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,**
,3,,
*
North Shore-Long Island Jewish Research Institute, Manhasset, NY 11030;
Department of Otolaryngology, Long Island Jewish Medical Center, The Long Island Campus of Albert Einstein College of Medicine, New Hyde Park, NY 11040;
**
Biological Sciences, St Johns University, Jamaica, NY 11530;
Department of Radiation Oncology, Long Island Jewish Medical Center, The Long Island Campus of Albert Einstein College of Medicine, New Hyde Park, NY 11040; and
Developmental and Molecular Biology and
Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461
4To whom correspondence should be addressed. E-mail: kauborn{at}nshs.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX 1APPENDIX 2LITERATURE CITED |
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KEY WORDS: • diindolylmethane • apoptosis • humans • DNA microarray • human papilloma virus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX 1APPENDIX 2LITERATURE CITED |
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,2
), I3C, DIM and diets rich in cruciferous vegetables (e.g., broccoli, cabbage) are being used clinically for prevention and treatment of the human papillomavirus (HPV)-induced pathologies such as laryngeal papillomas (3
–5
), which are associated with HPV types 11 and 6, and precancerous lesions of the cervix (6
), which are associated with many HPV types, most commonly type 16. I3C and DIM initiate a variety of cellular responses incompatible with tumors. Although I3C and DIM are antioxidants and could in theory protect against the deleterious effects of biologically active electrophiles and free radicals (7
,8
), much of the prophylactic effect of I3C and DIM for cancer can be ascribed to their ability to induce a variety of enzymes that detoxify carcinogens. Induction of a number of Phase I and Phase II enzymes occurs via the aryl hydrocarbon receptor (AhR), for which DIM is a weak ligand (9
–12
). Some of the Phase I enzymes alter the metabolism of estrogen (13
–15
) and thus affect the establishment and progression of estrogen-responsive tumors. In addition to inducing the AhR battery of enzymes, I3C (or some in vitro conversion product of I3C) is also a ligand for the estrogen receptor (15
), and I3C has been shown to be a negative regulator of estrogen signaling in cell culture (16
). This ability to alter estrogen effects has been the major rationale for the use of I3C/DIM for treatment of papillomavirus-induced lesions (1
,17
,18
).
More recently, I3C and DIM have been shown to decrease proliferation and induce apoptosis of cervical (19
), prostate (20
) and breast cancer cells, independent of estrogen signaling (21
,22
). I3C also suppresses invasion and migration of breast cancer cells (23
,24
). Thus, I3C and DIM appear to affect multiple, disparate cellular pathways, many of which probably involve the modulation of gene expression. Cover et al. (21
) determined that I3C down-regulates cyclin-dependent kinase (CDK)-6 transcriptionally, and recent studies indicate that this requires the Sp1 binding site and an adjacent region in the CDK-6 promoter (25
). Considering that I3C is being used clinically for HPV-related tumors (3
–6
) and is being investigated for prevention of breast cancer (26
), it is critical to determine which genes have altered expression in response to this agent. It is also important to determine whether the effect of DIM on gene expression depends upon cell type or other relevant factors such as the presence and expression of viral genes.
We have used microarray profiling to determine which genes are up- or down-regulated by DIM in cervical cancer cells, which are derived from keratinocytes in the cervical epithelium, and whether expression of these genes is also affected in an immortalized epithelial cell line and in normal primary human keratinocytes. DIM was selected because it is a major condensation product to which I3C is converted in the stomach and it is a biologically active compound in vitro and in vivo (19
,27
). By contrast, I3C is apparently devoid of activity when injected intravenously (28
), requiring conversion to active molecules such as DIM by acid-catalyzed condensation reactions that occur rapidly in the stomach and slowly in cell culture (29
). Consequently, in cell culture, the effects of I3C and DIM are indistinguishable except that DIM works more quickly and at a lower concentration that I3C (19
). We therefore used DIM to obtain rapid responses in microarray profiling experiments, but used both DIM and I3C in experiments that validated these results and characterized potential biological consequences of these responses.
