HEMATOLOGICAL ONCOLOGY, VOL. 4,13-20 (1986)

HUMAN T-CELL LEUKEMIA VIRUS: CAUSATIVE ROLES IN DEVELOPMENT OF ADULT T-CELL

LEUKEMIA AND PROVIRUS INTEGRATION INTO LEUKEMIC CELL DNA

M. YOSHIDA AND M. SEIKI

Department of Viral Oncology, Cancer Instituie, Kami-Ikebukuro, Toshima-ku, Tokyo 170, Japan

INTRODUCTION

Human T-cell leukemia virus (HTLV) was isolated from a cutaneous T-cell lymphoma patient by Gallo and his colleagues (Poiesz et al., 1980) and adult T-cell leukemia virus (ATLV) was isolated from patients with adult T-cell leukemia (ATL) in Japan (Yoshida et al., 1982). Originally, these two viral isolates were thought to be different since they were isolated from apparently different tumors. However, we have shown that HTLV in the Caribbean and ATLV in Japan are the same virus (Watanabe et al., 1983, 1984) and the patient from which HTLV was isolated was identified as an atypical case of ATL (Bunn et al., 1983).

The HTLV is exogenous for humans (Reitz et al., 1981; Yoshida et al., 1982; Yoshida, 1983) and distinct from known animal retroviruses with respect to the nucleotide sequence of the pro- virus genome (Seiki et al., 1982, 1983). However, HTLV replicates by the same mechanism as known animal retroviruses (Seiki et al., 1982). Furthermore, HTLV was shown to be closely associated with a unique T-cell malignancy, ATL (Uchiyama et al., 1977), by extensive surveys of antibodies against the viral proteins (Hinuma et al., 1981, 1982; Robert-Guroff et al., 1982; Blattner et al., 1982). The association of the virus with ATL was also demonstrated by detecting the provirus genome in the leukemic cells of ATL patients (Yoshida et al., 1982; Wang-Staal et al., 1983). Analysis of the total nucleotide sequence of the viral genome lead to the conclusion that HTLV has no typical transforming gene (Seiki et al., 1983). This was consistent with the finding that no part of the HTLV genome showed significant hybridization with normal human DNA (Seiki et al., 1983).

Based on this information, it seemed important to study whether HTLV is directly involved in the leukemogenesis of ATL and whether the virus is associated with any other types of lym- phomas or leukemias. Therefore, we looked for the provirus genome in fresh tumor cells from 210 cases of lymphoma or leukemia obtained from endemic areas in Japan. The results showed that the provirus genome was integrated in the tumor cells from all 122 cases of ATL tested, but not in those of other types of lymphomas or leukemias, indicating that HTLV integration in the target cells is a prerequisite for ATL development. However, no common site for HTLV integration was found in fresh leukemic cells. Thus, the results do not support direct activation of cellular one gene by the LTR sequence of the integrated proviral genome. A trans-acting factor as proposed for induction of ATL development.

0278-0232/86/010013-08$05.00 Q 1986 by John Wiley & Sons, Ltd.

Received 14 May 1984

14 M. YOSHIDA AND M. SEIKI

MATERIALS AND METHODS

Preparation of cellular DNA

Patients with typical ATL or ATL-like clinical features were from the Kyushu area where HTLV is endemic (Hinuma et al., 1982). Lymphocytes were prepared from the fresh peripheral blood of leukemic patients by centrifugation on a Ficoll-Conray gradient (Yoshida et al., 1982). To pre- pare DNA for several blotting analyses, lymphocytes or lymphnode suspensions from lymphoma patients containing more than lo7 cells were used. High molecular weight DNA was extracted by phenol extraction after treating the cells overnight with SDS-proteinase K.

Detection of provirus sequence integrated in cellular DNA

Cellular DNA as digested with EcoRI, Sst I or Pst I and the digest as analysed by a blotting procedure using cloned viral [32P]-DNA (Seiki et al., 1982, 1983), under relatively stringent conditions (Yoshida et al., 1982) in buffer containing 4 x SSC (SSC: 0.1 5 M NaCI, 0.01 5 M sodium citrate, pH 7-59, 5 x Denhardt solution, 100 pg/ml of sonicated and heat denatured E. coli DNA and 40 pg/ml of poly (A) at 65°C for 2440 h. The filter was washed several times with 0-5 x SSC at 65"C, and then exposed to X-ray film at - 70°C.

Detection of antibodies against viral components

Antibodies against ATLA (ATL -associated antigens) (Hinuma et al., 1981) were examined by indirect immunofluorescence with MT-1 cells, as described by Hinuma et al. (1981).

