Review paper: cancer stem cells and cancer nonstem cells: from adult stem cells or from reprogramming of differentiated somatic cells.

Two opposing hypotheses of the origin of cancer have existed for many decades. One hypothesis postulates that the adult stem cell is needed to initiate the carcinogenic process, whereas the other claims a somatic differentiated cell can dedifferentiate or be reprogrammed to regain properties associated with cancer cells. Recent reemergence of the cancer stem cell hypothesis and the isolation of presumptive cancer stem cells from many types of cancer have forced a reexamination of these 2 hypotheses of the origin of cancer. In addition, normal embryonic and adult stem cells have now been isolated and partially characterized. Furthermore, the demonstration of embryonic-like stem cells, being isolated from adult-differentiated skin fibroblast cells of mice, monkey, and human beings, provides a newer opportunity to determine which of these 2 hypotheses might explain the cell type for initiating the carcinogenic process. The goal of this review is to integrate these recent findings, concerning the isolation of normal and cancer stem cells, with several of the classical concepts of carcinogenesis (initiation/promotion/progression; mutation/epigenetic; stem cell theory/dedifferentiation hypotheses; oncogene-tumor suppressor theory). Although the weight of the evidence in this review seems to support the stem cell hypothesis, only future studies, probably using comparative animal and human oncologic studies, will determine if targeting the cancer stem cell, with individualized medical approaches, will improve cancer prevention and therapy.

Modulation of connexin 43 in rotenone-induced model of Parkinson's disease.

Gap junctional communication plays an important role in various models of brain pathology, but the changes of gap junctions in Parkinsonism are still not understood. In this study, we show that a major gap junctional protein, connexin43 (Cx43), in astrocytes is enhanced both in a rat Parkinson's disease (PD) model induced with rotenone, a widely used pesticide that inhibits mitochondrial complex I, and in vitro in cultured astrocytes stimulated with rotenone. Enhancement of Cx43 protein levels in rotenone-treated cultured astrocytes occurred in parallel with an increase in gap junctional intercellular communication, but was not accompanied with an increase in Cx43 mRNA levels. Furthermore, the rotenone-induced increase of Cx43 protein levels both in vitro and in vivo was associated with increased levels of phosphorylated Cx43, which is required for gap junctional intercellular communication. In our rat PD model, phosphorylated Cx43 was selectively enhanced in the basal ganglia regions, which contain DA neurons or their terminal areas. The increase of Cx43 levels was lower in the substantia nigra pars compacta and the striatum than in the substantia nigra pars reticulata and the globus pallidus. Our findings indicate that modulation of Cx43 protein, and consequently gap junctional cellular communication, in astrocytes may play an important role in PD pathology.

Role of diet and nutrition on the alteration of the quality and quantity of stem cells in human aging and the diseases of aging.

An integrative synthesis of concepts and an explosion of experimental and epidemiological findings allow new insights as to how the interactions of genetic, environmental, dietary, cultural (social, psychological, economic) factors can influence the aging and diseases of aging processes. Although the net effect of the best dietary maintenance of homeostatic control of cell proliferation, cell differentiation and apoptosis, systems breakdown of the human being and death will inevitably be the ultimate end result. Reduction of the quantity of the stem cell pool in any tissue will affect the "aging" of that organ. This, in turn, will affect the homeostatic maintenance of the organ systems of the human. Clearly, not all organs of the body age uniformly. The quality of the stem cells in any organ, depending on circumstances, can contribute to various disease pathogeneses. In the case where the quality of the stem cells is altered in utero or early postnatal development by some mutagenic mechanism that could lead to the initiation step of carcinogenesis, then the individual can, to some degree, control the fate of those prenatally and early postnatally-derived initiated stem cells by choosing those environmentally and dietary factors that either enhance or prevent the clonal expansion of these initiated stem cells during the promotion phase of carcinogenesis. This might explain the Barker hypothesis which suggests that prenatal and early postnatal exposures to toxic agents can lead to diseases later in life.

Non-dioxin-like polychlorinated biphenyls induce a release of arachidonic acid in liver epithelial cells: a partial role of cytosolic phospholipase A(2) and extracellular signal-regulated kinases 1/2 signalling.

