Differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus

Cell, Vol. 10, 313-320,

February

1977, Copyright

0 1977 by MIT

Differential Genetic Susceptibility of Cultured Human Skin Fibroblasts to Transformation by Kirsten Murine Sarcoma Virus Lawrence M. Pfeffer* and Levy Kopelovich Memorial Sloan-Kettering Cancer Center 1275 York Avenue New York, New York 10021

Summary Hereditary adenomatosis of the colon and rectum (ACR), an autosomal dominant trait, is associated with a predisposition to neoplasia. The present study describes the differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus. Primary cutaneous outgrowths were derived from normal appearing subepidermal biopsies of ACR phenotypes and appropriate controls. Exponentially growing cell cultures from ACR subjects and a portion of the clinically asymptomatic ACR progeny subjected to the viral probe were 100-1000 fold more susceptible to transformation than were normal skin fibroblast cultures. The virally transformed human skin fibroblasts showed a loss of anchorage dependency in carboxymethylcellulose suspension and formed tumors in athymic mice. The results suggest that skin fibroblasts obtained from individuals genetically predisposed to colon neoplasia are preferentially transformed by the Kirsten murine sarcoma virus.

used for the elucidation of phenotypic alterations and biochemical mechanisms associated with neoplasia (Bader, 1972; Tooze, 1973; Risser and Pollack, 1974; Sanford, 1974). Transformation of human cells by murine sarcoma virus (MSV), although relatively inefficient, has been observed in several instances (Aaronson and Todaro, 1970; Klement et al., 1971; Rhim et al., 1973). Variation in susceptibility to transformation by the Kirsten strain of MSV (Ki-MSV) has been demonstrated in individual cell strains and cell lines derived from normal subjects or fetuses, as well as from individuals with genetic abnormalities or neoplasia (Klement et al., 1971). The present report describes the transformation of skin fibroblasts from ACR phenotypes and normal subjects by a rat-adapted Ki-MSV. The Kirsten strain of MSV, a sarcomagenic isolate from a ratpassaged murine erythroblastosis virus, shares not only the group-specific antigen of all MSV, but also many of the in vivo biologic properties of Moloney and Harvey strains of MSV (Rhim et al., 1973). The results show that cell cultures from ACR subjects and a portion of the clinically asymptomatic ACR progeny were considerably more susceptible to tranformation by Ki-MSV than were control cultures. This may possibly be related to the presence of early and previously undetected metabolic lesions in skin fibroblasts from ACR genotypes. Results

Introduction A heritable propensity to colonic neoplasia is found in individuals with familial adenomatosis of the colon and rectum (ACR), an autosomal dominant trait (Morson and Bussey, 1970; Alm and Licznerski, 1973). As part of a study of the phenotypic expressions in cultured skin fibroblasts derived from normal appearing cutaneous biopsies of ACR phenotypes, we have demonstrated (Pfeffer et al., 1976) that skin fibroblasts from ACR subjects and a number of their clinically asymptomatic progeny (first filial generation, abbreviated as F,), but not from spouses of ACR individuals or normals, showed lack of contact inhibition and decreased serum requirement. These skin fibroblasts, however, did not grow in carboxymethylcellulose (methocel), nor did they form palpable tumors in athymic mice. Thus skin fibroblasts obtained from ACR phenotypes provide a model for the analysis of biochemical and genetic parameters which may help to elucidate the inherited differential susceptibility of human subjects to neoplastic transformation (Kopelovich, Pfeffer, and Lipkin, 1976). Viral transformation of cells in culture has been ’ L.M.P. is a predoctoral student sity Graduate School of Medical

with L.K. at the Cornell Sciences.

Univer-

Viral Transformation of Human Skin Fibroblasts Viral transformation was carried out at day 1 postplating on exponentially growing skin fibroblasts. Focal areas of transformed cells became apparent within 7-9 days and were scored 14 days postinfection. The morphological alterations of the transformed foci were characterized by refractile spindleshaped and round cells which grew on top of the monolayers, exhibiting large cytoplasmic vacuoles (Figure 1). The number of foci induced by various samples from the same virus pool in several individual cultures representing one subject did not vary by > 30% in 2-4 different experiments. The infection of the skin fibroblast cultures with serially diluted viral stock resulted in a titration curve consistent with a “single-hit” dose response (Figure