Preparation of cellular flanking probes

EcoRI DNA fragment containing HTLV provirus was cloned in the Charon 4A vector from fresh leukemic cell DNA which contains only one copy of the provirus (Seiki et al., 1983). The cellular flanking sequences adjacent 3'LTR were subcloned into pBR322. Among these sub- clones, a clone containing a unique sequence (probe A) was selected by screening with Alu DNA and total cellular DNA as probes. With this unique cellular probe A, a human gene library was surveyed and two clones containing wider regions were isolated. Overlapping the restriction patterns of these clones, a restriction map of a region of human DNA surrounding 5'- and 3'- flankings of HTLV in leukemic cell DNA was constructed. Two more cellular clones, B and C , were also isolated from unique sequences in these clones and used for detection of DNA rearrangements. A similar strategy was repeated for another DNA of an ATL patient.

RESULTS

Survey of provirus genome in primary tumor cells

In this study, fresh tumor cells were used to avoid detection of artificial infections as follows: HTLV produced from only a few non-leukemic cells in a primary specimen can be transmitted into cells in culture and the infected cells can grow similar to the transformed T-cells (Miyoshi et

HUMAN T-CELL LEUKEMIA VIRUS

1 2 3 4 5 6 7 8 9 1 0

15

Figure 1. Monoclonality of leukemic cells with respect to the sites of HTLV provirus integration. DNA samples from lymphocytes of peripheral; blood of ATL patients (1-10) were digested with EcoRI and subjected to blot- ting analysis using representative proviral [32P]-DNA as probe. Since EcoRI does not cleave the provirus sequence, each band should contain the provirus genome and cellular flanking sequences at both ends. Thus, multiple bands in a lane indicate multiple copies of the

provirus integrated in cells

at., 1981; Popovic et at., 1983). Thus, cell lines or cells maintained in culture might not represent primary tumor cells. In fact, the available cell lines contained numerous copies of the integrated proviruses (Watanabe et al., 1984), whereas fresh leukemic cells contained only one or two copies (Yoshida et al., 1982). Thus, we have no direct evidence that the established cell lines were derived from the ATL cells.

For detection of the provirus sequence, representative probes (Seiki et al., 1983) were used in the blot-hybridization assay. Cellular DNA samples were digested with EcoRI, which does not cleave the proviral sequence. By this assay, the provirus sequence integrated at a certain locus of cellular DNA was detected as a discrete band (Figure 1).

The results of 210 cases of lymphoma and leukemia obtained from the endemic area in Japan are summarized in Table 1 . They were divided into three groups as follows: (i) patients with antibodies and the provirus genome in their tumor cells; (ii) patients with antibodies, but with no detectable provirus genome in their leukemic cell DNAs; and (iii) patients who had neither pro- virus or antibodies. All 122 patients with typical ATL are included in group (i) in Table 1 without any exception. Furthermore, all these ATL cases gave one or two, or in some cases three discrete bands in digests with EcoRI, as shown in Figure 1. Since EcoRI does not cut the proviral

16 M. YOSHIDA AND M. SEIKI

Table 1. Association of the HTLV genome with ATL and related diseases

Viral* Anti? Cell Case Group genome ATLA Diagnosis tY Pe No. Sum

ATL 1 + +

11

... 111

Non-Hodgkin CLL M F

+ CLL CLL Hodgkint Non-Hodgkin AML

- CLL CLL Sezary ALL Non-Hodgkin Hodgkinf Myeloma C M L AML

Total

T T T T

T B

(-1 (-1 (-1 T

B and N T

TBN TBN (-1 (-1 (-1 (-1

122 1 ] 137

I

1 16

23 1 1 64

“J 4

210

*Provirus genome in tumor cells. ?Serum antibodics against ATL-associated antigens (ATLA). $The number of tumor cells in the specimen might not be sufficient to detect the provirus sequence. (-), not analysed. Non-Hodgkin, non-Hodgkin’s lymphoma; Hodgkin, Hodgkin’s disease; CLL, chronic lymphocytic leukemia; AML, acute myelogenous leukemia; Sezary, Sezary syndrome; CML, chronic myelogenous leukemia.

sequence, th&e discrete bands clearly indicate that the leukemic cells were monoclonal, originat- ing from a single cell infected with HTLV, because integration sites in non-leukemic cell DNA were found to be random (Yoshida et af., 1984a, b). The monoclonal expansion of infected cells in all 122 cases of ATL strongly suggests that HTLV directly infects the target cell which becomes leukemic, suggesting that HTLV infection is a prerequisite step in the development of ATL (Yoshida et al., 1984a).

