Increasing cell membrane potential and GABAergic activity inhibits malignant hepatocyte growth

Increasing hepatocyte membrane potentials by augmenting GABAergic activity inhibits nonmalignant hepatocyte proliferative activity. The objectives of this study were to document 1) potential differences (PDs) of four malignant hepatocyte cell lines, 2) GABAA receptor mRNA expression in the same cell lines, and 3) effects of restoring malignant hepatocyte PDs to levels approximating those of resting, nonmalignant hepatocytes. Hepatocyte PDs were documented in nonmalignant and malignant (Chang, HepG2, HuH-7, and PLC/PRF/5) hepatocytes with a fluorescent voltage-sensitive dye and GABAA receptor expression by RT-PCR and Western blot analyses.Compared with nonmalignant human hepatocytes, all four malignant cell lines were significantly depolarized (P _0.0001, respectively). Only PLC/PRF/5 cells had detectable GABAA-_3 receptor mRNA expression and all cell lines were negative for GABAA-_3 receptor protein by Western blot analysis. Stable transfection of Chang cells with GABAA-_3 receptor cDNA resulted in significant increases in PD and decreases in proliferative activity as manifest by decreased [3H]thymidine and bromodeoxyurieine incorporation rates, 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3- benzene disulfonate activity, a lower mitotic index, prolongation of cell-doubling times, and attenuated growth patterns compared with cells transfected with vector alone. Colony formation in soft agar and the number of abnormal mitoses were also significantly decreased in GABAA-_3 receptor transfected cells. The results of this study indicate 1) relative to healthy hepatocytes, malignant hepatocytes are significantly depolarized, 2) GABAA-_3 receptor expression is absent in malignant hepatocyte cell lines, and 3) increasing the PD of malignant hepatocytes is associated with less proliferative activity and a loss of malignant features.

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Carcinogenesis and the plasma membrane

Summary
Presented is a two-stage hypothesis of carcinogenesis based on: (1) plasma membrane defects that produce abnormal electron and proton efflux; and (2) electrical uncoupling of cells through loss of intercellular communication. These changes can be induced by a wide variety of stimuli including chemical carcinogens, oncoviruses, inherited and/or acquired genetic defects, and epigenetic abnormalities. The resulting loss of electron/proton homeostasis leads to decreased transmembrane potential, electrical microenvironment alterations, decreased extracellular pH, and increased intracellular pH. This produces a positive feedback loop to enhance and sustain the proton/electron efflux and loss of intercellular communication. Low transmembrane potential is functionally related to rapid cell cycling, changes in membrane structure, and malignancy. Intracellular alkalinization affects a variety of pH-sensitive systems including glycolysis, DNA synthesis, DNA transcription and DNA repair, and promotes genetic instability, accounting for the accumulation of genetic defects seen in malignancy. The abnormal microenvironment results in the selective survival and proliferation of malignant cells at the expense of contiguous normal cell populations.

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Contact inhibition of division: Involvement of the electrical transmembrane potential

Clarence D. Cone , Max Tongier Journal of Cellular Physiology Volume 82, Issue 3, Pages 373 - 386

Measurements of simultaneous mitotic activity, electrical transmembrane potential (Em), and cell density levels in both 3T3 and Chinese hamster ovary (CHO) cell cultures reveal that a 5- to 6-fold increase in the Em level is associated with development of mitotic arrest at saturation densities. This rise occurs both in confluent monolayers and in interior areas of isolated colonies, and is independent of the rate at which confluence is attained. The Em rise is accompanied by a substantial decrease in intracellular Na. Electron microscopy of saturated CHO monolayer sections shows from 46 to 63% of the cell surfaces to be in close apposition (<300 Å spacing). These results for contact inhibited cultures support the hypothesis that mitotic activity may be functionally coupled with the Em level and associated ionic concentration levels. It is suggested that contact inhibition of mitosis may result from a reduction in synthesis of mitogenically essential RNA following a decrease in intracellular Na produced by contact-induced alteration of surface ion-transport activity.

  
Cellular potentials of normal and cancerous fibroblasts and hepatocytes.

Binggeli R, Cameron IL Cancer Research  1980 Jun;40(6):1830-5.

Several lines of investigation point to differences in electrical properties between normal and cancerous cells. Several tumor lines have low-resting membrane potentials. A few comparisons have been made between normal and tumor cells within the same tissue cell type. This study compares the cellular or transmembrane potential of hepatocytes and fibroblasts in both normal and tumor cells. High-impedance micropipets were used to record intracellularly in vivo in Buffalo rat hepatocytes and Morris 7777 hepatoma cells, as well as A/J mouse corneal fibroblasts and poorly differentiated fibrosarcoma cells. Rat hepatocytes had a mean membrane potential of -37.1 +/- 4.3 (S.D.) mV compared to -19.8 +/- 7.1 mV in the hepatoma cells. Mouse corneal fibroblasts measured -42.5 +/- 5.4 mV, while cells of mouse fibrosarcoma were -14.3 +/- 5.4 mV. The membrane potentials of the tumor cells were lower in both instances than in their normal counterpart (statistically significant at p = 0.001 for both tissue cell types). This supports the notion that lower cellular or membrane potentials may play a significant role in the altered physiology of the tumor cell.

Deficits in elevating membrane potential of rat fibrosarcoma cells after cell contact

Binggeli R, Weinstein RC Cancer Research  1985 Jan;45(1):235-41.

Most cancer cells are known to have lower resting cellular potentials than do their normal counterparts. This study investigates how these potentials establish themselves during growth and cellular contact in tissue culture. Normal quail embryonic fibroblasts and quail fibrosarcoma (QT-35) and normal rat kidney cells and rat fibrosarcoma (from rat fibroblasts chemically transformed by nitroquinoline oxide) were recorded intracellularly using high-impedance micropipets. In high-density high-contact cultures, both quail and rat cancer cells had lower potentials than did normal cells (-20.7 compared to -40.1 mV for quail and -30.7 compared to -61.9 mV for rat). In low-density mitotically synchronous cultures, the rat cells were recorded every 4 hr for 96 hr. Starting at a low density, normal cell membrane potential is maintained at a low level through subsequent cell divisions. Without any additional change in cell density, the potential suddenly elevates to a high level. The membrane potential of cancer cells is by contrast unrelated either to cell density or to time. Cancer cells maintained an intermediate potential from low to very high densities and never elevated their potential to high values. The failure of cancer cells to reach high potentials may be linked to their uncontrolled cell division.

Calcium ion and the membrane potential of tumor cells

Binggeli R, Weinstein RC, Stevenson D Cancer Biochemisry Biophysics 1994 Oct;14(3):201-10.

Calcium ion affects ion permeability and membrane potential among many other aspects of cell function. Initial effects of increasing extracellular calcium upon membrane potential were studied in a quail fibrosarcoma (QT35) where calcium had a dose dependent effect, and normal quail fibroblasts, where there was little effect. Comparisons were then made in six different human hepatocellular carcinomas (Tong, HepG2, Hep3B, PLC/PRF/5, Mahlavu, and HA22T) in response to smaller changes in concentration. There were insignificant changes in membrane potential in two cell lines and significant elevations in four. Cytolysis by natural killer cells also declined in rough proportion to the increase in membrane potential. The less differentiated hepatocellular carcinoma cells have both higher baseline membrane potentials and a greater potential increase to increased calcium. By contrast, more highly differentiated tumor cells had paradoxically smaller membrane potentials and along with normal cells had small potential responses to calcium increases.