Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail

Cell,

Vol. 11, 95-103,

May 1977,

Continuous Chemically

Copyright

0 1977 by MIT

Tissue Culture Cell Lines Derived from Induced Tumors of Japanese Quail

Carlo Moscovici, M. Giovannella Moscovici and Humberto Jimenez Tumor Virology Laboratory Veterans Administration Hospital Gainesville, Florida 32601 and Department of Pathology College of Medicine University of Florida Gainesville, Florida 32601 Michael M. C. Lai, Michael J. Hayman and Peter K; Vogt Department of Microbiology University of Southern California School of Medicine 2025 Zonal Avenue Los Angeles, California 90033

Summary Several continuous tissue culture cell lines were established from methylcholanthrene-induced fibrosarcomas of Japanese quail. The lines consist either of fibroblastic elements, round refractile cells or polygonal cells. They show transformed characteristics in agar colony formation and hexose uptake, and most are tumorigenic. Their cloning efficiency in plastic dishes is not increased over that of normal quail embryo fibroblasts. The quail tumor cell lines do not produce endogenous avian oncoviruses and fail to complement the Bryan high titer strain of Rous sarcoma virus; those tested lack the p27 protein of avian oncoviruses. Most of the cell lines are susceptible to subgroup A avian sarcoma viruses, but are relatively resistant to viruses of subgroups C, E and F as compared to normal quail embryo fibroblasts. Introduction Avian embryo fibroblasts have a limited life span in tissue culture, and attempts to develop permanent cell lines have been generally unsuccessful (for review, see Gey et al., 1974). Carrel and Ebeling (1921) claimed continuous growth of chick embryo fibroblasts, but it is probable that fresh cells were inadvertently added to his cultures with the embryo extract which he found an obligatory component of the medium. More recently, several chicken lymphoblastoid cell lines have been established. These were derived either from neoplasms induced by a lymphoid leukosis virus (Siegfried and Olson, 1972; Hihara, Shimizu and Yamamoto, 1974) or from tumors induced by Marek’s disease herpes virus (Akiyama, Kato and Iwa, 1973; Akiyama and Kato, 1974; Powell et al., 1974). Long-term growth of normal chicken embryonic fibroblasts on a collagen sub-

strate has been described by Gey et al. (1974), and a fibroblast line of ring-necked pheasant was recently established by Linial (1976). In this report, we describe several continuous cell lines derived from methylcholanthrene-induced tumors of Japanese quail. The properties of these lines make them potentially useful tools in virology and cell genetics. Results Chemical Induction of Tumors in Various Avian Species Young birds aged between 6 and 24 days received intramuscular injections of one of the following carcinogens: 20-methylcholanthrene (20 MCA), 7,12-dimethylbenzathracene (DMBA) or N-methylN-nitroso-N-nitroguanidine (NTG) (Table 1). Tumors were induced with 20 MCA in Japanese quail (Coturnix coturnix japonica), in ring-necked pheasants (Phasianus colchicus torquatus) and in Bantam chicken (Gallus gallus). The time of first tumor appearance varied from 28 days in Japanese quail to 44 days in pheasants. All tumors were histologically fibrosarcomas. Primary cell cultures were prepared from the chemically induced tumors. Cultures obtained from tumors in Japanese quail grew very slowly for 1-2 weeks, then began to replicate actively. Some of the cultures degenerated after a few weeks in vitro, while others (a total of 13 out of 50 originally seeded) gave rise to continuous cell lines designated by “QT” followed by an identifying number (Table 2). These QT lines have been kept in culture for 3 years and have undergone from 70120 transfer generations. The lines were tested for mycoplasma contamination in the laboratory of Dr. George Kenny (Seattle). The most recent transfer generations were found to be negative for mycoplasma in direct culture and in the uracil incorporation test (Kenny, 1973). Attempts to establish similar cell lines from the chicken and pheasant tumors were not successful, even when tumor pieces were placed directly in the culture dish without prior enzymatic digestion. Morphology and Karyotype of QT Lines Figure 1 and Table 2 show that by cell shape and growth pattern, the QT lines could be divided into three morphological types. In cultures of QT4, QT24 and QT35, the cells were predominantly of fusiform-fibroblastic shape. Cultures of QT5 and QT29 showed many flat polygonal cells and, especially QT29 at high population densities, numerous polykarocytes. The rest of the cell lines consisted of overtly transformed fibroblasts with varying proportions of round and of swollen, refractile, spindle-shaped cells. In these cultures; there was con-

