• connexin 36;
  • telomerase;
  • PAH;
  • gap junction


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Smoking is a well-documented risk factor for the development of pancreatic adenocarcinoma. Although the most abundant polycyclic aromatic hydrocarbons (PAHs) in cigarette smoke are methylated anthracenes and phenanthrenes, the epigenetic toxicity of these compounds has not been extensively studied. We previously showed that methylanthracenes, which possess a bay-like structure, affect epigenetic events such as an induced release of arachidonic acid, inhibition of gap junctional intercellular communication (GJIC) and induction of mitogen-activated protein kinases in a pluripotent rat liver epithelial stem cell line. Anthracenes with no bay-like structures were inactive. These biological effects are all molecular events associated with the promotional phase of cancer. A human immortalized, nontumorigenic pancreatic ductal epithelial cell line, H6c7, was examined to study the epigenetic toxicity of PAHs related to pancreatic cancer by using scrape-loading dye transfer, immunostaining, RT-PCR and telomerase assay methods. H6c7 cells were GJIC-incompetent and exhibited high telomerase activity when grown in growth factor and hormone-supplemented medium. In the presence of the cAMP elevating drugs (forskolin and IBMX) the cells became GJIC competent and expressed connexins. Telomerase activity was also decreased by cAMP elevating drug treatment. After induction of cAMP, 1-methylanthracene with bay-like structures inhibited GJIC, whereas the 2-methylanthracene lacking a bay-like structure had no effect on GJIC. Telomerase activity remained high in 1-methylanthracene treatment but not with 2-methylanthracene. These results indicate that a prominent component of cigarette smoke, namely methylanthracenes with distinct structural configurations, could be a potential etiological agent contributing to the epigenetic events of pancreatic cancer. © 2007 Wiley-Liss, Inc.

Pancreatic cancer is the fourth most common cause of cancer-related death in the United States.1, 2 Incidence in the developed world parallels the United States, and in undeveloped nations the incidence is lower, which could be due to underreporting.3 There are no early screening tests and no universally-effective treatment options, making this disease one of the most fatal cancers known, with a 5-year survival rate of less than 5%.1, 3, 4, 5 A recent article even suggested no patients are actually “cured” of this disease.6 Although several risk factors, such as diet, have been associated with pancreatic cancer, cigarette smoking is the only unequivocal risk factor that has been identified.1, 7, 8, 9 The incidence of pancreatic cancer is 50–90% higher among blacks in the United States than whites. This disparity has been linked primarily to cigarette smoking—and to a lesser extent diabetes—in men,10 again indicating the significance of tobacco smoke in the incidence of pancreatic cancer.

Although cigarette smoke has been identified as one etiological cause of pancreatic cancer, the underlying molecular mechanism causing the neoplastic development is not known. A fundamental question as to the cause of cigarette smoke-induced pancreatic cancer is as follows: at what stage or stages of cancer does cigarette smoke contribute to this disease? Compounds thought to work as initiators, such as 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), were measured at high levels in the pancreatic juice of smokers.2 The level of DNA adducts, in response to polycyclic aromatic hydrocarbons (PAHs) and aromatic amines, has been interpreted as being positively correlated with the spectrum of k-ras mutations in tumors of pancreas.5 The mutation profiles of p53 and K-ras genes in pancreatic cancer resemble the profiles found in bladder and lung cancers and the proportion of G to A transition in pancreatic cancer suggests the involvement of nitrosamines.5 Although this is not the proof that cigarette smoke contributes to the initiating phase, it does indicate likelihood.

Initiating events alone are not sufficient for the development of cancer, therefore understanding whether mixtures or specific compounds found in cigarette smoke can contribute to the promoting and progression phase of cancer is also very important. This is important, particularly when numerous studies have shown that cigarette smoke contributes more to the promoting than the initiating phase of cancer.11 A recent review article strongly suggested that point mutations contributing to cancer arise primarily as errors during turnover of undamaged DNA and environmental conditions, including cigarette smoke, select rather than induce oncomutations.12 The strong promoting and cocarcinogenic activity of cigarette smoke and its weak activity as a complete carcinogen11 is consistent with the observations that the risk of pancreatic cancer of former smokers approaches the risk of “never” smokers within a few years subsequent to quitting.4, 13 V.R. Potter has said that14 “The cancer problem is not merely a cell problem, it is a problem of cell interaction, not only within tissues, but with distant cells in other tissues.” Therefore, focusing on the question of how tobacco-relevant compounds cause a premalignant cell in multicellular tissue to become a tumor is probably more relevant than asking how tobacco causes a normal cell to initiate the cancer process.

The promoting phase of cancer involves a series of epigenetic events that allow the clonal expansion of an initiated cell into a tumor.15, 16 Epigenetic events regulating the expression of genes involve transcriptional, translational and posttranslational changes that alter the phenotype of the cell.15, 17, 18, 19, 20 These epigenetic events are controlled by intra-, inter- and extracellular communication mechanisms.21 Considering that the number of initiated cells far exceeds the number of tumors that develop in vivo,22, 23, 24 it is reasonable to assume that the clonal expansion of an initiated cell or cells is actively suppressed by normal cells.19, 25 Central to this suppression by normal cells is gap junctional intercellular communication (GJIC).26, 27, 28, 29 One hypothesis concerning the carcinogenic process applicable to solid tissues has been the idea that GJIC is reversibly down- regulated by endogenous (growth factors or hormones) or exogenous (chemical tumor promoters) agents during the tumor promotion phase, and then stably downregulated by alterations in activated oncogenes (e.g., ras, src, neu), or by the loss of tumor suppressor genes during the progression phase of carcinogenesis.30 Understanding the complex, multistage, multimechanism process of carcinogenesis, including that of human pancreatic cancer, also necessitates characterizing the genotype and phenotype of those cells that give rise to cancers. One of the oldest ideas on the origin of cancers is that of “cancer as a disease of differentiation,”31, 32 a “stem cell disease”33 or “oncogeny as partially blocked ontogeny,”14 suggesting that the target cells are the pluripotent stem cells. The isolation and immortalization of normal human pancreatic ductal epithelial cells with human papilloma virus (HPV) type 16 E6E7 genes (HPDE6-E6E7c7) has been reported.34 This cell line was shown to retain stem-like characteristics and have the potential to differentiate into duct-like structures and perhaps insulin-producing cells that were dependent upon the induction of functional gap junction genes.35 A recent study reported NNK, the tobacco-specific carcinogen, stimulates proliferation of HPDE6-c7 cells.36 To determine a potential mechanistic effect of cigarette smoke on biologically relevant cell type, we determined specific structure–activity effects of methylanthracene isomers, which are some of the most prevalent polycyclic aromatic hydrocarbons found in cigarette smoke condensates,37 on GJIC and stem cell markers in the pluripotent pancreatic stem cell line (HPDE6-E6E7c7).

