A malignant transformation of human cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibits altered expressions of growth regulatory factors

Jae-Ho Yang2, Christoph Vogel1 and Josef Abel1

Department of Pharmacology and Toxicology, School of Medicine, Catholic University of Taegu-Hyosung, 3056-6 Taemyong-4-dong, Namgu, Taegu 705-034, Korea and
1 Department of Toxicology, Medical Institute of Environmental Hygiene at the Heinrich-Heine University of Dusseldorf, Dusseldorf, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The neoplastic transformation of human cells in culture with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has recently been reported. In this study, expressions of growth regulatory factors were analyzed to examine their possible roles in TCDD-induced malignant transformation of human cells. Reverse transcription–polymerase chain reaction (RT–PCR) and immunoblot analysis were performed to detect altered expressions of genes associated with dioxin responses. The RT–PCR analysis showed that expressions of the growth regulatory factors, such as transforming growth factor-ß1 (TGF-ß1), plasminogen activator inhibitor-2 (PAI-2) and tumor necrosis factor-{alpha} (TNF-{alpha}), were significantly changed in the transformed cells as compared with the parental cells. Whereas parental cells showed a dose-dependent increase of PAI-2 mRNA levels following TCDD exposure, the transformed cells did not show any significant induction. In addition, constitutive levels of PAI-2 mRNA were 25 times lower in the transformed cells than in the parental cells. The mRNA stability assay suggests that downregulation of PAI-2 mRNA in the transformed cells may be associated with the post-transcriptional control. Expression of TGF-ß1 mRNA in the transformed cells was also four times lower than the parental cells. However, levels of TNF-{alpha} mRNA in the transformed cells were increased 3-fold. These results suggest that dysregulation of growth regulatory factors may be involved in TCDD-induced cellular transformation. Whereas plenty of studies demonstrated a number of immediate toxic effects by TCDD, this study revealed an initial evidence that altered expression of growth regulatory genes, such as PAI-2, TGF-ß1 or TNF-{alpha}, are some of the genetic events fixed in the genome following the successive cell divisions of TCDD-damaged cells. It is suggested that these changes may be associated with TCDD-induced malignant transformation of human cells.

Abbreviations: AHH, aryl hydrocarbon hydroxylase; AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; CYP, cytochrome P450; DMEM, Dulbecco's modified essential medium; DMSO, dimethyl sulfoxide; DRE, dioxin responsive element; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; PAI-2, plasminogen activator inhibitor-2; PMSF, phenylmethylsulfonyl fluoride; RT–PCR, reverse transcription–polymerase chain reaction; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TGF, transforming growth factor; TNF, tumor necrosis factor; TPA, phorbol 12-myristate 13-acetate; u-PA, urokinase-type plasminogen activator.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent carcinogens ever tested in animal bioassays and bioaccumulates in animals and humans (1). Recent epidemiological data from occupationally exposed workers have established an association between exposure of TCDD and several types of human cancers (24). Researchers now seem to agree that TCDD is a human carcinogen at least at high doses. Despite increasing evidence on human cancers, mechanisms of TCDD-induced carcinogenesis in humans remains uncertain. TCDD forms few DNA adducts and is negative in mutagenicity assays. TCDD is reported to induce tumor promotion in a fashion similar to TPA, which acts through alteration of growth regulatory genes (5,6). Thus, it is suggested that this compound may act by epigenetic mechanism such as dysregulation of cellular proliferation and differentiation. However, due to the limited cellular system, it has been difficult to study the roles of growth regulatory factors involved in TCDD-induced carcinogenesis of human cells.

Numerous studies report TCDD-mediated modifications of growth factors and cytokines in experimental animals and cell systems (7). Some of the TCDD-mediated alterations observed include changes in TGF-{alpha}, TGF-ß, TNF-{alpha}, IL-1ß and PAI-2. Because these growth regulatory factors are involved in cell proliferation and differentiation, disruption of these pathways by TCDD are suggested to be a plausible mechanism of carcinogenic action. However, most of the alterations in growth regulatory factors are observed with a short-term exposure to TCDD and they tend to return to normal levels when the chemical exposure is removed. In addition, the malignancy usually requires fixation of altered gene expression through clonal expansion of the damaged cells. Thus, it has been difficult to know whether the alterations of growth regulatory factors observed immediately after the exposure play a significant role in TCDD-induced carcinogenic action. We have recently reported a malignant transformation of immortalized human keratinocytes cell line with a 2 week exposure of TCDD and six subsequent subcultures (8). Since the cell line used in our previous study is the only human in vitro system ever reported to show carcinogenicity by TCDD, it may provide a unique opportunity to look into effects of growth regulatory factors expressed after clonal expansion of the TCDD-damaged cells.

