virus type 2*

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(recombinant plasmids/Syrian hamster embryo cells/continuous passage assay/escape from .... ose (Baltimore Biological Laboratory) was assayed by suspend-.
Proc. Nati. Acad. Sci. USA Vol. 80, pp. 5902-5906, October 1983

Cell Biology

Immortalization and neoplastic transformation of normal diploid cells by defined cloned DNA fragments of herpes simplex virus type 2* (recombinant plasmids/Syrian hamster embryo cells/continuous passage assay/escape from senescence/tumorigenic conversion)

RAXIT J. JARIWALLAt, LAURE AURELIANt§,

AND

PAUL 0. P. Ts'ot

tLaboratory of Molecular Carcinogenesis, Armand Hammer Cancer Research Center of the Linus Pauling Institute of Science and Medicine, 440 Page Mill Road, Palo Alto, California 94306; and Divisions of fBiophysics and §Comparative Medicine, The Johns Hopkins University, Baltimore, Maryland 21205 Communicated by Linus Pauling, June 23, 1983

ABSTRACT Diploid Syrian hamster embryo (SHE) cells were passaged after transfection with recombinant plasmids containing herpes simplex virus type 2 (HSV-2) DNA inserts Bgl II focusforming fragment N, Bgl II transforming fragment C, and EcoRI/ HindIll fragment AE. Cultures transfected with salmon DNA or with 0.1-5.0 ,ug of Bgl HI fragment N reached crisis and senesced. Those transfected with 0.1-0.5 ,ug of Bgl H fragment C or its lefthand 64% subclone EcoRI/HindIll fragment AE escaped senescence and formed continuous lines. At early passages, these lines as well as isolated clones grew in 2% serum but formed small (0. 1 mm) colonies in 0.3% agarose and were nontumorigenic. Serial passaging of Bgl II fragment C-induced cultures and isolated clones resulted in the appearance of large (>0.25 mm) colonies in agarose followed by tumorigenicity. This behavior was not exhibited by the EcoRI/HindIH fragment AE-induced cultures that remained nontumorigenic after 53 passages. DNA from normal SHE cells exhibited homology to Bgl HI fragment C but, under relatively stringent conditions, DNAs from transformed and tumor-derived lines exhibited discrete hybridizing -bands comigrating with authentic viral fragments. These results indicate that neoplastic transformation of normal diploid SHE cells by HSV-2 DNA fragments involves at least two distinct steps-i.e., immortalization and conversion to tumorigenicity. EcoRI/Hindlll fragment AE representing the left 64% of Bgl II fragment C is sufficient to induce immortalization. However, DNA sequences from both lefthand 64% and right-hand 36% subfragments of Bgl HI fragment C are required for tumorigenic transformation.

Inactivated herpes simplex virus type 2 (HSV-2) (1-4), herpes simplex virus type 1 (HSV-1) (5), and native, uncleaved HSV2 DNA (6) induce the neoplastic transformation of normal diploid mammalian cells in culture. Transfection with isolated restriction-enzyme fragments of viral DNA has identified one set of transforming sequences in HSV-1 DNA (7). On the other hand, two distinct transforming regions were identified in HSV2 DNA (8-10). One of these, the Bgl II fragment C, was found to transform normal diploid Syrian hamster embryo (SHE) cells in the continuous passage assay (8). The other (Bgl II fragment N) was identified on established, heteroploid murine cell lines and Wistar rat cells by a direct one-step assay [focus-formation in liquid medium (9, 10) or colony-formation in methocel

(10)].

Our studies were designed to determine whether HSV-2 DNA sequences that affect initial events in the transformation process-namely, escape from senescence (immortalization)-are identical to those that induce tumorigenic potential. Accordingly, we selected to study SHE cells that, unlike the estab-

