Inhibition of EGFR nuclear shuttling decreases irradiation resistance in HeLa cells.

INTRODUCTION: Cervical cancer is a leading cause of mortality in women worldwide. The resistance to irradiation at the advanced stage is the main reason for the poor prognosis and high mortality. This work aims to elucidate the molecular mechanism underlying the radio-resistance. MATERIAL AND METHODS: In this study, we determined the pEGFR-T654 and pDNA-PK-T2609 expression level changes in irradiated HeLa cells treated with T654 peptide, a nuclear localization signal (NLS) inhibitor, to inhibit EGFR nuclear transport. Cell viability, cell cycle and migratory capacity were analyzed. Xenograft animal model was used to evaluate the effect of EGFR nuclear transport inhibition on the tumor growth in vivo. RESULTS: The enhanced translocation of nuclear EGFR in the irradiated HeLa cells correlated with the increasing level of pEGFR-T654 and pDNA-PK-T2609. Inhibition of EGFR nuclear translocation by NLS peptide inhibitor attenuated DNA damage repair in the irradiated HeLa cells, decreased cell viability and promoted cell death through arrest at G0 phase. NLS peptide inhibitor impaired the migratory capacity of irradiated HeLa cells, and negatively affected tumorigenesis in xenograft mice. CONCLUSIONS: This work puts forward a potential molecular mechanism of the irradiation resistance in cervical cancer cells, providing a promising direction towards an efficient therapy of cervical cancer.

Genomewide analysis of TCP transcription factor gene family in Malus domestica.

Teosinte branched 1/cycloidea/proliferating cell factor 1 (TCP) proteins are a large family of transcriptional regulators in angiosperms. They are involved in various biological processes, including development and plant metabolism pathways. In this study, a total of 52 TCP genes were identified in apple (Malus domestica) genome. Bioinformatic methods were employed to predicate and analyse their relevant gene classification, gene structure, chromosome location, sequence alignment and conserved domains of MdTCP proteins. Expression analysis from microarray data showed that the expression levels of 28 and 51 MdTCP genes changed during the ripening and rootstock-scion interaction processes, respectively. The expression patterns of 12 selected MdTCP genes were analysed in different tissues and in response to abiotic stresses. All of the selected genes were detected in at least one of the tissues tested, and most of them were modulated by adverse treatments indicating that the MdTCPs were involved in various developmental and physiological processes. To the best of our knowledge, this is the first study of a genomewide analysis of apple TCP gene family. These results provide valuable information for studies on functions of the TCP transcription factor genes in apple.

TM6, a novel nuclear matrix attachment region, enhances its flanking gene expression through influencing their chromatin structure.

Nuclear matrix attachment regions (MARs) regulate the higher-order organization of chromatin and affect the expression of their flanking genes. In this study, a tobacco MAR, TM6, was isolated and demonstrated to remarkably increase the expression of four different promoters that drive gusA gene and adjacent nptII gene. In turn, this expression enhanced the transformation frequency of transgenic tobacco. Deletion analysis of topoisomerase II-binding site, AT-rich element, and MAR recognition signature (MRS) showed that MRS has the highest contribution (61.7%) to the TM6 sequence-mediated transcription activation. Micrococcal nuclease (MNase) accessibility assay showed that 35S and NOS promoter regions with TM6 are more sensitive than those without TM6. The analysis also revealed that TM6 reduces promoter DNA methylation which can affect the gusA expression. In addition, two tobacco chromatin-associated proteins, NtMBP1 and NtHMGB, isolated using a yeast one-hybrid system, specifically bound to the TM6II-1 region (761 bp to 870 bp) and to the MRS element in the TM6II-2 (934 bp to 1,021 bp) region, respectively. We thus suggested that TM6 mediated its chromatin opening and chromatin accessibility of its flanking promoters with consequent enhancement of transcription.

A genome-wide analysis of the RNA helicase gene family in Solanum lycopersicum.

