The isolation and preliminary characterization of temperature-sensitive transformation mutants of Moloney sarcoma virus

VIROLOGY

95,

303-316

(1979)

The isolation and Preliminary Characterization Sensitive Transformation Mutants of Moloney D. G. BLAIR,2 Cancer

Research

Laboratory,

M. A. HULL,

AND E. A. FINCH

University of Western Ontario, Accepted January

of TemperatureSarcoma Virus1

London, Ontario,

Canada

12, 1979

Temperature-sensitive (ts)transformation mutants of Moloney murine sarcoma virus were isolated from uv-irradiated viral stocks using a selection and screening procedure based on the ability of MSV-transformed NRK cells to grow in methyl cellulose or agar suspension. Mutants isolated by this procedure formed colonies in agar lOOO-to 10,000-fold more efficiently at 34” than at 39“. They also exhibited a transformed morphology and elevated hexose transport levels at 34”, but were phenotypically normal at 39”. Both morphology and hexose transport showed transformed + normal and normal + transformed conversion within 12-43 hr of a temperature shift from 34 to 39” and 39 to 34” respectively. In contrast, tstransformed cells suspended in agar and incubated at 39’ for 24 hr showed a 90% reduction in colony-forming ability when the plates were returned to 34”. Superinfection of nonproducer tstransformed cells with leukemia virus resulted in the rescue of ts MSV. Rescued supernatants also contained a high proportion (10%) of wt MSV. Repeated cloning of ts mutants, either as virus or cells, did not significantly affect the proportion of ts or wt virus rescued. The ability of &transformed cells to express the transformed phenotype at 39” could be restored by wt MSV superinfection, but not by MLV superinfection. INTRODUCTION

The function and genetic organization of mammalian sarcoma viruses have been less extensively studied than the corresponding avian viruses (for review, see Hanafusa, 19’77; Vogt, 1977). Studies have suggested, however, that significant differences may exist between avian and murine sarcoma virus-induced transformation, most notably the apparent ability of nontransforming murine leukemia viruses to complement defective MSV for transformation (Bassin et al., 1971; Scolnick et al., 1974; MeCarter, 1977). The correlation of biological and biochemical function of mammalian transforming viruses has also been hampered by the lack of stable conditionally defective mutants, since very few isolations of such * This work was supported by funds provided by the National Cancer Institute of Canada. * Author to whom reprint requests should be addressed. Present address: Laboratory of Viral Carcinogenesis, Building 41, Suite 400, National Cancer Institute, Bethesda, Md. 20205. 303

mutants have been reported (Scolnick et al. , 1972; Somers and Kit, 1973; Carchman et al., 1974; Yuasa and Shimogo, 1977). We undertook the studies described in this report in order to isolate and characterize mutants of the Moloney strain of MSV. Previous attempts to isolate temperaturesensitive mutants of transforming viruses have for the most part used the altered morphology of the transformed cell as a selective marker (Vogt, 1977). We were unsuccessful in our attempts to isolate stable MSV ts mutants using morphological screening techniques. It seemed possible that (1) stable MSV ts mutants might arise at a very low frequency, requiring some selective procedure before they could be isolated from the bulk of cells transformed by normal MSV; (2) initial isolates of ts mutants might be only slightly temperature sensitive, requiring several cycles of cloning before a workable mutant could be obtained; or (3) ts isolates might remain sufficiently transformed at the nonpermissive tempera0042-6822/79/080303-14$02.00/O Copyright Q I979 by Academic Press, Inc. All rights of reproduction in any form reserved.

304

BLAIR,

HULL,

ture to escape detection in procedures based on morphological screening. This report describes the isolation and preliminary characterization of ts mutants of Moloney MSV using an enrichment and selection procedure based on the ability of transformed cells to grow in semisolid media (MacPherson and Montagnier, 1964). The procedure allowed the isolation of temperature-sensitive viral mutants at relatively high frequency. Analysis of the stability and phenotype of these mutants and of their progeny, after multiple cloning both as virus and as cells, revealed a number of interesting properties. The mutants were stable when maintained as transformed, nonproducer clones, but upon super-infection with MLV of various types, the ts-transformed cells produced a relatively high proportion (- 10%) of phenotypically wt MSV. Furthermore, although morphological transformation and rate of hexose uptake were readily reversible when the cells were shifted from the nonpermissive to the permissive temperature, the ability of the cells to form colonies in agar suspension was strongly inhibited by a short period of incubation at the nonpermissive temperature. MATERIALS

AND

METHODS

Cells and viruses. Normal rat kidney (NRK) cells (Due-Nguyen et al., 1966) were obtained from Dr. J. Stephenson, National Cancer Institute (USA). They were cloned, and were regrown from frozen stocks whenever the passage level exceeded 25. The origins of TB cells and the MSV-transformed TB clone 349 cells (349) have been described (Ball et al., 1973). XC cells (Svoboda et al., 1963) were originally obtained from Dr. J. Hartley. All cells were grown in EGM [Eagles minimal essential medium supplemented with 10% heat-inactivated fetal calf serum (Gibco), 1% glutamine, bicarbonate, and antibiotics]. Mutagenesis

and selection of ts mutants.

