Presence of divalent cation is not mandatory for the formation of intramolecular purine-motif triplex containing human c-jun protooncogene target.

Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine•pyrimidine (Pu•Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F., Monnot, M., Merle, A., Malvy, C., and Fermandjian, S. (1995) Nucleic Acids Res. 23, 3831-3836). Herein, we show that a designed intramolecular version of the 11-bp core sequence of the said targets, which also constitutes an integral, short, and symmetrical segment (G(2)AG(5)AG(2))•(C(2)TC(5)TC(2)) of human c-jun protooncogene forms a stable triplex, even in the absence of magnesium. The sequence d-C(2)TC(5)TC(2)T(5)G(2)AG(5)AG(2)T(5)G(2)AG(5)AG(2) (I-Pu) folds back twice onto itself to form an intramolecular triple helix via a double hairpin formation. The design ensures that the orientation of the intact third strand is antiparallel with respect to the oligopurine strand of the duplex. The triple helix formation has been revealed by non-denaturating gel assays, UV-thermal denaturation, and circular dichroism (CD) spectroscopy. The monophasic melting curve, recorded in the presence of sodium, represented the dissociation of intramolecular triplex to single strand in one step; however, the addition of magnesium bestowed thermal stability to the triplex. Formation of intramolecular triple helix at neutral pH in sodium, with or without magnesium cations, was also confirmed by gel electrophoresis. The triplex, mediated by sodium alone, destabilizes in the presence of 5'-C(2)TC(5)TC(2)-3', an oligonucleotide complementary to the 3'-oligopurine segments of I-Pu, whereas in the presence of magnesium the triplex remained impervious. CD spectra showed the signatures of triplex structure with A-like DNA conformation. We suggest that the possible formation of pH and magnesium-independent purine-motif triplexes at genomic Pu•Py sequences may be pertinent to gene regulation.

Immediate-early inducible function in upstream region of junB gene.

OBJECTIVE: To analyze the upstream region of radiation-induced junB gene. METHODS: Four plasmids containing 250 bp, 590 bp, 900 bp and 1650 bp, and CAT reporter gene were constructed separately and introduced to L8704 cells. The cells were irradiated with 2 Gy X-rays and incubated at different intervals. Total RNA was extracted from the cells and fluctuation of the CAT mRNA level was assessed by the RNA ratio of CAT/beta-actin measured by quantitative Northern blot hybridization. RESULTS: CAT mRNA expression containing 900 bp and 1560 bp junB promoter remarkably and rapidly increased, and reached its peak 30 min after 2 Gy X-ray irradiation. CONCLUSIONS: 590-900 bp fragments located in the upstream region of junB gene play an important role in the early process of cells against radiation.

Preferential induction of c-fos versus c-jun protooncogene during the immediate early response of pig skin to gamma-rays.

The involvement of the nuclear protooncogenes c-fos and c-jun in the immediate early response of pig dermis cells was studied after in vivo gamma-irradiation. Following high radiation doses (8 to 48 Gy), the two protooncogenes were concomitantly induced, although c-fos induction was preferential. Both inductions were time and dose dependent. Therefore, the early response of the skin to high doses of radiation might involve heterodimeric activator protein 1 composed of c-Fos and c-Jun proteins. Following low radiation doses (0.5 to 2 Gy), c-jun was not induced. By contrast, dramatic c-fos induction was observed after 0.5 Gy, suggesting a specific role for c-fos at low doses.

Role of tyrosine phosphorylation in radiation-induced activation of c-jun protooncogene in human lymphohematopoietic precursor cells.

