Cell, Vol. 10. 669-678,
April
1977, Copyright
0 1977 by MIT
In Vitro Traits of Adenovirus-Transformed Cell Lines and Their Relevance to Tumorigenicity in Nude Mice Phillip H. Gallimore Department of Cancer Studies University of Birmingham The Medical School Birmingham B15 2TJ, England James K. McDougall Cold Spring Harbor P.O. Box 100 Cold Spring Harbor,
and Lan 60 Chen Laboratory New York
11724
Summary Six independently isolated adenovirus P-transformed rat cell lines and one adenovirus 54ransformed human cell line have been examined in vitro for serum growth requirements, saturation density, anchorage-independent growth, proteolytic enzyme activity and the presence of LETS glycoprotein and T antigen. This series of adenovirus-transformed cell lines exhibits an oncogenic spectrum ranging from being tumorigenic in immunocompetent rats through to nontumorigenic in adult nude mice. The relevance of the in vitro findings to growth potential in vivo is discussed. Introduction Cells transformed in culture by viruses exhibit a number of in vitro biological characteristics which distinguish them from their normal parent cell types: induction of proteolytic enzymes (Unkeless et al., 1973), changes in cell morphology (Gallimore, 1974; Risser and Pollack, 1974). alterations in growth behavior-that is, increased saturation density, reduced serum requirement, decreased doubling time and anchorage-independent growth (Gallimore, 1974; Risser and Pollack, 1974)-modified cell surface components (Hynes, 1974) and the intracellular expression of virus neo-antigens-for example, T antigens. Many attempts have been made to unravel from this plethora of transformed cell characteristics one or more phenotypes that correlate with the ability of a cell line to proliferate in vivo. The most recent studies in this area have been carried out using the athymic nude mouse mutant, which lacks T cell-mediated cellular immunity. Virus-transformed cell lines, tumor lines of xenogeneic and allogeneic origin (Shin et al., 1975; Stiles et al., 1976a, 1976b), and cell lines expressing tumor-specific transplantation antigens (TSTA) (Harwood and Gallimore, 1975) produce tumors in this biological rarity. Freedman and Shin (1974) and Shin et al. (1975) have reported that one transformed cell phenotype-anchorage-independent growth - is the only
virus-induced alteration that correlates with tumorigenicity in the nude mouse. Shields (1976) has recently challenged this assertion and criticized the use of “fibroblasts” as suitable model systems for the study of cancer. In this paper, we assess the importance of a number of in vitro traits for the in vivo growth potential for six adenovirus 2 (AdP)-transformed rat embryo cell lines and one adenovirus 5 (AdS)-transformed human cell line. The six Ad2-transformed lines were selected from a series of 46 independently isolated Ad2-transformed epithelioid lines because five of them (Ad2/F4, F17, F19, ASREM and T2/C4) have been examined by reassociation kinetics for integrated (Gallimore, Sharp and Sambrook, 1974) and expressed (Flint, Gallimore and Sharp, 1975) Ad2 genetic information; the sixth rat line (Ad2/50A) is the most tumorigenic line of this series (Harwood and Gallimore, 1975). These cell lines cover the malignancy spectrum from tumor production with extensive invasion in syngeneic rats (Ad2/50A) through to being nontumorigenic in adult nude mice (Ad2/F17). Graham et al. (1974) transformed human embryo kidney cells with sheared Ad5 DNA and established a single cell line (Ad5/HEK FG 293-31), and because of its uniqueness, this line was included in this study. As anchorage-independent growth (Freedman and Shin, 1974; Shin et al., 1975) and reduced levels of the large external transformation-sensitive glycoprotein (LETS) (Chen, Gallimore and McDougall, 1976a) are phenotypes that have most recently been proposed as changes correlating with malignancy. Our observations concerning these traits are discussed in detail. Results In Vitro Characteristics Doubling Time, Saturation Density and Serum Dependency The doubling times and saturation densities for six Ad2-transformed rat embryo lines and one Ad5transformed human cell line grown in medium containing 5% FCS are shown in Table 1. Serum dependence was measured by a comparison of doubling time and saturation density at two serum levels for each cell line, and expressed as a ratio of the results obtained with 5% FCS and 0.5% FCS (Table 1). Ad2/F17 is the only transformed line showing a significant level of serum dependence; however, this line doubled twice in low serum medium during the 12 day period, while rat embryo control lines only maintain the initial cell plating number (2 X IO5 cells per 5 cm dish). Table 1 shows the data
Cell 670
Table
1. In Vitro
Line Passage
Transformed
on the Adenovirus-Transformed
Doubling Time (Hr) 5% FCS
Cell Lines Control AS.RE
Studies
9 30.6
Ratio Doubling Time in 5% FCS:O.5% FCS
0
Cell Lines
Saturation Density (Cells/ cm*) x lo* 5% FCS
1.1
Ratio Saturation Density in 5% FCS:O.5% FCS
Doubling Time (Hr) in Spinner Culture 5% DFCS
% Efficiency of Plating in MethylCellulose + 10% FCS
>11.12’
No growth
~0.002
5%
22.3 20.7 24.3 20.2 17.1 Not tested
5.25 0.02 1.06 73.60 3.24 36.60
23.8
30.30
Ad2 T-ag by Immunofluorescence
% LETS” ProteinPositive Cells by Immunofluorescence
0
100
76% 54% 46% 52% 61% 100%”
++++ +++ ++ + + ++
100 100 60 10 1 10
65%
+++
Fibrinolysis (16 Hr)
Rat
AdP/F17 Ad2/F19 Ad2lF4 AdPlASREM Ad2/T2lC4 Ad2/50A
24.0 20.1 25.6 21 .o 17.7 21.6
0.20 0.91 0.94 0.90 0.76 0.65
4.2 6.6 5.7 10.5 10.1 10.4
10.41 1.93 1.76 1.47 1.70 1.53
Transformed Human Ad5/HEK FG (293-31)
25.2
0.81
9.5
1.69
a 100% fibrinolysis in i 5 hr. ” Carried out at the same passage c No growth with 0.5% FCS.
level as the cells
inoculated
into nude
for one such control line ASRE at the ninth passage. Anchorage-Independent Growth Anchorage-independent growth was measured in two ways: first, by the ability of a cell line to initiate and maintain growth in spinner culture, and second, by the efficiency of plating (EOP) in Dulbecco’s modified Eagle’s medium (DME) containing 1.2% methyl-cellulose, supplemented with 10% FCS or 7.5% dog serum plus 2.5% FCS. Table 1 clearly shows that these two systems measure quite separate phenotypes-for example, Ad2/F19 grows poorly in methyl-cellulose (EOP 0.02%), whereas its growth in spinner culture is prolific. Five other adenovirus-transformed lines examined for growth in spinner culture share the latter phenotype with Ad2/F19. Adenovirus-transformed cell lines show considerable variation in their ability to form colonies in methyl-cellulose medium supplemented with 10% FCS, from AdP/ASREM with an EOP of 73.8% down to Ad2/F19 (0.02%). The one Ad5 transformed human line grew very well in methylcellulose. Dog serum was also studied because Pollack et al. (1974) showed that this significantly increased the EOP of three SV40-transformed rat embryo lines. All the adenovirus lines plated less well in methyl-cellulose medium supplemented with 7.5% dog serum plus 2.5% FCS as compared with 10% FCS alone, the EOP being reduced 201000 fold depending upon the cell line examined. All the studies in methyl-cellulose were controlled by using the SV40-transformed rat embryo line SVRE9 developed by Pollack et al. (1974); this line plated 4.6% (10% FCS) and 1.94% (7.5%dog serum
0
mice.