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) have been shown to act as tumor promoters in liver; however, the exact mechanisms of their action are still only partially understood. One of the interesting effects of NDL-PCBs is the acute inhibition of gap junctional intercellular communication (GJIC), an effect, which has been often found to be associated with tumor promotion. As previous studies have suggested that NDL-PCB-induced disruption of lipid signalling pathways might correspond with GJIC inhibition, we investigated effects of PCBs on the release of arachidonic acid (AA) in the rat liver epithelial WB-F344 cell line, a well-established model of liver progenitor cells. We found that both 2,2',4,4'-tetrachlorobiphenyl (PCB 47) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), but not the dioxin-like, non-ortho-substituted, 3,3',4,4',5-pentachlorobiphenyl (PCB 126), induce a massive release of AA. The AA release, induced by PCB 153, was partially inhibited by extracellular signal-regulated kinases 1/2 (ERK1/2) signalling inhibitor, U0126, and by cytosolic phospholipase A(2) (cPLA(2)) inhibitor, AACOCF(3). Although PCB 153 induced both ERK1/2 and p38 activation, the specific p38 kinase inhibitor, SB203580, had no effect on AA release. Inhibitors of other phospholipases, including phosphatidylcholine-specific phospholipase C or phosphatidylinositol-specific phospholipase C, were also without effect. Taken together, our findings suggest that the AA release, induced by non-dioxin-like PCBs in liver progenitor cell line, is partially mediated by cytosolic PLA(2) and regulated by ERK1/2 kinases. Our results suggest that more attention should be paid to cell signalling pathways regulated by AA or eicosanoids after PCB exposure, which might be involved in their toxic effects.

Expression of the embryonic transcription factor Oct4 in canine neoplasms: a potential marker for stem cell subpopulations in neoplasia.

Neoplastic cells and stem cells share several phenotypic characteristics. Recently, numerous studies have identified adult stem cells that have been hypothesized to be the cellular origin for cancer in several tissues. Oct4 has been consistently associated with pluripotent or stemlike cells, and it is hypothesized that Oct4 is necessary for the maintenance of pluripotency. We hypothesize that Oct4-positive cells are present in all canine neoplasms and that these subpopulations of neoplastic cells might represent "cancer stem" cells. To test this hypothesis, 83 canine neoplasms representing 21 neoplastic diseases were evaluated for Oct4 expression using immunohistochemistry. The results of this study showed that all tumors included in this study contained a subpopulation of Oct4-positive cells, although the proportion of Oct4-positive cells and the intensity of immunoreactivity varied both within and between tumor types. Subpopulations of Oct4-positive cells identified in these tumors are likely to represent "cancer stem" cells and therefore might be responsible for the maintenance and propagation of the tumors. If these cells represent cancer stem cells, and are therefore responsible for the maintenance and growth of the neoplastic cellular population, then these cells should serve as relevant therapeutic targets and offer the greatest potential for curative treatment.

The role of stem cells and gap junctions as targets for cancer chemoprevention and chemotherapy.

Since carcinogenesis is a multi-stage, multi-mechanism process, involving mutagenic, cell death and epigenetic mechanisms, during the "initiation/promotion/and progression" phases, chemoprevention must be based on understanding the mechanism(s) of each phase. Prevention of each phase could reduce the risk to cancer. Because reducing the initiation phase to a zero level is impossible, the most effective intervention would be at the promotion phase. Assuming the "target" cells for carcinogenesis are the pluri-potent stem cells and their early progenitor or transit cells, chemoprevention strategies for inhibiting the promotion of these two types of pre-malignant "initiated" cells will require different agents. A hypothesis will be proposed that involves stem cells, which lack gap junctional intercellular communication (GJIC-) or their early progenitor daughter cells, which express GJIC+ and are partially-differentiated, if initiated, will be promoted by agents that either inhibit secreted negative growth regulators or by inhibitors of GJIC. Chemopreventing agents to each of these two types of initiated cells must have different mechanisms of action. Assuming stem cells are target cells for carcinogenesis, an alternative method of chemoprevention would be to reduce the stem cell pool. Anti-tumor promoter chemopreventive agents, such as green tea components, resveratrol, caffeic acid phenethylene ester, that either up-regulate GJIC in stem cells or prevent the down regulation of GJIC by tumor promoters in early progenitor cells, will be provided. Human pluri-potent stem cell systems, that can be induced to form 3-dimensional "organoid" structures, will be discussed as a more realistic model system to screen for relevant chemopreventive agents.