Cell 96

Table

1. Tumor

Induction

in Domestic

Species

Fowl with

Chemical

Carcinogens

Carcinogen

Japanese

Quail

Amount

ZO-MCA/corn Corn

oil

emulsion

NTGIDMSO Bobwhite

Quail

Ring-Necked White

Pheasant

Leghorn

Bantam

Chicken

Chicken

20-MCA/corn

Table

2. Morphological

DMBA

and Growth

63

116

oil

4 m9

8/30

oil

4w

o/55

oil

4 m9

= 7,12-dimethylbenzathracene;

Characteristics

of QT Lines

Cell Line

Saturation Density (Cells per mm2 x 1o--3)

Prevalent Shape

QT2

1.4

r

No

QT4

1.4

f

Yes

Cell

Transfer Requires Trypsin

QT5

1.4

P

Yes

QT6

1.4

r+f

No

QT7

1.5

r

No

QT24

1.6

f

Yes

QT27

2.5

r+f

Yes

QT29

1 .I

P

Yes

QT35

1.7

f

Yes

QT46

2.7

r+f

Yes

QT51

1.2

r+f

No

QT53

ND

r+f

Yes

QT54

2.1

r+f

Yes

QEF

0.5

f

Cells were seeded at 3 x 106/100 mm dish and medium (81% FIO, 12% tryptose phosphate broth, 1% chicken serum, 1% dimethylsulfoxide) daily. cells was determined for duplicate dishes on days latter 2 days showed no significant difference. quail embryo fibroblasts; r = round cell shape: f = fusiform cell shape; p = polygonal cell shape with

Yes received fresh 5% calf serum, The number of 5, 7 and 9; the QEF = normal fibroblastic and polykaryocytes.

siderable heterogeneity and variety of cell types. Some of this variety persisted after initial cloning, but a rigorous clonal analysis of the different cell lines and cell types has not yet been performed. In the lines containing mainly rounded, transformed cells, clumps and single cells were shed into the medium when the cultures became confluent, and these could be transferred without enzymatic dispersion. They may also be suitable for spinner culture.

63

46

o/29

20-MCA/corn

Pays)

o/40 0120

20-MCA/corn

(27%)

Length of Observation

0.5 mg 0.1 mg

oil

with Tumor/ Injected

o/20

4 w

20-MCA/corn

20 MCA = 20-methylcholanthrene; ylsulfoxide.

66/241

4 m9

oil

DMBA/fat

Injected

Number Number

16/30

46 (27%)

79 44

(53%)

NTG = N-methyl-N-nitro-N-nitrosoguanidine;

55 DMSO

= dimeth-

Because there have been very few successes in establishing continuous avian cell lines, we wanted to verify the avian nature of these cells by karyotype analysis. The Japanese quail has 2n = 78 chromosomes in which 5 autosomal pairs are large (macrochromosomes), and the remainder are small and very difficult to identify (microchromosomes). Also among the macrochromosomes is a pair of sex chromosomes which is zz (male) or zw (female). The w chromosome is difficult to distinguish in conventionally stained metaphases (without banding patterns) from the chromosomes of pair number 5 (Figure 2) (Ohno et al., 1964; Talluri and Vegni, 1965; Bernischke and Hsu, 1971; Hartung and Stahl, 1974). In all QT lines, the typical macrochromosomes of the Japanese quail were identified as summarized in Table 3. However, there were significant deviations from diploidy. In practically all cell lines, there was a tendency toward trisomy or tetrasomy in chromosomes 1 and 2, and to a lesser extend affecting chromosomes 3 and 4. Only QT4 and QT35, two of the fibroblastic lines, showed significant numbers of diploid metaphases. In QT5, QT6 and QT46, marker chromosomes were regularly seen. For QT5 and QT46, these had the appearance of chromosome 1 with the shorter arm reduced to approximately one third its normal size. For QT6, the marker chromosome was metacentric with both arms the length of the longer arm of chromosome 1. Whether these marker chromosomes result from translocations or duplications and deletions will have to be decided by banding analysis. Parameters of Transformation: Saturation Density, Tumorigenicity, Sugar Uptake, Colonies in Agar and Cloning Table 2 shows that all QT lines had increased saturation densities as compared to normal Japanese

