Burra V. Madhukar

Chemical, Oncogene and Growth Factor Inhibition of Gap Junctional Intercellular Communication: An Integrative Hypothesis of Carcinogenesis

Most, if not all, cancer cells have some dysfunction in gap-junction-mediated intercellular communication, either because of defects in cell adhesion or inability to have functional gap junctional communication. In addition, most, if not all, tumor-promoting chemicals and conditions down-regulate gap junction function, while some antitumor-promoting chemicals can up-regulate gap junctional communication. Several oncogenes are associated with down-regulation of gap junction function and several hormone and growth regulators, known to be tumor promoters, are also able to down-regulate gap junction function. On the other hand, some tumor suppressor genes have been linked to the up-regulation of gap junctions. Based on these observations, it is hypothesized that, if a progenitor cell is unable to perform gap junctional intercellular communication, normal growth control and cell differentiation would not be possible, thereby favoring the development of malignant neoplasia.

Inhibition of connexin43 gap junctional intercellular communication by TPA requires ERK activation

The phorbol ester, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), is a potent inhibitor of gap junctional intercellular communication (GJIC). This inhibition requires activation of protein kinase C (PKC), but the events downstream of this kinase are not known. Since PKC can activate extracellular signal regulated kinases (ERKs) and these also downregulate GJIC, we hypothesized that the inhibition of GJIC by TPA involved ERKs. TPA treatment (10 ng/ml for 30 min) of WB‐F344 rat liver epithelial cells strongly activated p42 and p44 ERK‐1 and ‐2, blocked gap junction‐mediated fluorescent dye‐coupling, and induced connexin43 hyperphosphorylation and gap junction internalization. These effects were completely prevented by inhibitors of PKC (bis‐indolylmaleimide I; 2 μM) and ERK activation (U‐0126; 10 μM). These data suggest that ERKs are activated by PKC in response to TPA treatment and are downstream mediators of the gap junction effects of the phorbol ester. J. Cell. Biochem. 83: 163–169, 2001.

TCDD (2,3,7,8-tetrachlorodibenzo-P-dioxin) causes increases in protein kinases particularly protein kinase C in the hepatic plasma membrane of the rat and the guinea pig

To study the cause of TCDD-evoked changes in the functions of plasma membrane constituents TCDDs effects on protein kinase activities in the liver of rats and guinea pigs were investigated. TCDD was found to cause a sharp increase in both c-AMP independent and dependent protein kinase activities in plasma membrane preparations from rat liver within 48 hours from the time of administration. Such effects reached maxima around day 20, and were quite noticeable even 40 days after a single administration of TCDD. As a result of SDS-polyacrylamide gel-electrophoresis (SDS-PAGE) analysis several substrate proteins for these increased protein kinases were observed. Among them are 170 K - 150 K bands, representing EGF receptor protein. TCDD was found to particularly stimulate protein kinase C which is known to influence many enzyme and receptor functions through protein phosphorylation. The possible significance of such an action of TCDD is discussed.

Alteration of rat hepatic plasma membrane functions by 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)

In vivo administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to rats produced significant alterations in Na-K, Ca, Mg ATPase and protein kinase activity, and receptor binding activity to insulin, concanavalin A, glucagon and epidermal growth factor in the canaliculi-rich plasma membrane fraction of the hepatocytes. Some of the changes are dose-related, and generally follow the time course of the sign of TCDD poisoning in the rat (e.g., the loss in body weight). Such TCDD-induced changes in membrane proteins and subsequent alteration in membrane functions appear to be extensive.

Immortalized Hypothalamic Luteinizing Hormone-Releasing Hormone Neurons Express a Connexin 26-like Protein and Display Functional Gap Junction Coupling Assayed by Fluorescence Recovery after Photobleaching

Expression of gap junction proteins was studied in the LHRH neuronal cell line, GT1-7, as a first step in defining the signalling mechanisms responsible for the pulsatile secretion of LHRH. GT1-7 cells were found to express a connexin 26-like protein that comigrated with mouse liver connexin 26 and that reacted with connexin 26-specific antibodies on Western blots. Immunofluorescent staining revealed punctate staining in a fraction of the cells, often present at points of apparent contact with neighboring cell bodies or processes. Fluorescence recovery after photobleaching analysis of 5,6-carboxyfluorescein loaded GT1-7 cells showed dye coupling among 20-30% of cells that made contact with other cells, suggesting the presence of functional gap junctions in this cell line.

Redox-mediated enrichment of self-renewing adult human pancreatic cells that possess endocrine differentiation potential.