2). Differential Susceptibility of Human Skin Fibroblasts to Transformation Tables 1 and 2 show the difference in genetic susceptibility of the various human skin fibroblast cultures to transformation by Ki-MSV. Transformed foci were observed in 34 of the 36 cultures tested, albeit to varying degrees (Table 1). The most resist-

Cdl 314

I

I

I

10

102

103

DILUTION

OF

Figure 2. Kinetics of Focus Infected with Ki-MSV

Formation

Ki-MSV

in Human

STOCK Skin

Fibroblasts

Human skin fibroblast cultures were infected with serial dilutions of virus as described in Experimental Procedures and Table 1. Transformed foci were assayed at 14 days for human cells, and a titration curve for focus formation was plotted according to Hartley and Rowe (1966). The pattern observed is representative of cultures examined in this study.

Figure 1. Morphological Demonstration blasts Transformed by Ki-MSV

of Human

Skin

Fibro-

Cultures were examined under phase-contrast optics. (A) Uninfected human skin fibroblast culture at 17 days after plating; unstained; magnification 44x. (6) Virus-infected skin fibroblast culture at 17 days post-infection; unstained; magnification 47x. (C) A focal area in virus-infected skin fibroblast culture at 17 days post-infection; unstained; magnification 147x. Note numerous cytoplasmic vacuoles.

ant cultures (log 10 FFU/ml < 1.0) were derived from normal volunteers chosen at random, normal cultures obtained from the American Type Culture Collection (ATCC), and a portion of the clinically asymptomatic F, ACR progeny. In contrast, cultures susceptible to transformation by Ki-MSV (log 10 FFU/ml > 2.0) included all ACR individuals and

a number of their asymptomatic F, ACR progeny (Tables 1 and 2; FFU/ml as counted on human skin fibroblasts). Thus skin fibroblast cultures taken from ACR genotypes and a fraction of the clinically asymptomatic F, ACR progeny were 100-1000 fold more susceptible to transformation than were all other human cell cultures tested. Parenthetically, rat NRK cells were about 200 fold more susceptible to transformation by Ki-MSV than was the most sensitive human cell culture tested. It is of considerable interest that skin fibroblast cultures from several clinically asymptomatic ACR progeny were resistant to transformation (Table I), since we have previously shown a similar dichotomy within individuals of asymptomatic F, ACR progeny in regard to growth properties of the skin fibroblasts (Pfeffer et al., 1976). Specifically, skin fibroblasts from asymptomatic progeny of ACR individuals were classified as those which were not contact-inhibited and grew in low serum, and those which were contact-inhibited and did not grow in low serum. Skin fibroblasts from individuals in the first category were susceptible to viral transformation, while those from the second category were resistant to transformation by Ki-MSV (Table 1).

Transformation 315

Table

of Human

1. Differential

Susceptibility

Group (I) Symptomatic

(II) Asymptomatic (A) High

(8)

(Ill)

Skin

ACR

Fibroblasts

by Ki-MSV

of Human

Skin

Fibroblasts

to Transformation

by Ki-MSV

Individuals and/ or Cell Lines

Age

J.P.

28

2.81

H.S.

51

2.60

M.G.

58

3.25

J.S.

23

3.45

M.E.

17

3.34

A.V.

54

3.00

A.F.

17

3.00

V.F.

31

2.30

B.L.

24

3.20

Y.P.

43

2.90

B.M.

10

3.20

J.V.

24

2.78

P.F.

19

2.90

KS.

21

2.69

W.G.

25

3.30

R.M.

8

2.90

N.S.

18

0.30

V.E.

14

0.48

B.B.

9

1.60

R.E.

39

0.60

R.V.

50

0.48

B.P.

51

0.78

G.S.

54

0.60

A.M.

23

0.60

L.A.

20

0.48

F.B.

29

0.69

G.B.

28

0.60

Sex

Log Titer

Growth in Low Serum

Progeny

Susceptibility

Low Susceptibility

Normals

(A) Spouses

(B) Normal

(C) ATCC

Volunteers

Cell Lines

s.s

43

0.69

J.S.

