Materials and methods

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

Drug and cell culture

5F 203 was synthesised by the Cancer Research Laboratories at the University of Nottingham, UK and the Drug Synthesis and Chemistry Branch, NCI, following published methods (Hutchinson et al, 2001). The compound was dissolved in DMSO to make a 100 mM stock concentration and further diluted to the working concentration (1 nM–100 M) for experimental procedures. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was prepared as 10 M top stock dissolved in DMSO and diluted to the working concentration (10 nM) immediately prior to use. MCF-7 cells were obtained from the National Cancer Institute Repository (National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD, USA) and grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA). AhR-deficient AH^R100 cells were generated from wild-type MCF-7 cells by 6–9-month exposure to benzo[a]pyrene and subsequently demonstrated a 100-fold higher resistance to benzo[a]pyrene than the wild-type line. Additional characterisation indicated that AH^R100 cells contained reduced amounts of AhR and normal levels of the aryl hydrocarbon receptor nuclear translocator (ARNT) (Ciolino et al, 2002).

Assessment of cytotoxicity

Cells were inoculated in 96-well plates and maintained for 24 h before treatment with 5F 203, which consisted of five serial dilutions (1 : 10) ranging from 10 nM to 100 M. Treated cells were subsequently maintained for an additional 48 h, and cellular protein was measured by the sulphforhodamine-B (SRB) assay as described previously (Monks et al, 1991). Briefly, cells were fixed with trichloroacetic acid and stained with SRB. Protein-bound SRB was solubilised and measured spectrophotometrically to determine relative cell viability in treated and untreated cells.

4,6-Diamidino-2-phenylindole (DAPI) staining

Approximately 2 10⁵ cells were grown on coverslips overnight. Cells were exposed to DMSO (0.1%) or 5F 203 (1 M) for 24 h. Floating cells contained in the supernatant were collected by cytocentrifugation, and stained with 0.4% DAPI. Experiments were repeated at least three times. Stained cells were then visualised on a Zeiss Axiovert microscope using 63 objective and images were captured with an Optronics CCD camera (Loaiza-Pérez et al, 2002).

³²P-postlabelling assay

Pellets of control and treated (0.1% DMSO or 1 M 5F 203 for 24 h) MCF-7 and AH^R100 cells were used for DNA extraction using Qiagen DNA extraction columns according to the manufacturer's protocol. Of each extracted DNA 5 g was digested to deoxynucleoside 3'-monophosphates by incubating with micrococcal nuclease (175 mU) and calf-spleen phosphodiesterase (6 mU) at 37°C overnight. DNA adduct enrichment was carried out by butanol extraction (Gupta, 1985). Adducts were then radiolabelled by 5'-phosphorylation using 62.5 Ci of [-³²P]ATP and 5 U of T4 polynucleotide kinase.

HPLC detection and quantification of DNA adducts

³²P-labelled products were separated on a Hypersil BDS C18 analytical column (250 4.6 mm, 5 m; Shandon). The mobile phase consisted of 88% 2 M ammonium formate (pH 4.0) and 12% acetonitrile for 50 min, followed by a linear gradient of 20–45% acetonitrile for 15 min. Radioactivity was monitored by a radiochemical detector (Lab Logic, -RAM) lined to a Varian Star 9012 pump. Data analysis was done by Laura, an MS Windows package (Lab Logic Inc.). The relative adduct levels (RAL) were calculated by the method of Reddy and Randerath (1986), based on the specific activity of [-³²P]ATP and the amount of DNA used. RAL values were then translated into fmol adducts per g DNA (Gupta, 1985).

Cell cycle analysis

Cell cycle analysis on MCF-7 and AH^R100 cells was performed as described previously (Sherwood and Schimke, 1995). Briefly, exponentially growing cells were exposed to either DMSO (0.1%) or 5F 203 (1 M) for 24 h, then harvested, washed briefly, in cold PBS and fixed in 70% ethanol. DNA was stained by incubating the cells in PBS, containing propidium iodide (50 g ml^-1) and RNAase A (1 mg ml^-1) for 30 min at 37°C and fluorescence was measured and analysed using FACSCaliber (Becton Dickinson Immunocytometry Systems, San José, CA, USA) and ModFit (Verity Software, Topsham, ME, USA).