We examined the effects of ionizing radiation on c-jun expression in human lymphohematopoietic precursors. Radiation exposure increased the level of c-jun transcripts in a dose- and time-dependent manner, providing direct evidence that ionizing radiation can activate c-jun protooncogene in human lymphohematopoietic precursors. Notable gamma-rays failed to induce c-jun expression in cells pretreated with herbimycin, and the use of cycloheximide did not overcome the inhibitory effects of herbimycin. The lack of c-jun signal in herbimycin-treated cells was not due to nonspecific damage to the distal protein kinase C signaling pathway. Thus, protein tyrosine kinase activation precedes and perhaps mandates radiation-induced activation of c-jun protooncogene expression in human lymphohematopoietic precursors.

Prolonged c-jun expression in irradiated ataxia telangiectasia fibroblasts.

PURPOSE: Ataxia telangiectasia (AT) is an autosomal recessive disorder associated with radiation sensitivity and an increased incidence of leukemia, lymphoma, and some solid tumors. After exposure to ionizing radiation, cells from patients with AT demonstrate an attenuated G1-phase checkpoint. Because c-jun is known to regulate, in part, the exit from G1 and the onset of DNA replication, we analyzed c-jun transcription in irradiated AT fibroblasts. METHODS AND MATERIALS: AT5BI fibroblasts were irradiated and RNA was extracted and assayed for c-jun expression by Northern blot analysis. Transcriptional regulation of c-jun was evaluated by use of the 5' untranslated region of the jun promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene. Deletion mutants of the RSRF, SP-1, AP-1 and CCAAT domains within the jun promoter linked to the CAT reporter were transfected into AT5BI cells. Transfectants were irradiated, and CAT expression was quantified. After x-irradiation, nuclear protein binding to CCAAT was evaluated by an electrophoretic mobility shift assay. RESULTS: X-ray-mediated c-jun expression was sustained in AT5BI cells as compared to only transient expression in irradiated normal diploid fibroblasts. Mutation of either the AP-1 or CCAAT domains within the c-jun promoter reduced transcription by 50% and combined deletion of both AP-1 and CCAAT cis-acting elements entirely eliminated radiation-mediated transcriptional activation. Electrophoretic mobility gel shift assay of the nuclear proteins isolated from irradiated AT fibroblasts demonstrated their increased binding to the CCAAT sequence at 30 min after irradiation. Competition for nuclear protein binding to the CCAAT sequence with excess cold CCAAT demonstrated that protein binding to this sequence was specific. These findings were distinct from induction by phorbol esthers in that the RSRF cis-acting element and DNA segments upstream of -132 base pairs do participate in c-jun induction by phorbol esthers but not by radiation. CONCLUSIONS: Radiation-mediated transcriptional regulation of c-jun is prolonged in AT fibroblasts and is regulated in combinatorial control by the AP-1 and CCAAT domains, and transcriptional regulation is distinct from that induced by phorbol esthers.

Ketoconazole attenuates radiation-induction of tumor necrosis factor.

PURPOSE: Previous work has demonstrated that inhibitors of phospholipase A2 attenuate ionizing radiation induced arachidonic acid production, protein kinase C activation and prevent subsequent induction of the tumor necrosis factor gene. Because arachidonic acid contributes to radiation-induced tumor necrosis factor expression, we analyzed the effects of agents which alter arachidonate metabolism on the regulation of this gene. METHODS AND MATERIALS: Phospholipase A2 inhibitors quinicrine, bromphenyl bromide, and pentoxyfylline or the inhibitor of lipoxygenase (ketoconazole) or the inhibitor of cyclooxygenase (indomethacin) were added to cell culture 1 h prior to irradiation. RESULTS: Radiation-induced tumor necrosis factor gene expression was attenuated by each of the phospholipase A2 inhibitors (quinicrine, bromphenyl bromide, and pentoxyfylline). Furthermore, ketoconazole attenuated X ray induced tumor necrosis factor gene expression. Conversely, indomethacin enhanced tumor necrosis factor expression following irradiation. CONCLUSION: The finding that radiation-induced tumor necrosis factor gene expression was attenuated by ketoconazole suggests that the lipoxygenase pathway participates in signal transduction preceding tumor necrosis factor induction. Enhancement of tumor necrosis factor expression by indomethacin following irradiation suggests that prostaglandins produced by cyclooxygenase act as negative regulators of tumor necrosis factor expression. Inhibitors of tumor necrosis factor induction ameliorate acute and subacute sequelae of radiotherapy. We propose therefore, that ketoconazole may reduce acute radiation sequelae such as mucositis and esophagitis through a reduction in tumor necrosis factor induction or inhibition of phospholipase A2 in addition to its antifungal activity.