plus 2.5% FCS) in our hands. Ad2/F19 produces colonies of similar size to SV-RE9, while the colonies of all the other adenovirus lines were considerably larger. Ad2 lines T2/C4, ASREM and 50A and the Ad5/HEK FG line produce visible colonies in 10 days or less. Protease Activity Table 1 shows that all the transformed lines studied are high protease producers as measured by fibrinolytic activity; Ad2/50A is very active in this respect, hydrolyzing the fibrin clot in 5 hr. Ad2 T Antigen The morphology and location of T-ag fluorescence shows considerable variation within a line and between lines; however, all the cells within a line show some T-ag-specific fluorescence. The majority of the fluorescence is located on or within the nuclear membrane as flecks or blobs; but specific fluorescence was also seen in the cytoplasm of all the lines. Ad2/F17 has the most T-ag by immunofluorescence (Figure l), and all the other lines were graded by comparison with this line as shown in Tables 1, 2 and 3. Large External Transformation-Sensitive Glycoprotein (LETS) Normal rat embryo fibroblast cultures contain 100% LETS protein positive cells which produce an extensive fibrous network of LETS protein when confluent. The series of Ad2-transformed rat embryo cell lines studied here show a spectrum from lines containing 100% LETS protein positive cellsfor example, AdP/FlS-through to lines showing 1% LETS protein positive cells by immunofluorescence-for example, Ad2/T2/C4 (Table 1). Ad2/F17
Tumorigenicity 671
Figure
of Ad2 and Ad5 Cell Lines
1. Ad2 T Antigen
Fluorescence
in Nude
Shown
Mice
by Ad2/F17
and Ad2fF19 confluent cultures produce a fibrous LETS network similar to that of the rat embryo fibroblast cells (Figures 2A and 2B). Ad2/F4 (60% LETS protein-positive cells) produces this glycoprotein at some cell/cell contact areas but does not produce a network (Figures 3A and 3B). The Ad5transformed human embryo kidney line is negative for LETS protein. However, normal epithelial cells show a different pattern of LETS protein when compared with fibroblasts and do not produce a LETS protein network (Chen et al., 1976b). Tumor Production in Adult Nude Mice Of the adenovirus cell lines inoculated subcutaneously into adult nude mice, three growth patterns were observed. First, two lines, Ad2/F17 and Ad5/ HEK FG, did not produce tumors. Second, Ad2/F19 produced two slow growing nude mouse tumors which had few mitotic cells and were covered in a thick fibroblastic tumor capsule. Within the tumors themselves, the majority of cells were those typically found in adenovirus-induced tumors. Third, lines Ad2lF4, Ad2/50A, Ad2IASREM and Ad2/T2/ C4 produced tumors in 100% of the nude mice. The tumors appeared in less than 3 weeks and showed invasion of the local mouse tissues; however, no tumor metastases were observed. Tables 2 and 3 show all the in vivo data for the adenovirus-transformed cell lines, including the tumorigenicity of these lines when inoculated into newborn syngeneic rats and such rats immunosuppressed with anti-thymocyte serum (ATS). The Ad21 F19 and Ad2IASREM tumors grown in nude mice do not transplant in newborn syngeneic rats. All three Ad2lF4 tumors, however, do transplant into nonimmunosuppressed newborn syngeneic rats and show tumor invasion and metastatic spread in all the rats inoculated.
Tumor Production by AdS/HEK FG in 5 Day Old and X-Irradiated Adult Nude Mice Because the Ad5/HEK FG cell line had many of the characteristics in common with the Ad2 rat lines that were tumorigenic in adult nude mice, the finding that it was nontumorigenic even when 5 x 10’ cells were inoculated into the adult nude mice was initially a surprise. Experiments were therefore carried out in 5 day old nude mice and X-irradiated adult nude mice (300 rads whole body) in an attempt to provide different in vivo environments from the normal adult situation. 1 x lo6 Ad5/HEK FG cells inoculated subcutaneously into the two new in vivo environments produced tumors in 100% of the survivors as shown in Table 3. These tumors were classified as highly differentiated carcinomas and were shown to have been derived from the Ad5 line by G banding karyology (P. H. Gallimore and C. A. Macpherson, unpublished results). The Ad5/HEK FG nude mouse tumors all transplant to adult nude mice. One interesting feature of the histology of the AdS/HEK FG nude mouse tumors is that the carcinoma cells show differentiation most strikingly when tumor cells are in contact with intra-tumor stromal fibroblasts (Figure 4) or blood vessels. Differentiation occurred in rare areas of Ad2/F4 tumors, but did not occur in any of the tumors induced by the other adenovirus-transformed rat lines which produce extremely primitive anaplastic tumors. Examination of the Cell Lines for C Type Virus Particles and Reverse Transcriptase In view of the recent findings of Price et al. (1975), some of the transformed cell lines and most of the nude mouse tumors established as cell lines were examined by electron microscopy for the presence of C type virus particles and by Dr. Natalie Teich (ICRF, London) for murine RNA tumor virus reverse transcriptase. None of the adenovirus-transformed cell lines or their respective nude mouse tumors were positive by either test. One other line studied, however, served as a positive control; an Ad2 virustreated rat embryo line (AS lOOB), which does not have an adenovirus-transformed phenotype or express Ad2 T-ag and which produced fibrosarcomas in newborn syngeneic rats, induced a nude mouse tumor in 63 days that was positive for reverse transcriptase and budding C type virus particles. In Vitro Studies on Cells from Tumors Grown in Nude Mice Following Inoculation of Ad2Transformed Cell Lines One of the most interesting findings from the studies on the nude mouse tumors (nMT) was that in all
Figure
2A. lmmunofluorescence
Figure
28.
Phase-Contrast
Photomicrograph Photomicrograph
of Ad2/F19 of Confluent
Showing
Ad2/F19
cases, the tumor lines contain fewer LETS proteinpositive cells than their respective, parental transformed cell lines (Table 2). This was particularly true for the series of Ad2/F4 nude mouse tumors which have 5% LETS protein-positive cells compared with the original 60% LETS protein-positive
Massive
(Same
Network
Field as A) (Bar
of LETS
Protein-Specific
lmmunofluorescence
= 20 pm)
Ad2/F4 cells. The two Ad2/F19 nude mouse tumor lines still contained 50% LETS protein positive cells and failed to produce tumors when transplanted back into syngeneic rats. The Ad2-transformed cell nude mouse tumor lines plate in methyl-cellulose medium supple-
Tumorigenicity 673
of Ad2 and Ad5 Cell
Figure Areas
3A.
lmmunofluorescence
Figure
38.
Phase-Contrast
Lines
in Nude
Photomicrograph Photomicrograph
Mice
of Ad2/F4 of Confluent
Showing
Ad2/F4
mented with 10% FCS as well as or better than the parent line, Ad2/F19 nMT1 showing the most dramatic indication of selection in this respect. The Ad2/F4 nMT1 is the only adenovirus line studied that is not suppressed in the presence of dog se-
LETS
(Same
Protein-Specific
Field as A) (Bar
Fluorescence
at SOIT le Cell/Cell
Co1
= 20 pm)
rum, having plating efficiencies of 1 .06% in 1 0% FCS and 1.29% in 7.5% dog serum plus 2.5% Fcs methyl-cellulose medium. st udThere is strong evidence from cy togenetic ies (P. H. Gallimore and C. A. Macpl herson, unp ub-
Cell 674
Table
2. In Vivo and in Vitro
Cell Lines and Nude Mouse Tumor (nMT) Lines Rat Control Line AS.RE Passage 9 Transformed AdPIFl7 Ad2/F19 F19 nMT1 F19 nMT2 Ad2/F4
Data for AdP-Transformed Rats
Rat Lines
and Nude
Tumorigenicity Athymic Nude
Mouse
Tumor
Lines
in Adult Mice
% Tumors
in Syngeneic
Newborn 2 x 10” Cells per Rat SC
ATS lmmunosuppressed 2 x lo6 Cells per Rat SC
2 x IO’ Cells Inoculated SC Tumor +/ Total Inoculated
1 x 1O’Cells Inoculated SC Tumor +/ Total Inoculated
% EOP in MethylCellulose 10% FCS
Ad2 T- ag
% LETS Positive
0
0
015 (165 days)
015 (165 days)
zo.002
0
100%
Rat 0
0
O/5 (165 days)
O/5
5.25
++++
100%
0
0
015 (165 days)
2/5 (30 days)”
0.02
+++
100%
0
NT
NT
NT
3.55
+++
50%
+++
50%
1.06
++
60% 5%
0
NT
NT
NT
0
100”
5/5 (19 days) b
515
NT
F4 nMT1
100
NT
NT
NT
1.20
++
F4 nMT2
100
NT
NT
NT
3.16
++
5%
F4 nMT3
100
NT
NT
NT
++
5%
100
515 (6.5 days) b
Ad2lT2IC4
100
NT
515 (7.5 days)”
Ad2/50A
100
100
AdPfASREM
0
B Additional ATS required. b Mean latent period. SC = subcutaneous;
5/5 (16 days)b
nMT
= nude
mouse
lished observations), LETS protein immunofluorescence, EOP in methyl-cellulose and tumor transplantation data that cell selection has occurred in vivo in the absence of T cell-mediated immunity. Discussion Ad2 virus does not induce tumors in newborn rodents unless they are given extensive treatment with immunosuppressants (Gallimore, 1973). However, the semipermissive Ad2/rat embryo in vitro system (Gallimore, 1974) generates transformed cell lines with a number of characteristics shared by all the lines examined thus far and some phenotypes not expressed by all the lines. The common characteristics include epithelioid morphology, growth to high cell density, growth in spinner culture without adaption, elevated production of protease, immortality without “crisis” (there is no cessation of growth or extensive cell death during isolation of Ad2 lines), integrated Ad2 DNA sequences representing the left-hand 14% of the Ad2 physical and genetic map (Gallimore et al., 1974), and the presence of intracellular Ad2 T antigen. The phenotypes not uniformly expressed by all the Ad2-transformed rat lines are serum- and anchorage-independent growth, the synthesis of LETS protein fibrillar network and tumorigenicity in new-
tumor
NT 73.6 3.24 36.6
+ + ++
ProteinCells
10% 1% 10%
line; NT = not tested.