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References


The complete keratinocyte serum-free medium (KSFM), containing growth factors, hormones and bovine pituitary extract and the keratinocyte basal medium (KBM), was purchased from Invitrogen (Carlsbad, CA). Forskolin and the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), were purchased from Sigma (St. Louis, MO). 1-methyl and 2-methyl anthracenes were purchased from Aldrich (Milwaukee, WI). The antibodies against the Cx36 and 43 proteins were purchased from Zymed Laboratories (South San Francisco, CA). All other reagents and chemicals used were of the highest purity available.

Cell culture

The human pancreatic duct epithelial cell line, HPDE6-E6E7c7, (abbreviated as H6c7 in the text) was cloned from the HPDE6-E6E7 cell line that was previously established by Furukawa et al.34 The cells were routinely cultured in KSFM containing insulin, hydrocortisone, epidermal growth factor (EGF) and bovine pituitary extract (BPE) and incubated in a humidified atmosphere containing 5% CO2 and 95% air. For experiments, cells were grown in 35 mm culture dishes (Corning Costar Corp., Cambridge, MA) and the culture medium was changed to KBM (which does not contain insulin, hydrocortisone, T3, EGF and BPE) and the treated chemicals.

Gap junctional intercellular communication

The scrape loading/dye transfer (SL/DT) technique adapted from the method of EL-Fouly et al.38 was used to measure the GJIC of the cells. Detailed methods were previously described.39 PAHs (70 μM) were added to 2 ml of KBM plus 5 μM forskolin or 5 μM forskolin with the addition of 200 μM IBMX based on previous experiments.40 PAHs and forskolin/IBMX were made up in 100% acetonitrile. Acetonitrile is noncytotoxic and noninhibitory on GJIC up to a concentration of 2% (v/v).41 Acetonitrile was used as the vehicle control and the volumes added to the cell cultures were typically between 5 and 25 μl (0.25–1.25%). Lucifer yellow was loaded into the cells by making 2 or 3 scrape lines on the monolayer with a sharp scalpel. In GJIC-competent cells Lucifer yellow moves through gap junctions from the primary dye loaded cells to contacting neighboring cells, whereas in GJIC-incompetent cells the dye does not transfer from the primary dye loaded cells to the neighboring cells. Migration of the dye in the cells was observed using a Nikon epifluorescence microscope equipped with a Sony digital camera. The fluorescence area of the scrape line and surrounding cells is used to determine the extent of dye transfer within a monolayer and quantified using “Gel Expert” image analysis program (NucleoTech Corp., San Mateo, CA). A minimum of 3 plates per treatment was used in each experiment. All experiments were repeated at least 3 times.

Immunofluorescent staining

Cells were grown in multiwell chamber slides until they reach the 80% confluency. Subsequently, cells were gently rinsed 3 times with phosphate-buffered saline (PBS) and fixed with 5% acetic acid/95% methanol for about 20 min. Cells were then incubated in blocking buffer (PBS, 10% goat serum and 0.1% Tween 20) for 1 hr. Primary antibody was added to slides in PBS containing 1% bovine serum albumin (BSA) and incubated for 2 hr or overnight. After rinsing with PBS, cells were incubated with Cyanine 3-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) for 1 hr at room temperature. Negative controls were prepared by eliminating the primary antibody or adding the rabbit or mouse serum to the buffer. Slides were then washed with PBS and counterstained with DAPI for 5 min before finally embedding with fluorescent mounting medium. Fluorescent images were obtained using a Nikon Epi-fluorescent microscope equipped with a SPOT-RT digital camera (Diagnostic Instruments, Detroit, MI) and analyzed with Spot Advanced analysis software (Diagnostic Instruments).

RT-PCR analysis of the expression of connexin genes in H6c7 treated with PAHs

RT-PCR analysis was used to examine the expression of connexin genes in H6c7 cells after growth in various conditions. In short, total RNA was isolated using Trizol reagent (GIBCO-BRL, Gaithersburg, MD) according to the manufacturer's protocol. Total RNA (1 μg) was then incubated at 37°C for 10 min with 2 units of DNase 1 (Roche Molecular Biochemicals, Indianapolis, IN) and 2 units of RNase inhibitor (Roche Molecular Biochemicals). Samples were then heated to 75°C for 10 min to heat inactivated the DNase 1. One microgram of RNA was first reverse transcribed into cDNA in a volume of 20 μl for 1 hr at 42°C using 200 units MMLV reverse transcriptase (Clontech, Palo Alto, CA), and heated at 94°C for 5 min to stop cDNA synthesis reaction and the DNase activity.

