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Carcinogenesis |
Department of Experimental Pathology, Roswell Park Cancer Institute, Buffalo, New York 14263 [Y. D., C. S., C. I.], and Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706 [H. E. G.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is one major problem with performing in vitro studies using MSC. A ß-lyase-mediated conversion of MSC to methylselenol (CH3SeH) is required for this agent to express its anticancer activity (7) . Because ß-lyase is present in abundance in the liver and kidney, animals have an ample capacity to metabolize MSC systemically. Breast cells, on the other hand, have a low ß-lyase activity. Consequently, concentrations of MSC far above physiological levels are necessary to produce growth inhibition in cultured breast cells. MSA (CH3SeO2H) was developed specifically for in vitro studies because it obviates the need for ß-lyase to generate a monomethylated metabolite (8) . Once taken up by cells, MSA is easily reduced to CH3SeH through nonenzymatic and enzymatic processes involving glutathione and NADPH (8 , 9) . Our previous research with mouse mammary premalignant cell lines showed that 5 µM MSA was able to induce a 50% growth inhibition, whereas 50 µM of MSC produced only a marginal effect (8) . Contrary to the in vitro data, MSA and MSC have equal chemopreventive efficacy in vivo (8) , and they behave similarly in modulating a panel of cell cycle- and apoptosis-regulatory biomarkers in premalignant lesions of the rat mammary gland (10) . Thus, these two reagents are interchangeable when ß-lyase is not a limiting factor. However, because of the high sensitivity of cultured epithelial cells to MSA, we believe that MSA is an excellent agent for molecular mechanistic studies in vitro.
To date, MSA has been evaluated only in rodent mammary cells. It is critical to find out whether human cells respond equally well to a monomethylated selenium compound such as MSA. In this study, we examined the growth inhibitory effect of MSA on two premalignant human breast cell lines. Our results indicated that these cells are exquisitely sensitive to MSA with respect to growth inhibition, which is achieved mainly by G0-G1 arrest coupled with an induction of apoptosis. We also made use of the cDNA microarray technology to gain a global view of the pool of genes that might play an important role in growth inhibition by selenium. A set of 30 potential selenium-responsive genes was defined by this method. As an epilogue, these genes are then integrated into a schematic diorama of signaling pathways that provide the supportive framework for understanding the molecular mechanism of selenium chemoprevention.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MTT Cell Proliferation Assay.
The assay, based on the conversion of the yellow tetrazolium salt MTT to purple formazan crystals by metabolically active cells (12)
, provides a quantitative determination of viable cells. As part of the design to evaluate the activity of MSA in modulating cell growth, we also compared it with MSC (Sigma Chemical Co., St. Louis, MO). Cells were seeded in 24-well plates at a density designed to reach 70–80% confluency at the time of assay. At 48 h after seeding, cells were treated with various concentrations of MSC or MSA in triplicate. After 24, 48, or 72 h of treatment, 200 µl of MTT was added to each well of cells, and the plate was incubated for 4 h at 37°C. The medium was removed, and the MTT crystals were solubilized in isopropanol and subjected to centrifugation to pellet the cellular debris. Spectrophotometric absorbance of each sample was measured at 570 nm using a Spectra Microplate Reader (SLT, Australia).
Cell Cycle Analysis.
MCF10AT1 cells were plated at a density of 3000 cells/cm2 in T75 culture flasks and allowed to grow for 48 h to reach 50–60% confluency. To achieve synchronization, cells were starved in serum-free and growth factor-free medium for 48 h. On returning to regular growth medium for 6 h, cells were exposed to 5 µM MSA. After treatment for 6, 12, or 24 h, cells were trypsinized, washed in PBS, and fixed overnight in 70% ethanol at 4°C. At the time of harvest, the cultures were 70–90% confluent, although they were maintained for various lengths of time. The ethanol solution was subsequently removed after centrifugation, and cells were resuspended in a buffer containing 10 mM Tris (pH 7.5), 125 mM sucrose, 2.5 mM MgCl2, 0.185% NP40, 0.02 mg/ml RNase A, 0.05% sodium citrate, and 25 µg/ml PI. After incubation on ice for 1 h, cells were subjected to DNA content analysis using a FACScan cytometer (Becton Dickinson).
