Effect of Cell Cycle on Cell Surface Expression of Voltage-Gated Sodium Channels and Na+,K+-ATPase.

Voltage-gated sodium channels (VGSCs) are the target for many therapies. Variation in membrane potential occurs throughout the cell cycle, yet little attention has been devoted to the role of VGSCs and Na+,K+-ATPases. We hypothesized that in addition to doubling DNA and cell membrane in anticipation of cell division, there should be a doubling of VGSCs and Na+,K+-ATPase compared to non-dividing cells. We tested this hypothesis in eight immortalized cell lines by correlating immunocytofluorescent labeling of VGSCs or Na+,K+-ATPase with propidium iodide or DAPI fluorescence using flow cytometry and imaging. Cell surface expression of VGSCs during phases S through M was double that seen during phases G0-G1. By contrast, Na+,K+-ATPase expression increased only 1.5-fold. The increases were independent of baseline expression of channels or pumps. The variation in VGSC and Na+,K+-ATPase expression has implications for both our understanding of sodium's role in controlling the cell cycle and variability of treatments targeted at these components of the Na+ handling system.

The Role of Targeted Osmotic Lysis in the Treatment of Advanced Carcinoma in Companion Animals: A Case Series.

Background: Targeted osmotic lysis (TOL) is a novel technology that involves concomitant stimulation of voltage-gated sodium channels (VGSCs) and the pharmacological blockade of Na+, K+-ATPase causing lysis of highly malignant cancer cells. Hypothesis/Objectives. TOL offers an option for treating advanced carcinomas in companion animals. Animals. Two cats and 2 dogs that presented to veterinary hospitals for evaluation and treatment of one of several forms of carcinoma. Methods: Digoxin was administered to achieve steady-state, therapeutic concentrations. The animals were then exposed to pulsed electric field stimulation. Pre- and posttreatment assessments of tumor size and quality of life were compared. The treatment frequency and survivability varied, based on the patient's premorbid functioning and response to treatment. Results: Regardless of cancer type, TOL consistently increased survival beyond expected, often improving, but without compromising of quality of life. Conclusions and Clinical Importance. TOL warrants consideration as an option for managing advanced carcinomas.

Emergency Use of Targeted Osmotic Lysis for the Treatment of a Patient with Aggressive Late-Stage Squamous Cell Carcinoma of the Cervix.

Upregulation of voltage-gated sodium channels (VGSCs) and Na+/K+-ATPase (sodium pumps) is common across most malignant carcinomas. Targeted osmotic lysis (TOL) is a developing technology in which the concomitant stimulation of VGSCs and pharmacological blockade of sodium pumps causes rapid selective osmotic lysis of carcinoma cells. This treatment of cervical carcinoma is evidence that TOL is a safe, well-tolerated and effective treatment for aggressive advanced carcinomas that has the potential to extend life without compromising its quality. TOL is likely to have broad application for the treatment of advanced-stage carcinomas.

Targeted Osmotic Lysis of Highly Invasive Breast Carcinomas Using Pulsed Magnetic Field Stimulation of Voltage-Gated Sodium Channels and Pharmacological Blockade of Sodium Pumps.

Abstract: Concurrent activation of voltage-gated sodium channels (VGSCs) and blockade of Na+ pumps causes a targeted osmotic lysis (TOL) of carcinomas that over-express the VGSCs. Unfortunately, electrical current bypasses tumors or tumor sections because of the variable resistance of the extracellular microenvironment. This study assesses pulsed magnetic fields (PMFs) as a potential source for activating VGSCs to initiate TOL in vitro and in vivo as PMFs are unaffected by nonconductive tissues. In vitro, PMFs (0-80 mT, 10 msec pulses, 15 pps for 10 min) combined with digoxin-lysed (500 nM) MDA-MB-231 breast cancer cells stimulus-dependently. Untreated, stimulation-only, and digoxin-only control cells did not lyse. MCF-10a normal breast cells were also unaffected. MDA-MB-231 cells did not lyse in a Na+-free buffer. In vivo, 30 min of PMF stimulation of MDA-MB-231 xenografts in J/Nu mice or 4T1 homografts in BALB/c mice, concurrently treated with 7 mg/kg digoxin reduced tumor size by 60-100%. Kidney, spleen, skin and muscle from these animals were unaffected. Stimulation-only and digoxin-only controls were similar to untreated tumors. BALB/C mice with 4T1 homografts survived significantly longer than mice in the three control groups. The data presented is evidence that the PMFs to activate VGSCs in TOL provide sufficient energy to lyse highly malignant cells in vitro and to reduce tumor growth of highly malignant grafts and improve host survival in vivo, thus supporting targeted osmotic lysis of cancer as a possible method for treating late-stage carcinomas without compromising noncancerous tissues.

Depletion of the Insulator Protein CTCF Results in Herpes Simplex Virus 1 Reactivation In Vivo.

Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in host peripheral neurons, including the neurons of the trigeminal ganglia (TG). HSV-1 can reactivate from neurons to cause recurrent infection. During latency, the insulator protein CTCF occupies DNA binding sites on the HSV-1 genome, and these sites have been previously characterized as functional enhancer-blocking insulators. Previously, CTCF was found to be dissociated from wild-type virus postreactivation but not in mutants that do not reactivate, indicating that CTCF eviction may also be an important component of reactivation. To further elucidate the role of CTCF in reactivation of HSV-1, we used recombinant adeno-associated virus (rAAV) vectors to deliver a small interfering RNA targeting CTCF to peripheral neurons latent with HSV-1 in rabbit TG. Our data show that CTCF depletion resulted in long-term and persistent shedding of infectious virus in the cornea and increased ICP0 expression in the ganglia, indicating that CTCF depletion facilitates HSV-1 reactivation.IMPORTANCE Increasing evidence has shown that the insulator protein CTCF regulates gene expression of DNA viruses, including the gammaherpesviruses. While CTCF occupation and insulator function control gene expression in DNA viruses, CTCF eviction has been correlated to increased lytic gene expression and the dissolution of transcriptional domains. Our previous data have shown that in the alphaherpesvirus HSV-1, CTCF was found to be dissociated from the HSV-1 genome postreactivation, further indicating a global role for CTCF eviction in the transition from latency to reactivation in HSV-1 genomes. Using an rAAV8, we targeted HSV-1-infected peripheral neurons for CTCF depletion to show that CTCF depletion precedes the shedding of infectious virus and increased lytic gene expression in vivo, providing the first evidence that CTCF depletion facilitates HSV-1 reactivation.

Chelatable trace zinc causes low, irreproducible KDAC8 activity.

Acetylation is an important regulatory mechanism in cells, and emphasis is being placed on identifying substrates and small molecule modulators of this post-translational modification. However, the reported in vitro activity of the lysine deacetylase KDAC8 is inconsistent across experimental setups, even with the same substrate, complicating progress in the field. We detected trace levels of zinc, a known inhibitor of KDAC8 when present in excess, even in high-quality buffer reagents, at concentrations that are sufficient to significantly inhibit the enzyme under common reaction conditions. We hypothesized that trace zinc in solution could account for the observed variability in KDAC8 activity. We demonstrate that addition of chelators, including BSA, EDTA, and citrate, and/or the use of a phosphate-based buffer instead of the more common tris-based buffer, eliminates the inhibition from low levels of zinc as well as the dependence of specific activity on enzyme concentration. This results in high KDAC8 activity that is consistent across buffer systems, even using low concentrations of enzyme. We report conditions that are suitable for several assays to increase both enzyme activity and reproducibility. Our results have significant implications for approaches used to identify substrates and small molecule modulators of KDAC8 and interpretation of existing data.