Down-regulation of Myc as a Potential Target for Growth Arrest Induced by Human Polynucleotide Phosphorylase (hPNPaseold-35) in Human Melanoma Cells*

Devanand Sarkar {ddagger}, Magdalena Leszczyniecka {ddagger} §, Dong-chul Kang {ddagger}, Irina V. Lebedeva {ddagger}, Kristoffer Valerie ¶, Sonu Dhar ||, Tej K. Pandita || ** and Paul B. Fisher {ddagger} {ddagger}{ddagger} §§ ¶¶

From the Departments of {ddagger}Pathology, {ddagger}{ddagger}Neurosurgery, and §§Urology, Herbert Irving Comprehensive Cancer Center, Columbia University, College of Physicians and Surgeons, New York, New York 10032, §Novartis Pharmaceutical Corporation, One Health Plaza, East Hanover, New Jersey 07936-1080, the Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, and the ||Radiation and Cancer Biology Division, Washington University School of Medicine, St. Louis, Missouri 63108

Received for publication, March 10, 2003 , and in revised form, April 28, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Terminal differentiation and senescence share several common properties, including irreversible cessation of growth and changes in gene expression profiles. To identify molecules that converge in both processes, an overlapping pathway screening was employed that identified old-35, which is human polynucleotide phosphorylase (hPNPaseold-35), a 3',5'-exoribonuclease. We previously demonstrated that hPNPaseold-35 is a type I interferon-inducible gene that is also induced in senescent fibroblasts. In vitro RNA degradation assays confirmed its exoribonuclease properties, and overexpression of hPNPaseold-35 resulted in growth suppression in HO-1 human melanoma cells. The present study examined the molecular mechanism of the growth-arresting property of hPNPaseold-35. When overexpressed by means of a replication-incompetent adenoviral vector (Ad.hPNPaseold-35), hPNPaseold-35 inhibited cell growth in all cell lines tested. Analysis of cell cycle revealed that infection of HO-1 cells with Ad.hPNPaseold-35 resulted in arrest in the G1 phase and eventually apoptosis accompanied by marked reduction in the S phase. Infection with Ad.hPNPaseold-35 resulted in reduction in expression of the c-myc mRNA and Myc protein and modulated the expression of proteins regulating G1 checkpoint and apoptosis. In vitro mRNA degradation assays revealed that hPNPaseOLD-35 degraded c-myc mRNA. Overexpression of Myc partially but significantly protected HO-1 cells from Ad.hPNPaseold-35-induced growth arrest, indicating that Myc down-regulation might directly mediate the growth-inhibitory properties of Ad.hPNPaseold-35. Inhibition of hPNPaseold-35 by an antisense approach provided partial but significant protection against interferon-{beta}-mediated growth inhibition, thus demonstrating the biological significance of hPNPaseold-35 in interferon action.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There are two contrasting endpoints in the life of a replicating cell. One involves the normal physiological processes of differentiation or senescence. The other is the pathological process of neoplastic transformation characterized by uncontrolled proliferation and de-differentiation. Treatment of HO-1 metastatic human melanoma cells with fibroblast interferon (IFN-{beta})1 and the protein kinase C activator mezerein (MEZ) induces irreversible growth arrest and terminal differentiation characterized by changes in cell morphology, increase in melanin synthesis, modifications in gene expression, and alterations in surface antigen expression (15). Replicative or cellular senescence, a process leading to irreversible arrest of cell division, was first described in cultures of human fibroblasts that lost the ability to divide upon continuous subcultures (6). Replicative senescence can result from telomere shortening linked with a DNA end-replication problem, overexpression of certain oncogenes, or tumor suppressor genes, or it can be stress-induced premature senescence after exposure to a variety of oxidative stresses or DNA damaging agents (for a review, see Ref. 7).

Terminal differentiation and cellular senescence share several common traits including irreversible growth arrest and changes in gene expression profiles. To understand the molecular and biochemical basis of the complex physiological changes associated with these phenomena, an overlapping pathway screen was used to identify genes displaying coordinated expression as a consequence of both processes (8). A temporally spaced terminally differentiated human melanoma subtracted cDNA library was screened with cDNAs derived from senescent progeroid fibroblast cells. This led to the identification of old-35, which is human polynucleotide phosphorylase (hPNPaseold-35), a 3',5' exoribonuclease involved in RNA degradation (8). hPNPaseold-35 is a highly evolutionary conserved gene in plants, prokaryotes and eukaryotes having similar domain structure and functional properties in all species. In vitro assays confirmed that hPNPaseold-35 is involved in RNA degradation. Analysis of the expression profile of hPNPaseold-35 revealed that it is predominantly a type I interferon-inducible gene, and its expression is also induced in senescent fibroblasts in comparison with young fibroblasts. These findings indicate that hPNPaseold-35 might play an essential role in interferon- and senescence-induced growth arrest. Indeed, when hPNPaseold-35 is transfected by plasmid or transduced via a replication-incompetent adenovirus (Ad.hPNPaseold-35), there is a marked reduction in the colony-forming ability of HO-1 cells. The objective of the present study was to elucidate the molecular mechanism of the growth-suppressing property of Ad.hPNPaseold-35.

