ARSENITE DISRUPTS MITOSIS AND INDUCES APOPTOSIS IN PHENOTYPICALLY P53 NEGATIVE HUMAN SKIN FIBROBLASTS

J. Christopher States^*, Michael J. McCabe Jr.^#, John J. Reiners Jr. ^#, David J. Kaplan^#, Patricia Mathieu^#, Hans G. Sowder^#, Vincent J. Lumetta^# and Joel G. Pounds^%

^*Department of Pharmacology and Toxicology, 570 S. Preston St. Ste. 221, University of Louisville, Louisville, KY 40202, ^#Institute of Chemical Toxicology, Wayne State University, 2727 Second Avenue, Detroit, MI 48201, and ^%Pacific Northwest National Laboratory, Richland, WA

^*Corresponding author, jcstates@louisville.edu

ABSTRACT:

The mechanism by which chronic arsenic intoxication causes skin, bladder and liver cancer is unknown. Normal diploid fibroblasts treated with arsenic exhibit mitotic spindle disruption and prolonged M-phase. The responses of human skin fibroblasts deficient in p53 treated with 0 - 10 mM NaAsO₂ were studied. Both SV40-transformed and spontaneously immortalized p53 deficient (Li-Fraumeni derived) human skin fibroblasts were hypersensitive to arsenite. Cell proliferation was completely inhibited by 10 mM NaAsO₂. Flow cytometric analyses revealed that arsenite treatment induced an accumulation of G₂/M cells. Arsenite treated cells developed membrane blebs suggesting they were apoptotic. Apoptosis was confirmed by annexin V binding and TUNEL posistive staining. Expression of p53 from a transfected expression vector protected immortalized Li-Fraumeni syndrome fibroblasts from arsenite-induced apoptosis. These results suggest that arsenite disrupts mitosis and induces apoptosis in cells incapable of expressing p53.

INTRODUCTION

Arsenic (As³⁺, arsenite) is a human carcinogen with a poorly defined mechanism of action. Efforts to define the mechanism have shown that arsenic is not mutagenic but is clastogenic in in vitro test systems (Rudel 1997, Rossman 1980). Arsenite disrupts cell division and induces aneuploidy in human peripheral blood lymphocytes treated in vitro (Vega 1995). Low concentrations of arsenite disrupt normal spindle retraction, delay mitosis and induce aneuploidy in normal diploid human fibroblasts (Yih 1997). Aneupoloidy was a consequence of imperfect chromosomal segregation in the survivors. Similar results were reported for human peripheral blood lymphocytes (Ramirez 1997). Arsenite also causes severe mitotic disruption in HeLa S3 and KB cells (Huang 1998).

Preliminary experiments in our laboratory indicated that SV40-transformed human fibroblasts were extremely sensitive to arsenite-induced cytotoxicity compared with normal diploid fibroblasts. We tested the hypothesis that the sensitivity of SV40-transformed cells is caused by the lack of p53 function. Results indicating arsenite disruption of cell cycle progression in mitosis and induction of apoptosis in SV40-transformed and spontaneously immortalized p53^(-/-) Li-Fraumeni human fibroblasts are presented. These results suggest that phenotypically p53 negative cells are sensitive to induction of apoptosis consequent to arsenite-induced disruption of mitosis.

METHODS

Cells and culture conditions: SV40-transformed human skin fibroblasts (GM0637, GM4429) were obtained from the NIGMS Human Mutant Cell Repository, Coriell Institute, Camden, NJ. Spontaneously immortalized Li-Fraumeni fibroblasts transfected with a tetracycline controlled p53 expression vector (TR9-7; Bischoff 1990) were the kind gift of Dr. Michael A. Tainsky (Wayne State University). GM4429 and GM0637 cells were cultured in alpha-MEM (GIBCO/BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum product (Fetal Clone II, HyClone, Logan UT) and 10 mM HEPES pH 7.0. TR9-7 cells were cultured in Dulbecco's MEM or alpha-MEM supplemented with 10% fetal bovine serum, 300 mg/mL Geneticin (GIBCO/BRL, Gaithersburg, MD) and 25 mg/mL hygromycin B (Sigma Chemical Co., St. Louis, MO). To suppress p53 expression 100 mg/mL tetracycline was also added to the TR9-7 media.