This study used the parental cells and their TCDD-transformed cells as cellular models to study the roles of growth regulatory factors in TCDD-induced malignant transformation of human cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals and reagents
TCDD was obtained from KOR Biomedical (Cambridge, MA). The purity of this compound was >99% as assayed by analytical high-pressure liquid chromatography. TCDD was dissolved in DMSO, and aliquots (100 µM) were stored at –70°C. Various concentrations of TCDD were prepared by direct dilution of 100 µM aliquots into appropriate media. All media including the control group contained a final concentration of <0.1% DMSO. Triazol and Moloney murine leukemia virus-reverse transcriptase were supplied by Gibco BRL (Gaithersburg, MD); oligo(dT)15 primer, Taq DNA polymerase and DNase I by Boehringer (Mannheim, Germany); deoxynucleotide triphosphates and RNase inhibitor by Pharmacia (Freiburg, Germany) and [{alpha}-32P]dCTP by Amersham Life Science (Arlington Heights, IL). Other reagents were purchased from Sigma (St Louis, MO).

Cell cultures
The parental cell line, designated as RHEK-1, was established from human foreskin epidermal keratinocyte with infection of Ad12-SV40 hybrid virus (9). These cells did not produce a virus, had a `flat' epithelial morphology, expressed a number of markers associated with epithelial cells and were non-tumorigenic in nude mice. RHEK-1 was a generous gift from Dr Rhim, Frederick-NCI, USA. The TCDD-transformed cell line was obtained from the parental cell line after a 2 week exposure of TCDD and six subsequent subcultures (8). The transformed cell line showed a variety of cellular transformation properties such as increases of soft-agar colony formation and cell density. Subcutaneous injection of the transformed cells into the nude mouse subsequently developed a tumor. Maintenance media of these cell lines consist of Dulbecco's modified essential medium (DMEM) with 10% fetal bovine serum (FBS), hydrocortisone (5 µg/ml), penicillin G (50 U/ml) and streptomycin (50 µg/ml).

Treatment
Since it is possible that the addition of fresh medium plus serum alters the expression of the genes under investigation, we did not add fresh medium at the time of TCDD treatment. At 48 h after a change of the fresh medium, both the parental cells and the transformed cells were treated with 0.1, 1, 10, 100 nM TCDD or 0.1% DMSO for 24 h.

RT–PCR analysis
Total RNAs were prepared with a RNA isolation kit (Gibco BRL, Grand Island, NY), according to the manufacturer's instructions followed by digestion with RNase-free DNase I. RT–PCR was then performed as described previously (10). Briefly, for cDNA synthesis 1 µg total RNA was heated in a final volume of 10 µl with 2 µg oligo(dT)15 primer for 5 min at 60°C, then chilled on ice, and reverse transcribed in a final volume of 40 µl containing 1 mM of each dNTP, 8 µl 5x M-MLV buffer, 60 U RNase inhibitor, 10 mM DTT and 400 U M-MLV reverse transcriptase. Samples were incubated at 37°C for 1 h and subsequently denatured for 10 min at 70°C. PCR primers were synthesized with an Applied Biosystems 391 DNA synthesizer (Weiterstadt, Germany) and purified with NAP-5 columns (Pharmacia). Primer sequences were from published sources or chosen using a primer selection program (Oligo; National Biosciences, Plymouth, MN) and are given in Table IGo (1118). PCR reactions were carried out in a final volume of 50 µl containing 2.5 µl RT samples, 5 µl 10x Taq buffer, and 200 µM of each dNTP in the presence of 0.2 µM of each primer, 2.5 U Taq DNA polymerase and 1 µCi[{alpha}-32P]dCTP. Amplifications were performed using a DNA thermal cycler (Ericomp, San Diego, CA) for the indicated cycles with the following profile: 4 min at 94°C before the first cycle, 1 min for denaturation at 94°C, 1 min for primer annealing, 1 min for primer extension at 72°C and 7 min at 72°C after the last cycle. Linearity of amplification was controlled by three different cycle numbers for one cDNA concentration. PCR products were analyzed on 10% (w/v) polyacrylamide gels, and then the gels were dried and autoradiographed. Analysis of respective bands was performed by an image analyzer (Bio-Rad, Hercules, CA).


