Pax6 and its Drosophila homolog Eyeless (Ey) play essential roles during eye development. Ey/Pax6 contains two distinct DNA binding domains, a Paired domain (PD) and a Homeodomain (HD). While Ey/Pax6 PD is required for the expression of key regulators of retinal development, relatively little is known about the HD-dependent Ey function. In this study, we used the UAS/GAL4 system to determine the functions of different Ey domains on cell growth and on retinal development. We showed that Ey can promote cell growth, which requires the HD but not the PD. In contrast, the ability of Ey to activate Ato expression and induce ectopic eye formation requires the PD but not the HD. Interestingly, deletion of the HD enhanced Ey-dependent ectopic eye induction while overexpression of the HD only Ey forms antagonizes ectopic eye induction. These studies revealed a novel function of Ey HD on cell growth and a novel antagonistic effect of Ey HD on Ey PD-dependent eye induction. We further show the third helix of the Ey HD can directly interact with the RED subdomain in Ey PD and that deletion of the HD increased the binding of Ey PD to its target. These results suggest that the direct interaction between the HD and the PD potentially mediates their antagonistic effects. Since different Ey splicing forms are expressed in overlapping regions during normal development, we speculate that the expression ratios of the different Ey splice forms potentially contribute to the regulation of growth and differentiation of these tissues.
Animals ; Animals, Genetically Modified ; metabolism ; Binding Sites ; Cell Differentiation ; Cell Proliferation ; DNA-Binding Proteins ; metabolism ; Drosophila ; metabolism ; Drosophila Proteins ; antagonists & inhibitors ; metabolism ; Enhancer Elements, Genetic ; Eye Proteins ; antagonists & inhibitors ; metabolism ; Homeodomain Proteins ; antagonists & inhibitors ; metabolism ; PAX6 Transcription Factor ; Paired Box Transcription Factors ; antagonists & inhibitors ; metabolism ; Protein Structure, Tertiary ; Repressor Proteins ; antagonists & inhibitors ; metabolism ; Retina ; cytology ; metabolism ; Wings, Animal ; growth & development

OBJECTIVETo explore the anti-cancer effects of siRNAs targeting hTERT in SMMC-7721 cells.

METHODSTwo siRNAs targeting hTERT mRNA were designed and synthesized by T7 transcription system in vitro. MMT, RT-PCR and Western blot were applied to evaluate effects on inhibiting cell growth, hTERT mRNA and protein expression in SMMC-7721 cells.

RESULTSsiRNAs decreased cell proliferation in a dose-dependent manner. At a concentration of 100 nmol/L, siRNAs exhibited obvious effects on inhibiting hTERT mRNA and protein expression in SMMC-7721 cells.

CONCLUSIONsiRNAs targeting hTERT have significant inhibitory effects on hTERT gene expression in SMMC-7721 cells. siRNA has the possibility to become a new anti-cancer agent

DNA-Binding Proteins ; antagonists & inhibitors ; genetics ; Gene Targeting ; Genetic Therapy ; Humans ; Liver Neoplasms ; pathology ; therapy ; RNA, Small Interfering ; genetics ; Telomerase ; antagonists & inhibitors ; genetics

The low dose hyper-radiosensitivity (HRS) in human lung cancer cell line A549 was investigated, the changes of ATM kinase, cell cycle and apoptosis of cells at different doses of radiation were observed, and the possible mechanisms were discussed. A549 cells in logarithmic growth phase were irradiated with (60)Co gamma-rays at doses of 0-2 Gy. Together with flow cytometry for precise cell sorting, cell survival fraction was measured by means of conventional colony-formation assay. The expression of ATM1981Ser-P protein was examined by Western blot 1 h after radiation. Apoptosis was detected by Hoechst 33258 fluorescent staining, and Annexin V-FITC/PI staining flow cytometry 24 h after radiation. Cell cycle distribution was observed by flow cytometry 6, 12 and 24 h after radiation. The results showed that the expression of ATM1981Ser-P protein was observed at 0.2 Gy, followed by an increase at >0.2 Gy, and reached the peak at 0.5 Gy, with little further increase as the dose exceeded 0.5 Gy. Twenty-four h after radiation, partial cells presented the characteristic morphological changes of apoptosis, and the cell apoptosis curve was coincident with the survival curve. As compared with control group, the cell cycle almost had no changes after exposure to 0.1 and 0.2 Gy radiation (P>0.05). After exposure to 0.3, 0.4 and 0.5 Gy radiation, G(2)/M phase arrest occurred 6 and 12 h after radiation (P<0.05), and the ratio of G(2)/M phase cells was decreased 24 h after radiation (P<0.05). It was concluded that A549 cells displayed the phenomenon of HRS/IRR. The mode of cell death was mainly apoptosis. The activity of ATM and cell cycle change may take an important role in HRS/IRR.
Cell Cycle Proteins/genetics ; Cell Cycle Proteins/metabolism ; Cell Cycle Proteins/physiology ; Cell Line, Tumor ; DNA-Binding Proteins/antagonists & inhibitors ; DNA-Binding Proteins/metabolism ; DNA-Binding Proteins/*physiology ; Dose-Response Relationship, Radiation ; Lung Neoplasms/*pathology ; Protein-Serine-Threonine Kinases/*metabolism ; Radiation Dosage ; Radiation Tolerance/*physiology ; Tumor Suppressor Proteins/metabolism

