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Exit from arsenite-induced mitotic arrest is p53 dependent.

BACKGROUND: Arsenic is both a human carcinogen and a chemotherapeutic agent, but the mechanism of neither arsenic-induced carcinogenesis nor tumor selective cytotoxicity is clear. Using a model cell line in which p53 expression is regulated exogenously in a tetracycline-off system (TR9-7 cells), our laboratory has shown that arsenite disrupts mitosis and that p53-deficient cells [p[53.sup.(-)]], in contrast to p53-expressing cells [p[53.sup.(+)]], display greater sensitivity to arsenite-induced mitotic arrest and apoptosis.

OBJECTIVE: Our goal was to examine the role p53 plays in protecting cells from arsenite-induced mitotic arrest.

METHODS: p[53.sup.(+)] and p[53.sup.(-)] cells were synchronized in [G.sub.2] phase using Hoechst 33342 and released from synchrony in the presence or absence of 5 [micro]M sodium arsenite.

RESULTS: Mitotic index analysis demonstrated that arsenite treatment delayed exit from [G.sub.2] in p[53.sup.(+)] and p[53.sup.(-)] cells. Arsenite-treated p[53.sup.(+)] cells exited mitosis normally, whereas p[53.sup.(-)] cells exited mitosis with delayed kinetics. Microarray analysis performed on mRNAs of cells exposed to arsenite for 0 and 3 hr after release from [G.sub.2] phase synchrony showed that arsenite induced inhibitor of DNA binding-1 (ID1) differentially in p[53.sup.(+)]and p[53.sup.(-)] cells. Immunoblotting confirmed that ID1 induction was more extensive and sustained in p[53.sup.(+)] cells.

CONCLUSIONS: p53 promotes mitotic exit and leads to more extensive ID1 induction by arsenite. ID1 is a dominant negative inhibitor of transcription that represses cell cycle regulatory genes and is elevated in many tumors. ID1 may play a role in the survival of arsenite-treated p[53.sup.(+)] cells and contribute to arsenic carcinogenicity.

KEYWORDS: apoptosis, arsenite, ID1, microarray, mitotic arrest, p53.

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Arsenic is both a human carcinogen causing cancer in multiple tissues [National Research Council (NRC) 1999, 2001] and a chemotherapeutic agent used in the treatment of acute promyelocytic leukemia (Cohen et al. 2001). Arsenic trioxide shows promise for chemotherapy in treatment of solid tumors and inhibits tumor growth in an orthotopic prostate cancer model (Maeda et al. 2001). However, the mechanism by which arsenic selectively induces cell death in tumor cells is not understood.

Clinically achievable concentrations of arsenite induce cell death in a variety of cancer cell lines and virally immortalized cell lines that lack normal cell cycle control. Arsenic trioxide ([As.sub.2][O.sub.3]) induces apoptosis in several prostate and ovarian cancer cell lines (Du and Ho 2001; Uslu et al. 2000). Arsenite induces mitotic arrest and apoptosis in SV40-transformed human fibroblasts (States et al. 2002), spontaneously immortalized p53-deficient Li-Fraumeni human fibroblasts (Taylor et al. 2006), and in HeLa S3 and KB cells (Huang and Lee 1998). In contrast, these moderate concentrations of arsenite slow the growth of diploid human fibroblasts but do not induce cell death (States et al. 2002; Yih et al. 1997). However, arsenite disrupts normal mitotic progression and induces aneuploidy in human diploid fibroblasts (Yih et al. 1997) and peripheral blood lymphocytes (Vega et al. 1995).

A common feature of the cell lines in which arsenite induces apoptosis is a p53-deficient phenotype, either by mutation or viral oncogene inactivation of p53. p53 regulates cell cycle progression and apoptosis in response to genetic damage. The molecular mechanisms by which p53 arrests cell cycle progression in [G.sub.1] and [G.sub.2] phases in response to DNA damage are reasonably well understood (Jin and Levine 2001). p53 is activated in response to mitotic disruption by arsenic (Yih and Lee 2000). However, the signals for p53 activation and the role that p53 plays in the cellular response to mitotic disruption are not clear.

