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Characteristics of an infinite life span diploid human fibroblast cell strain and a near-diploid strain arising from a clone of cells expressing a transfectedv-myc oncogene
EXPERIMENTAL
CELL
RESEARCH
197,125-136
(1991)
Characteristics of an Infinite Life Span Diploid Human Fibroblast Cell Strain and a Near-Diploid Strain Arising from a Clone of Cells Expressing a Transfected v-myc Oncogene THOMAS L. MORGAN,’ DAJUN YANG, DENNIS G. FRY,’ PETER J. HURLIN,’ SUZANNEK. KOHLER, VERONICAM. MAHER, AND J. JUSTIN MCCORMICK* Carcinogenesis Laboratory-Fee Hall, Department of Microbiology and Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824
INTRODUCTION Diploid human fibroblasts were transfected with a plasmid carrying a v-myc oncogene linked to the neo gene or with a vector control carrying a neo gene. Drugresistant clones were isolated and subcultured as needed. All populations went into crisis and eventually senesced. But while they were senescing, viable-appearing clones were noted among the progeny of a transfected population that expressed the v-myc oncogene. After several months, these cells began replicating more rapidly. Karyotype analysis indicated that they were clonally derived since all of them had 45 chromosomes, including 2 marker chromosomes. This cell strain was designated MSU-1.1. Similar analysis showed that cells from an earlier passage were diploid. These cells were designated MSU-1.0. Both strains have undergone more than 200 population doublings since their siblings senesced, without any change in chromosome complement. Both strains express the vmyc protein and have the same integration site for the transfected v-myc and neo genes. The MSU-1.0 cells cannot grow without exogenously added growth factors. The MSU-1.1 cells grow moderately well under the same conditions and grow to a higher saturation density than MSU-1.0 cells. Since the chance of human cells acquiring an infinite life span in culture is very rare, the data suggest that MSU- 1.1 cells are derived from MSU- 1 .Q cells. The expression of v-m yc is probably required :for acquisition of an infinite life span, since this phenotype did not develop in populations not expressing this oncogene. However, expression of vmyc is clearly not sufficient, since all of the progeny of the clone that gave rise to the MSU-1.0 cells expressed this oncogene, but the vast majority of them senesced. 0 1991 Academic Press, Inc.
Our research is directed at understanding the mechanisms by which malignant human cells arise from populations of normal cells. Our early attempts and those of other investigators to transform diploid human fibroblasts in culture to carcinogens were unsuccessful [l]. This is to be expected if, as suggested by many more recent in uiuo studies, malignant cells arise as a result of a cell acquiring a series of essential changes and if these accumulate as the result of sequential clonal selection. Therefore, we tried to circumvent the need for a series of sequential changes by transfecting normal diploid fibroblasts with fully activated rus oncogenes in plasmids engineered to promote overexpression of the ras protein [2, 31. The progeny of the transfected clones exhibited many of the characteristics of malignant, tumor-derived cells, but did not form progressively growing tumors when injected into athymic mice. This failure to produce malignant tumors may simply reflect the fact that by the time the progeny population derived from a single transfectant clone was propagated to the size needed for tumorigenicity assays, the cells had reached the end of their capacity to proliferate in vitro. This suggested the need for an infinite, or at least greatly extended, capacity to proliferate, if an individual cell were to acquire a series of transformation-related changes as a result of sequential clonal selection. Clonal selection and expansion requires that a cell that has randomly acquired a particular change, e.g., activation of various oncogenes and/or loss of tumor suppressor genes, clonally give rise to a progeny population large enough to make it likely that one of the progeny will acquire a second rare random change in the series of
1 Present addresses: D.F., Abbott Laboratories, Abbott Park, IL 60064; P.H., Fred Hutchinson Research Cancer, Seattle, WA 98104; T.M., Battelle Pacific Northwest Laboratories, Richland, WA 99352.
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126
MORGAN ET AL.
required events. For example, if the chance of the sec- frequency, they do so at a much higher frequency followond event is 1 X 10e6,the cells with the first change must ing transfection with various myc oncogenes. Therefore, undergo about 20 population doublings so that among in the present study we transfected a population of foreits lo6 progeny, one can expect to find such a variant skin-derived diploid human fibroblasts with a plasmid cell. Clonal expansion by that variant cell is also needed carrying the myc oncogene from a chicken virus, MC29, before a third equally rare event can be expected to oc- and the neo gene coding for Geneticin resistance. Sevcur in the cell that has acquired the first two changes, eral independent transfectant clones were isolated on and so on. But finite life span cells in culture that have the basis of their drug resistance, and their progeny been cloned exhibit a reduced in vitro span [4]. There- were carried to the end of their life span in culture. One fore, such cells taken through two clonal sections in cul- clonal population was found to express the viral myc ture are nearing the end of their in vitro life span. If the gene; the other four did not. The progeny of all five process is to result in a progressively growing malig- clones went into crisis at approximately the same time nant, metastatic tumor, this means one of the earlier as the progeny of control plasmid transfectants and changes must confer an extended, or infinite, replicative senesced, but among the sibling cells in the senescing capacity. Whether such a change is also required for population of the cells expressing the viral myc gene, an cells in uiuo is not known, but human tumors placed in infinite life span diploid human fibroblast cell strain culture give rise to infinite life span cell lines, whereas arose. It has stably maintained its diploid chromosomal these never arise spontaneously in the culture of normal complement for >200 population doublings since the human cells [ 11. sibling cells senesced. A second, near-diploid cell strain Generation of cells with an infinite life span also ap- with an equally stable chromosomal complement was pears to be a multistep process [5]. The most common also found in the progeny of this clone. method for generating such cell lines is to infect them with Simian virus 40 (SV40) [6] or transfect them with MATERIALS AND METHODS the early region of SV40, coding for large T-antigen [7]. However, acquisition of the gene for large T-antigen is Cells and cell culture. A diploid line of fibroblasts, designated LGl, not sufficient. Wright and his colleagues [8, 91 showed was isolated as described [12] from foreskin material derived from a that human fibroblasts that express this antigen bypass healthy male neonate. The cells were cultured in Eagle’s minimal a mortality stage, designated MI, and exhibit a some- essential medium (MEM) (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 0.2 mA4 L-aspartic acid, 0.2 n&f what extended life span. But unless some other genetic L-serine, 1.0 n-&f Na pyruvate or in McM medium [13], a modified change subsequently takes place, the cells eventually go version of the MCDBllO base medium [14], which has been further into crisis and senesce at a second mortality stage, desig- modified by substituting 0.884 n&4 monobasic sodium phosphate for nated M2. From such senescing cultures, cells with an 3.0 mM dibasic sodium phosphate, adding 0.054 mA4 KCl, and adjustthe NaCl content so that the osmolarity was 285 & 5 mOsm/kg infinite life span have been found to arise, although at a ing H,O. Penicillin (100 units/ml) and streptomycin (100 ag/ml) were very low frequency, i.e., < 3 X lo-’ [8]. Wright et al. [9] added to the medium along with 10% supplemented calf serum (Hyshowed that even in the infinite life span cells that arise, clone) (SCS), unless otherwise noted, and cells were incubated in a continued T-antigen expression is required if the cells 95% sir/5% COx humidified incubator. A chicken hepatoma, MC29 are to continue to replicate. Human fibroblasts that ex- virus-producer cell line, LSCC-DU72, was provided by K. Nazerian of U.S. Department of Agriculture Poultry Laboratory (East Lanpress SV40 T-antigen exhibit changes in morphology, the sing, MI) and grown in the modified Eagle’s medium described above. become aneuploid, exhibit anchorage independence [6], Cell stocks were stored at -135°C in MEM containing 10% DMSO and can form tumors in appropriate host animals [lo]. and 15% FBS. The absence of mycoplasma contamination was veriIt is likely that the random changes in chromosomes fied by growing cells on a coverslip, fixing with Carnoy’s solution, are, in fact, responsible for a rare cell acquiring the ad- staining with Hoeschst 33258 fluorescent stain as described [15], and examining the cells by fluorescence microscopy for the presence of ditional change(s) that allows it to circumvent M2 [9, cytoplasmic staining. The absence of SV40 T-antigen expression was lo]. However, because the changes caused by expression verified using immunofluorescence [16]. of SV40 T-antigen are those characteristic of tumorConstruction of plasmid containing v-myc oncogene. The MC29 derived cells, such cells have limited usefulness in stud- provirus containing the v-myc oncogene was isolated from its parent ies, such as ours, designed to analyze the step-wise vector, pMC38, by EcoRI digestion. The resulting 5.5kbp fragment cloned into the EcoRI site of pSV2neo [ 171 using standard molecuchanges required for a normal human cell to become a was lar cloning techniques [18]. This vector contains the neo gene which malignant cell. confers resistance to Geneticin. The recombinant plasmid was desigTo be able to investigate the number and nature of nated pSV2neo-MC29. Both pMC38 and pSV2neo were provided by such genetic changes, we tried to generate human cells H-J Kung, Case-Western Reserve University. Transfection. Cells in exponential growth were transfected with with an infinite life span in culture without using agents DNA using the modified Polybrene/DMSO procedure [19]. that could destabilize the chromosome complement. plasmid After 18 h, the transfectants were selected for Geneticin-resistance Weinberg and his colleagues [ 111 showed that although using 200 rg of active drug per ml. Colonies were isolated using sterile cultures of primary rat embryo fibroblasts spontane- filter paper moistened with trypsin, transferred to individual dishes, ously give rise to infinite life span cells at a very low and propagated.