TUNEL Detection of Apoptosis.
MCF10AT1 cells were plated at a density of 4000 cells/cm2 in Lab-Tek chamber slides suitable for tissue culture (Nalge Nunc, Rochester, NY). At 48 h after seeding, cells were exposed to 5 or 10 µM MSA for 18, 21, or 24 h. Cells were then fixed in 1% paraformaldehyde, and apoptosis was detected by the TUNEL method using the ApopTag Peroxidase in Situ Apoptosis Detection kit (Intergen, Purchase, NY) according to the manufacturer’s instructions. Color pictures were taken with a camera mounted on top of a microscope under a x40 objective. All hard-copy images were scored by an observer blinded to the identity of the sample to avoid bias. The quantification of apoptotic cells was calculated as a percentage of the total cells evaluated (>200 cells/sample).
Quantitation of Apoptosis by Flow Cytometry.
MCF10AT1 cells were plated at a density of 4000 cells/cm2 in T175 culture flasks. At 48 h after seeding, cells were exposed to 5 µM MSA for 18, 21, or 24 h. Adherent cells harvested by mild trypsinization were pooled together with detached cells. Cells were stained with biotin-conjugated Annexin V, FITC-conjugated streptavidin, and PI using the Annexin V-Biotin Apoptosis Detection kit (Oncogene Research Products, Boston, MA) as per manufacturer’s protocol. Apoptotic cells were subsequently counted by flow cytometry, and the data were analyzed with the WinList software (Variety Software House, Topsham, ME).
cDNA Microarray Analysis.
MCF10AT1 cells were plated at a density of 4000 cells/cm2 in 15-cm culture dishes. Synchronization was achieved as described above. Again, the cultures for the microarray analysis were grown to 80–90% confluency at the time of harvest. After exposure to 5 µM MSA for 6 or 12 h, total RNA and protein were isolated using TRIzol (Life Technologies, Inc.). 32P-cDNA probes were generated from total RNA using the Atlas Pure Total RNA Labeling system (Clontech, Palo Alto, CA) as per manufacturer’s protocol. The probes synthesized from the treatment samples and their corresponding controls were hybridized side-by-side to two identical Atlas Human Cell Cycle Arrays or Apoptosis Arrays (Clontech, Palo Alto, CA). After overnight hybridization and a high-stringency wash, the arrays were scanned by a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA) after a 3-day exposure. The two array images were analyzed and compared using the Clontech AtlasImage 1.5 software as reported previously (13)
. The entire synchronization and treatment experiment was repeated, and the total RNA collected from the replicate was subjected independently to microarray analysis. Only those genes that were modulated in both experiments were considered to minimize intervariation errors.
Western Blot Analysis.
Western blot analysis was performed as described previously (14)
using the TRIzol isolated protein. Briefly, 
20 µg of protein was resolved over 10–15% SDS/PAGE and transferred to polyvinylidene fluoride membrane. The blot was blocked in blocking buffer [5% nonfat dry milk, 10 mM Tris (pH 7.5), 10 mM NaCl, and 0.1% Tween 20] overnight at 4°C, incubated with the primary antibody at 37°C for 1 h, followed by incubation with an antimouse or antirabbit horseradish peroxidase-conjugated secondary antibody (Bio-Rad, Hercules, CA) at 37°C for 30 min. Individual proteins were visualized by an enhanced chemiluminescence kit obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Immunoreactive bands were quantitated by volume densitometry using the ImageQuant software (Molecular Dynamics, Sunnyvale, CA), and normalized to actin. The following monoclonal antibodies were used in this study (source): anti-actin (Sigma Chemical Co., St. Louis, MO); anti-cdc6, DP1, and c-jun (Oncogene Research, Boston, MA); anti-cyclin A, cyclin D1, cdc25A, and cdk5 (NeoMarkers, Fremont, CA); anti-cdc2, cyclin B1, and proliferating cell nuclear antigen (Santa Cruz Biotechnology, Santa Cruz, CA). Polyclonal antibodies to four other proteins were obtained from Cell Signaling Technology (Beverly, MA): anti-MAPKK1/2, phospho-ATF-2, SAPK/JNK, and phospho-SAPK/JNK.
