Chin-Mei Chang-Liu

Differential modulation of specific gene expression following high- and low-LET radiations

Experiments were designed to examine the effects of radiation quality on specific gene expression within the first 3 h following radiation exposure in Syrian hamster embryo (SHE) cells. Preliminary work demonstrated the induction of c-fos and alpha-interferon genes following exposure to low-linear-energy-transfer (low-LET) radiations (X rays or gamma rays). More detailed experiments revealed induction of c-fos mRNA within the first 3 h following exposure to either X rays (75 cGy) or gamma rays (90 cGy). We could not detect induction of c-fos following exposure of SHE cells to fission-spectrum neutrons (high-LET) from the JANUS reactor administered at either high (12 cGy/min) or low (0.5 cGy/min) dose rates. Expression of alpha-interferon mRNA was similarly induced by low-LET radiations but only modestly by JANUS neutrons. The induction by gamma rays was dose-dependent, while induction by neutrons was specific for low doses and low dose rates. These experiments demonstrate the differential gene inductive response of cells following exposure to high- and low-LET radiations. These experiments suggest that these different qualities of ionizing radiation may have different mechanisms for inducing many of the cellular consequences of radiation exposure, such as cell survival and cell transformation.

Sunlight -induced mammalian cell killing: a comparative study of ultraviolet and near-ultraviolet inactivation.

Abstract— We have examined the relationship between the survival curves obtained with UV light (254 nm) and those obtained with a near‐UV sburce (Westinghouse Sun Lamps, FS20) simulating sunlight for cultured Chinese hamster cells, C3H mouse cells, and HeLa cells. In no case do the “sunlight” survival curves have the same shape as the UV survival curves. Also, the combined lethal effects of UV and near‐UV, sunlight‐like exposures are not entirely additive. Hence, we conclude that (1) the cell‐killing photolesions induced by these radiations are at most only partly the same, and (2) in view of (I), caution should be exercised in predicting near UV‐induced dose‐dependencies of other end points based upon observations with UV.

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.

Expression of cytoskeletal elements in proliferating cells following radiation exposure.

Previous work has demonstrated that radiation exposure modulates the expression of a series of genes, including those that encode cytoskeletal elements. The experiments reported here were designed to examine (1) the comparative effects of neutrons administered at high versus low dose-rates, (2) the comparative effects of neutrons on cycling versus resting cells and (3) the comparative effects of neutrons versus gamma-rays on beta- and gamma-actin mRNA accumulation in Syrian hamster embryo (SHE) cells 1 and 3 h post-irradiation. JANUS fission-spectrum neutrons from Argonne National Laboratorys JANUS reactor administered at high (12 cGy/min) dose-rates had little effect on resting cells, but at very low dose-rates (0.1 cGy/min) had a repressive effect on gamma-actin mRNA accumulation. Increased accumulation of beta-actin mRNA was detected following the exposure of cells to neutrons administered at high dose-rates, but repression of beta-actin mRNA was observed when neutrons were administered at low dose-rates. Cycling cells (unexposed and neutron irradiated) in all cases expressed higher levels of all actin-specific mRNAs than resting cells; beta-actin mRNA (but not gamma-actin mRNA) was induced to a greater extent in cycling cells than in resting cells during the first hour following neutron exposure. In resting cells, however, low dose-rate neutrons were more effective than low dose-rate gamma-rays at repressing both gamma- and beta-actin mRNA accumulation. These results demonstrate the differential effects of radiation quality (neutrons versus gamma-rays) and cell-cycle state on the modulation of actin isotype-specific gene expression.

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.

Modulation of calmodulin by UV and X-rays in primary human endothelial cell cultures.