INFINITE
LIFE
SPAN
DIPLOID
Life span analysis. The progeny of the isolated transfectant clones were subcultured in supplemented MEM containing Geneticin at 100 pg of active drug per ml using duplicate 250 ml flasks (T75s). Unless otherwise indicated, they were plated at a density of 5 X 10’ cells per flask, the medium was changed weekly and at regular intervals, and a portion of each population was stored frozen. Cells were considered senescent when no increase in cell number was observed in a flask over a period of 4 months. Cytogenetic analysis. G-banded chromosomes were prepared using the method of Yunis and Chandler [20]. C-banding was done according to the method of Salamanca and Armendares [21], i.e., treating the slides with 0.2 M HCl at room temperature for 30 min., immersing them in 0.07 M Ba(OH), at 37°C for 8-12 min., and then incubating them in 2X SSC at 65°C for 2 h and staining in 5% Giemsa. For each cell strain, at least 25 G-banded karyotypes were examined for chromosomal rearrangements, and 100 conventionally stained metapbases were counted to determine the modal chromosome number. DNA hybridization analysis. DNA was isolated as previously described [22] and analyzed by the method of Southern [23] for transfected DNA sequences, using the indicated probes. The methods used for analysis of restriction fragment length polymorphisms have been described [22]. Briefly, the DNA was digested with HaeIII, electrophoresed on a 1% agarose gel, blotted, and probed with “P-labeled Ml3 bacteriophage DNA. Immunohistochemical staining for the u-myc protein. Cells were cultured on glass coverslips, washed in phosphate-buffered saline containing 1 mM Ca ‘+ , fixed in acetone, and air-dried. Immunoperoxidase staining was performed to detect the cells expressing the myc protein. The primary antibody was a rabbit polyclonal antibody specific for the v-myc protein (Oncor, Gaithersburg, MD) which does not cross-react with the human c-myc protein. Binding of this antibody to the test cells was visualized by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Cappel) and reaction with Hanker-Yates reagent (0.8 mg/ml) in 10 mM Tris (pH 7.5)/0.1% H,O,. Stained cells were photographed using phase-contrast on a Olympus IMT-2 microscope. Zmmunoprecipitation analysis of rasp21 and v-mycpll0. Analysis was carried out as previously described [25]. Analysis of factors required for cell growth. Cells (5 X 106) were plated in McM medium supplemented with 1% SCS. After 24 h, the number of cells attached was determined and the medium was exchanged for McM medium containing 0.1 mMcalcium and the serum replacement supplements previously defined by Ryan et al. [13], but lacking any growth factors (SRJ [26]. As controls, we also tested cell growth in McM medium in the presence of 10% SCS or 1.0 n&f calcium with SR,. The medium was renewed after 3-4 days, and the number of cells was determined. After an additional 3-4 days, the cell number was again determined. Tumorigenicity of infinite life span cells. Cells to be tested were washed twice with PBS, trypsinized, and resuspended in serum-free medium at 10’ cells per ml. Three- to five-week-old athymic BALB/c nude mice were injected subcutaneously in the subscapular region with 0.1 ml of this suspension, i.e., the total cells per mouse was lo7 cells. The mice were palpated for evidence of subcutaneous tumors twice a week and sacrificed and necropsied after 6-12 months for internal tumors.
RESULTS
Effect of v-myc Transfection Cells
on the Life Span of LGl
LGl cells, the foreskin-derived, parental cell line, were plated into a series of dishes and transfected with
HUMAN
FIBROBLAST
127
CELL
pSV2neo-MC29 carrying the viral myc oncogene and the neo gene coding for Geneticin resistance or with the pSV2neo plasmid as a control. The transfectants were selected for resistance to Geneticin. After 14 days of selection, five large-sized colonies of pSV2neo-MC29transfected cells, each from a separate dish of transformants, were isolated and propagated independently. Two separate series of control clones from the pSV2neo-transfected cells were isolated, pooled, and propagated. To assess the effect of transfection of these plasmids on the residual life span on the cells, these isolates were propagated to the end of their life span. After several months of subculturing as described, each of the seven populations were split into two; one part was subcultured on a regular basis, while the other was allowed to attain confluence and was subcultured much less frequently. At various intervals, portions of these cultures were frozen. The progeny of all seven populations went into crisis and eventually senesced. (The control populations, which were originally composed of 10 to 12 pooled clones of cells transfected with pSV2neo alone, underwent approximately 35 population doublings (PDLs) following transfection before they ceased cell division. At this point the flasks were held for an additional four months with twice weekly refeeding. No further increase in cell number was observed. The five independent, clonally derived pSV2neo-MC29-transfected populations became senescent at approximately 30 PDLs post-transfection.) However, during this period a few patches of viableappearing cells were noted in flasks containing senescing progeny cells of one of the pSV2neo-MC29-transfected clones. These were first noted in the flasks that contained the cells that were being subcultured at regular intervals. However, when a flask containing the corresponding cells (siblings) that were being held in confluence was subcultured, several patches of viableappearing cells were also noted after several days. The cells in this part of this experiment were then stored frozen while we continued to work with the parts that were being subcultured at regular intervals. After several months, the viable cells in the latter flasks began replicating more rapidly. They were isolated from the flasks containing senescent population and have continued to replicate vigorously for more than 200 PDLs since their sibling cells underwent senescence. Karyotype
Analysis
of these Rapidly Dividing
Cells
The chromosome complement of these rapidly replicating, apparently infinite life span cells was determined and compared with that of the original parental cell line, LGl, and of the clonally derived, Geneticin-resistant, presenescent, pSV2neo-MC29-transfected LGl cells from which they arose. All three cell populations had the expected X-Y chromosome pattern of a male.
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6
7
ET AL.
9
6
10
16
20
21
22
11
12
17
X
18
Y
FIG. 1. Modal karyotype (G-banded) of the infinite life span cells first identified in these experiments. The chromosomal constitution these cells is: 45,XY, -11, -12, -15, +der(ll) t(l;ll) (lqter+lpll::llpl5+llpter) [Ml], +der(l5) t(12;15) (12qter+12q11::15p11-+15qter)
of
tM21.
The LGl cells and the presenescent pSV2neo-MC29transfected precursor cells were diploid. The infinite life span cells, examined several months after they were first detected in the flask of senescing cells and after they had begun proliferating rapidly, were near-diploid with a modal chromosome number of 45 (Fig. 1). All of
the cells in the population analyzed exhibited two characteristic chromosome markers, Ml and M2, along with monosomy of chromosomes 11, 12, and 15. This strongly suggests that they have a clonal origin. Analysis of the banding patterns of extended chromosomes gave information on the make-up of the markers
t (1;ll) (G-banded)
(lqter-->lpll
FIG. 2. Partial karyotypes break points.
Trisomy
Partial
llpi5->llqter)
l
Lost
411 ,
15
: : ISpll->15qter)
M2
(upside down)
Ml and M2. Arrows indicate the origin of
t (12;lS) (12qter-->12qil
is*4
12
(ic: 1
I *
and diagrams showing the make-up of the marker chromosomes
: :
P15
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MORGAN
Ml
M2
FIG. 3. C-banding of marker chromosomes Ml and M2. Note that Ml has two centromeric regions staining darkly with C-banding. This indicates that the pericentromeric region of chromosome 1 in Ml is at the site of translocation.
(Fig. 2). Marker 1 resulted from translocation between chromosomes 1 and 11 in which virtually an entire chromosome 11 (llplE+qter) was translocated to chromosome 1 at band pll. The data suggest that in the course of the l/11 translocation, the pericentromeric region of chromosome 1 was integrated into Ml along with the entire long arm. This conclusion is supported by the results of C-banding (Fig. 3) which demonstrate aprominent C band at the translocation site. Since conventional staining showed only one centromere in marker chromosome 1, this translocated centromere is inactive. Marker 2 resulted from a translocation between the long arm of chromosome 12 and virtually all of chromosome 15. It is likely that chromosome 15 supplied the M2 C band material. Thus, the two markers involve four chromosomes and were formed in the course of two different translocation events. Since these cells still contain two copies of chromosome 1, duplication of one copy of chromosome 1 must have occurred. We have not yet determined whether the two complete copies of chromosome 1 are the original chromosomes inherited from each parent, or whether the cells are now homozygous for chromosome 1. Nor do we know which chromosome 1 donated the long arm which is part of the Ml chromosome. Identifying an Infinite these Cells
Life span Diploid
Precursor of
We examined stocks of cells from this pSV2neoMC29 transfectant that had been frozen during the time that the cells were senescing to determine when the cells with the two unique marker chromosomes first appeared. We found that in a stock frozen late in the period, all cells had the marker chromosomes, but in an earlier stock, some of the cells had the marker chromosomes, while the rest of the cells were diploid. In another early stock, prepared from the cultures that had been maintained in confluence, there was a pure population of diploid cells. Because the chance of obtaining infinite life span diploid human fibroblasts is so low, we reasoned that the cells with the marker chromosomes probably arose from a diploid infinite life span cell. Therefore, we designated the latter cell strain, MSU-
ET AL.
1.0, and the former, MSU-1.1. Table 1 reports the karyotype studies on these cell strains relatively soon after crisis and many cell generations later, and shows that these are identical. It includes data for the parental cell line, LGl; for the progeny of the Geneticin-resistant transfectant that received the control plasmid, LGl SV2 neo; and for the progeny of the transfectant that received the pSV2neo-MC29 plasmid carrying both the neo gene and the v-rnyc oncogene, both precrisis and postsenescence. ChUFUCteFiZing
the MSU-1.0
and MSU-1.1
Cell Strains
Because both infinite life span cell strains, MSU-1.0 and MSU-1.1, were found in cultures of progeny cells clonally derived from a single Geneticin-resistant pSV2neo-MC29 transfectant, it was important to determine the relationship of these cell strains to the LGl cells and to each other. Although there are subtle morphological differences that distinguish them, all three have a typical spindle cell appearance and fall within the range of morphologies seen with normal diploid human fibroblasts. The first question addressed was whether MSU-1.0 and MSU-1.1 cell strains were progeny of LGl cells. Isozyme analysis (Table 2), carried out by Dr. Ward Peterson of Children’s Hospital, Detroit, as described previously with other cell lines [l, 221,demonstrated that all three cell strains exhibit the same pattern. “Fingerprint” analysis of the restriction fragment length polymorphisms of the DNA from these cell strains using Ml3 DNA as a probe [22], indicated that all three were identical (Fig. 4). Taken together, these assays indicate with high probability that MSU-1.0 and MSU-1.1 cell strains were derived from LGl cells. A second question was whether the MSU-1.0 and MSU-1.1 were progeny of the pSV2neo-MC29-transfected clone that gave rise to the population of progeny in which they were found. To address the question, Southern blot analysis of the flanking regions of the integrated plasmid in the MSU-1.0 and MSU-1.1 cell strains was carried out. The DNA from the cells was digested cut with Hind111 or BglII, and the blots were hybridized using labeled pSV2neo as a probe (Fig. 5). Unique 2.7 and 18.5 kb fragments were found in the MSU-1.0 and MSU-1.1 cells digested with HindI that were not in LGl cells. Similarly, unique 5.3 and 9.4 kb fragments were found in the MSU-1.0 and MSU-1.1 cells digested with BglII that were not in LGl cells. The unique bands common to MSU-1.0 and MSU-1.1 cells are presumed to be junction fragments and indicate that the site of integration of the plasmid is identical in the two cell strains. Since integration is random, this supports the conclusion that the MSU-1.0 cell strain and the MSU-1.1 strain were derived from the same transfectant.