Semiquantitative RT-PCR.
Total RNA from the TRIzol isolate was treated with RNase-free DNase I (Roche, Indianapolis, IN). After removal of the DNase I, cDNA was reverse transcribed from 1 µg of RNA using an oligo(dT)12–18 primer. The genes of interest (GADD153, E2F1, or E2F5) and the invariant housekeeping gene control, GAPDH, were amplified for 18 cycles from 5% of synthesized cDNA. Primer pairs for each gene spanned at least an intron to distinguish amplified cDNA products from amplified genomic DNA. The following primer pairs were used to amplify GADD153, E2F1, E2F5, and GAPDH respectively: (sense) 5'-GTCATTGCCTTTCTCCTTCG-3'/(antisense) 5'-GCTAGCTGTGCCACTTTCCT-3'; (sense) 5'-TCGCAGATCGTCATCATCTC-3'/(antisense) 5'-CTCAGGGCACAGGAAAACAT-3'; (sense) 5'-GGGCTGCTCACTACCAAGTT-3'/(antisense) 5'-GCCACTGTTTTGATGACCTG-3'; (sense) 5'-TCGGAGTCAACGGATTTGGTCG-3'/(antisense) 5'-AACTGTGAGGAGGGGAGATTCAG-3'. At the end of 18 cycles, 5 µl of each PCR reaction were removed, and the rest of the reaction subjected to five more PCR cycles. This step was repeated until 38 cycles of amplification was reached. The 5-µl aliquots removed from each reaction after 18, 23, 28, 33, and 38 cycles were analyzed on a 2% agarose gel, and the intensity of each band was quantitated with AlphaImager 1220 Documentation and Analysis system (Alpha Innotech, San Leandro, CA). An optimal number of PCR cycles that provided a linear range of amplification was determined by plotting the intensity of each band against the number of PCR cycles. The RT-PCR signal obtained with the optimal cycling parameters for each gene was normalized to the one for the GAPDH gene.
Statistical Analysis.
Student’s t test was used to determine the significance between treatments and untreated controls, and P < 0.05 was considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and 2
, MSA was able to significantly suppress the growth of both cell lines in a time- and dose-dependent manner. The inhibitory response was apparent as early as 24 h of treatment with a concentration range between 2.5 and 10 µM. At 48 h, concentrations of 2.5 and 5 µM MSA were found to reduce the cell number of both cell lines to 30% and 12% of the untreated control, respectively. In contrast, a concentration of 200 µM MSC was required to produce at best a very modest decrease in cell number after 72 h. This concentration of MSC was much higher than that required for MSA to produce growth inhibition. We decided to choose the MCF10AT1 cell line for additional studies because we felt that a less pathologically advanced premalignant cell line would be more appropriate for investigating the molecular targets of selenium chemoprevention.
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clearly show a significant block in G0-G1 phase of the cell cycle, especially at 12 h after treatment with 5 µM MSA. The increase in the G0-G1 population was accompanied by a delay of passage of cells to S phase. Synchronized control cells, but not the treated population, moved into S phase. The enhancement in the proportion of G0-G1 cells was most prominent after 12 h of treatment (70 versus 26%); the effect was attenuated after 24 h (56 versus 27%), as cells moved into G2-M and G1 and became more randomized in their progression through the cell cycle.