Oxidative stress in liver and brain of the hatchling chicken (Gallus domesticus) following in ovo injection with TCDD.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was injected into chicken eggs prior to incubation to study possible mechanisms of toxicity and teratogenicity. One of the suggested mechanisms of teratogenicity is oxidative stress. Eggs were injected simultaneously with TCDD and cotreatment compounds in an attempt to prevent oxidative stress or to block cytochrome P450 activity. Indicators of oxidative stress were assessed in livers and brains of hatchling chicks. In ovo, exposure to TCDD caused significant effects on indicators of oxidative stress in liver, but not in the brain of the hatchling chicks. TCDD did not significantly affect superoxide production. In liver, TCDD treatment caused a decrease in glutathione content and glutathione peroxidase activity and an increase in the ratio of oxidized to reduced glutathione. TCDD increased the susceptibility to lipid peroxidation and oxidative DNA damage in liver. Administration of the antioxidants vitamin E and vitamin A provided partial protection against TCDD-induced oxidative stress in liver. The lack of effect of TCDD in chicken brain could be due to the low cytochrome P4501A activity in this tissue and little accumulation of TCDD in brain compared to liver. Phenytoin, a known inducer of oxidative stress, caused a decrease in glutathione content and an increase in susceptibility to lipid peroxidation in both liver and brain and increased oxidative DNA damage in brain. Responsiveness varied among individual animals, but measures of the oxidative stress were correlated.

Oxidative stress in laboratory-incubated double-crested cormorant eggs collected from the Great Lakes.

Double-crested cormorant (Phalacrocorax auritus) eggs were collected in 1998 from three sites on Lakes Huron and Superior and either analyzed for 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-like residues or artifically incubated. Some of the incubated eggs were injected with vitamin E (antioxidant) or piperonyl butoxide (CYPIA blocker) to examine the role of CYPIA and oxidative stress in normal bird development. Embryos (day 23) were analyzed for hepatic ethoxyresorufin O-deethylase (EROD) activity and different measures of oxidative stress. Glutathione-related parameters were also measured in brain. In contrast to the historical data, there were no statistically significant differences in concentrations of chlorinated dioxins, furans, dioxin-like PCBs, or total TCDD-equivalents (TEQs) in eggs among sites. Survival and incidence of abnormalities were comparable at all study sites. Slight differences in liver, heart, and egg weight were observed among sites. A greater incidence of eye deformities was observed in embryos treated with vitamin E. Treatment with the CYPIA blocker, piperonyl butoxide, decreased the body weights of embryos. EROD activities were similar at all locations, but measures of oxidative stress varied among locations. There were greater levels of oxidized glutathione and oxidative DNA damage at Little Charity Island in Saginaw Bay. There was relatively great interindividual variation in biochemical responses and significant interrelation of the parameters of oxidative stress. While exposure to PCDD/DF and PCBs does not seem to explain the observed oxidative stress, the potential of these compounds to cause the observed effects can not be completely excluded.

Gap junctions as targets for cancer chemoprevention and chemotherapy.