INFINITE
LIFE
SPAN
DIPLOID
HUMAN
TABLE Karotype
of LGl,
LG1 LGlpSV2neo (presenescent) pSV2neo-MC29-transfectant (precrisis precursor cells) MSU-1.0 (postsenescent) MSU-1.0 (postsenescent) MSU-1.1 (postsenescent) (postsenescent)
MUS-1.0,
and MSU-1.1
Cells Percentage of cells with marker chromosomead
Karyotype’
19 35 60
46 46 46
46, XY 46, XY 46, XY
6 98 9
46 46 45
109
45
46, XY 46, XY 45, XY, -11, -12, -15, +Ml, +M2 45, XY, -11, -12, -15, +Ml, +M2
’ For the postsenescent cells, this refers to population doublings b Determined from 100 conventionally stained metaphases. ’ Determined from the G-banded chromosomes of 25 cells. d Determined from G-banded chromosomes.
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CELL
1
Modal chromosome number*
Population doublings level”
Cell strain
MSU-1.1
Analysis
FIBROBLAST
0 0 0 0 0 100 100
after crisis.
Determining if the Cell Strains Expressed the v-myc Protein
Since MSU-1.1 cells appear to be missing the p15 region of chromosome 11, which contains the coding region for the H-ras-1 gene (llp15.5), and the short arm of chromosome 12, which contains the coding region for the K-ras 2 gene (12p12.1), we reasoned that they might have reduced expression for these two proteins. Immunoprecipitation analysis with ras pal-specific monoclonal antibody Y13-259 indicated that MSU-1.1 cells express markedly less of the upper p21 band (Fig. 8, arrow) than do the MSU-1.0 or LGl cells. There was no difference in the density of the lower ras band. The Kras and H-ras protein products have been determined to localize to the upper band.
The MSU-1.1 cells were tested for the presence of v-myc protein and exhibited distinct nuclear staining (Fig. 6E). Figures 6A-6C show LGl cells, MSU-1.1 cells, and an MC29-virus-transformed chicken hepatoma cell line (LSCC-DU72) used as a positive control, stained with Giemsa. Figures 6D-6F show the same cells stained for expression of v-myc protein by an immunohistochemical procedure. The pattern of this staining in the MSU-1.1 cells was identical to that seen in the positive control. In contrast, the LGl cells exhibited only a faint, uniform background level of staining and no distinct nuclear staining (Fig. 6D). To verify that the myc gene expressed was the chicken viral myc gene, immunoprecipitation analysis was carried out. As shown in Fig. 7, a ~110 v-myc protein was expressed in the MSU-1.1 and the MSU-1.0 cells as well as in the LSCC-DU72 cells. No such band was seen in the parental LGl cells.
Comparing LGl, MSU-1.0, and MSU-1.1 Cells for Growth Factor Independence Since the MSU-1.1 cells grow better in culture than either the MSU-1.0 or LGl cells, we examined the ability of these three cell strains to grow in serum-free medium. As shown in Fig. 9, LGl and MSU-1.0 do not grow
TABLE
2
Isozyme Phenotypes of LGl, MSU-1.0, and MSU-1.1 Cells Isozyme phenotypes Cells
LDH”
GGPD*
LGl MSU-1.0 MSU-1.1
Human Human Human
B B B
’ Lactic dehydrogenase. *Glucose-6-phosphate chondrial. #Adenylate kinase. “Glyoxalase 1.
PGMl’ 1 1 1 dehydrogenase.
PGM3d
ESD’
Me-2’
AK-l8
GLO-l*
2 2 2
1 1 1
l-2 1-2 1-2
l-2 l-2 1-2
1-2 1-2 l-2
“Phosphoglucomutase-1.
dPhosphoglucomutase-3.
“Esterase
D. f Malic mito-
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ET AL.
type, i.e., subcutaneous or on internal organs, have been seen, indicating that these cell strains are not tumorigenie. As a positive control, more than 40 malignant sarcomas have been produced in athymic mice subcutaneously injected with MSU-1.1 cells that had been malignantly transformed by transfection of the v-K-rus oncogene [24] or the H-ras or N-rus oncogene [25,26] in a high expression vector.
DISCUSSION
FIG. 4. Restriction fragment length polymorphisms pattern analysis of parental cell line LGl and the two infinite life span cell strains, using labeled Ml3 DNA as a probe. The DNA from all three cell types exhibit equivalent patterns. The methods used are described in Ref.
WI.
in McM medium containing 0.1 mM calcium without the addition of a protein growth factor or serum. However, MSU-1.1 cells grow moderately well in medium containing 0.1 mm calcium without the addition of a protein growth factor or serum. These cells are, however, still responsive to serum or calcium since they replicate more rapidly after its addition. These data suggest that MSU-1.1 cells either synthesize their own protein growth factor(s) or get around the need for such factor(s) by some other mechanism. If the MSU-1.1 cells are partial growth factor variants, one would expect that they would grow to higher density than the LGl or MSU-1.0 cells at confluence. When cells were placed in 1 or 10% SCS and allowed to reach confluence as determined by electronic cell counts of the cells, we obtained the results shown in Table 3. The MSU-1.1 cells grew to a higher density than the LGl or MSU-1.0 under both sets of conditions which is consistent with the data on their growth in growth factor-free medium. We are analyzing MSU-1.1 cells to determine whether they synthesize various growth factors.
The data demonstrate for the first time that apparently diploid, infinite life span human fibroblasts cell strains can arise in vitro and that they do so probably as a result of a multistep process. The diploid cell strain, MSU-1.0, responds normally to protein growth factors, medium containing normal concentrations of calcium (1.0 n&f) and serum. It does not exhibit a growth rate advantage over the finite life span LGl parental cells under any conditions we have been able to devise. This is an important point because it indicates that regardless of whether such infinite life span cells arise early or late in the life span of the population, the only selection for infinite life span cells is to grow cells to crisis and search for cells that do not senesce. As more and more of the cells in the population cease to replicate, i.e., the culture enters “crisis,” one will expect to see, as we did, a small number of replicating cells on a background of senescing cells. The task of detecting such cells is especially difficult since human fibroblast cell populations typically are in crisis over a period of several weeks to
Hind A
m BC
Tumorigenicity
To date, more than 40 athymic mice have been subcutaneously injected with 10’ cells of these various cell strains, i.e., LGl cells, MSU-1.0 cells, MSU-1.1 cells, and 10 mice have been injected with lo7 of the presenescent siblings of the two infinite life span cell strains to assess their tumorigenic potential. Animals have been observed for 6 to 12 months. No growths of any
FIG. 6. Southern LGl cells probed with with HindIII; D-F with B and E; LGl cells, C
blot of DNA from MSU-1.0, MSU-1.1, and pSV2neo-MC29. DNA in lanes A-C was cut BglII. MSU-1.0 cells, A and D; MSU-1.1 cells, and F.
INFINITE
LIFE SPAN DIPLOID
HUMAN
FIBROBLAST
CELL
133
FIG. 6. Expression of chicken v-myc protein in MSU-1.1 and LSCC-DUT72 chicken hepatoma cells. Cells were stained with Giemsa, A-C; or antibody against the v-myc protein, D-F. LGl cells, A and D; MSU-1.1 cells, B and E; LSCC-DU72 cells, C and F.
more than a year and during this period cultures replicate very slowly and have a high frequency of morphologically aberrant senescing cells. It should, perhaps, be noted that the situation with human fibroblasts differs from that of carcinogen-treated Syrian hamster embryo cells that give rise to infinite life span cells which arise at a low but measurable frequency [27]. The latter infinite life span rodent cells exhibit a clear growth rate
advantage over the finite life span parental cells and so are readily selected for this. This raises another possible problem in detecting cells with an infinite life span. In cell fusion experiments in which hybrids of finite life span human fibroblasts and infinite life span human cells are generated, the finite life span phenotype is seen to be dominant, i.e., the hybrid cells exhibit a finite life span. It is possi-
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Schwab and Bishop [28] shows even more clearly that N-myc or c-myc expression is sufficient to cause rat em-
FIG. 7. Expression of v-myc-encoded ~110 in MSU-1.0 and MSU-1.1 cell strains, their parental LGl cell line, and in LSCCDU72, a chicken hepatoma, MC29 virus-producer cell line. v-myc ~110 was immunoprecipitated from [36S]methionine-labeled cell lysates with an antibody that reacts with proteins of the myc family and analyzed by electrophoresis in 10% polyacrylamide gels and fluorography. The location of the v-myc-encoded ~110 is indicated by the arrow.