lished cell lines, exhibit a normal diploid karyotype and have a defined life-span in vitro. They were transfected with defined cloned viral DNA fragments and continuously passaged as described (4, 6, 8). The findings described in this report indicate that distinct fragments of HSV-2 DNA affect different stages in the multistep process of neoplastic transformation of normal diploid SHE cells. MATERIALS AND METHODS Cells. SHE cells, prepared from 11-day embryos, were grown in ERM medium/10% fetal bovine serum as described (6). They were used for transformation studies at passage 3-6. Recombinant Plasmid DNAs. Plasmids derived from pBR322 that contain fragments of HSV-2 DNA in the 0.419-0.625 region (Fig. 1) were obtained from Gary Hayward (The Johns Hopkins School of Medicine). They were propagated in Escherichia coli HB101. Their properties have been described by Peden et al. (11). Plasmid DNA was purified from clear lysates by centrifugation in cesium chloride/ethidium bromide density gradients (12). Restriction Enzyme Digestion and Gel Electrophoresis. DNAs were digested with the restriction enzymes HindIII or BamHI (New England BioLabs) in buffer recommended by the manufacturer at 370C for 3 hr. Cleaved DNAs were analyzed by electrophoresis on horizontal slab gels of 0.6% agarose in Tris acetate buffer (10 mM Tris, pH 7.8/20 mM sodium acetate/2 mM EDTA) containing 0.5 jig of ethidium bromide per ml at 40 V for 22 hr. The DNA was visualized with mid-wave (300 mm) UV light and photographed. Establishment of Immortal Lines. Cleaved plasmid DNAs were heated at 650C for 10 min to inactivate restriction enzyme, mixed with 10 ,ug of salmon sperm DNA per ml, and precipitated with CaCl2 (13) as described (6). Aliquots (0.5 ml) of the precipitate were applied to subconfluent monolayers of SHE cells (5 x 105 cells per 60-mm dish). After 8 hr at 370C, cells were refed growth medium (ERM medium/10% fetal bovine serum) and, at confluency, were trypsinized and seeded at 1:3 split ratio. Thereafter they were serially passaged (at confluency) at 1:5 or 1:10 split ratios. Cultures that developed refractile, rapidly proliferating cells were propagated at higher (1:25, 1:50) split ratios. Those entering crisis were refluided until recovery, at which time they were further subcultured and grown into cell lines. Cultures that senesced were discarded. Cell lines SDNA-578 (14) and SDNA-CD (8) established by this Abbreviations: HSV-2, herpes simplex virus type 2; HSV-1, herpes simplex virus type 1; CE, cloning efficiency; PTP, post-treatment passage(s); PIsP; post-isolation passage(s); m.u., map unit; kb, kilobase(s). * A preliminary report of these results was presented at the Sixth Meeting on Herpesviruses, Cold Spring Harbor, NY, September 1982.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5902

Cell Biology:

jariwalla et al.

Proc. Natl. Acad. Sci. USA 80 (1983)

protocol by using mechanically sheared HSV-2 DNA and the Bgl II/Hpa I CD viral fragment, respectively, were used as controls in the study of clonal derivatives. Phenotypic Alterations Associated with Transformation. Cloning efficiency (CE) in 2% serum was determined by seeding 100-500 cells per 60-mm dish as described (6). CE in agarose (Baltimore Biological Laboratory) was assayed by suspending cells in 0.3% agarose/0. 1% peptone on top of a basal layer of 0.6% agarose/0.2% peptone in ERM medium/10% fetal bovine

serum.

