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J Biol Chem, Vol. 275, Issue 3, 1846-1854, January 21, 2000

Serine/Threonine Protein Phosphatase Type 11 Is Required for the Completion of Cytokinesis in Human A549 Lung Carcinoma Cells^*

Aiyang Cheng, Nicholas M. Dean^§, and Richard E. Honkanen^¶

From the  Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, Alabama 36688 and the ^§ Department of Pharmacology, ISIS Pharmaceuticals, Carlsbad, California 92008

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In lower eukaryotic organisms, the loss of serine/threonine protein phosphatase type 1 (PP1) results in growth arrest after the onset of mitosis. In humans, four highly homologous isoforms of PP1 (PP1, PP1, PP11, and PP12) have been identified. Determining the roles of these phosphatases, however, has proven difficult due to the lack of subtype-specific inhibitors. In this study, we developed chimeric antisense 2'-O-(2-methoxy)ethylphosphothioate oligonucleotides targeting human PP11 that specifically inhibit PP11 gene expression. Two potent antisense oligonucleotides (ISIS 14435 and 14439; IC₅₀ ~ 50 nM) were then employed to elucidate the cellular functions of PP11 during cell cycle progression. In A549 cells, the inhibition of PP11 expression resulted in a dose-dependent inhibition of cellular proliferation, with growth arrest occurring after ~36-48 h, when PP11 mRNA expression was inhibited by >85%. Fluorescence-activated cell sorter analysis revealed that ISIS 14435/14439-induced growth arrest was associated with an increase in the number of cells containing 4N DNA. Immunostaining of treated cells revealed that the inhibition of PP11 expression had no apparent effect on the formation of mitotic spindles. However, decreased expression was associated with the failure of cell division in a late stage of cytokinesis and the formation of dikaryons.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In eukaryotic cells, the reversible phosphorylation of proteins determines the biological activity of many protein complexes and is recognized as a major mechanism controlling cell cycle progression. Therefore, the protein kinases that mediate progression through the cell cycle have been extensively investigated. Because many of the protein kinases that play key roles in the regulation of cell cycle progression are Ser/Thr kinases, it seems logical that the regulation of serine/threonine protein phosphatase (PPase)¹ activity will also contribute to growth control processes. Indeed, the addition of semiselective PPase inhibitors, such as okadaic acid (1), cantharidin (2), and fostriecin (3, 4), to rapidly growing cells affects several aspects of cell cycle progression (for a review, see Refs. 5-7). When applied at low concentrations (2-8 nM), okadaic acid induces premature entry into S phase. In contrast, when added to cells at slightly higher concentrations (10-50 nM), okadaic acid impedes the completion of mitosis, arresting cell growth after the appearance of multiple aberrant mitotic spindles (4, 8, 9). Similar results are produced by cantharidin and fostriecin when applied at concentrations that inhibit PPase activity to a comparable extent (4). These findings suggest that the okadaic acid/fostriecin/cantharidin-sensitive PPases also make critical contributions to the regulation of cell cycle progression (for a review, see Refs. 5-7). However, elucidation of the roles played by individual mammalian PPases has proven difficult.

Traditionally, mammalian PPases have been classified based on their biochemical characteristics, their sensitivity to inhibitors, and a limited amount of substrate specificity that can be demonstrated in vitro. Accordingly, four subtypes, PP1, PP2A, PP2B, and PP2C, have been established (for a review, see Refs. 5, 7, and 10). More recent structural data, however, indicate that PP1, PP2A, and PP2B are related, while the primary amino acid structure of PP2C is distinct. In addition, three isoforms of PP1 that demonstrate >90% identity (11-13), two isoforms of PP2A with >97% identity (14, 15), three isoforms of PP2B with >80% identity (16-18), and several structurally related phosphatases, designated PP4 (19, 20), PP5 (21-23), PP6 (24), and PP7 (25, 26), have been identified. Based on a comparison of their primary amino acid sequences, these mammalian PPases can be placed into five distinct subfamilies termed PP1, PP2A (which includes PP4 and PP6), PP2B, PP5, and PP7 (6, 25). The PP1, PP2A, and PP5 families of PPases are all sensitive to inhibition by okadaic acid, while PP2B, PP2C, and PP7 are resistant to inhibition (1, 3, 6, 21, 25).

Studies designed to determine the roles of individual PPases in the control of cell cycle progression have been most successful in lower eukaryotes. In Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Aspergillus nidulans, mutations in the genes encoding PPases homologous to mammalian PP1 result in abnormalities during mitosis or the completion of anaphase (27-31). Thus, PP1 is believed to play an essential role in the progression through mitosis, possibly contributing to the regulation of a mitotic/spindle checkpoint control mechanism. This theory is supported by genetic experiments in Drosophila, studies of microtubule dynamics during the transitions into and out of mitosis in Xenopus egg extracts, and studies with PP1-neutralizing antibodies in rat embryo fibroblasts, which all indicate that PP1 activity is necessary for the completion of mitosis (32-34).