Many of the same genes coding for bZip proteins and additional proteins involved in signaling, stress response and growth were transcriptionally altered by DIM in all keratinocyte cell types investigated. To explore the significance of some of the changes in gene expression, we examined the consequences of the up-regulation of GADD153, the protein most robustly induced by DIM, for HPV expression because of its potential interaction with other bZip proteins known to regulate HPV11 (30
–38
). Our results confirm that DIM can suppress expression of HPV oncogenes by down-regulating transcription from the viral upstream regulatory region (URR) and that a potential mechanism for this effect involves induction of GADD153.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX 1APPENDIX 2LITERATURE CITED |
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I3C was purchased from Sigma Chemical (St. Louis, MO). DIM was a gift from Dr. M. Zeligs, BioResponse, Boulder, CO. Plasmids containing sequences from 36B4 and HPV16 E6E7 in pGem4Z have been described previously (32
). The firefly luciferase reporter gene driven by the HPV11 viral E6 promoter was obtained from B. Steinberg (Long Island Jewish Medical Center, New Hyde Park, NY). The Renilla luciferase expression vector driven by the herpes simplex virus-thymidine kinase (HSV-TK) promoter was purchased from Promega (Madison, WI). Expression vectors driven by the cytomegalovirus promoter were from S. Akira, Osaka University, Japan for nuclear factor-interleukin 6 (NF-IL6) and A. J. Fornace, National Institutes of Health, Bethesda, MD for GADD153.
Cell lines and cell culture.
The cervical cancer cell lines CaSki (containing multiple copies of integrated HPV16 DNA), C33A (HPV negative, mutant p53), and HaCat (a spontaneously immortalized, p53-negative, nontransformed human epithelial cell line) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). All cells were maintained as monolayer cultures at 37°C, 7% CO2, in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L glucose and bicarbonate (GIBCO-BRL, Gaithersburg, MD), supplemented with 110 mg/L sodium pyruvate, 200 mmol/L glutamine, 100g/L fetal bovine serum and 1 x 105 6U/L each of penicillin and streptomycin. For selected experiments, charcoal-stripped fetal bovine serum was used. Normal human foreskin keratinocytes (HFK), from explants of foreskin (surgical discards from circumcisions) were grown in F12-DMEM on feeder layers by the method of Rheinwald and Green (39
) as described previously (32
).
Gene chip hybridization.
Cells were treated with 100 µmol/L DIM in dimethyl sulfoxide (DMSO), the equivalent amount of DMSO or left untreated for 6 h unless specified differently. The oligonucleotide microarray hybridization used the HG-U95a gene chip from Affymetrix (Santa Clara, CA) containing 12,000 known genes and expressed sequence tags (EST). Reagents and procedures were those recommended by Affymetrix. Briefly, total RNA was prepared using reagents from Qiagen (Valencia, CA) followed by cDNA synthesis using a T-7 linked oligo (dT) primer (regents from GIBCO BRL, Grand Island, NY). Amplification of cDNA used biotinylated UTP and CTP (Bioarray High Yield Transcript Labeling kit from Enzo Diagnostics, Farmingdale, NY), followed by fragmentation into 50–150 nt oligomers and hybridization to the microarray. Hybridizations were scanned using Affymatrix equipment and software.
Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR).
Semi-quantitative RT-PCR for the analysis of GADD153 mRNA was conducted as described previously (40
). Briefly, RNA was isolated from treated and untreated cells using TriPure reagent (Boehringer Mannheim, Indianapolis, IN) following the manufacturer’s instructions. Extracted RNA was treated with DNase and further purified by phenol/chloroform extraction. Total RNA (5 µg) was reverse transcribed using Superscript II (GIBCO). Finally, aliquots of cDNA corresponding to 0.5 µg of original RNA were used for PCR amplification in a Perkin-Elmer DNA thermal cycler. The cDNA was first denatured for 3 min at 94°C, then amplified using cycles of 1 min at 94°C, 2 min at 50°C, and 2 min at 72°C, with a final 7-min incubation at 72°C. The sequences of Gadd153 PCR primers were 5'-CCA ACT GCA GAG ATG GCA GCT GAG-3' (forward); 5'-GCA GTC AGC ACC GAG ACA GCT-3' (reverse) and the expected size of PCR products was 600 bp. The PCR products were visualized on a 2% agarose gel containing ethidium bromide.