For PCR reaction, MgCl2 (50 mM) was added to 5 μl DNA (1:5 dilution with DEPC-treated water from earlier preparation) for a final concentration of 1.5 mM along with 5 μl 10× PCR buffer (200 mM Tris-HCl, pH 8.4, 500 mM KCl), 1 μl of each 10 mM dNTP, AmpliTaq Gold polymerase (2 units, Perkin–Elmer) and 5 pmol of sense and antisense primers were added to a final volume of 50 μl. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard for Cx36 RT-PCR. The 2 primer sequences of human Cx36, 43 and human GAPDH were previously described.35

Western blot analysis of gap junction proteins of H6c7 cells

To identify the type of gap junction proteins expressed in H6c7 cells and examine the effects of various growth conditions on the expression of these proteins Western blotting was employed using antibodies against connexin 43 and 36. H6c7 cells cultured under different growth conditions were lysed in a buffer containing 62.5 mM Tris-HCl, pH 7.4, 20% SDS, 5.0 mM EDTA, 2.0 mM PMSF and 10 μg/ml leupeptin. The protein concentration of the samples was determined using a Bio-Rad DC protein assay kit. Fifty micrograms of whole cell lysate proteins of each sample were separated on 12.5% SDS-PAGE and transferred to nitrocellulose membrane.42 Connexin 43 and 36 immunoreactivity was detected with specific antibodies and visualized with a SuperSignal West Pico chemiluminescent kit (Pierce).

PCR-based telomerase assay

Telomerase activity of the cells was examined by the TRAP assay43 using TRAPez Telomerase Detection Kit (Intergen, Purchase, NY). This protocol includes primers of a 36-bp internal control (I.C.) for quantifying the amplification efficiency, thus providing a positive control for accurate quantification of telomerase activity. Each analysis included a negative (lysis buffer instead of cell lysate) and a positive cell line control provided from the kit. TRAP assays were resolved by electrophoresis in a 12.5% nondenaturing polyacrylamide gel in a buffer containing 54 mM Tris-HCL (pH 8.0), 54 mM boric acid, and 1.2 mM EDTA. The gel was stained with SYBR Green (Molecular Probes, Eugene, OR) and visualized by a 302 or 254 nm UV transilluminator and recorded in Kodak EDA image system (Kodak, Rochester, NY).

Statistical analyses

All data were expressed as the Mean ± Standard Error of the Mean (SEM). Data were analyzed using one-way analysis of variance (ANOVA). Significant differences between solvent control and forskolin with or without IBMX treatment were evaluated using Dunnett's method.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Gap junctional intercellular communication competence of H6c7 cells and the effect of c-AMP and PAHs

The human immortal pancreatic ductal cell line, H6c7 cells, was GJIC incompetent when cultured in complete growth medium (KSFM with growth factors, hormones and bovine pituitary extract) (Fig. 1). When cultured in the basal medium devoid of growth factors, hormones or bovine pituitary extract, plus the treatment with agents that increase c-AMP (forskolin only or forskolin plus IBMX) for 24–72 hr, significantly enhanced the extent of GJIC of these cells compared with the GJIC of H6c7 cells treated with vehicle control (Fig. 1). The increase in GJIC of the cells in KBM plus forskolin or forskolin and IBMX was very similar (not statistically different). There were 1.9- and 1.8-fold increases in the level of GJIC at 24 hr, 2.5-and 2.3-fold increases at 48 hr and 2.4- and 2.3-fold increases at 72 hr, respectively, with treatment of forskolin only or forskolin plus IBMX in comparison with vehicle control.35

thumbnail image

Figure 1. Gap junctional intercellular communication (GJIC) in H6c7 cells as measured by the scrape-loading dye transfer (SL/DT) technique. Cells were treated with 0.25% acetonitrile (solvent control) or 5 μM forskolin ± 200 μM IBMX for 24–72 hr. Scale bar: 100 μm.

Download figure to PowerPoint

The increase of GJIC in the cells with forskolin and IBMX treatment was blocked by 1-methylanthracene but not 2-methylanthracene (Fig. 2). Quantitative analysis of GJIC in the cells treated with 1-methylanthracene was significantly decreased by 1.8-, 2.1- and 2.4-fold at 24, 48 and 72 hr, respectively. There was no significant difference in the 2-methylanthracene and vehicle control treatment of the cells (Fig. 3).

thumbnail image

Figure 2. GJIC in H6c7 cells treated with PAHs. H6c7 cells were cultured in basal medium without growth factors or hormones in the presence of 5 μM forskolin plus 200 μM IBMX for 24–72 hr to increase their GJIC (top panel). 1-methylanthracene decreases GJIC in cells treated with forskolin/IBMX (middle panel). 2-methylanthracene had no impact on GJIC in cells treated with forskolin/IBMX (bottom panel). Scale bar, 100 μm.

Download figure to PowerPoint

thumbnail image

Figure 3. (a) Structural formula of 1-methylanthracene and 2-methylanthracene. 1-methlyanthracene contains a bay-like structure. (b) Quantitative analysis of GJIC of H6c7 cells treated with PAHs. H6c7 cells were treated with 5 μM forskolin plus 200 μM IBMX ± 1-methylanthracene or 2-methylanthracene for 24–72 hr. GJIC was assayed by Lucifer yellow dye transfer method at 24, 48 and 72 hr and quantified using an image analysis program (see method). GJIC data were reported as % of the 0 hr control. Each datum represented Mean ± SEM. Significantly different from the control (*), p < 0.001 (n = 4).

Download figure to PowerPoint

Forskolin and IBMX treatment increases CX43 immunostaining

Figure 4 shows the immunofluorescent images of untreated H6c7 cells or cells treated with forskolin plus IBMX for 48–72 hr that were labeled with the Cx43 polyclonal antibody. The untreated H6c7 cells exhibited few fluorescent-labeled Cx43 punctate plaques compared to cells treated with foskolin/ IBMX combination. Dramatic increase in the number of fluorescent-labeled cells were observed after a 72 hr treatment with forskolin + IBMX.

thumbnail image

Figure 4. Immuno-fluorescent staining of H6c7 cells with Cx43-specific antibody. H6c7 cells were treated with forskolin/IBMX for 48–72 hr. The Cx43 protein fluorescent staining showed an increase level of punctate staining on the cell membranes when cells were treated with foskolin/IBMX than vehicle control. Scale bar: 20 μm.