In mammals, there are four highly homologous isoforms of PP1 (PP1, PP1, PP11, and PP12). These isoforms share >89% identity and are encoded by three distinct genes, with PP11 and PP12 produced from the alternative splicing of the same primary transcript (6, 35). PP12 is highly enriched in testis, while PP1, PP1, and PP11 are expressed in most, if not all, tissues. The functions of the individual isoforms are unknown, and no type-selective inhibitors of PP1 have been identified to date. Furthermore, due to the high degree of homology, it is impossible to draw conclusions about the roles of mammalian PP1 isoforms from comparative studies in lower eukaryotes. To study the role of individual PP1 isoforms in the regulation of cell cycle progression, in the present study we developed antisense oligonucleotides targeting PP11 that specifically inhibit PP11 gene expression in human cells. Using these antisense oligonucleotides, we demonstrate that the expression of PP11 is necessary for A549 cell proliferation and that the inhibition of PP11 expression leads to the formation of dikaryons following the failure of cytokinesis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Tissue culture medium, Lipofectin^®, and TRIzol^® were purchased from Life Technologies, Inc. DECAprime^TM II DNA labeling, MAXIscript^TM in vitro transcription, and HybSpeed^TM RPA kits were purchased from Ambion Inc. (Austin, TX). PP11-specific antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antibody against -tubulin, fluorescein isothiocyanate (FITC)-conjugated phalloidin, and FITC- and horseradish peroxidase-conjugated secondary antibodies were obtained from Sigma. [-³²P]dATP and [-³²P]UTP were purchased from NEN Life Science Products.

Cell Culture-- Human A549 lung carcinoma cells obtained from the American Type Tissue Collection were grown in Dulbecco's modified Eagle's medium containing 1 g of glucose/liter (DMEM) and 10% heat-inactivated fetal bovine serum. Cells were routinely passed when 90-95% confluent.

Oligonucleotide Synthesis-- 2'-O-(2-Methoxy)ethylphosphothioate oligonucleotides were synthesized and purified as described previously (36). The sequence of the oligonucleotides tested is provided in Table I.

Assays for Oligonucleotide Inhibition of PP11 Expression-- A549 cells were plated in 60-mm dishes and cultured in DMEM containing 10% fetal bovine serum. When the cultures were ~70% confluent, they were treated with the indicated oligonucleotides as described previously (36). Briefly, cells were washed with DMEM. A solution (1 ml) of DMEM containing 15 µg/ml DOTMA/DOPE (Lipofectin®) and the oligonucleotides at the indicated concentration were then added. After incubating the cells for 4 h at 37 °C, the cells were washed and cultured in fresh DMEM containing 10% fetal calf serum for 18-20 h. The cells were then harvested, and total RNA was isolated with TRIzol® reagent according to the methods provided by the manufacturer. The total RNA (20 µg) was then separated on 1% agarose gels containing formaldehyde and transferred to Duralon-UV^TM membrane (Stratagene, La Jolla, CA). Following UV cross-linking, the membranes were hybridized with a ³²P-labeled probe for PP11. The PP11 probe employed was generated from the full-length coding region of human PP11 and was ³²P-labeled by polymerase chain reaction amplification in the presence of [-³²P]dATP using a DECAprime II^TM labeling kit (Ambion) according to the protocol of the manufacturer. Hybridization was performed at 42 °C for 16 h in 50% formamide, 0.1 M Pipes, 0.8 M NaCl, 5× Denhardt's solution, 100 µg/ml denatured herring sperm DNA, 1 × 10⁶ cpm/ml -³²P-labeled probe, and 10% dextran sulfate. Following hybridization, the filter was washed twice in 2× SSC, 0.1% SDS for 20 min at room temperature and then twice in 0.1× SSC, 0.1% SDS for 20 min at 55 °C. Hybridization was visualized by autoradiography, and the filters were then stripped and reprobed with a ³²P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe to confirm equal loading. Quantification of hybridization signals was achieved by analysis of the scanned autoradiograms using the NIH Image program (ImagePC).

Analysis of Cell Growth-- A549 cells were seeded in 60-mm tissue culture plates at a density of 5.0 × 10⁴ cells/dish. On the next day, the cells were treated with PP11-specific antisense oligonucleotides (ISIS 14435 and 14439) or scrambled mismatch control oligonucleotides (Table I) at a final concentration of 500 nM as described above. On each of the next 4 days, the cell cultures were treated briefly with trypsin to detach the cells from the dish (three wells from each test group). The number of cells was determined by counting using a hemacytometer. Cell viability was determined with trypan blue staining. The percentage of viable cells was calculated by dividing the number of cells excluding trypan blue by the total number of cells.