Western analysis.
Cells treated with I3C, DIM or vehicle controls were lysed at room temperature in buffer containing 10 mmol/L NaH2PO4, 20 g/L Triton X-100, 12 g/L SDS and 10 g/L dissolved organic carbon, supplemented just before use with 2 µmol/L aprotinin, 100 µmol/L phenylmethylsulfonyl fluoride and 1 mmol/L EDTA, boiled for 2 min, and centrifuged for 10 min at 12,000 x g at 4°C. Supernatants were stored at -80°C until use. Extract protein (100 µg) in sample buffer (125 mmol/L Tris-HCl, pH 6.8, 10 g/L SDS, 20 g/L ß-mercaptoethanol and 0.01% bromophenol blue) was loaded onto a 12% SDS-polyacrylamide gel. After electrophoresis at 32 V for 3 h at room temperature, protein bands were transferred to an Immobilon-P membrane from Millipore (Bedford, MA) by electroblotting overnight in Transfer Buffer (192 mmol/L glycine, 25 mmol/L Tris and 200 g/L methanol). Before incubation with antibodies, the membrane was blocked with TBST/milk (20 mmol/L Tris-HCl, 137 mmol/L NaCl, 15 g/L nonfat dry milk and 1 g/L Tween20, pH 7.6) for 1 h. GADD153, NF-IL6 and AFT3 were detected with specific rabbit polyclonal antibodies from Santa Cruz Biotechnology (Santa Cruz, CA) (diluted 1/500–1/1000) for 1 h. After washing in TBST/milk, the filters were incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody (Santa Cruz) at 1/2000 dilution for 1 h at room temperature. Antibody bound to protein was detected using the enhanced chemiluminescence system (Amersham LIFE Science, Piscataway, NJ). Protein concentration was measured with the MicroBCA kit (Pierce, Rockford, IL) using a bovine serum albumin standard.
Luciferase assays.
Subconfluent proliferating cells plated in 24-well culture dishes were cotransfected with experimental firefly luciferase reporter constructs driven by the HPV11 E6 promoter, expression vectors for GADD153 or NF-IL6 (selected experiments) and a Renilla luciferase reporter internal control construct driven by the HSV-TK promoter. Transfections were done using lipofectant (GIBCO) according to manufacture’s instructions. For experiments with DIM, transfection medium was changed after 8 h to medium with DIM or DMSO, and luciferase was measured 16 h later. This abbreviated transfection time was necessary to minimize the reduction in overall transfection efficiency resulting from apoptotic cell death induced by DIM (19
). For experiments with bZip expression constructs, the medium was changed 24 h after transfection and luciferase assayed 24 h later. Luciferase was assayed using a Turner Design 20/20 Luminometer. Experiments were done in triplicate and multiple times. The luminescence emitted by the firefly luciferase was normalized to that of Renilla luciferase to correct for variation in transfection efficiency and for cell death as a result of DIM treatment.
RNase protection assay.