CYP1A1 activity in intact MCF-7 and AH^R100 cells

MCF-7 and AH^R100 cells were grown in 24-well plates to 60–70% confluence prior to treatment with 0.1% DMSO (control) or 5F 203 (1 nM–1 M). At the end of the incubation period, the medium was aspirated and the cells were washed with PBS. CYP1A1 enzyme activity was subsequently determined by 7-ethoxyresorufin-O-deethylase (EROD) activity in intact cells as described by Kennedy and Jones (1994). Briefly, the fluorescence of resorufin generated from the conversion of ethoxyresorufin by CYP1A1 was measured spectrophotometrically and resorufin content was derived from a standard curve.

Real-time RT–PCR

Evaluation of CYP1A1 and CYP1B1 gene expression in MCF-7 and AH^R100 cells was performed by real-time RT–PCR, using 15 cycles of PCR, primers and probes described in a previous report (Loaiza-Pérez et al, 2002). RNA was isolated using Qiagen kits (Qiagen, Valencia, CA, USA). PCR efficiencies were validated by means of a standard curve.

Transfections

Cells were transfected using LipofectAMINE (Invitrogen), with 0.5 g Renilla reniformis luciferase (pRL-TK) (Promega, Madison, WI, USA), and 1.5 g of pTX.Dir or pT81 as described previously (Loaiza-Pérez et al, 2002). Luciferase activity was measured by the Dual-Luciferase Assay System (Promega, Madison, WI, USA) following the manufacturer's instructions, and transfection efficiency was monitored by the activity of the R. reniformis plasmid.

Electrophoretic mobility shift assay (EMSA)

Synthetic oligonucleotides containing the AhR-binding site of the human CYP1A1 promoter (5'-CTC CGG TCC TTC TCA CGC AAC GCC TGG GCA-3') (Invitrogen) were used as probes. Electrophoretic mobility shift assays were performed as described previously (Loaiza-Pérez et al, 2002). Briefly, nuclear extracts (20 g) from control (0.1% DMSO) and treated (1 M 5F 203 for 1 h, or 10 nM TCDD for 1 h) MCF-7 and AH^R100 cells were incubated in binding buffer with the [³²P]DNA probe and the resulting DNA/protein DNA complexes were separated on a 6% polyacrylamide gel (Novex) under nondenaturing conditions and high ionic strength. Gels were dried and imaged by autoradiography.

Results

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

5F 203-induced cytotoxicity correlates with AhR activation in sensitive MCF-7 cells

Previous observations have shown that cytotoxicity of benzothiazoles is mediated via activation of the AhR signalling pathway (Kashiyama et al, 1999; Chua et al, 2000; Loaiza-Pérez et al, 2002). In addition, cytotoxicity across a large panel of human tumour cell lines correlates with CYP1A1 inducibility (Hose et al, 2001). We then sought to investigate responsiveness of MCF-7 wild-type cells to the clinical candidate 5F 203 as compared to that of MCF-7-derived AhR-deficient cells (AH^R100). Specifically, we sought to establish whether AhR-mediated signalling could account for all the cytotoxic potential of this agent. Figure 2A indicates that MCF-7 cells are sensitive to this compound (GI₅₀=121 nM, 48 h exposure). In contrast, AH^R100 cells show a decreased growth inhibition after treatment with high drug concentration (GI₅₀>100 M) and can therefore be considered resistant.

Figure 2.

(A) Selective cytotoxicity of 5F 203 to MCF-7 cells. Growth inhibition induced by 5F 203 was assessed by sulphforhodamine B assay as detailed in Materials and Methods. Values are meanss.d. (n=10). (B) 5F 203 induces changes in nuclear morphology of 5F 203-sensitive MCF-7 breast cells but not AH^R100 cells. Cells prepared as described in Materials and Methods were exposed for 24 h to 0.1% DMSO (control) or 1 M 5F 203, and stained with 4,6-diamidino-2-phenylindole (DAPI).