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Table I. Oligonucleotide primers used for gene expression analyses by RT–PCR
 
mRNA stability assay
Cells were cultured and treated as described above. After incubation for 12 h, the medium was removed from control or TCDD-treated cultures and pooled. Actinomycin D (5 µg/ml) was added to the pooled medium to block new transcription initiation and the medium was then added back to the cultures. At the indicated times after addition of actinomycin D, total RNA was prepared for RT–PCR analysis as described above.

Immunoblotting
Cells at 70% confluency were lysed in lysis buffer (50 mM HEPES, 150 mM NaCl, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 1% NP-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin and 10 µg/ml leupeptin, adjusted to pH 7.5), then sonicated twice for 10 s on ice. Proteins (25 µg) of the lysates from the parental cells or the transformed cells were then prepared on 8% SDS–PAGE. The nitrocellulose sheet was blocked with 3% non-fat dry milk in Tris-buffered saline. The AhR antibody bound to the protein on the nitrocellulose sheet were detected with goat anti-rabbit IgG conjugated with peroxide, using the ECL system of Amersham Life Science. Affinity purified polyclonal antibody to human AhR and the corresponding pre-immune immunoglobin G fraction were kindly provided by Dr Oliver Hankinson, UCLA.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Expression of PAI-2 mRNA
RT–PCR analysis was performed in the parental cells and the transformed cells to measure mRNA levels of PAI-2, one of the dioxin-responsive genes. The parental cells showed a dose-dependent increase of PAI-2 mRNA level following a 24 h exposure to TCDD. A maximum induction was observed at 100 nM: the level of PAI-2 mRNA expression was increased 3.5-fold, relative to the control (3.5 ± 0.8; based on the densitometric results from three separate experiments) (Figure 1AGo). However, when the basal level of PAI-2 mRNA expression was analyzed, the transformed cells showed a 25-fold decrease of mRNA expression as compared with the parental cells (25.0 ± 2.4; based on the results from three separate experiments) (Figure 1BGo). In addition, when the transformed cells were treated with TCDD for 24 h, there was no significant dose-dependent increase of PAI-2 mRNA expression. To examine whether downregulation PAI-2 in the transformed cells is under post-transcriptional control, the stability of PAI-2 mRNA in presence of actinomycin D, an inhibitor of mRNA synthesis, was measured. The result showed that the rate of decay of PAI-2 mRNAs in the parental cells was slower than in the transformed cells. A maximum difference of decay as expressed in percentage of initial PAI-2 mRNA was observed 6 h after actinomycin D treatment (parental cells, 65 ± 14; transformed cells, 20 ± 17). Experiments using ß-actin as a control gene showed no significant difference between the parental cells and the transformed cells on the rate of decay of ß-actin mRNAs (Figure 2Go).



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Fig. 1. Expression of PAI-2 mRNA in the parental cells and the transformed cells. (A) Parental cells were treated for 24 h with 0.1% DMSO (1), 0.1 nM (2), 1 nM (3), 10 nM (4) or 100 nM TCDD (5). (B) The parental cells were treated with 0.1% DMSO only (1). The transformed cells were treated for 24 h with 0.1% DMSO (2), 0.1 nM (3), 1 nM (4) or 10 nM TCDD (5). A typical result of three separate experiments is shown.

 


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Fig. 2. Comparison of PAI-2 mRNAs stability between the parental cells and the transformed cells. Cells were treated as described in Materials and methods. RNA was collected at the indicated time points after actinomycin D treatment. Quantification was based on densitometric tracings normalized to GAPDH. Results from three separate experiments are presented. Student's t-test was used to determine if differences of PAI-2 mRNA between the parental cells and the transformed cells were significant (*P < 0.05). Bars, ±SD.

