Analysis of intestinal cell proliferation after guanethidine-induced sympathectomy. II. Percentage labelled mitoses studies

Neonatal administration of guanethidine-sulfate results in an alteration of the cell proliferative pattern of the small intestinal epithelium of the young adult rat. Sympathectomy with guanethidine has previously been shown to depress mitotic, labelling, and total cellular migration indices while increasing the generation cycle time (Tc) of small intestinal crypt cells as measured by a stathmokinetic method. The present study showed that the G1, S and G2 phases of the crypt cell cycle are altered by sympathectomy, G1 accounting for most of the increase in Tc. In addition, the percentage of [3H]-thymidine labelled crypt cells is reduced and the duration of crypt cell transit is lengthened by guanethidine-induced sympathectomy.     

Selective migration of terminally differentiating cells from the basal layer of cultured human epidermis

How terminally differentiating cells are selectively expelled from the basal layer of epidermis has been a source of interest and speculation for many years. The problem can now be studied in culture, using involucrin synthesis as an early marker of terminal differentiation in human keratinocytes. When keratinocytes are forced to grow as a monolayer by reducing the calcium ion concentration of the culture medium, they still begin to synthesize involucrin. Raising the level of calcium ions induces stratification, and cells that are synthesizing involucrin are selectively expelled from the basal layer. I have found that during calcium-induced stratification no new proteins or glycoproteins are synthesized, and the rate of cell division does not change. Movement of involucrin-positive cells out of the basal layer was found to be unaffected by cycloheximide, tunicamycin, or cytosine arabinoside. These results suggest that keratinocytes growing as a monolayer already have the necessary properties to determine their position when stratification is induced. Addition of calcium simply allows formation of desmosomes and other intimate cell contacts required for stratification. The properties of involucrin-positive cells that determine their suprabasal position include a reduced affinity for the culture substrate and preferential adhesion to other cells at the same stage of terminal differentiation. The molecular basis of these adhesive changes is discussed.     

Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation

In epidermis the onset of terminal differentiation normally coincides with inhibition of integrin function and expression, thereby ensuring that differentiating cells are selectively expelled from the basal layer. However, when stratification of cultured human epidermal keratinocytes is prevented by reducing the calcium concentration of the medium to 0.1 mM, keratinocytes initiate terminal differentiation while still attached to the culture substrate. We have examined the mechanism by which differentiating keratinocytes adhere to extracellular matrix proteins in low calcium medium and the consequences of inducing stratification by raising the calcium ion concentration to 1.8 mM (Standard Medium). In low calcium medium keratinocytes co-expressed integrins and terminal differentiation markers such as involucrin and peanut lectin-binding glycoproteins: differentiating cells contained integrin mRNA, synthesized integrin proteins de novo and expressed functional mature integrins. There were no differences in integrin synthesis, maturation or break down in low calcium or standard medium, although the level of beta 1 integrins on the surface of proliferating cells was higher in standard medium. Within 6 h of transfer from low calcium to standard medium integrin mRNA was no longer detectable in terminally differentiating cells, integrins were being lost from the cell surface, and selective migration out of the basal layer had begun. Antibodies to P- and E-cadherin, which block calcium-induced stratification, prevented the selective loss of integrin mRNA and protein from terminally differentiating cells. This suggests that cadherins may play a role in the down-regulation of integrin expression that is associated with terminal differentiation.     

Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells

The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm-2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra-peritoneally with 50 microCi tritiated thymidine [( 3H]-TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [3H]-TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four-fold increase of the high initial number of labelled cells in UVB-exposed epidermis within 18 h of the pulse injection, and a six-fold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four-fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10-12 h for UVB-exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB-exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [3H]-TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [3H]-TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.     

Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice

The proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (less than 0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture medium from 1 to 5 mM stimulated the proliferation of newborn mouse (1-3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2-3-fold higher in the magnesium-enriched cultures. Exposure to high magnesium caused 3-4-fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10-15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2 +M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)-induced differentiation.     

Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells

Abstract. The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm^-2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra-peritoneally with 50 /iCi tritiated thymidine ([³H]-TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [³H]-TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four-fold increase of the high initial number of labelled cells in UVB-exposed epidermis within 18 h of the pulse injection, and a sixfold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four-fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10–12 h for UVB-exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB-exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [³H]-TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [³H]-TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.     

Changes in cell cycle and chromatin distribution in C6 glioma cells treated by dibutyryl cyclic AMP

C6 glioma cells in culture were treated with 1 mM dibutyryl cyclic AMP (Db-cAMP) for 5, 8, 24 and 72 h. The cells were labelled with [3H]-thymidine before either the end, or the beginning, of the Db-cAMP treatment. The cell cycle passage was monitored by the simultaneous determination of DNA content and DNA synthesis in propidium iodide stained autoradiograms. The data revealed an early (t less than or equal to 3-8 h) and moderate inhibitory effect of Db-cAMP on all phases of the cell cycle except mitosis; some cells (2%) were completely blocked in the S phase. Later (8 less than t less than 24-72 h), the cycling of a substantial part of the population became inhibited in G1 phase. Microdensitometric texture analysis of Feulgen-stained nuclei, performed 24 h after administration of Db-cAMP, showed a higher inhomogeneity of the DNA distribution in cell nuclei, caused by the condensation of a part of the chromatin. This may reflect either changes in genome expression taking part in the process of cAMP induced differentiation or transit of some cells into quiescent G0 or S0 phases.     

Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium

In this study we examined the different aspects of the pathway leading to the differentiation of keratinocytes as a function of time in culture and calcium concentration of the culture medium. Human neonatal foreskin keratinocytes were grown in a serum-free, defined medium containing 0.07, 1.2, or 2.4 mM calcium and assayed for the rate of growth and protein synthesis, involucrin content, transglutaminase activity, and cornified envelope formation at preconfluent, confluent, and postconfluent stages of growth. We observed that keratinocytes grown to postconfluence in all calcium concentrations showed an increased protein/DNA ratio and an increased rate of membrane-associated protein synthesis. Extracellular calcium concentrations did not have a clear influence on these parameters. However, preconfluent and confluent keratinocytes grown in 0.07 mM calcium showed markedly retarded differentiation at all steps, i.e., involucrin synthesis, transglutaminase activity, and cornified envelope formation. Within 1 week after achieving confluence, these keratinocytes began synthesizing involucrin and transglutaminase and developed the ability to form cornified envelopes. Cells grown in 1.2 and 2.4 mM calcium synthesized involucrin and transglutaminase prior to confluence and were fully competent to form cornified envelopes by confluence. Thus external calcium-regulated keratinocyte differentiation is not an all or none phenomenon, but rather it is the rate at which keratinocytes differentiate that is controlled by calcium. We conclude that either or both higher extracellular calcium concentration and the achievement of cell-cell contacts lead to a coordinate increase of at least two precursors--involucrin content and transglutaminase activity--required for cornified envelope formation. We speculate that a critical level of cytosolic calcium, achieved by increased extracellular calcium or by achievement of intercellular communication established by cell-cell contact, may trigger mechanisms required for initiation of keratinocyte differentiation.     

Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free medium

Growth and differentiation of keratinocytes in a serum-free medium (keratinocyte growth medium or KGM) was studied and compared to that under conditions in which serum and feeder cell layers were used. Cells were grown in KGM containing 0.1 mM calcium (KGM/low calcium), KGM containing 1.2 mM calcium (KGM/normal calcium), or Dulbecco's modified Eagles medium containing 5% fetal calf serum and 1.8 mM calcium in presence of mitomycin treated 3T3 M cells (DMEM/5% FCS). Plating efficiency and rate of growth were similar in the three media till confluence. In postconfluent cultures, protein and DNA content of cells attached to the plate in KGM/low-calcium dishes decreased as an increased number of cells were shed into the medium. Cell shedding was much less evident in the presence of normal calcium. Cells grown in KGM/low calcium had a higher rate of cell proliferation (3H-thymidine incorporation into cellular DNA) than cells grown in normal calcium. Transglutaminase activity, involucrin content, and cornified envelope formation were greatest in cells grown in KGM/normal calcium, intermediate in cells grown in DMEM/5% FCS, and least in cells grown in KGM/low calcium. Keratin profiles from cells grown in KGM/low calcium showed a lower percentage of high molecular weight bands compared to the keratin profiles from cells grown in the presence of normal calcium. Keratinocytes in KGM/low calcium grew as a monolayer of cuboidal cells with few features of differentiation, whereas cells grown in KGM/normal calcium stratified into multilayered islands (3-5 layers) surmounted by 2-4 layers of enucleated cells with thickened cornified envelopes. Cells grown in KGM/normal calcium also contained tonofilaments and lamellar bodies unlike cells grown in KGM/low calcium. Cells grown in DMEM/5% FCS also formed stratified layers comparable to cells grown in KGM/normal calcium but lacked cornified cells, keratohyalin granules, tonofilament bundles, and lamellar bodies. These studies indicate the usefulness of serum-free conditions for the culture of human keratinocytes and confirm the importance of extracellular calcium in keratinocyte differentiation.     

Age-related changes in the cell kinetics of rat foot epidermis

The durations of the cell cycle and its component phases have been determined for the basal layer of the epidermis of the skin from the upper surface of the hind foot of the rat using single pulse [3H]-thymidine labelling and the percent labelled mitosis (PLM) technique. Rats of three age groups were used, namely 7, 14 and 52 weeks. The duration of DNA synthesis (Ts) and the G2 plus M phase (TG2 + M) were comparable in 7-week and 52-week-old rats (P greater than 0.1). The major difference between 7-week and 52-week-old rats was in the duration of the G1 phase (TG1). In 7-week-old rats TG1 was 15.0 +/- 0.8 h and in 52-week-old rats TG1 was 31.2 +/- 3.5 h. A consequence of this variation was that the overall duration of the cell cycle was longer in 52-week-old rats (53.9 +/- 5.3 h) than in 7-week-old rats (30.1 +/- 1.3 h). Difficulties were found in fitting a simple curve to the PLM data for 14-week-old rats. This suggests that the proliferative cell population of the epidermis of rats of this age group may be heterogeneous. A satisfactory fit to the data was obtained using a computer model which assumed that the proliferative population of the epidermis of 14-week-old rats was a mixture of cells with cell cycle parameters the same as those of the 7-week and the 52-week-old rats. These two sub-populations of relatively slowly and rapidly proliferating cells were present in the ratio of 2:1.     

Rabbit embryo-fetal fluid decreases the cell cycle activities of DU-145 cells

Successful reproduction requires tight control of cell proliferation and differentiation. Rabbit blastocoelic fluid contains such regulatory factors. For instance, it inhibits tumour or transformed cell proliferation. In this study, DU-145 cells have been used to characterize further this inhibitory activity. Maximal inhibition of cell proliferation is observed at day 12 of embryo-fetal development and this is accompanied by a strong reduction of [3H]-thymidine incorporation. DNA specific staining and analysis by flow cytometry show that cells are not stopped at any specific stage of the cell cycle. Using bromodeoxyuridine incorporation in combination with propidium iodide labelling, it has been possible to estimate the percentage of labelled cells, the duration of the S phase of the cell cycle derived from their relative movement and also the proportion of cells participating to the cell cycle. In the presence of embryonic and fetal fluids collected on day 12 (EFF D-12) the duration of the S phase and the doubling time are considerably increased and the percentage of cells participating in the cell cycle is decreased. The results also show that treatment with EFF D-12 induces the release of the cells from the monolayer. Taken altogether, these results suggest that EFF D-12 increases the duration of the cell cycle. This reduction of the mitotic activities lead up to cell death with subsequent release of cells into the culture medium.     

