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,
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.