ble that metabolic cooperation, i.e., the sharing of metabolites between cells by physical contact between normal human fibroblasts and newly arising infinite life span human fibroblasts causes the death of the latter cells. If so, this could account in part for the fact that infinite life span human fibroblasts have never been seen to arise spontaneously. Studies that support this idea have been carried out by Schwab and Bishop [28]. They found that in rat embryo cells, eight of eight clones of cells transfected with the human N-myc oncogene and selected by drug resistance gave rise to infinite life span cells. The cells had normal growth rates. However, if drug selection was not used to isolate the original transfected colonies, but instead the transfectants were just grown along with the nontransfectants, no infinite life span cells were obtained. These rat embryo cells differ significantly from human fibroblasts in that expression of a deregulated myc apparently is sufficient to create the infinite life span phenotype, but the data show that, even in this case, the expression of the infinite life span phenotype is blocked, or the infinite life span cells are killed, when the latter are grown in the presence of a majority of finite life span cells. The role of the v-myc gene in generating our infinite life span fibroblasts remains unclear. Expression of the v-myc oncogene clearly was not sufficient to confer the infinite life span since all the progeny cells of the original transfectant selected for Geneticin resistance contained the v-myc oncogene and expressed its protein, but only one or, perhaps, a small number of cells survived senescence. We employed the v-myc oncogene because in rat embryo fibroblasts, the expression of MC29 v-myc as well as other myc genes caused infinite life span fibroblasts to develop [ll]. The recent study by
bryo cells to develop an infinite life span. These data are supported by a study by Tavassoli and Shall [29] who found that spontaneously immortalized mouse and rat fibroblasts expressed a 3- to 20-fold higher level of cmyc than finite life span cells. However, as noted above, expression of the v-myc protein clearly is not sufficient to immortalize human cells since the siblings of MSU1.0 and MSU-1.1 cells senesced. Nevertheless, we consider that the v-myc oncogene played some role because the immortal cell strains spontaneously developed in cultures of cells expressing this oncogene, and no such cell strains have arisen in nontransfected cultures [ 11. The hypothesis that integration of a plasmid carrying the v-myc oncogene disrupted expression of an essential gene for senescence in the LGl transfectant cannot explain the immortalization event that gave rise to MSU1.0 cells, since all of its siblings had the same integration site. A likely explanation for the acquisition of an infinite life span is that during expansion of the Geneticinresistant transfectant clone, a rare, spontaneous mutation occurred in a v-myc-expressing finite life span cell allowing it to circumvent senescence. If so, mutagen treatment of human fibroblasts clonally selected for drug resistance and expressing a transfected v-myc gene is expected to increase the frequency of the chance event. Such experiments are being carried out. The recent study of Kinsella et al. [30] supports the suggestion that an unregulated myc oncogene can play some role in the acquisition of an infinite life span in human fibroblasts. In their study, diploid human fibroblasts were infected with a retrovirus carrying the hu-
P21Z
FIG. 8. Analysis of ras p21 expression in MSU-1.0 and MSU-1.1 cell strains and in their parental cell line LGl. The ras-encoded p21 proteins were immunoprecipitated with antibody Y13-259 from [3SS]methionine-labeled cell lysates and analyzed by electrophoresis and fluorography as described [2]. The location of the endogenous ras-encoded ~21 protein (doublet) is indicated by the bar. The upper band (arrow) clearly is less prominent in the MSU-1.1 cell strain.
INFINITE
I
LIFE
LGI I
SPAN
DIPLOID
HUMAN
FIBROBLAST
MSU- 1.0 I
135
CELL
MSU-I.1
-r-----l
ll-..-JU
0
2
TIME
4
6
00
2
4
6
0
(DAYS)
FIG. 9. Dependence of MSU-1.0 and MSU-1.1 cell strains and their parental cell line LGl on the presence of exogenously added growth factors. Cells were plated into 21 cm2 dishes at 5 X lO’/dish in McM medium supplemented with 1% FBS. Twenty hours later (Day 0) the number of cells which had attached to the dish was determined, and the medium was replaced with 0.1 mM Ca’+-McM medium supplemented with S&, 1.0 n&f Ca*+-McM medium supplemented with S&, and 1.0 mM Ca’+-McM medium supplemented with 10% FBS. Cells were fed with the designated media on Days 3 and 5, and their number was determined on the days indicated.
man c-myc gene under the control of a viral LTR, and infinite life span cells arose in the infected population. Our results support the multistep hypothesis for immortalization proposed by Wright et al. [9]. They show that to acquire an infinite life span, the cells must not only express the large T-antigen, but also acquire some other rare change. They propose that the expression of large T-antigen allows the cell to bypass mortality point one (Ml), and a rare chance event allows these cells to bypass mortality point two (M2). However, it is not possible to interpret the events that gave rise to the MSU1.0 cells directly in terms of this hypothesis. The MSU-1.1 cell strain was the one first detected in our cultures and shown to have an infinite life span. Therefore, we used it as the target cell strain used in an attempt to obtain malignant transformation of human cells, using transfection of an H-rus [25] or an N-rm [26] oncogene in a plasmid engineered for high expression of the oncogene, or a viral K-rus oncogene [24]. In each case, the transfectants became malignantly trans-
formed. In previous studies the H-rus [2] and N-ras [3] oncogenes in the same plasmid constructs caused finite life span diploid human fibroblasts to acquire many of the characteristics of tumor-derived cells, but the progeny cells did not produce progressively growing invasive tumors and retained their finite life span. These results support the hypothesis that acquisition of an infinite life span is required for malignant. transformation in vitro. Similar transfection studies have recently been begun using the MSU-1.0 cell strain. We thank our colleague, Dr. Ward D. Peterson, for carrying out the isoenzyme analyses of the cell strains. The excellent technical assistance of Lonnie Milam and Clay Spencer is gratefully acknowledged. We also thank Mrs. Connie Williams for manuscript preparation. This research was supported in part by Department of Health and Human Services Grant CA21289 from the National Cancer Institute, by Department of Energy Grant DE-FG02-87ER-60524, and by Contract ES65152 from the National Institute of Environmental Health Sciences. REFERENCES 1.
TABLE Cell Number
3
at Satuation Density
Cell strain LG1 MSU-1.0 MSU-1.1
2.
Density (cells/cm’
X lo-‘)
10% scs
1% scs
9
3.2
5.4
2.8
19.9
7.7
3. 4. 5. 6.
7. Note. Cells were plated at 5000 cells/cm2 in culture dishes and refed two times a week with appropriate medium. At 3 weeks, the cells had reached a constant density. The counts are an average of two dishes.
8.
McCormick, J. J., and Maher, V. M. (1988) Mutat. Res. 199, 273-292. Hurlin, P. J., Fry, D. G., Maher, V. M., and McCormick, J. J. (1987) Cancer Res. 4’7,5752-5757. Wilson, D. M., Fry, D. G., Maher, V. M., and McCormick, J. J. (1989) Carcinogen&s 10, 635-640. Goldstein, S. (1990) Science 249, 1129-1133. Holliday, R., Huschtscha, H. E., Tarrant, G. M., and Kirwood, T. B. L. (1977) Science 198, 366-372. Sack, G. H., Jr. (1981) In Vitro 17, 1-19. Chang, P. L., Gunby, J. L., Tomkins, D. J., Mak, I., Rosa, N. E., and Mak, S. (1986) Exp. Cell Res. 167,407-416. Shay, J. W., and Wright, W. E. (1989) Exp. Cell Res. 184,109118.
136 9. 10.
12.
13.
15. 16. 17. 18.
MORGAN Wright, W. E., Pereira-Smith, 0. M., and Shay, J. W. (1989) Mol. Cell. Biol. 9, 3088-3092. Ray, F. A., Peabody, D. S., Cooper, J. L., Cram, L. S., and Kraemer, F. M. (1990) J. Cell. Biochem. 42, 13-31. Land, H., Chen, A. C., Morgenstern, J. P., Parada, L. F., and Weinberg, R. A. (1986) Mol. Cell. Biol. $1917-1925. McCormick, J. J., and Maher, V. M. (1981) in DNA repair: A Laboratory Manual of Research Procedures (Friedherg, E. C., and Hanawalt, P. C., Eds.), Vol. lB, pp. 501-521, Marcel Dekker, Inc., New York. Ryan, P. A., McCormick, J. J., and Maher, V. M. (1987) Exp. Cell Res. 172,318-328. Bettger, W. J., Boyce, S. T., Walthall, B. J., and Ham, R. B. (1981) Proc. Natl. Acad. Sci. USA 78, 5588-5592. Chen, T. R. (1977) Exp. Cell. Res. 104, 255-262. Deppert, W. (1980) Virology 104,497-501. Southern, P. J., and Berg, P. (1982) J. Mol. Appl. Genet. 1,327341. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Morgan, T. L., Maher, V. M., and McCormick, J. J. (1986) In Vitro Cell. Deu. Biol. 22, 317-319.
Received May 27,199l Revised version received July 24, 1991
ET AL. 20. 21. 22.
23. 24. 25. 26. 27.
28. 29. 30.
Yunis, J. J., and Chandler, M. E. (1977) Prog. Clin. Pathol. 7, 267-288. Salamanca, F., and Armendares, S. (1974) Ann. Genet. 2,135 136. McCormick, J. J., Yang, D., Maher, V. M., Farber, R. A., Newman, W., Peterson, W. D., Jr., and Pollack, M. S. (1988) Carcinogenesis 9, 2073-2079. Southern, E. M. (1975) J. Mol. Biol. 5,503-517. Fry, D. G., Milam, L. D., Dillberger, J. E., Maher, V. M., and McCormick, J. J. (1990) Oncogene 5, 1415-1418. Hurlin, P., Maher, V. M., and McCormick, J. J. (1989) Proc. N&l. Acad. Sci. USA 86, 187-191. Wilson, D. M., Yang, D., Dillberger, J. E., Dietrich, S. E., Maher, V. M., and McCormick, J. J. (1990) Cancer Res. SO, 5587-5593. Bols, B. L. M. C., Naaktgeboren, J. M., and Simons, J. W. 1. M. (1989) in Cell Transformation and Radiation Induced Cancer (Chadwick, K. H., Seymour, C., and Barnhart, B., Eds.), pp. 109-116, Adam Hilger, Bristol, U.K. Schwab, M., and Bishop, J. M. (1989) Proc. N&l. Acad. Sci. USA 85,9585-9589. Tavassoli, M., and Shall, S. (1988) Oncogenc 2,337-345. Kinsella, A. R., Fiszer-Maliszewska, L., Mitchell, E. L. D., Guo, Y., Fox, M., and Scott, D. (1990) Carcinogenesis 11,1803-1809.
