Role of intracellular redox status in apoptosis induction of human T-cell leukemia virus type I-infected lymphocytes by 13-cis-retinoic acid.

We have shown that cell cycle progression of human T-cell leukemia virus type I (HTLV-I)-transformed T-cell lines was inhibited by 13-cis-retinoic acid (13cRA). In the present study, we report that 13cRA inhibited proliferation and induced cell death of peripheral blood mononuclear cells obtained from four patients with acute adult T-cell leukemia but not of mitogen- or interleukin 2-activated peripheral blood mononuclear cells from HTLV-I-negative healthy donors. Because HTLV-I-infected lymphocytes are susceptible to oxidative stress, we examined the role of the intracellular redox state in 13cRA-induced cell death using a HTLV-I-positive T-cell line, ATL2, as a model. 13cRA induced apoptosis in ATL2 cells within 48 h in a dose-dependent manner. The ability of 13cRA to induce apoptosis was more potent than that of all-trans-retinoic acid. Apoptosis induction by 13cRA was significantly enhanced by buthionine sulfoximine (BSO), which decreased the levels of intracellular reduced glutathione, although 13cRA by itself did not alter them, suggesting that intracellular reduced glutathione may modulate 13cRA-induced apoptosis. In addition, flow cytometric analysis revealed that 13cRA increased intracellular peroxides in 24 h and that the addition of BSO further enhanced them. Although N-acetylcysteine had only a marginal effect, pretreatment with catalase markedly inhibited 13cRA-induced apoptosis. These results suggest that peroxide generation, ie., oxidative stress, may play a crucial role in the induction of apoptosis by 13cRA and further demonstrate that combined treatment with 13cRA and BSO induces apoptosis of HTLV-I-positive lymphocytes even more potently.

Transactivation of an inducible anti-oxidative stress protein, human thioredoxin by HTLV-I Tax.

Adult T-cell leukemia derived factor (ADF)/human thioredoxin (TRX), which has thiol reducing and radical scavenging activities, plays an essential role on cellular protection against oxidative stress and cell death. TRX itself is induced by various oxidative stress as well as the Human T-cell lymphotropic virus type I (HTLV-I) Tax protein. To investigate the mechanism of this induction, the promoter region of the TRX gene was analyzed. Chloramphenicol acetyltransferase (CAT) reporter constructs containing the TRX promoter sequences responded to the overexpression of the Tax protein, whereas various oxidative agents activated the TRX promoter through a newly identified oxidative responsive element. Moreover, TRX was translocated from the cytoplasm into the nucleus by ultraviolet irradiation, suggesting its possible role on sensing and transducing oxidative signals.

Thiol-mediated redox regulation of lymphocyte proliferation. Possible involvement of adult T cell leukemia-derived factor and glutathione in transferrin receptor expression.

The proliferative response of PBMC to PHA, Con A, OKT3 mAb and IL-2-dependent proliferation of PHA-blasts was examined in a thiol-free environment (cultured in a L-cystine- and GSH-free medium). [3H]TdR incorporation assay and cell cycle analysis revealed that stimulated PBMC could not enter the S phase when deprived of these thiol compounds. In thiol-free cultures, an increase in intracellular free Ca2+ concentration and IL-2R alpha-chain/p 55 (Tac) induction was still observed, whereas transferrin receptor induction was markedly reduced, suggesting that the proliferative response of mitogenically stimulated PBMC was arrested in the late G1 phase in which transferrin receptor is induced. In GSH-depleted cultures, a similar reduction of the proliferative response of PBMC and PHA-blasts was observed when the concentration of L-cystine was lowered, in a dose-dependent manner. Each reduction or loss of proliferative response was partially restored by supplementation of 2-ME or adult T cell leukemia-derived factor (ADF)/human thioredoxin which is considered to be an endogenous dithiol-reducing factor. L-Cystine transport analysis showed that mitogenically stimulated PBMC and PHA blasts incorporated L-cystine, whereas resting PBMC did not. Furthermore, ADF as well as 2-ME exhibited an enhancing activity on the L-cystine transport in PHA blasts. Together with the fact that L-cystine transport is a limiting step in glutathione synthesis, these findings suggest that GSH and ADF might cooperate in the thiol-mediated redox regulation process and might also play key roles in cell cycle (late G1 to S) progression of activated lymphocytes.