 
Alterations of TGF-ß and TNF-{alpha}
TGF-ß1 involves growth regulation of human keratinocytes and TNF-{alpha} is associated with stimulation of cancer cells. To test whether expression of TGF-ß1 or TNF-{alpha} is associated with the transformation process, mRNA levels of TGF-ß1 and TNF-{alpha} were measured by the RT–PCR technique. The transformed cells showed a 4-fold decrease of TGF-ß1 mRNA relative to the parental cells (4.5 ± 1.2; based on the results from the three separate experiments) (Figure 3Go). mRNA expression of TNF-{alpha} in the transformed cells was three times higher than in the parental cells (Figure 3Go). However, when the parental cells or the transformed cells were treated with TCDD for 24 h, neither of these cell lines showed any significant change of TGF-ß1 or TNF-{alpha} mRNA expression (data not shown).



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Fig. 3. Constitutive expressions of TGF-ß1 and TNF-{alpha} in the parental cells (lanes 1, 2 and 3) and the transformed cells (lanes 4, 5 and 6). Cells at 70% confluency were used for mRNA extraction.

 
Expression of CYP1A1, AhR and ARNT
Since CYP1A1 is the most sensitive gene in human keratinocyte in response to TCDD, we investigated CYP1A1 mRNA expression in the parental cells and the transformed cells to detect possible effects in the transformation process. The parental cells showed a dose-dependent increase of CYP1A1 mRNA following TCDD exposure for 24 h (Figure 4AGo). The maximum induction observed was at 10 nM. However, when the transformed cells were treated with TCDD for 24 h, the cells did not show induction of the gene except for doses of 100 nM, at which only a marginal increase was detected (Figure 4BGo). The basal level of CYP1A1 mRNA expression was similar between the parental cells and the transformed cells. Since induction of CYP1A1 gene is a AhR- and ARNT-mediated response, effects of AhR and ARNT mRNAs were analyzed by RT–PCR. Whereas induction of CYP1A1 mRNA by TCDD was dramatically different between the parental cells and the transformed cells, the analysis revealed that the basal levels of AhR or ARNT were similar between the two cell lines (Figure 5Go). In addition, there were no significant changes of AhR or ARNT mRNA levels in the parental cells that were treated with TCDD for 24 h (Figure 5Go). Western blot analysis was performed to measure the expression of Ah receptor at the protein level. This analysis also showed no significant difference of the basal AhR protein amount between the parental cells and the transformed cells (Figure 6Go).



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Fig. 4. Dose-dependent expressions of CYP1A1 mRNA in the parental cells (A) and the transformed cells (B). Cells at 70% confluency were treated with TCDD for 24 h. (1) 0.1% DMSO; (2) 0.1 nM TCDD; (3) 1 nM TCDD; (4) 10 nM TCDD; (5) 100 nM TCDD. A typical result of three separate experiments is shown.

 


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Fig. 5. Expressions of AHR and ARNT mRNAs in the parental cells and the transformed cells. The parental cells were treated for 24 h with 0.1% DMSO (1), 0.1 nM TCDD (2), 1 nM TCDD (3), or 10 nM TCDD (4). The transformed cells were treated with 0.1% DMSO only (5).

 


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Fig. 6. Immunoblot analysis of AhR protein in the parental (C) and TCDD-transformed cells (T). Samples were obtained from the cells at 70% confluency. Immunoblotting was performed as described in Materials and methods. The position of the molecular makers are indicated on the left.