The selection procedure used is based on one described by Wyke (1971, 19’73). MSV 349 virus was 70% inactivated by uv irradiation (General Electric 30-W germicidal lamp) and was then used to infect NRK

AND

FINCH

cells at an m.o.i. of less than 0.5. After incubation at 34” overnight, the cells were trypsinized and suspended in EGM containing 0.8% methyl cellulose, at a density of lo5 cells/ml (Vogel and Pollack, 1974). Cells were grown in methyl cellulose EGM for 3 weeks at 34” to guarantee that all potential mutants would be present at the start of the selection in multiple copies, reducing the chance of accidental loss. The semisolid medium containing the colonies was then diluted with 4 vol of EGM, repeatedly pipetted, and centrifuged at low speed to pellet the cells and separate them from the methyl cellulose. The cell pellets were washed in fresh EGM until a suspension free of methyl cellulose and consisting predominantly of single cells was obtained. The cells were then grown in tissue culture for 7-10 days at 34” before they were subjected to the selection procedure. For selection, the cells were suspended in methyl cellulose EGM as before, seeded at 5 ml/plate in 60-mm petri dishes (bacterial plates) and incubated at 39” for 24 hr. Onehalf milliliter of a stock solution of cytosine, arabinoside (Sigma) at 20 pg/ml in H,O (sterilized by filtration) was added to each plate and the plates were then incubated at 39” for an additional 48 hr. The cells were separated from the methyl cellulose as was described above and grown at 34” for 7- 14 days to allow survivors to recover after the drug treatment. The selection process was repeated a total of three times. Cells were then suspended at 2 x lo4 cells/ml or less in EGM-0.35% agar and plated either in 60-mm petri dishes or in 60-mm tissue culture dishes containing a base layer of 3 ml of EGM and 0.7% agar. The plates were incubated at 34” for 3-4 weeks, until macroscopic colonies had developed. Fresh agar medium was added to the plates at weekly intervals. Individual colonies were picked and tested for temperature-sensitive behavior as is described under Results. Assay procedures for MST-infected cells. Viral supernatants were assayed for focusforming ability on NRK cells according to established procedures. Polybrene (4-8 pg/ml, Aldrich Chemical Co.) was used to enhance the efficiency of infection.

ts MSV TRANSFORMATION

To assay for colony formation in agar, cells were removed from plates by trypsinization 24 hr after infection, suspended at the appropriate concentration in 2-5 ml EGM containing 0.35% agar, and quickly transferred to a 35- or 60-mm culture dish which contained a hardened base layer of EGM and 0.7% agar. Plates were incubated 14 days (39”) or 21 days (34”) before colonies were counted microscopically. Plates were fed with EGM and 0.7% agar at weekly intervals. The colonies which develop vary widely in size, but are readily distinguished from single cells. No colonies have ever been observed in numerous control assays of uninfected NRK cells. Measurement of 2-deoxyglucose uptake. The ability of various cell lines to take up 2-deoxyglucose was measured using a modification of the procedure of Martin (Martin et aE., 1971). 2-deoxy[3H]glucose was obtained from New England Nuclear, Lachine, Quebec. Protein concentration was determined using either the procedure of Lowry (Lowry et al., 1951) or the Bio-Rad protein assay [Bio-Rad Laboratories (Canada) Ltd.].

MUTANTS

305

any possessed a ts phenotype. A number of the surviving clones (59/336) formed colonies between 3- and 270-fold less efficiently at 39” than they did at 34”. The average 34”/39” colony forming ratio for 46 randomly selected wild-type transformed clones was 0.5. Figure 2 compares typical regions of agar plates containing a presumptive temperature-sensitive mutant after incubation at 34 and 39”. In most cases the temperaturesensitive phenotype was readily apparent even prior to counting the colonies under a microscope. Three colonies of each presumptive ts mutant were picked from 34” agar