Proto-oncogene mRNA levels and activities of multiple transcription factors in C3H 10T 1/2 murine embryonic fibroblasts exposed to 835.62 and 847.74 MHz cellular phone communication frequency radiation.

This study was designed to determine whether two differently modulated radiofrequencies of the type generally used in cellular phone communications could elicit a general stress response in a biological system. The two modulations and frequencies studied were a frequency-modulated continuous wave (FMCW) with a carrier frequency of 835.62 MHz and a code division multiple-access (CDMA) modulation centered on 847.74 MHz. Changes in proto-oncogene expression, determined by measuring Fos, Jun, and Myc mRNA levels as well as by the DNA-binding activity of the AP1, AP2 and NF-kappaB transcription factors, were used as indicators of a general stress response. The effect of radiofrequency exposure on proto-oncogene expression was assessed (1) in exponentially growing C3H 10T 1/2 mouse embryo fibroblasts during their transition to plateau phase and (2) during transition of serum-deprived cells to the proliferation cycle after serum stimulation. Exposure of serum-deprived cells to 835.62 MHz FMCW or 847.74 MHz CDMA microwaves (at an average specific absorption rate, SAR, of 0.6 W/kg) did not significantly change the kinetics of proto-oncogene expression after serum stimulation. Similarly, these exposures did not affect either the Jun and Myc mRNA levels or the DNA-binding activity of AP1, AP2 and NF-kappaB in exponential cells during transit to plateau-phase growth. Therefore, these results suggest that the radiofrequency exposure is unlikely to elicit a general stress response in cells of this cell line under these conditions. However, statistically significant increases (approximately 2-fold, P = 0.001) in Fos mRNA levels were detected in exponential cells in transit to the plateau phase and in plateau-phase cells exposed to 835.62 MHz FMCW microwaves. For 847.74 MHz CDMA exposure, the increase was 1.4-fold (P = 0.04). This increase in Fos expression suggests that expression of specific genes could be affected by radiofrequency exposure.

Apoptosis and delayed expression of c-jun and c-fos after gamma irradiation of Jurkat T cells.

The purpose of this study was to determine the role of radiation-induced expression of c-jun and c-fos in radiation-induced apoptosis of cells of the Jurkat T-cell line. Doses of 10-20 Gy caused a massive number of cells to undergo apoptosis within the first 24 h. This was accompanied by extensive increases in c-jun mRNA levels and moderate increases in c-fos levels, both occurring at the time of onset of internucleosomal DNA fragmentation. Increased c-jun and c-fos expression was maximum at 8 h after irradiation with a 10-fold increase in c-jun and a 2-fold increase in c-fos mRNA levels. In comparison, stimulation of the Jurkat cells with PMA resulted in rapid induction of c-jun and c-fos within 1 h. The late induction of c-jun and c-fos was not preceded by induction of tumor necrosis factor-alpha (TNF-alpha) or the bifunctional repair endonuclease and nuclear redox factor Ref-1; rather a slow decrease in Ref-1 mRNA levels was found over the first 24 h. Our results showed that radiation-induced c-jun and c-fos expression is a late response in Jurkat cells, and is most likely a secondary effect not necessary for radiation-induced apoptosis. Furthermore, apoptosis was induced by the RNA synthesis inhibitor actinomycin D, which does not induce c-jun or c-fos expression. This demonstrates that massive late induction of c-jun and c-fos is not a general requirement for apoptosis in Jurkat cells.