CELL
RESEARCH
197,125-136
(1991)
Characteristics of an Infinite Life Span Diploid Human Fibroblast Cell Strain and a Near-Diploid Strain Arising from a Clone of Cells Expressing a Transfected v-myc Oncogene THOMAS L. MORGAN,’ DAJUN YANG, DENNIS G. FRY,’ PETER J. HURLIN,’ SUZANNEK. KOHLER, VERONICAM. MAHER, AND J. JUSTIN MCCORMICK* Carcinogenesis Laboratory-Fee Hall, Department of Microbiology and Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824
INTRODUCTION Diploid human fibroblasts were transfected with a plasmid carrying a v-myc oncogene linked to the neo gene or with a vector control carrying a neo gene. Drugresistant clones were isolated and subcultured as needed. All populations went into crisis and eventually senesced. But while they were senescing, viable-appearing clones were noted among the progeny of a transfected population that expressed the v-myc oncogene. After several months, these cells began replicating more rapidly. Karyotype analysis indicated that they were clonally derived since all of them had 45 chromosomes, including 2 marker chromosomes. This cell strain was designated MSU-1.1. Similar analysis showed that cells from an earlier passage were diploid. These cells were designated MSU-1.0. Both strains have undergone more than 200 population doublings since their siblings senesced, without any change in chromosome complement. Both strains express the vmyc protein and have the same integration site for the transfected v-myc and neo genes. The MSU-1.0 cells cannot grow without exogenously added growth factors. The MSU-1.1 cells grow moderately well under the same conditions and grow to a higher saturation density than MSU-1.0 cells. Since the chance of human cells acquiring an infinite life span in culture is very rare, the data suggest that MSU- 1.1 cells are derived from MSU- 1 .Q cells. The expression of v-m yc is probably required :for acquisition of an infinite life span, since this phenotype did not develop in populations not expressing this oncogene. However, expression of vmyc is clearly not sufficient, since all of the progeny of the clone that gave rise to the MSU-1.0 cells expressed this oncogene, but the vast majority of them senesced. 0 1991 Academic Press, Inc.
Our research is directed at understanding the mechanisms by which malignant human cells arise from populations of normal cells. Our early attempts and those of other investigators to transform diploid human fibroblasts in culture to carcinogens were unsuccessful [l]. This is to be expected if, as suggested by many more recent in uiuo studies, malignant cells arise as a result of a cell acquiring a series of essential changes and if these accumulate as the result of sequential clonal selection. Therefore, we tried to circumvent the need for a series of sequential changes by transfecting normal diploid fibroblasts with fully activated rus oncogenes in plasmids engineered to promote overexpression of the ras protein [2, 31. The progeny of the transfected clones exhibited many of the characteristics of malignant, tumor-derived cells, but did not form progressively growing tumors when injected into athymic mice. This failure to produce malignant tumors may simply reflect the fact that by the time the progeny population derived from a single transfectant clone was propagated to the size needed for tumorigenicity assays, the cells had reached the end of their capacity to proliferate in vitro. This suggested the need for an infinite, or at least greatly extended, capacity to proliferate, if an individual cell were to acquire a series of transformation-related changes as a result of sequential clonal selection. Clonal selection and expansion requires that a cell that has randomly acquired a particular change, e.g., activation of various oncogenes and/or loss of tumor suppressor genes, clonally give rise to a progeny population large enough to make it likely that one of the progeny will acquire a second rare random change in the series of
1 Present addresses: D.F., Abbott Laboratories, Abbott Park, IL 60064; P.H., Fred Hutchinson Research Cancer, Seattle, WA 98104; T.M., Battelle Pacific Northwest Laboratories, Richland, WA 99352.
‘To whom dressed.
correspondence
and reprint
requests
125 All
Copyright 0 1991 rights of reproduction
should
be ad-
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126
MORGAN ET AL.
required events. For example, if the chance of the sec- frequency, they do so at a much higher frequency followond event is 1 X 10e6,the cells with the first change must ing transfection with various myc oncogenes. Therefore, undergo about 20 population doublings so that among in the present study we transfected a population of foreits lo6 progeny, one can expect to find such a variant skin-derived diploid human fibroblasts with a plasmid cell. Clonal expansion by that variant cell is also needed carrying the myc oncogene from a chicken virus, MC29, before a third equally rare event can be expected to oc- and the neo gene coding for Geneticin resistance. Sevcur in the cell that has acquired the first two changes, eral independent transfectant clones were isolated on and so on. But finite life span cells in culture that have the basis of their drug resistance, and their progeny been cloned exhibit a reduced in vitro span [4]. There- were carried to the end of their life span in culture. One fore, such cells taken through two clonal sections in cul- clonal population was found to express the viral myc ture are nearing the end of their in vitro life span. If the gene; the other four did not. The progeny of all five process is to result in a progressively growing malig- clones went into crisis at approximately the same time nant, metastatic tumor, this means one of the earlier as the progeny of control plasmid transfectants and changes must confer an extended, or infinite, replicative senesced, but among the sibling cells in the senescing capacity. Whether such a change is also required for population of the cells expressing the viral myc gene, an cells in uiuo is not known, but human tumors placed in infinite life span diploid human fibroblast cell strain culture give rise to infinite life span cell lines, whereas arose. It has stably maintained its diploid chromosomal these never arise spontaneously in the culture of normal complement for >200 population doublings since the human cells [ 11. sibling cells senesced. A second, near-diploid cell strain Generation of cells with an infinite life span also ap- with an equally stable chromosomal complement was pears to be a multistep process [5]. The most common also found in the progeny of this clone. method for generating such cell lines is to infect them with Simian virus 40 (SV40) [6] or transfect them with MATERIALS AND METHODS the early region of SV40, coding for large T-antigen [7]. However, acquisition of the gene for large T-antigen is Cells and cell culture. A diploid line of fibroblasts, designated LGl, not sufficient. Wright and his colleagues [8, 91 showed was isolated as described [12] from foreskin material derived from a that human fibroblasts that express this antigen bypass healthy male neonate. The cells were cultured in Eagle’s minimal a mortality stage, designated MI, and exhibit a some- essential medium (MEM) (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 0.2 mA4 L-aspartic acid, 0.2 n&f what extended life span. But unless some other genetic L-serine, 1.0 n-&f Na pyruvate or in McM medium [13], a modified change subsequently takes place, the cells eventually go version of the MCDBllO base medium [14], which has been further into crisis and senesce at a second mortality stage, desig- modified by substituting 0.884 n&4 monobasic sodium phosphate for nated M2. From such senescing cultures, cells with an 3.0 mM dibasic sodium phosphate, adding 0.054 mA4 KCl, and adjustthe NaCl content so that the osmolarity was 285 & 5 mOsm/kg infinite life span have been found to arise, although at a ing H,O. Penicillin (100 units/ml) and streptomycin (100 ag/ml) were very low frequency, i.e., < 3 X lo-’ [8]. Wright et al. [9] added to the medium along with 10% supplemented calf serum (Hyshowed that even in the infinite life span cells that arise, clone) (SCS), unless otherwise noted, and cells were incubated in a continued T-antigen expression is required if the cells 95% sir/5% COx humidified incubator. A chicken hepatoma, MC29 are to continue to replicate. Human fibroblasts that ex- virus-producer cell line, LSCC-DU72, was provided by K. Nazerian of U.S. Department of Agriculture Poultry Laboratory (East Lanpress SV40 T-antigen exhibit changes in morphology, the sing, MI) and grown in the modified Eagle’s medium described above. become aneuploid, exhibit anchorage independence [6], Cell stocks were stored at -135°C in MEM containing 10% DMSO and can form tumors in appropriate host animals [lo]. and 15% FBS. The absence of mycoplasma contamination was veriIt is likely that the random changes in chromosomes fied by growing cells on a coverslip, fixing with Carnoy’s solution, are, in fact, responsible for a rare cell acquiring the ad- staining with Hoeschst 33258 fluorescent stain as described [15], and examining the cells by fluorescence microscopy for the presence of ditional change(s) that allows it to circumvent M2 [9, cytoplasmic staining. The absence of SV40 T-antigen expression was lo]. However, because the changes caused by expression verified using immunofluorescence [16]. of SV40 T-antigen are those characteristic of tumorConstruction of plasmid containing v-myc oncogene. The MC29 derived cells, such cells have limited usefulness in stud- provirus containing the v-myc oncogene was isolated from its parent ies, such as ours, designed to analyze the step-wise vector, pMC38, by EcoRI digestion. The resulting 5.5kbp fragment cloned into the EcoRI site of pSV2neo [ 171 using standard molecuchanges required for a normal human cell to become a was lar cloning techniques [18]. This vector contains the neo gene which malignant cell. confers resistance to Geneticin. The recombinant plasmid was desigTo be able to investigate the number and nature of nated pSV2neo-MC29. Both pMC38 and pSV2neo were provided by such genetic changes, we tried to generate human cells H-J Kung, Case-Western Reserve University. Transfection. Cells in exponential growth were transfected with with an infinite life span in culture without using agents DNA using the modified Polybrene/DMSO procedure [19]. that could destabilize the chromosome complement. plasmid After 18 h, the transfectants were selected for Geneticin-resistance Weinberg and his colleagues [ 111 showed that although using 200 rg of active drug per ml. Colonies were isolated using sterile cultures of primary rat embryo fibroblasts spontane- filter paper moistened with trypsin, transferred to individual dishes, ously give rise to infinite life span cells at a very low and propagated.