born syngeneic rats, immunosuppressed rats or adult athymic nude mice (Tables 1 and 2). Since these cell lines were all derived from independent transformation events in cultures isolated from rat embryo tissues, it is possible that the normal parental cell type was different for each line. Nevertheless, comparison of the properties of lines transformed by a single agent yet showing diverse in vivo growth behavior may be invaluable in determining characteristics important in oncogenicity. In the present series of experiments, a number of in vitro characteristics was examined in an attempt to define one or more correlating with the ability to produce tumors in adult athymic nude mice. The results indicate that high saturation density, increased protease activity, decreased doubling time and anchorage-independent growth are frequently features of Ad2-transformed cell lines but are not essential for a cell line to be tumorigenie. This conclusion is in agreement with the recent findings of Jones et al. (1975) and Stiles et al. (1976b), but not with those of Freedman and Shin (1974) and Shin et al. (1975), in that three of the seven adenovirus-transformed lines reported here failed to uphold their conclusion that anchorage-independent growth correlates with tumor formation in adult athymic nude mice. Ad2/F17 (EOP 5.25%) and AdS/HEK FG (EOP 30.3%) both show
Tumorigenicity 675
Table
of Ad2 and Ad5 Cell Lines
3. Tumorigenicity
of Ad5/HEKFG
in Nude
(293-31)
Mice
Cell Line Inoculated
Subcutaneously
into Athymic
Nude
Mice
T-ag Status
Cell Dose per 0.1 ml Inoculated
Adult (IO-12 Weeks)
5 Day Old
(293-31)
+++
5 x 10’
O/6 (165 days)
NT
NT
MSIHEKFG
(293-31)
+++
1 x 106
O/6 (165 days)
213” (30 days)c
4/6b (36 days)’ days)’
Ad5lHEKFG
nMT1
+++
5 1 5 1 5
5/5 (10 days)’ 5/5 (9.5 days)’ 3/5 (11.2 days)< o/5 o/5
NT NT NT NT NT
NT NT NT NT NT
AdS/HEKFG nMT3
X-Irradiated
+++
1 x 106
3/3 (12.5
NT
NT
Cell Line and Nude Tumor Lines (nMT) Ad5IHEKFG
Mouse
a One mouse died day 10. h Two mice died days 7 and 12 post-irradiation. c Mean latent period. nMT = nude mouse tumor
x x x x x
106 106 105 10” 10’
days)’
X-Irradiated Adults (300 rads Whole Body)
line; NT = not tested.
anchorage-independent growth in methyl-cellulose but were nontumorigenic in the adult nude mouse, while Ad2/F19 (EOP 0.02%) shows anchorage dependency in this system but produced two slow growing tumors which, although benign in nature, did not regress, being excised when 2.5 cm in diameter. If anchorage-independent growth reflects a potential for growth in vivo, then there should be a correlation between both plating efficiency and/or colony size in methyl-cellulose and the latent period and/or tumor incidence in the nude mouse. This was not observed in our study even for Ad2 lines that show anchorage-independent growth. From their study of SV40-transformed rat embryo cell lines, Pollack et al. (1974) provided evidence that elevated levels of plasminogen activator accompanied loss of anchorage dependency, and they suggested that this proteolytic activity may have a significant role in tumor invasion and metastatic spread. The significance of fibrinolytic activity to malignancy has recently been questioned by the observation of Pearlstein et al. (1976) that some normal cells-for example, cells from calf bladder-produce high levels of protease as measured by fibrinolysis. In the same report, Pearlstein et al. observed that loss of LETS protein from transformed cells was not due to their increased synthesis of protease. We have confirmed this finding in that the nontumorigenic line Ad2/F17 has high fibrinolytic activity and yet produces a massive three-dimensional network of LETS protein. It is possible that proteolytic enzymes different from plasminogen activator may be associated with a cell line’s malignant behavior, and with this in mind, we have recently further investigated Ad2/ 50A. This cell line is the most malignant and the highest producer of fibrinolytic activity of the Ad2-
transformed rat lines; however, exponentially growing cultures of Ad2/50A do not hydrolyze overlays of purified rat tail collagen even after prolonged incubation (10 days at 37°C). This preliminary finding implies that collagenase activity is not associated with this cell line’s agressive infiltration of normal tissues. Our data shown in Table 2 suggest that Ad2transformed cells expressing normal levels of LETS protein either grow poorly in vivo or respond to the control processes that inhibit the growth of normal cells in vivo. This theory could explain a previous observation that tumorigenicity in syngeneic rats did not always appear to be a reflection of the antigenic status of a cell line (Harwood and Gallimore, 1975). Cell line Ad2/F4 is heterogeneous for the expression of LETS protein, and it can be postulated that the reason why additional anti-thymocyte serum-induced immunosuppression is required before this line produces tumors in syngeneic rats is a reflection of the time it takes for LETS protein-negative cells to establish themselves in vivo, the latent period of this line in nude mice being 19 days. The finding that all three Ad2lF4 nude mouse tumor lines show a dramatic reduction in LETS protein-positive cells and are highly tumorigenic when inoculated into newborn syngeneic rats lends some support to this theory. LETS protein therefore appears to have a major role in determining the in vivo behavior of Ad2transformed cell lines. Pearlstein et al. (1976), however, have reported that three sarcomas, derived 1976; Chen et al., 1976a, 1976b) express low or undetectable levels of LETS protein. Preliminary observations on subclones of Ad21 F4, F17 and F19 isolated from either spinner culture or methyl-cellulose indicate that a strong selection of LETS-negative cells occurs in these an-
Cell 676
Figure
4. Haematoxylin-
(A) photomicrograph (B) photomicrograph fibroblasts.
and Eosin-Stained showing showing
Histological
tumor cells. epithelial differentiation
Sections of carcinoma
from a spontaneously transformed mouse line, produced appreciable levels of LETS protein, and they therefore concluded that loss of LETS protein was not essential for malignancy. This finding could indicate that some fibroblasts after transformation retain their differentiated capacity to produce LETS protein. Recently, Chen et al. (1976b) have observed that cultured normal rodent epithelial cells
of AdWHEK cells where
FG (293-31) tumor
cells
Nude
Mouse
are in direct
Tumor contact
1 with
intra-tumc
jr stromal
from liver and kidney express LEITS protein, but the distribution is different from th at of fibrob lasts in that no fibrillar network of LE TS protein is produced by epithelial cells. These ? observatio ns may provide a clue as to the origi nal cell typ Fe from which cell lines such as Ad21 F17 and Fl 9 have been derived. All the experimen Ital and bun ran carcinema lines studied thus far (Pearlstein et al.,
Tumorigenicity 677
of Ad2 and Ad5 Cell Lines
in Nude
Mice
chorage-independent environments, thus mimicking our findings with in vivo selection. These subclones are presently being examined for tumorigenicity in both syngeneic rats and nude mice. In common with a number of SV40-transformed human cell lines (Stiles et al., 1975), the Ad5transformed human cell line AdS/HEK FG is not tumorigeneic in adult nude mice. However, Ad5/HEK FG produces differentiated carcinomas when inoculated into 5 day old nude mice or X-irradiated adult nude mice. This finding may indicate that the in vivo proliferation of this cell line is dependent upon the level of expression of factors, such as growth hormone. A recent observation (C. D. Stiles and G. Sato, personal communication) that an epithelial cell line produces tumors only in newborn nude mice is similar to our finding. The histological evidence that Ad5/HEK FG tumor cells differentiate only when they are in contact with blood vessels or intra-tumor stromal fibroblasts clearly indicates that some cells in this transformed human cell line have not lost the ability to react normally to the nontransformed mouse cell component of the carcinomata. Although the molecular biology of the adenovirus-transformed rat cells have been accurately mapped in terms of integrated virus DNA (Gallimore et al., 1974) and expressed virus-specific messenger (Flint et al., 1975), our understanding of the role played by the virus genes in the diversity of phenotypes observed in our study remains unresolved. However, a number of explanations for this spectrum of transformed rat lines are possible: in the semi-permissive transformation system, there may be a number of different virus host interactions promoting heterogeneity; Ad2 can transform different differentiated cell types in rat embryo cultures; they may be the consequence of integration of the Ad2 “transformation” gene(s) at different chromosomal locations (D. Galloway and J. Sambrook personal communication); initially, the transformed cells have an inherent instability giving rise to cell lines with different in vivo and in vitro characteristics. Further progress in this area will depend upon the development of adenoviruses with a conditioned lethal mutation in the transforming gene(s). Finally, this study was initiated to ascertain the relevance of a number of in vitro characteristics, expressed by Ad2-transformed cell lines to tumorigenicity. We found that reduced levels of LETS protein was the only trait correlating with malignancy. We believe that some of the disagreements reported by us and other groups as to the relative importance of different in vitro traits to malignancy may simply be a reflection of the type of normal
parental cell giving rise to experimentally established cell lines. We agree with Shields (1976) and Stiles et al. (1976b), who have recently emphasized the need for investigators to study in vitro transfer: mation and malignancy using normal epithelial cells as the starting parental cell type. Experimental
Procedures
Tissue Culture Transformed cell lines Ad2/F4, F17, F19 and ASREM were derived from cells cultured from the same pool of AS rat embryos; the other two AdP-transformed rat embryo lines were derived from different Hooded Lister rat embryo material. Each of these lines was established from a single raised focus containing epithelioid cells which had been initiated and isolated as previously described (Gallimore, 1974). Examination of the chromosomes using G banding (P. H. Gallimore, C. Ft. Richardson and C. A. Macpherson, manuscript in preparation) confirms that each line is derived from a separate transformation event. Saturation density and doubling time analysis for each cell line was carried out using minimal Eagle’s essential medium (MEM) supplemented with either 5% or 0.5% fetal calf serum (FCS). These experiments were performed using three 5 cm dishes with an initial seeding level of 2 x lo5 cells per dish for each point. Dishes were medium changed on days 3, 6, 6 and IO, and cell counts were made daily for 12 days using a Coulter counter. Doubling times were calculated on the exponential phase of growth, and saturation density was expressed as cells per cm2 using the figure of maximum cell number. The results shown in Table 1 are the mean values of two experiments. Spinner cultures were set up as 100 ml cultures of Joklik’s modified Eagle’s medium supplemented with 5% dialyzed FCS (DFCS) at a cell concentration of 2 x IO5 cells per ml. An equal volume of fresh medium was added every 46 hr. and doubling times were established over a 12 day period. Colony Formation in Methyl-Cellulose The efficiency of plating (EOP) in methyl-cellulose was carried out exactly as described previously by Risser and Pollack (1974). After 3 weeks of incubation, colonies containing 100 cellsor more were counted using a stereoscopic microscope. Protease The activity measured occurring procedure Chen and
Activity of membrane-bound and/or excreted proteases was by the amount of fibrinolysis of purified fibrin clots over an 16 hr period. The materials and experimental used for this study were the same as those used by Buchanan (1975).