Effects of ionizing radiation and beta-adrenergic stimulation on the expression of early response genes in rat parotid glands.

There is little known about the regulation of gene expression in rat parotid glands after exposure to ionizing radiation. The present studies investigate the effects of in vivo ionizing radiation, with subsequent stimulation of beta-adrenergic receptors by isoproterenol, on parotid gland function and on the expression of the early response genes, c-fos, c-jun, and jun B. Ionizing radiation diminished parotid gland weight and saliva output. Treatment of irradiated rats with isoproterenol increased the gland weight to levels similar to those in nonirradiated rats. However, such treatment had no effect on saliva output as indicated by measurements of parotid salivary flow rate. Irradiation alone increased the expression of c-fos, c-jun, and jun B. The combination of irradiation and isoproterenol had an additional effect on the levels of c-fos and jun B mRNAs and proteins particularly at earlier experimental times (1 to 8 h). Isoproterenol alone induced high levels of c-fos and jun B mRNA but not of c-jun mRNA. However, c-jun mRNA was induced markedly by radiation and 8 h of isoproterenol treatment, indicating a combined effect on c-jun gene expression. These observations suggest that the expression of the proto-oncogenes c-fos, c-jun, and jun B is probably regulated through differential signal transduction pathways which may be activated by these external stimuli and may be associated with functional changes induced in the rat parotid gland by ionizing radiation and by ionizing radiation and isoproterenol.

Effect of ionizing radiation on wild-type and mutant mouse leukemia L1210 cells.

Wild-type (WT) mouse leukemia L1210 cells express steady-state levels of p53 mRNA and protein. However, the p53 expressed by the wild-type L1210 cells was found to be a mutant form of p53 (relative to normal mouse fibroblast p53 sequence) having a point mutation in the DNA binding domain of p53. A deoxyadenosine-resistant L1210 cell line (Y8) derived from the parental WT cells had previously been shown to lack the expression of p53 but to respond to cycloheximide (CHX) treatment by superinduction of p53 mRNA. The mRNA for p53 induced by CHX had the same sequence as the p53 from normal mouse fibroblasts. Although the Y8 cells had no constitutive levels of p53 mRNA or protein, the Y8 cells expressed constitutive levels of WAF1 mRNA and protein. Gadd45 mRNA was also present in the Y8 cells. Subjecting the WT or Y8 cells to ionizing radiation did not result in a G0/G1 cell cycle block; the cells blocked in G2/M. The Y8 cells were much more sensitive to the irradiation treatment than the WT cells, resulting in marked increases in apoptosis in the Y8 cells. Although radiation treatment induced p53 mRNA, but no p53 protein, in the Y8 cells, WAF1 mRNA was induced in the Y8 cells. These data indicate that there are p53-independent pathway(s) that may still involve WAF1 and Gadd45 with respect to cell cycle control and apoptosis.

Ultraviolet-radiation induced c-jun gene transcription: two AP-1 like binding sites mediate the response.

In HeLa cells transcription of the c-jun gene is activated strongly and rapidly by ultraviolet (UV) irradiation and, to a somewhat lesser extent, by treatment with phorbol ester tumor promoters. In the same cells UV and phorbol esters only marginally enhance the abundance of RNA transcribed from the jun D gene and from the gene coding for the serum response factor (which in turn acts on the UV and phorbol ester response element of the c-fos gene). In contrast to c-jun, jun B transcription is induced more efficiently by phorbol ester than by UV irradiation, suggesting that the members of the jun family are differently regulated. The promoter of c-jun carries two enhancer elements resembling AP-1 binding sites: the jun1 UV response element (URE-71 TGACATCA -64) and the jun2 URE (-190 TTACCTCA-183). These elements act independently in the UV induced expression of c-jun. In the context of the complete c-jun promoter they seem not to be required for c-jun induction by phorbol esters. When fused to the Herpes simplex thymidine kinase promoter, however, the isolated elements mediate induction by both UV and phorbol esters. UV and phorbol ester treatment of cells increases the binding of transcription factors to both elements. Both elements bind factors different in modification or/and constitution from AP-1, the heterodimeric transcription factor composed of c-Fos and c-Jun that controls the activity of the UV and phorbol ester response element (-72 TGAGTCA-66) of the human collagenase gene.