RESULTS

Isolation of Temperature-Sensitive MSVtransformed NRK clones by selection with Cytosine Arabinoside Wyke had utilized BUdR treatment of transformed cells at 39” in methyl cellulose followed by exposure to visible light, to select for cells transformed by ts avian sarcoma viruses (Wyke, 1973). In our hands, cytosine arabinoside gave a more efficient and reproducible killing of transformed cells growing in methyl cellulose suspension than did BUdR and visible light. Figure 1 shows that cytosine arabinoside concentrations of up to 50 pg/ml had little effect on the viability of normal rat cells (NRK) while reducing the viability of transformed rat cells (XC) by 3 logs. Similar patterns are observed with MSV-transformed NRK cells. NRK cells transformed by uv-irradiated MSV-349 virus and subjected to the selection procedure (see Materials and Methods) were screened for their ability to form colonies in agar at 34 and 39” to see if

DAYS

AFTER

SEEDING

FIG. 1. Survival of normal and transformed rat cells following treatment with cytosine arabinoside. Normal (NRK) and transformed (XC) rat cells were seeded in EGM + methyl cellulose (see Materials and Methods) and incubated 24 hr at 37”. Cytosine arabinoside was then added at varying concentrations. After 24 or 48 hr exposure to the drug, the cells were removed from the methyl cellulose and varying numbers of cells were plated on 60-mm tissue culture dishes in EGM. Surviving cells which formed colonies were counted 14 days later. The ratio of survivors after drug treatment to survivors of a parallel control plate where no drug was used is shown. Normal rat cells (solid lines) treated with 50 pg/ml (O), 10 pg/ml (A), and 2 pg/ml (D). Transformed rat cells (dashed lines) treated with 50 pg/ml (O), 10 pg/ml (A), and 2 pg/ml cm).

306

BLAIR,

HULL,

AND FINCH

FIG. 2. Colony formation by a temperature-sensitive NRK transformed clone in agar at 34 and 39”. Equal numbers of cells were seed in EGM + 0.35% Difco purified agar and incubated at 34 and 39”. Plates were photographed after 21 days. (A) NRK tsll0, 34”; (B) NRK tsll0, 39”.

assay plates, grown up in liquid culture at 34”, and then retested for colony formation at 34 and 39”. Thirteen of the colonies of 12 individual isolates were selected for further study. ts-Transfcrrmed ts MSV

Cells Contain

a Rescuable

Table 1 shows the agar growth properties of several temperature-sensitive clones at various passage levels after isolation. The clones showed a wide range of plating efficiencies at different passage levels, but in all cases, they plated much more efficiently at 34 than at 39”. It was noted that ts clones assayed for growth in agar suspension immediately after cloning often showed strong temperature sensitivity for colony growth with few if any colonies growing at 39”. As a first step in the characterization of these presumptive ts MSV mutants, it was necessary to show that the ts character of these cells was virus dependent. Preliminary tests of tissue culture supernatants indicated that the ts-transformed cell clones did not release biologically active MSV or MLV, or

detectable reverse transcriptase activity. Each clone was therefore super-infected with Moloney MLV at high multiplicity (m.o.i. 2 1.0) and after one cell passage at 34”, a 24-hr viral harvest was taken and assayed on NRK cells. Table 2 shows that although rescued-MSV transformed NRK cells less effectively at 39 than at 34”, the efficiency was only reduced 2- to lo-fold when measured by either parameter. The parental ts isolates had shown a much greater temperature dependence (see Table 1). The table indicates, however, that there is fairly good agreement between the relative temperature sensitivity of focus formation and colony formation, suggesting that the ts defect involves a true transforming function. In order to investigate the nature of the 39” colony-forming ability which appeared after rescue and reinfection further, individual colonies of transformed cells growing at 34” were picked, grown up in liquid culture at 34”, and then tested in an agar colony assay at both 34 and 39”. Table 3 shows the results obtained for 1’7 subclones transformed by MSV which had been rescued from 4 mutant clones. Two were

ts MSV TABLE

TRANSFORMATION

are plated in agar at 34 and 39” (12.5%, see Table 2).

1

STABILITY OF TEMPERATURE-SENSITIVE PHENOTYPE ON CELL CULTURE

NRK

Repeated Cloning Does Not Increase Stability of ts Virus upon Rescue

Relative colony formationb Clone

Passage levela

34’

39”