The development of the most efficacious strategy for the prevention and treatment of cancers is based on understanding the underlying mechanisms of carcinogenesis. This includes the knowledge that the carcinogenic process is a multi-step, multi-mechanism process and that no two cancers are alike, in spite of some apparent universal characteristics, such as their inability to have growth control, to terminally differentiate, to apoptose abnormally and to have an apparent extended or immortalized life span. The multi-step process, involving the "initiation" of a single cell via some irreversible process, with the clonal expansion of this initiated cell by suppressing growth control and inhibiting apoptosis (promotion step), leads to a situation whereby additional genetic and epigenetic events can take place (progression step) to confer the necessary phenotypes of invasiveness, and metasis (neoplastic stage). While it is clear that, in principle, prevention of each of these three steps is possible, in practical terms, while it would make sense to minimize the initiation step, one can never reduce this step to zero. On the other hand, since the promotion step is the rate-limiting step of carcinogenesis, intervening to block this step makes the most sense. Also, by understanding the ultimate biological function that confers growth control, terminal differentiation or apoptosis for cells, there is even some hope of treating some, but not all, malignant cells such that they can regain some ability to perform these vital cellular functions. Gap junctional intercellular communication (GJIC) has been speculated to be a necessary, if not sufficient, biological function of metazoan cells for the regulation of growth control, differentiation and apoptosis of normal progenitor cells. Normal, contact-inhibited fibroblast and epithelial cells have functional GJIC, while most, if not all, tumor cells have dysfunctional homologous or heterologous GJIC. Cancer cells are characterized by the lack of growth control, inability to terminally differentiate or apoptose under normal conditions and have extended or immortalized life spans. Chemical tumor promoters, growth factors and hormones have been shown to inhibit GJIC. Several oncogenes and anti-sense connexin genes have been shown to down-regulate GJIC function. Anti-oncogene drugs, anti-tumor promoting natural and synthetic chemicals, as well as GJIC-deficient neoplastic cells, transfected with various connexin genes, have been shown to re-gain GJIC and growth control with the loss of tumorigenicity. Therefore, the hypothesis for a rational approach to identify anti-tumor promoting chemopreventive drugs and anti-carcinogenic treatments is to use the prevention of the down regulation of GJIC by the tumor promoters and the restoration of GJIC in neoplastic cells. While previous and many current strategies for chemoprevention and therapy have been based on treating specific oncogene products or cell signalling mechanisms, as well as advance molecular modifications of older strategies, none have specifically used the prevention of GJIC by agents during the rate limiting step of carcinogenesis or the restoration of GJIC in neoplastic cells which are deficient in GJIC. Since there are multiple mechanisms by which GJIC is down regulated during the tumor promotion phase and since stable GJIC deficiencies in neoplastic cells can be the result of transcriptional, translational or posttranslational mechanisms, it should be clear there would not be one "golden bullet" approach to resolve either the chemoprevention or therapeutic approach. Even with the hypothesis that GJIC, which depends on the transcription of normal connexin genes, their normal translation, trafficking, assembly and function, it should be clear that cells with normal connexin genes and potentially normal GJIC might not have functional GJIC because of dysfunction of other defects in cancer cells, namely cell-adhesion or cell-matrix problems (both of which are necessary for GJIC to occur). In essence, if dietary or chemopreventive/therapy is to be effective, the strategy must either ameliorate the growth stimulatory effects of exogenous chemicals, growth factors or hormones, that trigger various mitogenic/anti-apoptotic signal transducing systems that inhibit GJIC.

Ozonation of chrysene: evaluation of byproduct mixtures and identification of toxic constituent.

The effects of chrysene and the ozonated byproducts on in vitro gap junctional intercellular communication (GJIC) were evaluated using the scrape loading/dye transfer (SL/DT) technique. A 1 mM solution of chrysene was ozonated at dosages of 1.75, 3, 4.25, and 5 mol O3/mol chrysene (Chr). The early ozonation mixture, 1.75 mol O3/mol Chr, exhibited greater inhibition to GJIC than chrysene and irreversible damage to cells leading to cell death. To determine the compounds potentially responsible for the increase in toxicity, the byproducts formed upon treatment with 1.44 mol O3/mol Chr were separated into 14 fractions using RP-HPLC. The major compounds identified in the fractions were 2-(2'-formyl) phenyl-1-naphthaldehyde, 2-(2'formyl) phenyl-1-naphthoic acid, and 2-2-carboxyphenyl-1-naphthoic acid. 2-(2'-Formyl) phenyl-1-naphthaldehyde was determined to be the compound causing GJIC inhibition in sample fractions and byproduct mixtures.

From bacteria to humans: lessons learned from a reductionist's view of ultraviolet light-induced DNA lesions.

