lymphoma virus type-I integration in cultured lymphoma cells

VIROLOGY

154,67-‘75 (1986)

Dynamic and Nonspecific Dispersat of Human T-Cell Leukemia/Lymphoma Virus Type-t tntegration in Cultured Lymphoma Cells LEONARD J. SEIGEL,* WILLIAM G. NASH,? BERNARD J. POIESZ,§ JANET L. MOORE& AND STEPHEN J. O’BRIENpl *Laboratory of Tumor CeUBiology, National Cancer Institute, National Institutes of Health, Bethesda, iUarp!and 20205;~Section of Genetics, National Cancer Institute, Frederick, Maryland 21701;$De-partments of Medicine and Microbiology, State University of New York, Upstate Medical Center, Symcuse; anal 8Veterans Administration Medical Center, Syracuse, New York l.%?lO Received February 3, 1986;accepted May 19, 1986 The progression of HTLV-I proviral integration over a I-year period of in vitro culture was examined in two human lymphoma lines, Hut 102 and MJ. Using specific HTLV-I molecular clones and a Southern analysis at different cell passages, Hut 102 increased from 2 to 19 integrated proviral integrations while MJ increased to at least 25 different integrations by passage 43. During the progress of increased superinfection and novel integration in vitro some of the previous proviral integrations were lost from the cultures. The 19 integrations of late passage Hut 102 cells were shown to be dispersed to 19 different human chromosomes by analysis of 34 distinct rodent X Hut 102 somatic cell hybrids which segregated human chromosomes (and included proviral integrations) in different combinations. The two primary integrations in Hut 102 were located on human chromosomes 4 and 20, respectively. A similar pattern of nonspecific integration was observed in somatic cell hybrid analysis of the 25 proviral integrations of MJ. The dynamic infectionreintegration process in vitro revealed in these studies may confuse experimental verification of potential cis acting functions of HTLV-I in the as yet poorly understood mechanism 8 1996 Academic Press. IX. of neoplastic transformation. INTRODUCTION

(Saxinger et d, 1984). HTLV-I is strongly implicated in the etiology of a distinct Human T-cell leukemia/lymphoma virus clinical syndrome, adult T-cell lymphoma, type I (HTLV-I), was first isolated from mature lymphocytes cultured from a pa- ATL (Gallo et cd, 1983). Various isolates of HTLV-I cause rapid tient with acute T-celI leukemia (ATL) transformation of T lymphocytes in vitro (Poiesz et al, 1980). Comparative protein (Miyoshi et al, 1981; Popovic et cd., analysis and nucleotide sequence determination have revealed that HTLV-I is 1983a, 1983b) but the mechanism by which unique from all previously described HTLV-I induces neoplastic transformation in viva and in vitro is unknown. HTLV-I mammalian retroviruqes, but is distantly related to bovine leukemia virus (BLV) does not contain any oncogene sequences (Kalyanaraman et a& 1981; Manzari et aL, which are recognizable in uninfected hu1983; Seiki et al, 1983). Subsequent man DNA (Seiki et a& 1983) and similar HTLV-I isolates have been reported to chronically transforming viruses (like worldwide (Popovic et al, 1983b) but the feline leukemia virus or Moloney murine virus appears to be endemic in certain leukemia virus), HTLV-I is replication areas, such as southern Japan (Robert- competent and is clonally integrated in tuGuroff et a& 1982), the Caribbean basin mors (Manzari et uL, 1983; Wong-Staal et uL, 1983). The genome of HTLV-I contains (Blattner et c& 1982), and Central Africa certain open reading frames whose tran‘To whom requests for reprints should be adscription may participate in the transfordressed. mation process (Seiki et uL, 1983; Goh et 67

0042-6822/86 $3.00 Copyright All righta

@ 1986 by Academic Press. Inc. of reproduction in any form reserved.

