Activation of the human immunodeficiency virus (HIV), the etiologic agent responsible for the acquired immune deficiency syndrome (AIDS), is a complex process involving both viral and cellular factors (1,2). Although virally encoded proteins such as tat and rev are known to regulate viral replication by both transcriptional and posttranscriptional mechanisms, less is understood about activation of HIV through changes in the cellular environment (3-5). It has been demonstrated that T-cell activation by phytohemagglutinin, CD-3/T-cell receptor stimulation, viral genes, and calcium ionophores can induce viral transcription through stimulation of the HIV long-terminal repeats (LTR) (6-8). Recent data also suggest that certain cytokines, such as granulocyte macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor, can induce HIV LTR activation (9-11). For the most part, however, factors external to the HIV genome that activate a relatively dormant provirus are not well understood.
Much attention has recently surrounded the observation that UV light can activate the HIV LTR (12-15). Although the mechanism by which UV-induced LTR activation occurs has yet to be identified, investigators have generally implicated DNA damage as the initiating stimulus for cellular responses that ultimately lead to LTR activation (13). Because patients with AIDS often develop malignancies (e.g., non-Hodgkin's and Hodgkin's lymphoma, primary central nervous system lymphoma, Kaposi's sarcoma) and are treated with chemotherapy and radiotherapy, we were interested in exploring whether genotoxic stimuli other than UV light could activate the HIV LTR and stimulate virus replication. The results of this study demonstrate that although certain chemotherapeutic agents do strongly induce HIV LTR activation and virus replication, this is not a general consequence of DNA damage, as many genotoxic drugs and ionizing radiation have little or no effect on the LTR.
Cells and Culture Conditions
HeLa cells were obtained from American Type Culture Collection (ATCC, Rockville, MD, U.S.A.) and grown in Eagle's minimal essential media with 10% nonessential amino acids and 10% fetal bovine serum. Jurkat cells, clone EG-1, MT-4 cells, a human T-lymphotropic virus type 1 (HTLV-1)-transformed T lymphoid cell line, and U937 human monocytic leukemia cell lines were obtained from ATCC and grown in RPMI with 10% fetal bovine serum (6).
The 1 × 107 HeLa or Jurkat cells were cotransfected by electroporation (Gene Pulser, Biorad: 25 V, 960 μF) with 20 μg of pT219 III chloramphenicol acetyltransferase (CAT) plasmid encoding the CAT gene driven by the HIV LTR or pT219 Δ III CAT (the same construct except for an internal deletion of the NF-κB binding site; base pairs - 109 to - 79 in the HIV LTR) (16) and 0.5 μg of the pSV2-neo plasmid containing the neomycin 3' phosphotransferase gene driven by the SV40 promotor. Clones were selected for 2 weeks in the presence of 400 μg/ml (active drug) geneticin sulfate and then screened for the presence of CAT activity. Further cloning was performed by limiting dilution using 96-well microwell plates (Microtest III, Becton Dickinson Co, Lincoln Park, NJ, U.S.A.).
Cells were treated with various agents as described in the text. Mitomycin C (MMC) methotrexate, cisplatinum (II) diammine dichloride (CDDP), daunorubicin, doxorubicin, methylmethane sulfonate (MMS), hydrogen peroxide (H2O2), VP-16 (etoposide), and 1-β-D-arabinofuranosylcytosine (ara-C) were all purchased from Signa Chemical Co. (St. Louis, MO, U.S.A.); 1, 3-bis (2-chloroethyl)-1-nitrosourea (BCNU) was purchased from Bristol-Myers U.S. Pharmaceutical and Neutral Groups (Evansville, IN, U.S.A.). Ionizing radiation was delivered with a gamma cell 1,000 U (Atomic Energy of Canada Ltd., Ontario) with a 137Cs source emitting at a fixed dose rate of 14.3 Gy as determined by dosimetry.
As previously described, UV irradiation was delivered in a specifically constructed sterile chamber (17). Cells were washed twice with phosphate-buffered saline (PBS) and irradiated without PBS (HeLa cells) or with a minimal amount of PBS plated on a 10-cm-diameter culture dish (Jurkat cells) with a dose of 10 and 20 J/m2. The original medium was added back to the cells. The same procedure was performed for control cultures.
Forty-two hours after treatment, CAT assays were performed in a final volume of 50 μl containing 1 μl of 50 μM Ci/mmol D-threo-(dichloroacetyl-1-2-14C)-chloramphenicol (DuPont NEN Research Products, Boston, MA, U.S.A.), 200 μg acetylcoenzyme A lithium salt (Sigma Chemical Co, St. Louis, MO, U.S.A.), and 0.04 M tris-HCl (pH 8.0) with 200 μg of freeze-thaw fractionated cell lysates as previously described (18). Reaction mixtures were incubated at 37°C for 1 h and developed in a solvent of 95% chloroform and 5% methanol on thin-layer chromatography plate. Nonacetylated and acetylated chloramphenical were counted by β-emitter counter (CS500TD, Beckman, Palo Alto, CA, U.S.A.).