Ratio 34739

ts101

2 9

0.060 0.062

0.0040 0.0090

15 6.9

ts102

4

0.056

0.0008

70

2

10

0.200 0.063

0.0030 0.0025

67 25

2 9

0.160 0.0025


2 3

0.026 0.013

0.0002

2 7

0.008 0.002

<0.0001

80 <20

6 8

0.012 0.092

0.0003 0.0028

40 33

ts109

7 9

0.017 0.023

0.0001 0.0017

170 14

ts110

2 8

0.0430 0.0580

0.0001 0.0040

430 15

9 18

0.066 0.049

0.1520 0.1080

ts103

ts104 ts105

ts106

ts10s

wt

cl 606Al

0.0002

0.0001 0.0001

307

MUTANTS

<‘1600 13 260 65

the

The virally transformed ts subclones shown in Table 4 were superinfected with Moloney MLV and MSV-MLV virus stocks prepared in an attempt to identify a clone which might stably produce a high proportion of ts MSV. Table 4 shows the results obtained when harvests taken after MLV superinfection of 20 subclones of ts 110 were tested for their ability to transform NRK cells (as measured by colony formation in agar) at 34 and 39”. All harvests obtained from ts clones formed colonies with a reduced efficiency at 39”. Again, clones which showed lOOO- to lO,OOO-fold reductions in TABLE

2

PROPERTIES OF MSV RESCUED

FROM~S NRKCELLLINES

0.43 0.45

Relative plating, 34”/390b Clone”

1.3

ts104

ts105

10

ts106 ts108 ts109

11

16 13

ts110

able to form colonies at 39” and are presumably wild type, while 15 were temperature sensitive for colony growth. Table 4 shows a more extensive analysis of 1 mutant, tsll0, indicating that 2 of the 20 colonies picked at random at 34” were able to form colonies at 39”, while the remaining 18 were temperature sensitive. The proportion of wild-type colonies observed (10%) was consistent with the ratios observed directly when cells newly infected with tsll0 MSV-MLV mixtures

10 8 2

B

606Al

Agar colony assay

10

ts101 ts102’

ts103A” n Number of passages after initial clonal isolation. Cells were passed at 7- to lo-day intervals at 34”. In some instances, cells were frozen at -7o”, then revived and cell growth continued. b Fraction of cells seeded which gave rise to colonies at the indicated temperature.

Focus assay

14

(wt)

1.5

2

>l 7 7 2.5 6.5 3 7 5 8 0.6

n ts clones were infected with wt MuLV at 34”, passed, and a 24-hr harvest was taken from semiconfluent plates. b Focus assay plates were either left at 34” for 21 days, with weekly media changes, or were shifted to 39” l-2 hr after infection and left 14 days with weekly media changes. Foci were counted after fixing and staining with Geimsa. Agar colonies were counted after 14 days (39”) or 21 days (34”). r Rescue of this clone was poor; plating efficiency is based on t10 foci and colonies per plate. d A and B are two independent clones of ts103 which were rescued separately.

308

BLAIR, TABLE

HULI ;. AND FINCH

3

TABLE

4

ABILITY OF NRK CLONES TRANSFORMED BY ts MSV TO GROW IN AGAR AT 34 AND 39

PROPERTIES OF VIRUS RESCUED FROM SUBCL~NES DERIVED FROM NRK tsll0

Number of colonies

Relative colony-forming ability

Subclone

34””

39

340b

Rescued MSV supernatants’ Ratio of ability to transform colonies NRK cells at 34” 34°/390b relative to 39”

1 2 3 4

810 37 402 324

0 210 0 0

NT’ NT NT NT 0.013 <10-d 0.011 10-h

1 2 3 4 5 6

112 984 480 0 28 16

0 0 0 0 0 0

Parent clone ts101

ts105

39”

ts110

NT NT 0.024
ts106

1 2 3 4

330 0 1428 1980 35 0 1740 0

0.030 < 10-4 0.016 0.019 NT NT 0.022 <10-a

ts107

1 2 3

1620 33 75

0.046 0.0002 0.036
0 0 0

Parent

(1Equal numbers of cells were plated at each ternperature. * Represents the relative number of cells giving rise to colonies when lo4 cells were plated in GM + 0.35% agar and incubated at 34 and 39”. r Not tested.

wt606A1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

>lOOO > 1000 54 1 > 1000 2.5 > 1000 170 38 1400 > 1000 1600 43 > 1000 > 1000 > 1000 4.3 >lOOO > 1000 1 0.3 1

wt349

agar growth potential, produced MSV-MLV stocks which were only 2- to l&fold less efficient at inducing agar colony formation at 39”. A summary of the data obtained from 70 subclones is given in Table 5. Although several clones showed higher plating ratios than the original parental cells, all superinfected clones produced a substantial fraction of phenotypically wild-type MSV. The clones which had originally been classified as wt, however, all produced virus upon superinfection which appeared able to transform cells at both 34 and 39”. Several clones have been followed through four cycles of rescue and reinfection without any significant reduction in the fraction of wild-type virus

Subclone”