Zinc finger protein 133 (ZNF133) is composed of a Kruppel-associated box (KRAB) domain and 14 contiguous zinc finger motifs. ZNF133 is regarded as a transcriptional repressor because the KRAB domain has potent repressor activity and the zinc finger motifs usually act in binding to DNA. However, we found that the zinc finger motifs of ZNF133 also possessed transcriptional repressor activity. By two-hybrid screening assay, we found that the zinc finger motifs of ZNF133 interacted with protein inhibitor of activated STAT1 (PIAS1). PIAS1 enhanced the transcriptional repression activity of ZNF133 through the zinc finger motifs. This effect of PIAS1 was relieved by an inhibitor of the histone deacetylases (HDACs). These results demonstrate that the transcriptional repressor activity of ZNF133 is regulated by both the KRAB domain and the zinc finger motifs, and that the repressive effect by zinc finger motifs is mediated by PIAS1.
Cell Line ; DNA-Binding Proteins/*metabolism ; Histone Deacetylases/antagonists & inhibitors/metabolism ; Humans ; Protein Binding ; Protein Inhibitors of Activated STAT/*metabolism ; Protein Structure, Tertiary ; Repressor Proteins/*metabolism ; Small Ubiquitin-Related Modifier Proteins/*metabolism ; Transcription, Genetic ; Two-Hybrid System Techniques ; Zinc Fingers

Animals ; Antiviral Agents ; pharmacology ; therapeutic use ; DNA-Directed RNA Polymerases ; antagonists & inhibitors ; Drug Discovery ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; metabolism ; Humans ; Influenza A virus ; drug effects ; genetics ; metabolism ; Influenza, Human ; drug therapy ; Molecular Targeted Therapy ; Neuraminidase ; antagonists & inhibitors ; RNA-Binding Proteins ; antagonists & inhibitors ; Signal Transduction ; drug effects ; Viral Core Proteins ; antagonists & inhibitors ; Viral Matrix Proteins ; antagonists & inhibitors

BACKGROUNDTransforming growth factor-beta1 (TGF-beta1) exerts strong fibrogenic potential in culture-activated HSCs. Smad4 is a key intracellular mediator for the transforming growth factor-beta (TGF-beta) superfamily of growth factors. The aim of this study was to assess the effects of the antisense Smad4 gene on Ito cell line, LI90.

METHODSThe recombinant retroviral vector pLXSN-Smad4 was constructed by cloning the rat antisense Smad4 cDNA into the retroviral vector pLXSN. Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad4 and pLXSN vectors into PA317 cells. Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418. The expression of Smad4 was detected by Northern and Western blots. Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed.

RESULTSmRNA and protein expressions of Smad4 in LI90 cells transfected with retrovirus containing the antisense Smad4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells. Cells hypoexpressing the Smad4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline (P < 0.01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus.

CONCLUSIONSThe antisense Smad4 gene can suppress the expression of the Smad4 gene, reduce endogenous production of Smad4 mRNA and protein, block TGF-beta1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM). Our results may provide a basis for the development of antifibrotic gene therapy.

Cell Line ; DNA, Antisense ; pharmacology ; DNA-Binding Proteins ; antagonists & inhibitors ; genetics ; Genetic Therapy ; Genetic Vectors ; genetics ; Humans ; Liver Cirrhosis ; therapy ; Retroviridae ; genetics ; Smad4 Protein ; Trans-Activators ; antagonists & inhibitors ; genetics ; Transfection ; Transforming Growth Factor beta ; physiology ; Transforming Growth Factor beta1

OBJECTIVETo assess the telomerase activity inhibitory effect of ham merhead ribozyme targeting the 5'-end of human telomerase reverse transcriptase mRNA (hTERT-5'RZ), to compare it with the effect of another ribozyme teloRZ, and the combine the applications of the two ribozymes.

METHODShTERT-5'RZ gene was synthesized and cloned into pcDNA3.1(+); the ribozyme was produced by in vitro transcription. The teloRZ ribozyme was produced in the same way by in vitro transcription of p(SPT19-teloRZ) which had been constructed by the present authors. The ribozymes were transiently transfected into HeLa cells by liposome every 24 hours. After 72 hours, the cells were collected and their telomerase activities were assayed.