The decay of autoradiographic grain number over crypt base columnar cells in murine ileum as a measure of their generation time

The decay in the number of grains over [3H]-thymidine labelled crypt base columnar cells (BCC) in autoradiographs of the ileum of BDF1 mice has been studied. The results revealed that using the conventional grain count halving (GCH) method it is possible to obtain an estimation of the generation time (Tc) of the proliferative BCC cells in the Paneth cell zone (PC-zone) of 18.8 +/- 0.74 h. This lies within the range obtained by the percent labelled mitoses (PLM) method, but is shorter than most values obtained by stathmokinetic methods. The present data show no evidence for a shortening of the cell cycle 3 days after irradiation (8 Gy) which is contrary to some earlier observations. Some reasons for this discrepancy are discussed. The comparatively high labelling index of the BCC allows a larger amount of data to be easily collected, compared with the PLM technique, and correction factors which take into account the complicated shape of the bottom of the crypt are not required.     

Effects of foscarnet on the cell kinetics of Madin-Darby canine kidney cells

The antiherpes compound, foscarnet (trisodium phosphonoformate), showed concentration-dependent effects on the cell kinetics of Madin-Darby canine kidney cells. At 1 mM, only minor effects could be seen on cell proliferation and cell cycle distribution, as measured by flow cytometry DNA analysis. Treatment with 5 mM foscarnet resulted in an accumulation of cells in the S-phase although no complete cell cycle block was evident. At 10 mM foscarnet, cells accumulated earlier in the S phase, probably at the G1/S border. However, at both 5 and 10 mM foscarnet the block was not established until after 15 h incubation. Upon removing 10 mM foscarnet after 24 h incubation, G1 cells rapidly entered the S phase, whereas the progression through S and G2 + M was delayed considerably. The DNA synthesizing S phase seems, therefore, to be the main cell cycle phase affected by foscarnet.     

The extracellular calcium-sensing receptor is required for calcium-induced differentiation in human keratinocytes

In cultured keratinocytes, the acute increase of the extracellular calcium concentration above 0.03 mM leads to a rapid increase in intracellular calcium concentration ([Ca(2+)]i) and inositol trisphosphate production and, subsequently, to the expression of differentiation-related genes. Previous studies demonstrated that human keratinocytes express the full-length extracellular calcium-sensing receptor (CaR) and an alternatively spliced variant lacking exon 5 and suggested their involvement in calcium regulation of keratinocyte differentiation. To understand the role of the CaR, we transfected keratinocytes with an antisense human CaR cDNA construct and examined its impact on calcium signaling and calcium-induced differentiation. The antisense CaR cDNA significantly reduced the protein level of endogenous CaRs. These cells displayed a marked reduction in the rise in [Ca(2+)]i in response to extracellular calcium or to NPS R-467, a CaR activator, whereas the ATP-evoked rise in [Ca(2+)]i was not affected. Calcium-induced inhibition of cell proliferation and calcium-stimulated expression of the differentiation markers involucrin and transglutaminase were also blocked by the antisense CaR cDNA. When cotransfected with luciferase reporter vectors containing either the involucrin or transglutaminase promoter, the antisense CaR cDNA suppressed the calcium-stimulated promoter activities. These results indicate that CaR is required for mediating calcium signaling and calcium-induced differentiation in keratinocytes.     

