Yanina A. Assef

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.

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.

xshroom1 Regulates the Number of ENaC Channels Inserted in the Membrane of Oocytes From XENOPUS Laevis

Shroom is a family of proteins linked to the actin cytoskeleton. We studied its effect upon the currents through ENaC channels (INa amil) in oocytes (X. laevis) injected with α, β, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We observed a strong reduction in INa amil with the xShroom1 antisense: inward conductances (Ginward) (−160 to 0 mV) were 36 ± 12 µS and 1.80 ±.50 µS with xShroom1 sense and antisense. Similar results were obtained in oocytes expressing a mutant β-mENaC subunit (β-S518K) with a open probability of 1 (Ginward 65 ± 10 µS and 1.80 ± 2.0 µS for oocytes with xShroom1 sense or xShroom1 antisense. The negative effects of xShroom1 antisense can not be reversed with forskolin which reduced the rate of ENaC retrieval: Ginward : 124 ± 27 µS and 7.0 ± 1.9 µS with xShroom1 sense or xShroom1 antisense. Trypsin in the range of ng/ml activates the membrane-resident ENaC channels (Bengrine et al.2007), being this effect dependent on activation of G-proteins. Addition of 20 ng/ml of trypsin led to a slow increase in INa amil with xShroom1 sense and it had no effect in most of the oocytes coinjected with ENaC and xShroom1 antisense (2 out of 20). Trypsin were without effects on the endogenous conductances. These data are consistent with the idea that the reduced INa amil when xShroom1 is blocked is most probably due to a lack of functional ENaC channels in the plasma membrane.Acknowledgements: ENaC cDNAs were provided by Dr M. Carattino (Pittsburgh, Pa). and the set for oocytes was a gift of Dr C. Peracchia (Rochester, NY). Supported by grants MO35 (UBA) and PICT 38181.