Expression of alternatively spliced human T-lymphotropic virus type I ...

Proc. Natl. Acad. Sci. USA Vol. 89, pp. 3005-3009, April 1992 Medical Sciences

Expression of alternatively spliced human T-lymphotropic virus type I pX mRNA in infected cell lines and in primary uncultured cells from patients with adult T-cell leukemia/lymphoma and healthy carriers (adult T-cell leukemia/alternative splicing/gene expression/human retrovirus/regulatory genes)

Zwi N. BERNEMAN*, RONALD B. GARTENHAUS*, MARVIN S. REITZ, JR.*, WILLIAM A. BLATTNERt, ANGELA MANNSt, BARRIE HANCHARDt, OSAMU IKEHARA§, ROBERT C. GALLO*, AND MARY E. KLOTMAN* *Laboratory of Tumor Cell Biology and tEnvironmental Epidemiology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; tUniversity of the West Indies, Kingston, Jamaica; and §Department of Internal Medicine, Okinawa Chubu Hospital, Okinawa, Japan

Contributed by Robert C. Gallo, January 2, 1992

Although human T-cell lymphotropic virus ABSTRACT type I (HTLV-I) is the etiologic agent of adult T-cell leukemia/lymphoma (ATL), the role of viral gene expression in the progression to and maintenance of the leukemic state in vivo is unclear because of the inability of most previous studies to readily detect HTLV-I RNA in infected individuals. By using the reverse transcriptase-polymerase chain reaction, we detected spliced messages for the HTLV-I pX regulatory genes in primary uncultured cells from ATL patients and healthy asymptomatic carriers. In addition to the expected doubly spliced pX message, three alternatively spliced mRNAs were demonstrated (pXA17, pX-p21rex, and pX-orflI mRNAs, where orf = open reading frame). The same splice sites were shown in the messages from uncultured ATL cells and from the HTLV-I-producing C1O/MJ cell line. Alternatively spliced pX mRNAs have the potential to code for known and putative pX gene products. Among the transcripts is a monocistronic mRNA likely to code for p2l'rx (pX-p2lrx mRNA). Since alternative splicing of HTLV-I pX mRNA can be found in primary uncultured cells, it is likely to have a functional significance in vivo. This suggests possible roles for HTLV-I gene expression in the progression to and maintenance of ATL, as well as in the phase preceding it.

MATERIALS AND METHODS Cell Lines. Several HTLV-I-infected human T-cell lines were used. They included the interleukin 2 (IL-2)independent lines MT-2 (2), C10/MJ (4), HUT 102 (14), and NS1 (15) and the IL-2-dependent lines G11/MJ (3), EC155 (16), N1185, and N1186. The N1185 and N1186 cell lines were established by infecting cord blood lymphocytes by coculture with thawed peripheral blood mononuclear cells (PBMC) from two ATL patients. The H9 cell line (14, 17) served as a negative control. Patient Samples. PBMC from 10 ATL patients (5 from Jamaica and 5 from Okinawa, Japan) and 3 healthy HTLVI-seropositive subjects (from Jamaica) were separated on a Ficoll/Hypaque gradient and stored viably frozen in liquid nitrogen until analysis. All ATL cases had leukocytosis and/or abnormal lymphocytes in the peripheral blood, 6 of 10 had cutaneous involvement, and 7 of 10 had hypercalcemia during the course of the disease. Most died within 1 yr of blood sampling. RNA Extraction. Total cellular RNA was extracted from the patients' samples and the cell lines by the method of Chomczynski and Sacchi (18). RNA was also extracted from the C10/MJ and N1186 cell lines by the method of Chirgwin et al. (19). Cytoplasmic RNA was extracted from the cell lines by a standard protocol (20). Oligonucleotides. Oligonucleotide primers and probes (Table 1) were synthesized by using an Applied Biosystems Synthesizer. Reverse Transcriptase (RT)-PCR. PCR amplification was performed after reverse transcription of RNA to cDNA. Two micrograms of RNA were incubated in a 20-A.l reaction mixture containing 10 ng of the antisense primer MZ1 (Table 1 and Fig. 2), 50 mM Tris-HC1 (pH 8.3), 3 mM MgCI2, 75 mM KCI, and 10 mM dithiothreitol. The mixture was heated at 65'C for 3 min and cooled slowly to room temperature, which was followed by the addition of 1 pul of 25 mM dATP, dCTP, dGTP, and dTTP; 1 ,Al of 0.2 M dithiothreitol; and 60 units of RT (AMV Super RT; Molecular Genetics Resources, Tampa, FL). The RT reaction was at 50'C for 30 min. After standard extractions with phenol-chloroform mixtures and precipitation with ethanol, the cDNA was redissolved in 20 pul of distilled water; 2.5 1.l was used for PCR amplification in a 100-1.l reaction mixture containing 10 mM Tris HCI (pH 8.3); 50 mM KCI; 1.5, 2.0, or 2.5 mM MgCI2; 0.01% gelatin; 0.2 mM dATP, dCTP, dGTP, and dTTP; 1 AtM of primers MZ4

