2002 - University of Washington Bone and Joint Sources

(A)
(B)
Figure 2: GFP expression was monitored under laser light (488 nm) in induced cells (A) and uninduced
cells (B). Only those cells that were induced show significant expression of GFP.
implications as a general mechanism
for the initiation of tumor
development.
Our results showed that the EWS/
Fli-1 fusion protein was successfully
down-regulated by the expression of
antisense EWS/Fli-1 RNA in Ewing’s
cells. This inducible system permits us
to study genetically identical cells that
differ only in the presence or absence
of the EWS/FLI-1 fusion protein. As
appropriate control constructs are also
used, any changes in splicing patterns
in these cells should therefore be
attributable to the effect of the fusion
protein itself.
Alternatively spliced forms of
CD44 and Bin-1, proteins important in
cell growth and differentiation, were
identified by comparison of splicing
patterns before and after the EWS/Fli-
1 fusion protein was down-regulated.
CD44 splice variants have been
associated with increased invasiveness
and loss of contact inhibition in tumor
cells. Alternatively spliced forms of Bin-
1 no longer possess the ability to
interact with the protein myc, resulting
in the inability of myc to function as a
tumor suppressor. Our findings
indicate that aberrant splicing of premRNA,
as mediated by the EWS/Fli-1
fusion protein, may explain the myriad
changes in gene expression that
transform a benign mesenchymal cell
into a malignant sarcoma.
RECOMMENDED READING
Figure 3: The proliferation rates of induced and uninduced cells were measured over 72 hours. Equal
numbers of cells were initially seeded and counted at each time point using tryphan blue staining to
identify viable cells. Uninduced cells exhibited a greater rate of proliferation than the induced cells
since the fusion protein confers a growth advantage in Ewing’s cells.
known about its functional effects on
cells. This study explored the possibility
that the fusion protein could promote
tumorigenesis by interfering with
mRNA splicing in Ewing’s cells.
Aberrant splicing of pre-mRNA is a
poorly understood but common
feature of human tumors. Because
alternative splicing can lead to the
expression of nonfunctional proteins or
proteins with altered activity, this
process might have important
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2002 ORTHOPAEDIC RESEARCH REPORT 31