiplication of the virus

.<

⁴⁶) Flourescent: antibody staining of hamoter cells infected with high

concentrationc; of virnt:J indicateo that a small proportion - - perhaps

1~~·-- does oyntheoi~e viral capsid antigen, and the amount of virus multiplication observed supports thi!3 conclusion. (Sl) Unlike infected mouse cultures, infected hamster <1nd rat cultures can be subcultured, but they too are eventually ovet·grown with tranoformed cells. The delay between infection and overgrowth of the culture with transformed cello is about three to four we~k.; for hamster culturee.

The transformed cella which finally overgrow Py infected mouse . 1 h . - (SZ, 53) Th and hamster -cultures have been extenr:ave y c aracterlzea. ey tend to crow in interwoven, netlilte arrays and form multi -layered mate when they come to coniluency. Uninfected cells, on the other hand. grow in parallel bundles and do not continue to divide when the cell sheet covers tl"le available area of the petri dish. 'Iransforrr.ed cellB

-12-

arising from infected hamster cultures are generally free of virus and are completely refractory to reinfection. Transformed mouse embryo cells, on the other hand, continue to release virus at about 1 to 10 per cent of the rate observed during the initial steady state period of degeneration. The analysis of the yields of single Py- infected transformed mouse cells shows that Py is released in bursts -- each infected cell releasing about one thousand plaque forming units {pfu) of virus. .A.lthough the proportion of infected cells in transformed mouse cultures (about 1 to 2 per cent) was not reduced by treatment with anti-viral antiserum , transformed, non- virus releasing cultures could be obtained by the expedient of picking a single cell from an infected culture and growing it into a clone.

Neither the hamster nor the virus-free mouse transformed cultures could be induced to release virus by treatments known to be effective in inducing phage development in lysogenic bacteria, or by super-

infection by a mutant of Py. In addition, it has thus far been impossible to extract infectious nucleic acid from such cultures or to demonstrate the presence of virus capsid antigens.

This situation is quite distinct from that of cells transfor med by RSV. In the latter case, it has been shown that every transformed cell is capable of releasing virus when superinfected by a helper virus, and that this ability is transmitted to its progeny.

The finding that Py-transformed cells cannot be induced to pro- duce virus shows that these cells do not behave like a population of

-13-

bacteria lysogenic for an ir.ducible prophage. Nevertheless, it has been pointed out by Vogt a:td J)ulbecco(53

) L"J.at some or all ·of the genetic material of the virus may exist in the transformed cells in a firmly integrated (i.e., non-inducible) state. Some support for the idea that viral genetic material is present in transformed cells has come from experiments on the transplantability, in mice and hamsters, of Py-induced tumors and of cells transformed in vitro. These experi- ments indicate that there is a new and apparently virus specific antigen in these cells. Some of the work which has led to this conclusion will be reviewed in detail.

!t has already been noted that adult mice and hamsters usually do not develop tumors when infected with Py. It has been found that these tumor -free, infected animals are more resistant to subsequent

grafts of Py-induced tumors and of transformed cells than are un- infected animals. <⁵⁴• 55

• 56

) This resistance is specific: Py-infected mice do not become more resistant than uninfected mice to transplants of isologous spontaneous or chemically induced tumors. (S4

) Nor do mice immunized with othe1· tumor viruses acquire resistance to Py induced tumors. Such mice do, however, show resistance to grafts of

. . . d d b h . . . . (57. 58. 59) tumors or1g1nally tn uce y t e 1mmun1z1ng Vlrus.

It has been shown

by

^{Sjogren (60)} that neither the resistance nor the new antigen depend or.. the presence of antiviral antibodies in the graft recipient or on infectious virus in the tumor cells. This was done by demonstrating that mice which had been immunized by homo- grafts of a virus-free. Py-induced tumor were resistant to subsequent

-14-

ioografts of other virus free F)r-induced tumors. (Hornografts are gratto between individuals of the same species; isografts, between individuals of the same inbred strain.) This experiment also provides clh·ect evidence that different Py-induced tumors are antigenically croes- related. The Py specific antigen ia present in most Py-induced tumors and ic maintained even when the tumors are serially transferred in P y- immunized animals. (l.-l) For all the8e reasons, it has been suggested that the event leading to the production of the new virus- specific antigen i£J intimately connected with the initiation and maintenance o!: the neo- plastic state. Moreover. this event n1ay be analogous to the phenomenon

~ . ' . 1 . b . ( b2.)

or conver r31on 1n y sogen1c acter1a.