Full figure and legend (19K)

As a complementary approach, MCF-7 and AH^R100 cells treated with 0.1% DMSO (control) or 5F 203 (1 M) for 1 h were subsequently stained by DAPI (Figure 2B). Only MCF-7 cells treated with the drug showed altered nuclear morphology, which included chromatin condensation and marginalisation to the nuclear membrane. AH^R100 cells failed to show any significant morphological changes. Taken together, Figure 2A and 2B suggests that activation of the AhR signalling pathway participates in the antiproliferative activity of 5F 203, which results in growth inhibition and modified nuclear features.

5F 203 induces DNA adduct formation in MCF-7 cells

We next questioned whether DNA adducts could be produced in MCF-7 and AhR-deficient AH^R100 cells. DMSO-treated MCF-7 and AH^R100 cells showed a similar profile of DNA adducts, with a few adducts eluting within the early retention time (20 min) (Figure 3). After treatment with 1 M 5F 203, no significant changes in these peaks were found in either cell lines, while several other adducts with longer retention time were detected. The major adduct formed in AH^R100 cells (adduct 17 in Figure 3Bii) is chromatographically equivalent to an adduct detected in MCF-7 cells (adduct 7 in Figure 3Aii). Although DNA adduct profiles appear similar in the two cell lines, the number of total adducts/10⁸ nucleotides induced by drug treatment was greatly decreased in AH^R100 cells compared to wild-type MCF-7 cells. These findings suggest that impairment of the AhR signalling pathway may result in reduced activation of 5F 203 into reactive species able to damage DNA.

Figure 3.

DNA adduct profiles in (A) MCF-7 and (B) AH^R100 cells. Cells were incubated with (i) 0.1% DMSO or (ii) 1 M 5F 203 for 24 h. ³²P-labelled adducts were analysed using 2 g digested DNA on a Hypersil C18 column with a gradient of acetonitrile in 2 M ammonium formate as detailed in Materials and Methods. The early peak is due to the presence of free [-³²P]ATP.

Full figure and legend (41K)

5F 203 causes altered cell cycle distribution

As our results in Figures 2 and 3 indicated that 5F 203 was inducing the formation of DNA adducts, we investigated whether this could result in an altered cell cycle profile. For this approach, cells sensitive (MCF-7) and insensitive (AH^R100) to 5F 203 were exposed to 1 M of the drug or 0.1% DMSO for 24 h and subsequently processed for cell cycle analysis. As illustrated in Figure 4, 5F 203 treatment of MCF-7 cells caused a notable increase in G₁ (37 to 52%) and S (20 to 27%) phase cells, which coincided with a decrease in G₂/M (42 to 16%) phase cells. Accumulation of cells in sub-G₁ was minimal when compared to control. In contrast, 5F 203-insensitive cells (AH^R100) failed to demonstrate any notable effect on the cell cycle, in particular S, G₂/M and sub-G₁ phases. The data demonstrate that 5F 203-induced DNA damage may lead to cell accumulation in G₁ and S phase concomitant with growth inhibition. As MCF-7 cells are p53 wild type, their response to 5F 203 treatment is consistent with the operation of a G₁- and possibly S-phase checkpoint to cell cycle progression after DNA adduct formation.

Figure 4.

5F 203 induces accumulation of cells in S phase. Exponentially growing cells (MCF-7 and AH^R100) were exposed to either 0.1% DMSO (control) or 5F 203 (1 M) for 24 h, harvested, washed in PBS, and fixed in 70% ethanol. DNA was stained by incubating the cells in PBS containing propidium iodide and fluorescence measured and analysed as described in Materials and Methods.

