Upregulation of gap junctional intercellular communication and connexin 43 expression by cyclic-AMP and all-trans-retinoic acid is associated with glutathione depletion and chemosensitivity in neuroblastoma cells.

PURPOSE: Downregulation of gap junctional intercellular communication (GJIC) has been implicated in carcinogenesis. This is a result of altered expression of connexins, the proteins that mediate GJIC, including connexin 43 (Cx43). Our aim was to evaluate the effect of known inducers of Cx43 on the chemosensitivity of the human neuroblastoma cell line IMR-32 to chemotherapeutic agents. METHODS: We examined the effect of dibutyryl-cyclic AMP (db-cAMP) and all-trans-retinoic acid (tRA) on Cx43 and GJIC, glutathione (GSH) and gamma-glutamyl-cysteine-synthetase (gamma-GCS) levels, and glutathione S-transferase (GST) activity. Finally, we performed cell survival assays to measure the response of IMR-32 cells to the chemotherapeutic drugs doxorubicin, melphalan and bis-chloronitrosourea (BCNU), after treatment with db-cAMP and/or tRA. RESULTS: Exposure to db-cAMP led to the upregulation of GJIC and Cx43 expression and phosphorylation. On the other hand, exposure to tRA led to the upregulation of GJIC but Cx43 expression and phosphorylation were not greatly affected. The combination of both agents was more potent in inducing GJIC in comparison to treatment with db-cAMP or tRA alone. Treatment with db-cAMP, but not with tRA, was associated with a significant increase in the cytotoxic effects of the anticancer drugs doxorubicin, melphalan and BCNU as shown by a decrease in their IC50 values. Concomitant exposure to db-cAMP and tRA, however, had a more pronounced effect on cell sensitization to chemotherapy drugs (particularly doxorubicin) than exposure to db-cAMP or tRA alone. Under the db-cAMP and tRA treatment conditions (which upregulate GJIC and modulate drug response), GSH levels were significantly reduced while the levels of GST and gamma-GCS activities remained unchanged. CONCLUSIONS: This study suggests that GJIC plays a role in cellular drug resistance, and highlights the potential use of GJIC modulators in combination with chemotherapy. Also, this is the first study exploring the ability of both db-cAMP and tRA to enhance cell chemosensitivity.

The role of connexin-mediated cell-cell communication in breast cancer metastasis.

Gap junctional intercellular communication (GJIC) is a form of cell-cell communication mediating the exchange of small molecules between neighboring cells. Gap junctions (GJs) are formed by connexins (Cxs), and are subject to tight and dynamic regulation. They are involved in the cell cycle, differentiation, and cell signaling. The loss of Cxs and GJs is a hallmark of carcinogenesis, while their induction in cancer cells leads to a reversal of the cancer phenotype, induction of differentiation, and regulation of cell growth. On the basis of the observations about Cx loss in breast cancer, this review examines Cxs' involvement in breast cancer metastasis. Previous work indicates that Cx expression is inversely correlated to metastatic potential. This is probably because of the loss of cooperation between neighboring cells, leading to cell heterogeneity and cell dissociation in the tumor. The possible involvement of Cx activity during metastasis will be discussed.

Deficiency of connexin43 gap junctions is an independent marker for breast tumors.

Gap junctions are intercellular channels that are formed from members of a family of proteins, the connexins (Cxs). Gap junctions play an important role in vital functions, including the regulation of cell growth and cell differentiation. Here, we examined the expression of Cx43, a major Cx in breast tissue, in 32 surgical specimens obtained from breast cancer patients who underwent a primary surgical resection prior to chemotherapy or radiotherapy treatments. The expression of Cx43 gap junctions was compared to the levels of estrogen, progesterone, and erbB2 tyrosine kinase receptors. In addition, a panel of breast cancer cell lines and a series of normal rat mammary tissues and rat mammary tumors induced in vivo by dimethylbenz(a)anthracene were studied. We demonstrated that the lack of Cx43 gap junctions is a common feature of human mammary cancer tissues compared to nonneoplastic breast tissues surrounding primary tumors. Cx43 gap junctions were not observed in ductal carcinomas in situ, infiltrating ductal carcinomas, and infiltrating lobular carcinomas, and they seem to be independent of estrogen, progesterone, and erbB2 receptor status. In breast cancer cell lines and rodent mammary carcinoma tissues, down-regulation of Cx43 occurs at the mRNA level, suggesting a transcriptional mechanism for the decrease of Cx43 protein in breast cancer. In summary, this study provides evidence of decreased expression of Cx43 gap junctions in breast cancer at various stages of progression as well as breast cancer cell lines and raises the possibility that Cx43 may be a useful marker for detecting early oncogenesis in the breast. Because Cx43 gap junctions are lacking in breast cancer and restoration of Cx43 has been shown to reverse the malignant phenotype in vitro, pharmacological up-regulation of Cx43 may prove beneficial in cancer therapeutics.