Download figure to PowerPoint

RT-PCR analysis of the expression of connexin genes in H6c7 cells treated with PAHs

To reveal the effect of PAHs on the connexin gene expression of H6c7 cells, we performed the RT-PCR analysis of the cells treated with 1-methylanthracene or 2-methylanthracene plus forskolin and IBMX. Cx43, which is a constitutively expressed connexin gene, had no changes in their expression level when treated with 1- or 2-methylanthracene by RT-PCR (Fig. 5). The expression of Cx36 was inducible by forskolin and IBMX treatment previously reported.35 When the cells were treated with 1-methylanthracene, their Cx36 expression was decreased at 48-hr treatment. 2-methylanthracene did not affect Cx36 gene expression.

thumbnail image

Figure 5. RT-PCR analysis of Cx36 and Cx43 gene expression. H6c7 cells were treated with forskolin/IBMX ± 1-methlyanthracene and 2-methlyanthracene for 24–48 hr. GAPDH expression was served as a house keeping gene control.

Download figure to PowerPoint

1-methlyanthracene but not 2-methylanthracene decreases forskolin/IBMX-induced Cx43 protein expression

The hemi-channels or connexons are the subunits of gap junctions, and consist of 6 subunit of connexin proteins. Upregulation of Cx43 expression can induce a significant enhancement of GJIC in H6c7 cells. H6c7 cells express low levels of nonphosphorylated Cx43 (P0) and its phosphorylated forms (P1 and P2). Forskolin/IBMX induced phosphorylated Cx43 (P2) expression in H6c7 cells by Western blot detection (Fig. 6). 1-methylanthracene but not 2-methylanthracene blunts forskolin/IBMX-induced Cx43 P2 expression at 72 hr. Since 1-methylanthracene did not change the steady-state expression of Cx43 mRNA, functional GJIC depends on the ability of forskolin/IBMX to phsophorylate Cx43.

thumbnail image

Figure 6. Cx43 protein expression in H6c7 cells treated with forskolin/IBMX and PAHs. Cx43 protein levels were measured at 48 and 72 hr for control and treated cells. P0, nonphosphorylated Cx43; P1 and P2, phosphorylated forms of Cx43. Representative western blot (n = 3).

Download figure to PowerPoint

Effect of forskolin/IBMX on telomerase activities

H6c7 cells are normal pancreatic duct epithelial cells immortalized with human papilloma virus (HPV) type 16 E6E7 genes. E6 is an early HPV gene product that is involved in immortalization and transformation by the virus. E6 has been shown to increase telomerase activity when expressed in a variety of cell types, through transcription activation of the TERT gene, which encodes the catalytic subunit of the telomerase enzyme.44, 45 H6c7 cells, as many immortal and tumor cell lines, were high in telomerase activity when cultured in keratinocyte medium supplemented with grow factors (Fig. 7, lane 1). Forskolin and IBMX were cAMP elevating reagents. Treatment of H6c7 cells with forskolin and IBMX resulted in inhibition of telomerase activity observable at 72 hr (Fig. 7, lane 9). Telomerase activities in H6c7 cells were dependent on enzyme activity from the catalytic and RNA template, as shown by elimination of the activity by heat inactivation (lane 2, 4, 6, 8 and 10).

thumbnail image

Figure 7. Telomerase activities in H6c7 cells treated with forskolin/IBMX for 48–72 hr. Lane 1 showed the high telomerase activity in immortalized H6c7 cells cultured in KSFM. No change in telomerase activities when cells were treated for 48 hr (lane 5). Telomerase activities were diminished when cells were treated for 72 hr (lane 9). Lane 11 and 12 were TSR8 template positive control and CHAPS buffer negative control of the assay. I.C., the internal control of TRAP assay. ΔH, the heat-inactivated control.

Download figure to PowerPoint

Effect of PAHs on telomerase activities

Next the impact of 1-methyl and 2-methylanthracene on telomerase activities in H6c7 cells was tested. Figure 8 Lane 1 shows the high telomerase activities in immortalized H6c7 cells cultured in keratinocyte medium supplemented with growth factors. Telomerase activities were slightly decreased in the cells cultured in basal keratinocyte medium without growth factors for 72 hr (Fig. 8, lane 8). This may be due to H6c7 cells which began to differentiate and show low level of GJIC (data not shown) when cultured in the basal medium. Telomerase activities were found to be further diminished when cells were treated with forskolin and IBMX for 72 hr (Fig. 8, lane 9). Telomerase activities were found in the H6c7 cells treated with 1-methylanthracene plus forskolin and IBMX (Fig. 8, lane 10). 2-methlyanthracene, which is an inactive isomer of 1-methylanthracene, had no effect on telomerase activity (lane 11). No change in telomerase activity was seen either when cells were treated with foskolin/ IBMX in combination with 1-methyl or 2-methylanthracene for 24 or 48 hr (Fig. 8, data not shown).

thumbnail image

Figure 8. Telomerase activities in H6c7 cells treated with PAHs. Lane 1 showed the high telomerase activities in immortalized H6c7 cells cultured in KSFM. Telomerase activities were slightly decreased when cells were cultured in KBM for 72 hr (lane 8). Telomerase activities were further diminished when cells were treated with forskolin/IBMX for 72 hr (lane 9). 1-methlyanthracene blocked the effect of forskolin and IBMX on telomerase activities. High level of telomerase activities was found in the cells treated with 1-methlantracene plus forskolin/IBMX for 72 hr (lane 10). 2-methlyanthracene treatments had no effect on telomerase activities (lane 11). No change in telomerase activity when cells were treated for 24 hr (lane 2–6) and 48 hr (data not shown). Lane 12 and 13 were positive and negative control of the assay. I.C., the internal control of TRAP assay.