In asynchronous TR9-7 cells (human fibroblasts with tetracycline-regulated p53 expression), arsenite treatment induces a p53-independent accumulation of cells with [G.sub.2]/M phase DNA content (Taylor et al. 2006). However, accumulation of mitotic cells occurs in p53-deficient [p[53.sup.(-)]] but not in p53-expressing [p[53.sup.(+)]] TR9-7 cells. Furthermore, the mitotically arrested p[53.sup.(-)] cells undergo apoptosis, whereas most p[53.sup.(+)] cells exit [G.sub.2]/M phase and arrest in [G.sub.1] phase. Therefore, we hypothesized that p53 did not prevent delayed [G.sub.2] transit but did protect against apoptosis subsequent to the arsenite-induced mitotic arrest.

To investigate the role of p53 in the arsenite-induced disruption of [G.sub.2]/M phase progression, we examined the response of p[53.sup.(+)] and p[53.sup.(-)] TR9-7 cells to arsenite treatment after release from [G.sub.2] phase synchrony. We show that in synchronized TR9-7 cells arsenite delayed entry into mitosis independently of p53 expression but that the exit from mitosis in arsenite-treated cells was p53 dependent. Furthermore, we show that the apoptotic process occurred while the cells attempted cytokinesis. We hypothesized the difference in exit kinetics and resistance to apoptosis between p[53.sup.(+)] and p[53.sup.(-)] cells was due to p53-mediated alterations of gene expression profiles. Microarray analysis of [G.sub.2] phase-synchronized p[53.sup.(+)] and p[53.sup.(-)] cells treated with arsenite for 3 hr revealed p53-dependent induction of inhibitor of DNA binding-1 (ID1). Negative transcriptional regulation of cyclin-dependent kinase inhibitors by ID1 may have implications for cell cycle progression in p[53.sup.(+)] cells surviving arsenite-induced mitotic perturbation and may play a role in arsenic-induced carcinogenesis.

Materials and Methods

Cell culture and synchronization. TR9-7 cells were the gift of M.A. Tainsky (Wayne State University, Detroit, MI). Cells were cultured at 37[degrees]C at 5% C[O.sub.2] in a humidified incubator in alpha modification of minimal essential media (GIBCO/BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 10 mM HEPES (pH 7.0), 200 [micro]g/mL geneticin (Invitrogen Corp., Carlsbad, CA), 40 [micro]g/mL hygromycin B (Sigma Chemical Co., St. Louis, MO), 100 U/mL penicillin, and 10 U/mL streptomycin.

Cells were synchronized in [G.sub.2] phase of the cell cycle according to adaptation of published procedures (Kuhholzer and Prather 2001; Tobey et al. 1990). Briefly, we plated cells in the presence of 15 ng/mL tetracycline (Fisher Scientific, Pittsburgh, PA) to allow moderate p53 expression. We replaced media with media containing 5 [micro]g/mL aphidicolin (Sigma) and incubated the cells for 24 hr. We then washed the cells with phosphate-buffered saline (PBS) and incubated them in media containing 0.1 [micro]g/mL Hoechst 33342 (Sigma) for 12 hr. After 6 hr of incubation with Hoechst 33342, p53 expression was suppressed in half the cultures by direct addition of tetracycline (1 mg/mL) to the media to a final concentration of 1,500 ng/mL. Cells were released from [G.sub.2] phase by washing with PBS and refeeding with fresh drug-free media.

Arsenic compounds. We prepared fresh working aqueous solutions of sodium arsenite (NaAs[O.sub.2]) (Sigma) on the day of treatment and filter sterilized prior to use.

Flow cytometry. For DNA content analysis, cells were harvested by trypsinization and fixed in 1 mL ice-cold 70% ethanol. Following fixation overnight at 4[degrees]C, we centrifuged the samples at 1,500 x g and resuspended the resulting cell pellets in PBS containing propidium iodide and 100 U/mL RNase A (Sigma) for 30 min at room temperature. We analyzed propidium iodide-stained samples on a FACSCalibur (Becton Dickinson and Co., San Jose, CA) using doublet discrimination. Propidium iodide fluorescence was collected on channel two (FL2) (585/42 nm) using linear amplification. A minimum of 20,000 events/sample was analyzed. Data were collected using CellQuest software (Becton Dickinson) and analyzed for cell cycle distribution using Modfit software (Verity Software House, Inc., Topsham, ME).

