No abstract

Krüppel-like factor 4 (KLF4), a transcription factor that regulates cell fate in a context-dependent fashion, is normally induced upon growth arrest or differentiation. In many cancer cells there is dysregulation, with increased expression in proliferating cells. To identify sequence elements that mediate KLF4 suppression in normal epithelial cells, we utilized a luciferase reporter and RK3E cells, which undergo a proliferationdifferentiation switch to form an epithelial sheet. A translational control element (TCE) within the KLF4 3-untranslated region interacted with microRNAs (miRs) 206 and 344-1 to promote or inhibit KLF4 expression, respectively, in proliferating epithelial cells. Overall, the TCE suppressed expression in proliferating primary human mammary epithelial cells, but this suppressive effect was attenuated in immortalized mammary epithelial MCF10A cells, in which Dicer1 and miR-206 promoted KLF4 expression and TCE reporter activity. In contrast to MCF10A cells, in breast cancer cells the activity of miR-206 was switched, and it repressed KLF4 expression and TCE reporter activity. As miR-206 levels were KLF4 dependent, the results identify a KLF4-miR-206 feedback pathway that oppositely affects protein translation in normal cells and cancer cells. In addition, the results indicate that two distinct miRs can have opposite and competing effects on translation in proliferating cells.

We describe two enhancements of the planar bilayer recording method which enable low-noise recordings of single-channel currents activated by voltage steps in planar bilayers formed on apertures in partitions separating two open chambers. First, we have refined a simple and effective procedure for making small bilayer apertures (25-80 micrograms diam) in plastic cups. These apertures combine the favorable properties of very thin edges, good mechanical strength, and low stray capacitance. In addition to enabling formation of small, low-capacitance bilayers, this aperture design also minimizes the access resistance to the bilayer, thereby improving the low-noise performance. Second, we have used a patch-clamp headstage modified to provide logic-controlled switching between a high-gain (50 G omega) feedback resistor for high-resolution recording and a low-gain (50 M omega) feedback resistor for rapid charging of the bilayer capacitance. The gain is switched from high to low before a voltage step and then back to high gain 25 microseconds after the step. With digital subtraction of the residual currents produced by the gain switching and electrostrictive changes in bilayer capacitance, we can achieve a steady current baseline within 1 ms after the voltage step. These enhancements broaden the range of experimental applications for the planar bilayer method by combining the high resolution previously attained only with small bilayers formed on pipette tips with the flexibility of experimental design possible with planar bilayers in open chambers. We illustrate application of these methods with recordings of the voltage-step activation of a voltage-gated potassium channel.

We previously reported that MCF-7 cells were arrested in the G0/G1 phase of the cell cycle by agents known to block the activity of ATP-sensitive potassium channels (Woodfork et al., 1995, J. Cell Physiol. 162:163-171). The goal of our current study was to determine if MCF-7 cells undergo changes in membrane potential during the cell cycle that might be linked to changes in K permeability. The resting membrane potentials of unsynchronized MCF-7 cells during exponential growth phase were measured using sharp glass microelectrodes, and they ranged from -58.6 mV to -2.7 mV. The distribution of membrane potentials was best fitted by the sum of four Gaussian distributions with means of -9.0 mV, -17.4 mV, -24.6 mV, and -40.4 mV. These membrane potential groups were designated D (depolarized), ID (intermediate depolarized), IH (intermediate hyperpolarized), and H (hyperpolarized), respectively. The membrane potential was sensitive to the substitution of external K and Na but not Cl. The K:Na permeability ratio increased in proportion to the negativity of the membrane potential. MCF-7 cells pharmacologically arrested in G0/G1 phase were depolarized compared to control, with cells shifted from the H and IH groups to the D group. Tamoxifen-arrested cells chased from G0/G1 into S phase by the addition of mitogenic concentrations of 17 beta-estradiol were not depolarized, and these cells were shifted from the D group back to the IH and H groups. We conclude that MCF-7 cells hyperpolarize during passage through G0/G1 and into S phase, and this hyperpolarization probably results from an increase in the relative permeability of the plasma membrane to K.

The purpose of this study was to determine if potassium channel activity is required for the proliferation of MCF-7 human mammary carcinoma cells. We examined the sensitivities of proliferation and progress through the cell cycle to each of nine potassium channel antagonists. Five of the potassium channel antagonists produced a concentration-dependent inhibition of cell proliferation with no evidence of cytotoxicity following a 3-day or 5-day exposure to drug. The IC50 values for these five drugs, quinidine (25 microM), glibenclamide (50 microM), linogliride (770 microM), 4-aminopyridine (1.6 mM), and tetraethylammonium (5.8 mM) were estimated from their respective concentration-response curves. Four other potassium channel blockers were tested at supra-maximal channel blocking concentrations, including charybdotoxin (200 nM), iberiotoxin (100 nM), margatoxin (10 nM), and apamin (500 nM), and they had no effect on MCF-7 cell proliferation, viability, or cell cycle distribution. Of the five drugs that inhibited proliferation, only quinidine, glibenclamide, and linogliride also affected the cell cycle distribution. Cell populations exposed to each of these drugs for 3 days showed a statistically significant accumulation in G0/G1 phase and a significant proportional reduction in S phase and G2/M phase cells. The inhibition of cell proliferation correlated significantly with the extent of cell accumulation in G0/G1 phase and the threshold concentrations for inhibition of growth and G0/G1 arrest were similar. The G0/G1 arrest produced by quinidine and glibenclamide were reversed by removing the drug, and cells released from arrest entered S phase synchronously with a lag period of approximately 24 hours. Based on the differential sensitivity of cell proliferation and cell cycle progression to the nine potassium channel antagonists, we conclude that inhibition of ATP-sensitive potassium channels in these human mammary carcinoma cells, reversibly arrests the cells in the G0/G1 phase of the cell cycle, resulting in an inhibition of cell proliferation.