Effect of drugs affecting synthesis or degradation of connexin on onset or completion of ethylene glycol induced inhibition of intercellular gap junctional communication.

The role of a connexin synthesis and degradation in the onset or completion of the ethylene glycol-induced inhibition of the gap junctional intercellular communication (GJIC) in V79-4 Chinese hamster cell line was studied as a model of an interaction between the cells and a potential tumor promoter. GJIC was assessed on two levels: on the cytophysiological level - the dye coupling method, and on the immunocytochemical level - the immunolabeling of connexin43. The specific activator of connexin synthesis - Dibutyryl cAMP made the onset of the EG-induced inhibition of GJIC slower, but its effect was only temporary. On the other hand it also speeded up the re-establishment of standard values of GJIC after the removal of EG. Although the non-specific inhibitor of protein degradation via proteasomes - leupeptin increased the amount of connexin plaques on cell membranes, its effect on GJIC remained insignificant. The non-specific inhibitors of transcription - actinomycin D and translation - cycloheximide significantly inhibited the re-establishment of the standard values of GJIC after the removal of EG. The results indicate that although the storage of connexins in Golghi complex probably plays the principal role in the control of the gap junctional communication, the extensive changes in GJIC activity depend on the de novo synthesis of connexin per se.

Selective gap junctional communication within the V79-4 Chinese hamster cell line.

The probability of cell-to-cell coupling between directly adjacent cells (communication capability) in the V79-4 Chinese hamster cell line was evaluated under standard conditions or after 18-h treatment with EG. The cell monolayer did not form a continuous network of cells interconnected via gap junctions, but an average cell was coupled to only one half of its directly adjacent neighbours under standard conditions, or to one third of its directly neighbouring cells after 18-h exposure to EG. The rest of the directly adjacent neighbours did not establish functional gap junctions with an injected cell, although they were competent to couple to other cells with a probability similar to that of the coupling between the injected cell and its direct neighbours. Moreover, all the cells possessed the identical connexin--cx43, present on all cell membranes. The results indicated that the choice of a cell to which neighbour be coupled was rather random in the standard cell population as a whole, although the population contained some clones whose capability to couple was more or less different from that of the original cell population. Ethylene glycol reduced the gap junctional communication by increasing the frequency of cells not coupled to any of their direct neighbours from 1% for untreated cells to 23.3% for cells exposed to EG, and consequently by reducing the number of directly adjacent cells coupled to communicating injected cells. The communication capability of the cell population appeared to be unstable. It varied slightly in time and so did the response of the cells to EG. The results indicate that a cell can control its coupling to different directly adjacent neighbours independently, being able to control the gap junctional communication not only in time but in space as well. All control mechanisms of GJIC, known so far, affect a cell as a whole, while our results indicate that another regulatory mechanism may exist, controlling the gap junctional communication to different adjacent neighbouring cells independently.

The biological conditions of assessment of ethylene glycol-induced inhibition of gap junctional intercellular communication by metabolic cooperation assay.

Ethylene glycol (EG) has been previously shown to inhibit gap junctional intercellular communication. In this paper we examine conditions under which the effect of EG on gap junctional communication is assessed by metabolic cooperation assay. The later, after the start of metabolic cooperation assay, EG was added, the lower its inhibitory effect was. If treatment with EG began 12 h or even later after plating of cells, no significant effect on gap junctional communication was observed. Short EG treatments (2-8 h) induced a reversible inhibition of cell-to-cell communication, provided that the cells could communicate freely after the drug was removed. However, if further cell-to-cell communication was excluded, the effect of short exposures was irreversible. Using Scrape loading method we observed that after a 60 min exposure to EG the standard gap junctional intercellular communication was completely restored in a few hours.

The behavior of cells during the evaluation of gap junctional intercellular communication by metabolic cooperation assay.

