JOURNAL OF CELLULAR PHYSIOLOGY 145:23&243 (1990)
Establishment of a Cyclic Adenosine Monophosphate-Dependent Growing Human T-cell Line Derived From an Interleukin-2-Dependent CelI Line TOSHIKAZU TAKESHITA, KAZUYUKI OHBO, MASATAKA NAKAMURA, YUSO GOTO, AND K A Z U O SUGAMURA* Department of Microbiology, Tohoku University School of Medicine, 2- I Seiryo-machi,
Sendai 980, japan An adenosine 3‘,5‘-cyclicmonophosphate (CAMP)-dependentgrowing cell line called CT-Mat was established by the long-term cultivation of an interleukin-2 (IL-2)-dependent human T-cell line, ILT-Mat, in the presence of cholera toxin instead of IL-2. CT-Mat cells can grow in the medium containing either cholera toxin or forskolin or cAMP derivatives. Although the CT-Mat cell line can still grow dependent on IL-2, the forskolin-induced growth of CT-Mat cells was demonstrated not to be mediated by an autocrine mechanism of IL-2 or any other growth factor. The intracellular cAMP level was elevated by treatment with the chemical agents but little by treatment with IL-2. These suggest that cAMP transduces intracellular growth signals different from those through the IL-2 receptor in an IL-2-dependent T-cell line CT-Mat.
Clonal expansion of T lymphocytes activated by antigen is mediated by extracellular lymphotropic hormones such as interleukin-2 (IL-2) (Morgan et al., 1976; Smith, 1980). The IL-2 action is triggered by its interaction with the specific high-affinity IL-2 receptor (IL-BR),which is a complex consisting of at least ci (p55) and p (p75) chains (Sharon et al., 1986; Tsudo et al., 1986; Teshigawara et al., 1987; Robb et al., 1987; Dukovich et al., 1987; Takeshita et al., 1989). Recently, the structural and genetical studies of the IL-2Rp chain have been extensively developed. The results indicate that the IL-2Rp is expected to be a molecule essential for signal transduction (Hatakeyama et al., 1989a, b). However, there are some arguments about the mechanism of signal transduction from IL-2RP. So far, at least two biochemical events were detected in accordance with IL-2 stimulation. One is the activation of phosphotidylinositol turnover, resulting in the activation of Ca2+phospholipid-dependent kinase, protein kinase C (PKC),and the other is the modulation of intracellular adenosine 3’,5’-cyclic monophosphate (CAMP) level. However, it has been demonstrated that PKC is not necessary for the IL-2 signaling because IL-2 can transduce the growth signal in PKC-less mutant cells (Mills et al., 1988). We have also shown that phorbol esters known as PKC-activators can persistently replace IL-2 for the growth of an IL-2-dependent T-cell line named TPA-Mat (Takeshita et al., 19881, but the signaling pathway of phorbol ester-induced growth was different from that of IL-2-induced growth in TPA-Mat cells (Goto et al., 1988). On the other hand, the increasing of an intracellular cAMP level is known to accompany with not only growth promotion but also growth suppression of various mammalian cells (reviewed in Friedman, 1982). In T cells, CAMP analogues and 0 1990 WILEY-LISS, INC.
cAMP increasing agents such as cholera toxin and forskolin antagonize IL-2 action (Johnson et al., 19881, and furthermore IL-2 inhibits cAMP production (Becker and Farrar, 19861,although there is a contrary report that IL-2 elevates an intracellular cAMP level after IL-2 stimulation (Wickremasinghe et al., 1987). These apparently controversial observations make it different to understand the biochemical mechanisms involved in the signal transduction through IL-2R. We have previously investigated the effects of intracellular cAMP level on IL-2-dependent cell growth and found that the growth of an IL-2-dependent cell line, ILT-Mat, was transiently stimulated by cholera toxin. Based on this observation, we attempted to establish a cholera toxin-dependent cell line from ILT-Mat, and we have succeeded in this. Using this cell line, we show here that the elevation of intracellular cAMP level induces the persistent growth signaling of T cells. MATERIALS AND METHODS Cell culture An IL-2-dependent human T-cell line, ILT-Mat, which is carrying human T-cell leukemia virus type I (HTLV-I), was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, antibiotics, and 0.9 nM recombinant human IL-2 (provided by Shionogi Pharmaceutal Co., Osaka, Japan). A cholera toxin-dependent cell line, CT-Mat, which was established here, was maintained in the same medium as the above, excepting replacement of IL-2 with 100 nglml of cholera toxin. Received March 29, 1990; accepted July 3, 1990. *Towhom reprint requests/correspondence should be addressed.
CAMP-DEPENDENTT-CELL LINE
Incorporation of 3H-thymidine (3H-TdR) Murine IL-2-dependent CTLL-2 cells and CT-Mat cells preincubated with cholera toxin-free medium for 6 days were used for 3H-TdR incorporation assays. They were incubated in microplates for 48 hr and 1 pCi of 3H-TdR was added to each well at 4 hr before harvesting. The incorporated 3H-TdRwas counted as described elsewhere (Takeshita et al., 1988). Values are means for triplicate determinations.
cAMP measurement Intracellular cAMP of CT-Mat cells was measured as described previously (Goto et al., 1988). In brief, CTMat cells preincubated in the absence of cholera toxin for 6 days were incubated with the forskolin- or IL2-containing medium for the indicated times. After harvesting the cells, their intracellular cAMP was measured by the radioimmunoassay method with a Yamasa cAMP assay kit. Southern and Northern blot hybridization High-molecular-weight genomic DNA extracted from cells was digested with EcoRI, electrophoresed through a 0.7% agarose gel, and transferred to a nitrocellulose membrane for Southern blot hybridization. Total cellular RNA extracted from cells was electrophoresed through a 1%agarose gel and transferred to a nitrocellulose membrane for Northern blot hybridization. The probes used for hybridization were the HTLV-I pX probe from pHTD2 (Ohtani et al., 1987), T-cell receptor (3 Chain (TCRP) constant region cDNA robe from 86T5 (Hedrick et al., 1984), which were abeled by nick translation. The Southern and Northern blot hybridizations were performed as described previously (Takeshita et al., 1988).