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, a concentration-dependent induction of apoptosis was apparent at 18 h of MSA treatment; this occurred at a time point succeeding the G0-G1 block seen at 12 h. The increase in apoptosis continued at 21 and 24 h. The TUNEL data were confirmed by flow cytometric analysis of cells labeled with annexin V and PI (Fig. 3)
. Phosphatidylserine externalization is a characteristic of cells undergoing apoptosis. Annexin V has a strong affinity for phosphatidylserine. Staining cells simultaneously with annexin V and PI allows the resolution of intact cells (double-negative), early apoptotic cells (annexin V-positive and PI-negative), and late apoptotic or necrotic cells (double-positive), which can be located in the lower left, lower right, and upper right quadrants of the cytograms of Fig. 3A
, respectively. Because only cells that are annexin V-positive and PI-negative are truly representative of apoptotic cells, the percentage of this cell population was quantitated from four individual experiments and shown in a bar graph form in Fig. 3B
. It is clear that MSA was able to induce progressive apoptosis in a time-dependent manner, resulting in a 5-fold increase in the number of apoptotic cells at the 24-h time point. This magnitude of change was comparable with the 4-fold induction detected by the TUNEL assay (Fig. 2)
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2 or 
0.5 in expression level in two independent experiments were scored as significant and are reported in Table 3
. Of the 30 genes identified, 22 were significantly repressed and 7 significantly induced at either or both time points. Cyclin D1 was the only gene that was repressed at the early time point but induced at the later time point.
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Our next step was to confirm the changes in expression of a subset of 15 genes by Western blot (if the antibody were available) or by semiquantitative RT-PCR analyses. This subset of genes was chosen based on their key roles in regulating cell proliferation and programmed cell death. We examined 12 genes by Western blots (cyclin A, cyclin B1, cyclin D1, cdc2, cdc6, cdc25A, cdk5, c-jun, DP1, JNK2, MAPKK1, and PCNA) and three genes by semiquantitative RT-PCR (GADD153, E2F1, and E2F5). We were able to verify eight by Western analysis and one (GADD153) by RT-PCR. This represents a success rate of 60% (9 out of a total of 15). The Western blot and RT-PCR results are shown in Fig. 4
and 5
, respectively. The lack of complete concordance could be attributable to either false positive signals of the array data or the discrepancy between transcript and protein expression. There were two other noteworthy observations. First, we found a decreased level of c-jun protein at the 6-h time point, but an up-regulation of c-jun expression detected by array and Western analyses at the 12-h time point. Cells lacking c-jun are known to stay in cell cycle arrest and not to undergo apoptosis (15)
. It is possible that at the early time point, the repression of c-jun was necessary for initiating MSA-mediated cell cycle arrest, whereas at the later time point when G0-G1 arrest was firmly established, cells produced more c-jun in order for apoptosis to advance. Second, although the array data showed a down-regulation of JNK2, we were unable to detect altered JNK2 expression at the total protein level. Instead, we found that the amount of phosphorylated JNKs was elevated at 12 h post-MSA treatment (Fig. 4)
. Clearly, both observations highlighted a fundamental limitation of the array technology in that changes in gene expression that occur posttranscriptionally would not be identifiable.