INFINITE
LIFE
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DIPLOID
Life span analysis. The progeny of the isolated transfectant clones were subcultured in supplemented MEM containing Geneticin at 100 pg of active drug per ml using duplicate 250 ml flasks (T75s). Unless otherwise indicated, they were plated at a density of 5 X 10’ cells per flask, the medium was changed weekly and at regular intervals, and a portion of each population was stored frozen. Cells were considered senescent when no increase in cell number was observed in a flask over a period of 4 months. Cytogenetic analysis. G-banded chromosomes were prepared using the method of Yunis and Chandler [20]. C-banding was done according to the method of Salamanca and Armendares [21], i.e., treating the slides with 0.2 M HCl at room temperature for 30 min., immersing them in 0.07 M Ba(OH), at 37°C for 8-12 min., and then incubating them in 2X SSC at 65°C for 2 h and staining in 5% Giemsa. For each cell strain, at least 25 G-banded karyotypes were examined for chromosomal rearrangements, and 100 conventionally stained metapbases were counted to determine the modal chromosome number. DNA hybridization analysis. DNA was isolated as previously described [22] and analyzed by the method of Southern [23] for transfected DNA sequences, using the indicated probes. The methods used for analysis of restriction fragment length polymorphisms have been described [22]. Briefly, the DNA was digested with HaeIII, electrophoresed on a 1% agarose gel, blotted, and probed with “P-labeled Ml3 bacteriophage DNA. Immunohistochemical staining for the u-myc protein. Cells were cultured on glass coverslips, washed in phosphate-buffered saline containing 1 mM Ca ‘+ , fixed in acetone, and air-dried. Immunoperoxidase staining was performed to detect the cells expressing the myc protein. The primary antibody was a rabbit polyclonal antibody specific for the v-myc protein (Oncor, Gaithersburg, MD) which does not cross-react with the human c-myc protein. Binding of this antibody to the test cells was visualized by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (Cappel) and reaction with Hanker-Yates reagent (0.8 mg/ml) in 10 mM Tris (pH 7.5)/0.1% H,O,. Stained cells were photographed using phase-contrast on a Olympus IMT-2 microscope. Zmmunoprecipitation analysis of rasp21 and v-mycpll0. Analysis was carried out as previously described [25]. Analysis of factors required for cell growth. Cells (5 X 106) were plated in McM medium supplemented with 1% SCS. After 24 h, the number of cells attached was determined and the medium was exchanged for McM medium containing 0.1 mMcalcium and the serum replacement supplements previously defined by Ryan et al. [13], but lacking any growth factors (SRJ [26]. As controls, we also tested cell growth in McM medium in the presence of 10% SCS or 1.0 n&f calcium with SR,. The medium was renewed after 3-4 days, and the number of cells was determined. After an additional 3-4 days, the cell number was again determined. Tumorigenicity of infinite life span cells. Cells to be tested were washed twice with PBS, trypsinized, and resuspended in serum-free medium at 10’ cells per ml. Three- to five-week-old athymic BALB/c nude mice were injected subcutaneously in the subscapular region with 0.1 ml of this suspension, i.e., the total cells per mouse was lo7 cells. The mice were palpated for evidence of subcutaneous tumors twice a week and sacrificed and necropsied after 6-12 months for internal tumors.
RESULTS
Effect of v-myc Transfection Cells
on the Life Span of LGl
LGl cells, the foreskin-derived, parental cell line, were plated into a series of dishes and transfected with
HUMAN
FIBROBLAST
127
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pSV2neo-MC29 carrying the viral myc oncogene and the neo gene coding for Geneticin resistance or with the pSV2neo plasmid as a control. The transfectants were selected for resistance to Geneticin. After 14 days of selection, five large-sized colonies of pSV2neo-MC29transfected cells, each from a separate dish of transformants, were isolated and propagated independently. Two separate series of control clones from the pSV2neo-transfected cells were isolated, pooled, and propagated. To assess the effect of transfection of these plasmids on the residual life span on the cells, these isolates were propagated to the end of their life span. After several months of subculturing as described, each of the seven populations were split into two; one part was subcultured on a regular basis, while the other was allowed to attain confluence and was subcultured much less frequently. At various intervals, portions of these cultures were frozen. The progeny of all seven populations went into crisis and eventually senesced. (The control populations, which were originally composed of 10 to 12 pooled clones of cells transfected with pSV2neo alone, underwent approximately 35 population doublings (PDLs) following transfection before they ceased cell division. At this point the flasks were held for an additional four months with twice weekly refeeding. No further increase in cell number was observed. The five independent, clonally derived pSV2neo-MC29-transfected populations became senescent at approximately 30 PDLs post-transfection.) However, during this period a few patches of viableappearing cells were noted in flasks containing senescing progeny cells of one of the pSV2neo-MC29-transfected clones. These were first noted in the flasks that contained the cells that were being subcultured at regular intervals. However, when a flask containing the corresponding cells (siblings) that were being held in confluence was subcultured, several patches of viableappearing cells were also noted after several days. The cells in this part of this experiment were then stored frozen while we continued to work with the parts that were being subcultured at regular intervals. After several months, the viable cells in the latter flasks began replicating more rapidly. They were isolated from the flasks containing senescent population and have continued to replicate vigorously for more than 200 PDLs since their sibling cells underwent senescence. Karyotype
Analysis
of these Rapidly Dividing
Cells
The chromosome complement of these rapidly replicating, apparently infinite life span cells was determined and compared with that of the original parental cell line, LGl, and of the clonally derived, Geneticin-resistant, presenescent, pSV2neo-MC29-transfected LGl cells from which they arose. All three cell populations had the expected X-Y chromosome pattern of a male.
128
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7
ET AL.
9
6
10
16
20
21
22
11
12
17
X
18
Y
FIG. 1. Modal karyotype (G-banded) of the infinite life span cells first identified in these experiments. The chromosomal constitution these cells is: 45,XY, -11, -12, -15, +der(ll) t(l;ll) (lqter+lpll::llpl5+llpter) [Ml], +der(l5) t(12;15) (12qter+12q11::15p11-+15qter)
of
tM21.
The LGl cells and the presenescent pSV2neo-MC29transfected precursor cells were diploid. The infinite life span cells, examined several months after they were first detected in the flask of senescing cells and after they had begun proliferating rapidly, were near-diploid with a modal chromosome number of 45 (Fig. 1). All of
the cells in the population analyzed exhibited two characteristic chromosome markers, Ml and M2, along with monosomy of chromosomes 11, 12, and 15. This strongly suggests that they have a clonal origin. Analysis of the banding patterns of extended chromosomes gave information on the make-up of the markers
t (1;ll) (G-banded)
(lqter-->lpll
FIG. 2. Partial karyotypes break points.
Trisomy
Partial
llpi5->llqter)
l
Lost
411 ,
15
: : ISpll->15qter)
M2
(upside down)
Ml and M2. Arrows indicate the origin of
t (12;lS) (12qter-->12qil
is*4
12
(ic: 1
I *
and diagrams showing the make-up of the marker chromosomes
: :
P15
130
MORGAN
Ml
M2
FIG. 3. C-banding of marker chromosomes Ml and M2. Note that Ml has two centromeric regions staining darkly with C-banding. This indicates that the pericentromeric region of chromosome 1 in Ml is at the site of translocation.
(Fig. 2). Marker 1 resulted from translocation between chromosomes 1 and 11 in which virtually an entire chromosome 11 (llplE+qter) was translocated to chromosome 1 at band pll. The data suggest that in the course of the l/11 translocation, the pericentromeric region of chromosome 1 was integrated into Ml along with the entire long arm. This conclusion is supported by the results of C-banding (Fig. 3) which demonstrate aprominent C band at the translocation site. Since conventional staining showed only one centromere in marker chromosome 1, this translocated centromere is inactive. Marker 2 resulted from a translocation between the long arm of chromosome 12 and virtually all of chromosome 15. It is likely that chromosome 15 supplied the M2 C band material. Thus, the two markers involve four chromosomes and were formed in the course of two different translocation events. Since these cells still contain two copies of chromosome 1, duplication of one copy of chromosome 1 must have occurred. We have not yet determined whether the two complete copies of chromosome 1 are the original chromosomes inherited from each parent, or whether the cells are now homozygous for chromosome 1. Nor do we know which chromosome 1 donated the long arm which is part of the Ml chromosome. Identifying an Infinite these Cells
Life span Diploid
Precursor of
We examined stocks of cells from this pSV2neoMC29 transfectant that had been frozen during the time that the cells were senescing to determine when the cells with the two unique marker chromosomes first appeared. We found that in a stock frozen late in the period, all cells had the marker chromosomes, but in an earlier stock, some of the cells had the marker chromosomes, while the rest of the cells were diploid. In another early stock, prepared from the cultures that had been maintained in confluence, there was a pure population of diploid cells. Because the chance of obtaining infinite life span diploid human fibroblasts is so low, we reasoned that the cells with the marker chromosomes probably arose from a diploid infinite life span cell. Therefore, we designated the latter cell strain, MSU-
ET AL.
1.0, and the former, MSU-1.1. Table 1 reports the karyotype studies on these cell strains relatively soon after crisis and many cell generations later, and shows that these are identical. It includes data for the parental cell line, LGl; for the progeny of the Geneticin-resistant transfectant that received the control plasmid, LGl SV2 neo; and for the progeny of the transfectant that received the pSV2neo-MC29 plasmid carrying both the neo gene and the v-rnyc oncogene, both precrisis and postsenescence. ChUFUCteFiZing
the MSU-1.0
and MSU-1.1
Cell Strains
Because both infinite life span cell strains, MSU-1.0 and MSU-1.1, were found in cultures of progeny cells clonally derived from a single Geneticin-resistant pSV2neo-MC29 transfectant, it was important to determine the relationship of these cell strains to the LGl cells and to each other. Although there are subtle morphological differences that distinguish them, all three have a typical spindle cell appearance and fall within the range of morphologies seen with normal diploid human fibroblasts. The first question addressed was whether MSU-1.0 and MSU-1.1 cell strains were progeny of LGl cells. Isozyme analysis (Table 2), carried out by Dr. Ward Peterson of Children’s Hospital, Detroit, as described previously with other cell lines [l, 221,demonstrated that all three cell strains exhibit the same pattern. “Fingerprint” analysis of the restriction fragment length polymorphisms of the DNA from these cell strains using Ml3 DNA as a probe [22], indicated that all three were identical (Fig. 4). Taken together, these assays indicate with high probability that MSU-1.0 and MSU-1.1 cell strains were derived from LGl cells. A second question was whether the MSU-1.0 and MSU-1.1 were progeny of the pSV2neo-MC29-transfected clone that gave rise to the population of progeny in which they were found. To address the question, Southern blot analysis of the flanking regions of the integrated plasmid in the MSU-1.0 and MSU-1.1 cell strains was carried out. The DNA from the cells was digested cut with Hind111 or BglII, and the blots were hybridized using labeled pSV2neo as a probe (Fig. 5). Unique 2.7 and 18.5 kb fragments were found in the MSU-1.0 and MSU-1.1 cells digested with HindI that were not in LGl cells. Similarly, unique 5.3 and 9.4 kb fragments were found in the MSU-1.0 and MSU-1.1 cells digested with BglII that were not in LGl cells. The unique bands common to MSU-1.0 and MSU-1.1 cells are presumed to be junction fragments and indicate that the site of integration of the plasmid is identical in the two cell strains. Since integration is random, this supports the conclusion that the MSU-1.0 cell strain and the MSU-1.1 strain were derived from the same transfectant.
