* Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Institutes of Health, Bethesda, Maryland 20892;
Department of Biomedical Sciences and
Quantitative and Computational Toxicology Group, Center for Environmental Toxicology and Technology, Department of Environmental and Radiological Sciences, Colorado State University, Fort Collins, Colorado 80523
Received January 27, 2003; accepted April 17, 2003
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ABSTRACT |
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Key Words: arsenic; metal mixture; gene expression; cDNA microarray; real-time RT-PCR; human keratinocytes; RHEK-1 cells.
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INTRODUCTION |
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Although, based upon epidemiological evidence, As, Cd, and Cr are human carcinogens, the exact mechanisms involved in transformation by these agents are unclear. The primary target for As in exposed individuals is the skin; in fact, squamous and basal cell carcinomas have reached epidemic proportions in countries with As-contaminated water supplies (Chen et al., 1985; Tseng et al., 1968
). Studies designed to characterize the transforming potential of As have historically proven difficult, as the metal has generally failed to act in as a carcinogen in skin model systems. In recent years, several investigators were able to demonstrate that As can act as a carcinogen or copromoter in defined skin carcinogenesis protocols in mice (Germolec et al., 1998
; Ng et al., 1999
). In vitro, although both As and Cr, a well-known skin sensitizer, have substantial effects on growth and differentiation of epidermal keratinocytes (Germolec et al., 1996
, 1997
; Ye et al., 1995
), they have not acted to directly transform this cell type under culture conditions (Bae et al., 2002
). That transformation by arsenic is cell-type specific is evident in the fact that the metal acts as an anticancer agent for some hematopoietic malignancies (Achanzar et al., 2002
; Zhang et al., 1998
). Clearly, the intracellular milieu, dictated by gene expression patterns, is extremely important in the ability of As to participate in the transformation process. Characterization of the relationship among As exposure, gene expression alterations, and transforming potential of the metal under different conditions should help in delineating important molecular events that are mechanistically linked to the carcinogenic process.
New technologies in expression analysis at the RNA and protein levels have led to the development of the field of toxicogenomics, i.e., the use of genetic information to address issues that are crucial in toxicology. Microarray analysis provides the advantage of being able to investigate expression of thousands of genes simultaneously in chemically treated versus control cells; thus enabling one to gain a more complete view of batteries of genes affected by exposure. When combined with cluster analysis, the qualitative effect of a chemical on multiple molecular pathways can be determined. One limitation to the microarray technology, however, is that it is less sensitive and quantitative than the traditional technologies that it has replaced. Both RT-PCR and Northern analysis allow for the relative quantification of expression of individual genes. Therefore, studies of global gene expression using microarrays are best if used in conjunction with these other more highly quantitative technologies.
Advances in polymerase chain reaction (PCR) instrumentation and use of fluorescent labels for detection of amplification products have facilitated several approaches to real-time RT-PCR quantification of input target copies. In particular, hot-start PCR methodology with the glass capillary format of the LightCycler has been used for amplification and quantitative detection of a single copy of bacterial DNA of Chlamydia spp. (Huang et al., 2001a). Rajeevan et al. demonstrated that real-time RT-PCR based on LightCycler technology is well suited to validate DNA array results because it is quantitative, rapid, and requires 1000-fold less RNA than conventional assays (Rajeevan et al., 2001
). Real-time RT-PCR has been used to confirm the results of microarray analysis on global gene expression in a variety of systems (Huang et al., 2001b
; Shultz et al., 2001
; Waters et al., 2001
).
We describe here an extension of a previous work comparing microarray analysis with real-time RT-PCR on a defined subset of genes altered in their expression by transformation enhancing and inhibiting chemical treatments in immortalized human keratinocytes.
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MATERIALS AND METHODS |
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Cell culture.
The AD12/SV40 immortalized keratinocyte cell line (RHEK-1) was originally obtained from Dr. J. Rhim (Center for Prostate Disease Research, Rockville, MD; Rhim et al., 1985). RHEK-1 cells and derived malignant populations were cultured in Dulbeccos Modified Eagles medium supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 10 mM L-glutamine, and 10% fetal bovine serum (Summit Biotechnology, Ft. Collins, CO).
Cell line establishment following exposure to MNNG, As, or As-containing mixture.