Download figure to PowerPoint


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The most abundant PAHs in cigarette smoke are the low molecular weight compounds. These compounds include the methylated anthracenes and phenanthrenes and are 62-fold higher than benzo(a)pyrene and benzo(e)pyrene.37 The fact that the fraction of cigarette smoke containing the 3-ringed PAHs is highly cocarcinogenic when applied to the skin of mice treated with benz(a)pyrene (BaP),46 and that cigarette smoke is a strong promoter and weak complete carcinogen11, 47, 48, 49 suggest that this fraction could significantly contribute to cancer. Ten to 15 years after giving up smoking, the exsmoker faces the same low risk of developing cancer of the upper digestive tract, the lung, the pancreas, and the urinary tract as the nonsmoker.50 This strongly suggests that cigarette smoke contributes to the nongenotoxic and reversible phases of cancer. Therefore, understanding the biological effects of these 3-ringed PAHs on cell signaling pathways relevant to the epigenetic, nongenotoxic phase of cancer is important. We previously reported that three- and four-ringed PAHs, which contain an angular pocket (Fig. 3)—formed with either a benzyl (bay region) or alkyl group (bay-like region), were biologically active in the induction of arachidonic acid release, inhibition of GJIC and the activation of mitogen activated protein kinase pathways in a rat liver epithelial cell line with oval cell characteristics.51, 52, 53 This structure–activity relationship was also observed in the induction of arachidonic acid release from endothelial cells54 and the inhibition of GJIC in rat cardiac myocytes.55

Consistent with these previous observations, 1-methylanthracene, which has a bay-like region, inhibited forskolin-induced GJIC in the H6c7 pancreatic epithelial ductal cells, whereas the linear isomer, 2-methylanthracene, had no effect on GJIC. Although these cells constitutively expressed Cx43 mRNA, and 1-methylanthracene had no further effect on the levels of Cx43 RNA message, functional GJIC was strongly correlated with the treatment of the cells with the adenylate cyclase inducer, forskolin, and the phosphodiesterase inhibitor, IBMX. We have previously shown that the activation of adenylate cyclase was necessary for the expression of the Cx43 protein,35 suggesting that cAMP-dependent pathways are needed in the translation of the constitutively expressed Cx43 mRNA. The immunofluorescent staining of Cx43 protein indicated that forskolin not only induced the expression of the protein, but that it was also properly trafficked to the plasma membrane, which correlated with active GJIC. In contrast, Cx36 mRNA was not constitutively expressed, and forskolin was necessary for the induction of this connexin RNA message. The induction of Cx36 mRNA expression correlated with the activation of GJIC and the expression of Cx43 protein. Conversely, inhibition of Cx43-dependent GJIC with 1-methylanthracene correlated with reduced Cx36 mRNA expression. These results suggest that Cx43 is an essential early connexin gene needed for the initial establishment of GJIC, which is subsequently needed for the expression of the Cx36 gene normally observed in differentiated pancreatic islet tissue.56, 57, 58, 59 Connexin 26 (Cx26)—which was reported to be expressed in islets of Langerhans60—was not detected in H6c7 cells by Western Blot analysis in our previous study.61 Cx26 protein or gene expression was not upregulated by cyclic AMP.61 The linear PAH isomer, 2-methylanthracene, did not inhibit GJIC and also did not block the expression of Cx36 mRNA, further indicating that functional GJIC in H6c7 cells might be dependent on the expression of this connexin. These differences between the linear vs. the bay-like isomers of methylanthracene suggest the involvement of a receptor. Currently we have not identified a putative receptor but this receptor appears to be affiliated with a phosphatidylcholine specific phospholipase C (PC-PLC).62 Inhibition of PC-PLC prevents inhibition of GJIC by 1-methylanthracene, and 1-methylanthracene but not 2-methylanthracene activates PC-PLC.62

Normal somatic cells have a limited life span in which telomere shortening has been proposed as a mitotic clock for cellular senescence.63, 64, 65, 66 The telomerase enzyme is capable of maintaining telomere length and the proliferative activity of cells,67, 68, 69 and high activity of this enzyme is found in stem cells70, 71, 72, 73, 74 as well as malignant cells.75, 76 As stem and progenitor cells become more differentiated-or when tumor cells are induced to differentiate, the activity of this enzyme sharply decreases.71, 77, 78 In essence, telomerase activity is an inverse marker of differentiation. Consistent with this telomerase phenomenon, the induction of differentiation in the H6c7 pancreatic cells with the adenylate cyclase stimulator, forskolin and IBMX, induced a sharp decline in telomerase activity. This activity correlated with the induction of GJIC; and inhibition of GJIC by 1-methylanthracene prevented this sharp decline in telomerase activity, while 2-methylanthracene, which does not inhibit GJIC, had no effect on preventing the decrease of telomerase activity.

One of the oldest ideas on the origin of cancers is that of “cancer as a disease of differentiation,”31, 32 a “stem cell disease”33 or “oncogeny as partially blocked ontogeny”,14 suggesting that the target cells are the pluripotent stem cells. Our results are consistent with this hypothesis. As indicated above, induction of early differentiation of these pancreatic cells, which possess stem-like characteristics,35 results in the decrease of telomerase activity (an inverse differentiation marker) and the expression of Cx36 (a differentiation marker of pancreatic islets). These events correlated with GJIC activity and the inhibition of GJIC with 1-methylanthracene, an extremely prevalent PAH found in cigarette smoke, prevented inactivation of telomerase and the expression of Cx36 (suggesting a potential cause and effect relationship). The specificity of this response relative to a strict structural criterion of the PAH molecule is evident in the lack of an effect by the 2-methyl isomer of anthracene. Apparently, Cx43 is an important gene needed to establish intercellular communication that is needed in the early induction of differentiation of pancreatic stem cells.

