Book of abstract 2008

Reduction of plasminogen activity in breast tumour cells by anticytokeratin
monoclonal antibody
Bojan Doljak, Nataša Obermajer, Janko Kos
University of Ljubljana, Faculty of Pharmacy, Dept. of Pharmaceutical Biology, Aškerčeva 7, 1000
Ljubljana, Slovenia
Cytokeratins (CKs) are ubiquitous structural proteins in cells of epithelial origin. Although
they mainly form cytoplasmic structures, they are also localized at the plasma membrane
or secreted from the cells. Some CKs are over-expressed in tumour cells and are used as
diagnostic and prognostic biomarkers. Moreover, CKs have been reported to participate in
cell invasion by enhancing plasminogen activation on their surface. Cell-surface activation
of plasminogen leads to the activation of other proteases and finally to the degradation of
the proteins of extracellular matrix, a process that is pivotal for migration, invasion and
metastasis of tumour cells.
A stable hibridoma cell line producing monoclonal antibody (anti-CK MAb) was
prepared after immunisation of mice with breast cancer MCF-7 cell extract. As shown
by 2D-electophoresis immunoblotting and mass spectroscopy, the monoclonal antibody
recognizes CK1, CK2, CK8, CK10 and CK18 in the lysate of MCF-7 cells. Using the
aligned sequences of the recognized CKs, a consensus sequence of 27 AA was used to
synthesize three overlapping short peptides of 12 AA. In ELISA, the anti-CK antibody
expressed high affinity towards the VKIALEVEIATY dodecapeptide. The specificity
of the interaction was verified by surface plasmon resonance. Plasmin formation from
plasminogen was monitored in the presence of different concentrations of anti-CK MAb.
In both MCF-10A neoT cells and MCF-7 cells, plasmin generation was diminished by
anti-CK MAb in a dose dependent manner.
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