Quail 97

Figure (A) (B for (D)

Cell

Lines

1. Morphology

of QT Cell Lines;

Bright

Field Optics,

QT24 consists mainly of fibroblastic elements. and C) QT6 and QT2 are examples of overtly transformed cell clumping. QT29 contains many vacuolated cells with polygonal

100x cultures

outline

quail fibroblasts. For several of the lines, there was no true saturation density, as clumps of rounded cells floated off in the tissue culture medium and were replaced by new cell growth. Table 4 shows the results of injecting QT lines into the pectoral muscle of young Japanese quail. Of the lines tested, all except QT5 induced progressively growing tumors. Whether the failure of QT5 to induce sarcomas is due to an enhanced antigenicity of the tumor cells or to reduced neoplastic properties is not known. Measurements of hexose uptake were carried out on a few selected QT lines and are summarized in Table 5. The rate of uptake was increased 3-5 fold relative to normal quail embryo fibroblasts and was similar to that seen with quail embryo fibroblasts transformed by avian sarcoma virus. An exception was one of the fibroblastic lines, QT24, which showed near normal rates of hexose uptake; these became increased if the QT24 cells were infected

with

refractile

and epitheloid

spindle-shaped growth

and round

cells,

and a marked

tender

pattern.

and transformed by avian sarcoma virus. On the other hand, infection of QT6, an already fully transformed line, with avian sarcoma virus did not enhance hexose uptake further. Table 6 lists the efficiency of colony formation by QT lines in agar. Even the fibroblastic cell lines QT4, QT24 and QT35 were colony formers, which for QT4 and QT35 is in accord with their tumorproducing potential. Agar colonies were from 1-3 mm in diameter on day 14 after seeding; they could be picked and grown into mass cultures. The cloning efficiencies of QT lines are compiled in Table 7. On plastic dishes, none of the lines showed a cloning efficiency above that of secondary quail embryo fibroblasts, and some cloned at substantially lower efficiencies. However, if cloning was carried out on a feeder layer of mitomycin Ctreated quail fibroblasts, cloning efficiencies were increased 5-20 fold, including the cloning efficiency of normal quail embryo fibroblasts.

Cell 98

Tests for Avian Leukosis Virus in QT Lines We have screened, several of the QT lines for the presence of protein p27 of the avian leukosis and sarcoma complex (Table 8). All were negative with the exception of a high passage of QT24. However, when earlier passages of QT24 were tested, these were also negative. The late passage QT24 was found to release infectious subgroup A avian leukosis virus, and since nucleic acid sequences of this virus are absent from Japanese quail fibroblasts (Varmus et al., 1972), we assume that the virus in late passage QT24 is an accidental laboratory contaminant. QT3.5, QT7, QT4 and QT27 have also been tested by conventional complement fixation using the procedure of Friis, Toyoshima and Vogt (1971). All were negative. The possible production of virus particles was monitored in QT2, QT6, QT7, QT24, QT35 and QT46 by labeling with 3H-uridine, but no particles sedimenting at the density expected for oncoviruses in

a sucrose gradient were found. Tests for reverse transcriptase in the supernatants of QT2, QT4, QT5, QT6, QT7, QT24, QT29 and QT35 were also performed with negative results. The possible presence of viral glycoprotein in the QT lines was tested in two ways. In the first, QT cells were infected with avian sarcoma virus 877 or Prague strain Rous sarcoma virus of subgroup C (PR-C). After 2 weeks, virus harvests were tested for their ability to plate on C/C cells. These cells are resistant to viruses with glycoprotein specificity of subgroup C, such as B77 or PR-C, but are susceptible to all other avian tumor virus subgroups. Thus if B77 or PR-C had acquired a new glycoprotein during passage in QT lines, it would be expected to allow plating on C/C cells. With one exception (QT29-B77),!the subgroup C avian sarcoma viruses passaged in QT lines did not show detectable plating on C/C (Table 9). Virus titers were generally low, however, and very small amounts of a new host range variant would not have been detected. The nature of the QTPS-passaged 877 which plates on C/C is now being investigated. Preliminary data suggest that it may carry glycoprotein of the subgroup E specificity, possibly derived from the initial B77 inoculum. As a control, subgroup C avian sarcoma viruses were passaged in line 6 chick embryo fibroblasts which produce subgroup E avian leukosis virus glycoprotein. The virus yields from these passages show the expected change of host range; B77 and PR-C plated on C/C cells with an efficiency of 16 and 29%, respectively. In the second test for endogenous viral glycoprotein, all the QT lines were co-cultivated with quail cells transformed by the glycoprotein-defective