Although the discovery that benzo(a)pyrene (BaP) formed DNA-adducts in codons that correlated with the major mutational hotspots found in human lung cancers using in vitro cell systems,79 this PAH is less prevalent in cigarette smoke and might be interpreted as contributing to the initiating phase of cancer rather than the promotion phase. Our results using an in vitro stem cell system showed biological effects of a PAH, which is much more prevalent than BaP, could contribute to cell signaling events affiliated with the promotional phase of cancer. This observation is significant considering that cigarette smoke contributes more to the promoting than the initiating phase of cancer.11 Our results do not explain the entire carcinogenic nature of the complex mixtures found in cigarette smoke, however, these in vitro studies do indicate that a much overlooked class of compounds found in cigarette smoke that can induce molecular events consistent with the tumor promoting phase of cancer.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This research was supported by the National Institute of Environmental Health Science—Superfund Basic Research Program (grant #P42 ES04911-17) and a research gift from L. Van Camp, both funded to James E. Trosko, and ES013268-01A2 to B. Upham. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHs. NIH R21 DK57173-01 and Michigan Life Science Corridor Fund GR178 to L. Karl Olson also supported this research.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Risch HA. Etiology of pancreatic cancer, with a hypothesis concerning the role of N-nitroso compounds and excess gastric acidity. J Natl Cancer Inst 2003; 95: 94860.
  • 2
    Prokopczyk B, Hoffmann D, Bologna M, Cunningham AJ, Trushin N, Akerkar S, Boyiri T, Amin S, Desai D, Colosimo S, Pittman B, Leder G, et al. Identification of tobacco-derived compounds in human pancreatic juice. Chem Res Toxicol 2002; 15: 67785.
  • 3
    Ahlgren JD. Epidemiology and risk factors in pancreatic cancer. Semin Oncol 1996; 23: 24150.
  • 4
    Nilsen TI, Vatten LJ. A prospective study of lifestyle factors and the risk of pancreatic cancer in Nord-Trondelag, Norway. Cancer Causes Control 2000; 11: 64552.
  • 5
    Li D. Molecular epidemiology of pancreatic cancer. Cancer J 2001; 7: 25965.
  • 6
    Trede M, Richter A, Wendl K. Personal observations, opinions, and approaches to cancer of the pancreas and the periampullary area. Surg Clin North Am 2001; 81: 595610.
  • 7
    Ghadirian P, Lynch HT, Krewski D. Epidemiology of pancreatic cancer: an overview. Cancer Detect Prev 2003; 27: 8793.
  • 8
    Lynch HT, Brand RE, Lynch JF, Fusaro RM, Kern SE. Hereditary factors in pancreatic cancer. J Hepatobiliary Pancreat Surg 2002; 9: 1231.
  • 9
    Silverman DT. Risk factors for pancreatic cancer: a case-control study based on direct interviews. Teratog Carcinog Mutagen 2001; 21: 725.
  • 10
    Silverman DT, Hoover RN, Brown LM, Swanson GM, Schiffman M, Greenberg RS, Hayes RB, Lillemoe KD, Schoenberg JB, Schwartz AG, Liff J, Pottern LM, et al. Why do Black Americans have a higher risk of pancreatic cancer than White Americans? Epidemiology 2003; 14: 4554.
  • 11
    Rubin H. Selective clonal expansion and microenvironmental permissiveness in tobacco carcinogenesis. Oncogene 2002; 21: 7392411.
  • 12
    Thilly WG. Have environmental mutagens caused oncomutations in people? Nat Genet 2003; 34: 2559.
  • 13
    Fuchs CS, Colditz GA, Stampfer MJ, Giovannucci EL, Hunter DJ, Rimm EB, Willett WC, Speizer FE. A prospective study of cigarette smoking and the risk of pancreatic cancer. Arch Intern Med 1996; 156: 225560.
  • 14
    Potter VR. Phenotypic diversity in experimental hepatomas: the concept of partially blocked ontogeny. Br J Cancer 1978; 38: 123.
  • 15
    Trosko JE. The role of stem cells and gap junctional intercellular communication in carcinogenesis. J Biochem Mol Biol 2003; 36: 438.
  • 16
    Ruch RJ, Trosko JE. Gap-junction communication in chemical carcinogenesis. Drug Metab Rev 2001; 33: 11724.
  • 17
    Subarsky P, Hill RP. The hypoxic tumour microenvironment and metastatic progression. Clin Exp Metastasis 2003; 20: 23750.
  • 18
    Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 2003; 33 ( Suppl): 24554.
  • 19
    Barcellos-Hoff MH. It takes a tissue to make a tumor: epigenetics, cancer and the microenvironment. J Mammary Gland Biol Neoplasia 2001; 6: 21321.
  • 20
    Klaunig JE, Kamendulis LM, Xu Y. Epigenetic mechanisms of chemical carcinogenesis. Hum Exp Toxicol 2000; 19: 54355.
  • 21
    Trosko JE, Chang CC. Nongenotoxic mechanisms in carcinogenesis: role of inhibited intercellular communication. In: HartRW, HoergerFD, eds. Banbury Report 31: carcinogen risk assessment: new directions in the quantitative and qualitative assessment aspects. Cold Spring Harbor: Cold Spring Harbor Laboratory, 1988. 13970.
  • 22
    Terzaghi M, Nettesheim P. Dynamics of neoplastic development in carcinogen-exposed tracheal mucosa. Cancer Res 1979; 39: 400310.
  • 23
    Ullrich RL. The rate of progression of radiation-transformed mammary epithelial cells is enhanced after low-dose-rate neutron irradiation. Radiat Res 1986; 105: 6875.