This conclusion of the HTLV role in ATL development was also supported by surveying patients who were diagnosed as ‘pre-ATL‘ or ‘smoldering ATL’ (Yamaguchi et al., 1983). These patients showed only a small percentage of abnormal lymphocytes found at random by careful cytological examination of blood samples in hospital. From hematological and clinical features, these cases are thought to be a stage preceding typical ATL (Yamaguchi et al., 1983). Surveying these patients for HTLV sequences, monoclonal growth of HTLV-infected cells was detected in more than half of the cases although it was hard to detect the provirus sequence since only a small percentage of lymphocytes in peripheral blood were infected (data not shown). These findings suggest that ATL cells are of monoclonal origin from the HTLV cells infected at a very early stage of leukemogenesis.

In addition to ATL patients, group (i) in Table 1 includes some cases of T-cell malignancies diagnosed as non-Hodgkin’s lymphoma or chronic lymphocytic leukemia (T-CLL). However,

HUMAN T-CELL LEUKEMIA VIRUS 17

these cases were all clinically and pathologically similar to ATL (Yamaguchi et al., 1984) and thus could be classified as ATL.

No cases in groups ii and iii were concluded to be directly associated ith HTLV, since the pro- virus genome was not detected in their leukemic cells; e.g. their leukemic cells were not infected with the virus. Patients in group ii had antibodies against the viral proteins, but no provirus genome was detected in their leukemic cells. Thus no cases of lymphoma or leukemia in groups ii and iii appeared to be associated with HTLV.

Specificity of the provirus integration in leukemic cell DNA HTLV was shown to have no typical transforming gene in its genome (Seiki et al., 1983). How- ever, it was suggested that the virus plays a causative role in ATL development (Yoshida et al., 1984a, b). These findings suggest a simple molecular mechanism of leukemogenesis by ATL, i.e. insertional mutagenesis, in which the provirus genome is integrated into a specific locus on the chromosomal DNA and then directly activates an adjacent cellular onc gene. This mechanism has been demonstrated in avian lymphomas (Hayward et al., 1981; Payne et al., 1982) and the erythroblastosis (Fung et al., 1983) induced by avian leukosis viruses.

As previously reported, and shown in Figure 1 , the size of the EcoRI DNA fragment contain- ing the integrated provirus and cellular flanking sequences differs in different individuals (Yoshida et al., 1982, 1984b) suggesting that the sites of integration of the HTLV provirus in these patients are not identical. However, it is possible that proviruses integrate into one or a few common loci of cellular DNA, then activate a unique cellular gene. To test this possibility, we examined the rearrangements of cellular DNA by provirus integration, using specified cellular probes.

A cellular flanking sequence of HTLV genome in fresh leukemic cell DNA was isolated from an ATL patient (KKT) who had only one copy of the proviral sequence (Seiki et al., 1983), since the provirus should play some role in initiating or promoting the growth of the leukemic cells. Therefore, the cellular flanking sequence of this provirus could be used as a specific probe to test the significance of the provirus integration into the tumor cell DNA. Three Sst I fragments of cellular flanking DNA A, B and C were found to cover a region of about 28 kilobases (kbp) around the provirus integration site in the leukemic cell DNA of patient KKT, and unique sequences in these fragments were used as probes to detect DNA rearrangements in leukemic cell DNA from other ATL patients. DNA samples from 35 ATL patients were digested with Sst I and analysed by the blotting procedure using the probes A, B and C . Some typical examples of the screening using probe A are shown in Figure 2. The rearranged DNA fragment was detected only in the control DNA from patient KKT, from which the probes were isolated, but no such rearranged DNA fragments were detected in the other ATL patients. These results were con- firmed by rehybridization of the same filters with the viral probes detecting comigration of the provirus sequence with the rearranged fragment only in the case of patient KKT (Figure 2). With other probes B and C, no DNA rearrangements were detected (data not shown). These results clearly indicated that the provirus integration sites in these 35 ATL cases are not located within the locus of 28 kbp where patient KKT had provirus integration. We also isolated another flank- ing sequence from fresh leukemic cells of another ATL patient, MYK, who again had one copy of the HTLV provirus in the leukemic cells. Using this cellular probe which covers about 20 kbp, no rearranged DNA fragments were detected in other ATL patients (data not shown). From these results with two independent cellular flanking sequences, it was concluded that HTLV has no common site for provirus integration in leukemic cells (Seiki et al., 1984).

