Basilio A. Kotsias

Imatinib resistance in multidrug-resistant K562 human leukemic cells

The multidrug resistance phenotype (MDR) is one of the major causes of failure in cancer chemotherapy and it is associated with the over-expression of P-glycoprotein (P-gp or MDR1) in tumor cell membranes. A constitutive NF-kappaB activity has been observed in several haematological malignancies and this is associated with its anti-apoptotic role. In the present work, the relationship between NF-kappaB and MDR phenotype was evaluated in wild type K562 human leukemic cells (K562-WT) and in its vincristine-resistant counterpart, K562-Vinc cells. These data showed that K562-Vinc cells, which express an active P-gp, exhibited MDR phenotype. The resistant indexes (IC(50)(K562-Vinc)/IC(50)(K562-WT)) for structurally unrelated drugs like imatinib, doxorubicin and colchicine were 8.0+/-0.3, 2.8+/-0.4 and 44.8+/-8.8, respectively. The imatinib resistance was reversed by P-gp blockade suggesting the involvement of P-gp in imatinib transport. We observed that NF-kappaB was constitutively activated in both cell lines but in a lesser extent in K562-Vinc. The inhibition of NF-kappaB with BAY 11-7082 increased the cytotoxicity of imatinib in K562-Vinc cells but not in K562-WT. Further, the co-administration of imatinib and BAY 11-7082 sensitized multidrug-resistant K562 cells to cell death as detected by increased percentage of annexin V positive cells. The induced cell death in K562-Vinc cells was associated with activation of caspases 9 and 3. Finally, we provide data showing that BAY 11-7082 down-regulates the expression of P-gp suggesting that the activity of NF-kappaB could be functionally associated to this protein in K562 cells. Our results indicate that the vincristine-resistant K562 cells which developed MDR phenotype, exhibited resistance to imatinib associated with a functional P-gp over-expression. This resistance could be partially overcome by the inhibition of NF-kappaB pathway.

Mechanical and electrical properties of denervated rat skeletal muscles

Mechanical activity (twitch and tetanus) and electrical activity (single and repetitive action potentials) were recorded in vitro (34 degrees C) in control and denervated (3 to 14 days) soleus and extensor digitorum longus muscles of the rat. After denervation tetanic tension (100 to 200 Hz, 500 ms duration) was decreased in both types of muscles. Denervation reduced significantly the rates of rise and fall and the amplitude of the action potential in both types of muscle fibers. In denervated fibers with very low resting membrane potential no action potentials could be recorded: in these fibers only a slow response without overshoot was detected. Hyperpolarization of denervated fibers to -90 mV prior to application of the depolarizing pulse increased their excitability. Action potential amplitudes were well maintained during tetanic stimulation (200 Hz, 40 to 90 ms) in innervated fibers. Depolarization of the innervated fibers with cathodic current before the tetanic pulse hindered the generation of repetitive action potentials at 200 Hz. A proportion of denervated fibers stimulated at 100 to 200 Hz generated only one action potential or gave rise to an incomplete train. Hyperpolarization of the denervated fibers resulted in an improvement in the ability to generate a train of action potentials at 100 to 200 Hz. A group of denervated fibers exhibited well maintained action potentials during tetanus. We suggest that failure in the repetitive electrical activity of denervated fibers could be the reason for the reduced tension of tetanus. Depolarization of the fibers and/or the increment in the electrical time constant of the sarcolemma are suggested for the decrease in the electrical excitability of denervated fibers.

ENaC Channels in Oocytes from Xenopus laevis and their Regulation by xShroom1 Protein

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC). Oocytes were injected with α, β, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We used voltage clamp techniques to study the amiloride-sensitive Na+ currents (INa(amil)). We observed a marked reduction in INa(amil) in oocytes co-injected with xShroom1 antisense. Oocytes expressing a DEG mutant β-mENaC subunit (β-S518K) with an open probability of 1 had enhanced INa(amil) although these currents were also reduced when co-injected with xShroom1 antisense. Addition of low concentration (20 ng/ml) of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa(amil) in oocytes with xShroom1 sense but had no effect on the currents in oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with higher concentrations of trypsin (2 µg/ml) exposed during 2.5 min. In addition, fluorescence positive staining of plasma membrane in the oocytes expressing α, β and γ mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. On this basis, we suggest that xShroom1-dependent ENaC inhibition may be through the number of channels inserted in the membrane.