Purpose : Previous studies by the present authors and others have shown that the expression of many genes is modulated by radiation. The purpose of this study is to identify additional genes that are affected by UV and X-radiation. Identification of specific genes affected by radiation may allow the determination of pathways important in radiation responses as well as an examination of transcriptional elements that are involved in the process. Materials and methods : A modified differential display approach coupled with sequencing was used to identify genes that are modulated in response to UV and ionizing radiation, and Northern blot analysis was used to confirm specific gene modulation. Results : Treatment of human primary umbilical vein endothelial cells with UV radiation resulted in the differential expression of several genes. Sequencing of the bands revealed that one of these was calmodulin. There was a 30% reduction in accumulation of calmodulin-specific mRNA 1h post UV exposure, and a 50% decrease 3h after treatment. X-rays also repressed accumulation of calmodium mRNA. Radiation exposure of HeLa cells also resulted in a decrease in expression of this gene. Conclusions : UV and ionizing radiations cause a decrease in accumulation of calmodulin transcripts in the first 1-3 h following exposure. Repression of calmodium mRNA levels may be one mechanism of stress-induced intracellular Ca 2+ modulation.PURPOSE Previous studies by the present authors and others have shown that the expression of many genes is modulated by radiation. The purpose of this study is to identify additional genes that are affected by UV and X-radiation. Identification of specific genes affected by radiation may allow the determination of pathways important in radiation responses as well as an examination of transcriptional elements that are involved in the process. MATERIALS AND METHODS A modified differential display approach coupled with sequencing was used to identify genes that are modulated in response to UV and ionizing radiation, and Northern blot analysis was used to confirm specific gene modulation. RESULTS Treatment of human primary umbilical vein endothelial cells with UV radiation resulted in the differential expression of several genes. Sequencing of the bands revealed that one of these was calmodulin. There was a 30% reduction in accumulation of calmodulin-specific mRNA 1 h post UV exposure, and a 50% decrease 3 h after treatment. X-rays also repressed accumulation of calmodulin mRNA. Radiation exposure of HeLa cells also resulted in a decrease in expression of this gene. CONCLUSIONS UV and ionizing radiations cause a decrease in accumulation of calmodulin transcripts in the first 1-3 h following exposure. Repression of calmodium mRNA levels may be one mechanism of stress-induced intracellular Ca2+ modulation.

Changes in gene expression following EMF exposure

Experiments were designed to examine the effects of electromagnetic field (EMF) exposure on specific gene expression, an effect that can be deleterious, beneficial, or neutral, depending on the long-term consequences; however, the proof of a reproducible, quantitative biological effect (such as change in gene expression) will lead to latter experiments aimed at determining the relative contribution of these changes to cellular consequences. Past work by ourselves and by others has shown that measures of gene expression are extremely sensitive indicators of the cellular and biological effects of ionizing radiation, with transcriptional changes being detected by exposure of cells to doses of {gamma}-rays as low as 0.01 cGy that have no pronounced cellular consequences. On the basis of this work, the authors hypothesized that measures of gene expression will be equally sensitive to EMF effects on cells.

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.

Effect of passage number on cellular response to DNA-damaging agents: Cell survival and gene expression

The effect of different passage numbers on plating efficiency, doubling time, cell growth, and radiation sensitivity was assessed in Syrian hamster embryo (SHE) cells. Changes in gene expression after UV or {gamma}-ray irradiation at different passage numbers were also examined. The SHE cells were maintained in culture medium for up to 64 passages. Cells were exposed to {sup 60}Co {gamma} rays or 254-m UV radiation. Differential display of cDNAs and Northern blots were used for the study of gene expression. With increasing passage number, SHE cells demonstrated decreased doubling time, increased plating efficiency, and a decreased yield in the number of cells per plate. Between passages 41 and 48 a ``crisis`` period was evident during which time cell growth in high serum (20%) was no longer optimal, and serum concentrations were reduced (to 10%) to maintain cell growth. Sensitivity to ionizing radiation was no different between early- and intermediate-passage cells. However, after UV exposure at low passages (passage 3), confluent cells were more sensitive to the killing effects of UV than were log-phase cells. At intermediate passages (passages 43, 48), confluent cells were slightly more radioresistant- than were log-phase cells. By passage 64, however, both confluent and log-phase cells showed similar patterns of UV sensitivity. Expression of {gamma}-actin, PCNA, and p53 transcripts did not change following UV exposure. p53 mRNA was induced following {gamma}-ray exposure of the intermediate (passage 45) epithelial cells. Differential display, however, revealed changes in expression of several transcripts following exposure to ionizing and ultraviolet radiations. The observed differences in radiation sensitivity associated with increasing passage number may be influenced by radiation-induced gene expression. We are conducting experiments to identify these genes.

HIV transcription 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. 14 refs., 4 figs., 1 tab.