-

-

-

I

1.3 4.5 0.7 2.2 0.3 5.8 2.6 3.7 3:7 7.7 5.8 14.0 2.0 3.9 15.0 2.1 2.3 4.0 2.1 0.6 0.2 0.6 1.1 1.3 1.1 0.9

cIVirus rescued from the parental clone was used to infect NRK cells at 34”. After 21 days, individual colonies were picked at random from the 34” plates into separate wells of a 24-well microplate (Linbro Scientific Inc.). These colonies were grown up at 34” for subsequent testing. b Equal numbers of cells from each subclone were plated in agar. The ratio of the number of colonies on the 34” plate to the number on the 39” plate is given in the table. r Each subclone was infected with wt MuLV and a 24-hr harvest was taken from a subconfluent plate. This harvest was then used to infect NRK cells, and plates were left at 34” until foci could be seen. The cells were then removed from the plate and equal portions were tested for the ability to form colonies in agar at 34 and 39”. The ratio of the number of colonies obtained at the two temperatures is given.

ts MSV TRANSFORMATION

MUTANTS

309

TABLE 5 PROPERTIES OF MSV RESCUEDFROM~S NRK AND SUBCLONEDAS VIRUS Rescued virus-34”/39” Classification of subclone”

No. tested
ts tsiwt wt Parental clonesc

49 4 17 10

Not determined

Number with plating ratios* 0 0 9 0

1<2 4 0 6 1

2 < 10 27 3 0 8

10 < 20 9 0 0 0

>20 3 1 1 0

9 0 2 1

(2Clones were isolated, tested, and classified by their 34”/39” agar colony ratio: ts, >lO; tslwt, >2-10;

wt, <2. b Virus was rescued from each individual clone by MLV superinfection, and the rescued MSV-MLV mixture was assayed on NRK cells in an agar colony assay. The number of clones where the ratio of colonies induced at 34” relative to 39” falls within each range is indicated. c Data from virus rescued from parental clones, taken from Table 2.

produced upon rescue. The wild-type transformed NRK isolates studied thus far, in addition to their ability to form colonies at 39”, are fully morphologically transformed at both temperatures. Both MSV-MLV producers and nonproducer clones have been isolated, although most of the clones studied produced no infectious MSV or MLV as measured by a variety of criteria. Several clones of Moloney MLV, Rauscher MLV, xenotropic MLV (induced from TB cells by IUdR) and simian sarcoma-associated virus (SSAV) all rescue both ts and wt transforming activity at apparent ratios of between $1 and 2O:l from tsll0 NRK cells. In addition, harvests taken at various times between 1 day and 63 days after superinfection show only minor variations in the proportion of ts and wild-type MSV rescued. Although the other ts clones have not been studied as extensively, they behave similarly in rescue experiments using the Moloney strain of MLV. Expression of the Transformed Phenotype in NRK Cells Transformed by tsMSV The data in Table 6 indicate that although growth in agar suspension is temperature sensitive, growth on plates in liquid media is not significantly affected. The ability of these ts-transformed cells to form colonies when plated on monolayers of normal NRK cells is also temperature sensitive.

Cells growing in liquid culture at 34” are rounded, refractile, and loosely attached to the substrate, often by long processes (Fig. 3A). The morphology is essentially TABLE 6 RELATIVEABILITYOF~S-TRANSFORMEDNRKCELLS TOFORMCOLONIESUNDERDIFFERENT GROWTH CONDITIONS

Clone

Temperature (“)

Plating efficiency” Aw

Plate

<0.0001 <0.0001

0.43 0.52

0.310 0.001 0.006

0.11 0.09 0.09

Uninfected NRK

34 39

ts 110

34 39 34 t 24 hr

Is 109

34 39 34f24hr

0.060
0.13 NTb 0.19

ts 105

34 39 34 t 24 hr

0.100 eO.001
0.10 NT 0.08

wt MSVtransformed NRK

34 39

0.28 0.17

0.76 0.78

a Plating efficiency is defined as that fraction of the cells seeded which will give rise to a colony. Plates were counted after lo- 14 days at 39” and 15-21 days at 34”. * Not tested.

310

BLAIR,

HULL,

AND FINCH

FIG. 3. Morphological transition of tsllO-transformed NRK cells following a temperative shift. The cells were seeded at 100 cells per 60-mm dish and incubated for 6 days at either 34 or 39”. Selected colonies were marked and photographed. The plates were then shifted to 39 and 34”, respectively, and the marked colonies were photographed 24 hr later. (A) 39”; (B) same colony after 24 hr at 34”; (C) 39”; (D) same colony after 24 hr at 34”.

ide !ntical to that of NRK cells transformed by wild-type MSV. If cells growing at 34’ ’ are shifted to 39”, morphological changes bet zome apparent within 5-6 hr. The cells be1gin to spread out on the surface of the GUI.ture dish and become noticeably less

refractile. Within 18-24 hr the morpholog jcal changes are essentially complete (Fig. 3;B), with the cells now resembling uninfec ‘ted NRK control cells. The morphological st
ts MSV TRANSFORMATION

