Viveka Björklund

Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis

A neo‐epitope in cytokeratin 18 (CK18) that becomes available at an early caspase cleavage event during apoptosis and is not detectable in vital epithelial cells is characterized. The monoclonal antibody M30, specific for this site, can be utilized specifically to recognize apoptotic cells, which show cytoplasmic cytokeratin filaments and aggregates after immunohistochemistry with M30, while viable and necrotic cells are negative. The number of cells recognized by the antibody increases after induction of apoptosis in exponentially growing epithelial cell lines and immunoreactivity is independent of the phosphorylation state of the cytokeratins. The generation of the M30 neo‐epitope occurs early in the apoptotic cascade, before annexin V reactivity or positive DNA nick end labelling. In a flow cytometric assay, the majority of the M30‐positive cells appear in the ‘apoptotic’ subG1 peak. Tests with synthetic peptides define positions 387–396 of CK18, with a liberated C‐terminus at the caspase cleavage site DALD‐S, as the ten‐residue epitope of M30. This epitope starts at the end of coil 2 of the predicted CK18 structure, at a probable hinge region, compatible with the sensitivity to proteolytic cleavage. The definition of a specific caspase cleavage site in CK18 as a neo‐epitope can be used for quantification of apoptotic epithelial cells with immunocytochemical techniques and is applicable to both fresh and formalin‐fixed material. Copyright

The cytostat, an apparatus for automatically controlled continuous propagation of suspended cells.

Summary The cytostat and its operation have been described. It would seem to be a useful aid to further in vitro studies of cell populations.

Early apoptotic responses in transgenic mouse mammary carcinoma for photodynamic therapy.

BACKGROUND Male transgenic mice expressing the human RAS gene on an FVB strain background develop adenocarcinoma of the breast between 7 and 8 weeks of age. We have utilized this mammary tumour model to investigate apoptotic responses following photodynamic therapy (PDT) with a chlorin-based, water-soluble photosensitizer. METHODS Detection of apoptosis was accomplished by use of the antibody M30 against a neo-epitope of caspase-cleaved cytokeratin 18 that becomes available at an early stage of the apoptotic cascade. Mice bearing multiple tumours were injected with the photosensitizer intraperitoneally, and following a drug-light interval of 96h, 40J/cm(2) of 652nm laser light was applied to one tumour per animal, while the other tumours were protected from light to serve as host controls. The M30 antibody was used for standard immunohistochemistry of tumour sections and flow cytometric detection of epitope expression coupled to cell cycle analysis in tumour cell populations retrieved from paraffin blocks. RESULTS M30 staining was significantly increased within 2h following light treatment and persisted until 96h after treatment. Flow cytometric analysis for the S-phase fraction (SPF) of tumour cells post-PDT showed a substantial decrease in SPF at 2h post PDT, and recovery of SPF within 96h. CONCLUSIONS Cytokeratin 18 cleavage seems to be both an early and ongoing event during the cellular response to PDT. Calculating the M30/SPF ratio at both 2h and 96h suggested distinct cellular dynamics at early and late time points, and we propose the M30/SPF ratio as a tumour dynamic index (TDI) to monitor events post PDT.