Full figure and legend (13K)

CYP1A1 and CYP1B1 induction

5F 203 induces DNA damage and cell cycle arrest most obviously in MCF-7 cells with a fully functional AhR signalling pathway. The hypothesis that deficient activation of CYP1A1 might occur in AhR-deficient AH^R100 cells was then addressed. The enzymatic activity of CYP1A1 in intact MCF-7 and AH^R100 cells treated with 5F 203 was assayed by measuring EROD activity. Although both CYP1A1 and CYP1B1 can catalyse the dealkylation of ethoxyresorufin, the specific activity of CYP1A1 is approximately 40-fold higher than that of CYP1B1 (Doostdar et al, 2000). Incubation with 5F 203 for 24 h resulted in a concentration-dependent increase in EROD activity over a range of 50 nM to 1 M in MCF-7 cells, whereas the drug had no effect on EROD activity in AH^R100 cells (Figure 5A).

Figure 5.

(A) 5F 203 induces CYP1A1 activity in MCF-7 but not in AH^R100 cells. MCF-7 and AHR100 cells were incubated for 24 h with DMSO (0.1%) and 5F 203 (1 M) for 24 h and assayed for CYP1A1 enzyme activity by EROD assay as described in Materials and Methods. n=4s.d. (B) 5F 203 induces CYP1A1 and CYP1B1 mRNA levels in sensitive (MCF-7) cells. MCF-7 and AH^R100 cells were treated with 5F 203 (1 M) for 24 h, RNA was isolated from control and treated samples and CYP1A1 and CYP1B1 gene expression were measured by real-time RT–PCR as described in Materials and Methods. Data are shown as fold induction of treated cells relative to constitutive expression in control cells (s.d., n=7 samples from two independent experiments).

Full figure and legend (11K)

In order to compare the effect of 5F 203 on CYP1A1 and CYP1B1 gene expression, MCF-7 and AH^R100 cells were treated with the compound (1 M) for 24 h and mRNA levels for these two genes were measured by real-time RT–PCR. 5F 203 caused an increase in the mRNA levels of both cytochromes in MCF-7 cells, where relative levels of CYP1A1 mRNA were approximately 100-fold higher when compared to control; only a minimal increase of CYP1B1 mRNA (10-fold) was observed (Figure 5B). In contrast, in AH^R100 cells, levels of CYP1A1 and CYP1B1 mRNA after treatment remained similar to control (Figure 5B). These findings are in agreement with previous studies which described that the parent compound DF 203 causes induction of CYP1A1 protein and increase in mRNA levels in MCF-7 cells but not in DF 203 inherently resistant cells (Chua et al, 2000; Loaiza-Pérez et al, 2002).

5F 203 increases protein/DNA complex formation on the XRE sequence of CYP1A1 in MCF-7 but not AH^R100 cells

CYP1A1 and CYP1B1 promoters are regulated by the AhR, which forms a heterodimer with ARNT. Binding of the complete dimer to the XREs in the promoter region mediates transcription of AhR-responsive genes, including CYP1A1 and CYP1B1 (Whitlock, 1999). MCF-7 and AH^R100 cells were transfected with an XRE-luciferase reporter construct (pTX.Dir); as control, the same reporter construct but without the XRE (pT81) was used. Cells were then treated with DMSO (0.1%), TCDD (10 nM) or 5F 203 (1 M). TCDD was used as a prototypic compound activator of CYP1A1 transcription and included as a positive control. As shown in Figure 6A, in MCF-7 cells transfected with pTX.Dir, a 15-fold induction of luciferase activity was observed with TCDD, and 5F 203 treatment similarly resulted in a 15-fold induction. However, when AH^R100 cells were transfected with XRE and treated with 5F 203 (1 M), XRE-luciferase activity was demonstrated to be only approximately 1.7-fold higher than control. No induction in luciferase activity was observed when cells transfected with pT81 were treated with 5F 203 (1 M) or TCDD (10 nM). Similar results were obtained when cells were transfected with a fragment of mouse native CYP1A1 promoter (inclusive of four dioxin responsive elements (DREs)) (pGudLuc1.1) (data not shown) (Garrison et al, 1996). When pTX.Dir-transfected cells were pretreated with an antagonist of AhR, -naphthoflavone, before treatment with 5F 203, luciferase activity was reduced approximately 25%, suggesting competition between the two ligands for binding to the AhR (results not shown).