40

0.30

CRL 1101

43

0.30

CRL 1102

46

CRL

1185

55

0.84

CRL 1186

56

CRL 1220

15

0.30 f

CRL

38

1225

0.60

Human skin fibroblasts were plated at 2 x lo5 cells per 25 cm2 Falcon tissue culture flask 24 hr prior to infection with virus. Following treatment for 1 hr with DEAE-dextran (molecular weight >2 x 10e daltons) at a final concentration of 25 pg/ml in a total incubation volume of 4 ml, cultures were infected with IOfold serial dilutions of virus. The undiluted virus stock contained lOa.’ FFUH .O ml on rat NRK cells. Transformed foci were scored at 14 days post-infection. The titer on human skin fibroblasts was expressed as FFUlml. Results represent the average of duplicate flasks (variability < 20% in 3-4 different experiments). Asterisks denote no detectable foci in duplicate flasks infected with undiluted virus. The asymptomatic progeny was classified into either high susceptibility (log 10 FFU/ml >2.0) and low susceptibility (log 10 FFLl/ml ~1.0) groups according to our experimental results. Growth in low serum was determined as described by Pfeffer et al. (1976).

Cell 316

Table 2. Student t Analysis Skin Fibroblasts by Ki-MSV

Number Subjects

Group (I) Symptomatic (II) Asymptomatic

ACR

11

of Human

(B) Low Susceptibility

Table blasts

3. Adsorption

Mean Titer

P

3

Particles

3.00 2 0.09


Number Subjects

Group (I) Symptomatic

6

of Ki-MSV

by Human

of

Progeny

(A) High Susceptibility

(Ill)

for the Transformation

2.96 + 0.09 0.46

+ 0.09


(II) Asymptomatic (A) High

NS

ACR

(A) Spouses

5

0.61

? 0.05

(B) Volunteers

5

0.55

+ 0.07

(C) ATCC

6

0.51

* 0.13

(Ill)

Human skin fibroblast cultures were infected with virus as described in Experimental Procedures and Table 1. Mean titer was determined for each group based on the log titer values tabulated in Table 1. (+) denotes standard error of the mean. The P values represent group analyses and express the probability of obtaining a difference with respect to the average value of group III.

Whether loss of contact inhibition, growth serum, and susceptibility to transformation MSV appear concurrently due to a single tional event remains to be established.

in low by Kimuta-

Viral Adsorption to Human Skin Fibroblasts Previous studies have indicated (Vogt, 1967; Toyoshima and Vogt, 1969) that treatment of target cells with diethylaminoethyl dextran (DEAE-dextran) increased the focus-forming titer of MSV, a procedure which also increased the plating efficiency of murine leukemia virus (MLV). It is believed that polycations in general enhance the adsorption of murine type C particles (Tooze, 1973). In our hands, the efficacy with which Ki-MSV particles transformed human skin fibroblasts appeared to depend upon the molecular weight of the DEAEdextran. Treatment of human skin fibroblasts for 1 hr with a DEAE-dextran preparation of 2 x lo6 daltons was 20-100 fold more efficient for subsequent transformation by Ki-MSV than one of 5 x lo5 daltons, although either preparation was equally effective in enhancing the transformation of rat NRK cells by Ki-MSV. Under these experimental conditions, 99% of the virus originally inoculated was adsorbed by all skin fibroblast cultures tested (Table 3). Hence there was no correlation between the ability of the different skin fibroblast cultures to adsorb virus and their susceptibility to transformation. Evidence for Virus Production in Infected Human Skin Fibroblast Cultures Culture fluid from infected skin fibroblast cultures was collected at weekly intervals and assayed for focus formation on uninfected human skin fibro-

Normals

Fibro-

% Virus Adsorbed after 2 Hr Incubation

9

99.52

+ 0.09

5

99.55

* 0.09

3

99.65

-c 0.03

12

99.45

* 0.07

ACR

Susceptibility

(B) Low Susceptibility

Normals

of

Skin

Rat NRK cells were plated at a density of 2 x lo5 cells per 25 cm* Falcon flask at 24 hr prior to viral infection. Cultures were treated for 1 hr with DEAE-dextran (molecular weight >2 x lo6 daltons) at a final concentration of 25 pg/ml in a total volume of 4 ml, and then infected with serially diluted tissue culture fluid harvested from individual human skin fibroblast cultures, following a 2 hr period for viral adsorption. The rat NRK cells were assayed for focus formation at 7 days. The original virus stock contained 5 x lo5 FFU/ml when assayed on NRK cells. (?) denotes standard error of the mean.