What can be aaid about the role of the virus in the initiation oi these apparently specific transfor med cells? It has recently been shown that a small proportion oi transforrned cells appears very shortly after the infection of ham ster cell c'.lltures with P y. (63

• 64 )

These cells can be detected by the distinctive morphology and growth pattern of the colonies they form. Stoker and J:v1acPhGrson, plating ireshly isolated hamster cells after infection with about 24 plaque ior rdi.ng m'lits of vhus per cell, found that about 0. 006% of the cells

(63)

forn1ed transformed colonies. The nun"lber of transformed colonies we.s approximately proportional to the input r.nulti.plicity.

Vog'i: and Dulbecco, using colonial morphology and trans-

-15-

plantability as their criteria of transformation, did not fincl typical tra!'lsformed colonies when th~y plated hamster cells immedic.tely after infection. (65

• 66

) Instead, it was possible to ioolate "foci of

pereio~ent mitotic activity" ,;vhich, unlike colonies from uninfected hamater embryo cu.ltu~ ,~s. multiplied contii:moualy. But they did net show in full degreethe:rx:n-rcgulated gr owth which is characteri::;tic of cstabliohecl transformed cultures, nor did they produce rapidly growing tumors when implanted into hamsters. .As these "early"

transformed lines were transferred, a great many abnorn1al mitoses and chromatid breaks were observed - ··· events which caused a high frequency of dead cello to be thrown off. .1T'inally there arose typical

;

eatablishcd or "late'' trru'lsformed cells which formed dense, piled- up colonies, which had a low frequency of chromatid breakc c::.nd of dead cells. and wi;l.ich were fully neoplastic in the animal. Since the infecting virus had been diluted out, reinfection couid be excluded as the ir.,ducer of the late transforr.ned cells. Vogt and Dulbecc:o conclude that the late transforn1ed cells derived fron1 the early tranoformed

cells "by a oecondary variational process" as a consequence of the original infection with Py.

It is not known whether the transfo1·:mecl cells observec;l by Stoker and :t-IJ:acpherson were similar to the early or to the late transformed cells of Vogt and Dulbecco.

Stoker and hii!S co -worker s have also studied the Py-induced

. (67 , 68)

transformation of a permanent tiaBue culture hne of hamster cells.

-16-

Infection of this line induces transformed cells which display fully neoplastic behavior with reEJped to transplantability and tiGsue culture characteristics. This "one-step" transformation n0ed not be at variance with th.e two-step rroceGs proposed by Vogt ancl Dulbecco, since it i:::; quite poGsible that the first step had occurred spontaneoualy during the period needed to produce a perm anent line from freshly explanted tiBsue. (66) In addition, Stoker has

recently reported that several cell divicionB occur between infection of this line and the appearance in it of tranoformed cells. {69

) In conclusion, it can be Geen that the evento ot:>served after infection of either mouse or hamater cells with Py contrasto with the situation in cultures of chicken embryo cells infected with H :__ V. In the latter case, there ie an i:r'!'Jmediatc n'"lorphological transfor- mation of a large fraction of the cells, and in a matter of clays, virtually the entire culture consists of these altered cells. We have noted that traneformed F ous cells can t:ransndt virus, or the ability to produce virus, directly to their progeny without external trans- micsion. In addition, P. SV tra.nsforrned cells can release virus or can be induced to release viruffl. Finally, we may recall th..:: intra- cytoplasmic site of synthesis of viral antigen and the elow trickle of progeny virus released from infected cells.

In the case of Py, there appears to be a complete distinction between cells which produce virus and transformed cells. The

• 1. • ' • ,. • f t . 1 to 0

propertiero of the transfor:rnea cells wo~.·ncn ar1se :crorn 1n ec ca cu_.,ure

-17-

are maintained in the absence o:Z iniectiouo virus. Nevertheless, the transformed cells do show evidence of a prior interaction with the virus in the form of a new and specific transplantation antigen.

!?inp.lly, the relative inefficiency of Py in inducing neoplastic

transiorrnation should be pointed out this is shown, on the one hand, by the very small proportion of cells in hamster cultures infected with very high multiplicities which form transformed colonies, and, on the other hand, by the extended period between. infection and the overgrowth of transformed cells in infected mouse }.'lopulations.