Interference of a synthetic C18 juvenile hormone with mammalian cells in vitro. II. Effects on cell cycle

Interference of a synthetic C18 juvenile (JH) with the cell cycle of mouse embryo cells (ME-cells) and mouse cells of established cell line (L-cells) was examined. After 3 hour in the medium with JH (20 mg/ml) the cells were transfered to the regular culture medium and labelled with H3-thymidine then incubated for 1 to 48 hours before processing them for autoradiography. The percentage of labelled mitosis was then calculated for all cells samples examined and the labelled mitosis curves were drown and analyzed. It was shown that in contrast to the solvent which had no effect on duration of any of the component phases of the cell cycle of ME-cells, the juvenile hormone under conditions of these experiments prolonged G1 and G2 intervals what resulted in prolongation of the total cell cycle of these cells. On the other hand it shortened G1 and prolonged G2 intervals of L-cells without changing duration of the total cell cycle. Thus, in the examined mouse cells, they were the G1 and G2 intervals which are affected by JH. This findings are considered as an argument for pleiotropic nature of the juvenile hormone interference with mouse cells, the more so as it interfered with both protein and DNA synthesis in these cells.     

In vitro response of lymphocytes to phytohemagglutinin (PHA) as studied with antiserum to PHA : II. Cell cycle analyses

The cell cycle and phase times of human lymphocytes responding to PHA have been analysed with the percent labelled metaphases (PLM) technique. The range of generation times (13–18 h) and DNA synthesis times (6.5–10.5 h) reported here compare well with previous measurements in the literature. Cycle analyses of the early responding cells of the initial response, selected with partial anti-PHA serum inhibition, and of restimulated cells yield relatively well-defined PLM curves. The short cycle times measured from these curves may reflect the early cycles after stimulation or a subpopulation of responding cells. Analyses at two times during both the initial and restimulation responses suggest that cycles lengthen with time after stimulation. The poor PLM curves of the initial response and the restimulation response of cells released from anti-PHA inhibition indicate considerable intercellular variation in cycle times. Cells in the initial long G 1 phase contribute to this variation. PHA dose does not appear to affect the cycle time.     

Cell kinetics in mouse epidermis studied by bivariate DNA/bromodeoxyuridine and DNA/keratin flow cytometry.

Hairless mice were injected intraperitoneally with bromodeoxyuridine (Brd-Urd). Basal cells were isolated from epidermis, fixed in 70% ethanol, and prepared for bivariate BrdUrd/DNA flow cytometric (FCM) analysis. Optimum detection of incorporated BrdUrd in DNA was obtained by combining pepsin digestion and acid denaturation. The cell loss was reduced to a minimum by using phosphate-buffered saline containing Ca2+ and Mg2+ to neutralize the acid. The percentage of cells in S phase and the average uptake of BrdUrd per labelled cell in eight consecutive windows throughout the S phase were measured after pulse labelling at intervals during a 24 h period. Furthermore, the cell cycle progression of a pulse-labelled cohort of cells was followed up to 96 h after BrdUrd injection. In general the results from both experiments were in good agreement with previous data from 3H-thymidine labelling studies. The percentage of cells in S phase was highest at night and lowest in the afternoon, whereas the average uptake of BrdUrd per labelled cell showed only minor circadian variations. There were no indications that BrdUrd significantly perturbed normal epidermal growth kinetics. A cell cycle time of about 36 h was observed for the labelled cohort. Indications of heterogeneity in traverse through G1 phase were found, and the existence of slowly cycling or temporarily resting cells in G2 phase was confirmed. There was, however, no evidence of a significant population of temporarily resting cells in the S phase. Bivariate DNA/keratin FCM analysis revealed a high purity of basal cells in the suspensions and indicated that the synthesis of the differentiation-keratin K10 was turned on only in G1 phase and after the last division.     