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we previously established a TPA-dependent cell line, TPA-Mat, derived from ILT-Mat cell line (Takeshita et al., 19881, we attempted to establish a cholera toxindependent cell line in a similar manner to that of TPA-Mat. ILT-Mat cells were cultured in a medium containing 100 ng/ml of cholera toxin instead of IL-2. About a month later, most of the cells died, but some of them started to roliferate gradually. A cholera toxindependent cell f n e named CT-Mat was thus established. The pattern of TCRp rearrangement and the HTLV-I provirus integration sites of ILT-Mat were demonstrated to be identical to those of CT-Mat by Southern blot hybridization, although a few additional integration sites of HTLV-I provirus were detected in CT-Mat, confirming that CT-Mat was derived from ILT-Mat (Fig. 1).Cell-surface markers and expression of HTLV-I p4OtaX antigen were compared between CT-Mat and its parental ILT-Mat. Both cell lines were positive for CD2, Ia, IL-2Ra, and IL-2Rp, and 73% of CT-Mat cells and 16% of ILT-Mat cells expressed HTLV-I p4OtaXantigen. They were negative for CD3 and CD8 but positive for CD4 (data not shown). Figure 2 shows the growth curves of CT-Mat and the parental ILT-Mat cells. Proliferation of CT-Mat cells was observed in the presence of either cholera toxin or IL-2. The doubling time of cholera toxin-dependent cell growth was about 28 hr, which is longer than that of IL-2-dependent cell growth. The cholera toxin dependence and IL-2 dependence of CT-Mat have been retained for more than 10 months. Furthermore, when CT-Mat cells were maintained in the presence of IL-2 for more than 1 month, they did not lose their cholera
Monoclonal antibodies (mAbs) and chemical agents H-31 mAb is specific for human IL-2Ru (Fujii et al., 1986). TU27 mAb is specific for human IL-2Rp (Takeshita et al., 1989). Surface marker mAbs used were anti-CD2, -CD3, -CD4, -CD8, and -1a antibodies (Becton-Dickinson Monoclonal Center Inc., Mountain View, CA). Lt-4 mAb is specific for HTLV-I p40tax kindly provided by Dr. H. Tozawa (Kitasato University) (Lee et al., 1987). L-61 mAb specific for human IL-2 was provided by Shionogi Pharmaceutical Co. (Ide et al., 1987). Cholera toxin and its p subunit were purchased from Seikagaku Kogyo Co. (Jagan). 12-0tetradecanoylphorbol 13-acetate (TPA), N -2'-o-dibutyryladenosine 3' $'-cyclic monophosphate (db-CAMP), 8-bromoadenosine 3'5 '-cyclic monophosphate (8bCAMP), and forskolin were purchased from Sigma Chemical Co. (St. Louis, MO).
RESULTS ILT-Mat cell line was maintained in the medium containing IL-2 for more than a year. Effects of various chemical agents on 3H-TdR incorporation of ILT-Mat cells were examined first. Not only IL-2 but also TPA and cholera toxinsignificantly induced incorporation of 3H-TdRinto ILT-Mat cells, although the effects of TPA and cholera toxin was smaller than that of IL-2. Since
Fig. 1. Southern blot hybridization of cellular DNA. DNAs of ILTMat and CT-Mat cells were hybridized with gene probes of T-cell receptor p (TCRP) and HTLV-IpX as described in Materials and Methods.
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Fig. 2. Effects of IL-2 and cholera toxin on cell growth. ILT-Mat (A) and CT-Mat (B)cells were cultured in the medium supplemented with 200 Uiml of IL-2 (o),100 ngiml cholera toxin (*), or none (A),,andthe medium was renewed every 2 or 3 days by centrifugation and readjusting the cell number to 2 x 105/ml or less. Viable cells were determined by the dye exclusion staining.
toxin dependence (data not shown). In contrast, ILTMat cells proliferated in response to IL-2, but most died within 2 days in the presence of cholera toxin. These results suggest that CT-Mat is a stable variant derived from ILT-Mat cells. Cholera toxin is known to increase a cAMP level, but the purified p subunit of cholera toxin, which does not contain cAMP increasing activity, did not induce the growth of CT-Mat cells (data not shown). Other agents such as cAMP analogues and forskolin, known as an activator for adenylate cyclase, were examined for their effects on 3H-TdR incorporation of CT-Mat cells. The dose-response curves of the agents in 3H-TdR incorporation assays are shown in Figure 3. Forskolin, db-CAMP, and 8b-CAMPas well as cholera toxin and IL-2 stimulated 3H-TdR incorporation of CT-Mat cells in a dose-dependent manner. The maximum uptake of 3H-TdR was always higher in stimulation with IL-2 than with other agents. Next, CT-Mat cells were examined for their ability to produce IL-2 or other autccrine growth factors in response to cholera toxin and forskolin. The 3H-TdR uptake of CT-Mat cells was assayed in the presence of L61 mAb, which neutralizes the IL-2 activity. L61 mAb did not affect the forskolin-induced incorporation of 3H-TdR but significantly inhibited the IL-2-induced incorporation (Fig. 4).The supernatant of CT-Mat cells cultured in the presence of forskolin was assayed for activities of IL-2 and other growth factors (Table 1). IL-2 activity in the supernatant was not detected by a 3H-TdR incorporation assay using CTLL-2 cells, and significant activity of growth factors for CT-Mat cells was not detected either. The supernatant slightly stim-
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Fig. 3. Effects of various agents on 3H-TdR incorporation of CT-Mat cells. 3H-TdR incorporation assays of CT-Mat cells were performed in the presence of agents diluted twofold with 100 Uiml of IL-2 (01, 100 ngiml of cholera toxin (a), 20 pM forskolin ( A ) , 300 (LM db-CAMP ( A ) , and 300 (LM8b-CAMP(m) as described in Materials and Methods.
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Fig. 4. Effects of L61 mAb in 3H-TdR incorporation of CT-Mat cells. 3H-TdR incorporation assays of CT-Mat cells were performed in the presence of 5 Uiml of IL-2 (a) or 20 (LMforskolin (0)and various concentrations of L61 mAb as described in Materials and Methods.
ulated incorporation of 3H-TdR on CT-Mat cells. This stimulation should be due to residual forskolin, which was estimated at approximate 2.5 FM according to the dilution of medium. Furthermore, no mRNA of IL-2 was detected in CT-Mat cells in the presence of cholera toxin (data not shown). These results suggest that the
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CAMP-DEPENDENTT-CELL LINE TABLE 1. IL-2 and other growth factor activities in the culture suwmatant of CT-Mat cells
TABLE 2. Effect of I t 2 and forskolin on the CAMPlevel of CT-Mat cells'
~
Treatment with Medium Culture supernatant' Recombinant IL-2 Forskolin
%TdR incorporation (cpm) of CTLLS cells CT-Mat cells
*
1,319 f 836 727 394 287,383 f 6,815 1,598 f 410
2,886 f 327 6,555 f 792 76,476 f 2,781 28,208 f 400
'CT-Mat cells were cultured for 2 days in the presence of 20 FM forskolin. Their culture supernatant wasconcentrated 12-foldby vacuum dialysis and assayedat the final 1.5-foldconcentration. IL-2 and forskolin wereused atthe concentrationof 100 U/ml and 20 gM, respectively.