Helicases belong to a class of molecular motor proteins that are found in yeast, animals, and plants. The helicase family is divided into three subfamilies, including the DEAD-box, DEAH-box and DExD/H-box helicases, which are classified based on variations within a common motif, known as motif II. The RNA helicases are involved in every step of RNA metabolism, including nuclear transcription, pre-mRNA splicing, ribosome biogenesis, nucleocytoplasmic transport, translation, RNA decay, and organellar gene expression. The RNA helicase protein family plays a crucial role in plant growth and development as well as in response to biotic and abiotic stresses. However, unlike Arabidopsis, no detailed information regarding the RNA helicase family is currently available for tomato (Solanum lycopersicum) due to a limited number of whole-genome sequences. In this study, we identified a total of 157 RNA helicase genes in the tomato genome. According to the structural features of the motif II region, we classified the tomato RNA helicase genes into DEAD-box, DEAH-box and DExD/H-box helicase genes. But there are 27 RNA helicases not belonging to this three subfamilies, we called that "other helicase". We mapped the 157 RNA helicase genes onto the tomato chromosomes, which range from chr01 to chr12. Microarray and expressed sequence tag data showed that many of these RNA helicase proteins may be involved in diverse biological processes and responses to various stresses. To our knowledge, this is the first report of a genome-wide analysis of the tomato RNA helicase gene family. This study provides valuable information for understanding the classification and putative functions of the RNA helicase gene family in Solanaceae.

A DExD/H box RNA helicase is important for K+ deprivation responses and tolerance in Arabidopsis thaliana.

The molecular mechanism for sensing and transducing the stress signals initiated by K(+) deprivation in plants remains unknown. Here, we found that the expression of AtHELPS, an Arabidopsis DExD/H box RNA helicase gene, was induced by low-K(+), zeatin and cold treatments, and downregulated by high-K(+) stress. To further investigate the expression pattern of AtHELPS, pAtHELPS::GUS transgenic plants were generated. Histochemical staining indicated that AtHELPS is mainly expressed in the young seedlings and vascular tissues of leaves and roots. Using both helps mutants and overexpression lines, we observed that, in the low-K(+) condition, AtHELPS affected Arabidopsis seed germination and plant weight. Interestingly, the mRNA levels of AKT1, CBL1/9 and CIPK23 in the helps mutants were much higher than in the overexpression lines under low-K(+) stress. Moreover, under low-K(+) stress, the helps mutants displayed increased K(+) influx, whereas the overexpression line of AtHELPS had a lower flux rate in the roots by the noninvasive micro-test technique. Taken together, these results provide information for the functional analysis of plant DEVH box RNA helicases, and suggest that AtHELPS, as an important negative regulator, plays a role in K(+) deprivation stress.

Functional characterization of a tobacco matrix attachment region-mediated enhancement of transgene expression.

TM2, a new matrix attachment region (MAR) isolated from tobacco, increases transgene expression in plants. We have carried out a more detailed analysis of the DNA elements in TM2 with the aim of improving its effect on transcription activation. Our study of the location effect of individual MARs on the expression of the adjacent 35S:gusA cassette indicated that the TM2 functions in a bidirectional manner, with the 5'-MAR being more efficient in enhancing beta-glucuronidase expression than the 3'-MAR. The influence of 5'-MAR on different linked mini-promoters in transgenic tobacco cells suggested that the role of TM2 depends on the basic expression of the transgenes. Deletion analysis of one topo II site and two unwinding sites together with one T-box revealed that all these sites contribute most (93.3%) of the transcription activation mediated from the TM2 sequence. Additionally, micrococcal nuclease accessibility of the 35S promoter region can be strengthened by linked TM2, suggesting that the TM2 mediates the spreading of nucleosome opening. Taken together, our results reveal that the TM2 mediates a more open and accessible chromatin DNA structure for promoter-dependent active transcription, which in turn enhances transgene expression.