John Panozzo

Effects of gamma rays, ultraviolet radiation, sunlight, microwaves and electromagnetic fields on gene expression mediated by human immunodeficiency virus promoter.

Previous work by our group and others has shown the modulation of human immunodeficiency virus (HIV) promoter or long terminal repeat (LTR) after exposure to neutrons and ultraviolet radiations. Using HeLa cells stably transfected with a construct containing the chloramphenicol acetyl transferase (CAT) gene, the transcription of which is mediated by the HIV-LTR, we designed experiments to examine the effects of exposure to different types of radiation (such as gamma rays, ultraviolet and sunlight irradiations, electromagnetic fields and microwaves) on HIV-LTR-driven expression of CAT. These results demonstrated ultraviolet-light-induced transcription from the HIV promoter, as has been shown by others. Exposure to other DNA-damaging agents such as gamma rays and sunlight (with limited exposures) had no significant effect on transcription mediated by HIV-LTR, suggesting that induction of HIV is not mediated by just any type of DNA damage but rather may require specific types of DNA damage. Microwaves did not cause cell killing when cells in culture were exposed in high volumes of medium, and the same cells showed no changes in expression. When microwave exposure was carried out in low volumes of medium (so that excessive heat was generated) induction of HIV-LTR transcription (as assayed by CAT activity) was evident. Electromagnetic field exposures had no effect on expression of HIV-LTR. These results demonstrate that not all types of radiation and not all DNA-damaging agents are capable of inducing HIV. We hypothesize that induction of HIV transcription may be mediated by several different signals after exposure to radiation.

Low doses of neutrons induce changes in gene expression.

Studies were designed to identify genes induced in fibroblasts after exposure to low-dose neutron radiation but not after gamma rays. Our past work had shown similar modulation of transcripts for alpha-tubulin, beta- and gamma-actins, ornithine decarboxylase and interleukin 1 after exposure to either neutrons or gamma rays. However, differences in the expression of beta-protein kinase C and c-fos genes were observed, with both being induced after exposure to gamma rays but not neutrons. Recently we have identified two genes that are induced after exposure to neutrons but not gamma rays: Rp-8 (a gene associated with apoptosis) and the long terminal repeat (LTR) of the human immunodeficiency virus (HIV). Induction of Rp-8 mRNA was demonstrated in Syrian hamster embryo (SHE) fibroblasts and was found to be induced in cells exposed to neutrons administered at low (0.005 Gy/min) and high dose rate (0.12 Gy/min). No induction of other genes associated with apoptosis such as Rp-2, bcl-2 and Tcl-30 was observed. The induction of transcription from the LTR of HIV was demonstrated in HeLa cells bearing a transfected construct of the chloramphenicol acetyl transferase (CAT) gene driven by the HIV-LTR promoter. Measurements of CAT activity and CAT transcripts after irradiation demonstrated an unresponsiveness to gamma rays over a broad range of doses (0.1-3 Gy). Twofold induction of the HIV-LTR was detected after exposure to neutrons (0.48 Gy) administered at low (0.05 Gy/min) but not high (0.12 Gy/min) dose rates. Ultraviolet-mediated HIV-LTR induction, however, was inhibited by exposure to low-dose-rate neutron irradiation. These results are interesting in light of reports that Rp-8 is induced during apoptosis and that HIV causes apoptosis.

THE EFFECTS OF MULTIPLE UV EXPOSURES ON HIV‐LTR EXPRESSION

Abstract— Previous studies have shown that cellular stress agents such as UV radiation induce transcription from the long terminal repeat (LTR) of the human immunodeficiency virus (HIV). Using HeLa cells stably transfected with the HIV‐LTR sequence, which transcriptionally drives the chloramphenicol acetyl transferase (CAT) reporter gene, we examined the effects of multiple exposures to UVC (254 nm) on HIV‐LTR‐CAT expression. Low doses (± 5 J m‐2) had no effect on CAT expression, but up to 29‐fold induction was observed with 10 J m‐2 when cells were harvested 48 h after completion of the exposure. Little difference was noted in induction levels when cells were exposed to one 25 J m‐2 dose, viable cells were harvested at 24 h, 48 h or 72 h, and cell lysates were assayed for CAT expression. Two sequential 12.5 J m‐2 exposures, given 24 h apart, resulted in an additive effect on CAT expression; these two exposures produced CAT activity equivalent to that induced following a single 25 J m‐2 dose. This additive effect was not evident at the lower doses (≤5 J m‐2) or at the higher doses. Maximal induction was observed using doses from 25 to 37.5 J m‐2. Multiple exposures with either the low (≤5 J m ‐2) or high doses (>25 J m ‐2) did not result in an additive effect. Our data suggest that HIV‐LTR requires a specific threshold UV dose in order to elicit induction; a maximal induction dose is also evident; exposures higher than this maximal dose contribute no more to HIV‐LTR induction in viable cells.