Gene expression in skin tumors induced in hairless mice by chronic exposure to ultraviolet B irradiation.

We investigated the expressions of c-Ha-ras, c-jun, c-fos, c-myc genes and p53 protein in the development of skin tumors induced by chronic exposure to UVB without a photosensitizer using hairless mice. When mice were exposed to UVB at a dose of 2 kJ/m2 three times a week, increased c-Ha-ras and c-myc transcripts were detected after only 5 weeks of exposure, while no tumor appeared on the exposed skin. The increase in gene expression continued until 25 weeks, when tumors, identified pathologically as mainly squamous cell carcinomas (SCC), developed in the dorsal skin. In these SCC, overexpression of c-fos mRNA was also observed along with the increases in c-Ha-ras and c-myc. A single dose of UVB (2 kJ/m2) applied to the backs of hairless mice transiently induced overexpression of the early event genes c-fos, c-jun and c-myc, but not c-Ha-ras, in the exposed area of skin. Accumulation of p53 protein was determined by Western blotting analysis or immunohistochemistry using monoclonal antibodies PAb 240 or 246, which recognize mutant or wild type, respectively. In the SCC, a mutant p53 protein accumulated in the cytoplasm and nucleus. After single-dose irradiation, the increased wild-type p53 protein was observed in the nuclei of epidermal cells. The present results suggest that overexpression of the c-fos, c-myc and c-Ha-ras genes, and the mutational changes in p53 protein might be associated with skin photocarcinogenesis. Moreover, overexpression of the c-Ha-ras and c-myc genes might be an early event in the development of UVB-induced skin tumors in mice.

Photoprotective effect of black tea extracts against UVB-induced phototoxicity in skin.

In previous studies, we showed that green tea and black tea extracts and their major polyphenolic constituents protect against UVB light-induced carcinogenesis in murine skin. All of these studies required chronic administration of tea extracts or specific constituents either topically or orally. However, it is not known whether acute or subchronic administration of black tea extracts or constituents can ameliorate UVB-induced early effects in skin. In the present study, cultured keratinocytes and mouse and human skin were employed to assess the effect of both oral and topical administration of standardized black tea extract (SBTE) and its two major polyphenolic subfractions namely BTF1 and BTF2 against UVB-induced photodamage. In SKH-1 hairless mice, topical application of SBTE (0.2 mg/cm2) prior to UVB exposure (180 mJ/cm2) resulted in 40% reduced incidence and 64% reduced severity of erythema and 50% reduction in skinfold thickness by day 6 when compared to nontreated UVB-exposed animals. The SBTE was also effective in protecting against UVB-induced erythema in human volunteers. Administration of SBTE 5 min after UVB irradiation was similarly effective in reducing UVB-induced inflammation in both murine and human skin. The major polyphenolic subfractions, BTF1 and BTF2, were also effective in protecting in mouse skin. The SBTE subfractions inhibited UVB-induced tyrosine phosphorylation of epidermal growth factor receptor (EGFR). The UVB irradiation of human epidermoid carcinoma cells resulted in 3.3-fold induction of tyrosine phosphorylation of EGFR. Pretreatment with BTF1 and BTF2 reduced tyrosine phosphorylation of EGFR by 53% and 31%, respectively. The UVB-mediated enhanced expression of the early response genes, c-fos and c-jun in human epidermal keratinocytes was reduced in a dose-dependent manner by SBTE. Topical application of SBTE was also effective in reducing accumulation of c-fos and p53 proteins by 82% and 78%, respectively, in UVB-exposed mouse skin. These data provide evidence that constituents of black tea can abrogate UVB-induced erythema and associated early events in murine and human skin.