What follows is a personal remembrance of how Dr. Richard Setlow influenced me as a young postdoctoral fellow at Oak Ridge National laboratory between 1963 and 1966. The narrative tries to place my "maturation" as a young, inexperienced scientist in the context of the cultural upheaval caused by the Vietnam war, of a Northerner facing a "culture-shock" living in the South and in a revolution in molecular and radiation biology taking place at Oak Ridge National Laboratory at that time. The unique historic juxtaposition of Dr. Setlow's contribution of the discovery of UV-induced pyrimidine dimers in bacterial DNA, being potentially the molecular lesion responsible for cell killing and mutagenesis, occurring as I was at Oak Ridge, and the wonderful working relationship I had with William Carrier, his technician, led to our discovery with James Regan that normal human cells repaired these lesion from their DNA. Amazingly, because of Dr. Setlow's challenge to me about my thoughts of the implications of his findings in bacteria, the chance visit to Oak Ridge National Laboratory by Dr. James Cleaver and my background as a human geneticist provided me the extraordinary opportunity to carry out a collaboration to test if human cancer prone syndromes might be deficient in the repair of these UV-induced DNA lesions. With our finding that the direct demonstration of a lack of repair of UV-induced pyrimidine dimers in cells from the skin cancer prone syndrome, xeroderma pigmentosum, opened up a new paradigm for the understanding of the molecular mechanism of carcinogenesis of both radiation and chemical carcinogenesis. From this investigator's vantage point in the history of the understanding of carcinogenesis, which has led us to the present point of "oncogenes" and "tumor suppressor genes", the old adage by Newton, "I only saw further because I stood on the shoulder of giants", is so applicable here. Dr. Setlow's shoulders were indeed among those of all of us that have made some small contribution in trying to understand this extremely complex process of human carcinogenesis.

Radiation risk to low fluences of alpha particles may be greater than we thought.

Based principally on the cancer incidence found in survivors of the atomic bombs dropped in Hiroshima and Nagasaki, the International Commission on Radiation Protection (ICRP) and the United States National Council on Radiation Protection and Measurements (NCRP) have recommended that estimates of cancer risk for low dose exposure be extrapolated from higher doses by using a linear, no-threshold model. This recommendation is based on the dogma that the DNA of the nucleus is the main target for radiation-induced genotoxicity and, as fewer cells are directly damaged, the deleterious effects of radiation proportionally decline. In this paper, we used a precision microbeam to target an exact fraction (either 100% or < or =20%) of the cells in a confluent population and irradiated their nuclei with exactly one alpha particle each. We found that the frequencies of induced mutations and chromosomal changes in populations where some known fractions of nuclei were hit are consistent with non-hit cells contributing significantly to the response. In fact, irradiation of 10% of a confluent mammalian cell population with a single alpha particle per cell results in a mutant yield similar to that observed when all of the cells in the population are irradiated. This effect was significantly eliminated in cells pretreated with a 1 mM dose of octanol, which inhibits gap junction-mediated intercellular communication, or in cells carrying a dominant negative connexin 43 vector. The data imply that the relevant target for radiation mutagenesis is larger than an individual cell and suggest a need to reconsider the validity of the linear extrapolation in making risk estimates for low dose, high linear-energy-transfer (LET) radiation exposure.

Gap junctions and fluid flow response in MC3T3-E1 cells.

In the current study, we examined the role of gap junctions in oscillatory fluid flow-induced changes in intracellular Ca(2+) concentration and prostaglandin release in osteoblastic cells. This work was completed in MC3T3-E1 cells with intact gap junctional communication as well as in MC3T3-E1 cells rendered communication deficient through expression of a dominant-negative connexin. Our results demonstrate that MC3T3-E1 cells with intact gap junctions respond to oscillatory fluid flow with significant increases in prostaglandin E(2) (PGE(2)) release, whereas cells with diminished gap junctional communication do not. Furthermore, we found that cytosolic Ca(2+) (Ca) response was unaltered by the disruption in gap junctional communication and was not significantly different among the cell lines. Thus our results suggest that gap junctions contribute to the PGE(2) but not to the Ca response to oscillatory fluid flow. These findings implicate gap junctional intercellular communication (GJIC) in bone cell ensemble responsiveness to oscillatory fluid flow and suggest that gap junctions and GJIC play a pivotal role in mechanotransduction mechanisms in bone.

Inhibition of apoptosis by pentachlorophenol in v-myc-transfected rat liver epithelial cells: relation to down-regulation of gap junctional intercellular communication.

Pentachlorophenol (PCP), a promoter of murine hepatocarcinogenesis, inhibits gap junctional intercellular communication (GJIC) in rat liver epithelial cells in vitro. To test the hypothesis that both inhibition of GJIC and apoptosis contribute to tumor promotion, we investigated the effect of PCP on both GJIC and serum deprivation-induced apoptosis in v-myc-transfected rat liver epithelial cells. The results showed that PCP inhibited apoptosis, as measured by the TUNEL assay and DNA ladder formation. Inhibition of apoptosis was associated with a decrease in GJIC. The study demonstrated that PCP has a potential for inhibiting apoptosis and GJIC, supporting the hypothesis.