C. P lan of This Thesis

In Part I, experiments measuring the proportion of cells that yield virus and of cells that are killed in freshly infected r:10use embryo cultu...-es will be described. It will be shown that, even with very

high inputs of virus, it i3 impossible to convert all of the cells into

virus yielders or to kill all of the cells. ;,Jb:;h the aid of special antiviral tnedia, the ourvivors of a briei expo;3Ure to virus will be examined .in tha absence of the complications caused by reinfection. In this way, it will be shown that tran8~ormed cells appear in the population only after

a prolonged exposur:= of the culture to virus.

In Part Il. a ·Jtudy oi the response of clonal cultllre~» to virus infectioa will be described. The main purpose o.Z the study is to deten_line whether g0netic 'heterogend ty in the culture plays a role in the delayed appearance oi transfor med cellG. The properties of infected and uninfected clonal cultures will be examined with respect to their cell morphology,

-18-

their growth pattern, their ability to produce tumor£l upon implantation, ancl their antigenicity.

-19-

.t· .

Ma"erials 1. Media

.For ro"L~tine cuitul'ing of cells and for plaql.le ~.n:Ja]B, ~~:agle's

.\1ec:i~

^{.. u:n(?O) with a four} -fold increase in the concentration of amino acids and containing 10% c<?.lf serum was used. Other modifications of

.f;agle'~:.: 0riginal forr.:n.!la were alao u:Jed: th~y include an increace in the ~lucos<:: concentration to 0. -'!..:5% anc: in the bicarbonc-.te concentration to 0. 37%. ·:.~he parti&l preGeure of Co

2 in ou1· incubators was

adjusted to glve a [?~-i of ~1. -:1 - 7. 6 i~ th.::: ::nedium at thio bicnrbon~te

concentration. ?or cloning of cells, for ro,~tine maintenn.nce of cloned linea and, in later work, fo:;: plaque aliisayG, two media wer-::: used:

~- ... • ":"> '( •

c (

6 ( 71 ) ' tl • ^T ~ ,·~ ^{( )}

\~ne v1as ... .~.,' ... .t' • .:...~-.t . , w1 1 an J.'<a ... ~ ...

3 concentration of 0. 37% and with 12-14% ca.if Gerum added. The other (designated ~-=>r:-;7.'1:.) consisted

J: 4Zn~ ^''-T 16 (?Z) 4" ·rt. ·•~:· d -· l ¹ d' 4 ~c· l'l.:·~r-·~109 (?3 )

O!. ''1;, rv , t. ~<; trJOdl.!.le :..:.,ag e a me 1um , • "'to l-<1... 1 ·~ ,

anci

l2 o/.,

calf s~rum. '"l~he base of r~.:M wae Earle's balanced Galt colution<74

) (with the increased glucooe and bicarbon;o•.te noted above)

· • £ "" ,.

r

^{{? 5} >

111::1teaC! o ,;.:.a_lne . For dispersal and subculture of cells, 0. 05&/, trypein dissolved in tris buffered saline (53

) without divalent cation a

·wa.o used. Tris buffered caline (TDS) was uoe·' as a <lilt1ent for virus and to wash plates before virus infection.

2. ^·c·."'"· trus

Py wao originally obtained from Dr. Rowe of the .National

-20-

Institutes of ~Iealth. '.i.'his r.:train of vi:;.·us will be referred to as iarge plnque {lp) viras or, mo:;:oc f:-:equeatly, sirnply c:to ?y. In several experin:;ents two other strains of vi:ru:J were used: the small plaque ( sp ) 1:nu an.:, 1so t ,, . 1 a t ·eu 1n .. . t. ' . n.:to 1 a b oratory ' ( 52 ) an~ ^r1 th e: !:-' " 1 ~ o stra1n . { 6 8 ) whici:1 was :::ent to us by Proie:.eior :111. .:;toker of the Institute of Virolorgy. Glasgow. The latter strain is e. e;r.nall plaque forrr.ing variant of the cforonto strain c.f ~"....,Y·

Virus prepared by three different r~•ethodo war; used: (1) :Cissuc culture vi.rua WeJ.o the supernant mediu:~n of i!'..icctcd n10use

.,

embryo cultures. Thece stocks generally ccnt:<'l.ined 2 to 10 x 10' plaque fortxling uni~s {pfu) per cc; (2) Occasionally, tissue culture virus wae conce~1trated and partially pu:dfied by two cycleo of

adsorption to anci elution from euinea pig erythxocytes; (3) In some experiments, concentrate G. and purified viruo prepared according to the method of Vlinocom.J76

) was ue;ed.

Virus stockG were generally stored in a cJeep free:&e a·l: -20°C.