Colonies

were

counted at 3 wk and scored

ac-

cording to their size by using a calibrated microscope eyepiece. Colony size was: small (S), if it was sO. 1 mm; medium (M), if it was '0.2 mm; and large (L), if it was >0.25 mm. CE was expressed as the [no. of colonies (all sizes) 100]/no. of seeded cells. Tumorigenic potential was assayed in newborn (1-3-dayold) Syrian hamsters injected subcutaneously with 2 x 106 or fewer cells as specified. Hybridization Analysis. Cellular DNA was extracted from nuclei by lysing nuclear pellet with 0.1% NaDodSO4, followed by sequential digestions at 370C with RNase A (40 Ag/ml) for 1.5 hr and proteinase K (50 ,ug/ml) for 5 hr. DNA was extracted once with phenol/CHCl3/isoamyl alcohol and dialyzed extensively against 10 mM Tris.HCl/1 mM EDTA, pH 8.0. Cellular DNAs were cleaved with BamHI, electrophoresed through 0.6% agarose gels, denatured, and transferred to nitrocellulose filters as described by Southern (15). Filters were prehybridized in 0.6 M NaCl/0.06 M sodium citrate (Cit)/2.5x concentrated Denhardt's solution (16)/0.05 M NaPi, pH 7.1, at 60'C for 4.5 hr. Hybridization was carried out at 65°C for 14.5 hr in 0.6 M NaClI/0.06 M Cit/5x concentrated Denhardt's solution/0.05 M NaPO4, pH 7.1/10% dextran sulfate containing 32P-labeled BamHI-cleaved pGR140 (3 108 cpm in 15 ml of solution). After hybridization, filters were washed at 60°C twice in 0.3 M NaCl/0.03 M Cit and once each in 0.15 M NaCl/0.015 M Cit and 75 mM NaCl/7.5 mM Cit; filters were washed at 65°C once in 37.5 mM NaCl/3.75 mM Cit and twice in 15 mM NaCl/1.5 mM Cit. Filters were air-dried and exposed to Kodak XAR-5 film with intensifying screens at -70°C. X

X

RESULTS Immortality and Transformation-Associated Phenotypes. Previous studies from our laboratories described the establishment of rapidly proliferating, immortal cell lines after transfection of diploid SHE cells with native (6) or sheared (14) HSV2 DNA or the specific Bgl II/Hpa I fragment CD obtained by agarose gel fractionation of HSV-2 DNA (8). In all cases, the newly established, immortal lines were not tumorigenic. Acquisition of the tumorigenic potential required further in vitro passaging. These findings are consistent with the interpretation that the acquisition of neoplastic potential is a multistep process (17) in which immortalization represents a relatively early stage. To identify other phenotypic characteristics that might correlate better with tumorigenic potential, clones were isolated by low density seeding (100 cells per 60-mm dish) from the SDNA-CD and SDNA-578 mass cultures established with the Bgl II/Hpa I fragment CD (8) and total sheared HSV-2 DNA (14), respectively. They were examined for phenotypic alterations and tumorigenicity at increasing post-isolation passages

(PIsP) (Table 1). At PIsP 5-7, the three SDNA-CD clonal derivatives studied in these series exhibited different CEs in 0.3% agarose. Two of them (clone 1 and clone 3) had relatively high CEs; however, nontumorigenic. Serial passaging resulted in an inin their CEs (as compared to PIsP 5-7) but this increase did not appear to correlate with tumorigenic potential. On the all

were

crease

5903

Table 1. Progressive phenotypic changes with passage in clonal derivatives of cells transformed by fragments of HSV-2 DNA

Cloning Clonal line PIsP PDL* SDNA-CD 5-6 50-55 Clone lt 10-14 73-90

in agarose %CE Sizet 27.8 S, M 46.9 M, L

Tumorigenicity Inoculum Incidence 2 x 106 0/10 2 x 106 9/11

SDNA-CD Clone 2t

3.2 S 4.1 S 18.4 S, M

1 x 106 ND 2 x 106

6-7 55-60 14 80-85 17-21 90-105

0/4 ND 0/10

2 x 106 S, M S, M, L 2 x 106 M, L ND L 2 x 106

ND 10/10

145-150

2.6 M, L 4.2 L 12.0 L

2 x 106 1 x 104 ND

7/10 4/4 ND

SDNA-578 5-6 85-90 Clone 2§ 9-12 105-120 20-22 145-155

4.2 S 4.0 S, M 12.9 L

ND 1 x 105 1 x 105

ND 0/4 3/3

SDNA-578 5-6 85-95 Clone 3 13-23 115-160

3.08 M, L 3.7 M, L

2 x 106 2 x 106

5/8 3/3

SDNA-CD Clone 3t

5-6 10 14 20

50-55 70-75 90-95 110-115

SDNA-578 5-7 85-95 Clone 1 10-13 110-125 20

18.0 25.4 38.4 ND

0/5 1/5

3.9 S, M, L 2 x 106 SDNA-578 5 85 2/6 Clone 4§ 8-10 100-110 12.0 S, M, L 1 x 105 0/4 2 x 106 22-24 155-165 >15.0 L 4/4 ND, not done. * PDL, estimated number of post-treatment population doublings. tDefinition of the colony size as based on the diameter: small (S), -0.1 mm; medium (M), c0.2 mm; large (L), >0.25 mm. tClones were isolated from 60-mm dishes seeded with 100 SDNA-CD cells at post-treatment passage (PTP) 8 in ERM medium/10% fetal bovine serum. §Clones were isolated from 60-mm dishes seeded with 100 SDNA- 578 cells at PTP 17 in ERM medium/10% fetal bovine serum.