INFINITE
LIFE
SPAN
DIPLOID
HUMAN
TABLE Karotype
of LGl,
LG1 LGlpSV2neo (presenescent) pSV2neo-MC29-transfectant (precrisis precursor cells) MSU-1.0 (postsenescent) MSU-1.0 (postsenescent) MSU-1.1 (postsenescent) (postsenescent)
MUS-1.0,
and MSU-1.1
Cells Percentage of cells with marker chromosomead
Karyotype’
19 35 60
46 46 46
46, XY 46, XY 46, XY
6 98 9
46 46 45
109
45
46, XY 46, XY 45, XY, -11, -12, -15, +Ml, +M2 45, XY, -11, -12, -15, +Ml, +M2
’ For the postsenescent cells, this refers to population doublings b Determined from 100 conventionally stained metaphases. ’ Determined from the G-banded chromosomes of 25 cells. d Determined from G-banded chromosomes.
131
CELL
1
Modal chromosome number*
Population doublings level”
Cell strain
MSU-1.1
Analysis
FIBROBLAST
0 0 0 0 0 100 100
after crisis.
Determining if the Cell Strains Expressed the v-myc Protein
Since MSU-1.1 cells appear to be missing the p15 region of chromosome 11, which contains the coding region for the H-ras-1 gene (llp15.5), and the short arm of chromosome 12, which contains the coding region for the K-ras 2 gene (12p12.1), we reasoned that they might have reduced expression for these two proteins. Immunoprecipitation analysis with ras pal-specific monoclonal antibody Y13-259 indicated that MSU-1.1 cells express markedly less of the upper p21 band (Fig. 8, arrow) than do the MSU-1.0 or LGl cells. There was no difference in the density of the lower ras band. The Kras and H-ras protein products have been determined to localize to the upper band.
The MSU-1.1 cells were tested for the presence of v-myc protein and exhibited distinct nuclear staining (Fig. 6E). Figures 6A-6C show LGl cells, MSU-1.1 cells, and an MC29-virus-transformed chicken hepatoma cell line (LSCC-DU72) used as a positive control, stained with Giemsa. Figures 6D-6F show the same cells stained for expression of v-myc protein by an immunohistochemical procedure. The pattern of this staining in the MSU-1.1 cells was identical to that seen in the positive control. In contrast, the LGl cells exhibited only a faint, uniform background level of staining and no distinct nuclear staining (Fig. 6D). To verify that the myc gene expressed was the chicken viral myc gene, immunoprecipitation analysis was carried out. As shown in Fig. 7, a ~110 v-myc protein was expressed in the MSU-1.1 and the MSU-1.0 cells as well as in the LSCC-DU72 cells. No such band was seen in the parental LGl cells.
Comparing LGl, MSU-1.0, and MSU-1.1 Cells for Growth Factor Independence Since the MSU-1.1 cells grow better in culture than either the MSU-1.0 or LGl cells, we examined the ability of these three cell strains to grow in serum-free medium. As shown in Fig. 9, LGl and MSU-1.0 do not grow
TABLE
2
Isozyme Phenotypes of LGl, MSU-1.0, and MSU-1.1 Cells Isozyme phenotypes Cells
LDH”
GGPD*
LGl MSU-1.0 MSU-1.1
Human Human Human
B B B
’ Lactic dehydrogenase. *Glucose-6-phosphate chondrial. #Adenylate kinase. “Glyoxalase 1.
PGMl’ 1 1 1 dehydrogenase.
PGM3d
ESD’
Me-2’
AK-l8
GLO-l*
2 2 2
1 1 1
l-2 1-2 1-2
l-2 l-2 1-2
1-2 1-2 l-2
“Phosphoglucomutase-1.
dPhosphoglucomutase-3.
“Esterase
D. f Malic mito-
132
MORGAN
ET AL.
type, i.e., subcutaneous or on internal organs, have been seen, indicating that these cell strains are not tumorigenie. As a positive control, more than 40 malignant sarcomas have been produced in athymic mice subcutaneously injected with MSU-1.1 cells that had been malignantly transformed by transfection of the v-K-rus oncogene [24] or the H-ras or N-rus oncogene [25,26] in a high expression vector.
DISCUSSION
FIG. 4. Restriction fragment length polymorphisms pattern analysis of parental cell line LGl and the two infinite life span cell strains, using labeled Ml3 DNA as a probe. The DNA from all three cell types exhibit equivalent patterns. The methods used are described in Ref.
WI.
in McM medium containing 0.1 mM calcium without the addition of a protein growth factor or serum. However, MSU-1.1 cells grow moderately well in medium containing 0.1 mm calcium without the addition of a protein growth factor or serum. These cells are, however, still responsive to serum or calcium since they replicate more rapidly after its addition. These data suggest that MSU-1.1 cells either synthesize their own protein growth factor(s) or get around the need for such factor(s) by some other mechanism. If the MSU-1.1 cells are partial growth factor variants, one would expect that they would grow to higher density than the LGl or MSU-1.0 cells at confluence. When cells were placed in 1 or 10% SCS and allowed to reach confluence as determined by electronic cell counts of the cells, we obtained the results shown in Table 3. The MSU-1.1 cells grew to a higher density than the LGl or MSU-1.0 under both sets of conditions which is consistent with the data on their growth in growth factor-free medium. We are analyzing MSU-1.1 cells to determine whether they synthesize various growth factors.
The data demonstrate for the first time that apparently diploid, infinite life span human fibroblasts cell strains can arise in vitro and that they do so probably as a result of a multistep process. The diploid cell strain, MSU-1.0, responds normally to protein growth factors, medium containing normal concentrations of calcium (1.0 n&f) and serum. It does not exhibit a growth rate advantage over the finite life span LGl parental cells under any conditions we have been able to devise. This is an important point because it indicates that regardless of whether such infinite life span cells arise early or late in the life span of the population, the only selection for infinite life span cells is to grow cells to crisis and search for cells that do not senesce. As more and more of the cells in the population cease to replicate, i.e., the culture enters “crisis,” one will expect to see, as we did, a small number of replicating cells on a background of senescing cells. The task of detecting such cells is especially difficult since human fibroblast cell populations typically are in crisis over a period of several weeks to
Hind A
m BC
Tumorigenicity
To date, more than 40 athymic mice have been subcutaneously injected with 10’ cells of these various cell strains, i.e., LGl cells, MSU-1.0 cells, MSU-1.1 cells, and 10 mice have been injected with lo7 of the presenescent siblings of the two infinite life span cell strains to assess their tumorigenic potential. Animals have been observed for 6 to 12 months. No growths of any
FIG. 6. Southern LGl cells probed with with HindIII; D-F with B and E; LGl cells, C
blot of DNA from MSU-1.0, MSU-1.1, and pSV2neo-MC29. DNA in lanes A-C was cut BglII. MSU-1.0 cells, A and D; MSU-1.1 cells, and F.
INFINITE
LIFE SPAN DIPLOID
HUMAN
FIBROBLAST
CELL
133
FIG. 6. Expression of chicken v-myc protein in MSU-1.1 and LSCC-DUT72 chicken hepatoma cells. Cells were stained with Giemsa, A-C; or antibody against the v-myc protein, D-F. LGl cells, A and D; MSU-1.1 cells, B and E; LSCC-DU72 cells, C and F.
more than a year and during this period cultures replicate very slowly and have a high frequency of morphologically aberrant senescing cells. It should, perhaps, be noted that the situation with human fibroblasts differs from that of carcinogen-treated Syrian hamster embryo cells that give rise to infinite life span cells which arise at a low but measurable frequency [27]. The latter infinite life span rodent cells exhibit a clear growth rate
advantage over the finite life span parental cells and so are readily selected for this. This raises another possible problem in detecting cells with an infinite life span. In cell fusion experiments in which hybrids of finite life span human fibroblasts and infinite life span human cells are generated, the finite life span phenotype is seen to be dominant, i.e., the hybrid cells exhibit a finite life span. It is possi-
134
MORGAN
ET AL.
