Induction of transcription from the long terminal repeat of Moloney murine sarcoma provirus by UV-irradiation, x-irradiation, and phorbol ester.

Abstract

The long terminal repeat (LTR) of Moloney murme sarcoma virus (Mo-MuSV) was used as a model system to study the stress response of mammalian cells to physical carcinogens. The chloramphenicol acetyltransferase (CAT) gene was inserted between two Mo-MuSV LTRs, and the LTR-CAT-LTR construct was used for virus production and was integrated into the genome of NIH 3T3 cells in the proviral form. This construct was used to assure that the integrated CAT gene was driven by the promoter of the LTR. Expression of the CAT gene was stimulated 4-fold by UV irradiation, and the peak of activity was observed at 18 hr. In contrast, stimulation of the CAT expression after x-irradiation was 2-fold and occurred at -6 hr. Phorbol myristate acetate also stimulated CAT-activity 4-fold with a peak at 6 hr. Downregulation of protein kinase C blocked totally the response to x-irradiation brut only partially the response to UV. The protein kinase inhibitor-H7 blocked the response to treatment by UV, x-ray, and phorbol ester.Prokaryotic? cells respond to DNA damage by the induction ofa series of enzymes. This is known as the SOS response (1). In mammalian cells, the "stress response" is in a general sense similar in that a series of genes are activated under various conditions. Herrlich et al. (2) demonstrated that y-irradiation,'UV-irradiation, other agents that damage DNA or block DNA replication, and phorbol ester increased the levels of a series of mRNAs and proteins.The goals of this study were to establish a reporter system for stress response, to determine whether x-ray-and UVirradiation could trigger that response, and to investigate some aspects of the internal signaling system for that response. A retroviral long terminal repeat (LTR) was chosen as the sensor for signals arising from DNA damage. Previous work by others had demonstrated the induction of avian tumor virus production by carcinogens (3), the induction of avian tumor virus transcription by pyrrolizidine alkaloids (4), and the induction of endogenous murine retroviruses by aflatoxin B1 and 2-acetylaminofluorene (5). Also in transformed C3H/10T1/2 murine fibroblasts, transcription of Moloney murine sarcoma virus (Mo-MuSV) LTR sequences was observed that was absent or present at a very low level in untransformed cells (6). Thus, there was considerable evidence that an LTR might be transcriptionally activated due to a stress response from carcinogens.For experiments reported here, the chloramphenicol acetyltransferase (CAT) gene was inserted into a Mo-MuSV vector and stably integrated into the genome of NIH 3T3 cells in the proviral form. Increased CAT expression was observed following UV-irradiation, x-irradiation, and phorbol ester addition. Differences in the kinetics of expression were observed. Down-regulation of protein kinase C affected the response of the CAT gene to both x-ray-and UV-irradiation. The response to both these challenges was blocked by the protein kinase inhibitor H7. MATERIALS AND METHODSPlasmids. The retroviral-derived vector poly-pMV was...

Regulation of tumor necrosis factor gene expression by ionizing radiation in human myeloid leukemia cells and peripheral blood monocytes.

“…Exposure to ionizing radiation stimulated expression twofold, while treatment with H7, a nonspecific isoquinolinosulfonamide inhibitor of protein kinases, totally abrogated the response to x rays (34). Furthermore, preincubation with TPA to downregulate protein kinase C also abolished the response of x rays (34).…”

“…Although little is known about the intracellular signal transduction pathways activated by ionizing radiation, recent findings suggest the involvement ofprotein kinase C. In this regard, transcriptional activation of a construct containing the long terminal repeat ofMoloney murine sarcoma virus linked to the chloramphenicol acetyltransferase gene was examined after x irradiation (34). Exposure to ionizing radiation stimulated expression twofold, while treatment with H7, a nonspecific isoquinolinosulfonamide inhibitor of protein kinases, totally abrogated the response to x rays (34).…”

“…Exposure to ionizing radiation stimulated expression twofold, while treatment with H7, a nonspecific isoquinolinosulfonamide inhibitor of protein kinases, totally abrogated the response to x rays (34). Furthermore, preincubation with TPA to downregulate protein kinase C also abolished the response of x rays (34). Together with the results demonstrating the induction of TNF gene expression by phorbol esters (20,28), these findings suggest that the effects of ionizing radiation on TNF gene expression might also be mediated by a protein kinase C-dependent mechanism.…”

Ionizing radiation induces expression and binding activity of the nuclear factor kappa B.

“…In one study, transcriptional activation ofa construct containing the long terminal repeat of Moloney murine sarcoma virus linked to the chloramphenicol acetyltransferase gene was examined after gamma irradiation (39). Irradiation stimulated expression of the construct twofold, while treatment with H7, a nonspecific isoquinolinesulfonamide inhibitor of protein kinases, or preincubation with 12-O-tetradecanoylphorbol-1 3-acetate (TPA) to downregulate protein ki-nase C, abolished the response ofx rays (39). Similar inhibitory effects have been obtained with H7 and prolonged TPA stimulation during induction of TNF gene expression by ionizing radiation (unpublished data).…”

In vivo activation by ultraviolet rays of the human immunodeficiency virus type 1 long terminal repeat.

“…Like a number of eucaryotic promoters such as c-fos, metallothionein, and collagenase, known to be susceptible to DNA damaging agents and phorbol esters (7), retroviral promoters might be activated during the cascade of events triggered by the stress response. It has been recently reported that such activations by UV, x ray, and phorbol myristate acetate could occur in vitro for the Moloney murine sarcoma virus LTR (12). This effect is mediated by activation of the protein kinase C and would be independent of newly synthesized proteins.…”