Cyclic-AMP induction of gap junctional intercellular communication increases bystander effect in suicide gene therapy.

The phenomenon of the "bystander effect" (BE) observed in suicide gene therapy studies leads to the intriguing possibility that cytotoxicity can be achieved even in tumor cells that have not themselves been targeted with novel genetic material. There is considerable data suggesting the role of gap junction-mediated intercellular communication (GJIC) in the BE. Transfer of connexin (Cx)-encoding genes, the building blocks of GJIC, has been shown both in vitro and in vivo to increase the BE. Since the loss of GJIC is a common feature of cancer cells, we examined the consequence of GJIC up-regulation on the BE in suicide gene therapy. We used 8-bromo-cyclic-AMP to induce Cx43 and GJIC. In mixing assays, using various proportions of cells containing viral thymidine kinase delivered by an adenoviral delivery system or stably transduced by a retrovirus vector, 8-bromo-cyclic-AMP enhanced the BE of cell killing using ganciclovir. The induction in cell killing was more significant when a low percentage of the cell population was infected, which is the relevant clinical situation. We have demonstrated that this is not due to an effect on infectivity or suicide gene expression. Since decreased GJIC is part of the transformed phenotype, induction of Cxs provides an element of selectivity to suicide gene therapy. Our study adds strength to the rationale to develop clinically tolerable GJ inducers to potentiate the effect of suicide gene therapy via the BE.

Potential for selective modulation of glutathione in cancer chemotherapy.

Notwithstanding ongoing progress in anticancer therapeutics development, the persistent problem remains to selectively target tumors while sparing normal tissues. This is confounding largely because the differences between normal and tumor cells are often subtle and part of a gradient, where a gene product may be more or less expressed in tumor compared with the host normal tissue, but seldom expressed (or turned off) in tumors. The role of glutathione (GSH) and related enzymes in cellular resistance to xenobiotics, including chemotherapy is well established. This study is among those attempting to modulate GSH to therapeutic advantage. The authors briefly describe the experience with the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine, and then in greater detail outline recent evidence for a potentially more selective approach using the cysteine prodrug L-2-oxothiazolidine-4-carboxylate. This has led to a detailed study of the activating enzyme 5-oxo-L-prolinase, including enzymatic and immunocharacterization, as well as in vitro study of the effect of its modulators on anticancer drug toxicity. Using high affinity antibodies the authors have generated interesting information on the distribution of this enzyme in tumor versus normal human tissues. Finally, the authors have been studying the potential for modulating gap junctions as a part of anti-cancer therapeutics, since they transport GSH between cells and are generally deficient in tumor cells. Preliminary studies suggest that gap junction induction may dramatically deplete GSH concentration in tumor cells and sensitize them to a variety of treatments.

Vacuolar H(+)-translocating pyrophosphatase is induced by anoxia or chilling in seedlings of rice.

The present study was undertaken to determine whether vacuolar H(+)-pyrophosphatase (V-PPase) might replace vacuolar H(+)-ATPase under energy stress due to anoxia or chilling in anoxia-tolerant species such as rice (Oryza sativa L.) and corn (Zea mays L.). The relative transcript level of V-PPase in rice seedlings, like that of alcohol dehydrogenase 1, increased greatly under anoxia and declined again when the seedlings were returned to air. However, the distribution of transcripts in root, shoot, and seed differed somewhat from that of alcohol dehydrogenase 1. Immunoreactive V-PPase protein and V-PPase enzyme specific activity in a tonoplast fraction from rice seedlings increased progressively with time of anoxia or chilling at 10 degrees C, showing a 75-fold increase after 6 d of anoxia, compared with a 2-fold increase of vacuolar H(+)-ATPase activity. When the seedlings were returned to air, the specific activity returned to its initial level within 2 d. After 6 d of chilling at 10 degrees C, V-PPase specific activity reached a level 20-fold of that at 25 degrees C. In microsomes of corn roots, V-PPase specific activity did not respond to anoxia but was constitutively high. It is proposed that V-PPase can be an important element in the survival strategies of plants under hypoxic or chilling stress.