Mitotic indices. Cells were harvested for mitotic index analysis by trypsinization. Media, wash, and cells were collected together and centrifuged. We resuspended cell pellets in 150 [micro]L serum free media and added 2.5 mL of 0.4% KCl. We incubated the suspension for 10 min at 37[degrees]C, then added methanol:acetic acid fixative solution (3:1, vol/vol) to 2% (vol/vol) and collected cells by centrifugation. Cells were resuspended in 2.5 mL fixative solution and fixed at room temperature for 20 min. Samples were centrifuged; cell pellets were resuspended in 0.5 mL fixative and chilled on ice for 1 hr. Aliquots of the suspensions were dropped onto glass slides, air dried, and stained with Wright Giemsa solution (Fisher). We examined slides under a microscope and counted a minimum of 200 cells to determine mitotic index.

Western blot analyses. Media from culture dishes were removed and collected along with one PBS wash. Floating cells were pelleted via centrifugation, washed once with PBS, and lysed with lysis buffer [10 mM Tris-HCl (pH 7.4), 1 mM [Na.sub.2]-EDTA, 0.1% SDS, 180 [micro]g/mL phenylmethylsulfonyl fluoride]. Adherent cells were lysed directly in the plates. Lysates from floating and adherent fractions were then combined. Protein concentration in the lysates was determined by Bradford assay (BioRad Laboratories, Inc., Hercules, CA). Proteins were resolved by SDS-PAGE in 12% polyacrylamide gels and transferred to supported nitrocellulose membranes by electroblotting. p53, [beta]-actin, and ID1 were detected by probing membranes with antibodies for p53 (LabVision, Fremont, CA), [beta]-actin (Sigma), or ID1 (Santa Cruz Biotechnologies, Santa Cruz, CA), respectively. Blots were incubated with secondary anti-mouse or anti-rabbit antibody conjugated to horse radish peroxidase (Zymed Laboratories, San Francisco, CA) and visualized with enhanced chemiluminescence (Amersham Biosciences Inc., Piscataway, NJ).

Statistical analyses. Comparison of mitotic indices of arsenite-treated p[53.sup.(+)] and p[53.sup.(-)] cells was performed by Student t-test using Slide Write software (version 6.10; Advanced Graphics Software, Encinitas, CA) and confirmed by analysis of variance (ANOVA).

Microarray analyses. We harvested cells via trypsinization and prepared poly(A)[.sup.+] mRNAs using Micro-FastTrack (Invitrogen Corp.). SuperScript II (Invitrogen Corp.) was used with an oligo(dT) primer linked to the T7 RNA polymerase-binding site sequence to synthesize cDNAs. cDNAs were extracted with phenol:chloroforam:isoamyl alcohol (25:24:1), precipitated with N[H.sub.4]OAc and ethanol, and resuspended in RNase-free water. We synthesized biotin-labeled cRNA with an ENZO RNA transcript labeling kit (Enzo Life Sciences, Farmingdale, NY), using 0.7 [micro]g cDNA per reaction. cRNAs were purified with RNeasy purification columns (Qiagen, Valencia, CA). cRNA, 15 [micro]g per sample, was heated at 80[degrees]C for 33 min in fragmentation buffer (Affymetrix, Inc., Santa Clara, CA). Fragmented cRNAs were hybridized to U95Av2 GeneChips (Affymetrix) for 16 hr at 45[degrees]C. GeneChips were stained with streptavidin phycoerythrin stain solution (Affymetrix). Signal was amplified with goat anti-streptavidin antibody and biotinylated goat IgG (Affymetrix). We scanned stained GeneChips on an Agilent GeneArray Scanner (Agilent Technologies, Palo Alto, CA) and performed four biological replicates.

We performed ANOVA of the signal data with Bioconductor/R software using MAS (Microarray Suite software) background correction, quantiles normalization, MAS PM (perfect match) correction, MAS summary, and statistical significance defined as p < 0.05. Data were filtered on p53 status and arsenite exposure. Analysis was conducted with and without application of the Benjamini and Hochberg false discovery rate correction. Venn analysis was conducted on gene lists using GeneSpring software (Agilent Technologies).

Results

To determine the role that p53 plays in preventing arsenite-induced accumulation of mitotic cells and in preventing arsenite-induced apoptosis, TR9-7 cells were synchronized in [G.sub.2] using Hoechst 33342. Hoechst 33342 is a topoisomerase inhibitor that interferes with chromatin condensation, resulting in [G.sub.2] phase arrest (Kuhholzer and Prather 2001). The effects of arsenite treatment on the progression from [G.sub.2] phase and through mitosis of synchronized p[53.sup.(+)]) and p[53.sup.(-)] cells were evaluated after release from Hoechst 33342-induced [G.sub.2] phase synchronization.

