Ku protein levels, localization and association to replication ori gins in different stages of breast tumor progression.
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Ku protein levels, localization and association to replicationorigins in different stages of breast tumor progression.
. Khalil Abdelbaqi
Goodman Cancer Research Center, McGill University, Mo
ntreal, Quebec, Canada H3G 1Y6.
Human origins of DNA replication are specific sequences within th
e genome whereby DNA replication is initiated. A select group of proteins,known as the pre-replicative (pre-RC) complex, in which Ku was shown to p
lay a role in its formation, bind to DNA replication origins to initiate D
NA replication. In this study, we find that the Ku protein expression leve
ls in human breast metastatic (MCF10AC1a) cells were higher in the chromati
n fraction compared to hyperplastic (MCF10AT) and normal (MCF10A) human bre
ast cells, but remained constant in both the nuclear and cytoplasmic fract
ions. In contrast, in human intestinal cells, the Ku expression level wasrelatively constant for all cell fractions. Nascent DNA abundance and chro
matin association of Ku70/86 revealed that the c-myc origin activity in MCF
10AC1a is 2.5 to 5-fold higher than in MCF10AT and MCF10A, respectively an
d Ku was bound to the c-myc origin in MCF10AC1a approximately 1.5 to 4.2-fo
ld higher than in MCF10AT and MCF10A, respectively. In contrast, similar
nascent DNA abundance and chromatin association was found for all cell line
s for the lamin B2 origin. Electrophoretic mobility shift assays (EMSAs) pe
rformed on the nuclear extracts (NEs) of the three cell types revealed the
presence of protein-DNA replication complexes on both the c-myc and lamin B
2 origins, but an increase in binding activity was observed from normal,
to transformed, to cancer cells for the c-myc origin, whereas no such dif
ference was seen for the lamin B2 origin. These data suggest that Ku may b
e a valuable tumor marker and a potential new target for the development offuture breast cancer therapy.