3. ~cCt!?tor destroying enzyme (PDf!~)

F DE,

~n

enzyme which dest!"oys the receptor sites for Py(??) (c:md other hcr::agGluti::la.tine viruses) on the surface of eryt:.uocytes,

WiHJ obtained as a lyophilized powder frm-n Dehringwerke PG. The powder, supplied in Geru;.n bottles, wao dissolved in 2. 5 cc TBS I O. l~;s

c ac 1

2 and stored, for up to one month, in the refrigerator.

used, R DZ weH; add.~d directly to the culture medium. R D~-:; concen-

-21-

tra.tiono will be expreooed as the reciprocal of dilution.:; from the original solution. The preparations ofF DE obtained from Behring- wcrke were effective in inhibiting the agglutination of guinea pig

erythrocy~es by Py. at a concentration of 0, 002 units under conditions similLl.r to those described by E'mrnet: and Stone. ("IS)

4. l;,ntivirus serum (1~S)

1wo rabbito, No. 1 and !'-To. 2, were given two series of one intravenous followed by two subcutaneou~:~ injections of virus • over the course of two month&. 7ive -tenths to 1. 0 cc of a virus stock titerin.g 5 x 108

pfu/ cc, prepared by adsorption to and elution from guinea pig erythrocytes. wa!8 aciminh::tered at each injection. :Blood wae: collected by heart puncture 10 days after the last injection.

Eloocl wa.e collected once rno:re fron~ these rabbits after another series of three intravenouc injections spaced at intervals of three to five days.

In the later series of injcc:tionl!'l, 0. 5 cc of purified "empty shells'pb, 79 ) at a concentration of 5 x 105

hemagglutinating (I-LA) units was used as antigen.

The sera thus obtained were absorbed with 2 to 8 x 107

mouse en1bryo cello per cc of serum . Sera obtained from the second bleeding were aleo absorbed with 5 x 10 7

cell:J/ cc of a Py induced tumor.* After aboorption, the sera were heated at 56°C for one half hour, centrifugel'.

*

This tumor • designated S.ESF, was tJent to us by Dr • .'H. 0 . Sjogren.

-22-

to remove cell debris, and sterilized by filtration through a millipore filter. The sterile sera were distributed into tubes and Dtored e.t

.,cor

-._ "-·. Hcrnagglutination in."libition UTi) tests performed on these b t^h th d .r ^T> <· 1 (SO} ~~I · · ^1.

sera. y ... e me o o ... .r.-.owe, e .. a . gave .t.~ tltcrs 1n tne range of 1:40, 000 to 1:100, 000.

When the cerurn wao used f:o treat cells. the calf serum which is incorporated into our tissue cultut·e media wa~ heated at. 56 °C for one-half hour in order to destroy complement. .P S concen'i:rations will be expressed ao the reciprocal of dilutionc.

B. Methods

1. Primary mouGe c~mbryo cultures

Mouse embryo cells were obtained from 12 to 14 day old

e;nbryoG of nonin.brcd

~3wioa

^{or Py free Ah-:.;n ntrain mice* according}

to the method of :Culbecco and Freeman. (4

l) Generally, the d:i.a-

aggregated cells of one embryo would be e'.:planted on one or two 100 mrn polystyrene pe'i:ri dishes (obtained £rom Falcon Plaatics) in l:agle ¹8

medium. Cella irom these primary cultures were subcultured ~ to 6 days later, generally on 65 mm petri dis}les.

Z. Virus titration

Plaque assayo were performed according to. the method of Dulbecco and i"reeman(4

l) c>,cept that a one and one-half hour

*

'i'h\3SJe mice were kindly Bent to us by Dr. H. 0 . Sjogren from the colony of Dr. G. ~{lein at the lnBtitute for Tumor Biology, i.::.tockholm, : . .-~J. .y were kept and bred in a r~::stric:ted roorn on a different floor fror::; th.;::

laboratory where virus el!:perimento were pe:r.forr.ned. Eernngglutination inhibition tevts (see below) were regularly performed on ra.ncio::-.r:ly

selected •nice irom the colony• but no mouse with a flositi ve serum _ (according to the criteria of Sjogren and Hingertz(8 ) was ever found.

~23-

a.dsot·ption period wao used. If thE: infected cells were to be used subsequently for cloning or for an infective center assay, the

cultures were washed before infection with P .Slv!, and the virus was diluted in P.:>:M. Otherwise, TDG was usually used for these steps.