TR9-7 cells are a spontaneously immortalized human fibroblast cell line, derived from a Li-Fraumeni patient, and subsequently stably transfected with a tetracycline-regulated p53 expression vector (Yin et al. 1992). p53 expression in TR9-7 cells is inversely proportional to the tetracycline concentration in the media (tet-off). Absence of tetracycline strongly induces p53 and causes TR9-7 cells to arrest in [G.sub.1] and [G.sub.2] phases (Agarwal et al. 1995). Tetracycline at a concentration of 15 ng/mL induces p53 to a modest level that allows the cells to grow at a rate comparable to p[53.sup.(-)] TR9-7 cells (Taylor et al. 2006).

Synchronization of TR9-7 cells in [G.sub.2] phase using Hoechst 33342. Synchronization of TR9-7 cells in [G.sub.2] phase required plating cells with moderate p53 expression. Cells with high levels of p53 tended to arrest in [G.sub.1] or [G.sub.2] phase and not re-enter the cell cycle. In addition, induction of p53 by lowering the tetracycline concentration proved difficult to time reproducibly. We obtained reproducible changes in p53 expression by repressing p53 by addition of tetracycline (data not shown). The experimental design we developed is presented in Figure 1. Cells were synchronized in [G.sub.1] phase by addition of aphidicolin after allowing freshly plated cells to attach and to acclimate for 24 hr. Cells were released from [G.sub.1] phase synchrony after 24-hr aphidicolin exposure by washing the cells and applying fresh media containing Hoechst 33342, which induced [G.sub.2] phase synchrony. After 6 hr of incubation in Hoechst 33342 media, p53 transcription was suppressed in half the cultures by addition of tetracycline to 1,500 ng/mL. Cells were incubated for an additional 6 hr. At this point, media were changed again and half of each tetracycline group received media containing 0 or 5 [micro]M NaAs[O.sub.2] with tetracycline appropriate to maintain p53 expression levels. Samples were taken at each change of media and at several time points post-Hoechst 33342 incubation. Samples were analyzed with flow cytometry to determine cell cycle distribution (Figure 1B), by Western blot to determine p53 and ID1 expression (Figure 2), and for mitotic spread analysis to determine mitotic index (Figure 3A). We performed microarray analysis to determine changes in gene expression (Tables 1-3).

Cell cycle distribution of synchronized cells. Using the procedure depicted in Figure 1A, we were able to achieve reasonably good synchronization. Unsynchronized TR9-7 cells show a typical cell cycle distribution with a majority of the cells in [G.sub.1] phase and a distinct but small [G.sub.2]/M phase peak (Figure 1B). After aphidicolin treatment, > 90% of cells were in the [G.sub.1] phase (Figure 1B). After incubation with Hoechst 33342, approximately 60% of the cells were in [G.sub.2] phase, as characterized using ModFit software to analyze the flow cytometry data (Figure 1B). The approximately 40% of cells remaining in [G.sub.1] phase appeared irreversibly arrested in [G.sub.1].

p53 expression in cells after [G.sub.2] synchronization. p53 levels were assessed in cells during synchronization and after release from [G.sub.2] phase. Generally, p53 expression was maintained consistently in cultures with low tetracycline and suppressed by tetracycline at 1,500 ng/mL throughout the experiment (Figure 2). The achievement of a high level of synchrony in the cultures required the moderate expression of p53 that was suppressed by addition of tetracycline at 1,500 ng/mL 6 hr prior to release from [G.sub.2] phase blockade. A modest amount of residual p53 was detectable in 1,500 ng/mL tetracylcline cultures [p[53.sup.(-)]] at 0 and 3 hr after release, although p53 expression was much lower than that observed in cultures with only 15 ng/mL tetracycline [p[53.sup.(+)]].

