Sensitization of the HIV-1-LTR upon long term low dose oxidative stress.

Human lymphoid cell lines were transfected with HIV-1-LTR-CAT DNA and permanently transformed cell lines were obtained. These transformed cell lines were treated with 0.01 mM H2O2 for 25 days and the chloramphenicol acetyltransferase (CAT) activities of these cell lines were measured. The CAT activities of transformed cell lines were latent in normal culture conditions, but were activated by retreatment with 0.2 mM H2O2 for 1 h. On treatment with 0.05 mM H2O2 for 1 h, the CAT activity of these cell lines maintained in normal conditions remained latent, whereas cell lines pretreated with 0.01 mM H2O2 for 25 days were greatly activated by this treatment. Here, the HIV-1 promoter seemed latent in normal culture conditions, but it could be activated by a comparatively low concentration (0.05 mM) of H2O2 after treatment with a dilute H2O2 (0.01 mM) for about 1 month. Many patients infected with human immunodeficiency virus 1 (HIV-1) show a long latent period before development of AIDS. During this latent period, their infected cells may be subjected to oxidative stress due to metabolism and physical movement. The present results indicate that oxidative stress may cause activation of the HIV-1 promoter in patients with latent HIV-1.

Almost all patients infected with HIV-1 1 show a latent period of several years before development of acquired immunodeficiency syndrome (AIDS) (1). The production of HIV-1 has been found to be very low before onset of the disease, although the provirus DNA is inserted into the host cell DNA (2,3). The latent HIV-1 can be activated by superinfection with another DNA virus such as the cytomegalovirus or herpes virus (4), but little is known about this mechanism of activation of latent HIV-1. On the other hand, the nuclear factor -B (NF-B) is known to be a strong activator of the HIV-1 promoter (5), and activation of NF-B by hydrogen peroxide (H 2 O 2 ) has been reported (6). These findings suggest that oxidative stress such as hydrogen peroxide and superoxide may be important in the activation of latent HIV-1.
To examine this possibility, we transfected pCD12 (HIV-LTR-CAT) plasmid into human lymphoid cell lines and obtained stable transformants (cf. Refs. 4 and 7). These cell lines showed low CAT activity like latent HIV-1 in normal culture condition. The CAT activity was markedly elevated when these transformant cells were cultured in the presence of 0.2 mM H 2 O 2 for 1 h, but on treatment of 0.05 mM H 2 O 2 the CAT activity remained at a low level. However, when the transformed cells were pretreated with a dilute H 2 O 2 (0.01 mM) for about 25 days, the CAT activity of transformed cell lines could be markedly elevated by retreatment with 0.05 mM H 2 O 2 for 1 h. This activation of the HIV-1 promoter by a comparatively low concentration of H 2 O 2 seems interesting, because patients latently infected with HIV-1 presumably receive various oxidative stresses as results of physical movements and metabolism during the long latency period (8,9). In the early period of latency, the patients may have only cells of the "low CAT activity type" but when they undergo oxidative stress for a long time like cells during a long term culture with a dilute H 2 O 2 , the latent virus may be activated and comparatively low oxidative stress may cause marked activation of HIV-1 gene expression. From this view point, our transformed cell lines may act as a good model of lymphoid cells in patients infected with HIV-1 (cf. Refs. 10 and 11).
H 2 O 2 Treatment and CAT Assay-A part of the transformed cell lines were maintained in normal RPMI 1640 and treated with a low concentration of H 2 O 2 (0.01 mM) for 4 h per day for 25 days and maintained with or without 20 mM of N-acetyl-L-cysteine (NAC) for 24 h. These cell lines were treated with H 2 O 2 (0, 0.05, or 0.2 mM) for 1 h and then they were cultured in normal RPMI 1640 for 48 h, collected, and washed with phosphate-buffered saline. Samples of 5 ϫ 10 5 cells were suspended in 0.25 M Tris-HCl (pH 8.0), and cellular extracts were prepared by five cycles of freezing (Ϫ80°C) and thawing. Chloramphenicol acetyltransferase (CAT) activity was measured by incubating whole cell extracts with 14 C-labeled chloramphenicol and 5 mM acetyl coenzyme A at 37°C for 18 h. Acetylated chloramphenicol was separated from nonacetylated chloramphenicol by ascending thin-layer chromatography (16). Chromatograms were examined and quantitated with a Fuji image analyzer BA100.