Hemagglutinations v7ere performed according to the method of

'n t 1 (GO) Th . 1 • th h. h t d. 1 ' • . .t'i.OWe, e • a • e rec1proca o:t e • 1g es 1 utlon snow1ng

!,)ositive agglutination was tal·en as the concentration of that virus stock in Hi'- unit£:.

3. Determination of total virus yield

In order to determine the total virus content of an infected culture, it was necessary to aaoay the virus present in the supernatant medium and the virus acsociated with the cella. ...he latter virue

fraction ~- which consists of virus adsorbed to the cell surface and viruo present inside the cell -- iD known as cell asoociated virus (C.A V).

!t wao measured by removing the cells from the petri dish with a policeman, diarupting them by three cycles of rapid freeze-thawing, and assaying the lysed celle for plaque forrnero or for hemagglutinin. The supernatant medium, of course, could be aosayed directly.

in o::·der to determine virus production in an infected culture after a cinglc cycle of virus growth, its total virus content wao

meaaured at 40 to 44: h.ours after infection. This procedure is justified by the data.of \Vinocour and Sachs('l3) which show that the latent period for Py in mouae C€!llS ic 22 to 24 houre;.

4. Cell clm1ing

a) Feeder layers. :£0'eede:t" layers of secondary or tertiary mouse en~bry\..; cells were prepared afj follows: .F. dose of 5000 r was ctdministered to a celi suspension in 5 cc of medium in a petri dioh.

*

The irradiated cells were sedimented, resuspended in fresh m edium, counted, and 5 x 105

cells were plated per 65 mrn petri dish. Special precautions were taken to eliminate clumpta from the suspension as described below.

b) Cloning. One to two days afte? plating, t..he medium -.vas rernoved from the ieeder layerlj and 2 cc of PEM or CMP L -1066 were added. The suspension of cells to be cloned wc>.G added in a emall volume o£ medium an.d the plates were incubated foT 8 to 12 hours to allow cell attachment.

The following precautions were taken to avoid platin[; clumps:

the sut:,;pensions were e.llowed to stand in a centrifuge tube for 5 minutes, and the top lay~1· was removed and placed in a paraffin coated tt;;.be.

':i'he c.z:lls were counted uei.ng a technique wl'lica allowed the proportion of clumps to be determined. A minimum of 200 cella waG counted.

The number of clumpe largr;!r than 4 cells was always leso than 0. 5o/o.

,t. variable number of cells, generally les:;; than lOo/~ were in clumps of 2-4 ce1l3. On occaGion~ the cellr. to be cloned wer:a adcied to the feeder i':- The physical factors were 0. 38 mm .Al filtration, 50

l' ~ VP.

30 :MA,

S. 7 em target to sample distance, !vla.chlett o:::;G 60 tube with a beryllium window, and a dose rate of 2500 r /min.

-25-

plai:t:0 with a micropipette. Generally, only one cell wao added to eac!: t:>late in l;;.le:se caa,:;:s. The single cell wafl picked from a cell suorJencicn under the ciicsecting microscope.

!-iter the cellc had attached to the feeder plate, a molten solution. of 0.

69,

agar in ? ::~M or CMR L was added directly to the 2 cc of medium alreo.dy on the plate. ::Tour days later, 3 cc of liquid cloning medium was added and c~t the Sth to lOth day after plating, the agar was poured off and 5 cc o£ fresh medium wao added.

c) Pi c:.dng clor;.e o • One day after the agar was poured off, tr1e colonies were cou..""lted and theh position marked. 3everal control plates which received no cells were ahn-.ys counted to check the

efficiency of the irradiation. Clonir.~.g efficiencies of 7 to 20% were routinely obtained V"Jith mcmee embryo ~Jecondary cella.

On plates with four or fewer well-separnted colonies, as many

ns

three migh~ be picked l:'.nd transferred to a new plate with a feeder layer prepared as noted nbove. 'J.'he clones were picked using the following procedure: The medium was removed from the plate and a glass cylinder, lO mm in dinmeter, wo.G affixed around the colony with

"1" ( 8 z) ..,.. . dd d d ft t f th sterile s1 1cone grease. ^.!. rypfnn wa6 a e , an , a er mos o e cell(; had detached, they were transferred to the fresh plate with a pasteur pipette. ...:.'he feeding routine for these oecondary plates was t~'le same as fm.· the prin121.ry pla.te:s. ':ten days to two weeks later , rrw6t .;,£the clones could be trannfc:rred without a feeder layer. Generally, subcultivation codd be attempted from ~ to 6 weeks after the original

-26-

cloning. Overall, about 60% of the clones that were picked gave rise to continually growing cultures; most cf thocc that did not showed no cirowth at the-first transfc.-.