Figure 6.

(A) 5F 203 induces binding to the XRE sequence of CYP1A1. MCF-7 and AH^R100 cells were transfected with XRE-luciferase (pTX.Dir.) or pT81. A schematic of the respective construct is shown below the panel. Transfected cells were treated with DMSO, TCDD (10 nM) or 5F 203 (1 M) for 9 h. XRE-luciferase activity was determined normalizing to the amount of Renilla reniformis luciferase. The values are expressed as luciferase levels relative to control. (B) 5F 203 induces protein/DNA complexes on the XRE sequence of the CYP1A1 promoter. Nuclear extracts (20 g) prepared from MCF-7 cells treated with 0.1% DMSO (C) (lane 1), TCDD (10 nM, 1 h) (lane 2) or 5F 203 (1 M, 1 h) (lane 3) were incubated with labelled XRE sequence derived from the CYP1A1 promoter for 10 min at room temperature. Free and bound DNA were separated as described in Materials and Methods. In competition experiments, nuclear extracts from MCF-7 cells treated with 5F 203 (1 M, 1 h) were incubated with 4 g of anti-AhR antibody (lane 6), 100-fold excess of unlabelled XRE oligonucleotide (lane 7), 100-fold excess of unlabelled Sp1 oligonucleotide (lane 8) or 4 g of IgG antiserum (lane 9). Protein/DNA complexes from AH^R100 cells were resolved in the same gel. Nuclear extracts from these cells (20 g) treated with DMSO (lane 4) or 5F 203 (1 M, 1 h) (lane 5) were incubated with radioactive XRE and resolved by the same procedure.

Full figure and legend (175K)

In order to compare protein/DNA complex formation on the XRE of CYP1A1 promoter after treatment with 5F 203 in MCF-7 and AH^R100 cells, electrophoretic mobility shift assays were performed. Previous reports from our laboratory (Loaiza-Pérez et al, 2002) described that, in MCF-7 cells, three protein/DNA complexes were found in nuclear extracts of control cells, although the intensity of the uppermost complex was lower than the others (Figure 6B, lane 1). 5F 203 (1 M) treatment for 1 h (lane 3) caused a 15-fold induction of the binding of the uppermost complex. This induction exceeded that afforded by TCDD (10 nM, 1 h) treatment (lane 2). AH^R100 cells exposed for 1 h to 5F 203 (1 M) showed considerable attenuation of induction (lane 5). When nuclear extracts from cells treated with 5F 203 (1 M) for 1 h were pretreated with AhR polyclonal antibody (lane 6) or 100 unlabelled XRE probe (lane 7), the binding of the three complexes to labelled XRE sequences was greatly diminished. These protein/DNA complexes did not disappear when nuclear extracts from 5F 203-treated cells were preincubated with 100 unlabelled SP1 oligonucleotide (lane 8) or IgG antiserum (lane 9).

Discussion

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

We report here that treatment with 5F 203 causes accumulation in the G₁ and S phase, along with growth inhibition in wild-type MCF-7 cells, but it does not affect cell cycle progression of AH^R100 cells. We also confirm the production of DNA damage in these sensitive cells (Stevens et al, 2001), whereas the formation of DNA adducts is significantly reduced in the resistant subclone (Figure 3). These results corroborate the view that antitumour activity of 5F 203 is highly dependent on functional AhR signalling, whose impairment results in reduced efficacy of the agent.