 
Clonal variation of the transformed cells
To test whether effects observed in this study are dependent upon the clonal selection, the same analytical techniques were applied to two different clonal cells: one clonal cell line was isolated from the original foci detected after TCDD exposure and six subcultures, and the other was derived from solid tumor in nude mice following s.c. injection of the transformed cells. These cell lines share similar morphology and cellular properties as previously reported (8). The pattern of effects observed in these transformed cell lines was very similar to the transformed cells used in this study (data not shown). While it is possible that different clonal cells may exert different responses, no clonal variations on the effects were observed among the clonal cells examined in this study.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Whereas changes of growth regulatory factors with exposure to TCDD are reported in a variety of studies (1), it is not clear whether these changes are associated with TCDD-mediated carcinogenesis. Since TPA, the phorbol ester tumor promoter, is known to alter expressions of growth factors and cytokines in a fashion similar to TCDD and the alteration of expressions by TPA are correlated with tumor promotional activity (1921), it has been suggested that the altered regulation of growth regulatory factors, such as TGF-{alpha}, TGF-ß and PAI-2 by TCDD, may be associated with TCDD-mediated carcinogenesis (7). Therefore, this study has attempted to determine if alterations of growth factors or cytokines are associated with TCDD-mediated carcinogenesis of human cells by analyzing human epithelial cells malignantly transformed by TCDD.

PAI-2 is an efficient regulator of the urokinase-plasminogen activator (u-PA) system, which is involved in keratinocyte migration, proliferation and differentiation (22). Regulation of its expression is closely implicated with tissue degradation and cancer progression (23). PAI-2 is TCDD-responsive gene and its regulation by TCDD exposure has been well established in a human squamous carcinoma cell line (24). However, a link between carcinogenesis and TCDD-mediated regulation of PAI-2 expression remains unclear. This study demonstrated a significant reduction of PAI-2 expression in TCDD-transformed cells, suggesting that downregulation of PAI-2 expression may be involved in the transformation process. It is plausible that an imbalanced control of the u-PA/PAI-2 system may result from downregulation of PAI-2. This can lead to alteration of the PA/plasmin system, which may contribute to phenotypic changes of the parental cells. Whereas PAI-2 remained a sensitive target gene for TCDD exposure in the parental cells, the transformed cells did not show measurable increases of PAI-2 expression after TCDD exposure. Thus, it seems that loss of PAI-2 inducibility through clonal expansion rather than immediate induction after the exposure may be one of the genetic events responsible for the transformation. Further studies are warranted to identify the altered levels of u-PA and PAI-2 production in the transformed cells. The mRNA stability assay showed that PAI-2 mRNAs in the transformed cells were less stable than those in the parental cells. Thus, it is suggested that post-transcriptional modification in the process of cellular transformation may be a possible mechanism of downregulation of PAI-2 genes. Studies such as nuclear run-on transcription assay will be required in the future to examine the transcriptional regulation of the gene.

TCDD is known to increase TNF-{alpha} production and its toxicity is modulated by TNF-{alpha} in TCDD-treated mice (25). TNF-{alpha} stimulates growth of normal cells as well as certain cancer cells in culture (26). Its expression could contribute to tumor progression and spread. However, the role of TNF-{alpha} in TCDD-induced malignant transformation of human cells remains unknown. Whereas constitutively higher expression of TNF-{alpha} mRNA, as compared with the parental cells, was observed in the transformed cells (Figure 3Go), neither the parental cells nor the transformed cells showed induction of TNF-{alpha} mRNA level when they were exposed with TCDD for 1 day (data not shown). This finding suggests that TNF-{alpha} is not a TCDD-responsive gene in the current cellular system, but its modulation as a result of clonal expansion of the damaged cells may be associated with the transformation. Thus, it is speculated that modulations of growth factors and cytokines generally observed in the human keratinocytes after short-term exposure of TCDD may not be directly involved in phenotypic changes of the cells. However, they may contribute to triggering another set of genes that could be fixed in the genome after successive cell divisions of the damaged cells, thus ultimately being responsible for the transformation. A possibility of different genetic controls between responses after short-term exposure and cellular transformation has been previously reported in the present human cell system (8). AHH inducibility after short-term exposure of TCDD and a potency of TCDD-induced cellular transformation in the present cell system showed different patterns of dose–response, suggesting that these two events may be controlled by a different set of genes.