Adenovirus T Antigen (T-ag) lmmunofluorescence Cell lines were grown to subconfluency on glass coverslips, washed in phosphate-buffered saline (PBS) (pH 6.6) and fixed for 1.5 min in cold acetone (-20°C). The preparation of the Ad2 T-ag antiserum and of the FITC-labeled rabbit-anti-rat IgG have been reported by Harwood and Gallimore (1975). The cell line showing the strongest specific fluorescence, Ad2/F17. was given an arbitary 4+ rating, and all the other cell lines were compared with Ad2/F17 and given a plus rating O-4. LETS Protein Estimation by lmmunofluorescence Cell lines were grown on glass coverslips and fixed at subconfluent and postconfluent levels using 10% formol-saline. Coverslips were then washed in PBS and reacted with 10 ~1 of diluted (160) rabbit anti-human cold-insoluble globulin antibody (see Chen et al., 1976a) for 20 min at 37°C. After extensive washing
Cell 676
with PBS, coverslips were drained, layered with 10 ~1 of goat-antirabbit IgG, incubated for 20 min at 37”C, rinsed further in PBS and mounted on microscope slides with Elvanol. Animals Hooded Lister and AS rats were bred and maintained in the Department of Cancer Studies (Birmingham). Nude mice were obtained from the MRC Laboratory Animal Centre (Carshalton. Surrey, England) and maintained away from other animal stocks. Nude mice were also bred in the Department of Cancer Studies by mating homozygous nudes of Balb/c genetic background. Anti-Thymocyte Serum (ATS) Rabbit-anti-rat thymocyte serum (ATS) was produced and tested as previously described (Gallimore, 1972). Rats received 5 daily doses of 0.2 ml ATS intraperitoneally on days 6, 9, 10, 11 and 12 following inoculation of transformed cells into newborn animals. Acknowledgments The authors acknowledge the advice and support of Dr. James D. Watson and Professor D. G. Harnden; Dr. B. W. Codling, (Department of Pathology, Birmingham University) for his collaboration with tumor pathology; Dr. Natalie Teich (I.C.R.F. London) for carrying out reverse transcriptase assays; Dr. F. L. Graham for providing the cell line 293-31; and the following technicians for excellent assistance: Sylvia Green, Debbie Parton, Val Nash, Ann Maguire, Carol Morton-Harvey and Don Sammons at Birmingham University, and Shiela Levings and Elaine Paul at Cold Spring Harbor Laboratory. P. H. G. is supported by the Cancer Research Campaign (England) and was supported by a grant from the Robertson Research Fund while at Cold Spring Harbor Laboratory. L. B. C. is a fellow of the Muscular Dystrophy Association of America, and L. B. C. and J. K. M. are supported by the National Cancer Institute. Received
November
10, 1976;
revised
December
30. 1976
Chen. L. B., and Buchanan, J. M. (1975). Plasminogenindependent fibrinolysis by proteases produced by transformed chick bryo fibroblasts. Proc. Nat. Acad. Sci. USA 7.2, 1132-1136.
em-
Chen, L., B., Gallimore, P. H., and McDougall, J. K. (1976a). Correlation between tumor induction and the large external transformation sensitive protein on the cell surface. Proc. Nat. Acad. Sci. USA 73, 3570-3574.
Flint, S. J., Gallimore, of viral RNA sequences infected cells. J. Mol.
P. H.. and McDougall, protein on epithelial
P. H., and Sharp, P. A. (1975). in adenovirus P-transformed Biol. 96, 47-66.
J. K. cells.
Comparison and lytically
Freedman, V. H., and Shin, S. (1974). Cellular tumorigenicity in nude mice: correlation with cell growth in semi-solid medium. Cell 3, 355-359. Gallimore, P. H. (1972). Tumor production in immunosuppressed rats with cells transformed in vitro by adenovirus type 2. J. Gen. Virol. 76, 99-102. Gallimore, P. H. (1973). Birmingham, England.
Ph.D.
Thesis,
University
of Birmingham,
Gallimore, P. H. (1974). Interaction of Adenovirus type 2 with rat embryo cells: permissiveness, transformation and in vitro characteristics of adenovirus transformed rat embryo cells. J. Gen. Virol. 25. 263-273. Gallimore, P. H., Sharp, P. A., and Sambrook. in transformed cells: Il. A study of sequences
Graham, F. L., Abrahams, P. Warnaar, S. O., DeVries. F. A. (1974). Studies on the in vitro fragments of human adenovirus Harbor Symp. Quant. Biol. 39,
J. (1974). Viral DNA of Adenovirus 2 DNA
specific
fragments
Hynes, R. 0. (1974). tion: the importance 147-156.
of
J., Mulder, C., Heijneker, H. L., J., Fiers. W. and Van der Eb, A. J. transformation by DNA and DNA and simian virus 40. Cold Spring 637-650.
Harwood, L. M. J., and Gallimore. P. H. (1975). A study oncogenicity of adenovirus type 2 transformed rat embryo Int. J. Cancer 76, 496-508.
of the cells.
Role of surface alteration in cell transformaof proteases and surface proteins. Cell 7,
Jones, P. A., Rhim. J. S., Isaacs, H., and McAllister, The relationship between tumorigenicity, growth brinolytic activity in a line of human osteosarcoma Cancer 76, 616-621. Pearlstein, E., Hynes. R. O., Franks, (1976). Surface proteins and fibrolytic lian cells. Cancer Res. 36, 1475-1480.
R. M. (1975). in agar and ficells. Int. J.
L. M.. and Hemming, V. J. activity of cultured mamma-
Pollack. R.. Risser. R., Conlon, S., and Rifkin, D. (1974). Plasminogen activator production accompanies loss of anchorage regulation in transformation of primary rat embryo cells by simian virus 40. Proc. Nat. Acad. Sci. USA 77, 4792-4796. Price, J., Arnstein, P., Suk. W. A., Vernon, M. L., and Huebner, R. J. (1975). Type-C RNA viruses of the NIH nude mouse. J. Nat. Cancer Inst. 55, 1231-1232. Risser, R.. and Pollack, R. (1974). A non-selective analysis SV40 transformation of mouse 3T3 cells. Virology 59, 477-489. Shields, R. (1976). 262, 348.
Transformation
and tumourigenicity.
of
Nature
Shin, S.. Freedman, V. H.. Risser, R.. and Pollack, R. (1975). Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc. Nat. Acad. Sci. USA 72, 4435-4439. Stiles, Failure tumors 4975.
References
Chen, L. B., Maitland, N.. Gallimore, (1976b). Detection of surface LETS Exp. Cell. Res., in press.
in nine lines of transformed rat cells using the viral genome. J. Mol. Biol. 89, 49-72.
C. D., Desmond, W., Sato, G., and Saier, M. H. (1975). of human cells transformed by simian virus 40 to form in athymic nude mice. Proc. Nat. Acad. Sci. USA 72, 4971-
Stiles, C. D., Desmond, W., Chuman. L. M., Sato, G., and Saier, M. H. (1976a). Growth control of heterologous tissue culture cells in the congenitally athymic nude mouse. Cancer Res. 36, 13531360. Stiles, C. D., Desmond, W., Chuman, L. M.. Sato, G.. and Saier, M. H. (1976b). Relationship of cell behavior in vitro to tumorigenicity in athymic nude mice. Cancer Res. 36, 3300-3305. Unkeless, J. C., Tobia, A., Ossowski. L., Quigley, J. P., Rifkin, D. B.. and Reich, R. (1973). An enzymatic function associated with transformation of fibroblasts by oncogenic viruses. J. Exp. Med. 737. 85-111.