Total RNA was isolated using RNA STAT-60 Kit (TelTest, Friendswood, TX). Before analysis, RNA samples were treated with RNase-free DNase I to eliminate any contaminating DNA. The RNase protection assay used in this study was described previously (15
). Briefly, 10 µg of total cell RNA was hybridized simultaneously to a 32P-labeled HPV16 E6E7 antisense RNA fragment (nt115–nt882) and to labeled antisense RNA of the 36B4 gene (nt761–nt956) generated by in vitro transcription of these sequences cloned into the pGEM 4Z plasmid. After treatment with RNase T1, the remaining hybridization products were separated by electrophoresis on 5% acrylamide/8mol/L urea gels. The in vitro transcription and RNase protection assays used reagents and conditions of the Ambion MAXIscript and RNase Protection kits (Ambion, Austin, TX).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX 1APPENDIX 2LITERATURE CITED |
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As part of a comprehensive investigation of the effects of DIM on gene expression, we have begun DNA microarray profiling of cervical cancer cells lines, immortalized epithelial cells and primary human keratinocytes, evaluating the earliest observable effects of DIM on gene expression. Using the Affymetrix oligonucleotide microarray analysis system, we evaluated changes in gene expression in keratinocyte cell lines and in HFK after treatment with 100 µmol/L DIM, a concentration chosen to ensure an acute response in as many cells as possible (19
). We used C33A (without HPV transcripts) cells as a reference standard. Other analysis included CaSki cells (expresses HPV transcripts), HaCat, (a spontaneously immortalized human epithelial keratinocyte cell line that retains the capacity for differentiation) and HFK (normal human foreskin keratinocytes).
Multiple analyses were done after treating C33A cells with DIM for 6 h (Table 1
and Appendix 1
and 2
). Separate experiments using the oligonucleotide array compared treatment times of 4 and 12 h. Duplicate analyses were done on CaSki cells, HaCat cells and HFK, each treated for 6 h with DIM. All analyses used the Affymetrix microarray with >12,000 human sequences. In each case, we compared treated and untreated (DMSO solvent control) cells. The analysis software calculates the mean and SD of the intensity of the entire probe set for each gene excluding the highest and lowest values. Filter criteria for data analysis included a requirement that mRNA be called "present" and its expression level >200 (the defined threshold recommended for analysis) in at least one of the samples (treated or untreated). For comparison, in untreated cells, the expression level for the ribosomal protein gene L37a was 
105, whereas expression of the gene for the tumor necrosis factor receptor-associated protein TRADD was 500-1000. We focused our attention on genes whose expression was changed >1.8-fold relative to the untreated, solvent-control cultures. As an additional filter criterion, we looked only at those genes with sort scores >2.0 in the Affymetrix algorithm (similar to a measure of relative confidence). Briefly, the sort score is based on the fold change and the average difference change (used to determine the change in hybridization intensity between different experiments).
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, Appendix 1
). Results were comparable between C33A cells (without HPV sequences) and CaSki cells (with many copies of HPV16). As shown in Table 1
, a prominent subset of genes that included some of those most highly induced by DIM consisted of leucine-zipper (bZip) transcriptional regulatory proteins. GADD153 was induced >50-fold in C33A by 6 h, NF-IL6 (c/EBPß) >5-fold, c/EBP
nearly 5-fold, ATF3 >16-fold, c-Jun >9-fold, E4BP4 >3-fold, Nrf2 >2-fold and JunD >2-fold. The same set of bZip proteins was induced in HaCat cells and in normal keratinocytes by a 6-h exposure to DIM.
A major difference between the two tumor-derived cell lines vs. nontumor-derived cells was the induction of phase I enzyme transcripts for CYP1A1 and CYP1B1 by DIM (Table 1
, Appendix 1
). Transcripts for these genes were the most highly induced by 6 h of DIM treatment in HaCat and HFK, whereas these transcripts were not induced in C33A or CaSki cells by DIM exposure up to 12 h. As expected, comparatively few genes exhibited decreased transcript abundance after the short exposure to DIM (Appendix 1)
because mRNA stability would be expected to mask the effects of transcriptional inhibition. In keeping with this expectation, among the transcripts showing consistent decreases after 6 h of DIM exposure were those encoding cell cycle-related proteins with a short half-life such as cyclins D1 and F, and proliferating cell nuclear antigen (PCNA) and certain proteins involved in mRNA processing and function. Somewhat surprisingly, transcripts for cytoskeletal proteins, especially the tubulins, were consistently among those showing the largest decreases after DIM treatment. However, levels of at least some of these proteins did not change appreciably on Western blots (Figs. 1
and 2
), consistent with a protein half-life considerably longer than 6–12 h.
