Research project molecular targets for diagnosis and treatment of cancer
Research group Tumour Phenotype, Dept. of Molecular Cell Biology
Cyto- and nucleoskeleton behavior during apoptosis
The structures of the cytoskeleton and the nuclear matrix are not only subject to degradation during apoptosis. These structures do also provide structural support and a scaffold for the assembly and activation of different molecular structures that are pivotal in the initiation and execution of the process of programmed cell death.
The proper function and organization of the cyto- and nucleoskeletal structures in cancer cells can therefore influence the efficiency of anti-cancer therapies that are based on the induction of apoptosis. Indeed, the sensitivity for of apoptosis.
In this research line the mechanisms underlying the specific changes in the cyto- and nucleoskeleton during apoptosis are studied. Research is mainly focused on keratins, one of the components of the cytoskeleton. The keratin network is amongst the primary targets during the execution phase of apoptosis.
We have shown that the keratin network also plays a role in sensitizing cells to roscovitine-induced apoptosis by efficient recruitment of DEDD. In cells expressing high levels of DEDD, this protein is diubiquinated and shows a strong association with the cytokeratin scaffold. Cells showing high levels of DEDD are more sensitive to roscovitine-induced apoptosis in contrast to cells expressing low levels of DEDD, although both type of cells show similar procaspase-3 contents. We hypothesize that the DEDD-mediated recruitment of procaspases-3 and -9 leads to an increase in their local concentration, which renders cells more apoptosis-prone. When triggered, these cells respond in a rapid and efficient way to roscovitine-induced apoptosis in contrast to cells expressing low levels of DEDD.
We also showed that the C-terminal cytokeratin 18 peptide, liberated by early caspase-9 cleavage, plays a role in regulating early events during the execution phase of apoptosis. The C-terminal peptide, which shows basic properties, relocates specifically to the nucleoplasm as shown by confocal laser scanning microscopy using a specific antibody to this cytokeratin 18 region and is able to modulate topoisomerase activity in vitro as measured by relaxation of plasmid DNA. Based on our observations, we suggest that translocated C-terminal cytokeratin 18 peptide assists topoisomerase I in the adequate performance of nucleolar processes during the initial phase of apoptosis and indirectly supports protein biosynthesis via regulation of rDNA transcription and rRNA processing.
Role of p21 Waf1/Cip1 during cellular senescence
This research line is focused on the role of p21Waf1/cip1 during treatment induced senescence. Cellular senescence is a backup program for cell death and is characterized by altered gene expression and morphology. Although unable to proliferate, senescent cells interfere with cell growth by the secretion of a variety of growth factors. Using the parental HCT116 wt and the p53-/- and p21-/- knockout cell lines, we showed that p21Waf1/cip1 plays a pivotal role in the induction and maintenance of drug-induced senescence. In contrast to wild type cells the p21-/- knockout cells failed to undergo senescence after low dose treatment with the topoisomerase inhibitors etoposide or campthotecin, the cdk-inhibitor roscovitine was
unable to induce senescence.
Double labeling techniques were developed to detect chromosomal aberrations in combination with either cytoskeletal markers or the presence of incorporated BrdU
In this way we were able to discriminate between cells in the G2-phase of the cell cycle, tetraploid G1-phase cells, binucleated cells or aggregates of two G1-phase cells and we were able to show if genetically aberrant cells are capable of proliferating. We showed that during treatment with low dose topoisomerase inhibitors the number of tetraploid cells increased significantly in the p21-and p53- knockout cells as compared with the wild type cells. Similar observations were made when p53 knockout cells were exposed to short periods of anoxia. Furthermore we showed that these tetraploid cells are still able to proliferate as evidenced by their ability to incorporate BrdU. No obvious difference between wild type and knockout cells with respect to long term survival were observed after low dose drug treatment.
Preliminary results show that combined treatment of low dose topoisomerase inhibitors and the cdk-inhibitor roscovitine prevents the formation of tetraploid cells and senstitizes cells to low dose topoisomerase treatment as evidenced by a decrease long term survival.
Selected publications
Schutte B, Henfling M, Ramaekers FCS.
DEDD association with cytokeratin filaments correlates with sensitivity to apoptosis.
Apoptosis. 2006;11: 1561-72.
Targets for lung cancer vaccine development
Lung cancer is an important health problem worldwide. In the past decade, no significant improvement of the life expectancy of lung cancer patients has been achieved. Therefore, alternative treatment protocols that can be used next to the current therapies are highly needed for this type of malignancy. In the LCVAC project, a consortium of five academic and business partners, situated in the Netherlands, Great Britain and Germany, will therefore develop a therapeutic vaccination strategy for lung cancer.
The partners in the LCVAC consortium have combined their expertise in the following projects:
- Production of a vaccine based on cancer cells isolated from patient lung cancers. The cells will be propagated under pharmaceutical conditions and irradiated to eliminate their multiplying potential before use as an autologous vaccine.
- Production of a vaccine based on established lung cancer cell lines that are well characterized and still contain lung cancer specific characteristics.
- Production of a protein-based vaccine developed using the proteomics technology used to identify components in which the lung cancer cells differ from the normal lung cells. These components are being isolated, produced and their properties investigated in detail.
In the past year we identified and characterized NCAM-180 variant using differential expression methods and PCR. The NCAM-180 variant was showing to be associated specifically with small cell lung cancer and showed an upregulated expression pattern in cell lines derived from these
tumors. As a result the variant may be used as a target for diagnosis and therapy. We could show that the NCAM-180 is capable of inducing a
cytotoxic T lymphocyte (CTL) response in mice. Furthermore antibodies to this variant could be raised that are applicable in ELISA and immunocytochemistry.
Grant
EU (6th framework)
Ramaekers et al. LCVAC : new vaccination therapies for lung cancer
2004-2006
Euro 388.000
Research group
Prof. dr. Frans C.S. Ramaekers, project leader
Dr. Bert Schutte
Dr. Anton Hopman
Dr. Ann v.d. Borght
Dr. Jos Broers
Dr. Fons Verheyen
Technicians
Mieke Henfling
Monique Ummelen
Miriam Kamps
Students
Tanja Geelen
Jill Hagelstein
Leon Spijkers