Arsenite delays mitotic exit selectively in p[53.sup.(-)] cells. We examined arsenite effects on entry into and exit from mitosis in synchronized cells. Mitotic indices and p53 expression were determined in triplicate cultures exposed or not exposed to 5 [micro]M NaAs[O.sub.2] and harvested 0, 1, 3, 6, 12, 16, 20 and 24 hr after release from [G.sub.2] phase synchrony induced by Hoechst 33342 exposure. Mitotic indices were determined only in experiments in which the p53 expression was maintained as expected. The mitotic index of cells not exposed to arsenite peaked 3 hr after release from [G.sub.2] phase (Figure 3A). There was no difference between p[53.sup.(+)] and p[53.sup.(-)] cells. The mitotic index dropped sharply after 3 hr and was < 1% by 12 hr. The mitotic index started to increase again 24 hr after release from [G.sub.2] phase, indicating that the cells had started to cycle again. Arsenite treatment delayed the peak in mitotic index to 6 hr after release from [G.sub.2] phase in both p[53.sup.(+)] and p[53.sup.(-)] cells. The mitotic index of p[53.sup.(+)] cells treated with arsenite decreased after 6 hr and returned to baseline by 16 hr. The slope of the decrease in mitotic index of arsenite-treated p[53.sup.(+)] cells is similar to that of the decline in cells not exposed to arsenite. These results suggest that entry into mitosis is delayed in p[53.sup.(+)] cells but that mitotic exit is normal. In contrast, the mitotic index of p[53.sup.(-)] cells treated with arsenite dropped slowly and was still significantly elevated 16 hr (p < 0.001, n = four experiments) after release from [G.sub.2] phase arrest. The mitotic index dropped to nearly zero by 24 hr after release from [G.sub.2] phase.

Arsenite induces alteration of expression profiles. Microarray analysis was conducted on mRNAs prepared from p[53.sup.(+)] and p[53.sup.(-)] cells at 0 and 3 hr after release from [G.sub.2] phase synchrony with or without arsenite exposure. ANOVA of four biological replicates using a p < 0.05 was performed with Bioconductor/R software using MAS background correction, quantiles normalization, MAS PM correction, and MAS summary. Analysis was conducted with (Table 1) and without (Tables 2, 3) application of the Benjamini and Hochberg false discovery rate (FDR) correction. Table 1 is all inclusive, whereas Table 2 includes only those genes showing [greater than or equal to] 2-fold increase in either p[53.sup.(+)] or p[53.sup.(-)] samples. Arsenite induced several genes independently of p53 status. Heme oxygenase 1 (HMOX1) showed a dramatic > 25-fold increase in expression. Metallothioneines 2A and 1X and the zinc transporter SLC30A1 were also induced. Expression of the stress-responsive heat shock protein HSP70B was elevated > 7-fold. ID1 was induced 3.7- to 6.9-fold in p[53.sup.(+)] cells and 2.5- to 4.3-fold in p[53.sup.(-)] cells depending on which of three probe sets was evaluated. Although two-way ANOVA did not indicate that the difference in fold-change between p[53.sup.(+)] and p[53.sup.(-)] cells was statistically significant, Western blot analysis did show ID1 induction that was more extensive and sustained in p[53.sup.(+)] cells (Figure 2). Two-way ANOVA did not identify any genes or which expression was dependent on p53; therefore, Venn analysis was performed with GeneSpring software (Agilent Technologies) on lists of genes altered in p[53.sup.(+)] and p[53.sup.(-)] samples by arsenite treatment. The false discovery rate correction was not applied. This approach identified several genes altered by arsenite in only p[53.sup.(+)] or p[53.sup.(-)] (Table 3). For example, MAP kinase phosphatase 1 (MKP-1) was induced only in p[53.sup.(+)] cells and ubiquitin-conjugating enzyme E2N was induced only in p[53.sup.(-)] cells.

Arsenite induces cytokinesis failure and apoptosis. An example of a mitotic cell undergoing apoptosis in an arsenite-treated culture is shown in Figure 3B. Mitosis, from the start of prophase to the completion of cytokinesis, normally takes < 30 min in these cells. As can be observed in Figure 3B, 30 min elapsed from the time a cell was in late anaphase/early telophase (panel 1) to when the cell collapsed (panel 8). The metaphase plates holding the chromosomes did not change position while the membrane developed multiple blebs (panels 1-4). The chromosomes appeared to partially decondense (panel 5). The cell body collapsed between the plates but the membranes did not fuse to complete cytokinesis (panels 6-8).

