tretinoin and lucifer-yellow

Gap junctions play a central role in coordinating intercellular signal-transduction pathways to control tissue homeostasis. Deregulation of gap junctional intercellular communication is a common phenotype of cancer cells and supports its involvement in the carcinogenesis process. Many carcinogens, like environmental heavy-metal chemical pollutants, are known to activate various signal transduction mechanisms and modulate GJIC. They act as tumor promoters on preexisting "initiated" cells, rather than as genotoxic initiators, albeit their mode of action is often unknown. In this study we investigated the effect of Hg(II) (HgCl(2)) on GJIC in cultured human keratinocytes. It is shown that subcytotoxic concentrations of HgCl(2) as low as 10 nM cause inhibition of the GJIC, assessed by dye transfer assay, despite enhanced expression of connexins. In addition, HgCl(2)-treated keratinocytes exhibited a decrease of free thiols and accumulation of mitochondria-derived reactive oxygen species, albeit no effect on the respiratory chain activity was observed. Treatment of HgCl(2)-exposed keratinocytes with the PKC inhibitor calphostin C and with all-trans retinoic acid resulted in rescue of the mitochondrial ROS overproduction and full recovery of the GJIC. Similar results were obtained with the PKA activator db-cAMP. Overall, the presented results support a cross-talk between the altered intracellular redox tone and PKA- and PKC-mediated signaling in HgCl(2)-challenged keratinocytes. These events, although not cytotoxic, lead to inhibition of GJIC and possibly to carcinogenic priming.

Topics: Cell Communication; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Cytotoxins; Fluorescent Dyes; Gap Junctions; Glutathione; Glutathione Disulfide; Humans; Isoquinolines; Keratinocytes; Mercuric Chloride; Mitochondria; Oxidation-Reduction; Oxygen Consumption; Phosphorylation; Protein Kinase C; Reactive Oxygen Species; Signal Transduction; Tretinoin

In our previous study we evaluated the antitumor effect of herpes simplex virus-thymidine kinase gene (HSV-tk) on human medulloblastomas (MBs) in a therapeutic delivery system using the immortalized neural stem cell (NSC) line C17.2. However, our findings indicated that the bystander effect between C17.2tk and Daoy MB cells was weak compared to the bystander effect between NSCtk and C6 glioma cells. Gap junction intercellular communication (GJIC) is the main mechanism mediating the bystander effect in HSV-tk gene therapy. All-trans retinoic acid (ATRA) has been shown to up-regulate the expression of Connexin43 and GJIC. In this study we investigated the synergistic effect of ATRA and HSV-tk gene therapy in the treatment of MBs. We found that the expression of Connexin43 in Daoy cells was significantly increased when cells were exposed to 3μmol/l of ATRA (P<0.05). After co-culturing C17.2tk cells with Daoy cells at different ratios ranging from 1:1 to 1:16, ATRA significantly increased the bystander anti-tumor effect compared to ATRA-untreated cells (P<0.05). In intracranial co-implantation experiments, mice co-implanted with C17.2tk/Daoy cells and treated with a combination of ATRA and GCV had significantly smaller tumors compared to the animals treated with GCV alone (P<0.05). Together, our results show that ATRA enhanced the tumoricidal effect in HSVtk/GCV suicide gene therapy against Daoy MB cells by strengthening the bystander effect in vitro and in vivo.

Topics: Animals; Bystander Effect; Cell Line; Cerebellar Neoplasms; Connexin 43; Genes, Transgenic, Suicide; Genetic Therapy; Humans; Isoquinolines; Male; Medulloblastoma; Mice; Mice, Inbred BALB C; Simplexvirus; Stimulation, Chemical; Thymidine Kinase; Tretinoin; Up-Regulation

In the retina, all-trans retinoic acid (at-RA) could function as a light signal because its production increases with the level of illumination. Given the well-established effects of retinoic acid on cell coupling in other tissues, it is possible that the changing levels of at-RA modulate the gap junctional permeability between retinal neurons. This study examines the effects of retinoic acid on horizontal cell coupling, which is known to be modulated by the ambient light level. Single horizontal cells were injected under visual control with either Neurobiotin (mouse retina) or Lucifer yellow (rabbit retina) and the extent of tracer coupling or dye coupling was used to monitor the gap junctional permeability. In the mouse retina, the injection of Neurobiotin revealed a network of approximately 150-250 tracer-coupled horizontal cells. The tracer coupling was completely abolished by incubating the retina in 150 microM at-RA for 35 min. In the rabbit retina, the injection of Lucifer yellow into A-type horizontal cells revealed networks of approximately 15-30 dye-coupled horizontal cells. Incubation in 150 microM at-RA reduced the dye coupling within 12 min and complete uncoupling was achieved after 35 min. The uncoupling effects of at-RA in the mouse and rabbit retinas were concentration- and time-dependent and they were reversible after washout. The coupling was not affected by either the 9-cis form of retinoic acid or by at-RA that had been isomerized by intensive light. The uncoupling effect of at-RA persisted following treatment with a D1 receptor antagonist and thus was dopamine-independent. This study has established that at-RA is able to modulate the gap junctional permeability between horizontal cells in the mammalian retina, where its light-dependent release has already been demonstrated.