5. :Doutine culture methode

In view of the l··nown danger of crosCJ contamination of cell lines carried in the same laboratory (eJee reference 82, for instance), it may be well to give a b:def account of the methode used for cultivation of these cells. ~::very two to three days, pletes of each line were scanned unde1· the inverted microscope, and it was decided whether the plate s!"lould be transferred or fluid changed. Generally, plates were transferred wh.::n the cell sheet nea?:ed confluency. Only a fraction of the cell population \:"JaG used to reseed new petri dishes. Since the growth rate of :rnany cell lines fallc; off rathe1· sharply at suboptir.i"-.1 cell

den~itiea,

(S4

) several plateo were initiateC. at different cell densities at each trande:r. '.:.'he plate containing the minimum cell nm.1:1ber judged adequate to main.tain the maximal growth rate wa&

selected for the followir..g transfer.

_f\fter t..l-J.e plates we:o:-e scanned, media and trypsin solution were disCributed in tubes, one for each culture, in a section of the laboratory where no virue work was performed. 1>11 lines were fluid changed and transferred in the virus section, but no virus infected material was introduced into the working area before uninfected cells were trar.oferrcd. No instance of virus contamination was ever

-27--

detected. It is impoasible 'i:o Tule out contamination of one cell line with another by chromooome cytology since ali the cultures derived frorn r.nouse tisoue. i:-:iowev~r, whenever any suspicion of contamination arose -- that a pipette was not changed, for instance - ~ the lineo

concerned were irn:o.:..1ediately diocarded.

6. Overall growth curves

These curves(S2.) describe the net increase in cell mas of cultures which are being transferred at regular intervals as o function of time. When a confluent culture io transferred, generally only a fraction of the total cell population is used to seed the new plate (see 5 above). The net increas~ in cell mass in the intervale between transfers is set equal to the reciprocal of this fraction. If the cello are always maintain0d under optimal growth conditions, and if t!1ere is no long lag period after transfer, the elope of the o·..-erall growth curve will closely :reflect the averaee generation time of the cells.

7. Implantation teGto

Occaaionally, cultures derived from A/Sn ernbryoa were tested for their tumor-inducing potential by implantation into irradiated and

*

unirradiated p, strain mice. P confluent but not overcrowded plate of the culture to be tested wa.e trypsinized, centrifuged and resuspended

~ Most of the mice used for the implant?tion teat were irradiated with a whole-body dose of 425 r . The physical factors were: 1 rnm Al filtration, 2.50 :KV, 15 IV'.J·, and a close rate of 12.5 r I min.

-Z.8-

in 0.1 - 0. 2 cc of 'rEG. C:eH counts were not routinely made, but by experience it is known that fron1 2 to 6 million ceils can be recovered from auch cultures. The volume of the centrifuged cell suBpeneion wag always chedted, anci if a.ny doubt existed about the cell number.

a count was made. !.ny exceos over 6 million cells was discarded and fewer than l. 5 million cells were not injected. The cells were injected subcutaneously along the fla.P..k and the mice were regular ly observeci for at leal!lt 3 months after injection. ./1 mouse was judged

·-~

positive if a tumor arose at the site of implantation within 10 weeks of injection and grew to the size of a walnut (or killed the mouse before it did). Several of the tumors resulting from the injection of Py-infected transforrr.ed cells '"ere sent to a laboratory for examination. Dr. Dennis Shillam of the Pasadena Clinical Laboratory found that 5 out of 5 of these tumors could be diagnosed as chondrosarcomas of subcutaneous tis sues.

8. Detection of the Py specific antigen

The mice to be used were selected from cages containing no rnore than aboui five litters, of age one to two months. Half of these mice we~ce injected subcutaneously with P y. Generally three injections of 0. 1 cc of purified virus containing 5 x 108

pfu/ cc in TBS were administt~red at weekly intervals. P..t the time of the third injection, the control mice were given a !'lingle injection of 0.1 cc of TBS. The cells to be teo ted for the antigen were diaper s:ed by trypsin, centri-

-29-

iuged and rer;uspended in T:3S. One tenth cc of buffer containing

a

known number of cells waa injected subcutaneously into the virus-

infected and virus free groups of mice. In most cases, three different doS•!C of cells were employed.