Macrochromosomes of Coturnix coturnix japonica

1

2

3

4

zw

5

Figure 2. Schematic Representation of the Five Pairs Autosomes and the zw Sex Chromosomes in Coturnix (2n = 78)

Table

Cel I

3. Karyotyping

of Large japonica

of QT Lines

Number of Metaphases Analyzed

Macrochromosome 1

Copies 2

per Metaphase 3

4

X

QT2

4

4

4

4

2

2

QT4

a

4

4

2

2

2

Additional Marker Chromosome

22

2

2

2

2

2

QT5

4

3

4

4

3or4

2

Yes

QT6

16

3

3

2

2

1 or2

Yes

QT7

4

4

4

2

2

2

QT24

4

3

3

4

3

2

QT27

a

3

3

2

2

2

QT35

QT46

32

4

4

2

2

1

IO

2

2

2

2

1

15

3

4

2

2

2

The table lists the copies of macrochromosomes I-4 per metaphase-that is, 2 is disomic, chromosomes in QT5 and QT46 are subtelocentric, the size of chromosome 1 with its short chromosome in QT6 is metacentric with arms the size of the larger arm in chromosome 1,

3 trisomic, arm reduced

Yes 4 tetrasomic. to one third.

The The

marker marker

Quail 99

Cell

Table

4. Tumor

Cell Line QTl

Lines

Formation

Number of Cells lniected 3 x 105

with

Table

QT Lines Time First Tumor Appeared (Davs)

Number with Tumor/Number lniected 15120

5 x 105

13115

6

QT4

2 x 105

3111

47

QT5

3 x 105

o/21

>70

QT7

5 x 105

415

a

QT29

5 x 105

14118

9

QT35

5 x 105

213

QT46

5 x 105

213

QEF

5 x 105

o/20

Cell Line

28

QT2

5 6 270

Cells were suspended in 0.1 ml medium and injected into the right pectoral muscle of 1-2 day old Japanese quail. Birds were observed for 70 days.

Table

5. Uptake

of Deoxy-D-Glucose

by QT Lines

Cell

Deoxy-D-Glucose cpm/mg Protein

Uptake QEF

QEF

13,120

1 .o

SR-A-QEF

63,153

4.8

QT2

55,344

4.2

QT4

53,184

4.1

QT5

51,151

3.9

QT6

47,664

3.6

SR-A-QT6

42,035

3.2

MSR-A-QT6

46,015

3.5

QT24

16,959

1.3

PR-A-QT24

65,969

5.0

QT29

39,106

3.0

QT35

44,i la

3.4

QT46

45,922

3.5

Relative

6. Colony

to

The uptake of 3H-deoxy-D-glucose was measured as described by Martin et al. (1971). QEF = normal quail embryo fibroblasts; SR-A-QEF = QEF transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (SR-A); SR-A-QT6 = QT6 transformed by SR-A; tdSR-A-QT6 = QT6 infected by transformationdefective SR-A; PR-A-QT24 = QT24 transformed by Prague strain Rous sarcoma virus subgroup A.

Bryan high titer strain of Rous sarcoma virus for 21 days. Supernatants of such cultures were then tested for infectivity on chicken and quail embryo fibroblasts. None of the mixed cultures yielded infectious sarcoma virus, suggesting that the QT lines did not harbor a glycoprotein which could complement the envelope defect in the Bryan high titer Rous sarcoma virus.

Formation

by QT Lines Efficiency Agar (%)

QT2

0.9

QT4

0.5

QT5

6.2

QT6

13.0

QT7

1.3

QT24

0.9

QT35

15.0

QT51

6.1

W)Q

11.0

in Agar of Plating

in

Cells were suspended in cloning agar and seeded at concentrations of lo3 and lo4 per 60 mm dish. Colonies were counted 2 weeks after seeding. The plating efficiencies given are from a representative experiment. R(-)Q are Japanese quail fibroblasts transformed by the replication-defective Bryan high titer strain of Rous sarcoma virus.