The reciprocal changes can also be seen if cells are tist grown at 39” and then shifted to 34” (Figs. 3C and D). In the case of tsll0, cycloheximide (5 pg/ml) blocks the appearance of transformed, refractile cells when plates at 39” are treated with the drug and shifted to 34”. The morphologies of normal NRK cells and wt MSV-transformed cells are affected. Plates shifted from 34 to 39” in the presence of the drug show morphological changes similar to those seen in control plates shifted in the absence of the drug. The rate of hexose transport, which is characteristically elevated in transformed cells relative to their normal counterparts (Hatanaka and Gilden, 1970), was temperature dependent in NRK cells transformed by these ts mutants. At 34” ts-transformed NRK cells showed rates of 2-deoxy[3H]glucose uptake Z- to 3-fold higher than uninfected NRK cells or the same ts-transformed NRK cells grown at 39”. Table 7 indicates that shifting cells from 34 to 39” resulted in a 2.6-fold drop in the hexose uptake rate within 48 hr of the shift, while essentially the reverse occurs if the temperature is shifted from 39 to 34”. Two points should be noted, however; the shift from transformed to normal phenotype for this parameter is slower than that observed TABLE

MUTANTS

311

for the shift in morphology, and a clear lag of from 8-12 hr is seen before the rate of uptake changes significantly. The reason for the lags seen here is unknown, but both DNA and protein synthesis continue at normal rates during this period. ts-Transformed NRK Cells Lose Their Ability to Form Colonies at 34”in Agar if Incubated at 39” In contrast to the ready reversibility of the tranformation parameters described above, cells transformed by these ts viruses lose their ability to form colones at 34” if cells suspended in agar are first incubated at 39” and then returned to 34”. Table 8 shows that 90% of tsllO-transformed cells have lost the ability to grow in agar at 34” after 24 hr of incubation at 39”. Similar results were obtained for several other ts isolates tested. Table 8 also indicates that these mutants showed near normal colony formation at 37”, and shifting from 37 to 34” did not, as expected, affect the efficiency of colony formation. Similar behavior was obTABLE

ABILITY OF ~~~~O-TR~SF~RMED NRK CELLS TO FORM COLONIES IN AGAR AT 34” AFTER INCUBATION AT 37 AND 39”

7

Hours after shift

34” -3 39”

39” --, 34”

0 (not shifted) 2 4 8 12 18 24 48

1.00 1.21 1.13 0.92 NT NT 0.50 0.38

0.33 0.29 0.23 0.28 0.36 0.58 0.75 0.90

n Uptakes were calculated as cpm 2-deoxy[3H]glucose/mg protein00 min, and represent the average of two duplicate measurements.

Relative colony formation”

Shift

HEXOSE UPTAKE BY tsll0 MSV-TRANSFORMED NRK CELLS FOLLOWING A TEMPERATURE SHIFT Uptake relative to unshifted 34” sample”

8

Clone ts110

From

At

34”

37”

39

None -2hr -15hr -24hr -60hr 39 -2hr -15hr -24hr -60hr

1.0 0.9 0.8 0.8 0.8 1.0 0.2 0.1 0.1

0.6

0.0001

None -60hr -6Ohr

1.0 0.9 0.7

1.4

1.1

37”

wt

606Al 37” 39”

DThe number of colonies formed at 34” without any temperature shift is defined as 1.0. Colonies were counted after 21 days at 34”.

312

BLAIR,

HULL,

served for cells grown in methyl cellulose suspension, although cells exposed to 39” in methyl cellulose and then removed from suspension and returned to plate culture remain viable. Expression of the ts Phenotype in ts-transformed Cells Superinfected with WildType MSV and MLV To determine if the ts mutants exerted a dominant effect over the normal expression of MSV transforming genes, two of the ts clones were superinfected with wild-type MSV (MSV-349) and then tested for their ability to form colonies at 39”. Table 9 shows that the relative efficiency of transformation was similar whether NRK or ts-transformed NRK cells were infected by MSV, and the number of colonies formed was proportional to the amount of MSV added. On the other hand, NRK cells transformed by wt MSV showed the same relative colony-forming ability at both temperatures. Both the Moloney and Kirsten strains of MSV had similar effects. Superinfection of &s-transformed cells by wild-type leukemia virus produced a gradual increase in the ability of these cells to form colonies at 39”. Several weeks after the initial MLV infection the doubly infected TABLE TRANSFORMATION BY WILD-TYPE

