Virus-specific DNA sequences in mouse and rat cells transformed by the Harvey and Moloney murine sarcoma viruses detected by in situ hybridization

VIROLOGY 63, 40-47 (1975)

Virus-Specific

DNA Sequences

Transformed Sarcoma

and Rat Ceils

by the Harvey and Moloney

Viruses

Detected

MARIA CARLA LONI’ Saint Louis University

in Mouse

School of Medicine,

Murine

by ln Situ Hybridization AND

Institute

MAURICE GREEN

for Molecular

Virology,

St. Louis, Missouri

63110

Accepted August 12, 1974 The [SH]DNA product of the murine sarcoma-leukemia virus (MSV(MLV)) and viral 60-70 S [3H]RNA was annealed with cytological preparations of mouse and rat cells transformed by the Harvey and Moloney strains of MSV. With viral [SH]DNA as cytological probe, these in situ hybridization measurements detected from 25 to 30 autoradiographic grains per interphase nucleus of transformed cells and 1 to 3 grains per nucleus of uninfected rat and mouse cells. With viral 6070 S [3H]RNA of lower specific radioactivity as cytological probe, 12 grains per transformed cell nucleus were detected. These findings indicate that transformation of cells with MSV(MLV) produces a several-fold increase in the content of some virus-specific DNA sequences. Virus-specific sequences in transformed mouse cells were localized in the chromocenters of interphase nuclei. INTRODUCTION

In situ hybridization, i.e., molecular hybridization applied to cytological preparations (Gall and Pardue, 1969; John et al., 1969), has successfully detected and localized repetitive DNA sequences in normal cells (Pardue and Gall, 1970; Jones, 1970) and the large number of viral DNA molecules in mammalian cells productively infected with Shope papilloma virus (Orth et al., 1970) and with several human adenoviruses (McDougal et al., 1972). The detection of the far fewer viral DNA sequences in virus-free cells transformed by tumor viruses (Green, 1970) by cn situ hybridization requires the use of nucleic acid probes of very high specific radioactivity and long exposure to the photographic emulsion. Recent studies have detected viral DNA sequences in non-virus-producing cells transformed by several strains of human adenovirus (Dunn et al., 1973; Loni and Green, 1973). In this report, we describe ’ On leave from the University of Louvain, Faculty of Medicine 4, Avenue, Chapelle aux Champs, 1200 Brussels, Belgium. 40 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

the detection of viral DNA sequences in interphase nuclei of virus-producing mouse and rat cells transformed by the Harvey(H) and Maloney(M) strains of murine sarcoma virus (MSV), respectively. Two radioactive probes were used for these in situ hybridization experiments, the [3H]DNA product of the viral RNAdirected DNA polymerase and the viral 60-70 S [3H]RNA genome of the murine sarcoma-leukemia virus (MSV(MLV)). MATERIALS

AND METHODS

Cells and viruses. MSV-transformed rat cells producing M-MSV(MLV) (78Al cells) and MSV-transformed mouse cells producing H-MSV(MLV) (MEH ceils) were grown in monolayer culture and in suspension culture, respectively, for in situ hybridization experiments (Green et al., 1970). Normal rat (F-1853) and mouse cell lines (NIH/3T3 and NIH/3T6, clone 91) were grown in monolayer culture in Eagle’s minimal essential medium with 10% calf serum. For virus purification, HMSV(MLV) and M-MSV(MLV) were

