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Sezione III: Attività per progetti
ognizes endogenous amounts of Tyr380 phosphorylated Caspase-8 (14 and
data not shown). In the tissue microarray technology, small tissue cylinders are
removed from hundreds formalin-fixed and paraffin-embedded different
tumors, and placed into a single “ empty ” recipient block, using a custom-built
precision instrument (46) . Hundreds of serial sections are obtainable from each
array block. This technology provides a rapid, very large-scale molecular analysis
of hundreds of tumor samples. Multi-tumors (composed of the most common
different tumor types) as well as specific-tumor and progression-tumors
(that collect samples of a specific organ at different step of progression to the
malignancy) will be analyzed by immunohystochemistry with Caspase-8
phospho-specific antibodies.
These approaches might provide more insight on the tissue and stage
specificity of Caspase-8 Tyr380 phosphorylation and lead to the identification
of a new marker of tumorigenesis.
Milestone B2: Screening of human colorectal cancer cell lines for Src kinase
activation and Caspase-8 phosphorylation on Tyr380 – We have identified Caspase-8
phosphorylation on Tyr380 in human colon cancer (14) . Moreover we
were able to detect Caspase-8 phosphorylation in a colon cancer cell line, HT29
(data not shown), where Src kinase activity has been previously reported to be
aberrantly upregulated (28) . Therefore these cells may provide a good in vitro
system to develop approaches to study the role of Caspase-8 Tyr380 phosphorylation
in colon cancer.
However, to select the optimal in vitro colon cancer cellular system to
study the role of Caspase-8 phosphorylation on Tyr380, several colorectal
metastatic and non-metastatic colon cancer cell lines, including KM12L4,
KM12C, HCT116, HT29, SW480 and SW260 will be screened for the presence
of Caspase-8 Y380 phosphorylation. Moreover we will further investigate the
correlation between Tyr380 phosphorylation and Src kinase activity. For this
purpose we will take advantage of several cell lines that have been stably transfected
with constructs that allow the doxocycline inducible expression of: (a)
wild type Src, (b) constitutively active Src, (c) kinase dead Src, (d) the luciferase
reporter only vector control. An alternative approach will be based on
the use of specific Src kinase inhibitors.
These approaches, will be carried out in collaboration with Dr. Caroline
Dive at the Paterson Institute, Manchester, UK, and they will allow us to identify
the best colon cancer cell line where Caspase-8 is phosphorylated dependently
on Src kinase activity. These cells will be important for the development
of Milestones B3-B6.
Milestone B3: Caspase-8 tyrosine phosphorylation as a mechanism to modulate
cancer sensitivity to different chemiotherapic drugs – Src family kinase
activity is up-regulated in many tumors. We have shown that treatment with
the Src kinase inhibitor PP2, significantly sensitizes HT29 cells to Fas-induced
apoptosis. Interestingly, treatment with PP2 promotes Fas-induced Caspase-8
autoprocessing and activation (data not shown). These data suggest a role of
Caspase-8 tyrosine phosphorylation in resistance of colon cancer cells to Fasinduced
apoptosis.
634 2006