Members of the TGF-ß family are potent and reversible inhibitors of epithelial cell proliferation, including keratinocytes (28). Modulation of TGF-ß expression affects homeostasis of epithelia and is also associated with tumorigenesis (29). Unlike a human squamous carcinoma cell line, such as SCC-12F, which demonstrated altered mRNA expression of TGF-ß after TCDD exposure (30), our cell system did not show alteration of TGF-ß1 or TGF-ß2 expression after 24 h of exposure to TCDD (data not shown). However, constitutively, a lower level of TGF-ß1 mRNA was observed in the transformed cells. This finding suggests that while responses of TGF-ß after short-term exposure may not be associated with cell transformation, a substantial decrease of inhibitory action of TGF-ß1 following successive cell divisions may cause uncontrolled proliferation of epithelial cells, thus leading to tumorigenesis. Whether a reduced transcription of TGF-ß1 interferes with an autocrine inhibition of cell proliferation in the present cell system remains to be determined in the future. It is of particular interest to note that modulation of TGF-ß1 expression in the present cell system is dependent upon the transforming agents. The human papillomavirus-16-induced transformation increased TGF-ß1 mRNA, but the TCDD-induced transformation showed a decreased level of mRNA (31). In addition, a pattern of TCDD-induced transformation was different from that of the oncogene-derived transformation. Yang et al. reported a delayed appearance of the transformed foci in the present cell system with TCDD exposure as compared with K-ras transfection (8). Thus, it is plausible that TCDD-induced transformation in this cell system may take a transformation process different from other transforming agents.

Induction of CYP1A1 gene following TCDD exposure is the most-studied biochemical response (32,33). However, it remains controversial as to how the induction of this enzyme may lead to cancer or other toxic endpoints. While many CYP1A1 inducers, including dioxins, are carcinogenic in the long-term bioassays (34), induction of the enzyme following short-term exposure to dioxin can be, at least in part, protective against cancer by increasing the rate of detoxification of some carcinogens to a greater extent than it increases the rate of formation of DNA damaging metabolites (35). Thus, effects after short-term exposure may not be enough to explain the results after long-term exposure, such as the neoplastic transformation of the human cells. Compared with a dramatic increase of CYP1A1 mRNA in the parental cells following TCDD exposure, inducibility of the gene in the transformed cells was greatly dampened. In addition, the induced expression of the gene in the parental cells by TCDD was not observed in the transformed cells. These results suggest that the observed effects after short-term exposure, such as superinduction of the CYP1A1 gene, are not sustained during cell transformation and may not be directly involved in the carcinogenic process, which requires successive cell divisions. In contrast to the reduced inducibility of CYP1A1 in the transformed cells, both cell lines showed a similar level of AhR and ARNT expression. It is suggested that functional changes in the Ah receptor following clonal expansion may be associated with altered expression of growth regulatory factors or the CYP1A1 gene, as observed in this study. A gel shift mobility assay for DRE and AhR is required in the future. It is of particular interest to note that, whereas other in vitro systems demonstrated a rapid depletion of AhR levels following exposure to TCDD (36,37), the human cell system used in this study did not show a statistically significant decrease in AhR mRNA levels. The result suggests that there may be a tissue- and cell-type specificity in the regulation of the AhR gene.

While it remains unclear in this study whether the altered regulation of growth regulatory factors by TCDD caused the cell transformation, or that such altered regulation was made as a result of the cell transformation, the results indicate that altered regulation of cytokines and growth factors such as PAI-2, TNF-{alpha} and TGF-ß are some of genetic events fixed in the genome following the successive cell division of TCDD-damaged cells. Thus, it is suggested that dysregulation of growth regulatory factors that resulted from the clonal expansion of the damaged cells may be associated with a plausible mechanism of TCDD-induced carcinogenesis.

Multiple cell division is an essential component of chemical carcinogenesis. Without replication, DNA damage direct or indirect would not be fixed in the genome and the clonal expansion of genetically altered cells would not occur (38,39). Therefore, this study, which shows altered expressions of growth regulatory factors following the clonal expansion of the TCDD-damaged human cells, may help improve our understanding of the mechanism of TCDD-induced malignant transformation of human cells.


    Notes
 
2 To whom correspondence should be addressed Email: yangjh{at}cuth.cataegu.ac.kr Back


    Acknowledgments
 
The authors are grateful to Dr Linda S.Birnbaum, US EPA, for her critical review of the manuscript. This work was supported by a grant from Korean Science and Engineering Foundation #951-0709-096.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 19, 1998; revised June 8, 1998; accepted August 21, 1998.