blast cultures and rat NRK cells. Both the human cells and the rat cells were subsequently transformed, but the virus titer was considerably higher on rat cells, and in either case did not correlate with the susceptibility to transformation of the individual human skin fibroblast cultures originally infected. Transformed human skin fibroblasts were positive with regard to the murine leukemia groupspecific antigen and the RNA-dependent DNA polymerase. As shown in Figure 3, the group-specific antigen of the murine type C viruses appeared localized in the cytoplasm of cells at 21 days postinfection. Mock-infected cultures were negative by indirect immunofluorescence, nor did infected cultures stain with control rat serum. The results with virion polymerase would appear to indicate an increased activity during the post-infective period (Table 4). There was no correlation, however, between the susceptibility of skin fibroblasts to transformation by Ki-MSV and the levels of RNA-dependent DNA polymerase activity in the culture medium of these cells (Table 4). Experiments were carried out to establish whether skin fibroblasts from ACR individuals are specifically more susceptible to transformation as opposed to infection by Ki-MSV. Thus Ki-MSV-infected human cells were tested for their capacity to induce infectious centers on indicator rat NRK cells at day 1 post-infection. As shown in Table 5, production of infectious centers proceeded to a similar extent in skin fibroblasts derived from ACR individuals, their asymptomatic progeny, and normals. In addition, the XC syncytial assay on human skin fibroblasts 12 days post-infection by Ki-MLV dem-

Transformation 317

of Human

Skin

Fibroblasts

by Ki-MSV

onstrated that productive type C helper virus proceeds with equal efficacy on ACR and normal skin, fibroblast cultures (Table 6). The results of all these assays indicate that the differential susceptibility of skin fibroblasts derived from ACR phenotypes is probably due to differences in the transformation process rather than to an early stage defect in

penetration and uncoating, or to any steps involved in the post-penetration replication of the virus. Growth of Transformed Human Skin Fibroblasts in Methocel Suspension and Ability to Form Tumors in Athymic Mice Several susceptible skin fibroblast cultures infected with Ki-MSV (log 10 FFU >2.0) have been plated in a semi-solid medium (Risser and Pollack, 1974; Shin et al., 1975). As shown in Table 7, these cells formed spherical colonies at an efficiency of >2% of the initial cell number plated. Cell cultures from normal subjects which did transform also grew in methocel, but at lower efficiency (Table 7). Table 5. Infectious Fibroblasts

Center

Assay

Number Subjects

Group

Figure fected rum

3. Indirect Human Skin

lmmunofluorescent Fibroblasts with

Staining of Ki-MSV-lnAnti-MLV Group-Specific Se-

4. RNA-Dependent

Activity Number Subjects

Group (I) Symptomatic

(II) Asymptomatic

(Ill)

DNA Polymerase

Normals

ACR

Progeny

12

4

IO

in Medium

(I)

Symptomatic

(II)

Asymptomatic

(Ill)

Normals

ACR Progeny

of

Human

Skin

Infectious Centers (IC)1105 Cells

10

14.50

t 0.89

3

15.86

+ 2.14

6

15.86

f

1.56

Human skin fibroblasts were infected with Ki-MSV after treatment with DEAE-dextran (25 pg/ml) under the conditions described in Experimental Procedures. At day 1 post-infection, skin fibroblast cultures were trypsinized and plated on petri dishes preseeded with NRK cells (5 x 10’ cells per 60 mm dish) at densities of 1 x lo*, 5 x 102, 1 x 103, 5 x 103. 1 x IO’. 5 x 10’. and 1 x lo5 cells per dish. Following a 5 day incubation period, cultures were ultraviolet-irradiated at 1400 pWIcm* for 45 set, and XC cells were added (1 x lo* cells per dish). After 72 hr. cultures were fixed, stained, and scored for infectious centers by counting XC plaques. The number of infectious centers formed gave a linear titration pattern. (2) denotes standard error of the mean. Group II consisted of two highly susceptible skin fibroblast cultures and one of low susceptibility to transformation by Ki-MSV.

Human skin fibroblast cultures infected with Ki-MSVwere trypsinized 21 days post-infection, and a suspension of about 5 x lo3 cells was plated onto wells of immunofluorescent microscope slides (Hardy et al.. 1973). The slides were incubated overnight, washed 3 times in PBS, once with distilled water, and fixed in acetone. Wells were incubated with either rat anti-MLV groupspecific serum or normal rat serum, washed with PBS and water, and air-dried. The slides were then incubated with fluoresceinconjugated goat anti-rat serum, washed as described above, counterstained with Evan’s blue, and examined under fluorescent optics. Note the highly granular aspect of cytoplasmic fluorescence. Magnification 390x.