Schwab and Bishop [28] shows even more clearly that N-myc or c-myc expression is sufficient to cause rat em-
FIG. 7. Expression of v-myc-encoded ~110 in MSU-1.0 and MSU-1.1 cell strains, their parental LGl cell line, and in LSCCDU72, a chicken hepatoma, MC29 virus-producer cell line. v-myc ~110 was immunoprecipitated from [36S]methionine-labeled cell lysates with an antibody that reacts with proteins of the myc family and analyzed by electrophoresis in 10% polyacrylamide gels and fluorography. The location of the v-myc-encoded ~110 is indicated by the arrow.
ble that metabolic cooperation, i.e., the sharing of metabolites between cells by physical contact between normal human fibroblasts and newly arising infinite life span human fibroblasts causes the death of the latter cells. If so, this could account in part for the fact that infinite life span human fibroblasts have never been seen to arise spontaneously. Studies that support this idea have been carried out by Schwab and Bishop [28]. They found that in rat embryo cells, eight of eight clones of cells transfected with the human N-myc oncogene and selected by drug resistance gave rise to infinite life span cells. The cells had normal growth rates. However, if drug selection was not used to isolate the original transfected colonies, but instead the transfectants were just grown along with the nontransfectants, no infinite life span cells were obtained. These rat embryo cells differ significantly from human fibroblasts in that expression of a deregulated myc apparently is sufficient to create the infinite life span phenotype, but the data show that, even in this case, the expression of the infinite life span phenotype is blocked, or the infinite life span cells are killed, when the latter are grown in the presence of a majority of finite life span cells. The role of the v-myc gene in generating our infinite life span fibroblasts remains unclear. Expression of the v-myc oncogene clearly was not sufficient to confer the infinite life span since all the progeny cells of the original transfectant selected for Geneticin resistance contained the v-myc oncogene and expressed its protein, but only one or, perhaps, a small number of cells survived senescence. We employed the v-myc oncogene because in rat embryo fibroblasts, the expression of MC29 v-myc as well as other myc genes caused infinite life span fibroblasts to develop [ll]. The recent study by
bryo cells to develop an infinite life span. These data are supported by a study by Tavassoli and Shall [29] who found that spontaneously immortalized mouse and rat fibroblasts expressed a 3- to 20-fold higher level of cmyc than finite life span cells. However, as noted above, expression of the v-myc protein clearly is not sufficient to immortalize human cells since the siblings of MSU1.0 and MSU-1.1 cells senesced. Nevertheless, we consider that the v-myc oncogene played some role because the immortal cell strains spontaneously developed in cultures of cells expressing this oncogene, and no such cell strains have arisen in nontransfected cultures [ 11. The hypothesis that integration of a plasmid carrying the v-myc oncogene disrupted expression of an essential gene for senescence in the LGl transfectant cannot explain the immortalization event that gave rise to MSU1.0 cells, since all of its siblings had the same integration site. A likely explanation for the acquisition of an infinite life span is that during expansion of the Geneticinresistant transfectant clone, a rare, spontaneous mutation occurred in a v-myc-expressing finite life span cell allowing it to circumvent senescence. If so, mutagen treatment of human fibroblasts clonally selected for drug resistance and expressing a transfected v-myc gene is expected to increase the frequency of the chance event. Such experiments are being carried out. The recent study of Kinsella et al. [30] supports the suggestion that an unregulated myc oncogene can play some role in the acquisition of an infinite life span in human fibroblasts. In their study, diploid human fibroblasts were infected with a retrovirus carrying the hu-
P21Z
FIG. 8. Analysis of ras p21 expression in MSU-1.0 and MSU-1.1 cell strains and in their parental cell line LGl. The ras-encoded p21 proteins were immunoprecipitated with antibody Y13-259 from [3SS]methionine-labeled cell lysates and analyzed by electrophoresis and fluorography as described [2]. The location of the endogenous ras-encoded ~21 protein (doublet) is indicated by the bar. The upper band (arrow) clearly is less prominent in the MSU-1.1 cell strain.
INFINITE
I
LIFE
LGI I
SPAN
DIPLOID
HUMAN
FIBROBLAST
MSU- 1.0 I
135
CELL
MSU-I.1
-r-----l
ll-..-JU
0
2
TIME
4
6
00
2
4
6
0
(DAYS)
FIG. 9. Dependence of MSU-1.0 and MSU-1.1 cell strains and their parental cell line LGl on the presence of exogenously added growth factors. Cells were plated into 21 cm2 dishes at 5 X lO’/dish in McM medium supplemented with 1% FBS. Twenty hours later (Day 0) the number of cells which had attached to the dish was determined, and the medium was replaced with 0.1 mM Ca’+-McM medium supplemented with S&, 1.0 n&f Ca*+-McM medium supplemented with S&, and 1.0 mM Ca’+-McM medium supplemented with 10% FBS. Cells were fed with the designated media on Days 3 and 5, and their number was determined on the days indicated.
man c-myc gene under the control of a viral LTR, and infinite life span cells arose in the infected population. Our results support the multistep hypothesis for immortalization proposed by Wright et al. [9]. They show that to acquire an infinite life span, the cells must not only express the large T-antigen, but also acquire some other rare change. They propose that the expression of large T-antigen allows the cell to bypass mortality point one (Ml), and a rare chance event allows these cells to bypass mortality point two (M2). However, it is not possible to interpret the events that gave rise to the MSU1.0 cells directly in terms of this hypothesis. The MSU-1.1 cell strain was the one first detected in our cultures and shown to have an infinite life span. Therefore, we used it as the target cell strain used in an attempt to obtain malignant transformation of human cells, using transfection of an H-rus [25] or an N-rm [26] oncogene in a plasmid engineered for high expression of the oncogene, or a viral K-rus oncogene [24]. In each case, the transfectants became malignantly trans-
formed. In previous studies the H-rus [2] and N-ras [3] oncogenes in the same plasmid constructs caused finite life span diploid human fibroblasts to acquire many of the characteristics of tumor-derived cells, but the progeny cells did not produce progressively growing invasive tumors and retained their finite life span. These results support the hypothesis that acquisition of an infinite life span is required for malignant. transformation in vitro. Similar transfection studies have recently been begun using the MSU-1.0 cell strain. We thank our colleague, Dr. Ward D. Peterson, for carrying out the isoenzyme analyses of the cell strains. The excellent technical assistance of Lonnie Milam and Clay Spencer is gratefully acknowledged. We also thank Mrs. Connie Williams for manuscript preparation. This research was supported in part by Department of Health and Human Services Grant CA21289 from the National Cancer Institute, by Department of Energy Grant DE-FG02-87ER-60524, and by Contract ES65152 from the National Institute of Environmental Health Sciences. REFERENCES 1.
TABLE Cell Number
3
at Satuation Density
Cell strain LG1 MSU-1.0 MSU-1.1
2.
Density (cells/cm’
X lo-‘)
10% scs
1% scs
9
3.2
5.4
2.8
19.9
7.7
3. 4. 5. 6.
7. Note. Cells were plated at 5000 cells/cm2 in culture dishes and refed two times a week with appropriate medium. At 3 weeks, the cells had reached a constant density. The counts are an average of two dishes.
8.
McCormick, J. J., and Maher, V. M. (1988) Mutat. Res. 199, 273-292. Hurlin, P. J., Fry, D. G., Maher, V. M., and McCormick, J. J. (1987) Cancer Res. 4’7,5752-5757. Wilson, D. M., Fry, D. G., Maher, V. M., and McCormick, J. J. (1989) Carcinogen&s 10, 635-640. Goldstein, S. (1990) Science 249, 1129-1133. Holliday, R., Huschtscha, H. E., Tarrant, G. M., and Kirwood, T. B. L. (1977) Science 198, 366-372. Sack, G. H., Jr. (1981) In Vitro 17, 1-19. Chang, P. L., Gunby, J. L., Tomkins, D. J., Mak, I., Rosa, N. E., and Mak, S. (1986) Exp. Cell Res. 167,407-416. Shay, J. W., and Wright, W. E. (1989) Exp. Cell Res. 184,109118.
136 9. 10.
12.
13.
15. 16. 17. 18.
MORGAN Wright, W. E., Pereira-Smith, 0. M., and Shay, J. W. (1989) Mol. Cell. Biol. 9, 3088-3092. Ray, F. A., Peabody, D. S., Cooper, J. L., Cram, L. S., and Kraemer, F. M. (1990) J. Cell. Biochem. 42, 13-31. Land, H., Chen, A. C., Morgenstern, J. P., Parada, L. F., and Weinberg, R. A. (1986) Mol. Cell. Biol. $1917-1925. McCormick, J. J., and Maher, V. M. (1981) in DNA repair: A Laboratory Manual of Research Procedures (Friedherg, E. C., and Hanawalt, P. C., Eds.), Vol. lB, pp. 501-521, Marcel Dekker, Inc., New York. Ryan, P. A., McCormick, J. J., and Maher, V. M. (1987) Exp. Cell Res. 172,318-328. Bettger, W. J., Boyce, S. T., Walthall, B. J., and Ham, R. B. (1981) Proc. Natl. Acad. Sci. USA 78, 5588-5592. Chen, T. R. (1977) Exp. Cell. Res. 104, 255-262. Deppert, W. (1980) Virology 104,497-501. Southern, P. J., and Berg, P. (1982) J. Mol. Appl. Genet. 1,327341. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Morgan, T. L., Maher, V. M., and McCormick, J. J. (1986) In Vitro Cell. Deu. Biol. 22, 317-319.
Received May 27,199l Revised version received July 24, 1991
ET AL. 20. 21. 22.
23. 24. 25. 26. 27.
28. 29. 30.
Yunis, J. J., and Chandler, M. E. (1977) Prog. Clin. Pathol. 7, 267-288. Salamanca, F., and Armendares, S. (1974) Ann. Genet. 2,135 136. McCormick, J. J., Yang, D., Maher, V. M., Farber, R. A., Newman, W., Peterson, W. D., Jr., and Pollack, M. S. (1988) Carcinogenesis 9, 2073-2079. Southern, E. M. (1975) J. Mol. Biol. 5,503-517. Fry, D. G., Milam, L. D., Dillberger, J. E., Maher, V. M., and McCormick, J. J. (1990) Oncogene 5, 1415-1418. Hurlin, P., Maher, V. M., and McCormick, J. J. (1989) Proc. N&l. Acad. Sci. USA 86, 187-191. Wilson, D. M., Yang, D., Dillberger, J. E., Dietrich, S. E., Maher, V. M., and McCormick, J. J. (1990) Cancer Res. SO, 5587-5593. Bols, B. L. M. C., Naaktgeboren, J. M., and Simons, J. W. 1. M. (1989) in Cell Transformation and Radiation Induced Cancer (Chadwick, K. H., Seymour, C., and Barnhart, B., Eds.), pp. 109-116, Adam Hilger, Bristol, U.K. Schwab, M., and Bishop, J. M. (1989) Proc. N&l. Acad. Sci. USA 85,9585-9589. Tavassoli, M., and Shall, S. (1988) Oncogenc 2,337-345. Kinsella, A. R., Fiszer-Maliszewska, L., Mitchell, E. L. D., Guo, Y., Fox, M., and Scott, D. (1990) Carcinogenesis 11,1803-1809.