The detailed protocol used to obtain the RHEK-1 cell lines utilized in these studies is described in Bae et al.(2002). The RHEK-1 cell lines were designated as follows: (1) OM1: control cell population treated with 0.5% DMSO for 24 h and then cultured for 6 months; (2) OM3: cells treated with 0.1 µg/ml MNNG for 24 h and then cultured for 6 months; (3) As-Con: control population treated with sterile distilled water for 6 months; (4) As-High: cells treated chronically with 14 ppb As3+ for 6 months; (5) Mix-Con: control population treated with sterile distilled water for 6 months; and (6) Mix-High: cells treated with 14 ppb As3+, 104 ppb Cr3+ and 6+ (1:1), 618 ppb Cd2+, and 332 ppb Pb2+ for 6 months. These metal concentrations correspond to the LC10 for each metal as obtained by the MTT assay (Bae et al., 2001
). All control and test cultures were passaged at simultaneous time points and, other than for chemical exposure, were treated in an identical manner.
Real-time RT-PCR.
The real-time RT-PCR protocol was as adaptation of a previously published protocol (Solum and Handa, 2002). RNA from OM1 and OM3 cells at p2, p7, p13, p16 was analyzed. In order to eliminate one potential source of variability between the microarray and real-time RT-PCR, the same RNA samples from the different RHEK-1 populations established in Bae et al.(2002)
were utilized for both types of analysis. Thus, the gene expression data generated from these two methodologies can be directly compared. To ensure consistency across RNA samples from the same populations and to eliminate the complicating factors of cell density and other culture conditions, multiple flasks of cells from each control and treatment group were grown to approximately 70% confluency and were pooled prior to RNA preparation. For real-time RT-PCR analysis, 23 separate reverse transcriptase reactions were carried out on each pooled RNA sample to produce 23 individual cDNAs for further amplification. The genes analyzed by real-time RT-PCR and the primers utilized are shown in Table 1
. The sequences for gene-specific primers corresponding to the PCR targets on the Atlas Human Cancer 1.2 Array were obtained from Clontech. Primers themselves were synthesized and HPLC-purified by Genset (La Jolla, CA). External DNA standard was synthesized for each gene of interest by conventional PCR and the appropriate primer set. PCR products were run on an electrophoresis gel and purified using the Ambion spin columns (Austin, TX). The concentration of purified PCR product was determined by absorbance at 260 nm and was used to make a set of standard dilutions. The quantitative measurement of target transcript in chemically-treated and control cells was measured in a LightCycler (Roche, Indianapolis, IN) with FastStart DNA Master SYBR Green I and a hot-start PCR method as follows and as shown schematically for a representative DOC2 gene in Figure 1A
. Briefly: (1) A standard curve for each primer pair was created using the five to seven serially diluted known amounts of cDNA product; (2) from this standard curve, the absolute concentration of the unknown gene was determined; and (3) a subsequent melting curve analysis and length verification by gel electrophoresis were carried out to confirm the specificity of PCR products. As a negative control, template cDNA was replaced with PCR-grade water. As demonstrated in Figure 1A
, from our studies on the DOC2 gene, the estimated absolute amount of the DOC2 gene in the p25 OM1 sample was 36.5 fg which corresponds to a crossing point (Cp) of 20.9, that is the cycle number at which the amplified products of all the standards and unknowns emit the same fluorescence above the threshold/noise band (i.e., located at 0.5 in Fig. 1A
). A representative melting curve for the DOC2 gene is shown in Figure 1B
. The specific DOC2 product melts at 86°C, while the primer dimer melts at 80.5°C.
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RESULTS |
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In MNNG-treated OM3, the genes selected were interferon (IFN) inducible protein 927, melanoma associated antigen A32 (MAA A32), and the colon carcinoma laminin binding (CCLB) protein genes (which were all induced ≥ fourfold) and integrin ß4 (which was downregulated). No detectable changes in these four genes were observed in As- or mixture-treated RHEK-1 cells (data not shown). The genes chosen for further analysis from As-High, were DNA repair protein XRCC 1 (which was substantially upregulated in treated versus control cells) and those encoding the simple epithelial keratins 8 and 18 (K8 and K18); these latter two genes were representative of the many cytokeratins suppressed in As-High populations. There were no measurable changes in expression of these three genes in OM3 and mixture-exposed cells. As would be expected, RHEK-1 cells exposed to the four metal mixture (which included Cd) demonstrated increased mRNA levels for metallothionein 3 (MT 3). We chose this gene for further analysis and the gene encoding the important mitogenic signal transduction protein, mitogen activated protein kinase kinase 6 (MAPKK 6) as a representative of repressed genes in this cell population.