Discussion

Arsenite (as either NaAs[O.sub.2] or [As.sub.2][O.sub.3]) is known to activate the [G.sub.2] phase checkpoint (Chen et al. 2002) and to induce mitotic arrest and apoptosis in a variety of cell lines including HeLa (Huang and Lee 1998), U937 (Halicka et al. 2002; McCabe et al. 2000), SV40-transformed human fibroblasts (Halicka et al. 2002; McCabe et al. 2000; States et al. 2002), and several prostate and ovarian carcinoma cells (Uslu et al. 2000). A common feature of the cells in which arsenite induces both mitotic arrest and apoptosis is a functional loss of p53. We have shown that arsenite-induced mitotic arrest occurs in asynchronous cells lacking p53 expression but not in cells expressing p53 (Taylor et al. 2006). In the present study, we have determined whether p53 expression affects the progression of arsenite-treated cells from [G.sub.2] phase into M phase and/or the exit from mitosis.

TR9-7 cells are a well-characterized model to study p53-dependent processes, taking advantage of tetracycline-regulated p53 expression (Adimoolam and Ford 2002; Agarwal et al. 1995; Ford and Hanawalt 1997; Lloyd and Hanawalt 2002; Robles et al. 2001; Taylor et al. 2001) We synchronized these human fibroblasts in [G.sub.2] phase using a multi-step protocol developed by others (Kuhholzer and Prather 2001; Tobey et al. 1990) and adapted to our experiments. The fraction of the cells in [G.sub.2] phase was not as high as some investigators have achieved with normal diploid fibroblasts (Tobey et al. 1990) but higher than that achieved by another group (Kuhholzer and Prather 2001).

Our results indicate that arsenite exposure delays progression from [G.sub.2] phase to M phase in both p[53.sup.(-)] and p[53.sup.(+)] cells. This result is consistent with reported arsenite-induced [G.sub.2] phase delay (McCollum et al. 2005; Park et al. 2001). However, the exit from mitosis was delayed only in p[53.sup.(-)] cells. The mitotic index in arsenite-treated p[53.sup.(+)] cells declined with the same kinetics as in untreated cells. The mitotic index declined more slowly in arsenite-treated p[53.sup.(-)] cells, suggesting that these cells exit mitosis more slowly. The arsenite-treated cells entered apoptosis while futilely attempting cytokinesis. These data indicate that arsenite delays entry into mitosis and progression to anaphase in TR9-7 cells regardless of p53 expression but that exit from mitosis is p53 dependent in arsenite-treated cells. Thus, both entry into and exit from mitosis is delayed in p[53.sup.(-)] cells. Furthermore, the p[53.sup.(-)] cells undergo apoptosis as a result of failed or abnormal mitotic exit.

As p53 is a transcription factor, we hypothesized that expression profile changes could contribute to delayed mitotic exit kinetics seen in p[53.sup.(-)] cells. To test this, we performed microarray analysis of RNAs prepared from [G.sub.2] phase synchronized p[53.sup.(+)] and p[53.sup.(-)] cells harvested at 0-hr and after 3-hr exposure to 5 [micro]M sodium arsenite. HMOX1 was highly and rapidly induced, independently of p53. HMOX1 induction is a well-documented response to arsenite (Andrew et al. 2003; Liu et al. 2001). HMOX1 induction is associated with cellular response to oxidative damage, as the degradation products of heme are known to scavenge free radicals (Abraham 2003).

The induction of metallothioneins 2A and 3 may also serve a protective role, as metallothionein has been shown to bind arsenicals (Jiang et al. 2003) and metallothionein null mice are hypersensitive to arsenite toxicity (Liu et al. 2000). MKP1, a phosphatase, was induced in p[53.sup.(+)] cells and may serve a protective role as it opposes the activation of SAPK/JNK (Li et al. 2003), a kinase involved in the induction of apoptosis by arsenite (Davison et al. 2004), MKP1 is a transcriptional target of p53 (Li et al. 2003) and has been shown to be arsenite-induced (Li et al. 2001). The expression of ID1 was elevated by arsenite and Western blot analysis showed the extent of induction was greater and more sustained in p[53.sup.(+)] cells. ID1 is a dominant negative inhibitor of transcription that forms inactive heterodimers with basic helix-loop-helix transcription factors. Genes identified as targets of repression by ID1 include those encoding the cyclin-dependent kinase inhibitory proteins p[15.sup.INK4B], p[16.sup.INK4A], and p[21.sup.CIP1/WAF1] (Sikder et al. 2003). Our previous results indicate that the increased sensitivity to mitotic arrest associated apoptosis of p53-deficient cells is due to the lack of p[21.sup.CIP1/WAF1], a transcriptional target of p53 (Taylor et al. 2006). P[21.sup.CIP1/WAF1] may prevent mitotic arrest associated apoptosis in p[53.sup.(+)] cells by allowing for mitotic exit of cells arrested in mitosis. Mitotic exit requires the destruction of cyclin B or inhibition of the cyclin B/CDC2 complex. The promotion of mitotic exit by p[21.sup.CIP1/WAF1] would involve its direct inhibition of cyclin B/CDC2 (Taylor and Stark 2001). However, once p[53.sup.(+)] cells exit mitosis, cell cycle progression would require the inhibition of p[21.sup.CIP1/WAF1]. The induction of ID1 by arsenite may serve to repress p[21.sup.CIP1/WAF1] and allow for continued cell cycle progression (Figure 4).