RESULTSThe ribozyme targeting the 5'-end of hTERT mRNA exhibited a very strong telomerase-inhibitory activity, the combined use of hTERT-5'RZ and teloRZ also showed clear inhibitory activity, but the inhibitory effect of teloRZ used alone was not so strong.

CONCLUSIONThese observations suggest that the use of hTERT-5'RZ and the combined use of hTERT-5'RZ and teloRZ are more effective in telomerase inhibition as compare with the use of teloRZ alone. They may find applications in cancer therapy.

5' Untranslated Regions ; Cloning, Molecular ; DNA-Binding Proteins ; HeLa Cells ; Humans ; RNA, Catalytic ; metabolism ; RNA, Messenger ; Telomerase ; antagonists & inhibitors ; genetics ; Transcription, Genetic

To explore the effect of human telomerase reverse transcriptase (hTERT) gene antisense oligodeoxynucleotide (ASODN) on telomerase activity in primary acute myeloid leukemia (AML) cells and chronic myeloid leukemia (CML) cells, polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) was used to determine telomerase activity. The expression levels of hTERT protein were assayed by flow cytometry using immunofluorescence labeling. Immunofluorescence assay showed that the expression levels of hTERT protein in AML and CML cells decreased with time after hTERT ASODN treatment. There was no difference in hTERT protein levels between control and sense oligodeoxynucleotide (SODN)-treated cells. Telomerase activity decreased when AML and CML cells were treated with ASODN for 48 h. Telomerase activity of AML and CML cells was significantly inhibited when the cells treated with ASODN for 72 h. There was no difference in telomerase activity levels between control and hTERT SODN-treated cells. It was concluded that hTERT ASODN could inhibit hTERT protein expression level, thus decreasing the telomerase activity in primary AML and CML cells.
Adolescent ; Adult ; DNA-Binding Proteins ; Female ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; enzymology ; Leukemia, Myeloid, Acute ; enzymology ; Male ; Middle Aged ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Telomerase ; antagonists & inhibitors ; metabolism

BACKGROUNDPeptide nucleic acid (PNA) has many characteristics useful in molecular biology. This paper described an effective way to raise the cell ingestion rate of PNA so as to kill gastric cancer cells.

METHODSHeteroduplexes of PNAs and oligonucleotides, wrapped by Lipofectamine 2000, were used to infect SGC7901 cells. The inhibitive effect of heteroduplexes was evaluated by analyzing cell clone forming and cell growth rate. Telomerase activity of SGC7901 cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and silver staining assay.

RESULTSPNAs showed a dose-dependent inhibition of cell proliferation. The percentage of proliferation inhibition was 99.4% after 7 days; the rate of cloning inhibition was 98.2% after 8 days; whereas for oligonucleotide groups, at the same concentration, the percentages were 50.1% and 67.5% respectively. Antisense PNA-DNA-Lipofectamine 2000 group (AP-D-L group) exhibited significantly different percentages from the control groups (P < 0.05). The test result indicated that telomerase activity of the AP-D-L group was inhibited (P < 0.05). At the same time, the impact on cell morphology was observed.

CONCLUSIONSThe results showed that PNAs are potent antisense reagents. The telomerase-associated therapies are very promising for the treatment of malignant tumours.

Cell Division ; drug effects ; Cell Line, Tumor ; DNA-Binding Proteins ; Humans ; Peptide Nucleic Acids ; therapeutic use ; Stomach Neoplasms ; pathology ; therapy ; Telomerase ; antagonists & inhibitors ; metabolism ; Transfection

OBJECTIVETo clarify growth inhibition in pancreatic cancer cells by interference with the hTR component of the telomerase reverse transcriptase enzymatic complex.

METHODSA 593 bp full length hTR cDNA was subcloned into a mammalian expression vector pcDNA3.1(-) in the antisense orientation to construct an antisense hTR expression plasmid. These were introduced into panc1 cells, a human pancreatic carcinoma cell line, by lipofectin and G418-resistant stable transformants were expanded. Resulting stable clones were screened for the presence of the hTR insert by PCR with T7 and BGH reverse primers located on the flanks of the multiclonal site of the pcDNA3.1 vector. Cell growth rate, hTR expression, telomerase activity and anchorage-independent growth properties were analyzed.

RESULTSSignificant downregulation of endogenous hTR was evident in the antisense-hTR transformed cells and telomerase activity was markedly decreased compared to control cells in standard TRAP assays. Furthermore, cell proliferation and the anchorage-independent growth ability in antisense-hTR expressing cells were significantly decreased compared with control parental cells. However, no crisis or senescence phenomena were observed.

CONCLUSIONSThese data indicate that hTR may be a critical component of human telomerase activity and suggest that downregulation of the RNA component of human telomerase is a possible target for anticancer strategies.

DNA-Binding Proteins ; Humans ; Pancreatic Neoplasms ; pathology ; therapy ; RNA, Antisense ; therapeutic use ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; antagonists & inhibitors ; genetics ; Tumor Cells, Cultured