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KIM E. CREEK, PhD
Ohashi
Y, Creek KE, Pirisi L, Kalus R, Young SR. RNA degradation in
human breast tissue after surgical removal: a time-course
study. Exp Mol Pathol. 2004 Oct;77(2):98-103.
There is much interest in the study of
human malignancy using gene expression profiling techniques.
Expression profiles obtained from microarrays utilize RNA
extracted from the tissue in question. Currently, cell
cultures or fresh tissue processed "quickly" are used in
these studies. To our knowledge, there are no published
reports of a time-course of RNA degradation in surgically
removed breast tissue. Such a time-course study is
critically needed. We obtained normal breast tissue from
breast reduction surgery. Portions of breast tissue kept at
room temperature were sampled and placed into RNAlater to
preserve RNA at different time-points from 10 min to 3 h
after the surgical removal. We evaluated total RNA integrity
from each specimen using agarose gel electrophoresis and
real-time quantitative RT-PCR analysis of four genes.
Electrophoresis showed good-quality, intact RNA at all time
points up to 3 h. Quantitative RT-PCR showed no difference
in amplified products among all samples. Our study showed
that there was no loss of RNA integrity in normal breast
tissue for up to 3 h after surgical removal.
Baldwin A, Pirisi L, Creek KE. NFI-Ski
interactions mediate transforming growth factor beta
modulation of human papillomavirus type 16 early gene
expression. J Virol. 2004 Apr;78(8):3953-64.
Human papillomaviruses (HPVs) are present
in virtually all cervical cancers. An important step in the
development of malignant disease, including cervical cancer,
involves a loss of sensitivity to transforming growth factor
beta (TGF-beta). HPV type 16 (HPV16) early gene expression,
including that of the E6 and E7 oncoprotein genes, is under
the control of the upstream regulatory region (URR), and E6
and E7 expression in HPV16-immortalized human epithelial
cells is inhibited at the transcriptional level by TGF-beta.
While the URR contains a myriad of transcription factor
binding sites, including seven binding sites for nuclear
factor I (NFI), the specific sequences within the URR or the
transcription factors responsible for TGF-beta modulation of
the URR remain unknown. To identify potential transcription
factors and binding sites involved in TGF-beta modulation of
the URR, we performed DNase I footprint analysis on the
HPV16 URR using nuclear extracts from TGF-beta-sensitive
HPV16-immortalized human keratinocytes (HKc/HPV16) treated
with and without TGF-beta. Differentially protected regions
were found to be located around NFI binding sites.
Electrophoretic mobility shift assays, using the NFI binding
sites as probes, showed decreased binding upon TGF-beta
treatment. This decrease in binding was not due to reduced
NFI protein or NFI mRNA levels. Mutational analysis of
individual and multiple NFI binding sites in the URR defined
their role in TGF-beta sensitivity of the promoter.
Overexpression of the NFI family members in HKc/HPV16
decreased the ability of TGF-beta to inhibit the URR. Since
the oncoprotein Ski has been shown to interact with and
increase the transcriptional activity of NFI and since
cellular Ski levels are decreased by TGF-beta treatment, we
explored the possibility that Ski may provide a link between
TGF-beta signaling and NFI activity. Anti-NFI antibodies
coimmunoprecipitated endogenous Ski in nuclear extracts from
HKc/HPV16, confirming that NFI and Ski interact in these
cells. Ski levels dramatically decreased upon TGF-beta
treatment of HKc/HPV16, and overexpression of Ski eliminated
the ability of TGF-beta to inhibit the URR. Based on these
studies, we propose that TGF-beta inhibition of HPV16 early
gene expression is mediated by a decrease in Ski levels,
which in turn dramatically reduces NFI activity.
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