RNase Protection Assay-- DNA fragments of PP1, PP1, and PP11 that varied in size were amplified by polymerase chain reaction and subcloned into pGEM5Zf(+) T-vectors (Promega), and the integrity of each construct was verified by DNA sequencing. DNA templates for in vitro transcription, which contained T7 and SP6 promoter regions, were generated by polymerase chain reaction amplification with nested primers contained in the plasmid. DNA template for cyclophilin was purchased from Ambion Inc. ³²P-Labeled antisense RNA probes were prepared with MAXIscript^TM in vitro transcription kit following the instructions of the manufacturer. Total RNA (5 µg) from A549 cells was then analyzed employing a HybSpeed^TM RPA kit according to the methods of the manufacturer with a slight modification (i.e. the coprecipitation step was omitted, and the total RNA, ³²P-labeled probes, and hybridization buffer were combined prior to reducing the volume of the reaction to 10 µl by evaporation). Protected RNA probes were then separated on 5% denatured polyacrylamide gel and visualized by autoradiography.

Immunoblotting of PP11-- Western analysis was performed essentially as described previously using polyclonal goat antibodies against PP11 (37). Briefly, A549 cells grown in T-75 flasks were washed twice with ice-cold phosphate-buffered saline (PBS) and scraped from the plate in 1 ml of PBS. The cells were collected by centrifugation and then lysed by sonication in 0.1 ml of radioimmune precipitation buffer (10 mM Tris-HCl (pH 7.4) containing 1% (w/v) Nonidet P-40, 0.1% SDS, 0.1% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, and protease inhibitor mixture (Sigma)). Protein was determined using a Bio-Rad protein quantitation assay, with bovine serum albumin as a standard. Samples were prepared for electrophoresis by adding an equal volume of 2× sample buffer (120 mM Tris-HCl, pH 7.4, 200 mM dithiothreitol, 20% glycerol, 4% SDS, and 0.02% bromphenol blue) and boiling for 5 min. Protein samples (60 µg) were then separated on 10% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to Immobilon-P^TM membranes (Millipore Corp.). The membrane was then blocked for 1 h with Tris-HCl, pH 7.6, containing 150 mM NaCl and 5% nonfat milk. PP11 was detected with an anti-PP11 antibody (Santa Cruz Biotechnology, Inc.) diluted 1:1000 in Tris-HCl (pH 7.6) containing 150 mM NaCl, 0.2% Tween 20 (TBST) and 2% nonfat milk for 18 h. The membrane was then washed, and the primary antibody was detected employing ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), following the protocols of the manufacturer.

Fluorescence-activated Cell Sorter Analysis-- A549 cells were seeded in 10-cm culture dishes (1.5 × 10⁶ cells/dish) and grown in DMEM with 10% fetal calf serum for 22-24 h (70-80% confluent). The cells were then treated with oligonucleotides as described above. DNA content per cell was measured by flow cytometry in propidium iodide-stained cells. Cells (~10⁶) were harvested using trypsin/EDTA and suspended in 1 ml of 50 µg/ml propidium iodide in PBS. Cells in suspension were mixed with an equal volume of Vindelov's propidium iodide solution (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 10 µg/ml ribonuclease, 0.1% IGEPAL CA-630, and 50 µg/ml propidium iodide), incubated for 1 h at 4 °C, and analyzed by flow cytometry. Flow cytometry analysis was performed on a Becton Dickinson fluorescence-activated cell sorter as described previously (4).

Indirect Immunofluorescence Microscopy-- Cells were plated onto 60-mm dishes containing sterile coverslips in 4 ml of DMEM at a concentration of 0.5 × 10⁶ cells/dish and grown until the cultures were ~70% confluent. The cells were then treated with oligonucleotides as described above. After 18 h, the cell coverslips were then fixed by immersion of the coverslips in 20 °C methanol for 6-8 min and processed for immunofluorescence microscopy using previously published methods (4, 38). To collect mitotic cells, the cells from 60-mm dishes were harvested by "mitotic shake off" and deposited by centrifugation onto polylysine-coated coverslips. The coverslips were then fixed and processed as described above. Anti--tubulin (Sigma) was used at a 1:200 dilution. FITC-labeled secondary antibodies (Roche Molecular Biochemicals) were used at a 1:200 dilution. To visualize actin, the coverslips were washed twice with PBS and fixed in 3.7% formaldehyde in PBS for 10 min at room temperature. The fixing solution was removed by two washes with PBS. Each coverslip was placed in a glass Petri dish and extracted with a solution of 0.1% Triton X-100 in PBS for 4 min at room temperature. The coverslips were then washed two additional times with PBS and stained with Alexa 488 phalloidin (Molecular Probes, Inc., Eugene, OR) using the methods provided by the manufacturer. After processing, coverslips were mounted in PBS/glycerol (1:1) containing 25 µg/ml HOECHST 33258 dye. Cells were observed using an Olympus BX50 microscope. Images were recorded with a digital Optronics CCD camera system (Goleta CA).