Table

of Ki-MSV-Infected

Harvested

from

Mean

Polymerase

Ki-MSV-Infected Activity

Human

Skin

fibroblasts

at Day:

of 5

10

21

0.95

36.16

52.21

(0.56-3.92)

(0.82-100.98)

(2.35-l

1.30

25.03

45.01

11.15)

(0.96-4.25)

(2.64-60.01)

(2.87-132.53)

3.85

18.07

56.89

(0.11-6.69)

(4.10-83.91)

(3.68-122.56)

The medium of Ki-MSV-infected human cultures was harvested at 5. IO. and 21 days post-infection. The fluid was clarified by low speed centrifugation, and the virion particles were pelleted at 105,000 x g for 90 min. The pellets were resuspended in 0.1 M NaCI, 0.01 M Tris-HCI (pH 7.4) in one twenty-fifth of the original volume. The reaction mixtures contained 50 mM Tris-HCI (pH 7.8), 60 mM KCI, 2 mM dithiothreitol, 0.2 mM MnAc*, 0.02 Azau poly(rA) oligo (dT),,,_,,,, 0.02 mM ‘H-TTP (5000 cpm/pmole), and 0.1% (v/v) Triton X-100 in a total volume of 0.05 ml. The reaction mixtures were incubated at 37°C for 60 min. and the acid-insoluble material was counted in a Packard liquid scintillation spectrometer. Under these experimental conditions, polymerase activity was linear with respect to time and enzyme concentration, and was expressed as pmoles of TTP incorporated per 60 min per 1 x loo cells. The numbers in parentheses represent the range of polymerase activity for each group based on duplicate dishes. To date, all attempts to rescue viral activity from mock-infected human skin fibroblasts of ACR subjects by treatment with halogenated deoxynucleosides. steroids, and a combination thereof have not been successful.

Cdl 318

Table 6. Syncytial Formation Human Skin Fibroblasts

Number Subjects

Group (I) Symptomatic (II) Asymptomatic (Ill)

by Type

Normals

ACR Progeny

C Helper

of

Virus-Infected

Plaque Formation (log PFU/ml)

Table 7. Anchorage Independence Transformed bv Ki-MSV

Number Subjects

Group

6

3.54

k 0.02

(I) Symptomatic

4

3.59

2 0.02

(II) Asymptomatic

0

3.53

+ 0.03

Human skin fibroblast cultures were plated at 2 x IO’ cells per 60 mm petri dish at 24 hr prior to infection with virus. After treatment for 1 hr with DEAE-dextran, cultures were infected with IOfold dilutions of Ki-MSV stock. Following a 12 day incubation, cultures were ultraviolet-irradiated at 1400 pW/cmz for 45 sec. and XC cells were added (1 x IO’cells per dish). After 72 hr, cultures were fixed, stained, and scored for syncytia. The number of syncytia formed gave a linear titration pattern. (+) denotes standard error of the mean. Group II consisted of three highly susceptible skin fibroblast cultures and one of low susceptibility to transformation by Ki-MSV.

The latter result may possibly be due to the lower number of foci present in these cultures. Mockinfected cultures derived from ACR individuals, their progeny, and normals showed a plating efficiency of
ACR

(B) Low Susceptibility Normals

Fibroblasts

EOP in Methocel

6

2.08

+ 0.21

5

2.15

r 0.06

3

0.02

r 0.006

10

0.03

+ 0.003

Ki-MSV human skin fibroblast cultures were trypsinized at 21 days post-infection, and the anchorage requirement in carboxymethylcellulose (methocel) suspension was determined essentially as described by Risser and Pollack (1974). Following trypsinization, cells were plated in 1 ml of medium containing 1.2% methocel, 90% EMEM, and 10% FCS at densities of 1 x 105, 1 x lo’, and 1 x IO3 cells per 60 mm dish over a layer of 0.5% agarose. Cultures were fed with an additional 1 ml of methocel medium at 1 and 2 weeks, and colonies were scored at 3 weeks post-plating. The efficiency of plating (EOP) was tabulated as number of colonies formed (2 100 cells per colony) per 100 cells initially plated. (2) denotes standard error of the mean.