Cell cycle variation associated with feeder effects in cultures of Chinese hamster fibroblasts

Sub-confluent monolayer cultures of an established line of Chinese hamster fibroblast (Don) are shown to exhibit a density-dependent stimulation of growth. Evidence is presented that both long and short range ‘feeder effects’ are involved. Using the technique of autoradiography, cell cycle parameters have been studied in sub-confluent cultures seeded at different densities to identify the source of this density-dependent variation in growth rate. The durations of S phase, G2, and mitosis are constant as indicated by “percentage labelled mitoses” curves. A simple procedure has been developed for measurement of the fraction of a cell population in the G1 state, and this fraction is shown to be inversely related to the density of the culture. It is concluded that regulation of cell growth associated with feeder effects in cultured Don cells occurs within the G1 state. The data obtained from “percentage labelled mitoses” curves are shown to be highly consistent with the predictions of the Transition Probability model for cell cycle regulation.     

Calcium-induced reorganization of desmosomal components in cultured human keratinocytes

We used antibodies raised against individual desmosomal components to study calcium-induced desmosome formation in human keratinocytes. When keratinocytes are forced to grow as a monolayer by reducing the level of calcium ions in the culture medium, there is little contact between adjacent cells. Raising the level of calcium ions rapidly induces desmosome formation, and stratification occurs within 24 h. We found that before addition of calcium the 115,000- and 100,000-mol-wt core glycoproteins were distributed over the entire cell surface, whereas the plaque proteins (205,000 and 230,000 mol wt), the 82,000- and 86,000-mol-wt proteins, and the 150,000-mol-wt glycoprotein were located throughout the cytoplasm. 15 min after increasing the calcium ion concentration, all of these molecules appeared at the cell margins. The intensity of peripheral staining increased over the next 2 h and during this time the distribution of keratin filaments changed from predominantly perinuclear to extend throughout the cytoplasm. Keratinocytes could be dissociated with EDTA for up to 2 h after exposure to calcium. After 3 h of exposure to calcium the cells were no longer susceptible to EDTA dissociation and staining for desmosomal plaque antigens persisted in regions of intercellular contact. Desmosomal staining in stratified cultures became greatly reduced within 24 h of lowering the calcium ion concentration again. We have preliminary evidence that stratification occurs by breakdown of desmosomes at lateral surfaces and reformation at surfaces of contact between basal and suprabasal cells, rather than by rearrangement of existing desmosomes. Involucrin-positive cells in the monolayer appeared to contain more 205,000- and 230,000-mol-wt proteins free in the cytoplasm than involucrin-negative cells.     

Magnesium depletion causes growth inhibition,reduced expression of cyclin D1, and increased expression of P27KIP1 in normal but not in transformed mammary epithelial cells

In this study, we have evaluated the effects of extracellular magnesium restriction on the growth and cell cycle parameters of normal (HC11) and transformed (MCF-7) breast epithelial cell lines. Cells were incubated in medium with different concentrations of Mg²⁺ (from 0.5 to 0 mM) and the growth rates were determined by [³H]-thymidine incorporation and cell counting. The growth of the HC11 cells was drastically inhibited by Mg²⁺ depletion whereas the MCF-7 cells were only slightly inhibited (about 50% and 15%, respectively, after incubation in 0.05 mM Mg for 48 h). Cell cycle analyses showed a decrease in the percentage of cells in the S phase when both cell lines were incubated at low Mg²⁺ concentration. However, while the percentage of cells in both the G0/G1 and G2/M phases was increased in the HC11 cells, only the percentage of cells in the G2/M phase was increased in the MCF-7 cell line. Extracellular magnesium depletion was associated with increased expression of the cyclin-dependent kinase inhibitor p27^Kip1 and decreased expression of cyclin D1 in the HC11 but not in the MCF-7 cells. We also demonstrated that Mg²⁺ depletion does not inhibit kinase activities in the normal HC11 cells and that Mg²⁺-restricted HC11 cells are still responsive to the epidermal growth factor (EGF)- and insulin-mediated stimulation of cell growth. These data suggest that normal but not transformed mammary epithelial cells are inhibited by extracellular Mg²⁺ restriction and that this effect might be mediated by changes in the levels of expression of both cyclin D1 and p27^Kip1. J. Cell. Physiol. 180:245–254, 1999. © 1999 Wiley-Liss, Inc.