Time Lapse Video Microscopy-- A549 cells were plated and treated with oligonucleotides in 60-mm dishes as described above. Twenty-four hours after the treatment, cell cycle progression was monitored by time lapse video microscopy using an Axiovert 35 M microscope (Carl Zeiss, Inc.). A heated stage was employed to maintain the cells at a constant temperature of 37.0 °C, and CO₂ was maintained at 5% during the course of the experiments. Images were recorded at a rate of 1 frame/s for 24-48 h.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Antisense-mediated Inhibition of PP11 mRNA Expression-- Twelve oligonucleotides, 20 bases in length, predicted to hybridize to different regions of human PP11, mRNA were synthesized (Table I). The oligonucleotides tested were designed to target specific regions in the protein coding region, the 5'-untranslated, or the 3'-untranslated region of human PP11 mRNAs (Fig. 1A) and were "chimeric" 2'-O-(2-methoxy)ethylphosphothioate oligonucleotides, containing eight central phosphorothioate oligodeoxy residues ("oligodeoxy gap") flanked by six 2'-O-(2-methoxy) residues on the 3'- and 5'-ends. These modifications have been shown previously to enhance the potency of antisense oligonucleotides targeting mRNAs encoding other proteins (36, 39). Because phosphorothioate oligonucleotides commonly act through an RNase H-dependent mRNA cleavage mechanisms in cells (40), the ability of each oligonucleotide to specifically inhibit the expression of PP11 was determined by Northern blot analysis probing for levels of PP11 mRNA. For the initial screen, PP11 mRNA was detected using a PP11-specific cDNA probe that forms a hybrid with a single ~2.4-kilobase pair transcript.

View this table: [in this window] [in a new window]   Table I Design strategy for PP11 oligonucleotides Twelve 2'-O-(2-methoxy)ethylphosphothioate oligonucleotides 20 bases in length targeted to different regions of human PP11 were synthesized. Mismatch-scrambled control oligonucleotides corresponding to antisense oligonucleotide ISIS 14435 and 14439 were also synthesized. Sequences are shown 5' to 3', and the approximated location on the PP11 cDNA is indicated.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Inhibition of PP11 mRNA and protein expression by treatment with antisense oligonucleotides. A, relative positioning of the predicted hybridization sites within the human PP11 mRNA of 12 antisense oligonucleotides that were evaluated for their ability to inhibit PP11 mRNA expression in cultured A549 tumor cells. B, identification of antisense oligonucleotides that inhibit the expression of PP11 mRNA. A549 cells were treated with the indicated antisense oligonucleotides at a concentration of 300 nM. RNA was prepared 24 h later and analyzed for PP11 and GAPDH mRNA levels by Northern blot analysis. Control cells were treated with a random oligonucleotide. C, PP11 mRNA levels from Northern blot analysis (B) expressed as a percentage of the levels of PP11 mRNA in control cells following normalization to GAPDH. D, inhibition of PP11 mRNA levels by ISIS 14435. A549 cells were treated with increasing concentrations (25-500 nM) of ISIS 14435 (left panel) ISIS 14439 (center panel), or a 300-500 nM concentration of the indicated oligonucleotides (right panel). ISIS 15026 and ISIS 15030 are mismatched control analogues (Table I). Total mRNA was prepared 24 h later and analyzed for PP11 and GAPDH mRNA levels by Northern blot analysis. Each lane contained 15 µg of total RNA. E, quantification of PP11 mRNA levels (shown above) after normalization to GAPDH in A549 cells following treatment with increasing concentrations of ISIS 14435 () or 14439 (). F, Western blot analysis of PP11 protein levels in A549 cells 24 h after treatment with ISIS 14435 or a mismatch control (ISIS 15026). Cells were treated with ISIS 14435 at a concentration of 0-500 nM or with 500 nM ISIS 15026, and protein extracts were prepared 24 h later. Western analysis was then performed as described under "Experimental Procedures," with each lane loaded with 60 µg of protein. G, quantification of PP11 protein levels in A549 cells following treatment with increasing concentrations of ISIS 14435 determined by NIH Image analysis of the exposed film shown above. H, Western blot analysis of PP11 protein levels in A549 cells following a single treatment with ISIS 14435. Cells were treated with ISIS 14435 at a concentration of 500 nM or with 500 nM mismatch control oligonucleotides (ISIS 15026), and protein extracts were prepared at the time of treatment (0) and then each day for the next 4 days (1-4). Each lane contained 60 µg of protein.

A comparison of PP11 mRNA levels in A549 human lung carcinoma cells treated with antisense oligonucleotides targeting PP11 in the presence of cationic lipids is shown in Fig. 1, B and C. The cationic lipids (DOTMA/DOPE; Lipofectin®) were used to facilitate the uptake of the oligonucleotides (41), and the reduction in PP11 mRNA levels observed in response to treatment was varied. Some oligonucleotides had little or no effect on the inhibition of PP11 mRNA levels, while others had a moderate effect, and a few had pronounced effects. The two antisense oligonucleotides with activity against PP11 mRNA identified in this series and chosen for further analysis were ISIS 14435 and 14439, which target regions in the C-terminal coding and 3'-untranslated regions of PP11 mRNA, respectively. All of the effective oligonucleotides and several of the oligonucleotides with moderate or no effect were also tested against another human cell line (T-24 bladder carcinomas) for their effects on the expression of PP11 mRNA levels, with essentially identical results.

