Spectral imaging of MC540 during murine and human colon carcinoma cell differentiation

We studied the staining pattern of merocyanine 540 (MC540) by spectral imag ing of murine CT26 and human HT29 colon carcinoma cells incubated with the dye MC540. This dye, usually considered a potential membrane probe, localiz ed mainly in the cytoplasmic vesicles of the colon carcinoma cells. However , in cells incubated in an environment similar to that of a tumor (pH 6.7), high fluorescence was detected in the nuclear membrane and nucleoli. Under these acidic conditions, resembling the Krebs effect, a population of CT26 cells displayed fluorescent plasma membranes. In differentiating cells, ex hibiting cell cycle arrest at G(1)-phase and an elevated level of alkaline phosphatase, MC540 fluorescence was confined to cytoplasmic vesicles and wa s not detected in the plasma membrane or in the nucleoli. Cell sorting anal ysis of both cell types at pH 5.0 revealed higher fluorescence intensity in proliferating cells compared to differentiating cells. The fluorescence in tensity of MC540-stained cells reached a maximum at ph 5.0, although the fl uorescence of MC540 dye was maximal at pH 7.2. This phenomenon may result f rom increased binding of MC540 monomers to the cells because disaggregation of the dye with Triton X-100 produced similar results. We conclude that nu cleolar localization of MC540 and an elevated fluorescence intensity can be used as indicators for proliferating cells in the characteristically acidi c tumor environment.