68

SEIGEL

a& 1985;Lee et a.& 1984; Slamon et al, 1984, 1985). A second possibility is that HTLVI may induce transformation through an insertional mutagenesis mechanism, as has been demonstrated for some chronic transforming retroviruses (Neel et al, 1981; Varmus et aL, 1981). In order to study HTLV-I viral progression directly, we followed the appearance and persistence of integrated proviruses in a prototype human cell line, Hut 102, infected with HTLV-I over an extended period of tissue culture passages in vitro. Two diploid HTLV-I-infected human tumor lines (Hut 102 and MJ) were fused to rodent cells and the derived panels of somatic cell hybrids were examined for integration in various human chromosomes. The parental cell lines were characterized by multiple nonspecific integrations which were apparent by Southern analysis, as well as by multiple low level integrations which were heterogeneous and dynamic in the process of cell proliferation. MATERIALS

AND

METHODS

Cell lines and construction and characterization of somatic cell hgbricls. E36 is a hy-

poxanthine phosphoribosyl transferase deficient (HPRT-) Chinese hamster fibroblast cell line (Gillin et a& 1972). RAG is an HPRT- mouse fibroblast line (Klebe et aL, 1970). Hut 102 is an HTLV-I-infected cell line isolated from a patient with ATL (Poiesz et al, 1980). Hut 102 B2 is a clonal derivative of Hut 102 derived at passage 102. MJ is an HTLV-I-infected cell line derived by T-cell growth factor (TCGF) supplementation of peripheral blood obtained from a patient with a chronic cutaneous Tcell lymphoma (Popovic et d, 1983b). Two different panels of somatic cell hybrids were utilized in these studies. A panel of 35 Hut 102 B2 X rodent hybrid crosses was constructed by fusion of Hut 102 B2 passage 80 to either E36 (18 hybrids) or RAG (17 hybrids). Hybrids that retain the entire rodent genome but segregate different combinations of human chromosomes were selected in HAT (Littlefield, 1964) media supplemented with low5 M ouabain (Baker et a& 1974). One hundred ninety

ET AL.

primary hybrids were screened for their content of human chromosomes by electrophoretic resolution of 36 isoenzyme markers that have been previously assigned to specific human chromosomes (Harris and Hopkinson, 1976;O’Brien et al, 1982;Shows and MacAlpine, 1982). Hybrids that retained small numbers of human chromosomes but that gave strong isoenzyme signals for those chromosomes present, were selected for the panel. Panel hybrids were further characterized genetically by repeat isoenzyme analysis, G-11 chromosome staining (Bobrow and Cross, 1974), and in most instances by Giemsatrypsin banding (Lemons et al, 1978; O’Brien and Nash, 1982). G-11 chromosome staining permits the detection of the interspecific chromosomal rearrangements. Certain hybrid cell lines were subcloned by seeding 50-5000 cells per loo-mm plate in media containing either HAT or 6-thioguanine and picking individual colonies with cloning cylinders. A second panel of 30 MJ and rodent hybrid crosses was constructed by fusion of the cell line MJ passage 30 to either E36 (14 hybrids) or RAG (16 hybrids). Two hundred primary hybrids were selected in HAT media. Panel hybrids were characterized genetically by isoenzyme analysis and G-11 chromosome staining. Southern anal~ms. Two different molecular clones of HTLV-I were employed in these studies (Fig. 1); pCRQL1, a 0.55-kb 3’ LTR clone (Josephs et al, 1984) and pCH, a 2.3-kb clone which contains a portion of the viral env and x genes (Wong-Staal et al, 1983). Restriction endonuclease-digested genomic DNA was electrophoresed in 0.7% agarose gels, transferred to nitrocellulose filters,and hybridizd to a 32P-labeled, nick-translated probe in a solution containing 50% deionized formamide, 0.01% bovine serum albumin, 0.01% Ficoll, and 0.01% polyvinylpyrrolidine, 3X SSC (1X SSC is 0.15 M sodium chloride + 0.015 N sodium citrate), 20 mM sodium phosphate buffer (pH 6.5), and 10% dextran sulfate. Filters were hybridized at 42’ for 2448 hr, rinsed in 2X SSC for 60 min in three changes of 0.2X SSC, 0.1% SDS at 50”, rinsed in 0.2~ SSC, and exposed to X-ray