Infection of QT Lines with Avian Sarcoma Viruses Normal embryo fibroblasts of the Japanese quail are susceptible to avian sarcoma virus with envelope specificities A, E, F and G, and less so to subgroup C and D viruses. They are resistant to the envelope subgroup B (Duff and Vogt, 1969; Weiss, 1969; Hanafusa, Hanafusa and Miyamoto, 1970; Vogt and Friis, 1971; Fujita et al., 1974). We have tested the QT lines for their susceptibility to avian sarcoma viruses as host cells in direct focus assays, as infectious centers and by the amounts of virus released during long-term growth of infected cultures. In the fusiform-fibroblastic lines QT4, QT24 and QT35, multi-layered foci of rounded cells can be induced by avian sarcoma viruses. With subgroup A viruses, the efficiency of focus formation is comparable to quail embryo fibroblasts in QT4 and QT24, but about 100 fold reduced in QT35, probably due to overgrowth of noninfected cells. Infectious center assays of subgroup A, C, E and F sarcoma viruses were performed on QT2, QT5, QT6, QT7, QT46 and QT51 (Table 10). QT2, QT5 and QT51 were relatively resistant. The others showed a spectrum of susceptibility which was similar to normal quail fibroblasts-that is, high for subgroups A and E, low for subgroup C and intermediate for F. Table 11 summarizes the production of sarcoma viruses in QT lines. QT6 is the most susceptible line; QT2, QT5 and QT51 are again relatively resistant. Subgroup A viruses are synthesized at levels similar to those seen in normal quail embryo fibroblasts. Subgroup C, E and F sarcoma viruses are produced at considerably lower levels. The reason for this reduction remains to be investigated; preliminary data suggest that it may be due to an

Cell 100

Table

7. Cloning

Efficiency

of QT Lines

Cell Lines

Cloning

QT2

3.5

QT4

2.9

QT5

0.3

QT6

3.0

QT7

0.6

QT24

0.04

QT29

Efficiency

0.1

QT35

0.7

QT51

1.5

QT54

1.8

QEF

3.3

Cells were plated on 60 mm plastic dishes as described in Experimental Procedures. Clones were counted 2 weeks after seeding. Results are from a representative experiment. QEF = secondary quail embryo fibroblasts.

overgrowth of transformation defective viruses which appear more rapidly in QT lines. In several viral harvests, changes were noted compared to the original inoculum. The appearance of a host range variant in QT29-passaged B77 has already been mentioned. Schmidt-Ruppin strain of Rous sarcoma virus, subgroup E (SR-E) grown in QT29, QT4 and QT7 but not in QT5, QT6 and QT35 or in normal quail fibroblasts, gains plating ability for C/B cells. Bryan high titer RSV propagated in QT2 contains many fusiform focus formers. Whether these changes during QT passage represent a contribution of the host cell or whether they reflect the selection of a rare virus mutant present in the initial inoculum must be decided by future experimentation. Discussion The lines of Japanese quail tumor cells described in this paper are continuous; they have been in culture for over 3 years, have undergone about lo2 transfer generations and show vigorous growth. In ease of handling, they are comparable to continuous mammalian cell lines. However, they all clone on plastic dishes with a low efficiency, although some of them are relatively efficient colony formers in agar. The poor cloning efficiency is probably not an intrinsic property of the QT cells,‘but may reflect suboptimal composition of the medium. Efforts to improve the cloning efficiency are currently under way. If they are successful, avian oncovirus infections could be analyzed with the aid of cellular mutants. The QT lines are derived from fibrosarcomas, and the cells have properties characteristic of oncogenic transformation: they form colonies in agar,

Table 8. Radioimmunoassay for p27 Protein Leukosis and Sarcoma Complex Cell Line

p27 ng/mg

QEF

<20.0

QT2

19.6

QT6

111.5

QT7

<15.4

QT24

120.0

QT29

<21 .o

QT35

<8.5

QT51

<19.6

PR-C-QEF

950

of the Avian

Cell Protein

The amounts of p27 were determined in a competition radioimmunoassay asdescribed previously (Hayman and Vogt, 1976). QEF = quail embryo fibroblasts; PR-C-QEF = QEF infected by Prague strain Rous sarcoma virus of subgroup C. The limits of p27 in the QT lines were calculated from the fact that 2 ng of p27 give 20% inhibition; the amount of p27 in the PRC-QEF was computed from the fact that 20 ng of p27 give 50% inhibition in the radioimmunoassay.