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grown in suspension cultures of MEH and cytoplasm; therefore, no hybrid formation 78Al cells and purified from 6-liter outside the nucleus is expected. Cytologiamounts of culture fluid as previously cal preparations on slides were denatured described (Green et al., 1970; Rokutanda et with 0.07 N NaOH at room temperature for al., 1970). 2 min, and then annealed at 66” for 16-18 Preparation of viral [3H]DNA product. hr in 2 or 3 x SSC with 90-100 ~1 of either Single-stranded [3H]DNA from both virus (1) [‘H]DNA (2.4 x lo6 dpm/ml for Hstrains were prepared by incorporation of MSV(MLV) DNA and 3.3 x lo6 dpm/ml [3H]TTP into 0.02% Nonidet P-40 dis- ,for M-MSV(MLV) DNA), or (2) 60-70 S rupted virus in the presence of actinomycin [‘H]RNA (4 x lo6 dpm/ml). The specific D (20 &ml). The reaction mixture con- activity of [3H]DNA was 8.0 x lo7 dpm/pg, tained 0.1 mA4 each of dATP, dGTP, as calculated from the specific activity of dCTP, 30 mM NaCl, 5 mA4 dithiothreitol, [3H]TTP, assuming equimolar amounts of 2.5 mM MgCl,, 40 mA4 Tris. HCl (pH 8.3), the four deoxyribonucleotides in the DNA and 100 &i/ml of [‘H]TTP (50.8 Ci/ product. Slides were dipped in Kodak mmol). After 4- to 6-hr incubation at 37”, NTB-2 emulsion (diluted 1: 1 with distilled Na dodecyl SO, (1%) and EDTA (10 mM) water), air dried, and exposed for 16-19 wk were added, and the DNA product was in the dark at 4’. They were developed in purified as described (Green et al., 1971). Kodak D-19 for 4 min, fixed, and stained Labeled viral DNA hybridized 70-80% with Giemsa. with viral 60-70 S RNA in 2 x SSC (SSC = RESULTS 0.15 M NaCl-0.015 M Na, citrate) at 66” for 22 hr (Tsuchida et al., 1972). The DNA In Situ Hybridization of M-MSV(MLV)product produced under these reaction Transformed Rat Cells (78Al) with Mconditions with these viruses, even in the MSV(MLV) [3H]DNA and Viral 60-70 absence of actinomycin D, contains seS [3H]RNA quences derived from all or nearly all of the viral genome (Rokutanda et al., 1970; M-MSV(MLV) [3H]DNA was annealed Green, 1971; Tsuchida and Green, 1974). with fixed of Mpreparations Isolation and labeling of viral 60-70 S MSV(MLV)-transformed rat 78Al ceils RNA. Unlabeled MSV(MLV) 60-70 S and control F-1853 rat cells, as described in RNA was prepared from purified virus as Materials and Methods, and then exposed emulsion for 18 wk (Fig. described previously (Tsuchida et al., to photographic 1972). Viral 60-70 S [3H]RNA was pre- 1). The same [3H]DNA preparation was with slides of the Hpared from virus grown in 78Al cells la- annealed beled with [5,6-3H]uridine (20 pCi/ml, 36.8 MSV(MLV)-transformed mouse MEH cell Ci/mmole) for 22 hr. The specific activity line. The autoradiographic grain counts are could not be determined accurately be- given in Table 1. Background levels over cause of the small amounts of radioactive vacant areas were low, an average of 1 grain RNA that were isolated. per cell nucleus, and were constant for Isolation of cell DNA. DNA was isolated different cell preparations. An average of 28 grains (26-30) over background was from MEH cells, lysed with Na dodecyl SO,, digested with pronase, and extracted found for 78Al cell nuclei, while normal rat with chloroform-isoamyl alcohol (Fujinaga cell nuclei had an average of l-2 grains et al., 1973). After precipitation with ethaover background. Nuclei from transformed nol, the DNA was further purified by mouse MEH cells contained an average of centrifugation to equilibrium in CsCl den- 24 grains. Practically the same average sity gradients. grain count was obtained when MEH nuIn situ hybridization. The conditions for clei were annealed with the H-MSV(MLV) in situ hybridization are those described by [3H]DNA (see Table 2). This similarity is Gall and Pardue (1970). Fixation of cells consistent with the extensive homology was in acetic acid-methanol (Gall and between the viral RNA of these two Pardue, 1970) which removes most of the MSV(MLV) strains (Green et al., 1971).