Human T-cell lymphotropic virus type I (HTLV-I), a retrovirus, is the etiologic agent of adult T-cell leukemia/ lymphoma (ATL) (1). HTLV-I also transforms normal T cells in vitro (2-4). The pX region, which codes for the HTLV-I regulatory proteins tax (p4Otax) and rex (p27'X) and for a protein of unknown function, p21rex, is sufficient for immortalization of primary human T-lymphocytes in vitro (5). The tax gene can also transform NIH 3T3 and Rat-i fibroblasts (6). Previous studies have failed to readily detect HTLV-I antigens (7-9) and mRNA (10-12) in the leukemic cells of ATL patients, suggesting that HTLV-I gene expression is not required for the progression to and maintenance of the leukemic state and that the role of the virus may be limited to the induction of polyclonal T-lymphocyte proliferation years to decades before the development of ATL. This proliferation could lead to secondary events, increasing the chance for leukemia, at which stage HTLV-I gene expression would not be necessary (13). The recent development of the sensitive polymerase chain reaction (PCR) technology has allowed us to reassess the conclusions of the previous studies.

Abbreviations: HTLV-I, human T-cell lymphotropic virus type I; ATL, adult T-cell leukemia/lymphoma; RT, reverse transcriptase; PCR, polymerase chain reaction; IL-2, interleukin 2; PBMC, peripheral blood mononuclear cells; orf, open reading frame.

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Proc. Natl. Acad. Sci. USA 89 (1992)

Table 1. DNA oligonucleotide primers and probes used in this study Sequence Nucleotide localization Orientation Name Primers 5'-CATATGAATTCCCkTCCACGCCGGTTG>CGC-3' 399-420 (pX mRNA l1 exon) Sense MZ4 5'-CIGIGITGGGGCICCIGTCGCC-3' Antisense 7394-7373 (pX mRNA 3rd exon) MZ1 5'-CATATGCGGCCGCCIG ITGGGGCICCIGTCGCC-3' 7394-7373 (pX mRNA 3rd exon) Antisense MZ5 Probes 5'-TTTGGGkTCGGCGGGGCCTCCG-3' 5160-5139 (pX mRNA 21n exon) MZ6 Antisense 5'-GGGTCCICG&&CkkkCTGGCTGGG-3' 5333-5310 (env) MZ7 Antisense Splice junction probes 5'-GCGTCCGCCGTCTkG/CTTCCTGGTCTTkkT-3' 457-471/4993-5007 MZ2 Sense 5'-CCTCCkkCkCCkTGG/CCCkCTTCCCkGGGT-3' 5169-5183/7302-7316 MZ3 Sense 5'-CCTTTCCkCTGGCGG/CT&GkCGGCGGICTG-3' 5024-5010/471-457 Antisense MZ14 5'-kCCCTGGGkkGTGGG/CTkGkCGGCGGkCGT-3' 7316-7302/471-457 Antisense MZ10 5'-TGCCCGGkGGkCCTG/CT&GkCGGCGGACGT-3' 7241-7227/471-457 Antisense MZ9 The nucleotides derived from the HTLV-I sequence are indicated in boldface type. Slashes indicate the splice junction. Nucleotides underlined once represent an EcoRI site; those underlined twice represent a Not I site. The CATAT nucleotides in MZ4 and MZ5 were added to ensure restriction enzyme digestion. MZ4, MZ1, MZ5, MZ6, MZ7, MZ2, and MZ3 were designed on the basis of known HTLV-I structure and splicing, prior to this study; MZ14, MZ10, and MZ9 were synthesized when alternative splicing of pX mRNA was demonstrated by cloning and sequencing.