Incubation time (rnin)
IL2
0 5 15 30 60
Intracellular cAMP (pmole/106 cells) Forskolin
2.84
2.24
2.10
11 60
2.66 4.16 4.60
ND ND ND
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'CT-Mat cells wereincubatedin the presenceof 200U/ml of IG2or 20 pM forskolin. Their extracts were assayed for cAMP as described in Materials and Methods.
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DISCUSSION An IL-2-dependent human T-cell line, ILT-Mat, was x 15 characteristic of dependence on phorbol esters and cholera toxin for its transient growth. Based on these E, CJ observations, we previously succeeded in the establishv ment of a phorbol ester-dependent cell line called c TPA-Mat from the ILT-Mat cell culture with TPA 0 10 .c (Takeshita et al., 1988). Similarly, we have established 2 here a cholera toxin-dependent cell line called CT-Mat 0 from the same parental cell line, ILT-Mat. Four IL2-dependent human T-cell lines carrying HTLV-I were CJ .c 5 used to establish cholera toxin-dependent cell lines, and only one of them, ILT-Mat, was successful. Al% though the mechanism of acquisition of cAMP depenId I dence of CT-Mat is still unclear, this may be associated r m with additional integration of the HTLV-I proviral ----J-v\ ' I I genome observed in CT-Mat cells, because CT-Mat was 0 0 1 10 100 not frequently established from ILT-Mat cells. IL-2 ( U/ml 1 Cholera toxin is known to consist of a and p subunits, and the a subunit but not the p subunit harbors the Fig. 5. Mutual effects between IL-2 and Bb-CAMPon 3H-TdR incorability to activate the guanine nucleotide binding poration of CT-Mat cells. 3H-TdR incorporation assays of CT-Mat cells were performed in the presence ( 0 ) or absence ( 0 ) of 75 pM 8b-CAMP protein, which is involved in the elevation of cAMP level (Holmgren, 1981). The purified p subunit did not and various concentrations of IL-2 as described in Materials and Methods. induce the growth of CT-Mat cells, suggesting that the cholera toxin-induced growth was attributed to the elevation of intracellular cAMP level. This was further confirmed by cAMP analogues such as db-CAMP and forskolin-dependent growth of CT-Mat cells was not 8b-CAMPand forskolin, an activator of adenylate cymediated by the autocrine mechanism involving IL-2 clase that catalyzes the formation of CAMP, resulting in and other growth factors and that cAMP induces the elevation of cAMP level. All these chemical agents growth signals in CT-Mat cells. induced the growth of CT-Mat cells. The forskolinThe mutual effects between 8b-CAMPand IL-2 on induced growth of CT-Mat cells was not mediated by growth of CT-Mat cells were then examined to inves- the autocrine mechanism of IL-2 production, because tigate the difference in the signaling pathway between CT-Mat cells were found not to produce IL-2 in the IL-2-induced growth and CAMP-induced growth. As is presence of cholera toxin or forskolin. Furthermore, shown in Figure 5, any synergistic or cumulative effect any other factor supporting their own growth was not was not observed between 75 pM 8b-CAMPand various detected in the supernatant of CT-Mat cells cultured in doses of IL-2 from 0 to 100 U/ml. The 8b-CAMPseems the presence of forskolin. These results suggest two to be partly suppressive for the IL-Zinduced cell possibilities; one is that cAMP directly induces the growth. Furthermore, the effects of IL-2 on intracellu- intracellular growth signal in CT-Mat cells, and the lar cAMP level were examined with CT-Mat cells. other is that the ex ression of some cellular gene(s), CT-Mat cells were cultured with IL-2 for the indicated which induces the ce 1 growth, is promoted by HTLV-I times, and their intracellular cAMP levels were as- gene products such as p4OtaX,the expression of which is sayed (Table 2). The cAMP level was unaffected up t o at stimulated in CT-Mat cells as compared with ILT-Mat least 15 min after IL-2 stimulation, although it slightly cells, and cAMP is known t o promote the expression of increased after 30 min of IL-2 stimulation. On the other HTLV-I (Jeang et al., 1988; Nakamura et al., 1989).We hand, forskolin induced a significant elevation of CAMP cannot rule out the former possibility because the level within 5 min of treatment. elevation of intracellular cAMP leads to growth of
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yeasts (Matsumoto et al., 1982), and the transient promoting effects of CAMP on proliferation of cells have also been observed in nonlymphoid cells such as fibroblasts (Rozengurt et al., 1981) and liver epithelioid cells (Boynton and Whitfield, 19791, but consider that the latter possibility is more likely because the removal of cholera toxin from CT-Mat cells did not result in a rapid reduction of cell numbers, as shown in Figure 2. In lymphocytes, cAMP was recently suggested to be a second messenger for interleukin-1 (Shirakawa et al., 1988). Since CT-Mat cells are responsive to both IL-2 and cAMP for their growth, the question of whether the IL-2-induced growth is also sustained by the elevation of CAMP arises. However, the present study shows that 8b-CAMPpartially suppressed the IL-2-induced growth of CT-Mat cells and that the intracellular cAMP level was unaffected during the first 15 min of IL-2 stimulation. Similarly, there is evidence that the increased cAMP levels by cholera toxin or forskolin induces suppressive effects on the generation of IL-2 activity (Johnson et al., 1988) and that IL-2 induces a transient decrease of cAMP level in IL-2-dependent cells (Becker and Farrar, 1986). This suggests that cAMP does not play a substantial role in the IL-2-mediated signal transduction. We have previously shown that PKC activation by phorbol esters transduces the growth signal in an IL-2-dependent cell line, TPA-Mat (Takeshita et al., 1988). The phorbol ester-induced growth of TPA-Mat cells was strongly inhibited by forskolin, but their IL-2-induced growth was weakly inhibited, suggesting that the growth signal induced by IL-2 is transmitted through the pathway that is perhaps distinct from that involved in PKC activation (Goto et al., 1988). These data, together with the present study, suggest that the PKC and CAMP-dependent kinase may not play a crucial role in signal transduction directly from IL-2R. However, we (Ishii et al., 1987) and others (Benedict et al., 1987) have already demonstrated the IL-2-stimulated phosphorylation of cellular proteins that contain phosphoserine and phosphothreonine, indicating that IL-2 stimulates some protein kinase catalyzing phosphorylation of serine and threonine residues, although it is still vague whether there is a significant involvement of the protein kinase in the signal transduction pathway. Receptors for some growth factors such as epidermal growth factor, platelet-derived growth factors, insulin, insulin-like growth factor, and colonystimulating factor 1 are known to autophosphorylate on the tyrosine residues (reviewed in references Hanks et al., 1988). Interestingly, it has been reported that IL-2 also stimulates tyrosine phosphorylation on several proteins (Salzman et al., 19881, and we have recently demonstrated that IL-2 stimulates tyrosine phosphorylation of IL-2Rp molecules (Asao et al., 1990). These observations suggest the possibility that some tyrosine kinase associated with IL-2RP is stimulated by IL-2, and this phosphorylation is implicated in the transduction of the growth signal that is different from PKC- and CAMP-dependent pathways. CT-Mat showed here a possible existence of a pathway for cell growth signaling mediated by cAMP in T cells. The variant cell lines derived from an IL-2dependent ILT-Mat, such as CT-Mat and TPA-Mat,
should be useful for the study of the mechanisms of cell growth .