HTLV-I

INTEGRATION

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ANALYSIS

69

Southern analysis (Southern, 1975) of these digestions using specific subclones of HTLV-I showed an increase in prominent integrated proviral fragments with tissue RESULTS culture passage and upon cell cloning. The Genomic DNA was extracted from four earliest passage (p 13, viably frozen 8 weeks passage levels of HTLV-I-infected Hut 102 after the cells were placed in culture) discells and digested with EcoRI, which does played two major bands at 19.8 (visualized not cleave within the provirus (Fig. 1). A with both the LTR and env-pX probes) and 5.1 kb (visualized with the LTR probe only) and two less intense fragments at 14.0 and 9.0 kb. In addition, a blurred background was apparent in the lanes of each passage. The four early fragments persisted in later passages, but several new fragments were detected in later passages. Hut 102 B2 p 97, which had been in continuous culture for longer than 2 years, contained a minimum of 11 proviruses. Analysis of passage 141 Hut 102 B2 cells, which have been in culture - 23.7 for approximately 3 years, revealed a total 19.8of seven proviral integrations. Six of these were visualized in p 97 Hut 102 B2. One fragment (6.8 kb) appears to be novel, while - 9.7 four fragments (14.0, 13.2, 5.9, 4.8 kb) visualized in passage 97 Hut 102 B2 are no longer detected in passage 141 Hut 102 B2. - 6.8 A similar analysis of the MJ cell line also revealed a dynamic increase in viral integrations with later cell passages having a minimum of 25 integrations at p 43 (Fig. 2). - 4.4 A panel of 34 somatic cell hybrids was prepared by fusion of Hut 102 B2 p 80 with mutant rodent cells as described under Materials and Methods. These hybrid cells retain the entire rodent genome but lose - 2.3 human chromosomes in different combi- 2.0 nations. Genomic DNA from each of these hybrids was examined following EcoRI pCR3Ll PC” digestion using an LTR-specific subclone of HTLV-I as a molecular probe. The results pCRBL1 PCH -511 (Fig. 3) revealed that the 11 original fragments occurred in different combinations in different hybrid cells, suggesting that the proviruses were dispersed on multiple human chromosomes. In addition to the 11 I I I I I I I I I I original fragments evident in the Hut 102 0 12 3 4 5 67 6 9 Wbp) parental cell, eight additional fragments FIG. 1. Southern analysis of different passages of were revealed in the hybrid clones. These Hut 102 and ita clonal derivative Hut 102 B2 with the “novel” proviral bands might represent LTR probe, pCR321, (lane 1) and pal-env probe, pCH new integrations produced by the fusion (lanes 2-5). A simplified restriction map of HTLV-I event itself, but the occurrence of proviral and probes pCH and pCR3Ll is presented. film with an intensifier screen for 7-10 days at -70”.

SEIGEL

-Origln

- 23.7

-6.6

-4.4

-2.3 -2.0

FIG. 2. Southern analysis of MJ passage 43 and representative MJ X RAG hybrids (here labeled 92M) with the LTR probe, pCR3Ll.

bands of identical size in several hybrids suggests that they were present in the parent cell line. However, these low level integrations were apparently heterogeneous and present in only a fraction of the parental cells (unlike the prominent 11 original proviruses) at levels too low to be resolved by Southern analysis. The availability of a hybrid panel segregating 19 different retroviral integrations offered an unusual opportunity for examining the possibility of specific or targeted integration of HTLV-I. Hut 102 B2 is karyologically normal; thus, there are 46 chromosomal options for each integration. If two proviruses are integrated on the same chromosome homolog, they will always appear together in the hybrid DNAs, while proviruses on different chromosomes will have discordant appearances. The frequency of discordance in the hybrid panel between any 2 of the 19 proviruses was calculated for every combination. Of the 19

ET AL.

proviruses visualized in the hybrids (except in the few cases where a provirus could be scored in only a few hybrids), each was discordant with all of the others (Table 1). Thus, 19 proviral integrations visualized in Hut 102 B2 and derivative hybrids were dispersed among 19 chromosome homologs. Examination of the cell line MJ, passage 30, and its derivative hybrids revealed more than 30 proviral integrations (Fig. 2). Calculation of discordancy frequencies for each provirus was not technically feasible. However, the presence of different combinations of proviral integrations in different subsets of hybrids clearly demonstrated chromosomal dispersion of integrations in the MJ cell line as well (Fig. 2). Because of the possibility that the primary integrations in ATL were operative in the initial transformation, we genetically mapped two prominent proviruses which were present in the early passage Hut 102. This was achieved by Southern analysis of 35 Hut 102 B2 X rodent hybrids and 32 additional derivative subclones of certain members of the hybrid panel that had been genetically characterized for human chromosome composition. Chromosomal mapping of HTLV-I provirus integration was limited to hybrids that were positive for a particular provirus, since the presence of a chromosome homolog that did not contain the provirus in a hybrid could not preclude integration on the counterpart homolog. The 5.1-kb fragment was present in 13 of 35 panel members and was concordant with chromosomes 20 and X (Fig. 4). Every hybrid that contained the 5.1-kb fragment contained chromosomes 20 and X. Since the parental cell line was derived from a male (with a single X chromosome), the occurrence of several X-containing hybrids (not shown), which lacked the 5.1-kb fragment, excluded the X chromosome and permitted assignment of the 5.1-kb proviral fragment to one homology of chromosome 20. Chromosomal assignment of the 19%kb fragment was achieved in 13 Hut 102 X E36 hybrids and 32 subclones derived from two of these hybrids. The presence of the 19% kb fragment was 100% concordant with the presence of chromosome 4 and highly dis-