and MZ5; and 2.5 units of Taq DNA polymerase (PerkinElmer/Cetus). Thirty cycles of PCR amplification were performed in a DNA Thermal Cycler (Perkin-Elmer/Cetus) according to the following program: denaturation at 94°C for 1 min, annealing at 55°C or 60°C for 2 min, extension at 72°C for 2 min, lengthening of each extension cycle by 2 sec per cycle, extension of the final cycle at 72°C for 7 min. This initial RT-PCR, performed on C1O/MJ and N1186 RNA, was used for analysis (see Fig. 1) and cloning purposes (see Fig. 2). Subsequent RT-PCR, on primary samples was also performed with modifications (see Fig. 3). Subsequent amplification of total and cytoplasmic RNA from the cell lines was carried out with 30 cycles of PCR an annealing temperature of 60°C and 2.0 mM MgCl2. Analysis of RT-PCR Products. After electrophoresis on a 6% polyacrylamide gel, the RT-PCR products were denatured, neutralized, transferred to a nylon membrane by electroblotting at 20 V for 18 hr, and cross-linked by using a UV Stratalinker 1800 (Stratagene). The nylon filters were prehybridized at 37°C in Hybrisol I solution (Oncor, Gaithersburg, MD), and 106 cpm of a 32P-labeled DNA probe per ml was added. The genomic HTLV-I probe pMT-2 (21) was labeled by nick translation; oligonucleotide probes (Table 1) were end-labeled by using T4 polynucleotide kinase. Hybridization was overnight at 37°C. The filters were washed twice with 0.1 x SSC containing 0.5% SDS for 10 min at 60°C and autoradiographed. Cloning and Sequencing of the RT-PCR Products. The RT-PCR products of the C1O/MJ and N1186 cell lines and of ATL samples 1 and 3 (see Fig. 3) were cloned into the EcoRI-Not I sites of the pBluescript plasmid (Stratagene). Lysates from a representative number of bacterial colonies, transformed with plasmids containing C1O/MJ and N1186 RT-PCR products, were directly amplified by PCR as described above. These PCR products were resolved by electrophoresis alongside the original RT-PCR products on 6% polyacrylamide gels. The bacterial clones whose PCR products comigrated with the original RT-PCR products were selected. The cloned RT-PCR products from the ATL samples were selected by hybridizing nylon membrane replicas of recombinant bacterial colonies with 32P-labeled probes specific for splice junctions (see Fig. 2C and Table 1). The insert of the recombinant plasmids was sequenced by the dideoxynucleotide chain-termination method (22) with the Sequenase version 2.0 DNA sequencing kit (United States Biochemical).

RESULTS Alternatively Spliced HTLV-I pX mRNA in Cultured HTLV-I-infected Cells. RT-PCR performed on RNA extracted from the C1O/MJ cell line amplified a 381-base-pair (bp) DNA molecule (Fig. 1) corresponding to the doubly spliced pX mRNA that has been described (23, 24). In addition, other amplified DNA molecules were also shown after the gel was stained with ethidium bromide and after hybridization with the HTLV-I genomic pMT-2 probe (Fig. 1, lanes EB and pMT2). The structure of these amplified cDNAs was resolved after cloning and sequencing of the RT-PCR products from the C1O/MJ and N1186 cell lines (Fig. 2). Four alternatively spliced messages were found. All four (pX, pXA17, pX-p21lx, and pX-orflI, where orf = open reading frame) were cloned from the C1O/MJ RT-PCR products, and clones of pX and pX-p21lx were obtained from the N1186 cell line. At least two clones of each type of cDNA were sequenced. The cDNAs obtained from the N1186 cell line had the same splicing pattern as the equivalent messages in the C1O/MJ cell line. pX mRNA corresponded to the doubly spliced message characterized in the past (23, 24). It joins transcript from the 5' long terminal repeat (noncoding first exon up to nucleotide 471) to RNA from the downstream region of pol (second exon: nucleotides 4993-5183), which is spliced to transcript from the pX region (third exon: from nucleotide 7302 on); this pX mRNA has a coding capacity for tax, rex, and p211x (26). In the doubly spliced mRNA designated pXA17, the classical splice donor site of the first exon was spliced to a new splice acceptor of the second exon, located 17 bp downstream from the classical splice acceptor; that message still has the capacity to code for tax, rex, and p2lx. In the singly spliced pX-p21Ix mRNA, the classical splice donor site of the first exon was directly spliced to the 434 bp _

EB pMT2 MZ2 MZ3 MZ6 MZ14 MZ9 MZ1Q *

a Vs

pX----a 267 bp 192 bP

* l U

FIG. 1. Analysis of the RT-PCR products of C1O/MJ cell line pX cDNA. After autoradiography, the probe was stripped from the filter and hybridized with another probe. The positions of the amplified product of the expected pX cDNA and DNA size markers are indicated to the left. The probes are indicated above each lane. Hybridization with the env oligonucleotide probe MZ7 (Table 1) was negative. EB, ethidium bromide.