ACKNOWLEDGMENTS We thank Ms. E. Ohkubo for her help in preparation of the manuscript. This work was supported in part by Grants-in Aid for Scientific Research and Cancer Research from the Ministry of Education, Science and Culture, and by a grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research. LITERATURE CITED Asao, H., Takeshita, T., Nakamura, M., Nagata, K., and Sugamura, K. (1990) Interleukin 2 (IL-21-inducedtyrosine phosphorylation of IL-2 receptor p75. J. Exp. Med., 171:637-644. Becker, S.K., and Farrar, W.L. (1986) Interleukin 2 modulation of adenylate cyclase. J. Biol. Chem., 261:3043-3047. Benedict, S.H., Mills, G.B., and Gelfand, E.W. (1987) Interleukin 2 activates a receptor-associated protein kinase. J. Immunol., 139:169&1697. Boynton, A.L. and Whitfield, J.F. (1979) The cyclic AMP-dependent initiation of DNA synthesis by T51B rat liver epithelioid cells. J . Cell. Physiol., 101:139-148. Dukovich, M., Wano, Y., Thuy, L.B., Katz, P., Cullen, B.R., Kehrl, J.H., and Greene, W.C. (1987) A second human interleukin-2 binding protein that may be a component of high-affinity interleukin-2 receptors. Nature, 327:51&522. Friedman, D.L. (1982) Regulation of the cell cycle and cellular proliferation by cyclic nucleotides. J.W. Kebabian, and J.A. Nathanson, eds. Handbook of Experimental Pharmacology, 58,II, pp 151-187. Fujii, M., Sugamura, K., Nakai, S., Tanaka, Y., Tozawa, H., and Hinuma, Y. (1986) High- and low-affinity interleukin 2 receptors: Distinctive effects of monoclonal antibodies. J. Immunol., 137:15521556. Goto, Y., Takeshita, T., and Sugamura, K. (1988) Adenosine 3',5'cyclic monophosphate (CAMP) inhibits phorbol ester-induced growth of an IL-2-dependent T cell line. FEBS Lett., 239:165-168. Hanks, S.K., Quinn, A.M., and Hunter, T. (1988) The protein kinase family: Conserved features and deduced phylogeny of the catalytic domains. Science, 241 :42-52. Hatakeyama, M., Mori, M., Doi, T., and Taniguchi, T. (1989a) A restricted cytoplasmic region of IL-2-receptor p chain is essential for growth signal transduction but not for ligand binding and internalization. Cell, 59:837-845. Hatakeyama, M., Tsudo, M., Minamoto, S., Kono, T., Doi, T., Miyata, T., Miyasaka, M., and Taniguchi, T. (1989b) Interleukin-2 receptor p chain gene: generation of three receptor forms by cloned human a and p chain cDNAs. Science, 244:551-556. Hedrick, S.M., Nielsen, E.A., Kavaler, J., Cohen, D.I., and Davis, M.M. (1984) Sequence relationships between putative T-cell receptor polypeptides and immunoglobulins. Nature, 308:153-158. Holmgren, J . (1981) Actions of cholera toxin and the prevention and treatment of cholera. Nature, 292:413-417. Ide, M., Kaneda, K., Koizumi, K., Hojo, K., Murai, Y., Sagawa, K., Kono, M., and Sato, K. (1987) Neutralizing monoclonal antibodies against recombinant human interleukin-2. J. Immunol. Methods, 10157-62, Ishii, T.,Kohno, M., Nakamura, M., Hinuma, Y., and Sugamura, K. (1987) Characterization of interleukin 2-stimulated phosphorylation of 67 and 63 kDa proteins in human T-cells. Biochem. J., 242:211-219. Jeang, K.-T., Boros, I., Brady, J., Radonovich, M., and Khoury, G. (1988) Characterization of cellular factors that interact with the human T-cell leukemia virus type I p4OX-responsive21-base-pair sequence. J. Virol., 62:4499-4509. Johnson, K.W., Davis, B.H., and Smith, K.A. (1988) cAMP antagonizes interleukin 2-promoted T-cell cycle progression at a discrete point in early G1. Proc. Natl. Acad. Sci. USA, 85:6072-6076. Lee, B., Tanaka, Y., and Tozawa, H. (1989) Monoclonal antibody defining tax, protein of human T-cell leukemia virus type-I. Tohoku J . Exp. Med., 157:l-11. Matsumoto, K., Uno, I., Oshima, Y., and Ishikawa, T.(1982) Isolation and characterization of yeast mutants deficient in adenylate cyclase and CAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA, 79:2355-2359.