  • 24
    Kamiya K, Yasukawa-Barnes J, Mitchen JM, Gould MN, Clifton KH. Evidence that carcinogenesis involves an imbalance between epigenetic high-frequency initiation and suppression of promotion. Proc Natl Acad Sci USA 1995; 92: 13326.
  • 25
    Mehta PP, Bertram JS, Loewenstein WR. Growth inhibition of transformed cells correlates with their junctional communication with normal cells. Cell 1986; 44: 18796.
  • 26
    Enomoto T, Yamasaki H. Lack of intercellular communication between chemically transformed and surrounding nontransformed BALB/c 3T3 cells. Cancer Res 1984; 44: 52003.
  • 27
    Trosko JE, Chang CC. Adaptive and nonadaptive consequences of chemical inhibition of intercellular communication. Pharmacol Rev 1984; 36: 137S144S.
  • 28
    Loewenstein WR, Rose B. The cell–cell channel in the control of growth. Semin Cell Biol 1992; 3: 5979.
  • 29
    Trosko JE, Chang CC. Role of stem cells and gap junctional intercellular communication in human carcinogenesis. Radiat Res 2001; 155: 17580.
  • 30
    Trosko JE, Chang CC, Madhukar BV, Dupont E. Oncogenes, tumor suppressor genes and intercellular communication in the ‘Oncogeny as partially blocked ontogeny’ hypothesis. In: IversenOH, ed. New frontiers in cancer causation. Washington, DC: Taylor and Francis, 1993. 18197.
  • 31
    Markert CL. Neoplasia: a disease of cell differentiation. Cancer Res 1968; 28: 190814.
  • 32
    Pierce GB. Neoplasms, differentiations and mutations. Am J Pathol 1974; 77: 10318.
  • 33
    Till JE. Stem cells in differentiation and neoplasia. J Cell Physiol Suppl 1982; 1: 311.
  • 34
    Furukawa T, Duguid WP, Rosenberg L, Viallet J, Galloway DA, Tsao MS. Long-term culture and immortalization of epithelial cells from normal adult human pancreatic ducts transfected by E6E7 gene of human papilloma virus 16. Am J Pathol 1996; 148: 176370.
  • 35
    Tai M-H, Chang CC, Olson LK, Trosko JE. Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis. Carcinogenesis 2005; 26: 495502.
  • 36
    Askari MDF, Tsao M-S, Schuller HM. The tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone stimulateds proliferation of immortalized human pancreatic duct epithelia through β-adrenergic transactivation of EGF receptors. J Cancer Res Clin Oncol 2005; 131: 63948.
  • 37
    Severson RF, Snook ME, Higman HC, Chortyk OT, Akin FJ. Isolation, identification, and quantification of polynuclear aromatic hydrocarbons in tobacco smoke. In: FreudenthalRI, JonesPW, eds. Carcinogenesis—a comprehensive survey, vol. 1: polynuclear aromatic hydrocarbons: chemistry, metabolism, and carcinogenesis. New York: Raven Press, 1976. 25370.
  • 38
    El-Fouly MH, Trosko JE, Chang CC. Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication. Exp Cell Res 1987; 168: 42230.
  • 39
    Trosko JE, Chang CC, Wilson MR, Upham B, Hayashi T, Wade M. Gap junctions and the regulation of cellular functions of stem cells during development and differentiation. Methods 2000; 20: 24564.
  • 40
    Weis LM, Rummel AM, Masten SJ, Trosko JE, Upham BL. Bay or baylike regions of polycyclic aromatic hydrocarbons were potent inhibitors of gap junctional intercellular communication. Environ Health Perspect 1998; 106: 1722.
  • 41
    Upham BL, Yao JJ, Trosko JE, Masten SJ. Determination of the efficacy of ozone treatment systems using a gap junction intercellular communication bioassay. Environ Sci Technol 1995; 29: 29238.
  • 42
    Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1978; 76: 43504.
  • 43
    Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW. Specific association of human telomerase activity with immortal cells and cancer. Science 1994; 266: 20115.
  • 44
    Klingelhutz AJ, Foster SA, McDougall JK. Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature 1996; 380: 7982.
  • 45
    McMurray HR, McCance DJ. Human papillomavirus type 16 E6 activates TERT gene transcription through induction of c-Myc and release of USF-mediated repression. J Virol 2003; 77: 985261.
  • 46
    Hoffmann D, Schmeltz SS, Hecht SS, Wynder EL. Tobacco carcinogenesis. In: GelboinHV, Ts'oPO, eds. Polycyclic aromatic hydrocarbonds and cancer, vol. 1: environment, chemistry, and metabolism. San Diego: Academic Press, 1978. 85117.
  • 47
    Van Duuren BL, Sivak A, Langseth L. The tumor-promoting activity of tobacco leaf extract and whole cigarette tar. Br J Cancer 1967; 21: 4603.
  • 48
    Van Duuren BL, Sivak A, Katz C, Melchionne S. Cigarette smoke carcinogenesis: importance of tumor promoters. J Natl Cancer Inst 1971; 47: 23540.
  • 49
    Bock FG. Tumor promoters in tobacco and cigarette-smoke condensate. J Natl Cancer Inst 1972; 48: 184953.
  • 50
    Wynder EL, Hoffmann D. Tobacco and tobacco smoke. Semin Oncol 1976; 3: 515.
  • 51
    Weis LM, Rummel AM, Masten SJ, Trosko JE, Upham BL. Bay or baylike regions of polycyclic aromatic hydrocarbons were potent inhibitors of gap junctional intercellular communication. Environ Health Perspect 1998; 106: 1722.
  • 52
    Upham BL, Weis LM, Trosko JE. Modulated gap junctional intercellular communication as a biomarker of PAH epigenetic toxicity: structure-function relationship. Environ Health Perspect 1998; 106: 97581.