Hexamethylene bisacetamide protects peritoneal mesothelial cells from glucose.

BACKGROUND: Peritoneal dialysis causes damage to peritoneal mesothelial cells primarily because dialysis fluids have a high glucose concentration. This study examined the abnormalities of gap junctional intercellular communication (GJIC) in human peritoneal mesothelial cells (HPMCs) exposed to relatively high levels of glucose. Also, ability of hexamethylene bisacetamide (HMBA) to up-regulate GJIC in HPMCs exposed to high levels of glucose was measured. METHODS: An assay that monitors the recovery of fluorescence after photobleaching was used to measure GJIC in primary cultured HPMCs. The cells were exposed to a low (10 mmol/L) or high (50 or 90 mmol/L) glucose level for a total of six days, and some cells were also incubated with or without HMBA (1 or 6 mmol/L) from day 4. The effects of incubation in these various environments on expression of the connexin 43 (Cx43) gene were investigated by the reverse transcription-polymerase chain reaction (to detect Cx43 mRNA) or by immunofluorescence and Western blotting (to detect Cx43 protein). To evaluate the influence of protein kinase C (PKC) or mitogen-activated protein kinase (MAPK) on GJIC, specific inhibitors were added to cultures in a high glucose medium. RESULTS: Gap junctional intercellular communication was inhibited in a concentration- and time-dependent manner when cells were exposed to high glucose. The addition of 6 mmol/L HMBA to cultures significantly enhanced GJIC despite the presence of a high glucose concentration. High glucose also down-regulated Cx43 mRNA and protein expression, with the dose-dependent decrease of Cx43 protein at gap junctions paralleled by a decrease in the phosphorylation of this protein. As expected, treatment of cells with 6 mmol/L HMBA increased both Cx43 mRNA and protein levels despite exposure to high glucose. The addition of PKC or MAPK inhibitors to high glucose cultures did not restore GJIC, and there was no significant change of Cx43 phosphorylation in the presence of these inhibitors. CONCLUSIONS: High glucose down-regulates GJIC in human peritoneal mesothelial cells. It also decreases the levels of both Cx43 mRNA and Cx43 protein, with the latter becoming hypophosphorylated. HMBA appears to reverse all of these changes. These results are consistent with our hypothesis that HMBA protects HPMCs from the adverse effects of high glucose by reversing various processes that would otherwise lead to harmful loss of GJIC.

The epigenetic toxicity of pyrene and related ozonation byproducts containing an aldehyde functional group.

Gap junction intercellular communication (GJIC) was used to assess the epigenetic toxicity of pyrene, pure byproducts of pyrene ozonation, and other compounds similar in chemical structure. Byproduct mixtures collected from HPLC were also evaluated using GJIC. Of the 11 pure compounds studied, five inhibited GJIC completely. Two inhibiting compounds contained four rings and were the only compounds studied with greater than three rings. The remaining three compounds contained either two or three rings, and all three contained an aldehyde group. Toxicological evaluation and GC/MS of impure byproduct mixtures showed that two common compounds were found in inhibiting fractions. These common compounds contained both a bay region and at least one aldehyde group.

Mechanism of up-regulated gap junctional intercellular communication during chemoprevention and chemotherapy of cancer.