Objectives: The limited availability of transplantable human islets has stimulated the development of methods needed to isolate adult pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation. The objective of this study was to determine whether modulation of intracellular redox state with N-acetyl-l-cysteine (NAC) would allow for the propagation of pancreatic stem/progenitor cells from adult human pancreatic tissue. Methods: Cells were propagated from human pancreatic tissue using a serum-free, low-calcium medium supplemented with NAC and tested for their ability to differentiate when cultured under different growth conditions. Results: Human pancreatic cell (HPC) cultures coexpressed &agr;-amylase, albumin, vimentin, and nestin. The HPC cultures, however, did not express other genes associated with differentiated pancreatic exocrine, duct, or endocrine cells. A number of transcription factors involved in endocrine cell development including Beta 2, Islet-1, Nkx6.1, Pax4, and Pax6 were expressed at variable levels in HPC cultures. In contrast, pancreatic duodenal homeobox factor 1 (Pdx-1) expression was extremely low and at times undetectable. Overexpression of Pdx-1 in HPC cultures stimulated somatostatin, glucagon, and carbonic anhydrase expression but had no effect on insulin gene expression. HPC cultures could form 3-dimensional islet-like cell aggregates, and this was associated with expression of somatostatin and glucagon but not insulin. Cultivation of HPCs in a differentiation medium supplemented with nicotinamide, exendin-4, and/or LY294002, an inhibitor of phosphatidylinositol-3 kinase, stimulated expression of insulin mRNA and protein. Conclusion: These data support the use of intracellular redox modulation for the enrichment of pancreatic stem/progenitor cells capable of self-renewal and endocrine differentiation.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) causes reduction in the low density lipoprotein (LDL) receptor activities in the hepatic plasma membrane of the guinea pig and rat.

Administration of 1 micrograms/kg (single intraperitoneal injection and studied after 10 days) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to young male guinea pigs was found to cause a significant reduction in binding of low-density lipoprotein (LDL) to its receptor on the hepatic plasma membrane. This reduction in LDL binding is not caused by the decrease of food intake by treated animals since pair-fed control animals had significantly higher LDL binding than treated animals. It was also found that primary hepatocytes from treated animals had a reduced ability to internalize LDL than controls. Such a change in the plasma membrane function may explain the resulting hyperlipidemia particularly hypercholesterolemia which occurs in this species as a result of TCDD administration.

Influence of 2,3,7,8-TCDD on the protein composition of the plasma membrane of hepatic cells from the rat☆

Abstract To understand the mechanism of toxic action of TCDD, a serious environmental pollutant, rats were injected with 25 μg/kg and their livers removed 10 days post-treatment. A canaliculi-rich, plasma membrane fraction was prepared and its protein composition was examined using SDS-polyacrylamide gel electrophoresis. As a result of close comparison between untreated and treated preparations it was concluded that many protein levels were reduced in the membrane from the TCDD treated rats. This phenomenon was confirmed by the finding that there is an overall reduction in binding of 3 H-concanavalin A to the TCDD-treated plasma membrane.

Differences in the nature of induction of mixed-function oxidase systems of the rat liver among phenobarbital, DDT, 3-methylcholanthrene, and TCDD

Abstract A comparative study of the induction patterns of rat hepatic microsomal mixed-function oxidases was carried out in male Sprague-Dawley rats. The pattern of induction by DDT was different from that of phenobarbital. First, DDT induction was not acompanied by an increase in δ-aminolevulinic acid synthetase, and second, it was not affected by prior treatment with actinomycin D or α-amanitin in vivo . Otherwise, induction by these two chemicals as determined by differential increases in various drug-metabolizing activities and cytochrome P -450 was indistinguishable, indicating the basic similarities of these two induction patterns. Both TCDD and 3-methylcholanthrene (3-MC) induced a similar type of change in the cytochrome as judged by ethyl isocyanide- and CO-binding spectra. However, induction by TCDD and 3-MC was also different in two important areas: first, induction by 3-MC was inhibited by a prior or simultaneous treatment with actinomycin D, while that by TCDD was not. Second, the pH response of the induced cytochrome P -448 in terms of ethyl isocyanide-binding spectral changes was different in the two cases.

Characterization of gap junctional intercellular communication in immortalized human pancreatic ductal epithelial cells with stem cell characteristics.

Introduction Gap junctional intercellular communication has been implicated in the homeostatic regulation of cell growth, differentiation, and apoptosis. Cancer cells, which have been viewed as “partially blocked stem cells,” and which lack the ability for growth control, terminal differentiation, and apoptosis, also lack functional gap junctional communication. Aims and Methodology A clone of a human pancreatic ductal epithelial cell line, H6c7, derived after immortalization with human papilloma virus, was used to examine gap junctional intercellular communication and the ability to differentiate under different growth conditions. Results The cells showed characteristic epithelial morphology on standard tissue culture dishes. When placed on Matrigel they showed phenotypical changes with extensive ductal organization and budding structures. In growth medium containing hormones and growth factors, these cells were gap junctional intercellular communication (GJIC)–incompetent. In the presence of c-AMP elevating agents, isobutylmethylxanthine, and forskolin, in basal medium that did not contain the hormones and growth factors, the cells became GJIC-competent and expressed connexin43 gap junction protein within 48 hours after treatment. RT-PCR analyses of the cells under different growth conditions showed that the cells expressed connexin 32, 36, and 43 genes when cultured in the basal medium with c-AMP elevating agents. They also expressed the connexin 45 gene that did not change with c-AMP treatment. H6c7 cells also have the capacity to turn on an ectopic insulin promoter reporter gene. Conclusion Our data suggest that the immortalized H6c7 cells retain stem-like characteristics and have the potential to differentiate into duct-like structures and perhaps insulin-producing cells.