Medical Sciences: Bernernan et al. A

LTR

Proc. Natl. Acad. Sci. USA 89 (1992) pol

gag

pX

env

3007

LTR Proviral DNA Genomic mRNA Subgenomic mRNA pX mRNA

a

B

X9

40

471

pX mRNAmo pXCA 17 mRNA

pX-p21lX mRNA pX-orfll mRNA

4993

\B1 8;3

5010

471

5183

pXA 17 mRNA

9 473739

7302

471

7227

t 1 1

pX mRNA

3

7302

471

SD SA SA

C

\

SD

MZ2

MZ14

_

MZ6

MZ3

MZ6

MZ3

MZ10

pX-p21lex mRNA

pX-orfl/ mRNA

S SA

SA

MZ9

FIG. 2. Alternative splicing of HTLV-I pX mRNA. (A) Scheme of the proviral DNA and the messages that were characterized in the past. (B) Four pX messages that were found in this study. The exons spliced together are indicated with the solid bars. (C) Oligonucleotide probes used to identify and distinguish the different pX messages. Dotted lines represent the continuity in a single oligonucleotide probe. LTR, long terminal repeat; SD, splice donor; SA, splice acceptor. Nucleotide number 1 starts at the upstream end of the proviral genome and refers to the first sequence published (25).

classical splice acceptor of the third exon. Since the second exon, which contains the initiation codons for both tax and rex, was absent from that message, pX-p21lex mRNA is a prime candidate for a monocistronic message for p21leX. This

protein, which shares its carboxyl terminus with rex, has been shown to be translated independently from rex, starting at an internal ATG site (at nucleotide 7476) (26). In the mRNA designated pX-orflI, the classical splice donor site of the first exon was joined to a new splice acceptor site, located 75 bp upstream of the classical splice acceptor of the third exon. That message cannot be translated into tax or rex but could code for the pX-orflI (25) with the ATG initiation codon at nucleotide 7288; it also has coding potential for p21rex and for a polypeptide of 14 amino acids with the ATG codon at position 7241. The alternative splicing demonstrated here occurs in two different ways: alternative splice acceptor sites and the presence or absence of a cassette exon (the second pX mRNA exon) (27). The splice junction sequences were completely conserved among the different clones examined. In addition, the two nucleotides at the upstream and downstream boundaries of all of the spliced-out introns were the highly conserved GT and AG (data not shown), respectively (27).

Synthesis of the oligonucleotide probes was based on the of the splice junctions. Those probes (MZ2, MZ3, MZ14, MZ10, and MZ9) span 15 nucleotides on each side of the splice junction (Fig. 2C and Table 1). The stringency of the washes after hybridization was adjusted (0.1 x SSC containing 0.5% SDS at 60°C) so that binding would occur only when a probe recognized sequences present on both sides of the same splice junction. These conditions were derived by using the PCR products of the bacteria containing sequence

the recombinant plasmids, whose insert sequence was known, as well as the RT-PCR products from the cell lines (Fig. 1). In addition, pX-p21rex and pX-orfII could be distinguished on a polyacrylamide gel from pX and pXA17 cDNAs on the basis of the amplified-product size (respectively 190, 265, 381, and 364 bp, including the additional nucleotides containing the restriction sites). It was more difficult to distinguish pX from pXA17 cDNA on the basis of size alone because there is only a 17-bp difference between the two messages.

Using hybridization with the different splice junction probes, we found the four different pX messages in different HTLV-I-infected cell lines, both IL-2 dependent (N1186, G11/MJ, and EC155) and IL-2 independent (HUT 102, MT-2, C10/MJ, and NS1). One exception was the absence of pX-orflI mRNA in the EC155 cell line. This cell line contains a defective HTLV-I provirus with a deletion between nucleotides 5183 and 7302 (16), resulting in the absence of the splice acceptor site for pX-orflI mRNA. To rule out the possibility that the alternatively spliced messages are intermediate pathway products in the generation of mature mRNAs, cytoplasmic RNA extracted from the C10/MJ, NS1, N1185, and N1186 cell lines was analyzed. All four pX messages were present in the RT-PCR products of the cytoplasmic RNAs (data not shown). Expression of Alternatively Spliced pX mRNA in Primary Cells. To determine whether alternative splicing of pX mRNA is relevant to the in vivo situation, RT-PCR was performed on RNA extracted from uncultured, unstimulated PBMC from ATL patients and healthy HTLV-I-seropositive individuals. Seven of 10 ATL samples and three of three carrier samples were positive for one or more of the four alternatively spliced