Ectopic overexpression of ID1 immortalizes primary keratinocytes (Alani et al. 1999) and a portion of the ID1 localizes to centrosomes, causing abnormal centrosome number (Hasskarl et al. 2004). Centrosomes serve as the organizational units for the mitotic spindle apparatus. Therefore, an aberrant number of centrosomes may be aneuploidogenic by preventing proper chromosomal segregation. The induction of aneuploidy was posited as the most likely mechanism of arsenic carcinogenesis (NRC 1999, 2001). ID1 also protects against apoptosis through activation of the nuclear factor kappa B signaling pathway (Ling et al. 2003). Higher ID1 levels in p[53.sup.(+)] cells may therefore promote survival. ID1 basal levels are elevated in a variety of cancers, and ID1 is induced during malignant transformation of rat liver cells by arsenic (Chen et al. 2001). Consequently, ID1 induction by arsenite may play an important role in the mechanism of arsenic carcinogenesis by allowing for survival of cells progressing through aberrant mitosis and inducing abnormal chromosome numbers.

Loss of p53 function as seen in many tumors has been suggested to allow cells to bypass cell cycle checkpoints after DNA damage and to enter a nonmitotic state (Erenpreisa and Cragg 2001). Our results suggest p53 plays an important role in prevention of arsenite-induced mitotic arrest and also in apoptosis consequent to the arrest. The prevention of mitotic arrest-associated apoptosis is likely a key event in arsenic-induced carcinogenesis because it predisposes cells to aneuploidy. Understanding the link between p53 and prevention of apoptosis associated with arsenite-induced mitotic arrest will provide essential information regarding the mechanism of arsenic carcinogenesis and also help identify a key target for cancer chemotherapeutics.

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Samuel C. McNeely, (1) Xiaogiang Xu, (1) B. Frazier Taylor, (1) Wolfgang Zacharias, (1,2,3,4) Michael J. McCabe Jr., (5) and J. Christopher States (1,3,4)

(1) Department of Pharmacology and Toxicology, (2) Department of Medicine, (3) James Graham Brown Cancer Center, and (4) Center for Genetics and Molecular Medicine, University of Louisville, Louisville, Kentucky, USA; (5) Department of Environmental Medicine, University of Rochester, Rochester, New York, USA

Address correspondence to J.C. States, Dept. of Pharmacology and Toxicology, University of Louisville, 570 S. Preston St., Suite 221, Louisville, KY 40202 USA. Telephone: (502) 852-5347. Fax: (502) 853-2492. E-mail: jcstates@louisville.edu

This work was supported in part by National Institutes of Health grants R01 ES06460, R01 ES11314, F30 ES013372, T32 ES011564, and P30 ES01247, and the James Graham Brown Cancer Center.

The authors declare they have no competing financial interests.

Received 29 December 2005; accepted 22 June 2006.
Table 1. Arsenite-dependent gene induction detected by microarray
analysis at 0 and 3 hr after release from [G.sub.2] phase synchrony with
FDR applied. (a)

UniGene ID Probe set ID Gene title

517581 33802_at heme oxygenase (decycling) 1
504609 36618_g_at inhibitor of DNA binding 1
418241 39081_at metallothionein 2A
519469 34759_at solute carrier family 30 (zinc transporter),
 member 1
504609 36619_r_at inhibitor of DNA binding 1
504609 36617_at inhibitor of DNA binding 1
534330 36130_f_at metallothionein 1E (functional)
513626 31622_f_at metallothionein 2A
284141 33835_at TSPY-like 4
282326 32168_s_at Down syndrome critical region gene 1
 37055 1732_at fibroblast growth factor 5
440939 31623_f_at metallothionein 2A
105269 33369_at sterol-C4-methyl oxidase-like
467020 1700_at BCL2 binding component 3
503093 32588_s_at zinc finger protein 36, C3H type-like 2