Acute Infection of T-lymphoid Cells MT4
As previously described, 1 × 106 of MT4 cells were pretreated with different concentrations of drugs for 24 h and then infected with HIV at a multiplicity of infection (MOI) of 0.1 (19). Virus stocks were prepared from supernatants of MT4 cells transfected with the HxBH10 infectious molecular clone (19). Four hours postinfection, cells were washed and cultured in complete RPMI-1640 medium (RPMI + 10% fetal bovine serum) containing each drug concentration. At each time interval indicated, viable cells (as measured by their capacity to exclude trypan blue) were counted and culture supernatants collected for reverse transcriptase (RT) assay (20).
The chronically infected U-937 myelomonocytic cell culture was made as follows: 5 × 105 U937 cells chronically infected with HIV IIIB (named UHC-1) (21) were mixed with 5 × 106 of normal U937 cells. The percentage of infected cells in the total population was 10% as measured by anti-HIV immunofluorescence.
To investigate the effect of CDDP and Ara-C on the replication of HIV-1 and cell growth, 1 ml of 5 × 105/ml mixed cells were plated in each well of a 24-well plate in the presence of 0, 0.5, 2, 10, μM of each drug, respectively. Each day, the viable cells were counted (as assayed by trypan blue exclusion), and the cultured media was changed and collected for RT assay (20). The RT activity in the supernatants were calculated based on 1 × 106 cells.
Previous studies have evaluated the effects of UV light on HIV LTR activation by using transient transfection assays of reporter plasmids (10). Because only a relatively small number of cells take up plasmid DNA following transient transfections and DNA damage following genotoxic stimuli varies from cell to cell, we chose to use cell lines stably transfected with the pT219 III CAT constructs so that all target cells would contain the reporter plasmid (HeLa III CAT).
We first attempted to reproduce the finding that UV light induces HIV LTR. We found that UV irradiation of HeLa III CAT cell lines at doses of 10 and 20 J/m2(22) induced CAT activity by 3.6- and 13-fold over controls, respectively (Table 1). Based on the hypothesis that DNA damage was the precipitating event leading to induction of the LTR, we were interested in the effects of other DNA-damaging stimuli on LTR induction in pooled clones of HeLa cells. We first evaluated the ability of ionizing radiation to activate the LTR. Doses of radiation between 5 and 500 cGy had no detectable effect on CAT activity. Increasing doses of ionizing radiation between 1,000 and 10,000 cGy caused only a slight increase in CAT activity of 2 to 2.9 times baseline levels. Hydrogen peroxide (H2O2), like ionizing radiation, induces DNA damage through the formation of oxygen-free radicals. High concentrations of H2O2 (300 μM) also had little, if any, effect on CAT activity (1.8-fold induction) in the HeLa III CAT cells. It should be noted that for all doses of radiation and H2O2 tested, little cell death was seen, at least in the 2 days between treatment and harvesting of cells for CAT assays.
We next evaluated the ability of a number of genotoxic agents to activate the HIV LTR in the pooled clones of HeLa transfectants. We chose to evaluate drugs with different mechanisms of action. These drugs included the alkylating agents MMC, CDDP, and MMS, antimetabolites including methotrexate and Ara-C, a nitrosourea (BCNU), the topoisomerase 1 inhibitor camptothecin, the topoisomerase 2 inhibitor etoposide, and the anthracyclines doxorubicin and daunorubicin. We tested each drug at two doses, one that causes moderate cytotoxicity (approximately LD10), and a dose at which no cytotoxicity is observed (80% lower dose). As shown in Table 1, most agents, including VP-16 (etoposide), methotrexate, MMS, camptothecin, BCNU, and Ara-C, caused minimal (less than twofold) or no induction of the LTR. In contrast, MMC and CDDP caused significant induction of the LTR (>sevenfold induction) at the higher dose. The anthracyclines (daunorubicin and doxorubicin) were also effective in inducing HIV LTR, although at levels lower than that seen with MMC (fourfold induction at the highest doses evaluated).