This conclusion was further confirmed by specifying the chromosome into which HTLV pro- virus integrated (Seiki et al., 1984). The cellular flanking sequences described above were used for

18 M. YOSHIDA AND M. SEIKI

A B 1 2 3 4 5 6 7 8 9 1 0 1 2 3 4 5 6 7 8 9 1 0

kb

16

Figure 2. Survey of the integration sites of the proviral genome in primary tumor cells. A: Cellular DNAs were digested with Sst I and analysed by the blotting procedure. The probe used was a unique cellular flank- ing sequence adjacent to a provirus genome integrated into fresh leukemic cells in an ATL patient. Lane 10 represents DNA from which the probe was isolated. The bands of 16 kb are the normal DNA fragments and are expected even in tumor cells which contain provirus genome in this locus due to their diploid genomes. The band marked with the arrow is the rearranged DNA fragment containing the provirus sequence. B: The same filter as used in A was rehybridized with the representative viral probe. The filter was washed at 100"

for 5 min in distilled water and reused

detect'ion of specific human chromosomes in human x mouse cell hybrids. They demonstrated that the HTLV provirus was located on human chromosome 7 in one case and on chromosome 17 in another case. Thus, provirus integration has no specificity even at a chromosomal level (Seiki et al., 1984).

DISCUSSION

Leukemia cells from all 122 cases of ATL were found to contain the viral genome. Furthermore, the leukemic cells were all monoclonal with respect to the integration site of the provirus genome (Yoshida et al., 1984a). These results strongly suggest that HTLV directly infects the target cells which then grow as malignant transformed cells, although the infection does not necessarily induce ATL. If viral involvement was indirect, for example, mediated by a factor(s) released by infected cells, or just a coincidental infection of leukemic cells after transformation, some ATL cases should have leukemic cells in which the provirus genome is absent or is integrated at random sites. Therefore, these indirect mechanisms cannot explain the monoclonal origin of leukemic cells in all ATL cases. Alternatively, it is possible that if most of the T-cell population was infected with HTLV in a pre-leukemic state, proliferation of any T-cells induced by a factor(s) other than the virus would give a cell population with monoclonally integrated HTLV provirus. This seems unlikely because most of the T-cell populations of healthy carriers or ATL patients in remission were not infected with HTLV judging from intensity of the viral internal bands (data not shown).

Although some cases were not originally diagnosed as ATL but contained monodonally integrated proviruses in their leukemic cells, they were very similar to ATL. Thus the diagnosis of

HUMAN T-CELL LEUKEMIA VIRUS 19

these cases in group (i) in Table 1 can be changed into ATL. Other types of leukemias and lym- phoma are not directly related to HTLV infection. Consequently, HTLV appeared to be directly involved mainly in ATL, but not in other types of leukemia or lymphoma.

A molecular mechanism of leukemogenesis by animal retroviruses which have no transforming gene, promoter insertion model or insertional mutagenesis has been demonstrated in avian (Hayward et al., 1981; Payne et al., 1982) and murine (Nusse and Varmus, 1982) retroviral systems. But in human ATL, no common integration site was found within a 30 kbp region among 35 patients, and the integration differs even at a chromosomal level (Seiki et al., 1984).

All these data strongly suggest that provirus integration in tumor cells is random. Of course, it is still possible that some patient tumor cells might have proviruses integrated in specific regions, but separated from each other by 30 kbp or more. However, this situation is very different from the common sites for provirus integration of avian leukosis viruses or mouse mammary tumor virus, in which 80 per cent of ALV-induced lymphomas have the provirus within a 14 kb region (Payne et al., 1982) and 40 or 70 per cent of MMTV induced tumors within 24 kb or 35 kb regions (Nusse and Varmus, 1982). Since the direct activation of a cellular onc gene by a promoter or enhancer sequence in LTR of the provirus requires integration into a specific site, the results of random integration are not consistent with the idea of simple activation of a certain cellular one gene by the LTR sequence of the HTLV provirus. The situation is similar to that for induction of B-cell lymphomas by bovine leukemia virus, since bovine leukemia virus has no common integration sites within a 25 kb region of the lymphoma DNA (Kettmann et al., 1983). From the findings described above, the trans-acting function of HTLV, such as mediating the viral protein, was proposed for the mechanism of ATL development. Env gene products or protein(s) possibly encoded by the pX region can be candidates for the induction proliferation of the infected lym- phocytes. These genes are not onc genes, thus the products should not be enough to induce cellular transformation. Thus, these gene products may induce transient proliferation of the infected T-cells, consequently increasing the incidence of second genetic alterations which lead the cells into malignant cellular transformation. The studies of the identification and function of the viral proteins are in progress.

ACKNOWLEDGEMENTS

The authors thank Drs K. Yamaguchi and K. Takatsuki, Kumamoto University for supplying the clinical samples and also Drs R. Eddy and T. B. Shows, Roswell Park Memorial Institute for supplying DNA samples of human and mouse hybrid cells for analysing the human chromosome into which the provirus sequences are integrated.

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