LONI

AND

GREEN

FIG. 1. Autoradiographs of nuclei from M-MSV-transformed rat 78Al cells (a, b) and normal rat (F-1853) cells (c). M-MSV(MLV) [3H]DNA (8 x 10’ dpm/pg) was annealed with fixed preparations of transformed and normal rat cells for 18 hr. Exposure 18 wk (x 2080).

Competition experiments performed with unlabeled virion 60-70 S RNA reduced the labeling over 78Al nuclei by 70-75s (Table l), providing evidence for the specificity of in situ hybridization.

When viral 60-70 S [3H]RNA was hybridized with 78Al cells, an average of only 13 grains was observed over 78Al nuclei after 19 wk of exposure (Fig. 2, Table 1). The lower number of grains per nucleus as

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compared with the [3H]DNA probe reflect the lower specific activity of 60-70 S [3H]RNA and the slower rate of RNADNA hybridization. With [3H]RNA as probe, the number of grains over uninfected F-1853 nuclei was the same as background, and appeared to be randomly distributed. In Situ Hybridization of H-MSV(MLVjTransformed Mouse Cells (MEH) with H-MSV(MLVJ [3H]DNA An average of 26 grains per nucleus was detected when viral [3H]DNA was annealed with MEH cells (Fig. 3 and Table 2); background was 1 grain per nucleus. Uninfected mouse cells (NIH/3T3 and NIH/3T6, clone 91) contained an average of 1 and 3 grains above background. In competition experiments, unlabeled viral 60-70 S RNA reduced the autoradiographic grain count by 80%, while MEH cell DNA reduced the grain count to background levels. Human KB cell DNA at the same concentration as MEH cell DNA did not significantly reduce grain counts. In several experiments in which cytological preparations were pretreated with DNase (200 pug/ml of Worthington Corp., RNase-free DNase) in 10 mM TriseHCl (pH 7.3), 1 mM MgCl,, for 2 hr at-37”, grain counts were reduced to background levels. Localization quences

of

MSV(MLV)

DNA

Se-

Microscopic examination of gutoradiographs reveals that most nuclei in transformed rat and mouse cells were labeled. The labeling in MEH mouse cells was mostly associated with the dense Giemsastaining areas, the chromocenters (Fig. 3). The pattern of distribution over chromosomes showed that the centromeric heterochromatin regions were often labeled (Loni and Green, unpublished data), but grains were not restricted to this region. DISCUSSION

The viral specificity of these in situ hybridization measurements was verified by (1) the reduction in grain count upon competition with viral 60-70 S RNA, (2) the total eradication of grains upon compe-

43

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TABLE 1 MEAN GRAIN COUNT PER NUCLEUS OF M-MSV(MLV)TRANSFORMED RAT CELLY (78Al) AND HMSV(MLV)-TRANSFORMED MOUSE CELLS (MEH) HYBRIDIZED WITH M-MSV(MLV) [sH]DNA or 6070 S [SH]RNA No. of nuclei scored

l. M-MSV(MLV) [3H]DNA annealed with M-MSV transformed rat cells (78Al)

Background” Normal rat cells (F-1853) Background” Competition with unlabeled homologous viral W-70 S RNA (about 1 Pd Background” H-MSV transformed mouse cells (MEH) Background” 2. M-MSV(MLV) 60-70 S [3H]RNA annealed with M-MSV transformed rat cells (78Al)

Normal rat cells (F-1853)

Grain density per nucleus (mean f SEM)”

40

31 * 1.41

40 40 139 80 80 150

30 * 1.45 27 * 1.48 1 * 0.09 2 zt 0.16 3 * 0.20 1 i 0.08

80

11 i 0.42

150

2 * 0.11

40

25 * 1.40

130

1 * 0.11

40

13 * 0.83

40

13 * 0.07

110

1 f 0.10

GStandard error of the mean. h Comparable area on slide with no cellular tures.