Activation of the AhR signalling pathway may be an important factor determining sensitivity to 5F 203 because it triggers metabolic transformation of the drug through CYP1A1 into reactive species damaging DNA. Our data show that exposure of AH^R100 cells to 5F 203 produces markedly fewer DNA adducts, as compared to wild-type MCF-7 cells. The chemical structure of these adducts has not yet been characterised. Computational studies using frontier molecular orbital calculations point to the putative reactive species being a nitrenium ion (Dr SE O'Brien, personal communication), but further studies are required to confirm this hypothesis and identify the adducts. A more detailed description of the formation of DNA adducts induced by benzothiazoles in sensitive cells will be published separately.

MCF-7 cells have proven to be a valuable and extensively used model for studies involving the AhR and CYP1A1 expression (Dohr et al, 1995; Wang et al, 1995; Jorgensen and Autrup, 1996). Experiments reported elsewhere (Yeh et al, 2001) have demonstrated that resistance to aryl hydrocarbons in AH^R100 cells is a consequence of decreased expression of the AhR, which results in impaired induction of CYP1A1 and probably represents an adaptation to long-term culture with polycyclic aromatic hydrocarbons (PAHs). This leads to a diminished capacity to activate PAHs like DMBA, thereby decreasing the level of apoptosis caused by exposure to PAHs. Apoptosis induced by DMBA could be blocked by inhibitors of DMBA metabolism such as -naphthoflavone and diosmetin. These data demonstrated that PAH resistance in these cells is mediated by changes in the signal transduction pathway which regulates CYP1A1 expression (Ciolino et al, 2002). Previous reports have demonstrated that the growth-inhibitory properties of the parent compound, DF 203, were abrogated when MCF-7 cells were cotreated with an AhR antagonist like -naphthoflavone (Chua et al, 2000). These findings suggested that activation of AhR mediates sensitivity of MCF-7 cells to benzothiazoles. The fact that adriamycin-resistant MCF-7 cells (NCI/ADR-RES cells), which are also deficient in AhR signalling (Caruso and Batist, 1999), are resistant to benzothiazoles (Chua et al, 2000) further supports this hypothesis.

Activation of phase I enzymes, such as CYP1A1 and CYP1B1, usually occurs as a response to promote detoxification of xenobiotics; however, metabolic enzymes may convert inert compounds into potentially harmful products. This is the case for most PAHs, including benzo[a]pyrene, which induces CYP1A1 via the AhR signalling pathway and is metabolised into reactive species that cause DNA damage (Szeliga and Dipple, 1998; Burczynski and Penning, 2000) resulting in G₁ cell cycle arrest (Vaziri and Faller, 1997). In contrast, the environmental pollutant TCDD, which is the most potent AhR agonist, induces CYP1A1 and modulates CYP1B1 expression in MCF-7 cells (Dohr et al, 1995), but is not directly genotoxic. It has been hypothesised that TCDD-induced alterations in estrogen metabolism may lead to increased generation of reactive oxygen species and consequent induction of DNA adducts (Wyde et al, 2001).

In addition to selective metabolism, differential modulation of AhR functions could underlie specific cytotoxicity of the agent. Activation of the AhR signalling pathway consists of several steps, including translocation, pairing with nuclear factors, transactivation of gene transcription and degradation (Rowlands and Gustaffson, 1997), each of which might be differentially regulated in different cells. Other proteins, which integrate the function of the AhR, like heat-shock protein 90 or ARNT, may also be related to differential sensitivity to 5F 203. For example, Qin et al (2001) have demonstrated in patients that there is an association of ARNT splice variants with ER-negative breast cancer, poor induction of vascular endothelial growth factor under hypoxia, and poor prognosis. Further studies are necessary to determine, in greater detail, aspects of the pathway that may be altered in 5F 203-sensitive cells.

References

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

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Acknowledgements

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

This is part 21 of the series Antitumour 2-(4-aminophenyl)benzothiazoles). We are very grateful to Dr Tracey D Bradshaw (School of Pharmaceutical Sciences, University of Nottingham, UK) for the critical reading of the manuscript. We also thank Dr Peter B Farmer and his collaborators (MRC Toxicology Unit, University of Leicester, UK) for their assistance in DNA adduct studies.