In this study the effects of all-trans retinoic acid (ATRA) on cell cycle and apoptosis of MCF-7 human breast cancer cells were investigated to elucidate the mechanisms underlying the antineoplastic potential of this retinoid in breast cancer. The antiproliferative effect of ATRA was evaluated by DNA content measurements and dual-parameter flow cytometry of bromodeoxyuridine (BrdU) incorporation and of the expression of cell cycle-related proteins (Ki-67 as proliferation marker and statin as quiescence marker) vs DNA content. Apoptosis was also studied by flow cytometry of either DNA content or Annexin V labelling. After 10(-6) M ATRA treatment, the fraction of S-phase cells decreased significantly, and cells accumulated in the G0/G1 range of DNA contents. Dual-parameter flow cytograms showed a decrease in the percentage of Ki-67-labelled cells (after 10 days, only 20% of the cells were still positive for Ki-67 compared with 95% in controls), while the fraction of statin-positive cells increased slightly. From 3 days of treatment onwards, apoptosis was found to occur. These results show that ATRA-induced inhibition of MCF-7 cell growth is related to two mechanisms, i.e. the block of cell proliferation, mostly in a pre-S phase, and the induction of apoptosis. These results should be taken into account when attempting to design treatment programmes that associate ATRA with antineoplastic compounds of different cell cycle specificity.

Information concerning cell proliferation and differentiation in dental pulp may be important to understand tooth response to exogenous stimuli. Since few data concerning human dental pulp are available, we have investigated the growth fraction and the localization of proliferating cells in pulp tissue of third molars of young adult human males and females, using flow cytometry and immunohistochemistry. Flow cytometric analysis demonstrates a low proliferative activity of pulp tissue that appears to be confined to radicular pulp, as revealed by immunohistochemical detection of proliferating cells. No polyploid or aneuploid cell populations could be identified, and G2-blocked cells, if any, represented a negligible cell population. Odontoblasts, cells of the sub-odontoblastic layer, and cells of coronal pulp were found to be not proliferating under normal conditions. These data provide the basis for future investigations on proliferative activity and regenerative potentiality of human pulp cells in experimental and clinical situations.

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The phenanthridine dyes propidium iodide (PI) and ethidium bromide (EB) have contributed to make DNA quantitation a routine measurement by flow cytometry. As planar molecules they easily intercalate between base pairs of double stranded nucleic acids. When the quantity of dye exceeds the amount of DNA, the intensity of emitted fluorescence can be related to the amount of DNA. This is, in fact, the basic requirement of quantitative measurement. When staining is performed at low dye concentration and there are no sufficient molecules to saturate all the available DNA-binding sites, the emitted fluorescence, other than the true DNA quantity, may be dependent on the state of chromatin condensation. A staining procedure aimed to stress the influence of nuclear structure on the emitted fluorescence of PI is described. This is achieved by means of a very low dye concentration (< 0.01 gg/ml PI) to guarantee staining far below saturation condition. In this particular condition the staining rate slows down to be monitored by flow cytometry. As different experimental models the leucocytes of the normal human peripheral blood have been used taking into account the relatively condensed chromatin of granulocytes with respect to that of the mononuclear cells. Significantly higher fluorescence intensity have been obtained from mononuclear cells compared to that of the polymorphonuclear ones. Quiescent versus exponentially proliferating cell cultures had also been tested. In this staining condition low fluorescence intensity have been obtained by condensed chromatin structure (Go) in comparison to the more decondensed (G~) one.Abbreviations: PI = propidium iodide; PBS = phosphate buffered saline; BrdU = bromodeoxyuridyne; FCM = flow cytometry; PMNC = polymorphonuclear cells; MNC --mononuclear cells

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