Effects of ionizing irradiation and beta-adrenergic stimulation on gene expression pattern in rat submandibular glands.

Radiotherapy administrated to patients with head and neck malignancies and prior to bone marrow transplantation often results in severe xerostomia. We evaluated the expression of early response proto-oncogenes (c-fos and jun B), tissue specific genes (proline rich protein [PRP] and kallikrein), and proteolysis linked utiquitin gene following exposure to 15 Gy irradiation alone or in combination with beta-adrenergic stimulation of the rat submandibular glands. Head and neck irradiation resulted not only in dysfunction and tissue loss of the salivary glands but also in a systemic effect expressed as profound body weight loss. Irradiation alone was found to induce expression of the jun B but not the c-fos proto-oncogenes. The combination of irradiation and beta-adrenergic stimulation by isoproterenol induced earlier expression of jun B and profound expression of the c-fos proto-oncogene in comparison to irradiation alone. In contrast, the kallikrein and ubiquitin genes were expressed constitutively and were not affected by irradiation alone or in combination with beta-adrenergic stimulation. In addition, irradiation had no effect on submandibular gland mRNA translation. We observed that the expression of the genes whose regulation is associated with DNA damage (i.e. jun B and c-fos) was enhanced by irradiation alone or in combination with isoproterenol administration. In contrast, the expression of genes associated with the routine functional integrity of the cell (i.e. kallikrein, ubiquitin, and PRP) was unaffected. These findings, in addition to delayed gland dysfunction, leads us to believe that the irradiation induced injury to the submandibular glands is to be attributed to reproductive stem cell death which may be partly obliterated in the clinical setting by better understanding.

Influence of X-rays on early response gene expression in rat astrocytes and brain tumour cell lines.

The effects of ionizing radiation on c-fos, c-jun and jun-B mRNA levels were determined in cultures of rat perinatal type 1 astrocytes and two rat brain tumour cell lines, 175A and 9L. In astrocyte cultures X-ray doses as low as 1 Gy induced the expression of c-fos and jun-B but had essentially no effect on c-jun. The maximum increase in expression was found 1 h after irradiation, which then rapidly returned to control levels. These findings suggest that astrocytes may play a role in mediating the radiation response of the central nervous system via X-ray-induced changes in gene expression. In contrast, doses of up to 20 Gy had no effect on c-fos, c-jun and jun-B mRNA levels in the two brain tumour cell lines. In addition, whereas 12-O-tetradecanoylphorbol-13-acetate induced the expression of these genes in astrocytes, it had little or no effect on fos or jun expression in 9L or 175A cells. These results suggest that the signal transduction pathways mediating radiation-induced gene expression may be different in normal astrocytes and brain tumour cells.

The transcription-repair coupling factor CSA is required for efficient repair only during the elongation stages of RNA polymerase II transcription.

The known nucleotide excision repair (NER) defects of xeroderma pigmentosum (XP) and Cockayne syndrome (CS) cells can be exploited to analyze mechanisms of repair of UV-induced cyclobutane pyrimidine dimers (CPDs) at nucleotide (nt.) resolution. The two gene products of the CS complementation groups (CSA and CSB) have been implicated in the preferential repair of the transcribed strand of human genes. We had previously described very efficient repair of CPDs at sequences near the transcription initiation site of the human JUN gene in normal fibroblasts. Here, we have analyzed repair in a CSA fibroblast strain. CSA cells exhibited rapid repair near the transcription initiation site (positions -45 to +15) but were deficient in repair of sequences on the transcribed strand beginning around nt. +20. There was also no strand-selective repair of sequences further downstream of the start site (+260 to +450). The results suggest that the transcription-repair coupling factor (TRCF) CSA is required for efficient repair only during the elongation stages of RNA polymerase II transcription. We also discuss possible mechanisms of differential repair observed near the transcription initiation site in XP and CS cells and conclude that these in vivo repair data support some recent models obtained from nucleotide excision repair experiments in vitro.