Table Chick

9. 877 and PR-C Passaged Embryo Fibroblasts Efficiency

Cell

877

QT7

<3.9

QT29

in QT Lines:

of Plating

Plating

on C/C PR-C

x 10-S

<2.2

x IO-3

<1.7

x 10-3

<5.0

x IO-3

QT51

11.3

x IO-2

NT

QEF

<8.6

x 10-E


Not passaged

4.0 x 10-a

NT

QT35

CEF,LG

1.6 x 10-l <2.9

on C/C

x 10-C

x IO-5

2.9 x IO-’ c3.7

x IO-6

Avian sarcoma virus 877 and Prague strain Rous sarcoma virus subgroup C (PR-C) were passaged in various cell lines. After 2 weeks, harvests were taken and assayed on C/E and C/C chick embryo fibroblasts. The table lists the ratio of virus titers on C/C over those on C/E cells. QEF = normal quail embryo fibroblasts; CEF,LG = chick embryo fibroblasts, line 6 producing subgroup E avian leukosis glycoprotein; NT = not tested.

induce tumors in the animal host (exception: QT5) and shown an increased rate of hexose uptake. Most lines have round cell shape, but some are fibroblastic and can be “supertransformed” by infection with avian sarcoma viruses. The QT lines support the growth of subgroup A avian sarcoma viruses but are relatively resistant to subgroups C, E and F. QT2, QT5 and QT51 are also resistant to subgroup A. QT6 appears to be the most susceptible line, with virus yields exceeding those from normal quail embryo fibroblasts in many instances. The QT lines do not produce detectable quanti-

Quail 101

Cell

Lines

ties of infectious oncovirus and appear free of viral glycoprotein which could complement the envelope defect in the Bryan high titer strain of Rous sarcoma virus. They also lack the avian oncovirus Table

10.

Infectious

Center

Assays

Fraction Centers

on QT Lines

of FFU Registering

Cell

1

QEF

0.93

10.01

0.88

QT2

0.005


<0.003

QT7

0.83

10.01

0.42

0.02

QEF

0.95

<0.02

0.84

0.32

<0.02

3

PR-C

as Infectious

Experiment

2

PR-A

protein ~27. Some changes in host range were observed, however, with B77 and SR-E passaged in certain QT lines, but the possibility has not been ruled out that this represents selection of rare virus variants present in the initial inoculum. A similar argument can be made concerning the appearance of fusiform focus variants in QT2 passaged avian sarcoma virus (see Toyoshima et al., 1975).

SR-E

PR-F 0.04

QT6

0.53 0.11

0.53

0.39

0.31

0.12

QEF

0.14

QT7

0.93

1.2

QT51

0.06

0.02

QT5

0.04

0.17

<0.02

Ceils were infected with 0.5 to 3 x IO3 FFU per plate. On the next day, they were treated with 2 mg/ml of mitomycin C overnight, washed and plated on fresh layers of quail embryo fibroblasts or of C/B chicken embryo fibroblasts. Focus counts were taken 7 days after plating and compared to the number of foci induced by direct inoculation of quail embryo fibroblasts with the same amount of virus. PR-A, PR-C and PR-F = Prague strain Rous sarcoma virus subgroups A, C and F; SR-E = Schmidt-Ruppin strain of Rous sarcoma virus, subgroup E; B77 = avian sarcoma virus strain Bratislava 77.

Table

11.

Replication Virus

of Avian

and Relative

Sarcoma

Viruses

Procedures

Experimental Animals Japanese quail (Coturnix coturnix japonica), originally obtained from Dr. D. B. Parrish (Kansas State University, Manhattan, Kansas), and White Leghorn chickens of line K-137 (Kimber Farms, Niles, California) and of line 6 (Regional Poultry Research Laboratory, East Lansing, Michigan) have been maintained at the Veterans Administration Hospital in Gainesville for several years. Bobwhite quail (Colinus virginianus) were obtained from the Department of Poultry Science, University of Florida. Ring-necked pheasant (Phasianus colchicus) and Bantam chickens of the Black Rock breed were obtained from local dealers.