To develop a strategy for efficacious intervention in order to prevent or treat various cancers, one must understand the basic mechanism(s) by which various anticancer dietary factors prevent or reverse the tumor promotion or progression phases. Carcinogenesis is a multistage, multimechanism process, involving the irreversible alteration of a stem cell (the "initiation" phase), followed by the clonal proliferation of the initiated stem cell (the "promotion" phase), from which the acquisition of the invasive and metastatic phenotypes are generated (the "progression" phase). While intervention to prevent or treat cancer could occur at each step, the objective of this presentation will focus on the rate limiting step, the promotion phase.Gap junctional intercellular communication (GJIC) has been hypothesized to regulate growth control, differentiation and apoptosis. Most normal, contact-inhibited cells have functional GJIC, while most, if not all, tumor cells have dysfunctional homologous or heterologous GJIC. Cancer cells are characterized by the lack of growth control, by the inability to terminally differentiate and by resistance to apoptosis. Chemical tumor promoters (phorbol esters, DDT, phenobarbital, unsaturated fatty acids, saccharin, etc.) inhibit GJIC in a reversible fashion and at doses above particular chemical thresholds. Various oncogenes (e.g. ras, raf, neu, src, mos) down-regulate GJIC while several tumor suppressor genes can up-regulate GJIC. Antitumor promoters (retinoids, carotenoids, green tea components) and antioncogene drugs (i.e. lovastatin) can up-regulate GJIC. Transfection of gap junction genes ("connexins") into GJIC-deficient tumor cells can restore GJIC, growth control and reduce tumorigenicity. On the other hand, antisense gap junction genes can convert the phenotype of a non-tumorigenic cell to that of a tumorigenic one. Recently, a specific connexin knockout mouse was shown to have a higher frequency of spontaneous and induced liver cancers. Evidence from these studies clearly suggests that dietary factors can modulate GJIC by inducing various signal transducing systems. The modulation can either down-regulate GJIC and lead to tumor promotion or it can up-regulate GJIC and lead to suppression of the initiated cells. Multiple mechanisms of up- or down-regulation of GJIC exist, as well as multiple types of pre-malignant and malignant tumor cells that are unable able to have functional GJIC. GJIC can be down-regulated by mutations and by epigenetic means. Alteration of gene expression at the transcriptional, translational or post-translational levels would require specific dietary prevention or treatment of cancer. In conclusion, if dietary prevention or treatment of cancer is to occur, it must ameliorate the growth-stimulatory effects, above threshold levels, of chemicals, growth factors or hormones, that trigger various mitogenic/antiapoptotic signal transducing systems that block GJIC.

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 microM) and ERK activation (U-0126; 10 microM). 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.

Effect of selected pesticides and their ozonation by-products on gap junctional intercellular communication using rat liver epithelial cell lines.

The non-genotoxic effects of two commonly used pesticides, 1,1-bis (p-chlorophenyl)-2,2,2-trichloroethane (DDT) and malathion, and one widely used commercial insect repellent N,N-diethy-m-toluamide (DEET) on gap junction intercellular communication (GJIC) were determined using a rat liver epithelial cell line. Malathion and DDT reversibly inhibited GJIC in a treatment time- and dose-dependent manner at non-cytotoxic doses, whereas, DEET did not inhibit GJIC. Malathion was very reactive with ozone, while DEET and DDT did not react to any appreciable extent with ozone. The mixtures of ozonation products from malathion and DEET did not inhibit GJIC. The mixtures of ozonation by-products formed from DDT inhibited GJIC, but to a lesser extent than did DDT, itself. These results suggest that ozone can effectively remove malathion from solution without forming GJIC-toxic products, but is less effective in eliminating DEET and DDT from solution.

Commentary: is the concept of "tumor promotion" a useful paradigm?

Since the demonstration of the multistage nature of carcinogenesis in experimental work on mouse skin carcinogenesis (and subsequently on various other organ systems in other organisms), the concepts of "initiation", "promotion", and "progression" were operationally generated from empiric data. Because these early observations and concepts had no mechanistic explanations, various hypotheses have been generated to explain the unique characteristics of each phase (e.g., initiation, being irreversible, was ascribed as the result of DNA damage leading to mutagenesis; promotion, being interruptible or reversible, was believed to be caused by epigenetic mechanisms; progression, also being irreversible, was believed to be caused by genetic instability that led to mutagenic and epigenetic changes). In addition, many of the molecular, biochemical, and cellular experiments designed to investigate the mechanistic bases of these phases used technologies that did not always lead to unequivocal interpretations, and because "real-life" carcinogenesis does not mimic controlled experimental conditions of the initiation/promotion/progression experiments, many investigators believe that these concepts have lost their usefulness. In this commentary, I explain some of the confusion concerning the concept of promotion and suggest that, by understanding the limitations of many in vitro assays used to characterize mutagens, by integrating other theories of carcinogenesis (i.e., stem cell theory), and by recognizing the role of epigenetic agents, specifically, modulated gap-junctional intercellular communication, the concept of promotion can provide valuable insights into the carcinogenic process. Mol. Carcinog. 30:131--137, 2001.