Flowchart: Preparation: Ap1
 

 


Text Box: Egf


Text Box: Bcl2


Text Box: Nfkb


             

                                                                  

                                                        

Text Box: P53


                                         

Text Box: Ap1


Text Box: Jnk


Text Box: C-Jun


                                    

                                              

                                                          

 

Text Box: Gadd45
 


Text Box: Cell Death                                

 

 

 

 


Increased radiation-induced apoptosis of Saos2 cells via inhibition of NFkappaB: A role for c-Jun N-terminal kinase.

Eliseev RA, Zuscik MJ, Schwarz EM, O'keefe RJ, Drissi H, Rosier RN.

Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York.

To elucidate the possible effect of NFkappaB on radioresistance, we used the osteosarcoma cell line Saos2, stably expressing the NFkappaB constitutive inhibitor, mIkappaB (Saos2-mIkappaB) or stably transfected with the empty vector (Saos2-EV). Ionizing radiation induced "intrinsic" apoptosis in Saos2-mIkappaB cells but not in Saos2-EV control cells, with intact NFkappaB activity. We find as expected, that this NFkappaB activity was enhanced following irradiation in the Saos2-EV control cells. On the other hand, inhibition of NFkappaB signaling in Saos2-mIkappaB cells led to the upregulation of the pro-apoptotic systems, such as Bax protein and c-Jun N-terminal Kinase (JNK)/c-Jun/AP1 signaling. Inhibition of NFkappaB resulted in decreased expression of the DNA damage protein GADD45beta, a known inhibitor of JNK. Subsequently, JNK activation of c-Jun/AP-1 proteins increased radiation-induced apoptosis in these mutants. Radiation-induced apoptosis in Saos2-mIkappaB cells was inhibited by the JNK specific inhibitor SP600125 as well as by Bcl-2 over-expression. Furthermore, release of cytochrome-c from mitochondria was increased and caspase-9 and -3 were activated following irradiation in Saos2-mIkappaB cells. Antisense inhibition of GADD45beta in Saos2-EV cells significantly enhanced apoptosis following irradiation. Our results demonstrate that radioresistance of Saos2 osteosarcoma cells is due to NFkappaB-mediated inhibition of JNK. Our study brings new insight into the mechanisms underlying radiation-induced apoptosis of osteosarcoma, and may lead to development of new therapeutic strategies against osteosarcoma. J. Cell. Biochem. (c) 2005 Wiley-Liss, Inc.

PMID: 16167336 [PubMed - as supplied by publisher]

 

Promoter- and strain-selective enhancement of gene expression in a mouse skeletal muscle by a polymer excipient Pluronic P85.

Yang Z, Zhu J, Sriadibhatla S, Gebhart C, Alakhov V, Kabanov A.

Center for Drug Delivery and Nanomedicine and Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, 985830 Nebraska Medical Center, Omaha, NE 68198-5830, United States.

Amphiphilic triblock copolymers of ethylene oxide and propylene oxide (Pluronic) significantly enhanced expression of plasmid DNA in the skeletal muscle. In the presence of Pluronic P85 (P85) high levels of expression of a reporter gene (luciferase) were sustained for at least 40 days and the area under the gene expression curve increased by at least 10 times compared to the DNA alone. The effect of Pluronic depended on the strain of the mouse and the type of the promoter used. Thus, P85 enhanced luciferase expression by 17 to 19-fold in immunocompetent C57Bl/6 and Balb/c mice, while no enhancement was observed with athymic Balb/c nu/nu mice. Furthermore, P85 activated the expression of luciferase gene driven by CMV promoter, NFkappaB and p53 response elements. There was much less or no effect on the gene driven by SV40 promoter or AP1 and CRE response elements. Overall, the promoter selectivity suggested that Pluronic induced transcriptional activation of gene expression by activating the p53 and NFkappaB signaling pathways. In addition Pluronic increased the number of DNA copies and thus affected initial stages of gene transfer in a promoter selective manner.

PMID: 16154658 [PubMed - as supplied by publisher

 

Induction of human LTBP-3 promoter activity by TGF-beta1 is mediated by Smad3/4 and AP-1 binding elements.

Kantola AK, Keski-Oja J, Koli K.

Departments of Virology and Pathology, Haartman Institute and Helsinki University Hospital, University of Helsinki, Biomedicum Rm A506, P.O.Box 63, Haartmaninkatu 8, 00014 Helsinki, Finland.

Latent TGF-beta binding proteins (LTBPs) are extracellular matrix glycoproteins, which are essential for the targeting and activation of TGF-betas. LTBP-3 regulates the bioavailability of TGF-beta especially in the bone. To understand the regulation of LTBP-3 expression, we have isolated and characterized the promoter region of human LTBP-3 gene. The GC-rich TATA-less promoter contained several transcription initiation sites and putative binding sites for multiple sequence specific transcription factors including Sp1, AP-1, c-Ets, MZF-1, Runx1 and members of the GATA-family. Reporter gene analyses of the promoter indicated that it was more active in MG-63 than in Saos-2 osteosarcoma cells, suggesting that it is regulated as the endogenous gene. TGF-beta1 stimulated the transcriptional activity of LTBP-3 promoter in MG-63 cells, while certain other bone-derived growth factors and hormones were ineffective. TGF-beta1 increased LTBP-3 mRNA levels accordingly. Analyses of deletion constructs of the promoter and mutational deletion of specific transcription factor binding sites indicated that Smad3/4 and AP-1 binding sites mediated the TGF-beta1 response. The involvement of AP-1 activity was further indicated by decreased TGF-beta responsiveness of the LTBP-3 promoter in the presence of a MEK/Erk signaling pathway inhibitor. Our results suggest an important new role for TGF-beta1 in the regulation of its binding protein, LTBP-3.