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M Z10 /)X-p2I-'rey RNA

MZ2 (pX mRNA) r-

-

1 2

ATL----,

3 4 5 6

7 8 9 10 C

1

r ATL 2 3 4 5 6

-

7

9 10,

8

am.

a;-

a

-381 bp

l90bp-

-

I.

M Z9 i pX orfT 1rRNA

MZ14 (pXA 17 mRNA L -ATL 1 2 3 4 5 6 7 8 9 10 C

3 4

5

E.

-

0

'p

£

-36 4bpp 265 bp-

FIG. 3. RT-PCR amplification of pX mRNA in uncultured primary PBMC from ATL patients (ATL lanes 1-5: patients from Jamaica; ATL lanes 6-10: patients from Japan) and uninfected H9 cells (lanes C). The RT reaction was performed as described in text with the following modifications: 200 ng of the antisense primer MZ5 (Table 1 and Fig. 2) was used, and the reaction was at 42°C. PCR was directly performed on S ,l of the cDNA reaction as described in text with the following adaptations: 40 cycles of PCR amplification were performed with 0.5 ,uM of primers MZ4 and MZ5. The PCR products were treated as described in text. The same filters were sequentially hybridized with the different oligonucleotide probes indicated. The filters were washed twice with 0.1 x SSC containing 0.5% SDS at 60°C for 10 min and autoradiographed (MZ2 and MZ9, for 14 days; MZ14, for 10 days; MZ10, overnight). The position and length of the different pX RT-PCR products are indicated for each different probe. Faint bands were observed for ATL PBMC samples 1 (MZ2), 2 (MZ14, MZ9), and 3 and 10 (MZ9).

pX mRNAs (Fig. 3 and Table 2). pX-p21rex mRNA was the most prevalent message, both in the ATL and in the healthy carrier samples. The RT-PCR products of two ATL samples were cloned and sequenced. Clones of pX and pXA17 mRNAs were obtained from ATL 3, and clones of pX-p21'x and pX-orflI mRNAs were from ATL 1 (Fig. 3). At least two clones of each type of message were sequenced. The splice junctions were the same in the ATL samples as in the HTLV-I-producing cell lines. In two pX cDNAs from ATL 3, nucleotide changes were present, which could not be found in the cDNAs from the C10/MJ and N1186 cell lines (data not shown). Thus, it is unlikely that the RT-PCR products from the ATL samples were PCR contaminants. We could further exclude contamination, since the RT-PCR products of the negative controls analyzed simultaneously with the ATL samples, tested negative with the different HTLV-I oligonucleotide probes (Fig. 3). Table 2. Frequency of the alternatively spliced pX mRNAs in uncultured primary PBMC from ATL patients and

healthy carriers

Probe MZ2 MZ14 pXA17 pX-p21rcx MZ10 MZ9 pX-orflI *aa, Amino acids. mRNA

pX

(Potential) Protein(s) encoded by mRNA* tax, rex, p2lrex tax, rex, p21rex p2lrex 14 aa, pX-II, p21rex

Cases, no. with alternatively spliced pX/total ATL

Carriers

5/10 5/10 7/10 6/10

1/3 0/3 3/3

0/3

DISCUSSION Two conclusions can be drawn from this study. First, there is complex alternative splicing of HTLV-I pX mRNA. Second, there is expression of alternatively spliced pX mRNA in primary uncultured cells from ATL patients and healthy carriers. The complex alternative splicing of HTLV-I regulatory gene messages is similar to the situation with human immunodeficiency virus type 1 (HIV-1) (28-33). In fact, alternative splicing is a common feature of retroviruses (34). The finding of alternatively spliced pX messages in the cytoplasm of the cell lines demonstrates that they are exported to the cytoplasm, where they are likely to have a function. One of the main advantages of alternative splicing is to allow for a high level of protein diversity and abundance, starting from a limited genetic sequence (27). This biologically advantageous property could play a role in the expression of HTLV-I pX proteins. The translation of two orfs in the pX mRNA region, pX-I (orf I) and pX-II (orf II) (25), cannot be initiated in the classical pX mRNA. In this study, we have identified a message (pX-orfII mRNA) that can code for pX-II in addition to a small polypeptide of 14 amino acids. It is also striking that pX-p21rcx mRNA was the most prevalent message in the PBMC from both the ATL patients and the healthy carriers. This transcript is a prime candidate for a monocistronic message for p2lrex, whose function remains unknown. The common occurrence of its presumed mRNA suggests that p21rex may play a role in the biology of HTLV-I. HTLV-I pX mRNAs were detected in PBMC from ATL patients and healthy seropositives. We accomplished cloning and sequencing of RT-PCR products from ATL samples. Not