Specificity of PP11 Antisense Inhibition-- To assess the potency of ISIS 14435-/ISIS 14439-mediated inhibition of PP11 mRNA expression, dose-response studies were conducted with ISIS 14435, ISIS 14439, and mismatch control analogues. The mismatch control analogues, ISIS 15026/1530 and ISIS 15680/15682, contain the same base composition as ISIS 14435 and 14439, respectively; however, the sequences in mismatch controls are scrambled and noncomplementary to PP11 (Table I). The inhibition of PP11 expression following treatment of A549 cells with either ISIS 14435 or 14439 was apparent by 6-8 h and lasted for ~3 days. As seen in Fig. 1, D and E, by 24 h, treatment with ISIS 14435 or ISIS 14439 produced a dose-dependent reduction of PP11 mRNA levels with an apparent IC₅₀ of ~50 nM. No effect was observed following treatment with the mismatch controls, even when applied at concentrations 10 times that of the IC₅₀ for the active antisense oligonucleotides. To demonstrate equal RNA loading, the blots were probed for GAPDH. Studies with different human cell lines produced similar results (data not shown). Western analysis indicates that the treatment also effectively decreased PP11 protein levels in a dose-dependent manner in ~24 h, with PP11 protein levels remaining repressed for ~3 days (Fig. 1, F, G, and H).

The specificity of the antisense oligonucleotides targeting PP11 mRNA was then examined. Northern analysis revealed that ISIS 14435 had no effect on the expression of structurally related PPases (PP2A, PP5) or glyceraldehyde-3-dehydrogenase mRNA in A549 cells (Fig. 2A). Ribonuclease protection assays also showed that both antisense oligonucleotides targeting PP11 selectively inhibit the expression of PP11 and have no effect on the expression of PP1 or PP1 (Fig. 2, B and C). Since the sequence targeted by ISIS 14535 and ISIS 14439 is not contained in PP1, PP1_, PP2A, PP4, PP5, GAPDH, or cyclophilin (employed as a positive control to demonstrate that equal amounts of RNA were utilized in ribonuclease protection assays), neither ISIS 14439 or ISIS 14435 would be expected to inhibit the expression of these proteins if they inhibit the expression of PP11 mRNA via an antisense-mediated mechanism. The mismatch control oligonucleotides, ISIS 15026/1530 and ISIS 15680/15682, had no effect, and similar results were also observed with T-24 bladder carcinoma cells.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Target-specific inhibition of PP11 mRNA. A, A549 cells were treated with increasing concentrations (25-500 nM) of ISIS 14435. Total mRNA was prepared 24 h later and analyzed for PP2A, PP5, and GAPDH mRNA levels by Northern blot analysis. B, effects of ISIS 14435 and mismatched controls on the expression of PP11, PP1, and PP1 mRNA levels. A549 cells were treated with 0, 25, 50, 100, 200, or 300 nM ISIS 14435 (lanes 1-6, respectively) or 500 nM mismatch control oligonucleotides (ISIS 15026 and 15030), and the amount of mRNA encoding three PP1 isoforms (, , and 1) was revealed by RNase protection assay with 5 µg of total RNA as described under "Experimental Procedures." Cyclophilin was used as an internal control to ensure that uniform amounts of RNA were utilized in the assay. C, effects of ISIS 14439 and mismatched controls on the expression of PP11, PP1, and PP1 mRNA levels. A549 cells were treated with 0-500 nM ISIS 14439, 500 nM ISIS 14435, or 500 nM ISIS 15680 (a mismatch control), and the amount of mRNA encoding three PP1 isoforms (, , and 1) was revealed by RNase protection assay with 5 µg of total RNA as described above.

Antiproliferative Effects of PP11 Antisense Oligonucleotides-- Having developed a method to specifically inhibit the expression of PP11, we next explored the roles played by PP11 in human cells. To study the effects of antisense oligonucleotides targeting PP11 on cell growth and viability, A549 cells were treated one time with ISIS 14435 or its mismatched control at a concentration of 0-500 nM. Cell number and viability were then determined daily over a 4-day period. As seen in Fig. 3A, treatment resulted in a dose-dependent repression of A549 cell proliferation, with nearly complete inhibition of proliferation noted by 48 h after treatment with 500 nM ISIS 14435. In contrast, treatment with mismatched control oligonucleotides had no apparent effect on A549 cell growth (Fig. 3B). Similar results were obtained with ISIS 14439 (data not shown), and viability studies (Table II) indicated that >89% of cells treated cells were capable of excluding trypan blue 48 h after treatment. Therefore, the decrease in cell number was probably due to growth inhibition rather than cell death.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Antiproliferative effects of PP11 antisense oligonucleotides in A549 cells. A, dose-response for ISIS 14435 inhibition of cell proliferation. A549 cells in log phase growth were treated with a single dose (0-500 nM) at time 0 with ISIS 15534, and cell number was determined after days 1, 2, 3, and 4 following treatment. B, time course for the antiproliferative effects of ISIS 15534. A549 cells were treated with a single dose (500 nM) of ISIS 14435, mismatched control oligonucleotide analogues, or, as an additional control, Lipofectin alone (control; ). Cell number was then determined after days 1, 2, 3, and 4 following treatment. Each point represents the mean of triplicate cultures, with error bars representing the S.D.