April
1977, Copyright
0 1977 by MIT
In Vitro Traits of Adenovirus-Transformed Cell Lines and Their Relevance to Tumorigenicity in Nude Mice Phillip H. Gallimore Department of Cancer Studies University of Birmingham The Medical School Birmingham B15 2TJ, England James K. McDougall Cold Spring Harbor P.O. Box 100 Cold Spring Harbor,
and Lan 60 Chen Laboratory New York
11724
Summary Six independently isolated adenovirus P-transformed rat cell lines and one adenovirus 54ransformed human cell line have been examined in vitro for serum growth requirements, saturation density, anchorage-independent growth, proteolytic enzyme activity and the presence of LETS glycoprotein and T antigen. This series of adenovirus-transformed cell lines exhibits an oncogenic spectrum ranging from being tumorigenic in immunocompetent rats through to nontumorigenic in adult nude mice. The relevance of the in vitro findings to growth potential in vivo is discussed. Introduction Cells transformed in culture by viruses exhibit a number of in vitro biological characteristics which distinguish them from their normal parent cell types: induction of proteolytic enzymes (Unkeless et al., 1973), changes in cell morphology (Gallimore, 1974; Risser and Pollack, 1974). alterations in growth behavior-that is, increased saturation density, reduced serum requirement, decreased doubling time and anchorage-independent growth (Gallimore, 1974; Risser and Pollack, 1974)-modified cell surface components (Hynes, 1974) and the intracellular expression of virus neo-antigens-for example, T antigens. Many attempts have been made to unravel from this plethora of transformed cell characteristics one or more phenotypes that correlate with the ability of a cell line to proliferate in vivo. The most recent studies in this area have been carried out using the athymic nude mouse mutant, which lacks T cell-mediated cellular immunity. Virus-transformed cell lines, tumor lines of xenogeneic and allogeneic origin (Shin et al., 1975; Stiles et al., 1976a, 1976b), and cell lines expressing tumor-specific transplantation antigens (TSTA) (Harwood and Gallimore, 1975) produce tumors in this biological rarity. Freedman and Shin (1974) and Shin et al. (1975) have reported that one transformed cell phenotype-anchorage-independent growth - is the only
virus-induced alteration that correlates with tumorigenicity in the nude mouse. Shields (1976) has recently challenged this assertion and criticized the use of “fibroblasts” as suitable model systems for the study of cancer. In this paper, we assess the importance of a number of in vitro traits for the in vivo growth potential for six adenovirus 2 (AdP)-transformed rat embryo cell lines and one adenovirus 5 (AdS)-transformed human cell line. The six Ad2-transformed lines were selected from a series of 46 independently isolated Ad2-transformed epithelioid lines because five of them (Ad2/F4, F17, F19, ASREM and T2/C4) have been examined by reassociation kinetics for integrated (Gallimore, Sharp and Sambrook, 1974) and expressed (Flint, Gallimore and Sharp, 1975) Ad2 genetic information; the sixth rat line (Ad2/50A) is the most tumorigenic line of this series (Harwood and Gallimore, 1975). These cell lines cover the malignancy spectrum from tumor production with extensive invasion in syngeneic rats (Ad2/50A) through to being nontumorigenic in adult nude mice (Ad2/F17). Graham et al. (1974) transformed human embryo kidney cells with sheared Ad5 DNA and established a single cell line (Ad5/HEK FG 293-31), and because of its uniqueness, this line was included in this study. As anchorage-independent growth (Freedman and Shin, 1974; Shin et al., 1975) and reduced levels of the large external transformation-sensitive glycoprotein (LETS) (Chen, Gallimore and McDougall, 1976a) are phenotypes that have most recently been proposed as changes correlating with malignancy. Our observations concerning these traits are discussed in detail. Results In Vitro Characteristics Doubling Time, Saturation Density and Serum Dependency The doubling times and saturation densities for six Ad2-transformed rat embryo lines and one Ad5transformed human cell line grown in medium containing 5% FCS are shown in Table 1. Serum dependence was measured by a comparison of doubling time and saturation density at two serum levels for each cell line, and expressed as a ratio of the results obtained with 5% FCS and 0.5% FCS (Table 1). Ad2/F17 is the only transformed line showing a significant level of serum dependence; however, this line doubled twice in low serum medium during the 12 day period, while rat embryo control lines only maintain the initial cell plating number (2 X IO5 cells per 5 cm dish). Table 1 shows the data
Cell 670
Table
1. In Vitro
Line Passage
Transformed
on the Adenovirus-Transformed
Doubling Time (Hr) 5% FCS
Cell Lines Control AS.RE
Studies
9 30.6
Ratio Doubling Time in 5% FCS:O.5% FCS
0
Cell Lines
Saturation Density (Cells/ cm*) x lo* 5% FCS
1.1
Ratio Saturation Density in 5% FCS:O.5% FCS
Doubling Time (Hr) in Spinner Culture 5% DFCS
% Efficiency of Plating in MethylCellulose + 10% FCS
>11.12’
No growth
~0.002
5%
22.3 20.7 24.3 20.2 17.1 Not tested
5.25 0.02 1.06 73.60 3.24 36.60
23.8
30.30
Ad2 T-ag by Immunofluorescence
% LETS” ProteinPositive Cells by Immunofluorescence
0
100
76% 54% 46% 52% 61% 100%”
++++ +++ ++ + + ++
100 100 60 10 1 10
65%
+++
Fibrinolysis (16 Hr)
Rat
AdP/F17 Ad2/F19 Ad2lF4 AdPlASREM Ad2/T2lC4 Ad2/50A
24.0 20.1 25.6 21 .o 17.7 21.6
0.20 0.91 0.94 0.90 0.76 0.65
4.2 6.6 5.7 10.5 10.1 10.4
10.41 1.93 1.76 1.47 1.70 1.53
Transformed Human Ad5/HEK FG (293-31)
25.2
0.81
9.5
1.69
a 100% fibrinolysis in i 5 hr. ” Carried out at the same passage c No growth with 0.5% FCS.
level as the cells
inoculated
into nude
for one such control line ASRE at the ninth passage. Anchorage-Independent Growth Anchorage-independent growth was measured in two ways: first, by the ability of a cell line to initiate and maintain growth in spinner culture, and second, by the efficiency of plating (EOP) in Dulbecco’s modified Eagle’s medium (DME) containing 1.2% methyl-cellulose, supplemented with 10% FCS or 7.5% dog serum plus 2.5% FCS. Table 1 clearly shows that these two systems measure quite separate phenotypes-for example, Ad2/F19 grows poorly in methyl-cellulose (EOP 0.02%), whereas its growth in spinner culture is prolific. Five other adenovirus-transformed lines examined for growth in spinner culture share the latter phenotype with Ad2/F19. Adenovirus-transformed cell lines show considerable variation in their ability to form colonies in methyl-cellulose medium supplemented with 10% FCS, from AdP/ASREM with an EOP of 73.8% down to Ad2/F19 (0.02%). The one Ad5 transformed human line grew very well in methylcellulose. Dog serum was also studied because Pollack et al. (1974) showed that this significantly increased the EOP of three SV40-transformed rat embryo lines. All the adenovirus lines plated less well in methyl-cellulose medium supplemented with 7.5% dog serum plus 2.5% FCS as compared with 10% FCS alone, the EOP being reduced 201000 fold depending upon the cell line examined. All the studies in methyl-cellulose were controlled by using the SV40-transformed rat embryo line SVRE9 developed by Pollack et al. (1974); this line plated 4.6% (10% FCS) and 1.94% (7.5%dog serum
0
mice.