  • 53
    Blaha L, Kapplova P, Vondracek J, Upham B, Machala M. Inhibition of gap-junctional intercellular communication by environmentally occurring polycyclic aromatic hydrocarbons. Toxicol Sci 2002; 65: 4351.
  • 54
    Tithof PK, Elgayyar M, Cho Y, Guan W, Fisher AB, Peters-Golden M. Polycyclic aromatic hydrocarbons present in cigarette smoke cause endothelial cell apoptosis by a phospholipase A2-dependent mechanism. FASEB J 2002; 16: 14634.
  • 55
    Upham B, Davis JM, Trosko JE, Schwartz KA. Polycyclic aromatic hydrocarbons with bay-like structures inhibited gap junctional intercellular communication in neonatal rat cardiomyocytes and caused asynchronous beating. Toxicol Sci 2002; 66: 1410 (abstract).
  • 56
    Caton D, Calabrese A, Mas C, Serre-Beinier V, Wonkam A, Meda P. β-Cell crosstalk: a further dimension in the stimulus-secretion coupling of glucose-induced insulin release. Diabetes Metab 2002; 28: 3S453S53.
  • 57
    Calabrese A, Zhang M, Serre-Beinier V, Caton D, Mas C, Satin LS, Meda P. Connexin 36 controls synchronization of Ca2+ oscillations and insulin secretion in MIN6 cells. Diabetes 2003; 52: 41724.
  • 58
    Calabrese A, Guldenagel M, Charollais A, Mas C, Caton D, Bauquis J, Serre-Beinier V, Caille D, Sohl G, Teubner B, Le Gurun S, Trovato-Salinaro A, et al. Cx36 and the function of endocrine pancreas. Cell Commun Adhes 2001; 8: 38791.
  • 59
    Serre-Beinier V, Le Gurun S, Belluardo N, Trovato-Salinaro A, Charollais A, Haefliger JA, Condorelli DF, Meda P. Cx36 preferentially connects β-cells within pancreatic islets. Diabetes 2000; 49: 72734.
  • 60
    Pfeffer F, Koczan D, Adam U, Benz S, von Dobschuetz E, Prall F, Nizze H, Thiesen HJ, Hopt UT, Lobler M. Expression of connexin26 in islets of Langerhans is associated with impaired glucose tolerance in patients with pancreatic adenocarcinoma. Pancreas 2004; 29: 28490.
  • 61
    Tai M-H, Madhukar BV, Olson LK, Linning KD, VanCamp L, Tsao MS, Trosko JE Characterization of gap junctional intercellular communication in immortalized human ductal epithelial cells with stem cell characteristics. Pancreas 2003; 26: e18e26.
  • 62
    Blaha L, Trosko JE, Upham BL. The role of phospholipases in the inhibition of gap junction communication and the activation of MAPK by specific isomers of methylated anthracenes. Toxocol Sci Suppl 2006; 90: 961 (abstract).
  • 63
    Harley CB, Kim NW, Prowse KR, Weinrich SL, Hirsch KS, West MD, Bacchetti S, Hirte HW, Counter CM, Greider CW. Telomerase, cell immortality, and cancer. Cold Spring Harb Symp Quant Biol 1994; 59: 30715.
  • 64
    Counter CM, Gupta J, Harley CB, Leber B, Bacchetti S. Telomerase activity in normal leukocytes and in hematologic malignancies. Blood 1995; 85: 231520.
  • 65
    Counter CM, Botelho FM, Wang P, Harley CB, Bacchetti S. Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes. J Virol 1994; 68: 34104.
  • 66
    Harley CB, Vaziri H, Counter CM, Allsopp RC. The telomere hypothesis of cellular aging. Exp Gerontol 1992; 27: 37582.
  • 67
    Greider CW. Telomerase activity, cell proliferation, and cancer. Proc Natl Acad Sci USA 1998; 95: 902.
  • 68
    Hsiao R, Sharma HW, Ramakrishnan S, Keith E, Narayanan R. Telomerase activity in normal human endothelial cells. Anticancer Res 1997; 17: 82732.
  • 69
    Belair CD, Yeager TR, Lopez PM, Reznikoff CA. Telomerase activity: a biomarker of cell proliferation, not malignant transformation. Proc Natl Acad Sci USA 1997; 94: 1367782.
  • 70
    Wright WE, Piatyszek MA, Rainey WE, Byrd W, Shay JW. Telomerase activity in human germline and embryonic tissues and cells. Dev Genet 1996; 18: 1739.
  • 71
    Sun W, Kang KS, Morita I, Trosko JE, Chang CC. High susceptibility of a human breast epithelial cell type with stem cell characteristics to telomerase activation and immortalization. Cancer Res 1999; 59: 611823.
  • 72
    Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 11457.
  • 73
    Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Piatyszek MA, Shay JW, Ishioka S, Yamakido M. Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immunol 1995; 155: 37115.
  • 74
    Yui J, Chiu CP, Lansdorp PM. Telomerase activity in candidate stem cells from fetal liver and adult bone marrow. Blood 1998; 91: 325562.
  • 75
    Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW. Specific association of human telomerase activity with immortal cells and cancer. Science 1994; 266: 20115.
  • 76
    Rhyu MS. Telomeres, telomerase, and immortality. J Natl Cancer Inst 1995; 87: 88494.
  • 77
    Cerezo A, Stark HJ, Moshir S, Boukamp P. Constitutive overexpression of human telomerase reverse transcriptase but not c-myc blocks terminal differentiation in human HaCaT skin keratinocytes. J Invest Dermatol 2003; 121: 1109.
  • 78
    Lopatina NG, Poole JC, Saldanha SN, Hansen NJ, Key JS, Pita MA, Andrews LG, Tollefsbol TO. Control mechanisms in the regulation of telomerase reverse transcriptase expression in differentiating human teratocarcinoma cells. Biochem Biophys Res Commun 2003; 306: 6509.
  • 79
    Denissenko MF, Pao A, Tang M, Pfeifer GP. Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Science 1996; 274: 4302.