The mice were observed at four to seven day intervals !or the two r::1onths following the cell injection. Developing tumors were

measured with calipero and the m ean of ^t\'iiO r..neasurements was reported as the average tumor diameter .

9. Cell freezing

The cello to be fro?..en were suspended in a tube containing 3 to 4 cc oi medium pluo 6 to a·~c sterile glycerol. The tubes were placed in an alcohol bath at 5°C and the temperature was lowered by a

programmed temperature controller obtained from Conalco,

1":.

Y. at a rate of 1°C min. to -30°C. Thereafter, the temperature was

allowed to fall at an uncontrolled rate to the sublimation point of

co

₂. The cells were stored in a freezer at th.ie temperature.

The cells were thawed rapidly in a water bath at 37 0 C, and the contentn of the thawed tubes were immediately poured onto petri dishes. :.:;qual volumes of f:res h medium were slowly added and the plat~s were then placed in the incubator. They were generally fluid changed after 1 or 2 days. Cell recovery by this technique is somewhat variable -- it l'angeo from 10% to 90%. If, when the cell:3 were first fluid changed, a oubeta.ntial number had not attached, the old supernatant medimn was

-30-

centrifuged and the sedimented cells were resuspended in fresh medium and added back to the plate.

10. Antiviral medium

In scverz,l of the e>::perimento to be described later, ? y-infected cells were treated with medium containing antivirus serum and

:r:

DE (.AS and P D_!: treatment). 1lle function of the antiserum was to in- activate virus which was free in the medium (free virus) and virus

which was superficially associated with tbe cells (superficially adsorbed virus). R DE, by destroying the receptors for virus adsorption,

converts virus which is associated with cells to free virus(SS) and also prevents the infection of uniniected cells.

...

We shall summarize

several experiments performed to a.cses& the efficiency of .AS and F. D.=:

treatr!"lent in performing its functions.

a) Inactivation of free virus. The multiplicity curveG(Sb) for the two antivirus sera we have used have been presented in Fig. 1 . In the:;e experiments, variouo dilutions of serum were added to aliquots of a tiasue culture etoclc of virus at a. concentration of 5 x 10 7

pfu/ml.

This mixture was incubated at 37

°

for two hour o, then diluted and

plated for plaques. This incubation time should be long enough to allow the inactivation reaction to go to completion.

These reoults indicate that at the serum concentrations used, and at the virus concentrations we ohall encounter. we can expect to

1 he use of P D:S to prevent reinfection wall originally suggested to the author by .Mr. Michael Fried.

-31-

Yi'igurc 1. :i·.ful.ti;?licity of N~J:utrnli~ati0n Curves fox .Pntipolyoma .:era.

'\Yarious dilutionr.: of sera V~ere added to aliqaote cf a Py atock containing 5

~:

¹⁰⁷ pfu/cc. The mixtures were incubated at 37° for t\.vo hours, then diluted and plated for plaques.

a) .~ntise~um from rabbi\: l\·o. 1.

b) I ntiserum from rabbit No. 2. (Note that the sce.le of the abGciGon is dccreaeed by a. factor of ten. )

-

^tJ

::::.::..

-

:::::,..

l-. ::::J V)

V)

::::J

l...

-

:::::::..

~ C)

7

- _~

3:

l...

70

::::J

IJ) V)

::::J

l...

::;:

~

^C)

7

- 32 -

Serum

(a)

Ser urr. No 2

(b)

Fig . 1 70 - J

I

-33-

neutralize at leaot 99% of the free virus.

b) Inactivation of superficinlly adsorbed virus. Confluent monolayere We'L'e infected with high concentrations of viruG. p,fter adsorption, the rnonolayers were extensively washed to remove loosely bound virus. Nutrient :r..•edium containing high concentrations of .AS and

!·

F. DE was added to one eeriea of plates. and medium without AS and R DE was added Co a control eerie a. The cell sheets were washed and dis- perE>cd by tr)'-psin before any progeny viJ.·us had appeared, disrupted by free~e -thawing, and assayed for their virus content. It was iound that 80-90% of the superficially adsorbed virus could be eliminated by ],;;_: and F. IJ: . ..C tr~atment.

c) :::.:-.rotection of cella frorn infe::tion. Confluent plates of mouse embryo cells were treated with RD.!..': for five houre. then washed, in.fcctec and observed for plaqu c. The resulto (Table 1) show that 80 to 95°;:. o! the in:fcctible cells becz.me i'esiotant to infection at R Dl!:

concentrations of 0. 004. to 0. 02 units. In another experiment. at an I\ DE: concentration of 0. 006 units, the nu:.nbe1· of plaqueG after one hour of pretreatment was 15t;:S,anci after seven hours of pretreatment it was 10% that of an untreated control.