QT46

0.02

Experimental

877

Carcinogens and Tumor Induction 20-methylcholanthrene (20 MCA) was prepared as a 4% (w/v) solution by dissolving 400 mg of 20 MCA(Calbiochem. San Diego, California) in IO ml corn oil, USP (Fisher Scientific) and heating slowly until crystals were dissolved. 7,12-dimethylbenzanthracene (DMBA) was obtained at a concentration of 5 mg/g in a 15% fat emulsion from Upjohn Company (Kalamazoo, Michigan). Nmethyl-N-nitro-nitrosoguanidine (Calbiochem, San Diego, California) was dissolved at a concentration of 1 mg/ml in dimethylsulfoxide (Mallinckrodt, St. Louis, Missouri). Each carcinogen solution was injected in the amount of 0.1 ml in the right pectoral muscle of young birds. No further injections were given. The age of the injected birds was from 6-24 days.

in QT Lines

Yield

Cell

PR-A

SR-A

PR-C

877

SR-E

R(RAV-0)

PR-F

QEF

1 .o

1 .o

1 .o

1 .o

1 .o

1 .o

1 .o

(5.00)

(4.60)

(5.15)

(5.18)

(4.28)

(5.04)

(5.00)

1.0 x 10-h

NT

1.5 x 10-s

1.1 x 10-Z

NT

1.4 x 16

QT2

Cl.5

x 10-S

QT4

2.0

NT

5.0 x IO-3

NT

8.0 x lo-’

8.5 x 10-l

1.1 x 10-1

QT5

2.0 x 10-Z

<5 x IO-5

NT

6.8 x IO-4

2.9 x 10-a

5.0 x IO-3

8.0 x 1O-4

QT6

3.2

NT

1 .o x IO-2

2.3

2.8

1.3

8.3 x lo-’

QT7

3.7 x 10-l

5.0 x 10-l

1.4 x 10-q

4.6 x IO-*

2.2 x 10-Z

7.3 x IO-’

1.9 x 10-l

QT24

7.0 x IO-’

9.3 x lo-’

8.5 x 1O-3

5.4 x IO-’

6.8 x 10-Z

NT

5.2 x IO-’

QT27

4.7

NT

2.1 x 10-o

NT

NT

NT

NT

QT29

4.1 x 10-i

NT

2.9 x IO-4

2.0 x 10-Z

7.1 x 10-Z

5.9 x lo-’

1.2 x 10-S

QT35

1.3 x lo-’

2.0 x lo-’

2.1 X 10-Z

3.1 X 10-Z

6.9 x lo-’

1.5

6.7 x lo-’

QT46

5.0 x IO-’

NT

4.3 x IO-2

NT

1.8 x 10m2

NT

4.3 x lo-’

QT51

1.4 X IO-2

NT

1.2 x 10-a

NT

NT

NT


x IO-5

Cells were infected with a multiplicity of 0.1-l .O and were carried for 30 days. Medium was changed every second day. Harvests were taken on days 7,14,21 and 30. The titers given in the table represent plateau values reached with most cultures between 14 and 21 days. Titers are expressed as fractions of control yields in quail embryo fibroblasts (QEF). Yields in QEF (log,, FFU/ml) are in parentheses. Values are from a representative experiment. PR-A, PR-C and PR-F = Prague strain of Rous sarcoma virus subgroup A, C and F; 877 = avian sarcoma virus Bratislava 77; Sr-A and SR-E = Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A and subgroup E; R(RAV-0) = Bryan high titer strain of Rous sarcoma virus with helper virus RAV-0; NT = not tested.