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We wanted to evaluate the potential functional importance of the up-regulation by DIM of some of the genes whose expression was most dramatically altered. Because DIM induced NF-IL6 (c/EBPß), GADD153, ATF3 and a variety of other bZip proteins in all keratinocyte cell lines tested, we hypothesized that DIM (and, consequently, I3C) might directly affect transcription of HPV oncoproteins. We confirmed the up-regulation of several of these proteins, and have investigated a potential functional consequence of this change specifically with respect to GADD153 induction.
Agreement between multiple independent experiments, different time points and different cell lines itself constitutes a qualitative validation of the microarray profiling results. Because DIM is a major bioactive form of I3C (19
), we predicted that I3C would also induce the same genes in keratinocytes. We therefore tested whether I3C could induce GADD153, using semiquantitative RT-PCR (Fig. 1
A, B). Western analysis confirmed that specific protein content correlated with increased transcription of GADD153 (Fig. 1
C, D). Induction of GADD153 by I3C was both time and dose dependent. At the highest I3C concentration (300 µmol/L), increases in both RNA and protein could be detected as early as 4 h after addition of I3C to the cell cultures. We also validated the microarray profiling results by Western blot of extracts from DIM-treated cells. Induction of GADD153 by 100 µmol/L DIM was confirmed by Western blot after a 12-h treatment of C33A, HaCat and CaSki cells (Fig. 2
A). ATF3 and NF-IL6 protein levels were also robustly increased by DIM in both CaSki and C33A cells (Fig. 2
A). As shown in C33A cells, the very robust rate of induction of GADD153 was not maintained as was the case for NF-IL6 and ATF3 (Fig. 2
B). NF-IL6 was only slightly elevated in DIM-treated HaCat cells despite a twofold increase in transcript (Table 1
). This was most likely related to the high basal level of this protein in the untreated cells (Fig. 2
A). In contrast to the bZip proteins, actin and tubulin content did not change drastically after a 24-h treatment with I3C (Figs. 1
, 5
) or 12 h treatment with DIM (Fig. 2
A).
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Both RNase protection and reporter assays were used for analysis of HPV expression in cells exposed to either DIM or I3C. Using a reporter gene driven by the HPV11URR, we determined that DIM did indeed down-regulate expression from this HPV promoter in C33A, CaSki, HaCat and HFK cells (Fig. 3
). In contrast, DIM did not reduce transcription driven by the TK promoter (Fig. 3
, insert). To test whether DIM treatment could reduce expression of HPV genes in virally transformed cells, we used RNA from CaSki cells treated with I3C, which is slowly converted to DIM in vitro, in an RNase protection assay (Fig. 4
). In this case, HPV16-transformed cells (e.g., CaSki) had to be used, because of the absence of tumor-derived cell lines that express HPV11. In CaSki cells as expected, I3C had a minimal effect on the level of the ribosomal phosphoprotein 36B4, which is known to be regulated post-transcriptionally. This approximately twofold reduction in 36B4 RNA is consistent with the observation that exposure of cells in high concentrations of I3C results in general cytotoxicity. In contrast to 36B4, transcription of HPV16 was completely repressed by I3C. This result was not due to abrogation of the known estrogen enhancement of viral gene expression by I3C (15
) because the same result occurred in medium with charcoal-stripped serum. Thus, either I3C or DIM can decrease transcription from the URR of at least two HPV types in four different cell lines, which is consistent with a general effect on HPV transcription by I3C or its conversion products, including DIM.