CAMP-DEPENDENTT-CELL LINE Mills, G.B., Girard, P., Grinstein, S., and Gelfand, E.W. (1988) Interleukin-2 induces proliferation of T lymphocyte mutants lacking protein kinase C. Cell, 55:91-100. Morgan, D.A.,Ruscetti, F.W., and Gallo, R.C. (1976)Selective in vitro growth of T lymphocytes from normal human bone marrows. Science, 193:1007-1008. Nakamura, M., Niki, M., Ohtani, K., and Sugamura, K. (1989) Differential activation of the 2l-baseaair enhancer element of human T-cell leukemia virus type I by i’ts own trans-activator and cyclic AMP. Nucleic Acids Res., 17:5207-5221. Ohtani. K.. Nakamura. M.. Saito. S.. Noda. T.. Ito. Y.. Sueamura. K.. and Hinuma, Y. (1987)Identification of two distind’ele&ents in the long terminal repeat of HTLV-I responsible for maximum gene expression. EMBO J., 6:389-395. Robb, R.J., Rusk, C.M., Yodoi, J., and Greene, W.C. (1987)Interleukin 2 binding molecule distinct from the Tac protein: Analysis of its role information of high-affinity receptors. Proc. Natl. Acad. Sci. USA, 84: 2002-2006. Rozengurt, E., Legg, A., Strang, G., and Courtenay-Luck, N. (1981) Cyclic AMP A mitogenic signal for Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA, 78:4392-4396. Saltzman. E.M.. Thom, R.R., and Casnellie, J.E. (1988) Activation of a tyrosine protein kinase is an early event in the stimulation of T lymphocytes by interleukin-2. J. Biol. Chem., 263:6956-6959. Sharon, M., Klausner, R.D., Cullen, B.R., Chizzonite, R., and Leonard,
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Establishment of a Cyclic Adenosine Monophosphate-Dependent Growing Human T-cell Line Derived From an Interleukin-2-Dependent CelI Line TOSHIKAZU TAKESHITA, KAZUYUKI OHBO, MASATAKA NAKAMURA, YUSO GOTO, AND K A Z U O SUGAMURA* Department of Microbiology, Tohoku University School of Medicine, 2- I Seiryo-machi,
Sendai 980, japan An adenosine 3‘,5‘-cyclicmonophosphate (CAMP)-dependentgrowing cell line called CT-Mat was established by the long-term cultivation of an interleukin-2 (IL-2)-dependent human T-cell line, ILT-Mat, in the presence of cholera toxin instead of IL-2. CT-Mat cells can grow in the medium containing either cholera toxin or forskolin or cAMP derivatives. Although the CT-Mat cell line can still grow dependent on IL-2, the forskolin-induced growth of CT-Mat cells was demonstrated not to be mediated by an autocrine mechanism of IL-2 or any other growth factor. The intracellular cAMP level was elevated by treatment with the chemical agents but little by treatment with IL-2. These suggest that cAMP transduces intracellular growth signals different from those through the IL-2 receptor in an IL-2-dependent T-cell line CT-Mat.
Clonal expansion of T lymphocytes activated by antigen is mediated by extracellular lymphotropic hormones such as interleukin-2 (IL-2) (Morgan et al., 1976; Smith, 1980). The IL-2 action is triggered by its interaction with the specific high-affinity IL-2 receptor (IL-BR),which is a complex consisting of at least ci (p55) and p (p75) chains (Sharon et al., 1986; Tsudo et al., 1986; Teshigawara et al., 1987; Robb et al., 1987; Dukovich et al., 1987; Takeshita et al., 1989). Recently, the structural and genetical studies of the IL-2Rp chain have been extensively developed. The results indicate that the IL-2Rp is expected to be a molecule essential for signal transduction (Hatakeyama et al., 1989a, b). However, there are some arguments about the mechanism of signal transduction from IL-2RP. So far, at least two biochemical events were detected in accordance with IL-2 stimulation. One is the activation of phosphotidylinositol turnover, resulting in the activation of Ca2+phospholipid-dependent kinase, protein kinase C (PKC),and the other is the modulation of intracellular adenosine 3’,5’-cyclic monophosphate (CAMP) level. However, it has been demonstrated that PKC is not necessary for the IL-2 signaling because IL-2 can transduce the growth signal in PKC-less mutant cells (Mills et al., 1988). We have also shown that phorbol esters known as PKC-activators can persistently replace IL-2 for the growth of an IL-2-dependent T-cell line named TPA-Mat (Takeshita et al., 19881, but the signaling pathway of phorbol ester-induced growth was different from that of IL-2-induced growth in TPA-Mat cells (Goto et al., 1988). On the other hand, the increasing of an intracellular cAMP level is known to accompany with not only growth promotion but also growth suppression of various mammalian cells (reviewed in Friedman, 1982). In T cells, CAMP analogues and 0 1990 WILEY-LISS, INC.
cAMP increasing agents such as cholera toxin and forskolin antagonize IL-2 action (Johnson et al., 19881, and furthermore IL-2 inhibits cAMP production (Becker and Farrar, 19861,although there is a contrary report that IL-2 elevates an intracellular cAMP level after IL-2 stimulation (Wickremasinghe et al., 1987). These apparently controversial observations make it different to understand the biochemical mechanisms involved in the signal transduction through IL-2R. We have previously investigated the effects of intracellular cAMP level on IL-2-dependent cell growth and found that the growth of an IL-2-dependent cell line, ILT-Mat, was transiently stimulated by cholera toxin. Based on this observation, we attempted to establish a cholera toxin-dependent cell line from ILT-Mat, and we have succeeded in this. Using this cell line, we show here that the elevation of intracellular cAMP level induces the persistent growth signaling of T cells. MATERIALS AND METHODS Cell culture An IL-2-dependent human T-cell line, ILT-Mat, which is carrying human T-cell leukemia virus type I (HTLV-I), was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, antibiotics, and 0.9 nM recombinant human IL-2 (provided by Shionogi Pharmaceutal Co., Osaka, Japan). A cholera toxin-dependent cell line, CT-Mat, which was established here, was maintained in the same medium as the above, excepting replacement of IL-2 with 100 nglml of cholera toxin. Received March 29, 1990; accepted July 3, 1990. *Towhom reprint requests/correspondence should be addressed.
CAMP-DEPENDENTT-CELL LINE
Incorporation of 3H-thymidine (3H-TdR) Murine IL-2-dependent CTLL-2 cells and CT-Mat cells preincubated with cholera toxin-free medium for 6 days were used for 3H-TdR incorporation assays. They were incubated in microplates for 48 hr and 1 pCi of 3H-TdR was added to each well at 4 hr before harvesting. The incorporated 3H-TdRwas counted as described elsewhere (Takeshita et al., 1988). Values are means for triplicate determinations.
cAMP measurement Intracellular cAMP of CT-Mat cells was measured as described previously (Goto et al., 1988). In brief, CTMat cells preincubated in the absence of cholera toxin for 6 days were incubated with the forskolin- or IL2-containing medium for the indicated times. After harvesting the cells, their intracellular cAMP was measured by the radioimmunoassay method with a Yamasa cAMP assay kit. Southern and Northern blot hybridization High-molecular-weight genomic DNA extracted from cells was digested with EcoRI, electrophoresed through a 0.7% agarose gel, and transferred to a nitrocellulose membrane for Southern blot hybridization. Total cellular RNA extracted from cells was electrophoresed through a 1%agarose gel and transferred to a nitrocellulose membrane for Northern blot hybridization. The probes used for hybridization were the HTLV-I pX probe from pHTD2 (Ohtani et al., 1987), T-cell receptor (3 Chain (TCRP) constant region cDNA robe from 86T5 (Hedrick et al., 1984), which were abeled by nick translation. The Southern and Northern blot hybridizations were performed as described previously (Takeshita et al., 1988).
