Both the basal transcriptional activity of the GADD45A gene and its enhancement after ionizing irradiation are mediated by AP-1 element.

The growth arrest and DNA damage-inducible gene 45A (GADD45A) is involved in the DNA repair, maintenance of genomic stability, cell cycle control and apoptosis, and thus plays an important role in cellular response to DNA damage. The GADD45A gene is responsive to a variety of DNA-damaging agents, including ionizing radiation (IR), methyl methanesulfonate (MMS), and ultraviolet (UV) radiation. It is generally thought that induction of the GADD45A gene after IR exposure is principally p53-dependent, requiring binding of the p53 protein to the p53-recognition sequence in the third intron. However, the involvement of factors other than p53 in transcriptional regulation of the GADD45A gene after IR exposure has not been elucidated. In the present study, we show that the 5'-flanking region containing two OCT sites and a CCAAT box, as well as p53 and AP-1 sites in the third intron, are required for the basal transcriptional activity of the reporter gene. In addition, AP-1 recognition element was shown to be involved in the transcriptional enhancement of the GADD45A gene after X-ray irradiation. Electrophoretic mobility shift analysis (EMSA) and Chromatin immunoprecipitation (ChIP) assay revealed that JunD binds to the third intron of the GADD45A gene. These observations suggest that AP-1 complexes containing JunD, in addition to p53, play an important role not only in transcriptional enhancement by IR but also in basal expression of the GADD45A gene via binding to the AP-1 site in the third intron.

Low-dose radiation response of the p21WAF1/CIP1 gene promoter transduced by adeno-associated virus vector.

In cancer gene therapy, restriction of antitumor transgene expression in a radiation field by use of ionizing radiation-inducible promoters is one of the promising approaches for tumor-specific gene delivery. Although tumor suppressor protein p53 is induced by low doses (< 1 Gy) of radiation, there have been only a few reports indicating potential utilization of a p53-target gene promoter, such as that of the p21 gene. This is mainly because the transiently transfected promoter of p53-target genes is not much sensitive to radiation. We examined the response of the p21 gene promoter to low-dose radiation when transduced into a human breast cancer cell line MCF-7 by use of recombinant adeno-associated virus (rAAV) vectors. It was shown that the p21 gene promoter transduced by rAAV vectors was more highly radiation-responsive than that transiently transfected by electroporation. A significant induction of the p21 gene promoter by radiation of low doses down to 0.2 Gy was observed. When cells were transduced with the p21 gene promoter-driven HSVtk gene by rAAV vector, they were significantly sensitized to repetitive treatment with low dose radiation (1 Gy) in the presence of the prodrug ganciclovir. It was therefore considered that the p21 gene promoter in combination with a rAAV vector is potentially usable for the development of a low-dose radiation-inducible vector for cancer gene therapy.

The initiator motif is preferentially used as the core promoter element in ionizing radiation-responsive genes.

Recent improvements in DNA microarray technologies and bioinformatics have made it possible to look for common features of ionizing radiation-responsive genes and their regulatory regions. We analyzed the promoters of 217 radiation-responsive human genes, compiled from microarray databases available in the literature. Using the DBTSS database, the transcriptional start sites were determined, and the core promoter elements, such as the TATA-box, initiator (Inr), GC-box and CCAAT-box, were searched for in the -1000 bp/ +200 bp region of each gene by using MATCH. It was found that the frequency of Inr in radiation-responsive genes was higher than that in general genes, and the frequencies of the GC-box and CCAAT-box were significantly lower than those in general genes. Use of the GC-box and the CCAAT-box in radiation-responsive genes was found to be dependent on the TATA-box status; that is, GC-box frequency was low in TATA box-containing genes, and CCAAT-box frequency was also low in TATA-less genes. When correlations between gene functions and frequencies of core promoter elements were examined, no apparent biased use of the core promoter element in association with a specific gene function was observed. It may be speculated that use of Inr in the core promoter correlates with appearance of radiation-responsive enhancer (silencer) elements in the upstream (downstream) regulatory region.

The association of cyclin A and cyclin kinase inhibitor p21 in response to gamma-irradiation requires the CDK2 binding region, but not the Cy motif.