Pooled results from multiple real-time RT-PCR analyses on the nine genes selected from the chemical-specific gene expression profiles are shown in Table 2. Careful scrutiny of this data revealed that in approximately half of the cases, gene-specific comparative analysis between the three chemically treated samples and their respective controls gave very consistent absolute RT-PCR values in all samples analyzed, with SE ≤ 20%. In the other instances, however, while a consistent induction or suppression was observed in all three replicates from a treatment condition, the absolute fold alteration in expression varied by >20%. Table 2
also demonstrates for comparison, the alterations in expression of these genes in the three chemically treated RHEK-1 populations as measured initially by microarray analysis. From a qualitative standpoint, both types of analysis gave comparable results for these specific genes, that is, genes were consistently induced or suppressed under the different treatment regimens when measured by either technique. The absolute level of change in expression measured by microarray versus real-time RT-PCR varied in our hands by a maximum of approximately fourfold, with, in most cases, the values of induction or suppression being larger by microarray analysis.
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DISCUSSION |
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In OM3 versus OM1, the genes that were analyzed by real-time RT-PCR were the IFN inducible protein, integrin ß4, MAA A32, CCLB, and the DOC2 genes in both p25, when the OM3 cells were fully transformed, and in earlier passages in our time-course study. Of these, the MAA A32, CCLB protein, and DOC2 genes were regulated in a time-dependent manner consistent with a role in transformation. The more rapid induction of these genes in OM3 as compared to OM1 and/or the higher levels of expression in the more malignant cells at p25 suggest that these genes may be markers of the malignant process. Our findings are consistent with work from other laboratories on the MAA A32 and CCLB protein genes. MAA A32 is a marker of tumor progression in human melanoma (Lehmann et al., 1989). The CCLB protein is a mitotic phosphoprotein that is upregulated in cancer cells in correlation with their metastatic phenotype (Jackers et al., 1996
). Time-dependent changes in expression of the DOC2 gene in OM1 and OM3 suggest that overexpression might be a late event involved in acquisition of characteristics that support tumor formation in mice. Previous studies have linked the DOC2 gene to activation of the cell cycle (Xu et al., 1995
). Overexpression of DOC2 may reflect the aggressive growth characteristics of late passage, and tumorigenic, OM3 cells.
Although the genes for integrin ß4 and IFN-inducible protein were also altered in both OM1 and OM3 in a time-dependent fashion, these expression patterns indicated that neither gene is likely involved in malignant progression in RHEK-1. Decreased expression of integrin ß4 has previously been implicated in tumor cell growth and metastasis (Gille and Swerlick, 1996). It is not clear why expression of this gene increases in OM1 with time in culture, while the cells are spontaneously gaining more characteristics of the malignant phenotype. Likewise, in OM3, substantial suppression of the integrin ß4 gene occurs between p2 and p16; at p25, however, when the cells are their most tumorigenic, expression of the gene returns to basal. The IFN-inducible protein gene, while not significantly altered over the time course in OM3, decreased in OM1 with continued time in culture. The significance of our findings concerning this gene is unclear, as previous studies suggested that its protein product, the Leu-13 antigen, is often overexpressed in malignant conditions (Evans et al., 1990
).
Genes further analyzed in As and the metal-mixture treated RHEK-1 populations were XRCC1, K8, and K18 for As-High and MT3 and MAPKK6 for Mix-high cells. The human XRCC1 gene product is involved in DNA repair (Thompson et al., 1990); induction of this gene in cells treated with As may well be the normal response to the clastogenic activities of the metal. The two simple epithelial cytokeratins, K8 and K18, are highly expressed in several epithelial cancers and, are only expressed in keratinocytes under abnormal conditions (Trask et al., 1990
; Welsh et al., 2001
). Their suppression in As-treated RHEK-1 cells is consistent with the observed inhibition of spontaneous cell transformation. Induction of MT3 expression in Mix-High populations was not surprising due to the presence of Cd in the mixture (Hellemans et al., 1999
). MAPKK6 is involved in activation of the p38 MAP kinase signal transduction pathway (Raingeaud et al., 1996
) and has been linked to cellular transformation (Chiariello et al., 2000
). Repression of MAPKK6 in Mix-High cells correlates with the poor tumorigenic potential of this cell population.