The behavior of cells during the assessment of gap junctional intercellular communication (GJIC) by metabolic cooperation assay was studied, either under standard conditions, or with the simultaneous addition of an inhibitor of GJIC-ethylene glycol, and eventually plus dibutyryl cyclic adenosine monophosphate. Generally, the behavior of cells in the course of the assay did not depend significantly on the combination of drugs used. Cell locomotion during the assay was minimal, and consequently the results depended, almost solely on the probability of mutual contact between donor and recipient cells during the seeding. The first significant decrease in recovery of recipient cells, that is a detectable GJIC, was observed 30 min after the assay had started. Final results of the assay depended on the first 12 h of the experiment, in which the maximum decrease in the recovery of recipient cells, that is the maximum GJIC, was reached. The longer duration of the assay did not affect the results significantly.

Inhibition of intercellular gap junctional communication by alkyl ethers and its modulation by cAMP.

The inhibition of intercellular gap junctional communication (IGJC) by alkyl ethers (ethylene glycol, monomethyl ether, polyethylene glycol 1,000 and polyethylene glycol 6000) was examined using V79 Chinese hamster cells in vitro. Ethylene glycol and monomethyl ether inhibited IGJC very strongly, whilst the other agents inhibited IGJC only insignificantly. When the cells were treated with the combination of two agents, ethylene glycol and monomethyl ether, a significant increase in the inhibition of IGJC occurred. This was probably the result of potentiation rather than an addition effect. The effect of ethylene glycol was antagonized by dibutyryl cyclic adenosine monophosphate (DbcAMP). This effect was most intensive when the cells were treated with both agents at the same time and, in other experimental combinations, the effect was lower but also significant. Caffeine did not influence IGJC either in combination with DbcAMP or by itself.

Reliability of the "kiss of death" method for evaluation of intercellular communication: effect of cloning and culture technique.

I explored the effect of genetic (cloning) and non-genetic (arrangement of the method and quality of culture serum) factors upon variability of the results obtained by using the "kiss of death" method for evaluation of intercellular communication in Chinese hamster cells V79-4. Cloning had neither quantitative (intraspecies communication) nor qualitative (interspecies communication) effects. Of the non-genetic factors, the time interval of application of transferred agent (thioguanine) after plating the cells and the agent concentration were investigated. These factors did not influence significantly the results. Intercellular communication was more intensive in a less rich medium containing bovine serum than in a richer medium containing foetal calf serum. On the basis of the results obtained I conclude that the higher variability of intercellular communication values determined by the "kiss of death" method is not due to an error in the experimental method used but probably reflects the real changes in these values that occur with time in cell populations.

Population changes of human somatic cells in culture during short-term treatment with drugs.

The human somatic cell line VUPT was treated with 8-azaguanine or 5-bromo-2'-deoxyuridine for a week, then the drug was removed and cultivation continued in a standard medium. During or after the exposure to the drug various population parameters, such as morphological variability, frequency of multinuclear cells, total mitotic index, index of atypical mitoses, dispersion of chromosomal counts were evaluated. Increase in both morphological and karyological variability during the treatment with drugs was found but this increase was temporary and during one or two weeks of cultivation in a standard medium the initial phenotypic equilibrium of the population was restored. The meaning of such population changes is discussed.

Similarity in dynamics of single or double minute chromosomes incidence and number of chromosomal aberrations during long-term treatment of a human cell line with methotrexate.

The human cell line VUPT was treated with gradually increasing concentrations of methotrexate for 500 days. During this period of time the frequency of cells containing single or double minute chromosomes and the number of chromosomal aberrations were estimated. When the concentration of the drug increased, so did the frequency of cells with minute chromosomes, but after several days of adaptation to the given concentration of the drug the minute chromosomes disappeared again. The dynamics of the frequency of chromosomal aberrations resembled that of the minute chromosomes, but the change in the former cytogenetic parameter generally preceded corresponding changes in the latter. The results indicate a possible relationship between the development of chromosomal aberrations and the formation of minute chromosomes.