Arsenic uptake and cytotoxicity: Working solutions of NaAsO₂ (Sigma Chemical Co., St. Louis, MO) were prepared freshly. [⁷³As] was obtained from the Los Alamos National Laboratory as arsenate and was reduced to ([⁷³As]O₂^-) with sodium metabisulfite and sodium thiosulfate (Reay 1977) immediately before use. [⁷³As] was added to freshly prepared stock solutions of unlabeled arsenite to provide approximately 1-2 mCi/ml cell culture medium. Arsenite uptake was determined by counting the 53.4 keV gamma emission in lysates of washed cells. Parallel cultures without radioactivity were examined for cytotoxicity by phase contrast microscopy and by propidium iodide exclusion.

Flow cytometry: For DNA content analysis, cells were harvested by trypsinization and fixed in 1 mL of ice-cold 70% ethanol. Following fixation overnight at 4°C, samples were centrifuged at 1500 x g and the resulting cell pellets were resuspended in PBS containing propidium iodide and 100 units/mL RNase A (Sigma Chemical Co., St. Louis, MO) for 30 min at room temperature. Propidium iodide stained samples were analyzed on a FACSCalibur (Becton Dickinson, San Jose, CA) using doublet discrimination. Propidium iodide fluorescence was collected on FL2 (585/42 nm) using linear amplification. Phosphatidylserine externalization on apoptotic cells was determined following the recommendations detailed by van Engelund et al (1998). Green fluorescence (FL1 530/30 nm), indicative of annexin-V-FITC (Pharmingen, San Diego CA) binding, and red fluorescence (FL2 585/42 nm), indicative of propidium iodide uptake by damaged cells, were collected on a FACSCaliber using logarithmic amplification and electronic compensation for spectral overlap. Data for 20,000 events were collected for each sample.

TUNEL Assay: Cells grown on poly-L-lysine (Sigma Chemical Co., St. Louis, MO) precoated 4 -well glass slides (Nalge Nunc International, Naperville, IL) were treated with arsenite solutions and fixed with freshly prepared 4% formaldehyde (Sigma Chemical Corp. St. Louis, MO; made in PBS). The TUNEL assay was performed as described by the distributors of the In Situ Cell Death Detection Kit (Boehringer-Mannheim, Indianapolis, IN) and fluorescent cells were analyzed by confocal microscopy.

RESULTS AND DISCUSSION

Fig. 1. Concentration dependent [⁷³As] uptake by SV40-transformed human skin fibroblasts. Confluent cells were treated with the indicated concentrations of NaAsO₂ for 24 h. Total [⁷³As] and total cellular protein present in SDS-NaOH lysates of washed cells were determined. Means +/- SD of quadruplicate wells are plotted.

Fig. 2. Arsenite inhibits cell proliferation. GM4429 cells were treated with 10 mM NaAsO₂, harvested by trypsinization and cell number was determined using a Coulter counter. Means +/- SD are plotted.

Fig. 3. Viability of arsenite treated and untreated cells measured as ability to exclude propidium iodide by FACS analysis. Means of triplicates are plotted.

Fig. 4. Arsenite cell cycle disruption. The ratios of cells with G2/M relative to G1 DNA contents is plotted.

Arsenic accumulation per mg cell protein showed concentration-dependence in both SV40-transformed (GM4429, GM0637) human skin fibroblast lines tested (Fig.1). GM4429 cells accumulated slightly more arsenic per mg cell protein than GM0637 cells.