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we previously established a TPA-dependent cell line, TPA-Mat, derived from ILT-Mat cell line (Takeshita et al., 19881, we attempted to establish a cholera toxindependent cell line in a similar manner to that of TPA-Mat. ILT-Mat cells were cultured in a medium containing 100 ng/ml of cholera toxin instead of IL-2. About a month later, most of the cells died, but some of them started to roliferate gradually. A cholera toxindependent cell f n e named CT-Mat was thus established. The pattern of TCRp rearrangement and the HTLV-I provirus integration sites of ILT-Mat were demonstrated to be identical to those of CT-Mat by Southern blot hybridization, although a few additional integration sites of HTLV-I provirus were detected in CT-Mat, confirming that CT-Mat was derived from ILT-Mat (Fig. 1).Cell-surface markers and expression of HTLV-I p4OtaX antigen were compared between CT-Mat and its parental ILT-Mat. Both cell lines were positive for CD2, Ia, IL-2Ra, and IL-2Rp, and 73% of CT-Mat cells and 16% of ILT-Mat cells expressed HTLV-I p4OtaXantigen. They were negative for CD3 and CD8 but positive for CD4 (data not shown). Figure 2 shows the growth curves of CT-Mat and the parental ILT-Mat cells. Proliferation of CT-Mat cells was observed in the presence of either cholera toxin or IL-2. The doubling time of cholera toxin-dependent cell growth was about 28 hr, which is longer than that of IL-2-dependent cell growth. The cholera toxin dependence and IL-2 dependence of CT-Mat have been retained for more than 10 months. Furthermore, when CT-Mat cells were maintained in the presence of IL-2 for more than 1 month, they did not lose their cholera
Monoclonal antibodies (mAbs) and chemical agents H-31 mAb is specific for human IL-2Ru (Fujii et al., 1986). TU27 mAb is specific for human IL-2Rp (Takeshita et al., 1989). Surface marker mAbs used were anti-CD2, -CD3, -CD4, -CD8, and -1a antibodies (Becton-Dickinson Monoclonal Center Inc., Mountain View, CA). Lt-4 mAb is specific for HTLV-I p40tax kindly provided by Dr. H. Tozawa (Kitasato University) (Lee et al., 1987). L-61 mAb specific for human IL-2 was provided by Shionogi Pharmaceutical Co. (Ide et al., 1987). Cholera toxin and its p subunit were purchased from Seikagaku Kogyo Co. (Jagan). 12-0tetradecanoylphorbol 13-acetate (TPA), N -2'-o-dibutyryladenosine 3' $'-cyclic monophosphate (db-CAMP), 8-bromoadenosine 3'5 '-cyclic monophosphate (8bCAMP), and forskolin were purchased from Sigma Chemical Co. (St. Louis, MO).
RESULTS ILT-Mat cell line was maintained in the medium containing IL-2 for more than a year. Effects of various chemical agents on 3H-TdR incorporation of ILT-Mat cells were examined first. Not only IL-2 but also TPA and cholera toxinsignificantly induced incorporation of 3H-TdRinto ILT-Mat cells, although the effects of TPA and cholera toxin was smaller than that of IL-2. Since
Fig. 1. Southern blot hybridization of cellular DNA. DNAs of ILTMat and CT-Mat cells were hybridized with gene probes of T-cell receptor p (TCRP) and HTLV-IpX as described in Materials and Methods.
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Fig. 2. Effects of IL-2 and cholera toxin on cell growth. ILT-Mat (A) and CT-Mat (B)cells were cultured in the medium supplemented with 200 Uiml of IL-2 (o),100 ngiml cholera toxin (*), or none (A),,andthe medium was renewed every 2 or 3 days by centrifugation and readjusting the cell number to 2 x 105/ml or less. Viable cells were determined by the dye exclusion staining.
toxin dependence (data not shown). In contrast, ILTMat cells proliferated in response to IL-2, but most died within 2 days in the presence of cholera toxin. These results suggest that CT-Mat is a stable variant derived from ILT-Mat cells. Cholera toxin is known to increase a cAMP level, but the purified p subunit of cholera toxin, which does not contain cAMP increasing activity, did not induce the growth of CT-Mat cells (data not shown). Other agents such as cAMP analogues and forskolin, known as an activator for adenylate cyclase, were examined for their effects on 3H-TdR incorporation of CT-Mat cells. The dose-response curves of the agents in 3H-TdR incorporation assays are shown in Figure 3. Forskolin, db-CAMP, and 8b-CAMPas well as cholera toxin and IL-2 stimulated 3H-TdR incorporation of CT-Mat cells in a dose-dependent manner. The maximum uptake of 3H-TdR was always higher in stimulation with IL-2 than with other agents. Next, CT-Mat cells were examined for their ability to produce IL-2 or other autccrine growth factors in response to cholera toxin and forskolin. The 3H-TdR uptake of CT-Mat cells was assayed in the presence of L61 mAb, which neutralizes the IL-2 activity. L61 mAb did not affect the forskolin-induced incorporation of 3H-TdR but significantly inhibited the IL-2-induced incorporation (Fig. 4).The supernatant of CT-Mat cells cultured in the presence of forskolin was assayed for activities of IL-2 and other growth factors (Table 1). IL-2 activity in the supernatant was not detected by a 3H-TdR incorporation assay using CTLL-2 cells, and significant activity of growth factors for CT-Mat cells was not detected either. The supernatant slightly stim-
u 20
2-fold dilution
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Fig. 3. Effects of various agents on 3H-TdR incorporation of CT-Mat cells. 3H-TdR incorporation assays of CT-Mat cells were performed in the presence of agents diluted twofold with 100 Uiml of IL-2 (01, 100 ngiml of cholera toxin (a), 20 pM forskolin ( A ) , 300 (LM db-CAMP ( A ) , and 300 (LM8b-CAMP(m) as described in Materials and Methods.