Cell 102

Tumor Cell Culture Birds with tumors >I cm in diameter were killed by exsanguination. The tumors were excised, and pieces were fixed in 10% phosphate-buffered formalin for histology. About 1 g of tumor tissue to be used for primary cultures was placed in a petri dish, washed with Tris-buffered saline and cut finely with scissors. The pieces were transferred to a 30 ml beaker, washed 5 times with Tris-buffered saline and trypsinized 3 times for 3 min with 0.25% trypsin, as previously described for chick embryo fibroblasts (Vogt, 1989). Cells were seeded at a concentration of 1 x 10’ in 100 mm plastic dishes in 10 ml of Ham’s F-10 medium supplemented with 2% of a 2.8% sodium bicarbonate solution, 100 units of penicillin, 10% phosphate broth, 8% calf serum and 2% chicken serum. The latter was inactivated by heating at 58°C for 80 min. Primary cultures were incubated in a moist 5% CO% atmosphere at 37°C for about 2 weeks with medium changes every 3 days. For passage of the cultures, 0.05% trypsin was used; passaged cultures were fed every 3-4 days with F-10 medium supplemented as described above, except that only 1% chicken serum and 5% calf serum were used. Karyotyping Cultures containing cells on glass coverslips were treated for 4 hr with colcimide in growth medium at a concentration of 1O-6 g/ml. They were then treated for 20 min at room temperature with a hypotonic solution of 5 parts Tris-buffered saline and 7 parts water containing 1.25 g sodium citrate per 300 ml of solution. Fixation was in methanol glacial acetic acid (3:l) for 3 min. The preparations were stained and mounted in orcein lactic acid (Beerman, 1952). Metaphases were analyzed with a 40X objective and phase-contrast illumination. Viruses The following avian sarcoma viruses were used: Bryan high titer strain of Rous sarcoma virus (RSV) pseudotyped with Rous-associated virus 1 (RAV-l), and Rous-associated virus 0 (RAV-0); the Prague strain of Rous sarcoma virus of subgroup A (PR-A), subgroup C (PR-C) and subgroup F (PR-F); the Schmidt-Ruppin strain of Rous sarcoma virus of subgroup A (SR-A) and of subgroup E (SR-E); and avian sarcoma virus 877 (Duff and Vogt, 1989; Vogt and Friis, 1971; Weiss, Mason and Vogt, 1973; Fujita et al., 1974). Colony Formation in Agar Colony assays were performed as described by Silva, Dodge and Moscovici (1974), except that Ham’s medium F-12 was used and supplemented by primary growth medium (Vogt, 1989) conditioned by a 3 day incubation with primary fibroblasts of Japanese quail. Cloning of QT cells was carried out in thesame conditioned medium. Radiolmmunoassays The cells were washed 3 times with 5 ml amounts of ice-cold Trisbuffered saline [O.Ol M Tris-HCI (pH 7.4), 0.15 M NaCI]. They were then removed from the petri dish with a rubber policeman and resuspended in 2 ml of 0.5% NP 40, 0.5% deoxycholate, 25 mM Tris-HCI (pH 8.1), 50 mM NaCl, and sonicated for 30 set in a Bransonic model 12 sonicating bath. The insoluble material was removed by centrifugation at 10,000 g for 30 min. The supernatant fluids were taken, and the amount of p27 was determined by a competition radioimmunoassay, which was performed exactly as described previously (Hayman and Vogt, 1978). Protein p27 was isolated from the 877 strain avian sarcoma virus subgroup C. Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Hexose Uptake Hexose uptake was measured according tin et al. (1971) using *H-deoxy-D-glucose England Nuclear).

to the technique of Mar(10 Ci/mmol) (New

Tests for Virus Particles by Urldlne Incorporation and by Reverse Transcrlptase Activity For labeling with 3H-uridine, three 10 cm petri dishes of confluent cultures were used for each QT line. The cells were labeled with 5,8-3H-uridine (48 Ci/mmol, New England Nuclear) at 30 &i/ml. The medium was harvested at 12 hr intervals for 2 days. The combined media were purified for avian sarcoma virus according to published methods (Duesberg et al., 1988). One plate of B77infected chicken cells was included as a control. Screening of virus particles with the reverse transcriptase assay was performed as described by Aaronson, Todaro and Scolnick (1971). Acknowledgments This work was supported by USPHS research grants from the National Cancer Institute, by a Dernham Fellowship of the American Cancer Society and by the Medical Research Service of the Veterans Administration. The excellent technical assistance of Grace Schwider, Mark Anisman, Katrina Billingsley, Annie Chyung, Gordon Thompson and Pamela Farrington is gratefully acknowledged. Received

December

17. 1978

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