The effects of cisplatin and methotrexate on the expression of human immunodeficiency virus type 1 long terminal repeat

Previous work by many groups has documented the induction of HIV-LTR (human immunodeficiency virus-long terminal repeat) following exposure of cells or whole animals to ultraviolet (UV) light and other DNA damaging agents. In these experiments we set out to determine whether exposure to the cancer chemotherapeutic agents methotrexate and cisplatin had any effect on the expression of the HIV-LTR. Using HeLa cells stably transfected with a construct in which HIV-LTR drives the expression of the reporter gene chloramphenicol acetyl transferase (CAT), we demonstrated induction of HIV-LTR 24-48 h following exposure to 50 microM cisplatin. When UV exposure (10 Jm-2) was coupled with cisplatin (50 microM) treatment (which also causes DNA damage), HIV-LTR induction was additive relative to either treatment alone. Methotrexate, which depletes the medium of tetrahydrofolate and does not induce DNA damage, induced HIV-LTR at later (6-7 days) time points than cisplatin or UV treatments. When methotrexate (128 microM) and UV (10 Jm-2) treatments were combined, the agents were synergistic with regard to HIV induction. For both drugs, though, induction was not due to generalized transcriptional activation since both cisplatin and methotrexate induced a repression of total transcription as measured in nuclear run-on assays.

HIV expression is induced in dying cells.

Using HeLa cells stably transfected with an HIV-LTR-CAT construct, we demonstrated a peak in CAT induction that occurs in viable (but not necessarily cell-division-competent) cells 24 h following exposure to some cell-killing agents. gamma rays were the only cell-killing agent which did not induce HIV transcription; this can be attributed to the fact that gamma-ray-induced apoptotic death requires functional p53, which is not present in HeLa cells. For all other agents, HIV-LTR induction was dose-dependent and correlated with the amount of cell killing that occurred in the culture. Doses which caused over 99% cell killing induced HIV-LTR transcription maximally, demonstrating that cells that will go on to die by 14 days are the cells expressing HIV-LTR-CAT.

HIV transcription is induced with some forms of cell killing

Using HeLa cells stably transfected with an HIV-LTR-CAT construct`, we demonstrated a peak in CAT induction that occurs in viable (but not necessarily cell-division-competent) cells 24 h following exposure to some cell-killing agents. {Gamma} rays were the only cell-killing agent which did not induce HIV transcription; this can be attributed to the fact that {gamma}-ray-induced apoptotic death requires function p53, which is missing in HeLa cells. For all other agents, HIV-LTR induction was dose-dependent and correlated with the amount of cell killing that occurred in the culture.

The effects of 5-fluorouracil and doxorubicin on expression of human immunodeficiency virus type 1 long terminal repeat

Previous work by many groups has documented induction of the HIV-LTR following exposure of cells to ultraviolet light and other DNA damaging agents. Our experiments set out to determine the relative activation or repression of the HIV-LTR in response to two classes of chemotherapeutic agents: Doxorubicin is a DNA-damage inducing agent, and 5-fluorouracil has an antimetabolic mode of action. Using HeLa cells stably transfected with a construct in which HIV-LTR drives expression of the chloramphenicol acetyl transferase reporter gene, we demonstrated an up to 10-fold induction following doxorubicin treatment in 24 h post-treatment. This induction was repressed by treatment with salicylic acid, suggesting a role for prostaglandin/cyclo-oxygenase pathways and/or NFKB in the inductive response. Induction by 5-fluorouracil, in contrast, was more modest (two-fold at most) though it was consistently elevated over controls.

HIV transcription is induced with cell killing

In this report, we demonstrate that this induction of HIV-LTR transcription occurs when stably transfected HeLa cells are exposed to agents which mediate cell killing, such as UV radiation, electroporation of sucrose buffer, prolonged heating, and low and high pH. Cells cultured following UV exposure demonstrated a peak in CAT expression that is evident in viable (but not necessarily cell division-competent) cells 24 h after exposure; this inductive response continued until at least 72 h after exposure. HIV-LTR induction was dose-dependent, and the amount of CAT transcription induced was correlated with the amount of cell killing that occurred in the culture.