Earlier reports have implicated the NF-κB response element in the HIV LTR as the UV responsive element (15). To test whether this element was important for responsiveness to genotoxic drugs, we constructed HeLa and Jurkat (human T-lymphoblasts) cell lines with stably integrated HIV LTR CAT constructs containing an internal deletion of the NF-κB-responsive sequence (base pairs - 79 to - 109, pT219 Δ III CAT). Deletion of the NF-κB element did not appear to have any effect on the responsiveness of the LTR to either MMC or CDDP. Furthermore, although we confirmed the observation that this LTR deletion diminished induction by UV light, to our surprise, this effect appeared to be dose related. As seen in Table 2, 10 J/m2 of UV irradiation induced a 3.6 and 5.5 induction in wild-type LTR activation in the pooled clones of HeLa and Jurkat cells, respectively. There was no induction, however, in the HeLa and Jurkat cells transfected with the pT219 Δ III CAT plasmid. When the HeLa cell lines were irradiated with 20 J/m2 of UV light, however, both LTR constructs were induced to a similar extent (from 10- to 15-fold induction in three different experiments). No data could be obtained for the Jurkat cells irradiated with 20 J/m2 of UV light, as this amount of irradiation was cytotoxic to more than 90% of the cells. These findings suggested that elements other than NF-κB are responsive to high doses of UV irradiation as well as MMC and CDDP.
Although transcription of the HIV provirus is initiated at the LTR, control of HIV replication is a complex process regulated by viral and cellularly encoded proteins that work both in cis and trans. Thus, activation of the LTR, as assessed by induction of a reporter gene, may not necessarily correlate with increased virus production in infected cells. Only if drug-induced LTR activation led to increased virus production would these observations have potential clinical relevance. We therefore treated HIV-infected U-937 myelomonocytic cells with varying concentrations of Ara-C and CDDP. Because U-937 cells are more sensitive to the cytotoxic effects of the Ara-C and CDDP than HeLa cells, we used significantly lower doses of drugs than were used in the HeLa cell transactivation assay. As seen in Fig. 1A, all tested concentrations of CDDP caused a fourfold to fivefold increase in virus production as normalized for viable cells. Ara-C, which was roughly equivalent in cytotoxicity to U-937 cells, as was CDDP (Fig. 1), however, caused no significant increase in virus production. These data, along with previously reported data on the effects of UV light on virus replication, suggest that induction of CAT activity following drug exposure of the HeLa III CAT cells closely correlates with the effect of these drugs on virus replication.
Because HIV is not directly cytopathic for monocytic cells like U-937, we were interested in evaluating the effects of drug exposure on virus production and cell viability in lymphocyte cultures where HIV is cytopathic (23). Because CDDP was so toxic to the human lymphoblastic cell line MT-4, we chose to evaluate the effects of the less toxic MMC on acutely infected MT-4 cells. CD4-positive MT4 cell lines were treated 24 h before infection with noncytotoxic concentrations of MMC (0.03, 0.06, and 0.125 μM). The MMC-treated cells were infected with virus produced from an infectious molecular clone of HIV, HxBH10 at a MOI of 0.1. We monitored HIV infection by counting viable cells and measuring virus production by RT activity assayed at different intervals. As seen in Fig. 2A, virus production was significantly increased in cells treated at the highest concentrations of MMC. As shown in Fig. 2B, this increase in virus production was accompanied by a large increase in MT4 cytotoxicity. In contrast, uninfected MT4 cells treated with MMC experienced significantly less cytotoxicity.
This study supports previous reports that the HIV LTR can be activated by UV radiation. In addition, we demonstrate that several other DNA-damaging agents. including MMC, CDDP, and the anthracyclines, can similarly activate HIV LTR. This effect, however, is specific to these agents, and most of the genotoxic drugs/stimuli that we tested do not have this activity. Furthermore, although activation of the NF-κB element may be involved in UV-induced LTR transcriptional regulation, other factors are probably involved. Finally, we demonstrate that treatment of infected cell lines with MMC not only increase viral replication, but increase lymphocyte cytotoxicity.
Several reports over the last few years have documented the effect of UV irradiation on the induction of the HIV LTR (12-15,24). Valerie et al. demonstrated that UV irradiation and MMC could activate the LTR and that UV light increased virus production in chronically infected human T-cell lines (13). They concluded that cellular stress responses following DNA damage mediate this effect. Morrey et al. demonstrated that UV irradiation can similarly induce the HIV LTR in vitro using a transgenic mouse harboring a HIV LTR CAT minigene cassette (14). Although it has been shown that the LTR responsiveness to UV light is dependent on the NF-κB response element, our data suggest that this dependence can be overridden by increasing the dose of UV irradiation. Likewise, MMC and CDDP-induced LTR activation do not appear to depend on the presence of the NF-κB response element. Thus, it appears likely that other mechanisms must exist that allow induction of the HIV LTR following genotoxic stimuli.