9

OF &TRANSFORMED NRK CELLS MSV AT THE NONPERMISSIVE TEMPERATURE

AND FINCH TABLE EFFECT OF MLV AND COLONY FORMATION t~llO-TRANSFORMED

10

MSV SUPERINFECTION

ON

IN AGAR AT 39” BY NRK CELLS”

Relative colony-forming ability W/390* Hours after infection 24 48 72 144 144 hrs-no

+MLV

+MSV

0.002 0.0009 0.025 0.038 0.002

0.051 0.037 NT 0.039 -

MLV, MSV

a Cells were infected with either Moloney MLV (m.o.i. = 15) or 349 MSV (m.o.i. = 0.15). * Cells were trypsinized from individual plates at the indicated time and W and lo3 were suspended in agar and incubated at either 34” or 39” until colonies developed. The ratio of the number of colonies formed in duplicate plates incubated at 34” and 39” is given in the table.

cells plated in agar at 39” with 30 to 60% of the efficiency that these same cells exhibited at 34”. The gradual appearance of 39“ colony forming ability is demonstrated in Table 10. The number of cells capable of forming colonies at 39” increased about 20-fold in the first 5 days after incubation, but the level of colony formation at 39” was still only 4% of that observed at 34”. In contrast, the generation of 39” colonyforming ability was rapid in these cells following MSV superinfection, reaching a maximal level within 24 hr.

Number of colonies/W cells at 39”” CelI line NRK wt MSV-NRK ts105 NRK tsll0 NRK

-MSV 0 3 x 104 86 76*

+MSV - 10-l 2.5 3.0 4.1 2.9

x 104 x 10’

x 104 x 10’

+MSV - 10-Z 5.0 3.0 4.3 2.4

x 103

x 104 x 103 x lo3

a Cells were infected at 34“ with MSV at the indicated dilution and 43 hr later were transferred to agar suspension and incubated at 39”. Colonies were counted 10 days later. b Colonies were small, containing fewer cells than those seen on MSV-infected plates.

DISCUSSION

We have described a procedure for the selective isolation of murine sarcoma virus mutants conditionally defective in their ability to maintain the transformed state. The mutants were selected as transformed NRK cell clones which had lost the ability to form colonies in semisolid media at 39”, but which still grew well in such media at 34”. The cells could be maintained at the permissive temperature in liquid culture for many generations without losing the ts phenotype. Virus rescued from the ts-transformed cells

ts MSVTRANSFORMATIONMUTANTS could be used to transform previously uninfected NRK cells. The majority of this rescued MSV rendered the newly transformed cells temperature sensitive for the maintenance of the transformed state, indicating that a mutated viral genetic locus was involved. These mutants represent the first isolation of mammalian sarcoma virus ts mutants where the selection and screening was based on a phenotypic expression of transformation other than morphology. Nevertheless, all other phenotypic transformation markers tested so far have also been temperature sensitive. It was observed, however, that with continued tissue culture passages, both wild type- and temperature sensitive-transformed clones gradually became less morphologically transformed, although they retained their capacity to form colonies in agar suspension. Colony formation, at least in the NRK system used here, may be a more stable and reliable marker than morphology for MSV expression. It may also represent a more biologically significant marker, since it has been suggested that agar colony formation can be correlated with tumorigenicity (Shin et al., 1975). The ability of &transformed cells to form tumors in rats is under investigation. To be useful in biochemical and genetic studies, a mutant must possess both phenotypic and gentic stability. The mutants we have isolated confer a stable ts phenotype on NRK cells as long as the cells are maintained under permissive growth conditions (i.e., at 34”). In cultures maintained under permissive growth conditions less than 1 cell in 10,000 is capable of growth in agar at 39”. We are able, however, to isolate some stable revertants from cultures maintained for long periods (30-40 weeks) at 34”. In contrast to the relative stability of the &transformed cell, as much as 10% of the MSV rescued after super-infection appears to be wild type. The apparent leakiness of these mutants upon rescue and reinfection is due to the generation of z&transformed cells, rather than an increase in the tendency of ts-transformed cells to grow at the nonpermissive temperature. Indeed, ts-trans-