other hand, tumorigenic potential appeared to correlate well with the size of the agarose colonies. Thus, at PIsP 10-14, clone 1 exhibited a mixture of medium and large size colonies and was tumorigenic (incidence, 9/11), whereas clone 2 at PIsP 1721 formed only small and medium size colonies and was not tumorigenic (incidence, 0/10). At PIsP 10, clone 3 gave rise to a mixture of small, medium, and large colonies and it was poorly tumorigenic (incidence, 1/5), whereas by PIsP 20, it formed only large colonies and was highly tumorigenic (incidence, 10/ 10). The four SDNA-578 clones were isolated from the parental mass culture SDNA-578 at a later passage (PTP 17) compared to SDNA-CD clones, which were isolated from SDNA-CD at PTP 8. Therefore, some of the SDNA-578 clones were tumorigenic at earlier PIsP compared to SDNA-CD clones. Again, tumorigenicity correlated with colony size rather than CE in agarose. Thus, at PIsP 5-7, all SDNA-578 clones had similar CEs in 0.3% agarose but they differed in colony size and tumorigenic potential. Clones 1, 3, and 4 gave rise to large colonies and were tumorigenic. Clone 2 was not tumorigenic and it did not form large colonies in 0.3% agarose. Establishment of Immortal Lines with Cloned HSV-2 DNA Sequences. These studies were designed to determine whether distinct HSV-2 DNA fragments differ in their ability to immortalize normal diploid cells or to confer any of the other transformation-specific parameters (namely, cloning and colony

Cell Biology: Jariwalla et al.

5904

Proc. Natl. Acad. Sci. USA 80 (1983)

size in 0.3% agarose or tumorigenic potential). To this end, plasmid pBR322 derivatives pGR75, pGR140, and pGR62 (Fig. 1) containing HSV-2 (333) DNA inserts EcoRI/HindIII AE [0.419-0.525 map unit (m.u.)], Bgl II C (0.416-0.582 m.u.), and Bgl II N (0.582-0.625 m.u.), respectively, were first examined for their ability to induce continuously proliferating lines in the serial passage assay. Prior to transfection, DNAs were linearized by HindIII cleavage, based on a previous finding indicating that this treatment does not inactivate the transforming potential of HSV-2 DNA (¶). HindIll cuts once within pBR322 sequences but it does not cleave within the HSV-2 EcoRI! Hind1II AE fragment nor within the Bgl II N fragment (Fig. 1). This enzyme cuts once within HSV-2 Bgl II C (at 0.525 m. u.) to yield a left-hand 64% (0.416-0.525 m.u.) and a right-hand 36% (0.525-0.582 m.u.) subfragment (Fig. 1). Linearized DNAs were coprecipitated with CaCl2 in the presence of salmon sperm DNA and applied (0.1-5.0 Ag of DNA per dish) to subconfluent SHE cells (P6). Cultures (three per experimental group) were serially passaged at confluency. All cultures transfected with salmon DNA or with 0.1 ,ug of ¶Jariwalla, R. J. (1981) International Workshop on Herpesviruses, July 29-August 2, 1981, Bologna, Italy, p. 226 (abstr.).

X

NAN

i

cc

.Q,q'O 23.7

-

9.5 6.6 -

- 8.5

5.5 4.8

4.3 -

- 3.9

2.5

2.1 1.9 -

PGR 75

-

2.4

-

1.9

PGR 62

IPG R 140 He J

0

Y

J

E

T

A

0

0.1 I

0.2

0.3

0.5

0.