View this table: [in this window] [in a new window]   Table II Viability of A549 cells treated with antisense oligonucleotides targeting PP11 A549 cells were treated with ISIS 14435, ISIS 14439, or mismatched controls (ISIS 15026, ISIS 15030, ISIS 15680, ISIS 15682) at a concentration of 500 nM in the presence of DOTMA/DOPE liposomes (Lipofectin®). Additional control cells were treated with Lipofectin alone. Cell viability was estimated 48 h later by the ability of cells to exclude trypan blue dye as described under "Experimental Procedures."

Inhibition of PP11 Expression Inhibits a Late Stage in Cytokinesis Leading to the Formation of Dikaryons-- Studies in lower eukaryotic cells suggest that PP1 activity is necessary for the completion of mitosis, and the treatment of mammalian cells with okadaic acid or fostriecin at concentrations that partially inhibit the activity of PP1 in vitro results in the appearance of 4N cells that arrest in the G₂/M phase of the cell cycle (4, 8, 9). To determine if ISIS 14435-mediated growth arrest occurs during a specific stage of the cell cycle, we employed fluorescence-activated cell sorter analysis of propidium iodide-stained A549 cells 48 h after treatment with antisense oligonucleotides targeting PP11. These studies revealed that ~31% of the cells in which the expression of PP11 was suppressed contained twice the normal content of DNA (4N) (Fig. 4). In comparison, only 14-16% of the control cells (cells treated with either mismatch control oligonucleotides or Lipofectin alone) were 4N.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   DNA content flow cytometric histograms of PP11 antisense oligonucleotide-treated A549 cells. Logarithmically growing A549 cells were treated with Lipofectin alone (A), mismatched control oligonucleotides (B and C), or antisense oligonucleotides targeting PP11 (D) for 48 h. The cells were then harvested and stained with propidium iodide (10 µg/ml), and DNA content flow cytometry was preformed. DNA content is measured on the x axis as fluorescence, and 2N and 4N populations of cells are indicated. The numbers on the x axis represent flow cytometric channel units, and the results shown are representative of three independent experiments.

The G₂/M phase growth arrest observed following treatment with okadaic acid or fostriecin is associated with the appearance of multiple aberrant mitotic spindles (4, 8, 9). Thus, we next conducted immunostaining studies with antitubulin antibodies to determine if the inhibition of PP11 expression also affects the formation of mitotic spindles. For these studies, A549 cells were collected by mitotic shake after treatment with a single dose of 500 nM ISIS 14435 or ISIS 14439. As seen in Fig. 5, immunostaining with antitubulin antibodies revealed that the suppression of PP11 expression has no apparent effect on the formation of mitotic spindles or the appearance of prophase microtubules. However, there was a notable increase in the number of cells in the "late stages" of cytokinesis (Fig. 5A). Defining the "late stage of cytokinesis" as the presence of a cleavage furrow extending >50% of the diameter of the dividing cells (as indicated in Fig. 5), we then scored cells collected (>500 for each treatment) by mitotic shake off 36 h after treatment with ISIS 14439, ISIS 14435, and controls. These studies revealed that 49 ± 3.1% of the cells treated with ISIS 14435 and 51 ± 4.2% of the cells treated with ISIS 14439 were in late cytokinesis. In comparison, 15.1 ± 2.5, 14.6 ± 2.1, and 15.3 ± 2.4% of the control cells treated with ISIS 15026, ISIS 15680, or Lipofectin alone, respectively, were in late cytokinesis (Fig. 5A).

View larger version (52K): [in this window] [in a new window]   Fig. 5.   The suppression of PP11 expression inhibits cytokinesis leading to the formation of dikaryons. A, suppression of PP11 expression delays cytokinesis. A549 cells treated with ISIS 14435 or ISIS 14439 (500 nM) were collected by mitotic shake off 36 h after treatment and immunostained with anti--tubulin antibodies. Antibody-antigen complexes were then detected with FITC-conjugated anti-rabbit IgG and were visualized by fluorescence microscopy. The three arrows in the upper right-hand corner of the ISIS 13345-treated cells indicate mitotic cells, and the other arrows indicate cells in a "late stage of cytokinesis." The graph to the right shows the actual number (mean ± S.D.) of cells observed in late cytokinesis using data obtained from four independent experiments in which >500 mitotic cells were scored for each treatment group (LC represents an additional control treated with Lipofectin alone). B, formation of dikaryons following the suppression of PP11 expression. A549 cells treated with ISIS 14435, ISIS 14439, or mismatch controls (ISIS 15026 or ISIS 15680) (500 nM) were fixed 48 h after treatment and immunostained with anti--tubulin antibodies. Antibody-antigen complexes were then detected with FITC-conjugated anti-rabbit IgG (left column). DNA was visualized by staining with HOECHST 33258 stain (center column) and phase microscopy (right column). Examples of cells containing more then one nuclei are indicated by white arrows. The yellow arrow illustrates a characteristic observed in ISIS 14435/ISIS 14439-treated cells that was not observed in controls. The photographs are representative of three independent experiments, and the number of dikaryons present in each treatment group is provided under "Results."