The cyclin kinase inhibitor p21 associates with and inhibits cyclin-CDKs to retard the progress of the cell cycle in response to DNA damage. The recognition sites for cyclin binding on the various cell cycle-related molecules have been identified as RXL motifs. In the case of p21, the dependence of the Cy1 (18CRRL) or Cy2 (154KRRL) motifs on cyclin E, but not on cyclin A has been demonstrated by in vitro experiments. In this study, to clarify the mechanism of p21 association with cyclin A, we constructed a p21 expression system in mammalian cells. After transfection with an expression vector containing cDNA of various p21-mutants, cells were irradiated with 10 Gy of gamma-rays to introduce DNA damage, followed by quantification of the p21-cyclin A association. The p21-mutant constructs were single or multiple deletions in Cy1, Cy2, and the CDK2 binding region, and a nonphosphorylatable alanine mutant of the C-terminal phosphorylation site. We demonstrated that the association of p21 and cyclin A in response to gamma-irradiation requires the CDK binding region, 49-71 aa, but not the Cy motifs. We believe the mechanism by which p21 inhibits cyclin-CDKs is distinct in each phase of the cell cycle. Furthermore, the increase in the association of p21 and cyclin A was not correlated with the levels of p21. This suggests that DNA damage triggers a signal to the p21 region between 21 and 96 aa to allow cyclin A association.

Comprehensive search for X-ray-responsive elements and binding factors in the regulatory region of the GADD45a gene.

The growth arrest and DNA damage-inducible protein 45alpha (GADD45a) gene is responsive to a variety of DNA-damaging agents. It is known that induction of the GADD45a gene is regulated in a p53-dependent manner after ionizing irradiation. Our previous study showed that X-ray irradiation increased the transcription rate of the GADD45a gene much earlier than the maximum accumulation of stabilized p53 protein in human myeloblastic leukemia ML-1 cells. We hypothesized that some transcription factor(s) may cooperate with p53 in regulating the GADD45a gene early after the irradiation of ML-1 cells. This idea is supported by recent studies showing that the p53-dependent activation of several genes in human and mouse cells requires some additional transcription factors, such as Sp1, GKLF, Ets1, and IRF-1. To examine the possible involvement of cooperating factors in transcriptional regulation of the GADD45a gene by ionizing radiation, we comprehensively searched for the X-ray-inducible binding locus of the nuclear factor throughout the upstream region (-2244 bp/+89 bp) and the third intron (+1389 bp/+2488 bp) of the GADD45a gene by EMSA using 136 probes. The X-ray-responsive binding of nuclear factors was detected at eight loci. Oct, NF-kappaB, HNF, NF-AT, and KLF family transcription factors were identified by a competition assay. It is possible that some of these factors cooperate with p53 to mediate transcriptional regulation of the GADD45a gene after ionizing irradiation.

Functional characterization of the human spermidine/spermine N(1)-acetyltransferase gene promoter.

Spermidine/spermine N(1)-acetyltransferase (SSAT), the key enzyme of polyamine catabolism, is induced by antiproliferative stresses. We analyzed the 5' flanking region of the human SSAT gene, and clarified that the binding of Sp1 to the GC-box located 42 to 51 bp upstream from the transcription start site is essential for transcription in HeLa S3 cells. A polyamine-responsive element (PRE) seemed to be responsible for the elevated transcription after X-ray irradiation.

Identification of the regulatory region required for ubiquitination of the cyclin kinase inhibitor, p21.

The expression of cyclin kinase inhibitor p21 is regulated by the ubiquitin-proteasome protein degradation system, as well as by transcriptional regulation. Generally, ubiquitination is regulated by the phosphorylation of the substrate. In this study, we identified the region of p21 responsible for the regulation of ubiquitination. Since the phosphorylation sites of p21 are distributed in the C-terminal region, we constructed sequential C-terminal truncated fragments and examined their ubiquitination in eukaryotic cells. The ubiquitination was observed in the 1-164 (full length) and 1-157 fragments with the same efficiency, but not in the 1-147 fragment. The lack of ubiquitination in the 1-147 fragment was unlikely due to the removal of a Lys residue at position 154, since the p21 K154R mutant was ubiquitinated as efficiently as the full-length p21. Furthermore, the 148-157 deleted form of p21 was not ubiquitinated, just like the 1-147 fragment. Thus, the C-terminal 148-157 region, not a ubiquitination site by itself, should contain an essential regulatory region for the efficient ubiquitination of p21.

Early induction of CDKN1A (p21) and GADD45 mRNA by a low dose of ionizing radiation is due to their dose-dependent post-transcriptional regulation.

Previous studies have shown that induction of some genes by low-dose radiation has a different dependence on the time after irradiation than induction by high doses. To examine the mechanisms underlying this phenomenon, we investigated the changes in the time course of the rates of transcription of genes in cells of the human myeloblastic leukemia cell line ML-1 by a nuclear run-on assay. It is possible that the more rapid induction of the mRNA of the CDKN1A and GADD45 genes after exposure to 50 cGy of X rays than after 20 Gy is due to a lower level of stabilization of the mRNA of these genes after 50 cGy. In addition, our results show that 50 cGy of X rays increases the transcription rates of the CDKN1A and GADD45 genes, with a maximum induction at 0.5 to 1 h after irradiation, much earlier than the maximum accumulation of stabilized TP53 protein. We suggest the involvement of BRCA1 protein in the early induction of transcription of these two genes.