Topics: Animals; Biotin; Cell Communication; Coloring Agents; Dopamine; Gap Junctions; Isoquinolines; Mice; Microscopy, Confocal; Permeability; Rabbits; Receptors, Dopamine D1; Retina; Signal Transduction; Tretinoin

The controlled invasiveness of the trophoblast is based on the balance between invasive properties at implantation and the differentiation program of the developing placenta. During placental development in rats a switch of connexin gene expression has been observed in parallel to the switch from the invasive to the differentiated phenotype of trophoblast cells. To investigate the role of connexin expression for trophoblast invasion, proliferation, and differentiation, we studied one rat trophoblast (HRP-1) and one rat choriocarcinoma cell line (Rcho-1). The choriocarcinoma cells were characterized by expression of cx31 and a lack of E-cadherin, corresponding to the invasive trophoblast in vivo, whereas HRP-1 cells expressed cx43, normally found in the spongiotrophoblast and in late giant cells, and E-cadherin. Upon retinoic acid treatment, Rcho-1 cells irreversibly lost cx31 expression, accompanied by a loss of functional coupling. No changes in regard to connexin expression and cell-cell communication could be observed in HRP-1 cells. In addition, treatment of Rcho-1 cells with retinoic acid for 7 days upregulated expression of cx43 transcript, but no protein could be found. Proliferation was clearly reduced and the mean volume of cells doubled from Day 4 to Day 7 of retinoic acid treatment in Rcho-1 cells, while both parameters were not affected in HRP-1 cells. Both cell lines showed a similar invasion rate using a Matrigel invasion assay, and invasion was equally suppressed upon retinoic acid treatment. Thus the different connexin expression appears more likely to play a role in regulating proliferation and differentiation along the multilineage pathway than invasiveness of rat trophoblast cells.

Topics: Animals; Cadherins; Calcium; Cell Communication; Cell Division; Cell Movement; Choriocarcinoma; Collagen; Connexin 43; Connexins; Drug Combinations; Fluorescent Dyes; Gap Junctions; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Isoquinolines; Laminin; Proteoglycans; Rats; RNA, Messenger; Tretinoin; Trophoblasts; Tumor Cells, Cultured

The analysis of chimeras has shown that communication between germ-line and soma cells plays an important role during Drosophila oogenesis. We have therefore investigated the intercellular exchange of the fluorescent tracer molecule, Lucifer yellow, pressure-injected into the oocyte of vitellogenic follicles of Drosophila. The dye reached the nurse cells via cytoplasmic bridges and entered, via gap junctions, the somatic follicle cells covering the oocyte. The percentage of follicles showing dye-coupling between oocyte and follicle cells was found to increase with the developmental stage up to stage 11, but depended also on the status of oogenesis, i.e., the stage-spectrum, in the respective ovary. During late stage 10B and stage 11, dye-coupling was restricted to the follicle cells covering the anterior pole of the oocyte. No dye-coupling was observed from stage 12 onwards. During prolonged incubation in vitro, the dye was found to move from the follicle cells back into the oocyte; this process was suppressable with dinitrophenol. Dye-coupling was inhibited when prolonged in vitro incubation preceded the dye-injection. Moreover, dye-coupling was inhibited with acidic pH, low [K+], high intracellular [Ca2+], octanol, dinitrophenol, and NaN3, but not with retinoic acid, basic pH, or high extracellular [Ca2+]. Dye-coupling was stimulated with a juvenile hormone analogue and with 20-hydroxyecdysone. Thus, gap junctions between oocyte and follicle cells may play an important role in intercellular communication during oogenesis. We discuss the significance of our findings with regard to the electrophysiological properties of the follicles, and to the coordinated activities of the different cell types during follicle development and during the establishment of polarity in the follicle.