HTLV-I

INTEGRATION:

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15.0 14.0

FIG. 3. Southern analysis of Hut 102 B2 passage 30 (first panel), representative Hut 102 B2 p 80 X E36 hybrids (middle panel), and representative Hut 102 B2 p 80 X RAG hybrids (last panel), with probe pCR3Ll. Two different exposures of the parental cell line, Hut 102 B2 p 80, are presented. The molecular sizes of the 11 proviral integrations visualized in this cell line are shown.

cordant with all other human chromosomes (Fig. 4). This data clearly permits the assignment of the 19.8-kb fragment to one homolog of chromosome 4.

present in the parental cells, and not de nova infections caused by the fusion, because several of the novel integrations were present in multiple hybrid clones. In addition, several integrated proviruses which were present in an early passage of HTLVDISCUSSION I-infected cells apparently were lost during The results presented here demonstrate the progress of cell proliferation. Similar that HTLV-I integration is continual and dynamics of integration and exposure of dynamic during in vitro culture of two hu- multiple low abundance integrations were man T-cell lymphoma lines, Hut 102 and also observed in a second human T-cell MJ. The Hut 102 cells contained at least lymphoma line, MJ. Up to 30 distinct proviral fragments in genomic DNA of this four proviral integrations (two prominent and two less intense) at early passage (6- line have been resolved. A rather striking finding was that 19 in8 weeks). Upon continuous tissue culture passage, and after one cell cloning deri- tegrations detected in a later passage of vation, the number increased to 11 discrete Hut 102 B2 and over 30 proviruses present integrations. Derived clones following cell in MJ were nonspecifically dispersed over hybridization of these late passage cells the 46 possible human chromosome hodisplayed at least 19 different proviruses mologs. These data indicate that integraresolved by Southern analysis using mo- tion by HTLV-I is not restricted to one or lecular clones of HTLV-I LTR regions as even a few sites in the human genome. It probes. The eight additional proviral in- should be emphasized that this result obtegrations revealed in the hybrids are tained in vitro cannot be extended to conlikely to be low frequency integrations clude that site-specific integration and in-

0.00

0.35 0.33

0.00

0.19

0.00

0.26 0.00

0.33

0.27

18.0

0.25 0.00

0.29

0.26

0.30

16.5

0.00

0.20 0.27

0.27 0.20

0.20

16.0

0.33 0.00

0.21

0.18

0.19

0.21 0.29

14.0

0.00

0.36 0.03

0.16

0.20 0.19

0.23

0.22

13.2

0.00

0.29 0.27

0.27

0.13 0.14 0.00

0.20

0.27 0.33

0.27

0.33

0.13

10.5

0.33

0.27 0.20

0.13

0.20

0.27

12.0

0.00

0.36

0.09 0.21

0.15

0.23 0.57

0.28

0.23

0.25 0.21

9.0

size (EcoRI)

0.00

0.27 0.33

0.40

0.38 0.27

0.20 0.33

0.53 0.40

0.20

0.13

7.9

0.00

0.33 0.25

0.40

0.13

0.25

0.33

0.20

0.27

0.20 0.27

0.27

7.6

0.00

0.19

0.40 0.19

0.20

0.20

0.20

0.13 0.19

0.13

0.33 0.20

0.13

0.07

7.2

0.00

0.07

0.13 0.25

0.15

0.13

0.27

0.12 0.12

0.20

0.21

0.26 0.18

0.23

0.09

6.4

0.19 0.00

0.13

0.19

0.19

0.33 0.27

0.20

0.07

0.19

0.00 0.27

0.20

0.13 0.33

0.20

5.9

0.00

0.31

0.46 0.50

0.36

0.31

0.43

0.39 0.50

0.43

0.58 0.45

0.39

0.43

0.43 0.40

0.48

5.1

0.13 0.00

0.39 0.00

0.19 0.13

0.13

0.31

0.19

0.20 0.12

0.20

0.19 0.09

0.27

0.18

0.29 0.21

0.24

0.18

3.6

0.00 0.31

0.19

0.13

0.19

0.27 0.19

0.33

0.20

0.07

0.27 0.19

0.00

0.33 0.20

0.20 0.13

4.8

Note. Proviral bands are designated by molecular size and correspond to those presented in the left panel of Fig. 3. Numbers enclosed in parentheses indicate the number of hybrids (of 34 tested) which contained the proviral band. The * indicates additional proviral bands visualized in the hybrids but not in the Hut 102 B2 p 80 parent.