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. The 28 cycles of the PCR reaction fell within the linear range of amplification as determined by plotting the intensity of each band against the number of PCR cycles. An 8-fold increase was observed at 6 h of MSA treatment, when we normalized the GADD153 RT-PCR signal obtained with 28-cycle of amplification to the one for the GAPDH gene. Therefore, the microarray data and the RT-PCR data for GADD153 are very much consistent with each other (Table 3)
. ATF/cAMP-responsive-element-binding protein (CREB) binding site is one of the numerous cis-acting elements in the GADD153 promoter that is known to regulate the expression of this gene (16)
. Our Western blot analysis showed a substantial induction of phosphorylated ATF2 at 6 h post-MSA treatment (Fig. 4
, last panel). The coordinated up-regulation of GADD153 and the active form of ATF2 suggests a possible role of ATF2 in MSA-induced GADD153 expression. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our effort to identify selenium-responsive biomarkers and to understand the signal transduction pathways leading to MSA-induced cell cycle arrest and programmed cell death, we used the cDNA microarray technology to elucidate the changes in gene expression profile. On the basis of the collection of 200 genes screened by two Clontech arrays, we have identified 30 genes responsive to MSA treatment. Some of these genes are responsible for cell cycle arrest, whereas others are likely to play a role in apoptosis. We further characterized the signal from 15 genes by either Western blots or semiquantitative RT-PCR. We were able to verify the expression changes in 60% of the candidate genes with one or the other of the two methods. Because this is still an emerging technology and the reliability of the array data has not been widely studied and published in the literature, there is little guidance to compare our experience with that of other investigators. Armed with the proper precautions, the cDNA microarray technology can be used judiciously to examine global changes in gene expression as a consequence of a particular intervention.
On the basis of the results obtained from the present study, we propose a number of tentative signaling pathways that could mediate the outcome of MSA-induced cell cycle arrest and apoptosis. As shown in Fig. 6
, selenium treatment down-regulates the expression of DP1, which is a dimerization partner of E2F proteins. E2Fs are known to positively regulate the transcription of cdc25A (17)
. Thus, the decrease in the formation of E2Fs and DP1 dimers reduces the transcription of cdc25A. The phosphatase activity of cdc25A removes the inhibitory phosphate group from cdc2 (cdk1) and cdk2, allowing them to be activated by cdk-activating kinases, and to bind to cyclin A or B. These latter processes are expected to facilitate cell cycle progression. A deficiency in cdc25A would be expected to block cell cycle transition. In addition, the expressions of cdc2, cyclin A, and cyclin B1 are repressed by selenium, thereby providing an amplified effect on this cascade of events. Selenium also down-regulates cdk4 and its partner, cyclin D1 (at the early time point), both of which are required for G1-S transition. Furthermore, GADD153, which plays an essential role in DNA damage-induced cell cycle arrest and apoptosis (18)
, is induced by MSA. It should be noted that MSA does not exert a genotoxic effect on cultured cells (8)
. Hence the up-regulation of GADD153 is probably mediated by a novel mechanism involving the activation of ATF2 (16)
. In summary, Fig. 6
shows that different pathways modulated by selenium all converge to effect a G0-G1 block.
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represents our hypothesis of the pathways underlying the induction of apoptosis by selenium. At the later time point, selenium increases the expression of cyclin D1 and cdk5; these two proteins have been shown to be associated with an increase in apoptosis (19)
. Additionally, another known mediator of apoptosis, c-jun, is also up-regulated by selenium at the 12-h time point. Furthermore, c-jun could be activated through the action of phosphorylated JNKs, of which an induction by selenium was detected by Western blotting (see Fig. 4
). On the other hand, a key player of the survival pathway, AKT2, is down-regulated by selenium. Collectively, the switch-on of pro-apoptosis signals in conjunction with the switch-off of survival signals would produce a more powerful stimulus to programmed cell death.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grants CA 45164 and CA 27706 from the National Cancer Institute, Roswell Park Cancer Institute Core Grant CA 16056 from the National Cancer Institute, and AACR-Cancer Research Foundation of America Fellowship in Prevention Research. 
2 To whom requests for reprints should be addressed, at Department of Experimental Pathology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-8875; Fax: (716) 845-8100; E-mail: clement.ip{at}roswellpark.org
3 The abbreviations used are: MSA, methylseleninic acid; MSC, methylselenocysteine; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; PI, propidium iodide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ATF, activating transcription factor; MAPKK, mitogen-activated protein kinase kinase.
Received 9/24/01. Accepted 12/ 3/01.
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