Table 8. Cellular Tumorigenicity Infected Human Skin Fibroblasts

in Nude

Mice

of Ki-MSV-

Cell

Number of Cells Injected

Route

Tumor Incidence

(A) Virus-Transformed Human Skin Fibroblasts

5 x 106

ID

616

2 x 106

ID

517

1 x 10%

ID

416

5 x IO’

ID

2t7

1 x 105

ID

l/6

1 x IO’

ID

l/6

5 x 106

ID

017

2 x 108

ID

O/8

1 x 106

ID

o/7

5 x 105

ID

O/6

1 x 105

ID

o/9

1 x IO’

ID

017

Discussion Hereditary adenomatosis of the colon and rectum is a disorder in which numerous polyps develop in the gastrointestinal tract and where a close association with malignancy has been demonstrated (Morson and Bussey, 1970; Alm and Licznerski, 1973). Thus cell cultures derived from human subjects with an inherent propensity for colonic tumors may provide a model for study of the differential genetic susceptibility to malignant transformation and the biochemical events associated with neoplasia (Fraumeni, 1973; Knudson, Strong, and Anderson, 1973). Cell transformation by a viral agent has been used to study variations in susceptibility of human cell lines with genetic abnormalities or neoplasia (Todaro and Martin, 1967; Klement et al., 1971). The Kirsten strain of MSV has been used effectively to transform cell cultures derived from a number of mammalian species (Rhim et al., 1973), human cell cultures in particular (Aaronson and Todaro, 1970; Klement et al., 1971; Rhim et al., 1973). Aaronson and Todaro (1970) showed that Ki-MSV

of

Skin

Progeny

(A) High Susceptibility

(Ill)

of Human

(B) Mock-Infected Human Skin Fibroblasts

Ki-MSV-infected skin fibroblasts (log FFUlml B2.0) were harvested at 21 days post-infection, and the cells were injected intradermally in the dorsum of each nude mouse in a total volume of 0.02 ml of phosphate-buffered saline. The treated mice were scored for tumor formation during a 4 month period following intradermal (ID) inoculation. The numbers expressed represent 34 different experiments. Mock-infected cultures were treated in a manner similar to that used for virus-infected cultures.

was more efficient than Moloney murine sarcoma virus (M-MSV) in transforming human cell cultures. Further studies indicated that this was probably due to the Ki-MSV-associated xenotropic helper

Transformation 319

of Human

Skin

Fibroblasts

Table 9. Transformation of Human MLV Pseudotype of M-MSV

Skin

Number Subjects

Group (I) Symptomatic (II) Asymptomatic

ACR

9

by Ki-MSV

Fibroblasts

by the Ki-

of Mean 2.67

Titer

P

+ 0.12

0.001

Progeny

(A) High Susceptibility

4

2.70

k 0.16

0.001

(B)

2

0.38

k 0.08

NS

9

0.35

2 0.09

(Ill)

Low Susceptibility Normals

Human skin fibroblast cultures were infected with virus as described in Experimental Procedures. Mean titer values were determined for each group based on the average log titer values obtained on duplicate dishes in 2-3 different experiments. The undiluted virus stock contained 105.‘* FFU/ml on rat NRK cells. (et) denotes standard error of the mean. The P values represent group analyses and express the probability of obtaining a difference with respect to the average value of group III.

virus rather than to the sarcoma genome itself (Aaronson and Weaver, 1971). In this respect. the transformation of human skin fibroblasts by MMSV was found to be relatively inefficient with a titer of log 10 FFU/ml
trait where involvement of an autosomal dominant gene has been indicated. In one case, however, an asymptomatic child of an ACR subject, whose skin fibroblasts did not grow in 1% FCS, was highly susceptible to transformation. Previous studies suggest that transformation in vitro associated with tumorigenicity in athymic mice was dependent upon the ability of cells to proliferate in the absence of anchorage (Shin et al., 1975). This conclusion is supported by the present findings that selection for anchorage-independent cells resulted in the simultaneous selection of tumorigenic cells within the virally transformed human skin fibroblasts. Predisposition to neoplasia of ACR subjects would therefore appear to be correlated with loss of contact inhibition, decreased serum requirement for growth, and a heightened susceptibility to transformation by Ki-MSV of skin fibroblasts. Study of additional forms of inherent predisposition to cancer and of large familial aggregates with various neoplastic diseases is currently under way. Experimental