3.6 (1)

5.1 (13) *4.s (1)

*5.9 (1)

*6.4 (2)

*7.6 (5) 7.2 (1)

*7.9 (2)

9.0 (5)

*10.5 (4)

13.2 (2) *12.0 (5)

16.5 (5) *16.0 (3) 14.0 (4)

18.0 (6)

19.8 (10)

25.0 (5) 22.2 (7)

29.8

22.2

25.0

1 PROVIRAL FRAGMENTS IN HUT 102

Fragment

FREQUENCY OF DISCORDANCY OF H!lZV

TABLE

HTLV-I

INTEGRATION:

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ANALYSIS

a

1 2 3 4 5 6 7 8 9101112131415161718192~ HumanChmmosoonn

L

i 22 x

I

2 3 4 5 6 7 8 9 10III213141516171819202122 Human Chromowrm

X

FIG. 4. Analysis of the association of earlier proviral integration fragments (5.1 and 19.8 kb, see Figs. 1,3) with segregating human chromosomes in panels of Hut 102 X rodent somatic cell hybrids. Because viral integrations occurred on only one of two chromosomal homologs, only hybrids which retained the integration fragment were considered. For the 5.1-kb fragment, 13 hybrids were scored; for the 19.8-kb fragment, 45 hybrids were scored. Chromosome scores represent consensus of Gtrypsin analysis and isozyme scores for genes previously assigned to indicated chromosome (O’Brien et al, 1982; Seigel et al, 1984).

sertional mutagenesis are not operative in viva. In fact, for several retroviruses in which site-specific integration has been demonstrated in fresh tumors, chromosomal dispersion of multiple proviral integrations in cell lines obtained from mass infections or tumors has been previously reported (reviewed in Weinberg, 1980). Site-specific integration has been documented in several tumor systems (Dickson et aL, 1984; Fung et cc& 1983; Hayward et aL, 1981; Nusse et aZ.,1984; Nusse and Varmus, 1982; Peters et al, 1984; Tsichlis et al, 1983) and in every case, only a fraction of the multiple tumors examined displayed site specificity. Furthermore, if the locus of integration is important for tumorigenesis in the HTLV-I transformation, it is probable that only one of the multiple integrations is operative. We have concentrated on the two earliest integrations that were detected which map to homologs of chromosomes 4 and 20. The integration on chromosome 4, which also contains the gene for T-cell growth factor (TCGF), also known as interleukin-2 (IL-2) (Seigel et al, 1984), is of particular interest. IL-2 is required for the establishment of most HTLV-I-infected T-cell lines. These tumor cell lines often lose their dependence on IL-2 for growth and, further, in vitro transformation of normal T-cells

by HTLV-I is often accompanied by complete loss of IL-2 dependence. Current evidence, including the lack of expression of the IL-2 gene (Arya et a& 1984) and absence of rearrangements around the IL-2 locus (Seigel et aL, 1984), however, fails to support the concept of a direct regulatory role of the IL-2 gene in HTLV-I-mediated transformation. The presence of the human homolog of the src oncogene on chromosome 20 raises the prospect of the possible interaction of HTLV-I with this gene. Seiki et ccl. (1984) have approached this question of insertional mutagenesis by deriving a molecular clone from the cellular flanking DNA of HTLV-I integrations of two human tumors. A panel of somatic cell hybrids was employed to assign these proviral integrations to human chromosomes 7 and 17, respectively. When 35 tumors were examined with these same flanking probes, they failed to detect genomic rearrangements. While the data by Seiki et al. indicate that none of 35 tumors contains proviral integrations in common in either of the two chromosomal domains examined, the data does not permit the definite conclusion that there were no preferred integration sites present among these 35 tumors. Thus, the conclusion that insertional mutagenesis is not operative in ATL may be premature. Examination of addi-

74

SEIGEL ET AL.

tional integration sites will be required to determine whether integration in specific sites is important for transformation. ACKNOWLEDGMENTS We are grateful to Janice Simonson and Mary Eichelberger for excellent technical assistance and to Dr. R. C. Gallo and Dr. F. Wong-Staal for continuing support, biological reagents, and stimulating discussions.

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