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We next evaluated the effect of GADD153 and NF-IL6, both induced by DIM, on expression driven by the HPV11 URR (Fig. 5
). Previous studies have shown by a number of criteria that NF-IL6 suppresses HPV (30
,32
), and GADD153, the gene most highly induced by DIM, would also be likely to affect HPV oncogene expression, because GADD153 heterodimerizes with members of both the c/EBP and AP1 families of proteins (40
,41
). Expression constructs of NF-IL6 or GADD153 were cotransfected into cervical cancer together with the reporter construct driven by the HPV11 URR (Fig. 5
A, B). As expected, NF-IL6 decreased expression from the HPV11 URR (Fig. 5
A). GADD153 also decreased expression in C33A cells (Fig. 5
A, B). In this case, suppression of HPV11 transcription was accompanied by expression of GADD153 protein in the transfected C33A cells (Fig. 5
B, insert), as determined by Western blot analysis in parallel with luciferase expression assays. Ectopic expression of GADD153 also suppressed HPV11 transcription in CaSki, HaCat and HFK cells (Fig. 5
C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX 1APPENDIX 2LITERATURE CITED |
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This study was undertaken to determine how DIM alters gene expression in keratinocytes. It has been established that I3C and DIM affect many biochemical pathways. These compounds have also been shown to be beneficial in the treatment of cervical dysplasia containing HPV types 16/18, 6/11 or unknown types (6
) as well as laryngeal papilloma with HPV type 6/11 etiology (3
–5
). Published studies support the hypothesis that I3C and DIM help diminish the contribution of estrogen, a hormone that stimulates HPV pathology in these tissues (2
,42
). However, other effects of I3C and DIM such as growth arrest and induction of apoptosis could also be beneficial for prevention and treatment of these pathologies, and do not involve the interaction of these agents with estrogen metabolism or function. Indeed, the present study shows that early changes in gene expression induced by DIM included increased expression of a number of genes whose proteins cause growth arrest, e.g., the stress-related and DNA damage-associated proteins GADD153, GADD34 and GADD45, and decreased expression of proliferation-associated genes such as cyclin D1, cyclin F and PCNA. Not surprisingly, very few genes relating to apoptosis were induced during the first 6 h of DIM exposure because apoptosis occurs later (19
), potentially as an outcome of growth arrest.
An unexpected difference between the response of carcinoma cells and spontaneously transformed or primary keratinocytes was the robust, early induction of certain Phase I enzymes (e.g., CYP1A1 and CYP1A2) after exposure to DIM only in the nontransformed cells. In contrast, expression of these genes was not changed within 6–12 h in either C33A or CaSki cell lines. We interpret this to mean that induction of Phase I enzymes (as well as Phase II enzymes, which were not induced within 12 h in any cell line) is probably a secondary event in the carcinoma cells in vitro because Phase I enzymes have been shown to be induced by prolonged exposure to I3C in these same cell lines (15
). Consistent with this idea, Nrf2, as well as c-Jun and protein kinase C, which were all induced early by DIM (Table 1
), control both constitutive and inducible expression of Phase II enzymes involved in detoxification and glutathione biosynthesis (43
,44
). Other genes encoding transcription factors and signaling molecules were also induced by DIM. Together these changes would be expected to lead to subsequent changes in the expression of many genes as a secondary effect.