We were highly interested in genes that were altered in RHEK-1 by more than one chemical treatment. These genes, if expressed in a manner consistent with a role in transformation, i.e., oppositely regulated by MNNG, which enhances transformation, and the As/mixture treatments, which are inhibitory to the process, may potentially provide us with candidate molecular markers for malignant conversion. In our microarray studies, only the MIC1 gene was found to be expressed at high levels in OM3 and repressed in As-High and Mix-High. The MIC1 gene encodes a macrophage inhibitory cytokine; higher expression of this gene has recently been demonstrated in advanced and more aggressive prostatic tumors than in noncancerous tissues (Nakamura et al., 2003). Although it is unclear exactly how MIC1 may be involved in transformation, the protein is a divergent member of the TGFß superfamily. Many members of this family are highly active in keratinocytes (Bootcov et al., 1997
). Transformed cells often express TGFß; production of the cytokine by resistant tumor cells could potentially give them a substantial growth advantage over surrounding normal cells (Fynan and Reiss, 1993
).
Four genes, bikunin, MTS1, BMP4, and RAD23A were all significantly repressed in OM3 and induced in the populations derived from As or metal mixture treatment. Evidence would support a potential role for any of these genes in inhibition of the carcinogenic process in keratinocytes. Reduced expression of the bikunin gene has been implicated in progression of human glioblastoma (Hamasuna et al., 2001). MTS1 (p16-INK4) is an inhibitor of cyclin/cyclin-dependent kinase complexes (Luca et al., 1995
). Under normal conditions, the MTS1 gene is involved in induction of the growth arrest that accompanies terminal differentiation in keratinocytes; squamous cell carcinoma lines are often defective in induction of MTS1 in response to differentiating agents (Harvat and Jetten, 2001
). The gene is deleted or mutated with high frequency in human melanoma cell lines and familial melanoma patients (Luca et al.,1995
). The role of TGFß-like BMP4 in carcinogenesis is not yet defined. One function, however, suggested by Glozak and Rogers, is that BMP4 may induce apoptosis in some cell types (Glozak and Rogers, 1996
). Members of the BMP family, particularly BMP2 and BMP6, are important regulators of keratinocyte growth and differentiation. Altered expression of these proteins has been demonstrated in cancers of epidermal origin (Jin et al., 2001
). The remaining gene, RAD23A or HHR23A, is a nucleotide excision repair protein whose normal function is important for prevention of skin cancer (Masutani et al., 1994
).
The vimentin gene, along with the DOC2 gene as discussed above, was induced in OM3 and As-High populations, but repressed in cells treated with the mixture. Overexpression of vimentin, an intermediate filament protein, is frequently observed in cancer cells and correlates with malignancy (Dandachi et al., 2001). There are several possible explanations for the expression pattern we observe with the DOC2 and vimentin genes: (1) these two genes are unrelated to the process of transformation in RHEK-1 and their altered pattern of expression is the indirect result of other changes in chemically-treated cells; (2) expression of the gene(s) may be associated with, but not sufficient for malignant conversion, and other gene products present in OM3 are necessary for expression of the tumorigenic phenotype; or (3) other proteins present in As-H cells interfere in the process of transformation either through direct inhibition of DOC2 or vimentin activity or indirectly by blocking other steps in the pathway.
The overall goal of our studies is to identify potential target genes involved in carcinogenic activity of environmentally relevant chemicals in keratinocytes. To this end, we have utilized cDNA microarray analysis and real-time RT-PCR validation to correlate gene expression changes in chemically treated RHEK-1 cells with time-sensitive acquisition of transformation-associated characteristics. Presently, we are expanding our effort to study gene expression profiles of additional carcinogenic and noncarcinogenic chemicals in RHEK-1. Once candidate genes have been identified, mechanistic studies will be required to define the role of these genes in transformation. As RHEK-1 cells spontaneously transform in culture, one possibility is that the same "cancer-associated" gene expression alterations will eventually occur in chemically treated and control populations, albeit at very different rates. These alterations could read-out as perturbations in a common growth or differentiation pathway in keratinocytes such as those involving ras or protein kinase C. Alternatively, strong mutagens such as MNNG, by nature of their mode-of-action, could induce different gene expression changes each time. Control cells could either be genetically unstable and also undergo a variety of carcinogenic changes each time the cells are allowed to transform or could be altered along the same pathway each time, both possibilities would likely be due to viral infection and abrogated function of key tumor suppressor genes. These different possibilities will all be amenable to further study. This work should aid us in our pursuit of reliable toxicity markers for use in computational risk assessment approaches.
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ACKNOWLEDGMENTS |
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NOTES |
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