Topics: 1-Octanol; Animals; Calcium; Drosophila melanogaster; Female; Fluorescent Dyes; Hydrogen-Ion Concentration; Intercellular Junctions; Isoquinolines; Microscopy, Fluorescence; Octanols; Oocytes; Oogenesis; Ovarian Follicle; Potassium; Tretinoin

We have compared dye coupling in pairs of small (less than 10 microns in diameter) and large (greater than 20 microns in diameter) keratinocytes isolated from normal human epidermis, using Lucifer yellow microinjection. Under control conditions, dye coupling was found in only 1 out of the 25 small pairs tested, whereas it was evident in 75% of the large pairs (n = 52). After a 30-min incubation of the latter pairs in the presence of 10(-6) and 10(-4) M all-transretinoic acid (RA), the percentage of coupling was 53% (n = 15; NS) and 7% (n = 14; P less than 0.001), respectively. The almost complete uncoupling observed after 10(-4) M RA was not reversible even 30 min after return to control medium (n = 8). Dual whole-cell patch-clamp recordings from large keratinocyte pairs showed a macroscopic junctional conductance (gj) of 9 +/- 2 nS (n = 43), which was abolished by heptanol (3.5 mM) in a fully reversible way. Compared to heptanol, 10(-4) M RA abolished keratinocyte gj more slowly and irreversibly (n = 10). By contrast, 10(-6) M RA had no significant effect on gj (n = 8). Single-gap junctional channels were also identified between large keratinocytes. Events histograms of 152 transitions from three experiments revealed three main unitary conductances (gamma j) of 45 +/- 4, 78 +/- 4, and 106 +/- 7 pS. The dye coupling results indicate that junctional communication is markedly different in pairs of small and large cells, which showed the phenotype and keratin markers of basal and suprabasal keratinocytes, respectively. In the latter cell type, coupling is ensured by channels of three sizes and is blocked irreversibly by pharmacologic concentrations of RA.

Topics: Alcohols; Antibodies; Antibodies, Monoclonal; Cell Communication; Electrophysiology; Heptanol; Humans; Intercellular Junctions; Ion Channel Gating; Isoquinolines; Keratinocytes; Tretinoin

All-trans retinoic acid (RA), which was recently identified as a morphogen, affects gap junctional permeability in a dose- and time-dependent manner. In five different established mammalian cell lines (FL, BRL, BICR/M1Rk, HEL37, BT5C1) 100 mumol/liter RA reduced Lucifer yellow spreading within 30 min to 20-50% of the control. Ionic coupling, however, remained almost unaffected under the same conditions. Freeze-fractured membranes of untreated and RA-treated cells were similar with regard to frequency and sizes of gap junction plaques. With concentrations of less than 10 mumol/liter RA the dye spreading increased significantly in the human amniotic cell line FL, pointing to a possible modulatory effect of RA on junctional communication.

Topics: Animals; Cell Line; Cell Membrane Permeability; Fluorescent Dyes; Humans; Intercellular Junctions; Isoquinolines; Microscopy, Electron; Tretinoin

The role of gap junctional communication during patterning of the chick limb has been investigated. Affinity-purified antibodies raised against rat liver gap junctional proteins were used to block communication between limb mesenchyme cells. Co-injection of the antibodies and Lucifer yellow into mesenchyme cultures demonstrated that communication was inhibited almost immediately. When antibodies were loaded into mesenchyme tissue by DMSO permeabilization, [3H]nucleotide transfer was prevented for at least 16 h. Polarizing region tissue from the posterior limb bud margin causes digit duplications when grafted to the anterior margin. Quail polarizing region cells were loaded with gap junction antibody and grafted into chick wing buds. The antibody had no effect on growth or survival of the grafted cells. As very few polarizing region cells are required to initiate duplications, the number of polarizing region cells in the grafts was reduced by diluting 1:9 with anterior mesenchyme tissue. When either polarizing region or anterior mesenchyme tissue in the graft was loaded separately with antibody, there was little effect on respecification of the digit pattern. However, loading both tissues in the graft caused a significant decrease in duplications. This indicates that a major role of gap junctions in limb patterning may be to enable polarizing region cells to communicate directly with adjacent anterior mesenchyme. A role for gap junctional communication between anterior mesenchyme cells cannot be excluded. The results are discussed in relation to the role of retinoic acid as a putative morphogen.

Topics: Animals; Cell Communication; Cell Division; Cells, Cultured; Chick Embryo; Connexins; Dimethyl Sulfoxide; Extremities; Fluorescent Dyes; Freeze Fracturing; Intercellular Junctions; Isoquinolines; Membrane Proteins; Morphogenesis; Permeability; Quail; Tretinoin