Binding of Nuclear Proteins to the NF-B Binding Motif DNA-Nuclear proteins binding to the NF-B binding motif were detected by * This work is supported by a grant from Ministry of Health and Welfare, Research Committee on Prevention of Developing Illnesses and Therapy for HIV Infected Patients. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 The  (18) were used for binding assays. For supershift assays, 10-fold diluted rabbit antiserum against NF-B (p65) (Santa Cruz Biotechnology) was also added to the binding reaction. The complexes of NF-B motif DNA, nuclear protein, and antibody were identified by electrophoresis as described previously (19).
Binding of Nuclear Proteins with HIV-1-LTR DNA-HIV-LTR DNA was digested with SacI and PvuII to obtain a 120-bp DNA fragment (16). This 120-bp fragment, which contained the NF-B motif, was also end-labeled with [␥-32 P]ATP for binding with nuclear extracts. Band shift assays were performed as described above. After band shift assay, shifted bands were removed and eluted from the gel by electrophoresis and their radioactivities determined. The protein-DNA complexes obtained were immunoprecipitated with anti-NF-B p65 rabbit serum, and the recovery of radioactivity of [ 32 P]-labeled DNA was also determined.

RESULTS AND DISCUSSION
Effects of Short Term Treatment with H 2 O 2 on CAT Activity-Transformed cell lines were obtained by the protoplast fusion method as described previously (7). Three cell lines (HL60, U937, and MOLT4) transformed by pCD12 (HIV-LTR-CAT) plasmid were named HL60CD, U937CD, and MOLT4CD, respectively, and the same cells transformed by pSV2CAT plasmid were named HL60SV, U937SV, and MOLT4SV, respectively (cf. Ref. 7), and MOLT4 cells transformed by mutant pCD12 were named MOLT4CD*. For short term H 2 O 2 treatment, these cell lines were treated with 0, 0.05, or 0.2 mM H 2 O 2 for 1 h, then washed with normal medium (RPMI 1640, 10% fetal bovine serum), and cultured for 48 h. Samples of 5 ϫ 10 5 of these cells were then used for measurement of CAT activity by thin-layer chromatography (Fig. 1A, part a). The CAT activities were measured as percentage conversion of the acetylated form of chloramphenicol from extracts derived of transformed cell lines. The CAT activities of HL60CD cell line treated with 0, 0.05, and 0.2 mM H 2 O 2 were 0, 5.2, and 65.3%, respectively, and almost the same results were obtained in U937CD and MOLT4CD cell lines (Fig. 1A, part b). It was found that on short term (1 h) H 2 O 2 treatment with a moderate concentration of H 2 O 2 (0.05 mM), the CAT activity of pCD12transformed cell lines were only weak; however, a high concentration of H 2 O 2 (0.2 mM) treatment resulted in a marked acti-vation of CAT activity (cf. Ref. 6). The CAT activity of pSV2CAT-transformed cells were extremely high and did not change on treatment with H 2 O 2 , but the CAT activity of MOLT4CD* was low and did not change on treatment with H 2 O 2 (Fig. 1A).  Fig.  1A), but in long term treatment (25 days) the CAT activities were about 7.0, 68.4, and 71.0%, respectively (Fig. 1B). It was found that long term dilute H 2 O 2 treatment (4 h/day for 20 days or more) slightly elevated the CAT activity. And when these cells were retreated by 0.05 mM H 2 O 2 for 1 h, marked elevations of CAT activity were observed. Almost the same results were obtained in U937CD and MOLT4CD cell lines. This sensitization of CAT activity by dilute H 2 O 2 was abolished by the antioxidant (NAC, 20 mM) treatment (Fig. 1C). Marked elevation of CAT activity induced by a moderate concentration (0.05 mM) of H 2 O 2 after long term dilute H 2 O 2 treatment may indicate that the latent HIV-1 promoter in infected cells became sensitive on long term treatment with a low concentration (0.01 mM) of H 2 O 2 . In contrast CAT activity of MOLT4CD* cell remained at a low level after long term dilute H 2 O 2 treatment ( Fig. 1B; cf. Fig. 1A).  (Fig.  2A, parts a and b). It was found that the percentage shift of the 42-mer DNA fragment corresponded well with CAT activity in pCD12 transformed cell lines. The same band shift patterns were also obtained in the mutant pCD12 transformed cell line (MOLT4CD*), but the CAT activity of this cell was low ( Fig. 2A,  cf. Fig. 1B). These shifted bands disappeared on addition of 50-fold excess amounts of unlabeled 42-mer DNA fragments, but not on addition of 25-fold excess of unlabeled mutant NF-B motif (Fig. 2A, part a). When the above treated cells were also pretreated with NAC (20 mM), the shifted bands decreased in intensity or disappeared (Fig. 2B). On treatment with NAC, the percentage shift of the labeled 42-mer DNA fragment also corresponded with the CAT activity in pCD12 transformed cell lines (Fig. 2B, cf. Fig. 1C). The shifted bands from nuclear extract of 0.2 mM H 2 O 2 -treated cells were also supershifted by antisera against NF-B p65 protein, and several supershifted bands appeared (in Fig. 2B). These results suggest that pCD12 transformed cells potentially produce an NF-B-like transcription activating factor by long term dilute H 2 O 2 treatment, corresponding with the CAT activity.

Effects of Long Term Dilute H 2 O 2 Treatment on CAT Activity-For
Binding of the NF-B-like Factor to HIV-LTR DNA in Transformed Cells-To examine the binding activity of the NF-Blike factor with the transcription-activating motif in HIV-1-LTR, a SacI,PvuII fragment (120 bp) containing the NF-B binding DNA motif of HIV-1-LTR (20) was isolated, end-labeled, and incubated with nuclear proteins of transformed cell lines. The time courses of mobility shift on long term dilute H 2 O 2 treatment (Fig. 3A) and NAC treatment (Fig. 3B) were also examined. Essentially the same results were obtained as with the 42-mer NF-B DNA binding motif (Fig. 3, cf. Fig. 2). After band shift assay, shifted bands (except supershifted bands) were eluted from the gel and then DNA-protein complexes were immunoprecipitated with anti-NF-B p65 rabbit serum. The recoveries of radioactivity from shifted bands of transformed cells were about 75% or more. These results also strongly suggest that pCD12-transformed cell lines potentially produce an NF-B-like transcription-activating factor (which could bind to the HIV-LTR region) on long term dilute H 2 O 2 treatment corresponding with the CAT activity.
The transformed cells showed a latency for production of CAT activity, but when these cells were maintained in the medium with a low concentration of H 2 O 2 , they became potent producers of an NF-B-like factor, and the HIV-1 promoter was . Part a, a SacI and PvuII fragment of HIV-1-LTR DNA (120 bp) was end-labeled and used for binding assays. E, with 50-fold excess of unlabeled 120-bp DNA added during the band shift assay. q, with 25-fold excess of unlabeled mutant NF-B motif added during the band shift assay. The arrowhead indicates the shifted bands; part b, the percentage shift of the complex of 120-bp DNA and nuclear proteins (cf. Fig. 2). B, reduction of 120-bp shifted band by NAC (20 mM) treatment after long term (25 days) dilute H 2 O 2 (LDH) treatment. Part a, a 120-bp DNA fragment was end-labeled and used for band shift and supershift assay; part b, the percentage shift of the complex of 120-bp DNA and nuclear proteins (cf. Fig. 2). activated by even a moderate concentration of short term H 2 O 2 treatment. Patients infected with HIV-1 show a latency for a long time. The latency for the production of HIV-1 in transformed cell seems to correspond well with the latency of the cells of infected patients (8,9). It is interesting that the activation of transformed cells by treatment with a low concentration of H 2 O 2 for a long time seems to correspond with the activation of the latent HIV-1 virus in patients. Physical movement and normal metabolism should cause various types of oxygen stress in the body (9). As in transformed cells, this oxygen stress over a long period may result in activation of the latent virus by NF-B. Therefore, the present cell system may be a useful model of HIV-1 virus activation and production of AIDS.