plus 2.5% FCS) in our hands. Ad2/F19 produces colonies of similar size to SV-RE9, while the colonies of all the other adenovirus lines were considerably larger. Ad2 lines T2/C4, ASREM and 50A and the Ad5/HEK FG line produce visible colonies in 10 days or less. Protease Activity Table 1 shows that all the transformed lines studied are high protease producers as measured by fibrinolytic activity; Ad2/50A is very active in this respect, hydrolyzing the fibrin clot in 5 hr. Ad2 T Antigen The morphology and location of T-ag fluorescence shows considerable variation within a line and between lines; however, all the cells within a line show some T-ag-specific fluorescence. The majority of the fluorescence is located on or within the nuclear membrane as flecks or blobs; but specific fluorescence was also seen in the cytoplasm of all the lines. Ad2/F17 has the most T-ag by immunofluorescence (Figure l), and all the other lines were graded by comparison with this line as shown in Tables 1, 2 and 3. Large External Transformation-Sensitive Glycoprotein (LETS) Normal rat embryo fibroblast cultures contain 100% LETS protein positive cells which produce an extensive fibrous network of LETS protein when confluent. The series of Ad2-transformed rat embryo cell lines studied here show a spectrum from lines containing 100% LETS protein positive cellsfor example, AdP/FlS-through to lines showing 1% LETS protein positive cells by immunofluorescence-for example, Ad2/T2/C4 (Table 1). Ad2/F17
Tumorigenicity 671
Figure
of Ad2 and Ad5 Cell Lines
1. Ad2 T Antigen
Fluorescence
in Nude
Shown
Mice
by Ad2/F17
and Ad2fF19 confluent cultures produce a fibrous LETS network similar to that of the rat embryo fibroblast cells (Figures 2A and 2B). Ad2/F4 (60% LETS protein-positive cells) produces this glycoprotein at some cell/cell contact areas but does not produce a network (Figures 3A and 3B). The Ad5transformed human embryo kidney line is negative for LETS protein. However, normal epithelial cells show a different pattern of LETS protein when compared with fibroblasts and do not produce a LETS protein network (Chen et al., 1976b). Tumor Production in Adult Nude Mice Of the adenovirus cell lines inoculated subcutaneously into adult nude mice, three growth patterns were observed. First, two lines, Ad2/F17 and Ad5/ HEK FG, did not produce tumors. Second, Ad2/F19 produced two slow growing nude mouse tumors which had few mitotic cells and were covered in a thick fibroblastic tumor capsule. Within the tumors themselves, the majority of cells were those typically found in adenovirus-induced tumors. Third, lines Ad2lF4, Ad2/50A, Ad2IASREM and Ad2/T2/ C4 produced tumors in 100% of the nude mice. The tumors appeared in less than 3 weeks and showed invasion of the local mouse tissues; however, no tumor metastases were observed. Tables 2 and 3 show all the in vivo data for the adenovirus-transformed cell lines, including the tumorigenicity of these lines when inoculated into newborn syngeneic rats and such rats immunosuppressed with anti-thymocyte serum (ATS). The Ad21 F19 and Ad2IASREM tumors grown in nude mice do not transplant in newborn syngeneic rats. All three Ad2lF4 tumors, however, do transplant into nonimmunosuppressed newborn syngeneic rats and show tumor invasion and metastatic spread in all the rats inoculated.
Tumor Production by AdS/HEK FG in 5 Day Old and X-Irradiated Adult Nude Mice Because the Ad5/HEK FG cell line had many of the characteristics in common with the Ad2 rat lines that were tumorigenic in adult nude mice, the finding that it was nontumorigenic even when 5 x 10’ cells were inoculated into the adult nude mice was initially a surprise. Experiments were therefore carried out in 5 day old nude mice and X-irradiated adult nude mice (300 rads whole body) in an attempt to provide different in vivo environments from the normal adult situation. 1 x lo6 Ad5/HEK FG cells inoculated subcutaneously into the two new in vivo environments produced tumors in 100% of the survivors as shown in Table 3. These tumors were classified as highly differentiated carcinomas and were shown to have been derived from the Ad5 line by G banding karyology (P. H. Gallimore and C. A. Macpherson, unpublished results). The Ad5/HEK FG nude mouse tumors all transplant to adult nude mice. One interesting feature of the histology of the AdS/HEK FG nude mouse tumors is that the carcinoma cells show differentiation most strikingly when tumor cells are in contact with intra-tumor stromal fibroblasts (Figure 4) or blood vessels. Differentiation occurred in rare areas of Ad2/F4 tumors, but did not occur in any of the tumors induced by the other adenovirus-transformed rat lines which produce extremely primitive anaplastic tumors. Examination of the Cell Lines for C Type Virus Particles and Reverse Transcriptase In view of the recent findings of Price et al. (1975), some of the transformed cell lines and most of the nude mouse tumors established as cell lines were examined by electron microscopy for the presence of C type virus particles and by Dr. Natalie Teich (ICRF, London) for murine RNA tumor virus reverse transcriptase. None of the adenovirus-transformed cell lines or their respective nude mouse tumors were positive by either test. One other line studied, however, served as a positive control; an Ad2 virustreated rat embryo line (AS lOOB), which does not have an adenovirus-transformed phenotype or express Ad2 T-ag and which produced fibrosarcomas in newborn syngeneic rats, induced a nude mouse tumor in 63 days that was positive for reverse transcriptase and budding C type virus particles. In Vitro Studies on Cells from Tumors Grown in Nude Mice Following Inoculation of Ad2Transformed Cell Lines One of the most interesting findings from the studies on the nude mouse tumors (nMT) was that in all
Figure
2A. lmmunofluorescence
Figure
28.
Phase-Contrast
Photomicrograph Photomicrograph
of Ad2/F19 of Confluent
Showing
Ad2/F19
cases, the tumor lines contain fewer LETS proteinpositive cells than their respective, parental transformed cell lines (Table 2). This was particularly true for the series of Ad2/F4 nude mouse tumors which have 5% LETS protein-positive cells compared with the original 60% LETS protein-positive
Massive
(Same
Network
Field as A) (Bar
of LETS
Protein-Specific
lmmunofluorescence
= 20 pm)
Ad2/F4 cells. The two Ad2/F19 nude mouse tumor lines still contained 50% LETS protein positive cells and failed to produce tumors when transplanted back into syngeneic rats. The Ad2-transformed cell nude mouse tumor lines plate in methyl-cellulose medium supple-
Tumorigenicity 673
of Ad2 and Ad5 Cell
Figure Areas
3A.
lmmunofluorescence
Figure
38.
Phase-Contrast
Lines
in Nude
Photomicrograph Photomicrograph
Mice
of Ad2/F4 of Confluent
Showing
Ad2/F4
mented with 10% FCS as well as or better than the parent line, Ad2/F19 nMT1 showing the most dramatic indication of selection in this respect. The Ad2/F4 nMT1 is the only adenovirus line studied that is not suppressed in the presence of dog se-
LETS
(Same
Protein-Specific
Field as A) (Bar
Fluorescence
at SOIT le Cell/Cell
Co1
= 20 pm)
rum, having plating efficiencies of 1 .06% in 1 0% FCS and 1.29% in 7.5% dog serum plus 2.5% Fcs methyl-cellulose medium. st udThere is strong evidence from cy togenetic ies (P. H. Gallimore and C. A. Macpl herson, unp ub-
Cell 674
Table
2. In Vivo and in Vitro
Cell Lines and Nude Mouse Tumor (nMT) Lines Rat Control Line AS.RE Passage 9 Transformed AdPIFl7 Ad2/F19 F19 nMT1 F19 nMT2 Ad2/F4
Data for AdP-Transformed Rats
Rat Lines
and Nude
Tumorigenicity Athymic Nude
Mouse
Tumor
Lines
in Adult Mice
% Tumors
in Syngeneic
Newborn 2 x 10” Cells per Rat SC
ATS lmmunosuppressed 2 x lo6 Cells per Rat SC
2 x IO’ Cells Inoculated SC Tumor +/ Total Inoculated
1 x 1O’Cells Inoculated SC Tumor +/ Total Inoculated
% EOP in MethylCellulose 10% FCS
Ad2 T- ag
% LETS Positive
0
0
015 (165 days)
015 (165 days)
zo.002
0
100%
Rat 0
0
O/5 (165 days)
O/5
5.25
++++
100%
0
0
015 (165 days)
2/5 (30 days)”
0.02
+++
100%
0
NT
NT
NT
3.55
+++
50%
+++
50%
1.06
++
60% 5%
0
NT
NT
NT
0
100”
5/5 (19 days) b
515
NT
F4 nMT1
100
NT
NT
NT
1.20
++
F4 nMT2
100
NT
NT
NT
3.16
++
5%
F4 nMT3
100
NT
NT
NT
++
5%
100
515 (6.5 days) b
Ad2lT2IC4
100
NT
515 (7.5 days)”
Ad2/50A
100
100
AdPfASREM
0
B Additional ATS required. b Mean latent period. SC = subcutaneous;
5/5 (16 days)b
nMT
= nude
mouse
lished observations), LETS protein immunofluorescence, EOP in methyl-cellulose and tumor transplantation data that cell selection has occurred in vivo in the absence of T cell-mediated immunity. Discussion Ad2 virus does not induce tumors in newborn rodents unless they are given extensive treatment with immunosuppressants (Gallimore, 1973). However, the semipermissive Ad2/rat embryo in vitro system (Gallimore, 1974) generates transformed cell lines with a number of characteristics shared by all the lines examined thus far and some phenotypes not expressed by all the lines. The common characteristics include epithelioid morphology, growth to high cell density, growth in spinner culture without adaption, elevated production of protease, immortality without “crisis” (there is no cessation of growth or extensive cell death during isolation of Ad2 lines), integrated Ad2 DNA sequences representing the left-hand 14% of the Ad2 physical and genetic map (Gallimore et al., 1974), and the presence of intracellular Ad2 T antigen. The phenotypes not uniformly expressed by all the Ad2-transformed rat lines are serum- and anchorage-independent growth, the synthesis of LETS protein fibrillar network and tumorigenicity in new-
tumor
NT 73.6 3.24 36.6
+ + ++
ProteinCells
10% 1% 10%
line; NT = not tested.