struc-

tition with DNA from MSV-transformed mouse MEH cells, but not with DNA from human KB cells, and (3) the absence of grains when fixed cell preparations were treated with DNase prior to in situ hybridization. A rough estimate of the number of intracellular foci of viral DNA sequences detected in transformed cells by in situ by!bridization cau be made as follows. Based on a specific activity of [3H]DNA of 8.0 x lo7 dpmlpg, an 18-wk exposure should give 2.41 grains/78Al cell for each duplex viral

44

LONI AND GREEN

DNA genome of 20 x 106, assuming 10% efficiency for hybridization and a 10% efficiency autoradiographic detection (Gall and Pardue, 1970; Perry, 1964). For MEH cells exposed for 16 wk, 2.14 grains/cells were calculated. Dividing the mean grain density per nucleus (Table 1) by these values, 11.2 and 10.7 viral DNA copies per 78Al and MEH cell were calculated. Normal rat and mouse cells were estimated to possess approximately 1-2 copies per cell. It is obvious that these values are only order-of-magnitude estimates since the efficiencies of hybridization and autoradiographic detection are only approximations, and furthermore the size and arrangement TABLE 2 MEAN GRAINCOUNTPERNUCLEUSOF H-MSV(MLV)-TRANSFORMED MOUSE CELLS (MEH) ANNEALED WITH H-MSV(MLV) 13H]DNA”

(3H]DNAannealed with

H-MSV-transformed mouse cells H-MSV-transformed mouse cells Mouse cells (NIH/3T3] Mouse cells (NIH/3T6, clone 911 Competition with homologous viral 60-70 S RNA (about 1 pg per slide) Competition with homologous cell DNA (300 pg per slide)

Grain No. of nuclei density per nucleus (mean i SEMI

40 40 120 80 80 80

26 z+z1.46 26* 1.16 2 * 0.15 4 f 0.19 5 zt 0.20 6 f 0.27

145 140


a See footnote to Table 1 for experimental details. A background value of 1 grain per nucleus was not subtracted.

of viral DNA sequences within the chromosome is unknown. But it is interesting that these estimates of genome copies are of the same order of magnitude as those measured by reassociation kinetics (Varmus et al., 1972; Gelb et al., 1971). The grain density per nucleus of 78Al and MEH cells were compared by an analysis of variance. The comparison involved three groups of data: (1) the transformed rat cells (78Al) annealed with the M-MSV(MLV) [3H]DNA (Table l), (2) the transformed mouse cells (MEH) annealed with the M-MSV(MLV) [3H]DNA (Table l), and (3) the transformed mouse cells (MEH) annealed with the H-MSV(MLV) [‘H]DNA (Table 2). The grain density from the three groups was analyzed to detect differences that might be attributable to virus strains [(M-MSV(MLV) and H-MSV(MLV)] and to cell lines (rat versus murine cells). The results presented in Table 3 indicate that (i) the cell lines have significantly different grain densities and that (ii) the two virus strains do not differ from each other in the extent of hybridization with the same cell type. On the basis of previous observations (Green et al., 1971), a large extent. of homology was expected between the two strains of MSV(MLV). The differences in mean grain count found between the two cell lines might be related to the chromosome make up of the cells studied. The 78Al is an heteroploid cell line with a modal chromosome number in the range of

Fro. 2. Autoradiographs of nuclei from M-MSV-transformed rat cells (a) and normal rat (F-18531 cells (b). In situ hybridization was performed with viral 60-70 S [SH]RNA. Exposure 19 wk (x2080).

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FIG. 3. Autoradiographs of nuclei from H-MSV-transformed mouse MEH cells (a, b) and normal mouse (NIW3T3) cells (cl. H-MSV(MLV) [3H]DNA (8 x lo7 fpmlpg) was annealed with fixed preparations of transformed and normal mouse cells for 18 hr. The association of silver grain with the dense Giemsa-staining series is noted. Exposure, 16 wk (x 20801.