Medical Sciences: Bernernan et al. only was the classical pX mRNA found but also the other alternatively spliced mRNAs were found, demonstrating that alternative splicing of primary transcripts is present in primary cells and making it likely that it occurs in vivo. Our findings of HTLV-I pX message by RT-PCR in ATL samples are in agreement with those of Kinoshita et al. (35), while contradictory results exist (36). In support of pX gene expression in vivo, anti-tax antibodies are frequently found in ATL patients and in healthy carriers (37). The cell source of low-level HTLV-I message requiring PCR for detection and its biological relevance are unknown. Low-level viral mRNA in ATL could be the result of expression in polyclonally infected non-malignant T cells. Alternatively, the cells expressing HTLV-I message could be part of the proliferative compartment. Pertinent to the relevance of low-level viral message, recent data show that small amounts of retroviral regulatory proteins can stimulate the growth of certain target cells. The HTLV-I tax protein, exogenously provided in nanomolar concentrations, stimulates the proliferation of primary human lymphocytes in vitro (ref. 38; R.B.G., unpublished data). The HIV-1 transactivator tat stimulates the growth of cells derived from Kaposi sarcoma of AIDS patients when provided in picomolar amounts (39). Thus, a low level of message translating to low amounts of protein may induce a biological action. The findings of Kinoshita et al. (35) and our results, which demonstrate pX message in primary samples, make it possible to envisage a role for HTLV-I gene expression not only in initiating T-cell proliferation early in infection but also in the progression to and maintenance of ATL. We thank Drs. Suresh K. Arya and Leslie Bruggeman for careful review. This work was started when Z.N.B. was an International Research Fellow of the Fogarty International Center, National Institutes of Health. M.E.K. was supported by the Career Development Program of the Veteran's Administration Hospital. R.B.G. was supported by an Intramural Research Training Award of the National Cancer Institute. 1. Gallo, R. C. (1984) Cancer Surv. 3, 113-159. 2. Miyoshi, I., Kubonishi, I., Yoshimoto, S., Akagi, T., Ohtsuki, Y., Shiraishi, Y., Nagata, K. & Hinuma, Y. (1981) Nature (London) 294, 770-771. 3. Popovic, M., Sarin, P. S., Robert-Guroff, M., Kalyanaraman, V. S., Mann, D., Minowada, J. & Gallo, R. C. (1983) Science 219, 856-859. 4. Markham, P. D., Salahuddin, S. Z., Kalyanaraman, V. S., Popovic, M., Sarin, P. & Gallo, R. C. (1983) Int. J. Cancer 31, 413-420. 5. Grassmann, R., Dengler, C., Muller-Fleckenstein, I., Fleckenstein, B., McGuire, K., Dokhelar, M. C., Sodroski, J. G. & Haseltine, W. A. (1989) Proc. Nail. Acad. Sci. USA 86, 33513355. 6. Tanaka, A., Takahashi, C., Yamaoka, S., Nosaka, T., Maki, M. & Hatanaka, M. (1990) Proc. Nail. Acad. Sci. USA 87, 1071-1075. 7. Hoshino, H., Esumi, H., Miwa, M., Shimoyama, M., Minato, K., Tobinai, K., Hirose, M., Watanabe, S., Inada, N., Kinoshita, K., Kamihira, S., Ichimaru, M. & Sugimura, T. (1983) Proc. Nail. Acad. Sci. USA 80, 6061-6065. 8. Sugamura, K., Fujii, M., Kannagi, M., Sakitani, M., Takeuchi, M. & Hinuma, Y. (1984) Int. J. Cancer 34, 221-228. 9. Kitamura, T., Takano, M., Hoshino, H., Shimotohno, K., Shimoyama, M., Miwa, M., Takaku, F. & Sugimura, T. (1985) Int. J. Cancer 35, 629-635.

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