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Fig. 4. Effects of L61 mAb in 3H-TdR incorporation of CT-Mat cells. 3H-TdR incorporation assays of CT-Mat cells were performed in the presence of 5 Uiml of IL-2 (a) or 20 (LMforskolin (0)and various concentrations of L61 mAb as described in Materials and Methods.
ulated incorporation of 3H-TdR on CT-Mat cells. This stimulation should be due to residual forskolin, which was estimated at approximate 2.5 FM according to the dilution of medium. Furthermore, no mRNA of IL-2 was detected in CT-Mat cells in the presence of cholera toxin (data not shown). These results suggest that the
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CAMP-DEPENDENTT-CELL LINE TABLE 1. IL-2 and other growth factor activities in the culture suwmatant of CT-Mat cells
TABLE 2. Effect of I t 2 and forskolin on the CAMPlevel of CT-Mat cells'
~
Treatment with Medium Culture supernatant' Recombinant IL-2 Forskolin
%TdR incorporation (cpm) of CTLLS cells CT-Mat cells
*
1,319 f 836 727 394 287,383 f 6,815 1,598 f 410
2,886 f 327 6,555 f 792 76,476 f 2,781 28,208 f 400
'CT-Mat cells were cultured for 2 days in the presence of 20 FM forskolin. Their culture supernatant wasconcentrated 12-foldby vacuum dialysis and assayedat the final 1.5-foldconcentration. IL-2 and forskolin wereused atthe concentrationof 100 U/ml and 20 gM, respectively.
Incubation time (rnin)
IL2
0 5 15 30 60
Intracellular cAMP (pmole/106 cells) Forskolin
2.84
2.24
2.10
11 60
2.66 4.16 4.60
ND ND ND
~
~~
'CT-Mat cells wereincubatedin the presenceof 200U/ml of IG2or 20 pM forskolin. Their extracts were assayed for cAMP as described in Materials and Methods.
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DISCUSSION An IL-2-dependent human T-cell line, ILT-Mat, was x 15 characteristic of dependence on phorbol esters and cholera toxin for its transient growth. Based on these E, CJ observations, we previously succeeded in the establishv ment of a phorbol ester-dependent cell line called c TPA-Mat from the ILT-Mat cell culture with TPA 0 10 .c (Takeshita et al., 1988). Similarly, we have established 2 here a cholera toxin-dependent cell line called CT-Mat 0 from the same parental cell line, ILT-Mat. Four IL2-dependent human T-cell lines carrying HTLV-I were CJ .c 5 used to establish cholera toxin-dependent cell lines, and only one of them, ILT-Mat, was successful. Al% though the mechanism of acquisition of cAMP depenId I dence of CT-Mat is still unclear, this may be associated r m with additional integration of the HTLV-I proviral ----J-v\ ' I I genome observed in CT-Mat cells, because CT-Mat was 0 0 1 10 100 not frequently established from ILT-Mat cells. IL-2 ( U/ml 1 Cholera toxin is known to consist of a and p subunits, and the a subunit but not the p subunit harbors the Fig. 5. Mutual effects between IL-2 and Bb-CAMPon 3H-TdR incorability to activate the guanine nucleotide binding poration of CT-Mat cells. 3H-TdR incorporation assays of CT-Mat cells were performed in the presence ( 0 ) or absence ( 0 ) of 75 pM 8b-CAMP protein, which is involved in the elevation of cAMP level (Holmgren, 1981). The purified p subunit did not and various concentrations of IL-2 as described in Materials and Methods. induce the growth of CT-Mat cells, suggesting that the cholera toxin-induced growth was attributed to the elevation of intracellular cAMP level. This was further confirmed by cAMP analogues such as db-CAMP and forskolin-dependent growth of CT-Mat cells was not 8b-CAMPand forskolin, an activator of adenylate cymediated by the autocrine mechanism involving IL-2 clase that catalyzes the formation of CAMP, resulting in and other growth factors and that cAMP induces the elevation of cAMP level. All these chemical agents growth signals in CT-Mat cells. induced the growth of CT-Mat cells. The forskolinThe mutual effects between 8b-CAMPand IL-2 on induced growth of CT-Mat cells was not mediated by growth of CT-Mat cells were then examined to inves- the autocrine mechanism of IL-2 production, because tigate the difference in the signaling pathway between CT-Mat cells were found not to produce IL-2 in the IL-2-induced growth and CAMP-induced growth. As is presence of cholera toxin or forskolin. Furthermore, shown in Figure 5, any synergistic or cumulative effect any other factor supporting their own growth was not was not observed between 75 pM 8b-CAMPand various detected in the supernatant of CT-Mat cells cultured in doses of IL-2 from 0 to 100 U/ml. The 8b-CAMPseems the presence of forskolin. These results suggest two to be partly suppressive for the IL-Zinduced cell possibilities; one is that cAMP directly induces the growth. Furthermore, the effects of IL-2 on intracellu- intracellular growth signal in CT-Mat cells, and the lar cAMP level were examined with CT-Mat cells. other is that the ex ression of some cellular gene(s), CT-Mat cells were cultured with IL-2 for the indicated which induces the ce 1 growth, is promoted by HTLV-I times, and their intracellular cAMP levels were as- gene products such as p4OtaX,the expression of which is sayed (Table 2). The cAMP level was unaffected up t o at stimulated in CT-Mat cells as compared with ILT-Mat least 15 min after IL-2 stimulation, although it slightly cells, and cAMP is known t o promote the expression of increased after 30 min of IL-2 stimulation. On the other HTLV-I (Jeang et al., 1988; Nakamura et al., 1989).We hand, forskolin induced a significant elevation of CAMP cannot rule out the former possibility because the level within 5 min of treatment. elevation of intracellular cAMP leads to growth of