born syngeneic rats, immunosuppressed rats or adult athymic nude mice (Tables 1 and 2). Since these cell lines were all derived from independent transformation events in cultures isolated from rat embryo tissues, it is possible that the normal parental cell type was different for each line. Nevertheless, comparison of the properties of lines transformed by a single agent yet showing diverse in vivo growth behavior may be invaluable in determining characteristics important in oncogenicity. In the present series of experiments, a number of in vitro characteristics was examined in an attempt to define one or more correlating with the ability to produce tumors in adult athymic nude mice. The results indicate that high saturation density, increased protease activity, decreased doubling time and anchorage-independent growth are frequently features of Ad2-transformed cell lines but are not essential for a cell line to be tumorigenie. This conclusion is in agreement with the recent findings of Jones et al. (1975) and Stiles et al. (1976b), but not with those of Freedman and Shin (1974) and Shin et al. (1975), in that three of the seven adenovirus-transformed lines reported here failed to uphold their conclusion that anchorage-independent growth correlates with tumor formation in adult athymic nude mice. Ad2/F17 (EOP 5.25%) and AdS/HEK FG (EOP 30.3%) both show
Tumorigenicity 675
Table
of Ad2 and Ad5 Cell Lines
3. Tumorigenicity
of Ad5/HEKFG
in Nude
(293-31)
Mice
Cell Line Inoculated
Subcutaneously
into Athymic
Nude
Mice
T-ag Status
Cell Dose per 0.1 ml Inoculated
Adult (IO-12 Weeks)
5 Day Old
(293-31)
+++
5 x 10’
O/6 (165 days)
NT
NT
MSIHEKFG
(293-31)
+++
1 x 106
O/6 (165 days)
213” (30 days)c
4/6b (36 days)’ days)’
Ad5lHEKFG
nMT1
+++
5 1 5 1 5
5/5 (10 days)’ 5/5 (9.5 days)’ 3/5 (11.2 days)< o/5 o/5
NT NT NT NT NT
NT NT NT NT NT
AdS/HEKFG nMT3
X-Irradiated
+++
1 x 106
3/3 (12.5
NT
NT
Cell Line and Nude Tumor Lines (nMT) Ad5IHEKFG
Mouse
a One mouse died day 10. h Two mice died days 7 and 12 post-irradiation. c Mean latent period. nMT = nude mouse tumor
x x x x x
106 106 105 10” 10’
days)’
X-Irradiated Adults (300 rads Whole Body)
line; NT = not tested.
anchorage-independent growth in methyl-cellulose but were nontumorigenic in the adult nude mouse, while Ad2/F19 (EOP 0.02%) shows anchorage dependency in this system but produced two slow growing tumors which, although benign in nature, did not regress, being excised when 2.5 cm in diameter. If anchorage-independent growth reflects a potential for growth in vivo, then there should be a correlation between both plating efficiency and/or colony size in methyl-cellulose and the latent period and/or tumor incidence in the nude mouse. This was not observed in our study even for Ad2 lines that show anchorage-independent growth. From their study of SV40-transformed rat embryo cell lines, Pollack et al. (1974) provided evidence that elevated levels of plasminogen activator accompanied loss of anchorage dependency, and they suggested that this proteolytic activity may have a significant role in tumor invasion and metastatic spread. The significance of fibrinolytic activity to malignancy has recently been questioned by the observation of Pearlstein et al. (1976) that some normal cells-for example, cells from calf bladder-produce high levels of protease as measured by fibrinolysis. In the same report, Pearlstein et al. observed that loss of LETS protein from transformed cells was not due to their increased synthesis of protease. We have confirmed this finding in that the nontumorigenic line Ad2/F17 has high fibrinolytic activity and yet produces a massive three-dimensional network of LETS protein. It is possible that proteolytic enzymes different from plasminogen activator may be associated with a cell line’s malignant behavior, and with this in mind, we have recently further investigated Ad2/ 50A. This cell line is the most malignant and the highest producer of fibrinolytic activity of the Ad2-
transformed rat lines; however, exponentially growing cultures of Ad2/50A do not hydrolyze overlays of purified rat tail collagen even after prolonged incubation (10 days at 37°C). This preliminary finding implies that collagenase activity is not associated with this cell line’s agressive infiltration of normal tissues. Our data shown in Table 2 suggest that Ad2transformed cells expressing normal levels of LETS protein either grow poorly in vivo or respond to the control processes that inhibit the growth of normal cells in vivo. This theory could explain a previous observation that tumorigenicity in syngeneic rats did not always appear to be a reflection of the antigenic status of a cell line (Harwood and Gallimore, 1975). Cell line Ad2/F4 is heterogeneous for the expression of LETS protein, and it can be postulated that the reason why additional anti-thymocyte serum-induced immunosuppression is required before this line produces tumors in syngeneic rats is a reflection of the time it takes for LETS protein-negative cells to establish themselves in vivo, the latent period of this line in nude mice being 19 days. The finding that all three Ad2lF4 nude mouse tumor lines show a dramatic reduction in LETS protein-positive cells and are highly tumorigenic when inoculated into newborn syngeneic rats lends some support to this theory. LETS protein therefore appears to have a major role in determining the in vivo behavior of Ad2transformed cell lines. Pearlstein et al. (1976), however, have reported that three sarcomas, derived 1976; Chen et al., 1976a, 1976b) express low or undetectable levels of LETS protein. Preliminary observations on subclones of Ad21 F4, F17 and F19 isolated from either spinner culture or methyl-cellulose indicate that a strong selection of LETS-negative cells occurs in these an-
Cell 676
Figure
4. Haematoxylin-
(A) photomicrograph (B) photomicrograph fibroblasts.
and Eosin-Stained showing showing
Histological
tumor cells. epithelial differentiation
Sections of carcinoma
from a spontaneously transformed mouse line, produced appreciable levels of LETS protein, and they therefore concluded that loss of LETS protein was not essential for malignancy. This finding could indicate that some fibroblasts after transformation retain their differentiated capacity to produce LETS protein. Recently, Chen et al. (1976b) have observed that cultured normal rodent epithelial cells
of AdWHEK cells where
FG (293-31) tumor
cells
Nude
Mouse
are in direct
Tumor contact
1 with
intra-tumc
jr stromal
from liver and kidney express LEITS protein, but the distribution is different from th at of fibrob lasts in that no fibrillar network of LE TS protein is produced by epithelial cells. These ? observatio ns may provide a clue as to the origi nal cell typ Fe from which cell lines such as Ad21 F17 and Fl 9 have been derived. All the experimen Ital and bun ran carcinema lines studied thus far (Pearlstein et al.,
Tumorigenicity 677
of Ad2 and Ad5 Cell Lines
in Nude
Mice
chorage-independent environments, thus mimicking our findings with in vivo selection. These subclones are presently being examined for tumorigenicity in both syngeneic rats and nude mice. In common with a number of SV40-transformed human cell lines (Stiles et al., 1975), the Ad5transformed human cell line AdS/HEK FG is not tumorigeneic in adult nude mice. However, Ad5/HEK FG produces differentiated carcinomas when inoculated into 5 day old nude mice or X-irradiated adult nude mice. This finding may indicate that the in vivo proliferation of this cell line is dependent upon the level of expression of factors, such as growth hormone. A recent observation (C. D. Stiles and G. Sato, personal communication) that an epithelial cell line produces tumors only in newborn nude mice is similar to our finding. The histological evidence that Ad5/HEK FG tumor cells differentiate only when they are in contact with blood vessels or intra-tumor stromal fibroblasts clearly indicates that some cells in this transformed human cell line have not lost the ability to react normally to the nontransformed mouse cell component of the carcinomata. Although the molecular biology of the adenovirus-transformed rat cells have been accurately mapped in terms of integrated virus DNA (Gallimore et al., 1974) and expressed virus-specific messenger (Flint et al., 1975), our understanding of the role played by the virus genes in the diversity of phenotypes observed in our study remains unresolved. However, a number of explanations for this spectrum of transformed rat lines are possible: in the semi-permissive transformation system, there may be a number of different virus host interactions promoting heterogeneity; Ad2 can transform different differentiated cell types in rat embryo cultures; they may be the consequence of integration of the Ad2 “transformation” gene(s) at different chromosomal locations (D. Galloway and J. Sambrook personal communication); initially, the transformed cells have an inherent instability giving rise to cell lines with different in vivo and in vitro characteristics. Further progress in this area will depend upon the development of adenoviruses with a conditioned lethal mutation in the transforming gene(s). Finally, this study was initiated to ascertain the relevance of a number of in vitro characteristics, expressed by Ad2-transformed cell lines to tumorigenicity. We found that reduced levels of LETS protein was the only trait correlating with malignancy. We believe that some of the disagreements reported by us and other groups as to the relative importance of different in vitro traits to malignancy may simply be a reflection of the type of normal
parental cell giving rise to experimentally established cell lines. We agree with Shields (1976) and Stiles et al. (1976b), who have recently emphasized the need for investigators to study in vitro transfer: mation and malignancy using normal epithelial cells as the starting parental cell type. Experimental
Procedures
Tissue Culture Transformed cell lines Ad2/F4, F17, F19 and ASREM were derived from cells cultured from the same pool of AS rat embryos; the other two AdP-transformed rat embryo lines were derived from different Hooded Lister rat embryo material. Each of these lines was established from a single raised focus containing epithelioid cells which had been initiated and isolated as previously described (Gallimore, 1974). Examination of the chromosomes using G banding (P. H. Gallimore, C. Ft. Richardson and C. A. Macpherson, manuscript in preparation) confirms that each line is derived from a separate transformation event. Saturation density and doubling time analysis for each cell line was carried out using minimal Eagle’s essential medium (MEM) supplemented with either 5% or 0.5% fetal calf serum (FCS). These experiments were performed using three 5 cm dishes with an initial seeding level of 2 x lo5 cells per dish for each point. Dishes were medium changed on days 3, 6, 6 and IO, and cell counts were made daily for 12 days using a Coulter counter. Doubling times were calculated on the exponential phase of growth, and saturation density was expressed as cells per cm2 using the figure of maximum cell number. The results shown in Table 1 are the mean values of two experiments. Spinner cultures were set up as 100 ml cultures of Joklik’s modified Eagle’s medium supplemented with 5% dialyzed FCS (DFCS) at a cell concentration of 2 x IO5 cells per ml. An equal volume of fresh medium was added every 46 hr. and doubling times were established over a 12 day period. Colony Formation in Methyl-Cellulose The efficiency of plating (EOP) in methyl-cellulose was carried out exactly as described previously by Risser and Pollack (1974). After 3 weeks of incubation, colonies containing 100 cellsor more were counted using a stereoscopic microscope. Protease The activity measured occurring procedure Chen and
Activity of membrane-bound and/or excreted proteases was by the amount of fibrinolysis of purified fibrin clots over an 16 hr period. The materials and experimental used for this study were the same as those used by Buchanan (1975).