The increased expression of bZip proteins observed in DIM-treated cells should alter expression of HPV oncogenes. It is known that NF-IL6 negatively regulates both HPV11 (32
,34
), which is associated with laryngeal papillomas and exophatic tumors in the cervix, and HPV16 (30
), which is the major HPV type associated with cervical cancer. Additionally, the family of AP1 proteins, all bZip transcription factors, plays a particularly important role in regulating HPV gene expression, usually resulting in increased transcription (33
–37
). The ability of bZip proteins to form heterodimers with each other makes them acutely sensitive to changes in the expression of other bZip proteins. Expression of HPV genes, although relatively attenuated in infected cells, is positively regulated by the differentiation of keratinocytes [reviewed by (45
)]. NF-IL6 and GADD153, which are highly induced by DIM, normally appear to program the differentiation of keratinocytes (46
). In nontransformed, uninfected laryngeal epithelium (47
) and in mouse skin (45
), NF-IL6 is most abundant in the nuclei of basal and suprabasal cells, in which very little expression of HPV occurs. On the other hand, GADD153, a growth arrest protein, is expressed only in the more differentiated, spinous cells of both mouse skin (46
) and normal laryngeal epithelium (our unpublished data). The distribution of NF-IL6 is irregular in papillomas (47
), and NF-IL6 was already known to down-regulate HPV expression and replication (30
,32
). However, before this study, GADD153 (also known as CHOP) was not known to affect expression of HPV. Although known as a growth arrest protein (48
), overexpression of the GADD153 fusion protein TSL-CHOP that occurs in some cancers results in oncogenic transformation (49
). GADD153 does not bind to DNA, but instead forms heterodimers with other c/EBP and with the individual subunits of AP1 transcription factors (29
,49
). A result could be the sequestering of a particular bZip transcription factor such that it cannot bind DNA (40
), or, if the heterodimer still binds DNA, such that its transcriptional regulatory properties are altered (41
). The observations in the studies reported here are consistent with the hypothesis that the relative amounts of different bZip proteins, including GADD153, determine the extent of transcription from the HPV URR.
As often occurs with microarray profiling, we observed changes in the expression of many genes not previously known to be affected by treatment with DIM, allowing us to predict an effect of I3C/DIM on otherwise unsuspected cell processes. For example, the battery of genes induced by DIM resembles that induced by proteasome inhibitors, e.g., ATF3, GADD153 and MAD1 (50
) and by endoplasmic reticulum stress (51
), processes that are not necessarily mutually exclusive. Therefore, early transcriptional effects of DIM point to protein homeostasis as a potential mechanism by which this compound initiates antiviral and anticancer activities. Among the relatively few genes whose expression is consistently down-regulated by short exposure to DIM are those encoding cytoskeletal proteins, especially the tubulins; cell cycle-related proteins with a short half-life such as cyclins D1 and F, and PCNA; and proteins that are involved in mRNA processing and function (Appendix 2)
. As a result of these changes, cell division is likely to be compromised early after exposure to DIM, and the synthesis of some new proteins is likely to be disrupted. The mechanism by which DIM initiates such rapid and global changes in gene expression remains to be determined.
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APPENDIX 1 |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX 1APPENDIX 2LITERATURE CITED |
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APPENDIX 2 |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX 1APPENDIX 2LITERATURE CITED |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by RO1-CA733850, (K.J.A.), P50-DC00203 (K.J.A.), RO1-CA82599 (E.M.R.) from the National Institutes of Health and a gift from Theodore Danforth to the North Shore-Long Island Jewish Research Institute for establishment of a microarray facility. The contents are solely the responsibility of the authors.
3 Present address: Cellular Regulation and Transformation, Basic Research Section, National Cancer Institute, Bethesda, MD 20592.
5 Abbreviations used: AhR, aryl hydrocarbon receptor; CDK, cyclin-dependent kinase; DIM, diindolylmethane; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; EST, expressed sequence tags; HFK, human foreskin keratinocytes; HPV, human papillomavirus; HSV-TK, herpes simplex virus-thymidine kinase; I3C, indole-3-carbinol; NF-IL6, nuclear factor-interleukin 6; PCNA, proliferating cell nuclear antigen; RT-PCR, reverse transcriptase-polymerase chain reaction; URR, upstream regulatory region.
Manuscript received 3 May 2002. Initial review completed 12 June 2002. Revision accepted 13 August 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONAPPENDIX 1APPENDIX 2LITERATURE CITED |
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-actin mRNA by semiquantitative reverse transcriptase-polymerase chain reaction (panels A, B). Whole cell extracts were also analyzed for GADD153 and 