TABLE

3

ANALYSIS OF VARIANCE OF THE GRAIN DENSW PER NUCLEUS OF M-MSV(MLV)-TRANSFORMED RAT CELLS (78Al) AND H-MSV(MLV)-TRANSFORMED MOUSE CEU~ WEH] Source of variation

Among groups Among mouse cells Rat cells vs mouse cells Within groups I

Degrees of freedom

Mean square

F

2 1 1 237

347.64 35.27 660.01 78.81

4.41 0.45’ 8.37’ -

I

I

a The mean grain counts are from Tables 1 and 2. * Nonsignificant at P = 0.05. ( Significant at P < 0.01.

63-66, while most MEH cells have the normal diploid chromosome number (Loni and Green, unpublished data). If the virusspecific DNA sequences were associated with particular sites of insertion, or groups of chromosomes, the change in the balance of chromosomes carrying those sequences could result in differences between the number of grains detected. The 78Al and MEH cells are transformed by two different strains of virus and continuously produce relatively large quantities of MSV(MLV). The viral DNA sequences detected by in situ hybridization are probably responsible for the stable inherited viral genome that is template for

46

LONI

AND

progeny viral RNA synthesis and codes for the viral information associated wibh the transformed properties of the cell. The large increase in grain density in virus-producing transformed cells, as compared to uninfected mouse and rat cells, indicates that MSV(MLV) infection and cell transformation results in a large increase in the content of virus-specific DNA sequences in the cell nucleus. The possibility that the autoradiographic grains are due to hybridization with virus-specific RNA is excluded by the conditions of hybridization, the eradication of grains by pretreatment of cells with DNase, and the similar results obtained when viral 60-70 S RNA was used as probe (Table 1). Kinetic measurements of the reassociation of the duplex DNA product of the viral RNA-directed DNA polymerase in the presence of DNA from normal and virustransformed murine and avian cells have led to estimates of from 12 to 50 viral DNA equivalents per cell, the same copy number was found in normal and transformed cells (Varmus et al., 1972; Gelb et al., 1971). Our results by cytological hybridization with a single-stranded DNA probe indicate that MSV-transformed mouse and rat cells contain a several-fold higher content of some. virus-specific DNA sequences than do uninfected mouse and rat cells. The discrepancy between the results of in situ hybridization reported here and results of reassociation measurements with the duplex viral DNA probe cited above could possibly be due to (1) the absence from the duplex DNA probe of a portion of the viral genome that is represented with a higher frequency in transformed cells and/or (2) nonuniform representation of viral gene sequences in the duplex DNA probe which could lead to errors in estimating copy number by the standard treatment of reassociation kinetic data (Fujinaga, Sekikawa, Yamazaki and Green, unpublished data). The possibility that in situ hybridization ‘is detecting cell-specific DNA sequences that are amplified in transformed cells is unlikely since a similar large increase in grain count was obtained with viral 60-70 S [SH]RNA as the cytological probe. In addition, molecular hybridization

GREEN

studies using viral 60-70 S RNA as a probe have found a several-fold increase in viral DNA content in cells infected with avian oncornaviruses (Baluda, 1972; Baluda and Drahon, 1973; Nieman, 1972; Nieman, 1973). It is likely that the DNA sequences present in normal mouse and chicken cells which are detected by molecular hybridization measurements cited above and by the in situ hybridization measurements reported here represent DNA sequences shared with the endogenous viral genome present in murine and avian cells (Huebner and Todaro, 1969). The extent to which the increased viral DNA content of transformed cells, as detected by in situ hybridization, represents entirely new viral DNA sequences or sequences shared with the endogenous virus, is not known. ACKNOWLEDGMENTS We thank Dr. David Schlessinger for criticizing the manuscript. This work was supported by a Contract NO1 CP 43359 within the Virus-Cancer Program of the National Cancer Institute. MG is a recipient of a Research Career Award from the National Institutes of Health (5K6-AI-4739). MCL was supported by funds provided the University of Louvain, Belgium.

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