Adenovirus T Antigen (T-ag) lmmunofluorescence Cell lines were grown to subconfluency on glass coverslips, washed in phosphate-buffered saline (PBS) (pH 6.6) and fixed for 1.5 min in cold acetone (-20°C). The preparation of the Ad2 T-ag antiserum and of the FITC-labeled rabbit-anti-rat IgG have been reported by Harwood and Gallimore (1975). The cell line showing the strongest specific fluorescence, Ad2/F17. was given an arbitary 4+ rating, and all the other cell lines were compared with Ad2/F17 and given a plus rating O-4. LETS Protein Estimation by lmmunofluorescence Cell lines were grown on glass coverslips and fixed at subconfluent and postconfluent levels using 10% formol-saline. Coverslips were then washed in PBS and reacted with 10 ~1 of diluted (160) rabbit anti-human cold-insoluble globulin antibody (see Chen et al., 1976a) for 20 min at 37°C. After extensive washing
Cell 676
with PBS, coverslips were drained, layered with 10 ~1 of goat-antirabbit IgG, incubated for 20 min at 37”C, rinsed further in PBS and mounted on microscope slides with Elvanol. Animals Hooded Lister and AS rats were bred and maintained in the Department of Cancer Studies (Birmingham). Nude mice were obtained from the MRC Laboratory Animal Centre (Carshalton. Surrey, England) and maintained away from other animal stocks. Nude mice were also bred in the Department of Cancer Studies by mating homozygous nudes of Balb/c genetic background. Anti-Thymocyte Serum (ATS) Rabbit-anti-rat thymocyte serum (ATS) was produced and tested as previously described (Gallimore, 1972). Rats received 5 daily doses of 0.2 ml ATS intraperitoneally on days 6, 9, 10, 11 and 12 following inoculation of transformed cells into newborn animals. Acknowledgments The authors acknowledge the advice and support of Dr. James D. Watson and Professor D. G. Harnden; Dr. B. W. Codling, (Department of Pathology, Birmingham University) for his collaboration with tumor pathology; Dr. Natalie Teich (I.C.R.F. London) for carrying out reverse transcriptase assays; Dr. F. L. Graham for providing the cell line 293-31; and the following technicians for excellent assistance: Sylvia Green, Debbie Parton, Val Nash, Ann Maguire, Carol Morton-Harvey and Don Sammons at Birmingham University, and Shiela Levings and Elaine Paul at Cold Spring Harbor Laboratory. P. H. G. is supported by the Cancer Research Campaign (England) and was supported by a grant from the Robertson Research Fund while at Cold Spring Harbor Laboratory. L. B. C. is a fellow of the Muscular Dystrophy Association of America, and L. B. C. and J. K. M. are supported by the National Cancer Institute. Received
November
10, 1976;
revised
December
30. 1976
Chen. L. B., and Buchanan, J. M. (1975). Plasminogenindependent fibrinolysis by proteases produced by transformed chick bryo fibroblasts. Proc. Nat. Acad. Sci. USA 7.2, 1132-1136.
em-
Chen, L., B., Gallimore, P. H., and McDougall, J. K. (1976a). Correlation between tumor induction and the large external transformation sensitive protein on the cell surface. Proc. Nat. Acad. Sci. USA 73, 3570-3574.
Flint, S. J., Gallimore, of viral RNA sequences infected cells. J. Mol.
P. H.. and McDougall, protein on epithelial
P. H., and Sharp, P. A. (1975). in adenovirus P-transformed Biol. 96, 47-66.
J. K. cells.
Comparison and lytically
Freedman, V. H., and Shin, S. (1974). Cellular tumorigenicity in nude mice: correlation with cell growth in semi-solid medium. Cell 3, 355-359. Gallimore, P. H. (1972). Tumor production in immunosuppressed rats with cells transformed in vitro by adenovirus type 2. J. Gen. Virol. 76, 99-102. Gallimore, P. H. (1973). Birmingham, England.
Ph.D.
Thesis,
University
of Birmingham,
Gallimore, P. H. (1974). Interaction of Adenovirus type 2 with rat embryo cells: permissiveness, transformation and in vitro characteristics of adenovirus transformed rat embryo cells. J. Gen. Virol. 25. 263-273. Gallimore, P. H., Sharp, P. A., and Sambrook. in transformed cells: Il. A study of sequences
Graham, F. L., Abrahams, P. Warnaar, S. O., DeVries. F. A. (1974). Studies on the in vitro fragments of human adenovirus Harbor Symp. Quant. Biol. 39,
J. (1974). Viral DNA of Adenovirus 2 DNA
specific
fragments
Hynes, R. 0. (1974). tion: the importance 147-156.
of
J., Mulder, C., Heijneker, H. L., J., Fiers. W. and Van der Eb, A. J. transformation by DNA and DNA and simian virus 40. Cold Spring 637-650.
Harwood, L. M. J., and Gallimore. P. H. (1975). A study oncogenicity of adenovirus type 2 transformed rat embryo Int. J. Cancer 76, 496-508.
of the cells.
Role of surface alteration in cell transformaof proteases and surface proteins. Cell 7,
Jones, P. A., Rhim. J. S., Isaacs, H., and McAllister, The relationship between tumorigenicity, growth brinolytic activity in a line of human osteosarcoma Cancer 76, 616-621. Pearlstein, E., Hynes. R. O., Franks, (1976). Surface proteins and fibrolytic lian cells. Cancer Res. 36, 1475-1480.
R. M. (1975). in agar and ficells. Int. J.
L. M.. and Hemming, V. J. activity of cultured mamma-
Pollack. R.. Risser. R., Conlon, S., and Rifkin, D. (1974). Plasminogen activator production accompanies loss of anchorage regulation in transformation of primary rat embryo cells by simian virus 40. Proc. Nat. Acad. Sci. USA 77, 4792-4796. Price, J., Arnstein, P., Suk. W. A., Vernon, M. L., and Huebner, R. J. (1975). Type-C RNA viruses of the NIH nude mouse. J. Nat. Cancer Inst. 55, 1231-1232. Risser, R.. and Pollack, R. (1974). A non-selective analysis SV40 transformation of mouse 3T3 cells. Virology 59, 477-489. Shields, R. (1976). 262, 348.
Transformation
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of
Nature
Shin, S.. Freedman, V. H.. Risser, R.. and Pollack, R. (1975). Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc. Nat. Acad. Sci. USA 72, 4435-4439. Stiles, Failure tumors 4975.
References
Chen, L. B., Maitland, N.. Gallimore, (1976b). Detection of surface LETS Exp. Cell. Res., in press.
in nine lines of transformed rat cells using the viral genome. J. Mol. Biol. 89, 49-72.
C. D., Desmond, W., Sato, G., and Saier, M. H. (1975). of human cells transformed by simian virus 40 to form in athymic nude mice. Proc. Nat. Acad. Sci. USA 72, 4971-
Stiles, C. D., Desmond, W., Chuman. L. M., Sato, G., and Saier, M. H. (1976a). Growth control of heterologous tissue culture cells in the congenitally athymic nude mouse. Cancer Res. 36, 13531360. Stiles, C. D., Desmond, W., Chuman, L. M.. Sato, G.. and Saier, M. H. (1976b). Relationship of cell behavior in vitro to tumorigenicity in athymic nude mice. Cancer Res. 36, 3300-3305. Unkeless, J. C., Tobia, A., Ossowski. L., Quigley, J. P., Rifkin, D. B.. and Reich, R. (1973). An enzymatic function associated with transformation of fibroblasts by oncogenic viruses. J. Exp. Med. 737. 85-111.
