Daniel J. Belliveau

Retroviral Delivery of Connexin Genes to Human Breast Tumor Cells Inhibits in Vivo Tumor Growth by a Mechanism That Is Independent of Significant Gap Junctional Intercellular Communication

The mechanism by which gap junction proteins, connexins, act as potent tumor suppressors remains poorly understood. In this study human breast tumor cells were found to exhibit diverse gap junction phenotypes including (a) undetectable Cx43 and no intercellular communication (HBL100); (b) low levels of Cx43 and sparse intercellular communication (MDA-MB-231); and (c) significant levels of Cx43 and moderate intercellular communication (Hs578T). Although retroviral delivery of Cx43 and Cx26 cDNAs to MDA-MB-231 cells did not achieve an expected substantial rescue of intercellular communication, overexpression of connexin genes did result in a dramatic suppression of tumor growth when connexin-expressing MDA-MB-231 cells were implanted into the mammary fat pad of nude mice. Subsequent immunolocalization studies on xenograph sections revealed only cytoplasmic stores of Cx43 and no detectable gap junctions. Moreover, DNA array and Western blot analysis demonstrated that overexpression of Cx43 or Cx26 in MDA-MB-231 cells down-regulated fibroblast growth factor receptor-3. Surprisingly, these results suggest that Cx43 and Cx26 induce their tumor-suppressing properties by a mechanism that is independent of significant gap junctional intercellular communication and possibly through the down-regulation of key genes involved in tumor growth. Moreover, our studies show that retroviruses are effective vehicles for delivering connexins to human breast tumor cells, facilitating potential gene therapy applications.

Enhanced Neurite Outgrowth in PC12 Cells Mediated by Connexin Hemichannels and ATP

Gap junctions have traditionally been described as transmembrane channels that facilitate intercellular communication via the passage of small molecules. Connexins, the basic building blocks of gap junctions, are expressed in most mammalian tissues including the developing and adult central nervous system. During brain development, connexins are temporally and spatially regulated suggesting they play an important role in the proper formation of the central nervous system. In the current study, connexins 32 and 43 were overexpressed in PC12 cells to determine whether connexins are involved in neuronal differentiation. Both connexin 32 and 43 were appropriately trafficked to the cell membrane following overexpression and resulted in the formation of functional gap junctions. Connexin overexpression was found to cause enhanced neurite outgrowth in PC12 cells treated with nerve growth factor to initiate neuritogenesis. Surprisingly, however, enhanced neurite outgrowth was found to be the consequence of functional hemichannel formation as opposed to traditional intercellular communication. Additional analysis revealed that ATP was released into the media likely through hemichannels and acted on purinergic receptors to cause enhanced neurite outgrowth. Collectively, the results of the current study suggest that connexins may play an important role in neuronal differentiation by non-traditional mechanisms.

CELLULAR LOCALIZATION OF GAP JUNCTION MRNAS IN DEVELOPING RAT BRAIN

We investigated the developmental expression and cellular resolution of connexin32 and 43 mRNA in the rat brain using in situ hybridization. Utilizing 35S-labelled probes, in situ hybridization was performed on sections of embryonic day 20 and postnatal days 3, 10, 15, 30 and adult brain. Connexin32 mRNA was first detected in brainstem nuclei at postnatal day 3 and in the midbrain at postnatal day 15. The level of this message continued to increase to postnatal day 30 where the level of message reached a plateau or slightly decreased by adulthood. The distribution of signal included the medial vestibular nucleus, dorsal paragigantocellular nucleus and fibre tracts of the midbrain and brainstem such as the cerebellar white matter, spinal trigeminal tract, and decussation of the superior cerebellar peduncle, areas containing predominantly neurons or oligodendrocytes. Connexin43 mRNA was first detected much earlier than connexin32 and was found in the leptomeniges of the E20 brain. It was found at all ages examined and distributed homogenously in the regions of the brain examined, suggesting its presence in astrocytes. The connexin43 riboprobe also hybridized strongly to the ependymal cells of the fourth ventricle and cerebral aqueduct. These results describe the cellular resolution of connexin mRNAs during development and that a differential pattern of expression exists in the various cell populations in the central nervous system.

Regional differences in connexin32 and connexin43 messenger RNAs in rat brain.

The regional distribution of gap junction mRNAs was examined in the adult rat brain. The level of connexin43 mRNA is more abundant than connexin32, being homogeneously distributed throughout different regions of brain. In contrast, there is dramatic heterogeneity in the level of connexin32 mRNA, with the highest level in the hindbrain. These results suggest that the gap junction genes are differentially expressed in regions of the adult rat brain.

Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell-cell and cell-neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.

Changes in calcineurin expression induced in the rat brain by the administration of antipsychotics

Calcineurin (CN) was recently identified as a susceptibility gene for schizophrenia as well as showing altered RNA expression levels in the post‐mortem brains of individuals with schizophrenia. CN knockout mice show a number of behaviours associated with schizophrenia, including deficits in sensorimotor gating, suggesting a link between CN and psychosis. Concurrently, we found, using genome screening techniques, that antipsychotics alter CN expression levels. Therefore, western blotting, in situ hybridization, immunocytochemistry and phosphatase assays were employed to determine what effect antipsychotics have on CN. The results indicate that clozapine, risperidone and haloperidol cause substantial reductions in the A subunit of CN but not CN B at both the RNA and protein levels in the striatum and prefrontal cortex. The changes could only be observed after repeated treatment with antipsychotics but not after acute administration. The alterations in CN protein levels were specific to antipsychotics and mediated by D2 dopamine receptor antagonism. However, despite reductions in CN protein levels, the phosphatase activity of CN was significantly elevated after treatment with antipsychotics. Collectively the results suggest that CN may be a common target for antipsychotics and that antipsychotic‐induced alterations in CN may represent one of the mechanisms by which antipsychotics alleviate psychosis.