.I

1

0.6

0.7

Uam7 HI

ISq/Il

N

0.8

0.9

I

L

MAP UNIT

FIG. 1. (Lower) Physical map for the BamHI and Bgi II fragments within the 0.419-0.625 region of HSV-2 DNA. Plasmid pBR322 derivatives containing different inserts of HSV-2 DNA used for transfection are shown in brackets. (Upper) Ethidium bromide-stained gel of 0.6% agarose showing the restriction enzyme cleavage pattern of the recombinant plasmids digested with the indicated enzymes. HindilI-cleaved A DNA was used as a marker. Molecular sizes are indicated in kilobases.

pGR62 (Bgl II N) senesced at PTP 8-9. Those transfected with 1.0 pig or 5.0 ,ug of pGR62 continued to propagate past this stage, but their growth rate decreased with passage and they senesced at PTP 15-16. In contrast, cultures transfected with 0.1-0.5 ug of linearized pGR75 (EcoRI/HindIII AE) and HindIII-cleaved pGR140 (mixture of left 64% and right 36% subfragments of Bgl II C) developed colonies of refractile, rapidly dividing cells that overgrew the cultures within two to four further passages and formed continuous lines. Two cell lines, designated AEO1H and AE05H, established from cultures transfected with 0.1 pg and 0.5 Ag, respectively, of cloned EcoRI/HindIII fragment AE, exhibited moderate refractility and consisted predominantly of shortened fibroblasts (Table 2). One cell line, COSH, established after transfection with 0.5 Ag of cloned Bgl II fragment C, was very refractile, consisting of a mixture of small stellate and spindle-shaped cells (Table 2). Phenotypic Characteristics of Lines Established with Cloned Viral DNA Fragments. Cultures AE0lH, AE05H, COSH, and those transfected with 1.0 and 5.0 pg of cloned Bgl II fragment N (N1H and NSH, respectively) were examined for transformation-associated phenotypic alterations as a function of in vitro passage (Table 2). At PTP 5-7, all cultures formed colonies in 2% serum (1.2-30% CE) and in 0.3% agarose (2.9-11.8% CE). The agarose colonies were predominantly small and the cells were not tumorigenic. However, upon further passage, lines established with the various DNA fragments started to exhibit differences in phenotypic characteristics. Thus, the CE in 0.3% agarose of the N1H and N5H lines appeared to decrease with passage (90% lower at PTP 10-14 than at PTP 5) and at PTP 10-14 they formed only small colonies. Both cultures senesced at PTP 15-16. In contrast, the CO5H culture retained its cloning efficiency in 0.3% agarose and exhibited a progression towards the large size agarose colonies. CO5H formed a mixture of medium and large size colonies at PTP 13 and only large colonies at PTP 29. The cells were tumorigenic in 100% of injected animals (2 x 106 cells) within 5 wk of inoculation of the PTP 29 cells (7/7 animals) and within 23 wk of inoculation of the PTP 13 cells (7/7 animals). The AEO1H and AEO5H lines did not exhibit a similar increase in the size of the agarose colonies during continuous passage and the cells remained nontumorigenic as late as PTP 53. Normal SHE cells did not exhibit any of these phenotypic alterations and senesced at PTP 9. Viral DNA in Transformed Cells. DNAs extracted from transformed lines, tumor derivatives, and normal SHE cells were digested with BamHI, electrophoresed through a 0.6% agarose gel, and analyzed by Southern transfer hybridization with a 32p_ labeled BamHI-cleaved pGR140 Bgl II C probe. Hybridization was carried out at 65°C in 0.6 M NaCl/0.06 M Cit, followed by washing in decreasing salt concentrations (0.3 M NaCl/0. 03 M Cit, 0.15 M NaCl/0.015 M Cit, 75 mM NaCl/7.5 mM Cit) at 60°C and 37.5 mM NaCl/3.75 mM Cit and 15 mM NaCl/1.5 mM Cit at 65°C. Under these relatively stringent conditions, hybridization of the probe to BamHI-cleaved pGR140 revealed five hybridizing bands (Fig. 2, lane 1), consistent with expected products (Fig. 1) and published maps for BamHI cleavage of HSV-2 (18). Two other expected BamHI fragments [20 5-9 13-29

pGR62

NlHI

5 12-14 5 10-14 3-5tt

N5HII

ND 30 39 3.2 5.4 1.2 2