procedures

Cells Monolayered cells were grown and maintained in Eagle’s minimal essential medium with Earle’s balanced salt solution containing 10% (w/v) FCS, 2 mM glutamine, 100 U/ml penicillin, 100 rg/ ml streptomycin, and 1 x nonessential amino acids (Gibco. X114) (EMEM). Human skin fibroblast cultures were derived from normal appearing subepidermoid biopsies of individuals with ACR, their F, ACR progeny and spouses, as well as healthy volunteers having no family background of neoplasia. The biopsies were grown to confluency as explants, and the resultant cells were then subcultured and used within the first 10 passages in culture (Pfeffer et al., 1976). Several normal cell lines were obtained from the ATCC. The rat cell line, NRK. a Ki-MSV transformed NRK cell line (t-NRK) continuously producing Ki-MSV (MLV), and a Mok&V-transformed NRK cell line producing the Ki-MLV pseudotype of MO-MSV were provided by Dr. S. Aaronson (Viral Carcinogenesis Branch, National Cancer Institute). The Ki-MLV was a gift from Dr. P. O’Donnell (Memorial Sloan-Kettering Cancer Center). Virlls Ki-MSV was grown into stocks by harvesting tissue culture fluid from t-NRK cells 48 hr after a medium change. The fluid was clarified by low speed centrifugation. passed through 0.45 p millipore filter, and stored at -65°C. The virus stock was assayed for focus formation on rat NRK cells, and the titer was expressed as FFU/ml as described by Hartley and Rowe (1966). Virus Assays Assay of focus formation by the Ki-MSV stock was performed on cultures containing 2 x 10s cells in 25 cm2 Falcon flasks. Within 24 hr after cell plating, cultures were treated for 1 hr with 4 ml of 25 pg/ml of DEAE-dextran (Pharmacia; molecular weight >2 x lo6 daltons) with constant agitation. These were washed once with Earle’s balanced salt solution and incubated for 2 hr with 0.5 ml of appropriately diluted (10 fold serial dilutions) virus stock with frequent shaking. The multiplicity of infection was 1.2 for all experiments reported here. Medium was changed twice weekly, and cultures were scored for focus formation at 7 days for NRK cells and at 14 days for human skin fibroblasts. Virus replication in infected human cultures was assayed as

Cell 320

follows: first, detection of progeny virus by the assay of supernate RNA-dependent DNA polymerase activity (Stephenson, Reynolds, and Aaronson, 1972); second, immunofluorescent binding of an interspecies MSV (MLV) group-specific antigen using rat serum prepared against MLV and a fluorescein-conjugated goat anti-rat serum (Hardy, Hirshaut, and Hess, 1973); third, detection of transforming virus in tissue culture fluid by the focus assay (Hartley and Rowe, 1986); fourth, detection of type C helper virus replication in Ki-MSV-infected human skin fibroblasts by the XC syncytial assay (Rowe, Pugh, and Hartley, 1970; Stephenson et al., 1972); and fifth, induction of infectious centers by Ki-MSV-infected human skin fibroblasts on indicator rat NRK cells using an adaption of the XC syncytial assay. Neoplastic transformation of Ki-MSV-infected cultures was assayed by the following methods: growth in methocel suspension (Risser and Pollack, 1974; Shin et al., 1975), and ability of skin fibroblasts to form tumors in athymic mice (Freedman and Shin, 1974). Viral Adsorptlon DEAE-dextran-treated skin fibroblasts were infected with Ki-MSV stock. After a 2 hr adsorption period, the incubation medium was harvested, serially diluted, and incubated with DEAE-dextrantreated, virus-free NRK cells. Fresh medium was added after 2 hr, and the cultures were scored for focus formation 7 days postinfection. Acknowledgments We thank Mrs. P. Monaghan for her excellent technical assistance; Drs. E. Fleissner and R. Pollack for helpful discussions; and Dr. M. Lipkin for his advice and support of this project. This work was supported by a contract and a grant from the National Cancer Institute. Received

April 7. 1976;

revised

November

1, 1976.

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S., and Todaro,

Aaronson,

S.. and Weaver,

G. (1970). C. (1971).

Aim. T., and Licznerski. G. (1973). 2, R. McConnell, ed. (Philadelphia: Bader.

J. (1972).

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458-459. 73,245252.

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48, 494-501.

Fraumeni, J.. Jr. (1973). Frei. Ill, eds. (Philadelphia: Freedman,

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In Cancer Medicine, Lea and Febiger).

V. H. and Shin,

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