Proliferating cell nuclear antigen (PCNA) is a 36 kD nuclear protein associated with the cell cycle. A monoclonal antibody, PC10, that recognizes a fixation and processing resistant epitope has been used to investigate its tissue distribution. Nuclear PCNA immunoreactivity is found in the proliferative compartment of normal tissues. PCNA immunoreactivity is induced in lectin stimulated peripheral blood mononuclear cells in parallel with bromodeoxyuridine incorporation and the number of cells with PCNA immunoreactivity is reduced by induction of differentiation in HL60 cells. In non-Hodgkin's lymphomas a linear relation between Ki67 and PCNA staining was demonstrated. These data suggest that in normal tissues and lymphoid neoplasms, PCNA immunolocalization can be used as an index of cell proliferation. However, in some forms of neoplasia, including breast and gastric cancer and in vitro cell lines, the simple relation between PCNA expression and cell proliferation is lost. In some breast and pancreatic tumours there is apparent deregulation of PCNA with increased expression in tissues adjacent to the tumours. The over-expression in some tumours and in adjacent morphologically normal tissue may represent autocrine or paracrine growth factor influence on PCNA gene expression.

The expression of cyclin-dependent kinase inhibitor p27 kip1 in human tumors and normal tissues was investigated using a panel of novel anti-p27 kip1 mAbs. An inverse correlation between expression of p27 kip1 and cell proliferation was generally observed after analyzing its expression in 25 different normal human tissues. In some highly proliferative human breast cancer cells, however, high level p27 kip1 expression was seen, indicating the existence of a mechanism by which some growing tumor cells may tolerate this inhibitor of cell cycle progression. Detailed studies demonstrated a correlation between the high level expression of p27 kip1 and cyclin D1 in human breast cancer cells. There was also an inverse correlation between the expression of p27 kip1 and the degree of tumor malignancy in human breast and colorectal cancers, indicating that p27 kip1 may be a useful prognostic marker in these cancers.

We have used immunohistochemical staining to assess the expression of cyclin D I in formalin-fixed sections of 345 breast carcinomas, dating back 20 years. Clinical follow-up data were available on all patients. Approximately 50% of the tumours showed excessive nuclear staining for cyclin DI as compared with normal epithelium. Some tumours showed strong cytoplas-mic staining in the absence of nuclear staining, and around 25% of the tumours were judged to be negative for nuclear cyclin D I. Contrary to expectations, moderate/strong staining for cyclin D I was associated with improved relapse-free and overall survival relative to patients whose tumours stained weakly or negatively. Conversely, tumours that were considered negative for cyclin DI staining had an adverse prognosis, and the poor outcome was further accentuated if the tumours were also oestrogen receptor-negative. A possible explanation for our findings is that tumours in which cyclin D I levels are abnormally low may have sustained mutations in other genes, such as R 6 I and that it is this abnormality that has the more significant impact on survival from breast cancer. 8 1996 Wiley-Liss, Inc.

Spatially resolved fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM), provides a method for tracing the catalytic activity of fluorescently tagged proteins inside live cell cultures and enables determination of the functional state of proteins in fixed cells and tissues. Here, a dynamic marker of protein kinase Calpha (PKCalpha) activation is identified and exploited. Activation of PKCalpha is detected through the binding of fluorescently tagged phosphorylation site-specific antibodies; the consequent FRET is measured through the donor fluorophore on PKCalpha by FLIM. This approach enabled the imaging of PKCalpha activation in live and fixed cultured cells and was also applied to pathological samples.