Connexin Over-Expression Differentially Suppresses Glioma Growth and Contributes to the Bystander Effect Following HSV-Thymidine Kinase Gene Therapy

Neoplastic transformation is frequently associated with a loss of gap junctional intercellular communication and reduced expression of connexins. The introduction of connexin genes into tumor cells reverses the proliferative characteristics of such cells. However, there is very little comparative information on the effects of different connexins on cancer cell growth. We hypothesized that Cx26, Cx32, or Cx43 would display differential growth suppression of C6 glioma cells and uniquely modulate the bystander effect following transduction of C6 cells with HSVtk followed by suicide gene therapy. The bystander phenomenon is the death of a greater number of tumor cells than are expressing the HSVtk gene, presumably due to the passage of toxic molecules through gap junction channels. To test this hypothesis, we used retroviral vectors to infect C6 glioma cells producing connexin-expressing and HSVtk-expressing cell lines. All three connexin-expressing cell lines grew significantly slower than GFP-infected or native C6 cells. Cx32 and Cx26 were significantly more effective at mediating the bystander effect in cocultures of C6-connexin cells with C6-HSVtk cells. These studies indicate that connexins have unique properties that contribute to their tumor suppressive function.

Nerve growth factor increases connexin43 phosphorylation and gap junctional intercellular communication

The function of gap junctions is regulated by the phosphorylation state of their connexin subunits. Numerous growth factors are known to regulate connexin phosphorylation; however, the effect of nerve growth factor on gap junction function is not understood. The phosphorylation of connexin subunits is a key event during many aspects of the lifecycle of a connexin, including open/close states, assembly/trafficking, and degradation, and thus affects the functionality of the channel. PC12 cells infected with connexin43 (Cx43) retrovirus were used as a neuronal model to characterize the signal transduction pathways activated by nerve growth factor (NGF) that potentially affect the functional state of Cx43. Immunoblot analysis demonstrated that Cx43 and the mitogen‐activated protein kinase (MAPK), ERK‐1/2, were phosphorylated in response to TrkA activation via NGF and that phosphorylation could be prevented by treatment with the MEK‐1/2 inhibitor U0126. The effects of NGF on gap junction intercellular communication were examined by monitoring fluorescence recovery after photobleaching PC12‐Cx43 cells preloaded with calcein. Fluorescence recovery in the photobleached area increased after NGF treatment and decreased when pretreated with the MEK‐1/2 inhibitor U0126. These data are the first to show a direct signaling link between neurotrophins and the phosphorylation of connexin proteins through the MAPK pathway resulting in increased gap junctional intercellular communication. Neurotrophic regulation of connexin activity provides a novel mechanism of regulating intercellular communication between neurons during nervous system development and repair.

Aggregated DsRed-tagged Cx43 and over-expressed Cx43 are targeted to lysosomes in human breast cancer cells

To investigate if either wild-type or aggregated Cx43 is abnormally targeted to lysosomes in human breast tumor cells, we examined the fate of DsRed-tagged Cx43 and over-expressed Cx43 in communication-deficient HBL-100 and MDA-MB-231 cells. DsRed-tagged Cx43 was assembled into gap junctions in control normal rat kidney cells that express endogenous Cx43 but not in Cx43-negative HBL-100 cells. However, when HBL-100 cells were engineered to coexpress wild-type Cx43 a population of DsRed-tagged Cx43 was rescued and assembled into gap junctions. Co-expression of wild-type Cx26 failed to rescue the assembly of DsRed-tagged Cx43 into gap junctions. Immunolocalization studies revealed that DsRed-tagged Cx43 was aggregated and partially localized to lysosomes. Interestingly, when human MDA-MB-231 breast tumor cells over-expressed wild-type Cx43, Cx43 protein primarily localized to lysosomes. Together, these studies provide evidence for Cx43 being targeted to lysosomes as a result of misfolding and aggregation, while in other cases, the delivery of wild-type Cx43 to lysosomes appears to be due to defects innate to the breast tumor cell type.

PKC inhibition increases gap junction intercellular communication and cell adhesion in human neuroblastoma

Gap junction intercellular communication and cell–cell adhesion are essential for maintaining a normal cellular phenotype, including the control of growth and proliferation. Loss of either cell–cell adhesion or communication is common in cancers, while restoration of function is associated with tumor suppression. Protein kinase C (PKC) isozymes regulate a broad spectrum of cellular functions including growth and proliferation, and their overexpression has been correlated with carcinogenesis. Consequently, PKC inhibitors are currently undergoing clinical trials as an anti-cancer agents although the precise cellular alterations induced by PKC inhibitors remain to be elucidated. In the current study, the effects of PKC inhibitors on cell interactions were investigated using human neuroblastoma (IMR32, SKNMC, and SHSY-5Y) cell lines. An analysis of intercellular communication revealed an increase in gap junctional coupling with PKC inhibition. The observed increase in coupling was not associated with a change in Connexin43 distribution or an alteration of phosphorylation status of the protein. There was also an increase in cell–cell adhesion with PKC inhibitor treatment as indicated by a cell aggregation assay. Therefore, the growth suppressive abilities of PKC inhibition on tumors may be due to the cancer suppressive effects of increased gap junction intercellular communication and cell–cell adhesion.