HERG1 Currents in Native K562 Leukemic Cells

The human ether-a-go-go related gene (HERG1) K+ channel is expressed in neoplastic cells, in which it was proposed to play a role in proliferation, differentiation and/or apoptosis. K562 cells (a chronic myeloid leukemic human cell line) express both the full-length (herg1a) and the N-terminally truncated (herg1b) isoforms of the gene, and this was confirmed with Western blots and coimmunoprecipitation experiments. Whole-cell currents were studied with a tail protocol. Seventy-eight percent of cells showed a HERG1-like current: repolarization to voltages negative to −40 mV produced a transient peak inward tail current, characteristic of HERG1 channels. Cells were exposed to a HERG-specific channel blocker, E4031. Half-maximal inhibitory concentration (IC50) of the blocker was 4.69 nm. The kinetics of the HERG1 current in K562 cells resembled the rapid component of the native cardiac delayed rectifier current, known to be conducted by heterotetrameric HERG1 channels. Fast and slow deactivation time constants at −120 mV were 27.5 and 239.5 ms, respectively. Our results in K562 cells suggest the assembling of heterotetrameric channels, with some parameters being dominated by one of the isoforms and other parameters being intermediate. Hydrogen peroxide was shown to increase HERG1a K+ current in heterologous expression systems, which constitutes an apoptotic signal. However, we found that K562 HERG1 whole-cell currents were not activated by H2O2.

Effects of antinomycin D on fibrillation activity in denervated skeletal muscles of the rat

Abstract The effects of actinomycin D on fibrillation activity, acetylcholine sensitivity and resting membrane potential of denervated muscles of the rat was studied. Actinomycin D (0.7 mg/kg I.V.) administered 1 day after denervation delays the appearance of fibrillation for approximately 3 days. If this drug is given 5–7 days after denervation, it is also capable of blocking the already established fibrillation but fails to suppress extrajunctional cholinergic receptors and to reverse the fall in resting potential. The mechanical responses of denervated muscles are unaffected by actinomycin D. These results suggest that in fibrillation a genetic induction of newly formed RNA and protein is involved. It is also suggested that these molecules probably have a more rapid turnover than those required for the formation of extrasynaptic receptors in denervated muscle.

Cystic fibrosis transmembrane regulator (CFTR) in human trophoblast BeWo cells and its relation to cell migration

INTRODUCTION ENaC and CFTR are coexpressed in epithelia and have positive or negative functional interactions. In addition, ENaC and CFTR promote migration in placental trophoblastic cells and human airway cells, respectively. Here we tested the idea if CFTR is functionally expressed in BeWo cells, a trophoblastic cell line, and if it is involved in their migratory behavior. METHODS CFTR expression was studied in BeWo cells with RT-PCR, biotinylation and Western blot. Ion currents were analyzed with patch clamp, and cell migration with the wound healing method. RESULTS The mature CFTR 160-kDa band was present, and its localization at the surface membrane was confirmed. Forskolin (20 μM), an adenylate cyclase activator, was used for channel activation, and subsequently CFTR(inh)-172 (2 μM) for its inhibition. The conductances in the presence of CFTR(inh)-172 plus forskolin (16.0 ± 0.7 pS/pF and 32.6 ± 1.5 pS/pF) were significantly lower than in presence of only forskolin (29.7 ± 0.9 and 47.0 ± 2.0 pS/pF). The conductance of CFTR(inh)-172 inhibited currents was 14.9 ± 0.7 pS/pF with a linear I-V relationship illustrating the nonrectifying properties of the CFTR. Cell migration was measured and covered 11.2 ± 0.4, 24.0 ± 1.7 and 13.9 ± 1.0% of the wound when cells were cultivated under control, forskolin, and forskolin plus CFTR(inh)-172, respectively. Proliferation was not changed by any of the treatments. CONCLUSIONS Our results shows that BeWo cells functionally express the CFTR which plays a role in the wound healing increasing the cell migration process.

