Identification
of Novel Apoptosis Regulatory Molecules
Apoptosis,
a form of programmed cell death, is a process
which has only relatively recently been
recognized as fundamental to human development.
Programmed cell death is now known to be
integral to both tissue modeling and homeostasis,
and aberrant apoptosis contributes to many
diseases, including cancer. Work soon to
be initiated in my laboratory will involve
using an episomal system to functionally
screen for novel mammalian apoptosis regulatory
genes involved in cancer.
Mechanism
of action of LUCA-15 as a regulator of apoptosis
LUCA-15
was identified as an apoptosis regulatory
gene using the episomal functional expression
screening system. LUCA-15 maps to the short
arm of human chromosome three, a region
known to harbour at least one tumour suppressor
gene, and suspected to play a role in many
different cancers, including lung, renal,
breast, ovarian, and head and neck. Sequence
similarity searches identified some common
functional motifs within the protein coding
sequence, indicating LUCA-15 is a putative
RNA binding protein, involved in RNA splicing.
In confirmation, two groups have recently
published RNA binding assays which indicate
that LUCA-15 has the ability to specifically
bind poly(G) tracts in vitro.
My
work has focused on the role of LUCA-15
in mediating apoptosis, and its mechanism
of action. I have now demonstrated that
numerous products are transcribed from the
single LUCA-15 gene locus. At least 4 RNA
splice variants have been identified to
date, and more are likely. Overexpression
of full-length antisense LUCA-15 cDNA is
able to suppress receptor-mediated (and
staurosporine induced) apoptosis in the
human Jurkat E6 acute lymphoblastic leukemia
cell line. One alternative RNA splice variant
of LUCA-15, Clone 26, is able to augment
receptor-mediated apoptosis. And, interestingly,
a 300 bp cDNA fragment, Je2, which is antisense
to the 3'-untranslated region of Clone 26,
dramatically suppresses receptor-mediated
apoptosis, a fact which might bare investigation
for antisense therapy, should LUCA-15 be
proven to have a role in cancer.
The
mechanism of action of LUCA-15's ability
to regulate apoptosis is largely unknown.
This analysis is hindered by the fact that
the functional role of LUCA-15 and its family
members is unknown. However, preliminary
results from my work indicate that LUCA-15
may regulate apoptosis by indirectly modulating
expression of the known apoptosis inhibitory
molecule Bcl-2.
In
addition, each of the four known LUCA-15
variants lacks different consensus functional
motifs, suggesting that the putative RNA
binding function of LUCA-15 may be responsible
for its apoptosis regulatory role. I therefore
propose to conduct structure/function experiments
aimed at examining this question. Concentrating
on the mechanism of action of LUCA-15 and
its variants will be the main focus of my
immediate research efforts.
Role
of LUCA-15 in Breast Cancer
The LUCA-15 gene locus maps to human 3p21.3,
a region known to be important in breast
cancer. In a small study of human breast
cancer tissue, the aggressive cancers (those
overexpressing the Her-2/neu proto-oncogene,
and resistant to TNF-induced apoptosis)
demonstrated enhanced expression of LUCA-15
RNA, suggesting that the LUCA-15 gene locus
may be important in breast cancer development.
Breast cancer tissue, obtained from the
Sudbury Regional Hospital, is being used
to correlate LUCA-15 expression with Her-2/neu,
and a range of other known or suspected,
cancer genes. In addition, functional studies,
using breast cancer cell lines, are being
used to study the effect of induced LUCA-15
overexpression on death receptor-mediated
apoptosis.
Selected
Publications:
Rintala-Maki,
N.D., Burd, M., Abrasonis, V. and Sutherland,
L.C. Genetic instability of RBM5/LUCA-15/H37
in MCF-7 breast carcinoma sublines may affect
susceptibility to apoptosis. Cell Biochemistry
and Function. In press.
Sutherland, L.C.,
Rintala-Maki, N.D., White, R.D. and Morin,
C.D. RNA binding motif (RBM) proteins: A
novel family of apoptosis modulators? J.
Cell. Biochem. In press.
Rintala-Maki, N.D.
and Sutherland, L.C. LUCA-15/RBM5, a putative
tumour suppressor, enhances multiple receptor-initiated
death signals. Apoptosis 9, 475-484, 2004.[Pubmed]
Sutherland, L.C.,
Lerman, M., Williams, G.T. and Miller, B.A.
LUCA-15 suppresses CD95-mediated apoptosis.
Oncogene 20, 2713-2719, 2004. Corrigendum.
Mourtada-Maarabouni,
M., Sutherland, L.C., Meredith, J.M., and
Williams, G.T. Simultaneous acceleration
of the cell cycle and suppression of apoptosis
by splice variant delta-6 of the candidate
tumour suppressor LUCA-15/RBM5. Genes to
Cells 8: 109-119, 2003.
Mourtada-Maarabouni, M., Sutherland, L.C.,
and Williams, G.T. Candidate tumour suppressor
LUCA-15 can regulate multiple apoptotic
pathways. Apoptosis 7:421-432, 2002.
Sutherland, L.C., Lerman, M., Williams,
G.T. and Miller, B.A. LUCA-15 suppresses
CD95-mediated apoptosis. Oncogene 21:2713-2719,
2001.
Sutherland, L.C., Gill, S.E., Cable, H.C.,
Poirier, G.G., Miller, B.A., Cooper, C.S.
and Williams, G.T. LUCA-15-encoded sequence
variants regulate CD95-mediated apoptosis.
Oncogene 19:3774-3781, 2000.
Sutherland, L.C., Anderson, C.L. and Williams,
G.T. Zinc has no effect on IL-3-mediated
apoptosis in BAF-3 cells but enhances CD95-mediated
apoptosis in Jurkat cells. J. Immunol. Methods
234:43-50, 2000.
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