'l'hia reduction in cell infectibility was probably not due to some genera::. deleterioua effect of r~ DiC for the following reasons: {1) '.fhe plaque size on F Dt; treated plates was t:he same as the plaque size on

-34-

Table 1. P DE Treatment of Mouse Embryo Cells

Various dilutions of R DE in Eagle's medium containing lOo/o calf serum were added to confluent monolayers of mouse embryo cells. Five hours later, the medium was removed, the plates washed once with TBS, and the monolayers used for plaque assay in the usual manner. Three plates were used for each R DE dilution. No reduction in plaque size was noted when the plates were read.

-35-

Table 1

Dilution of F DE 1:10 1:50 1:250 1:6250 l:oo

.Average plaque 0. 04 0. 05 0.21 0.53 1. 00

number as a fraction of the untreated control

-36-

untreated plates and (2) cloning experiments performed in the presence and absence of R DE show that R DE does not lower the cloning efficiency of mouse cells.

C. Glossary of Descriptive Terms 1. Descriptive terms

a) Cell morphology and cell orientation. Mouse embryo cells which have been freshly explanted from the animal (Fig. 2c and d) have a rather broad, flat, irregular shape. They appear ~ refractile

•

when viewed with non-phase contrast optics. The cells tend to grow side by side when the cultures near confluency: this will be denoted as an oriented or parallel configuration.

Cells from cultures infected with Py (Fig. 2a and b) frequently have a rather elongated and generally triangular shape. They appear refractile under non-phase optics. The cells tend to lie across one another in a netlike array when the cultures near confluency: this will be called a random or netlike configuration. Cells from such cultures will be said to ha·ve transformed morphology.

b) Growth pattern. If a culture remains flat and two

dimensional after it has been confluent for at least a week, it will be said to have a regulated growth pattern. If, on the other hand, it

*

R efractility refer s to the property of some cells of acting as lenses. These cells appear light, and then dark, as one focuses through them with a microscope, and their edges are sharply outlined.

-37-

Figure 2.. Cell Morphology and Cell Orientation in Py Transformed Cultures and in Freshly Isolated Mouse Embryo Cultures.

(a) Transformed Culture. Phase contrast, x 90.

(b) Transformed Culture. Phase contrast, x 150.

(c) Freshly isolated, uninfected culture. Phase contrast.

X 90.

(d) Freshly isolated, uninfected culture. Phase contrast,

X 150.

The typical cris3-croas cGll orientation and sharply clefined cell shape oi the transformed cells contrasts with the parallel cell orh:::.t:c:.tion and rather ill-defined cell shape seen in ·che freshly isolated cultures.

- 3 8-

(a) (b)

(c) (d)

Figure 2

-39-

displays areas where cells are proliferating on top of the monolayer, it will be said to have a non-regulated or multilayered (ML) growth

pattern. (See Fig. 3 for examples). Cells with transformed morphology invariably have a non-regulated growth pattern.

c. Colonial morphology. Cells from freshly explanted cultures form colonies in which the cells grow strictly in two dimensions. Such colonies will be described as flat or regulated.

Cells from transformed cultures frequently form colonies with thick, multilayered ctnters. Unlike.flat colonies, these colonies are visible to the naked eye, without staining, by eight days after plating. They will be referred to as dense colonies. Two illustrations of a flat colony next to a dense colony are given in Figure 4.

2. P bbreviations

F. NP, ribonucleic acid; DNA, deoxyribonucleic acid; Py, polyoma virus; .RSV, Rous sarcoma virus; .f\S, antiserum; P DE, receptor destroying enzyme; C.AV, cell associated virus; MOl, multiplicity of infection; IC, infective center; ML, multilayered;

pfu, plaque forming unit,

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Figure 3. MicrophQtographs of Areas of ML Growth Appearing

in Confluent Cultures.

(a) Unstained, x 20.

(b) Unstained, x 60.

(c) Phase-contrast, x 130.

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r:--ig;ure 4. Microphotographs o[ Dense Colonies and F'lat Colonies.

Stained with Methylene Blue. x ~0.

(a) The dense clone is at the left of the photograph, and the flat clone to the right.

(b) The dense clone is at the top of the photograph, and tho flat clone toward the botton-J.

(c) The dense clone is at the top oi the photograph, and the flat clone at the bottorn.

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