Nucleofection induces non-specific changes in the metabolic activity of transfected cells.

Transfection has become an everyday technique widely used for functional studies in living cells. The choice of the particular transfection method is usually determined by its efficiency and toxicity, and possible functional consequences specific to the method used are normally overlooked. We describe here that nucleofection, a method increasingly used because of its convenience and high efficiency, increases the metabolic rate of some cancer cells, which can be misleading when used as a measure of proliferation. Moreover, nucleofection can alter the subcellular expression pattern of the transfected protein. These undesired effects are independent of the transfected nucleic acid, but depend on the particular cell line used. Therefore, the interpretation of functional data using this technology requires further controls and caution.

Study of membrane potential in T lymphocytes subpopulations using flow cytometry.

BACKGROUND: Ion channels are involved in the control of membrane potential (psi) in a variety of cells. The maintenance of psi in human T lymphocytes is essential for T-cell activation and was suggested to depend mostly on the voltage-gated Kv1.3 channel. Blockage of Kv1.3 inhibits cytokine production and lymphocyte proliferation in vitro and suppresses immune response in vivo. T lymphocytes are a heterogeneous cell population and the expression of Kv1.3 varies among cell subsets. Oxonol diBA-C4-(3) was used to determine psi by flow cytometry. The presence of distinct T cell subsets was evaluated by immunophenotyping techniques and the contribution of Kv1.3 channels for the maintenance of psi was investigated using selective blockers. RESULTS: The distribution of psi in T lymphocytes varied among blood donors and did not always follow a unimodal pattern. T lymphocytes were divided into CD3+/CD45RO- and CD3+/CD45RO+ subsets, whose peak channel values of psi were -58 +/- 3.6 mV and -37 +/- 4.1 mV, respectively. MgTX (specific inhibitor of Kv1.3 channels) had no significant effect in the psi of CD3+/CD45RO- subsets but depolarized CD3+/CD45RO+ cells to -27 +/- 5.1 mV. CONCLUSION: Combination of optical methods for determination of psi by flow cytometry with immuophenotyping techniques opens new possibilities for the study of ion channels in the biology of heterogeneous cell populations such as T lymphocyte subsets.

Ether à go-go potassium channel expression in soft tissue sarcoma patients.

BACKGROUND: The expression of the human Eag1 potassium channel (Kv10.1) is normally restricted to the adult brain, but it has been detected in both tumour cell lines and primary tumours. Our purpose was to determine the frequency of expression of Eag1 in soft tissue sarcoma and its potential clinical implications. RESULTS: We used specific monoclonal antibodies to determine the expression levels of Eag1 in soft tissue sarcomas from 210 patients by immunohistochemistry. Eag1 was expressed in 71% of all tumours, with frequencies ranging from 56% (liposarcoma) to 82% (rhabdomyosarcoma). We detected differences in expression levels depending on the histological type, but no association was seen between expression of this protein and sex, age, grade or tumour size. Four cell lines derived from relevant sarcoma histological types (fibrosarcoma and rhabdomyosarcoma) were tested for Eag1 expression by real-time RT-PCR. We found all four lines to be positive for Eag1. In these cell lines, blockage of Eag1 by RNA interference led to a decrease in proliferation. CONCLUSION: Eag1 is aberrantly expressed in over 70% sarcomas. In sarcoma cell lines, inhibition of Eag1 expression and/or function leads to reduced proliferation. The high frequency of expression of Eag1 in primary tumours and the restriction of normal expression of the channel to the brain, suggests the application of this protein for diagnostic or therapeutic purposes.

Overexpression of Eag1 potassium channels in clinical tumours.

BACKGROUND: Certain types of potassium channels (known as Eag1, KCNH1, Kv10.1) are associated with the production of tumours in patients and in animals. We have now studied the expression pattern of the Eag1 channel in a large range of normal and tumour tissues from different collections utilising molecular biological and immunohistochemical techniques. RESULTS: The use of reverse transcription real-time PCR and specifically generated monoclonal anti-Eag1 antibodies showed that expression of the channel is normally limited to specific areas of the brain and to restricted cell populations throughout the body. Tumour samples, however, showed a significant overexpression of the channel with high frequency (up to 80% depending on the tissue source) regardless of the detection method (staining with either one of the antibodies, or detection of Eag1 RNA). CONCLUSION: Inhibition of Eag1 expression in tumour cell lines reduced cell proliferation. Eag1 may therefore represent a promising target for the tailored treatment of human tumours. Furthermore, as normal cells expressing Eag1 are either protected by the blood-brain barrier or represent the terminal stage of normal differentiation, Eag1 based therapies could produce only minor side effects.

TRPM8 ion channels differentially modulate proliferation and cell cycle distribution of normal and cancer prostate cells.

Overexpression of the cation-permeable channel TRPM8 in prostate cancers might represent a novel opportunity for their treatment. Inhibitors of TRPM8 reduce the growth of prostate cancer cells. We have used two recently described and highly specific blockers, AMTB and JNJ41876666, and RNAi to determine the relevance of TRPM8 expression in the proliferation of non-tumor and tumor cells. Inhibition of the expression or function of the channel reduces proliferation rates and proliferative fraction in all tumor cells tested, but not of non-tumor prostate cells. We observed no consistent acceleration of growth after stimulation of the channel with menthol or icilin, indicating that basal TRPM8 expression is enough to sustain growth of prostate cancer cells.

Dynamic conformational changes in the FERM domain of FAK are involved in focal-adhesion behavior during cell spreading and motility.

Focal adhesion kinase (FAK) controls cellular adhesion and motility processes by its tight link to integrin- and extracellular-matrix-mediated signaling. To explore the dynamics of the regulation of FAK, we constructed a FRET-based probe that visualizes conformational rearrangements of the FERM domain of FAK in living cells. The sensor reports on an integrin-mediated conformational change in FAK following cellular adhesion. The perturbation is kinase-independent and involves the polybasic KAKTLR sequence in the FERM domain. It is manifested by an increased FRET signal and is expressed primarily in focal adhesions, and to a lesser extent in the cytoplasm. The conformational change in the FERM domain of FAK is observed in two consecutive phases during spreading - early and late - and is enriched in fully adhered motile cells at growing and sliding peripheral focal-adhesion sites, but not in stable or retracting focal adhesions. Inhibition of the actomyosin system indicates the involvement of tension signaling induced by Rho-associated kinase, rather than by myosin light-chain kinase, in the modulation of the FERM response. We conclude that the heterogeneous conformation of the FERM domain in focal adhesions of migrating cells reflects a complex regulatory mechanism for FAK that appears to be under the influence of cellular traction forces.