Blockade of the inward rectifier potassium currents by zinc and nickel ions in voltage-clamped toad muscles

The inward rectifier is one of the voltage-sensitive K+ channels present in several tissues: Its conductance increases under hyperpolarization and decreases with depolarization. In this work we studied the effects of Zn2+ and Ni2+ (5-30 mM) on the macroscopic K+ current through the inward rectifier system. The experiments were performed in the short muscle fibers of the lumbricalis muscle of toads with a two-microelectrode voltage clamp technique. The fibers were equilibrated in a control solution containing 68 mM K2SO4 and then exposed to Zn2+ or Ni2+. We found that both cations reduced in a reversible manner the current carried by K+ ions, and this reduction was prevented by decreasing the external pH of the solution (pH 5). The blockade of current was slightly dependent on the membrane potential and time independent. Two mechanisms may be involved in the blocking action of these cations: Zn2+ and Ni2+ may either be blocking the pore of the channels or acting at a regulatory binding site on the extracellular surface in an unspecified manner.

Caffeine contractures in denervated slow-twitch and fast-twitch muscles of the rat

Caffeine contractures (25 mM) and twitches were registered in vitro (34 degrees C) in normal and denervated soleus and extensor digitorum longus of the rat. Both muscles lost weight progressively after denervation although the loss of weight in soleus muscles was more manifest. Denervated extensor digitorum longus muscles showed an increase in the caffeine contracture tension (expressed in g/g muscle) whereas the response generated by denervated soleus muscles was smaller than the control values. The time to peak of the caffeine responses was shortened in both types of muscles after denervation. Twitches were larger and had a slower time course than in normal muscles, especially in the extensor digitorum longus muscles. These findings suggest that some changes in the excitation-contraction coupling could be responsible for the potentiation of the twitches in denervated muscles.

Muscle mechanical changes following denervation and their modification by the use of an inhibitor of RNA synthesis

Abstract Mechanical properties of soleus muscles from adult Wistar rats were studied “in vitro”. Contraction time, time for half relaxation, duration of the active state, fusion frequency and tetanus-twitch ratio were measured. In a first group of experiments normal innervated muscles were compared with muscles denervated for ten days. Significative differences in their contractile properties were found. In a second group of rats Actinomycin D was injected intravenously eight days after denervation and soleus contractile parameters compared 48 hours later with those obtained in the same muscle of denervated untreated rats. Treated muscles did not show significative differences with the untreated ones. However, when Actinomycin D was injected at the time of denervation differences in the duration of the active state were detected. It is suggested in the present paper that mechanical changes following denervation may be considered an induced phenomenon, as was demonstrated in other denervatory changes.

The effect of aminophylline on the contraction threshold of rat diaphragm fibers and its modification by 9-aminoacridine

We studied the effect of aminophylline (1mM) and 9-aminoacridine (100 microM) on the contraction threshold (CT) of rat diaphragm fibers (25 degrees C). The CT was measured by direct visualization (200 X) of the fiber under current-clamp conditions. The main findings are the following: 1) Aminophylline lowers the CT toward more negative values of the resting membrane potential (Vm). 2) 9-aminoacridine, a drug that diminishes Ca2+ release from the sarcoplasmic reticulum (SR), shifts the CT toward more positive values: 3) this effect is overcome by aminophylline. We suggest that the displacement in the CT to more negative Vm plays an important role in the potentiating effect of aminophylline. This could be the result of an enhancement of Ca2+ release from the SR.