Signal transduction in lymphocytes after low dose radiation.

Mice were given whole-body irradiation (WBI) with 75 mGy X-rays which had previously been found to stimulate immunologic functions. This low dose radiation (LDR) potentiated [Ca2+]i mobilization in thymic and splenic lymphocytes in response to Con A and anti-CD3 McAb and activated protein kinase C in T and B lymphocytes of the spleen. The expression of TCR/CD3 molecules on the thymocytes was enhanced after LDR indicating an expedited maturation and differentiation process in the thymus. The changes in TCR/CD3 expression and [Ca2+]i mobilization in response to McAb-CD3 after LDR was found to be highly correlated. Meanwhile the transcription of c-fos and c-jun genes was up-regulated beginning 3 hours after LDR. The expression of IL2R in active thymocytes was potentiated 24 hours after LDR which coincided with the previous finding of increased secretion of IL2 by splenocytes after WBI with 75 mGy X-rays. It is first reported in the present paper that LDR could stimulate immunologic functions through facilitation of the signal transduction process in the lymphocytes.

Tocopherol succinate: modulation of antioxidant enzymes and oncogene expression, and hematopoietic recovery.

PURPOSE: A class of naturally occurring isoforms of tocopherol (tocols) was shown to have varying degrees of protection when administered before radiation exposure. We recently demonstrated that α-tocopherol succinate (TS) is a potential radiation prophylactic agent. Our objective in this study was to further investigate the mechanism of action of TS in mice exposed to (60)Co γ-radiation. METHODS AND MATERIALS: We evaluated the effects of TS on expression of antioxidant enzymes and oncogenes by quantitative RT-PCR in bone marrow cells of (60)Co γ-irradiated mice. Further, we tested the ability of TS to rescue and repopulate hematopoietic stem cells by analyzing bone marrow cellularity and spleen colony forming unit in spleen of TS-injected and irradiated mice. RESULTS: Our results demonstrate that TS modulated the expression of antioxidant enzymes and inhibited expression of oncogenes in irradiated mice at different time points. TS also increased colony forming unit-spleen numbers and bone marrow cellularity in irradiated mice. CONCLUSIONS: Results provide additional support for the observed radioprotective efficacy of TS and insight into mechanisms.

Differential inhibition of UVB-induced AP-1 and NF-kappaB transactivation by components of the jun bZIP domain.

Potential targets for chemoprevention of nonmelanoma skin cancer include UV-induced nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1) activation in keratinocytes. Inhibition of both these ultraviolet light B (UVB)-induced transcription factors has been shown with the dominant-negative c-jun mutant, TAM67; however, its mechanism of action has not yet been determined. Here we demonstrated that transient transfection of a mouse keratinocyte cell line (308) with a dominant-negative phosphorylation mutant of c-Jun before exposure to 250 J/m(2) UVB inhibits transactivation mediated by both AP-1 and NF-kappaB transcription factors to levels below those of UVB exposed controls. Through the utilization of immunoprecipitation techniques, protein-protein interactions between NF-kappaB family members IkappaBalpha, IkappaBbeta, p50, and p65 (Rel-A) were identified with an Xpress tagged dominant-negative c-Jun (TAM67) protein. Expression of the leucine zipper domain of the TAM67 protein inhibited UVB-induced NF-kappaB transactivation but not AP-1 transactivation. Expression of the bZIP domain of the TAM67 protein was able to inhibit transactivation mediated by both transcription factors. These data demonstrate that TAM67 is able to inhibit two significant UVB-induced molecular targets AP-1 and NF-kappaB, and that the inhibition of these two transcription factor families is potentially due to protein-protein interactions between different regions of the dominant-negative c-Jun protein.