 Fold-change
UniGene ID Gene symbol p[53.sup.(+)] p[53.sup.(-)]

517581 HMOX1 27.2 26.1
504609 ID1 6.9 4.3
418241 MT2A 3.9 3.6
519469 SLC30A1 3.2 3.1
504609 ID1 3.8 2.7
504609 ID1 3.7 2.5
534330 MT1E 1.9 1.9
513626 MT2A 1.9 1.8
284141 TSPYL4 1.7 1.9
282326 DSCR1 1.6 1.8
 37055 FGF5 1.7 1.6
440939 MT2A 1.6 1.5
105269 SC4MOL 1.4 1.5
467020 BBC3 0.7 0.6
503093 ZFP36L2 0.4 0.3

(a) Gene annotations from NetAffx
(http://www.affymetrix.com/analysis/index.affx).

Table 2. Arsenite-dependent gene induction detected by microarray
analysis at 0 and 3 hr after release from [G.sub.2] phase synchrony with
FDR not applied. (a)

UniGene ID Probe set ID Gene title

517581 33802_at heme oxygenase (decycling) 1
 3268 35965_at heat shock 70k protein 6 (HSP70B')
504609 36618_g_at inhibitor of DNA binding 1
520028 1104_s_at heat shock 70kDa protein 1A
418241 39081_at metallothionein 2A
609521 34759_at solute carrier family 30 (zinc transporter),
 member 1
504609 36619_r_at inhibitor of DNA binding 1
504609 36617_at inhibitor of DNA binding 1
374950 39120_at metallothionein 1X
148340 41215_s_at inhibitor of DNA binding 2
 799 38037_at diphtheria toxin receptor
153863 1955_s_at SMAD, mothers against DPP homolog 6
 76884 37043_at inhibitor of DNA binding 3
491611 1137_at solute carrier family 20 member 2
517617 36711_at v-maf musculoaponeurotic fibrosarcoma oncogene
520819 35303_at insulin induced gene 1
196054 37483_at histone deacetylase 9

 Fold-change
UniGene ID Gene symbol p[53.sup.(+)] p[53.sup.(-)]

517581 HMOX1 27.2 26.1
 3268 HSPA6 8.5 6.9
504609 ID1 6.9 4.3
520028 HSPA1A 5.8 4.9
418241 MT2A 3.9 3.6
609521 SLC30A1 3.2 3.1
504609 ID1 3.8 2.7
504609 ID1 3.7 2.5
374950 MT1X 3.3 4.5
148340 ID2 3.1 2.8
 799 DTR 2.8 1.9
153863 SMAD6 2.4 2.0
 76884 ID3 2.4 2.5
491611 SLC20A2 2.2 2.3
517617 MAFF 2.2 1.9
520819 INSIG1 2.1 1.8
196054 HDAC9 2.1 1.7

(a) Gene annotations from NetAffx
(http://www.affymetrix.com/analysis/index.affx).

Table 3. p53-dependent gene induction by arsenite determined by Venn
analysis (FDR not applied). (a)

 Probe Gene Fold-
Unigene ID set ID Gene title symbol change

P[53.sup.(+)]
 cells
 520028 31692_at heat shock 70kDA protein 1A HSPA1A 4.7
 419 34585_at distal-less homeo box 2 DLX2 2.3
 171695 1005_at dual specificity phosphatase 1 DUSP1 2.1
 (MKP-1)
P[53.sup.(-)]
 cells
 142912 36799_at frizzled homolog 2 (Drosophila) FZD2 4.2
 50308 40121_at huntingtin interacting protein HIP2 2.5
 2
 93002 1651_at ubiquitin-conjugating enzyme UBE2C 2.2
 E2C
 524630 36604_at ubiquitin-conjugating enzyme UBE2N 2.2
 E2N

(a) Gene annotations from NetAffx
(http://www.affymetrix.com/analysis/index.affx).
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Title Annotation:Research
Author:States, J. Christopher
Publication:Environmental Health Perspectives
Date:Sep 1, 2006
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