After 48 h, the A549 cell cultures treated with either ISIS 14435 or ISIS 14439 contained numerous cells with two nuclei (Fig. 5B). Scoring of >1000 cells from three separate experiments revealed that 42.8 ± 4.8 and 37.6 ± 6% of the cells in cultures treated with 500 nM ISIS 14435 or ISIS 14439, respectively, contained two nuclei. In contrast, only <0.5 ± 0.01% of the control cells contained more than one nucleus. The appearance of cells with three and four nuclei was also noted in a small number (<2%) of the cells treated with antisense oligonucleotides that suppress the expression of PP11.

To gain insight into the mechanism by which the dikaryons are formed, we treated cells with antisense oligonucleotides that inhibited the expression of PP11 and followed cell cycle progression with time lapse video microscopy. These preliminary studies revealed that cell division in the cells treated with ISIS 14435 is delayed, with the most obvious delay occurring after the onset of cytokinesis. Although with video phase microscopy it is difficult to precisely define the onset and conclusion of a particular phase of the cell cycle, when the onset of cytokinesis is defined as the point when the cleavage furrow is first apparent and late cytokinesis is defined as the point when the cleavage furrow is >50% of the diameter of the cell, the inhibition of PP11 expression resulted in a delay in late cytokinesis. Scoring of 68 dividing cells in control cell cultures revealed that all 68 completed cytokinesis within 43 ± 4.3 min. In contrast, of the 72 dividing cells observed in cultures treated with ISIS 14435, 61 remained in cytokinesis for a prolonged period (139 ± 46 min). Of the 61 cells that were delayed in cytokinesis, cell division was unsuccessful in 24. In these 24 cells, after the prolonged delay in late cytokinesis, the cleavage furrow appeared to "relax," producing a single cell. Then two distinct nuclei reform, and the cells flatten. In contrast, 0 of the 68 dividing in cultures treated with the mismatch control oligonucleotides progressed to become dikaryons. During the course of these preliminary studies, most of the binucleated cells did not undergo an additional round of cell division. However, a small percentage (<1%) progress to produce multinucleated cells. Although additional more quantitative studies are clearly necessary to define the role of PP11 in cell cycle progression, Fig. 6 provides a simplified illustration outlining the observations made with time lapse video microscopy.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Diagram of cell division illustrating observations from time lapse video microscopy of cell division after treatment with ISIS 14435. Time lapse video microscopy revealed that a reduction on PP11 expression had no apparent affect on cell cycle progression into mitosis (1), the formation of mitotic spindles (2), or the onset of telophase (3). However, unlike cells treated with the mismatch control oligonucleotides (control), cells treated with the antisense oligonucleotides targeting PP11 arrest in cytokinesis after the formation of a deep cleavage furrow (4). After ~140 min, the contractile ring appears to relax, and two apparently normal nuclei reform (5). Most of the dikaryons die without further replication. However, a few (<1%) undergo a "second attempt at cellular replication" that fails and results in production of multinucleated cells (6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In eukaryotic organisms, cell cycle progression is regulated to a large extent by the reversible phosphorylation of proteins. Therefore, deciphering the complex mechanisms controlling the activity of the cellular kinases and phosphatases that participate in the control of cell cycle progression is a major challenge for understanding both normal and aberrant cellular proliferation. Determining the roles of individual PPases in human cells, however, has proven difficult due to the lack of truly specific inhibitors. To overcome this difficulty, in the present study we developed antisense oligonucleotides (ISIS 14435 and ISIS 14439) that potently inhibit the expression of human PP11 without having an affect on the expression of other structurally related PPases (i.e. PP1, PP1, PP2A, PP4, or PP5). These antisense oligonucleotides provided us with the ability to study the roles of a single PP1 isoform in human cells.