Some have suggested that HIV LTR activation is a general consequence of DNA damage. An important finding in our study, however, is that HIV LTR activation is not a generic effect of all DNA-damaging agents, but rather specific to only a few agents. DNA-damaging agents are known to cause specific types of damage. For instance, UV light causes thymidine dimer formation, whereas agents such as Ara-C and MMS cause single-stranded DNA breaks and DNA alkylation, respectively (25-28). The cellular responses to these types of damage are not very well understood, but it is likely that these responses may be specific to different types of DNA lesions (29-32). It appears that some of these processes, but not others, result in LTR activation. Consistent with this idea is a report by Valerie et al. suggesting that “bulky” DNA lesions, like those caused by UV light, result in a repair process (called “long patch” repair) that causes changes in DNA chromatin structure (15). This presumably allows better access to the HIV LTR by transcriptional factors, which is in contrast to ionizing radiation-mediated base excision repair (or “short patch” repair), where changes in DNA chromatin structure are less pronounced (34-38). Our data extend the hypothesis that genotoxic-induced HIV LTR activation is a function of long versus short patch repair. Our data are consistent with the idea that changes in chromatin structure rather than the specific DNA lesion or the actual repair process may be the important variable (15). In our study, the agents that activate the LTR, besides UV light and MMC (which clearly do induce long patch repair), were CDDP and the anthracyclines. Neither of the two latter agents cause long patch repair; however, both compounds physically interact with DNA. The anthracyclines intercalate into the DNA helix, whereas the active platinum metabolite forms DNA adducts (39-41). Both these interactions cause substantial distortion of DNA and/or chromatin tertiary structure (42,43). Thus, in agreement with Valerie et al., we speculate that the unifying factor that differentiates which genotoxic agent can activate the HIV LTR may be the ability of the agent to cause changes either directly (through physical interaction with DNA) or indirectly (through induction of DNA repair processes like long patch repair) in DNA and chromatin tertiary structure. This may allow greater accessibility to the LTR by transcriptional factors. Our group and others have demonstrated that a number of early response genes, including c-jun, c-fos, and EGR-1, are activated following genotoxic stimuli (44-46). This results in an increase in AP-1 activity, which is potentially important because the HIV-LTR contains AP-1 recognition sequences. Whether this relates to LTR activation remains speculative, however, and the final elucidation of the mechanism of drug-induced LTR activation awaits a better understanding of the cellular responses to genotoxic stress and further LTR deletional analysis.
Regulation of HIV replication is a complex process involving both viral and cellular regulatory proteins; thus, agents that activate the LTR in vitro may not necessarily increase viral replication (1,2). We therefore asked whether exposure of HIV-infected cells to different genotoxic agents would increase viral replication. CDDP significantly (fivefold) increased virus production in chronically infected U-937 cells compared with Ara-C. These results were consistent with the LTR-CAT activation experiments. Because Ara-C was equally cytotoxic to the U-937 cells as was CDDP, events associated with cell death could not have explained these differences. To ensure that our observations were not cell type specific and were pertinent to acute infections (as opposed to chronically infected cells), we examined the effects of MMC in an acutely infected human lymphocyte cell line (MT4). Again, MMC significantly increased viral production. Interestingly, there was significantly greater cytotoxicity at the 0.125 μM concentration of MMC in the infected cells compared with similarly treated uninfected MT4 cells (Fig. 2B). Based on the demonstration that MMC treatment significantly enhanced virus production from acutely infected MT4 cells (Fig. 2A), the most likely explanation for the accelerated cytotoxicity observed in these cells is that the enhanced virus replication led to increased HIV-mediated cytopathicity (23). It cannot be ruled out, however, that infection with HIV also sensitizes cells to drug-induced cell death by some other mechanism, as has been described for certain DNA viruses (i.e., adeno-associated virus) (47).
These observations have potential clinical significance. As antiviral therapy and improved supportive measures allow AIDS patients to live longer, a growing number of AIDS-associated malignancies (i.e., lymphoma, Kaposi's sarcoma, etc.) are being seen. These neoplasms are often treated with various chemotherapeutic agents. Given the results of this study, it is conceivable that certain treatment regimens may induce viral replication following each course of chemotherapy. This could be particularly important because antiviral therapy (i.e., AZT) is often terminated while patients are receiving chemotherapy. This could theoretically result in accelerated CD4 lymphocyte depletion and AIDS progression.
Acknowledgment: We thank Yichen Lu for the PT219IIICAT and PT219IIIΔCAT plasmid constructs. X.J.Y. is the recipient of a studentship from the National Health Research and Development Program (NHRDP) of Canada. E.A.C. is a recipient of a NHRDP AIDS career award. D.W.K. is supported from PHS grants CA 55241 and 38493. H.F. is supported by NIH K11 award #CA01467-03. E.A.C. is supported by grants from Medical Research Council (MRC) and NHRDP.
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Keywords:© Lippincott-Raven Publishers.
Human immunodeficiency virus (HIV); DNA damage; Long terminal repeat (LTR); Radiation; Ultraviolet light; Chemotherapy