Arsenic was cytotoxic as assessed by phase contrast microscopy, effect on cell growth and cell membrane permeability. Typical concentration-dependent morphological changes occurring in arsenite treated cells included cell rounding, and detachment of cells having the morphology of apoptotic cells. Arsenite concentrations up to 3 mM induced increased numbers of cells apparently arrested in anaphase. At higher arsenite concentrations (5 - 10 mM NaAsO₂), cultures showed an increased number of cells in mitosis (i.e. rounded cells with visible chromosomes) that did not appear to progress. By 48 hr most of these cells had detached, or were loosely attached to the culture dishes, and had developed large ground-glass blebs and shrunken bodies, characteristics of apoptotic cells.

The accumulation of mitotic cells suggested that proliferation was suppressed. Treating GM4429 cells with 10 mM NaAsO₂ inhibited proliferation (Fig. 2). Untreated control cells double twice in 48 h indicating rapid cell replication. The number of arsenite-treated cells increased by less than 30% in the same 48 h period.

Cell viability based on plasma membrane integrity was determined by propidium iodide (PI) exclusion. The percentage of viable cells in control cultures remained constant over time. In contrast, arsenite-treated GM4429 cultures progressively lost the ability to exclude propidium iodide (Fig. 3).

Visual analysis of arsenite treated cells suggested that arsenite induced an accumulation of mitotic cells and that these mitotic cells then underwent apoptosis. To determine if arsenite disrupted mitosis, cellular DNA contents of arsenite treated cells were determined by flow cytometry of propidium iodide stained cells. Cell cycle phase distribution was inferred from the distribution of cellular DNA contents. Treatment with ³5 mM NaAsO₂ induced a concentration-dependent accumulation of cells with G₂/M DNA content paralleled by a decrease in the fraction of cells with G₁ DNA content. These data are consistent with disruption of cell cycle in M-phase.

Kinetic analysis of cell cycle disruption revealed that significant accumulation of cells with G₂/M phase DNA content occurred within 12 h of treatment with 10 mM NaAsO₂ (Fig. 4). These cells were particularly enriched in the detached cell fraction. This fraction was also highly enriched in cells that bound annexin V and excluded propidium iodide indicating they were apoptotic. The detached fraction was also highly enriched in TUNEL positive cells. Thus, apoptosis is induced in mitotically arrested cells.

SV40 transformation results in a functional p53 null phenotype. Because normal diploid fibroblasts are quite refractory to 10 mM NaAsO₂ (Yih 1997), we hypothesized that p53 function protected against apoptosis subsequent to the arsenite-induced mitotic disruption. We tested this hypothesis using spontaneously immortalized Li-Fraumeni fibroblasts transfected with a tetracycline-regulated p53 expression vector (Yin 1992). Arsenite treated cells not expressing p53 were sensitive to arsenite and displayed cell cycle disruption similar to SV40 transformed cells. Expression of p53 in these cells prevented the arsenite sensitivity and cell cycle disruption.

We infer that p53 phenotype is an important determinant of arsenite sensitivity of mitotic cells. Arsenite-treatment disrupts mitosis and induces apoptotic cell death in both SV40-transformed and spontaneously immortalized Li-Fraumeni human fibroblasts (p53^-/-). Arsenite-treatment of normal diploid human fibroblasts (p53^+/+) induces a mitotic delay which is overcome by the cells (Yih 1997). Disruption of mitosis in HeLa S3 cells (p53 deficient, Liang 1995) with colcemid also induces apoptosis (Sherwood 1994). Arsenite-treatment induces p53 expression in lymphocytes and transfection of p53 null cells with a wild type p53 vector increases resistance to arsenite toxicity (Salazar 1997). These observations are consistent with a role for p53 in preventing apoptosis in response to mitotic disruption. A consequence of the SV40-transformed phenotype is loss of the spindle checkpoint response requiring p53 (Chang 1997). Thus, arsenite activation of the spindle checkpoint in the absence of functional p53 may trigger apoptosis. The resistance of p53^(+/+) cells to apoptosis in response to arsenite-induced mitotic disruption suggests that arsenic induced cancers may be p53 wild type. This speculation is supported by observations of elevated p53 in arsenic-induced cancers and in Bowen's lesions (Chang 1998).

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