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formed cells isolated after virus cloning appear more temperature sensitive, as measured by their colony-forming efficiency at 34 and 39”, than their parents. There are several possible sources for the wt MSV isolated after rescue and reinfection with MLV: (1) recombination of the ts viruses with wt transformation genes to yield wt recombinants; (2) rescue of a wt MSV genome present in the ts-transformed cell in which the expression of the ts genome is dominant; or (3) reversion of the mutant ts locus to wt. Recombination could occur with the helper leukemia virus used to effect the rescue or with ,endogenous viral-related information present in NRK cells. Rat cells harbor an endogenous virus (Chopra et al., 1970; Klement et al., 1971) and contain genetic information related to that responsible for the sarcomagenic potential of both Kirsten and Harvey sarcoma viruses (Scolnick and Parks, 1971; Scolnick et al., 1973). This latter information is present in the cell as an RNA which is known to be packaged along with viral RNA in rat cells producing C-type viruses (Scolnick et al., 1976). Recombination between MLV and cell sequences in tissue culture to produce biologically active transforming viruses has only recently been reported (Rasheed et al., 1978), but since tumor virus recombination appears to involve the formation of a heterozygote genome as an intermediate step (Weiss et al., 1973) and since heterozygotes between leukemia and sarcoma virus genome subunits have not been observed (Maisel et al., 1978), such recombinations may occur only very rarely. It is possible that recombination events between endogenous, sarcoma-related genetic information and MSV would occur more readily. It is clearly of interest to determine if wt MSV formed after virus rescue with MLV has acquired any rat-related genetic sequences. The fact that MLV can interact with MSV defectives in some way to correct the transformation defect is a frequently observed phenomenon in the MSV-MLV system. Earlier studies to ts MSV have described similar observations (Scolnick

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et al., 1972, 1974; Kit and Somers, 1973) mutagenized MSV which gave rise to and several leukemia virus focus assays mutants, but the fact that no mutants could (Bassin et al., 1971; McCarter, 1977) appear be isolated directly (i.e., without the cytosine to depend on the ability of leukemia virus arabinoside selection procedure) suggests to “complement” sarcoma viruses by an that it may be fairly low. Furthermore, unknown mechanism. not all of the mutants described here have The possibility that the wt MSV rescued been characterized as extensively as, for from ts-transformed cells represents a wt example, tsll0, and it is possible that some genome present in a suppressed state of them represent replicate isolations of the appears unlikely, in view of the fact that same mutant. It does appear, however, superinfection with wt MSV converts these on the basis of rescue behavior, morphology, cells to a wt transformed phenotype. How- and other criteria, that more than one ever, it cannot be ruled out completely, mutant phenotype is present in the collection since the conditions governing the expression we have described. Previous MSV ts mutant of two genomes entering together (i.e., as a isolates have been divided into two classes heterozygote) may be different from those on the basis of their ability to be “complegoverning the expression of a superinfecting mented” by MLV (Scolnick et al., 1974). genome entering a transformed cell at a Our mutants appear to represent a third later time. The data does indicate that at class, since the data suggest that MLV does not complement them directly, but through least two MSV genomes may be functionally integrated in a single transformed mammalian a process of rescue and reinfection. cell, although continued presence of the ts The lack of a stable, well characterized genome in these superinfected cell remains mutant of mammalian sarcoma viruses has hampered development of a thorough underto be rigorously established. Experiments standing of the biological and biochemical to do this are in progress. mechanism of transformation of these viruses. The possibility that there is reversion The complexity of the transformation process of the ts locus to wt during viral RNA synthesis or proviral DNA synthesis must in mammalian cells is indicated by the fact also be seriously considered. It seems that Kirsten MSV, Abelson lymphosarcoma unclear, however, why MSV ts mutants virus, and Moloney MSV, all of which would revert at such a high rate, while the induce morphological transformation and rate of reversion of ts MLV mutants has solid tumors, exhibit substantial nucleotide sequence differences (Scolnick et al., 1975; been observed to be relatively low (Wong Frankel et al., 1976). A collection of conand McCarter, 1973). Studies are underway ditional mutants of all three viruses would to determine the origin of these revertant be useful in elucidating the transformation viruses. mechanism utilized by each virus. We have The behavior of these mutants upon MLV infection and rescue, emphasizes the neces- begun to systematically isolate and characterize transformation mutants of the Moloney sity, first pointed out by Scolnick (Scolnick strain of MSV. Continued study of these et al., 1974) of isolating MSV mutants as nonproducers. These observations suggest mutants and similar ones of Kirsten MSV and Abelson virus should be extremely that our mutant isolation was probably facilitated greatly by our use of an MSV useful in correlating the biological and stock where the sarcoma virus was present biochemical changes induced by each virus. in a biologically vast excess over the leukemia helper. The selection procedures ACKNOWLEDGMENTS are theoretically applicable to any transforming virus capable of inducing colony We wish to thank Doris Little, Linda Vine, and growth in suspension culture as, for example, Barry Forbes for help in preparing the manuscript, Abelson virus or feline sarcoma virus. and Dr. J. A. McCarter for critically reading the No attempt was made during the selection manuscript. We also thank Dr. P. Fischinger for described here to measure the fraction of initially infecting tsll0 cells with SSAV.

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