In mammals, there are four isoforms of PP1 (PP1, PP1, PP11, and PP12), whereas in lower eukaryotes, PP1 homologues are apparently encoded by fewer genes. Studies in yeast indicate that PP1 plays an essential role in the normal progression through mitosis. In fission yeast, S. pombe, two genes (sds21 and dis2⁺/bws1⁺) encode PPases homologous to the catalytic subunit of mammalian PP1 (27-29). Mutants containing the semidominant dis2-11 allele of the major PP1 isoform enter, but fail to exit, mitosis (27). Loss-of-function mutations in both of these genes arrest cell growth in mitosis at the restrictive temperature, with the cells having condensed, unseparated chromosomes and short mitotic spindles (28, 42). In S. cerevisiae, a single essential gene (GLC7) encodes the catalytic subunit of PP1 (30, 43). Glc7p mutants are capable of DNA replication; however, growth arrest occurs before the onset of anaphase, with Glcp7 mutants containing short mitotic spindles (30, 43). Recent studies into the reasons for mitotic arrest produced by the absence of functional PP1 revealed that glc7-10-induced mitotic arrest is abolished in spindle checkpoint mutants (44). The glc7-10 mutants exhibit low kinetocore-microtubule binding activity and hyperphosphorylated Ndc10p, suggesting that defects in kinetocore-microtubule interactions caused by the hyperphosphorylation of kineticore proteins activate a spindle checkpoint (44). Studies with temperature-sensitive mutant strains of A. nidulans indicate that PP1 (the bimG gene product) is also required for normal mitotic spindle formation and the completion of anaphase in filamentous fungi (31). Similarly, genetic experiments suggest that one of the of PP1 isoforms in Drosophila is essential for mitosis (32). Results from studies of microtubule dynamics during the transitions into and out of mitosis in Xenopus egg extracts and studies with PP1-neutralizing antibodies in rat embryo fibroblasts also indicate that PP1 activity is necessary for the completion of mitosis in higher eukaryotes (33, 34).

To characterize the cellular roles of individual human PP1 isoforms and to determine if PP11 is necessary for the completion of mitosis in human cells, we treated A549 cells with ISIS 14435 and 14439 at concentrations that inhibited the expression of PP11. These studies indicated that the inhibition of PP11 expression inhibits cell proliferation. Fluorescence-activated cell sorting analysis of the treated cells revealed an increase in the number of cells containing 4N DNA. However, immunostaining to detect microtubules revealed no apparent abnormalities in the formation of mitotic spindles. In addition, there was no indication of growth arrest in mitosis or prior to the completion of anaphase. Therefore, it is likely that an isoform other then PP11 is necessary for the transition into or out of mitosis. Nonetheless, in A549 cells, when PP11 expression is suppressed cell division often fails. This failure is characterized by a delay in cytokinesis after the formation of the contractile ring and a deep cleavage furrow. Analysis of >1000 cells in which PP11 expression was inhibited by >85% revealed that ~41% of the growth-arrested cells then progress to become dikaryons, which preliminary studies suggest occurs when the contractile ring "relaxes" and two apparently normal nuclei reform. Therefore, although additional studies are needed to clarify the role of PP11 in human cells, it appears that PP11 has an essential function that is necessary for the completion of cytokinesis.

Although the molecular mechanisms regulating cytokinesis are poorly understood, other studies also suggest that the aberrant phosphorylation of proteins can impede the completion of cytokinesis. In NIH3T3 cells, the overexpression of p70 S6 kinase (p70^s6k) produces a similar phenotype (i.e. incomplete cytokinesis leading to the formation of dikaryons cells (45)). Similarly, in Chinese hamster ovary cells, the overexpression of protein kinase C, but not protein kinase C, protein kinase CII, or protein kinase C, produces cells with two nuclei following the inhibition of cell division in telophase (46). When the activation of cyclin-dependent kinase 1 (CDK1) is maintained through the introduction of a mutant form of cyclin B that is not degraded, cytokinesis is also inhibited (47). Although the inhibition of cytokinesis induced by prolonged CDK1 activity may be due to the inhibition of midzone microtubule formation or the phosphorylation of myosin II regulatory light chain (48), the precise reason(s) that aberrant or maintained activation of protein kinase C or p70^s6k inhibits cytokinesis are unclear. The mechanism(s) by which the inhibition of the PP11 expression is translated to a failure in cytokinesis is also not clear. All of the above mentioned kinases are serine/threonine kinases, and the phosphorylation of microtubule-associated proteins and myosin that occurs during cytokinesis is dynamic and highly regulated. Therefore, the activity of PP11 may be needed for the dephosphorylation of kinases involved in the phosphorylation of cytoskeletal components necessary for the completion of cytokinesis. Alternatively, PP11 activity may be required at a more distal site, such as the ubiquitin degradation pathway that mediates cyclin B degradation. Clearly, additional studies will be required to determine the substrates utilized by PP11 and how the regulation of PP11 activity is coordinated with that of cellular proteins that regulate the onset and progression of cytokinesis.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, MSB 2198, University of South Alabama, Mobile, AL 36688. Tel.: 334-460-6859; Fax: 334-460-6127; E-mail: honkanen@sungcg.usouthal.edu.

	ABBREVIATIONS

The abbreviations used are: PPase, serine/threonine protein phosphatase; PP1, serine/threonine protein phosphatase type 1; DMEM, Dulbecco's modified Eagle's medium; Pipes, 1,4-piperazinediethanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; UTR, untranslated region; FITC, fluorescein isothiocyanate; DOTMA/DOPE, N-[1-(2,3-dioleyloxy) propyl]-n,n,n-trimethylammonium chloride/dioleoylphosphatidylethanolamine.

	REFERENCES

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