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Friend leukemia cells from exponentially growing or differentiated (DMSO-induced) cultures were permealized and their DNA was stained with 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33342, acridine organge, ethidium bromide, propidium iodide, quinacrine, 7-amino-actinomycin D, mithramycin, or chromomycin AB. Accessibility of DNA to each of the above fluorochromes was compared in differentiated and nondifferentiated cells before and after nuclear proteins, mostly histones, were extracted with 0.1N HCl. A decrease in the accessibility of DNA to several dyes, especially pronounced in the case of some intercalators, was observed in differentiated cells. After extraction of nuclear proteins with HCl there was an increase in DNA accessibility, of varying degree depending on the fluorochrome and the difference between differentiated and nondifferentiated cells was abolished for most of the intercalating dyes. The increase was the lowest for DAPI (45%), the highest for 7-amino-actinomycin D (13-fold), and in general was higher for the intercalating dyes that unwind DNA than for dyes binding externally to the double helix. The results are discussed in terms of the mode of interactions between DNA and the fluorochromes and factors associated with chromatin structure that may affect accessibility of DNA in situ in exponentially growing and differentiated cells.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family (hence the alternative designation FGF-7). It is produced by stromal cells, but acts as a mitogen for epithelial cells. We examined the effects of topically applied KGF on healing of wounds in a porcine model. In partial-thickness wounds, KGF stimulated the rate of reepithelialization (p < 0.0002), associated with a thickening of the epidermis (p < 0.0001). Epidermis from KGF-treated full-thickness wound sites was significantly thicker (0.31 +/- 0.22 mm) compared with mirror image control sites (0.18 +/- 0.12 mm) (p < 0.0001). Moreover, the majority (77%) of KGF-treated wounds exhibited epidermis with a deep rete ridge pattern as compared with control sites. These effects were observed as early as 14 d and persisted for at least 4 wk. KGF treatment also increased the number of serrated basal cells associated with increased deposition of collagen fibers in the superficial dermis adjacent to the acanthotic epidermis. Electron microscopy revealed better developed hemidesmosomes associated with thicker bundles of tonofilaments in the serrated cells. The pattern of epidermal thickening observed in KGF-treated wounds resembled psoriasis. Psoriasis is a disease associated with epidermal thickening, parakeratosis as well as hyperproliferation that extends beyond the basal layer. In striking contrast to psoriasis, KGF-treated wounds exhibited normal orthokeratotic maturation, and proliferation was localized to the basal cells. Our present findings have significant implications concerning the role of KGF as a paracrine modulator of epidermal proliferation and differentiation.

The positively charged rhodamine analog rhodamine 123 accumulates specifically in the mitochondria of living cells. In the present work, the uptake of rhodamine 123 by individual lymphocytes undergoing blastogenic transformation in cultures stimulated by phytohemagglutinin was measured by flow cytometry. A severalfold increase in cell ability to accumulate rhodamine 123 was observed during lymphocyte stimulation. Maximal dye uptake, seen on the third day ofcell stimulation, coincided in time with the peak of DNA synthesis (maximal number of cells in the S phase) and mitotic activity. A large intercellular variation among stimulated lymphocytes, with some cells having fluorescence increased as much as 15 times in comparison with nonstimulated lymphocytes, was observed. Whereas the increased uptake ofrhodamine 123 also correlated with the increase in cellular RNA content, the correlation between the dye uptake and cell size (measured by light scatter) was less apparent. As observed by UV microscopy, the increased dye uptake during the blastogenesis was due, to a large extent, to an increase in number of mitochondria per cell. However, an additional increase in rhodamine 123 binding per mitochondrion or per unit of mitochondrial membrane in stimulated cells could not be excluded. The present data indicate that rhodamine 123 may be used as a supravital mitochondrial probe, discriminating between cycling and quiescent cells and having application in sorting functionally distinct cell subpopulations.Johnson et al. (1) have recently reported that the fluorescent laser dye rhodamine 123 directly and selectively stains mitochondria of living cells. These authors observed that the dye is taken up by mitochondria in a variety ofcell types without being accumulated, even transiently, in other cell organelles (i.e., lysosomes, endocytic vesicles). Changes in mitochondrial organization, distribution, and shape induced by viral transformation or colchicine treatment could be visualized easily after staining with rhodamine 123 (1). The dye, reported not to be cytotoxic at concentrations up to 10 ,g/ml, thus may be used supravitally as a mitochondrial probe.We report here that the fluorescence of individual cells stained with rhodamine 123 can be measured by flow cytometry, and we compare the rhodamine 123 binding of noncycling, quiescent lymphocytes with lymphocytes stimulated to proliferate by phytohemagglutinin (PHA). In particular, the uptake of rhodamine 123 is measured and correlated with progression of stimulated lymphocytes through the cell cycle, as analyzed by multiparameter flow cytometry based on simultaneous quantitation of cellular RNA and DNA (2).MATERIALS AND METHODS Cells. Blood was collected by venipuncture of healthy volunteers and the leukocytes were separated by gradient centrifugation on Ficoll/Isopaque (Lymphoprep; Nyegaardo, Oslo, Norway). The mononuclear cells were suspended in Eagle's basal medium containing 15% fetal bovine serum and subcultured in plastic dishes to remove most ofthe monocyte...