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yeasts (Matsumoto et al., 1982), and the transient promoting effects of CAMP on proliferation of cells have also been observed in nonlymphoid cells such as fibroblasts (Rozengurt et al., 1981) and liver epithelioid cells (Boynton and Whitfield, 19791, but consider that the latter possibility is more likely because the removal of cholera toxin from CT-Mat cells did not result in a rapid reduction of cell numbers, as shown in Figure 2. In lymphocytes, cAMP was recently suggested to be a second messenger for interleukin-1 (Shirakawa et al., 1988). Since CT-Mat cells are responsive to both IL-2 and cAMP for their growth, the question of whether the IL-2-induced growth is also sustained by the elevation of CAMP arises. However, the present study shows that 8b-CAMPpartially suppressed the IL-2-induced growth of CT-Mat cells and that the intracellular cAMP level was unaffected during the first 15 min of IL-2 stimulation. Similarly, there is evidence that the increased cAMP levels by cholera toxin or forskolin induces suppressive effects on the generation of IL-2 activity (Johnson et al., 1988) and that IL-2 induces a transient decrease of cAMP level in IL-2-dependent cells (Becker and Farrar, 1986). This suggests that cAMP does not play a substantial role in the IL-2-mediated signal transduction. We have previously shown that PKC activation by phorbol esters transduces the growth signal in an IL-2-dependent cell line, TPA-Mat (Takeshita et al., 1988). The phorbol ester-induced growth of TPA-Mat cells was strongly inhibited by forskolin, but their IL-2-induced growth was weakly inhibited, suggesting that the growth signal induced by IL-2 is transmitted through the pathway that is perhaps distinct from that involved in PKC activation (Goto et al., 1988). These data, together with the present study, suggest that the PKC and CAMP-dependent kinase may not play a crucial role in signal transduction directly from IL-2R. However, we (Ishii et al., 1987) and others (Benedict et al., 1987) have already demonstrated the IL-2-stimulated phosphorylation of cellular proteins that contain phosphoserine and phosphothreonine, indicating that IL-2 stimulates some protein kinase catalyzing phosphorylation of serine and threonine residues, although it is still vague whether there is a significant involvement of the protein kinase in the signal transduction pathway. Receptors for some growth factors such as epidermal growth factor, platelet-derived growth factors, insulin, insulin-like growth factor, and colonystimulating factor 1 are known to autophosphorylate on the tyrosine residues (reviewed in references Hanks et al., 1988). Interestingly, it has been reported that IL-2 also stimulates tyrosine phosphorylation on several proteins (Salzman et al., 19881, and we have recently demonstrated that IL-2 stimulates tyrosine phosphorylation of IL-2Rp molecules (Asao et al., 1990). These observations suggest the possibility that some tyrosine kinase associated with IL-2RP is stimulated by IL-2, and this phosphorylation is implicated in the transduction of the growth signal that is different from PKC- and CAMP-dependent pathways. CT-Mat showed here a possible existence of a pathway for cell growth signaling mediated by cAMP in T cells. The variant cell lines derived from an IL-2dependent ILT-Mat, such as CT-Mat and TPA-Mat,
should be useful for the study of the mechanisms of cell growth .
ACKNOWLEDGMENTS We thank Ms. E. Ohkubo for her help in preparation of the manuscript. This work was supported in part by Grants-in Aid for Scientific Research and Cancer Research from the Ministry of Education, Science and Culture, and by a grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research. LITERATURE CITED Asao, H., Takeshita, T., Nakamura, M., Nagata, K., and Sugamura, K. (1990) Interleukin 2 (IL-21-inducedtyrosine phosphorylation of IL-2 receptor p75. J. Exp. Med., 171:637-644. Becker, S.K., and Farrar, W.L. (1986) Interleukin 2 modulation of adenylate cyclase. J. Biol. Chem., 261:3043-3047. Benedict, S.H., Mills, G.B., and Gelfand, E.W. (1987) Interleukin 2 activates a receptor-associated protein kinase. J. Immunol., 139:169&1697. Boynton, A.L. and Whitfield, J.F. (1979) The cyclic AMP-dependent initiation of DNA synthesis by T51B rat liver epithelioid cells. J . Cell. Physiol., 101:139-148. Dukovich, M., Wano, Y., Thuy, L.B., Katz, P., Cullen, B.R., Kehrl, J.H., and Greene, W.C. (1987) A second human interleukin-2 binding protein that may be a component of high-affinity interleukin-2 receptors. Nature, 327:51&522. Friedman, D.L. (1982) Regulation of the cell cycle and cellular proliferation by cyclic nucleotides. J.W. Kebabian, and J.A. Nathanson, eds. Handbook of Experimental Pharmacology, 58,II, pp 151-187. Fujii, M., Sugamura, K., Nakai, S., Tanaka, Y., Tozawa, H., and Hinuma, Y. (1986) High- and low-affinity interleukin 2 receptors: Distinctive effects of monoclonal antibodies. J. Immunol., 137:15521556. Goto, Y., Takeshita, T., and Sugamura, K. (1988) Adenosine 3',5'cyclic monophosphate (CAMP) inhibits phorbol ester-induced growth of an IL-2-dependent T cell line. FEBS Lett., 239:165-168. Hanks, S.K., Quinn, A.M., and Hunter, T. (1988) The protein kinase family: Conserved features and deduced phylogeny of the catalytic domains. Science, 241 :42-52. Hatakeyama, M., Mori, M., Doi, T., and Taniguchi, T. (1989a) A restricted cytoplasmic region of IL-2-receptor p chain is essential for growth signal transduction but not for ligand binding and internalization. Cell, 59:837-845. Hatakeyama, M., Tsudo, M., Minamoto, S., Kono, T., Doi, T., Miyata, T., Miyasaka, M., and Taniguchi, T. (1989b) Interleukin-2 receptor p chain gene: generation of three receptor forms by cloned human a and p chain cDNAs. Science, 244:551-556. Hedrick, S.M., Nielsen, E.A., Kavaler, J., Cohen, D.I., and Davis, M.M. (1984) Sequence relationships between putative T-cell receptor polypeptides and immunoglobulins. Nature, 308:153-158. Holmgren, J . (1981) Actions of cholera toxin and the prevention and treatment of cholera. Nature, 292:413-417. Ide, M., Kaneda, K., Koizumi, K., Hojo, K., Murai, Y., Sagawa, K., Kono, M., and Sato, K. (1987) Neutralizing monoclonal antibodies against recombinant human interleukin-2. J. Immunol. Methods, 10157-62, Ishii, T.,Kohno, M., Nakamura, M., Hinuma, Y., and Sugamura, K. (1987) Characterization of interleukin 2-stimulated phosphorylation of 67 and 63 kDa proteins in human T-cells. Biochem. J., 242:211-219. Jeang, K.-T., Boros, I., Brady, J., Radonovich, M., and Khoury, G. (1988) Characterization of cellular factors that interact with the human T-cell leukemia virus type I p4OX-responsive21-base-pair sequence. J. Virol., 62:4499-4509. Johnson, K.W., Davis, B.H., and Smith, K.A. (1988) cAMP antagonizes interleukin 2-promoted T-cell cycle progression at a discrete point in early G1. Proc. Natl. Acad. Sci. USA, 85:6072-6076. Lee, B., Tanaka, Y., and Tozawa, H. (1989) Monoclonal antibody defining tax, protein of human T-cell leukemia virus type-I. Tohoku J . Exp. Med., 157:l-11. Matsumoto, K., Uno, I., Oshima, Y., and Ishikawa, T.(1982) Isolation and characterization of yeast mutants deficient in adenylate cyclase and CAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA, 79:2355-2359.
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