The protooncogene c-myc is involved in a wide range of cellular processes including proliferation, differentiation, and tumorigenesis (for a recent review, see Ref. 9). Myc belongs to the Max network, a group of transcription factors containing basic helix-loop-helix zipper motifs (10, 11). Myc heterodimerizes with Max and binds to the E-box sequence (CACGTG), thereby activating transcription (12). Max is constitutively expressed throughout the cell cycle (13), and it also heterodimerizes with the Mad family of transcription factors: Mad1, Mxi1, Mad3, and Mad4 (1416); however, in contrast to Myc-Max, the Mad-Max heterodimers act as transcriptional repressors at the same binding sites. The most important function of Myc is its essential role in controlling cell proliferation. Expression of exogenous Myc in cultured fibroblasts promotes S phase entry and shortens G1 phase of the cell cycle, whereas activation of a conditional Myc is sufficient to drive quiescent cells into the cell cycle (17, 18). The progression of cell cycle beyond the G1 phase is also augmented by the activities of the cyclin-dependent kinase (CDK) complexes cyclin D-CDK4 and cyclin E-CDK2, and the activities of these complexes are inhibited by CDK inhibitors, the Cip/Kip (CDK-interacting protein/kinase-inhibitory protein) family, including p27KIP1 and p21CIP1/WAF-1/MDA-6 and the INK (inhibitors of CDK4) family including p16INK4A and p15INK4B (19). Cyclins D and E are essential for G1-S progression in higher eukaryotic cells and when overexpressed are able to shorten the G1 interval (20, 21). The major pathway by which Myc induces cell cycle progression is by activating cyclin D2 and CDK4 (2225). An important consequence of the induction by Myc of cyclin D2 is its sequestration of p27KIP1 CDK inhibitor, permitting unfettered and prolonged activity of the cyclin E-CDK2 complex (26). Increased cyclin E-CDK2 activity shortens G1, whereas increased CDK2 and CDK4 activities result in hyperphosphorylation of the retinoblastoma (Rb) protein. This leads to release of E2F, a family of transcription factors that regulate a battery of genes necessary for cell cycle progression, from complexes with Rb and together with the direct induction of E2F2 by Myc, may further contribute to cell cycle progression (27). In addition Myc can directly repress p27KIP1 and p21CIP1/WAF-1/MDA-6 transcription (28, 29).

In the present study, we show that infection of HO-1 melanoma cells with Ad.hPNPaseold-35resulted in cell cycle arrest in the G1 phase and eventually apoptosis with marked reduction in DNA synthesis. Ad.hPNPaseold-35infection caused reduction in expression of the c-myc mRNA and Myc protein that was accompanied by induction of Mad1 protein. Overexpression of Myc protected HO-1 cells against Ad.hPNPaseold-35-mediated cell death. These findings argue that Myc might play a pivotal role in mediating the growth-inhibitory properties of Ad.hPNPaseold-35. We also show that inhibition of hPNPaseold-35 by an antisense strategy partially but significantly rescues HO-1 cells from interferon-{beta}-mediated growth inhibition, thereby documenting a potential role of hPNPaseold-35 in mediating interferon action.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Cell Viability Assays—Normal immortal human melanocyte (FM516-SV; FM516), WM35 early radial growth phase, and WM278 vertical growth phase primary human melanomas, and HO-1, FO-1, and MeWo metastatic melanoma cell lines and HEK-293 cells were cultured as previously described (30). HO-1-pREP4 and HO-1-hPNPaseold-35AS cell lines were generated by stable transfection of HO-1 cells with pREP4 (HO-1-pREP4) or antisense hPNPaseold-35 expressing pREP4 (HO-1-hPNPaseold-35AS), respectively, and selection with hygromycin. HO-1-Bcl-2 and HO-1-Bcl-xL cell lines were produced by stable transfection of HO-1 cells with Bcl-2 and Bcl-xL expression plasmids (kindly provided by Dr. John C. Reed) and selection with G418. Cell growth and viable cell numbers were monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) staining as described (30). Cultures were incubated with interferon-{beta} (2000 units/ml) and mezerein (10 ng/ml) for 5 days prior to assaying for cell viability.

Virus Construction and Infection Protocol—The construction and purification of hPNPaseold-35 expressing replication-defective adenovirus Ad.hPNPaseold-35were described previously (8, 31). A similar method was employed to generate an antisense hPNPaseold-35 expressing replication-defective adenovirus (Ad.hPNPaseold-35AS). The empty adenoviral vector (Ad.vec) was used as a control. Viral infections were performed as previously described (30).

Plasmid Construction, Transfection, and Colony Formation Assays— 3'-HA-tagged hPNPaseold-35 was created by PCR using the primers GCT AGC ATG GCG GCC TGC AGG TAC (sense) and GGA TCC TCA AGC GTA ATC TGG AAC ATC GTA TGG GTA CTG AGA ATT AGA TGA TGA (antisense). The authenticity of the amplified product was verified by sequencing, and it was cloned into the NheI/BamHI sites of pcDNA3.1 (Invitrogen) to generate hPNPaseold-35-HA. hPNPaseold-35AS was generated by ligating hPNPaseold-35 in an antisense orientation into BamHI/NotI sites of pREP4 (Invitrogen). The c-myc expression plasmid p290-myc (2, 3) was provided by Dr. Riccardo Dalla-Favera. HO-1 cells were plated at a density of 3 x 105 cells/6-cm dish and 24 h later were transfected with 5 µg of either empty vector or p290-myc (2, 3) using Superfect® (Qiagen, Hilden, Germany) transfection reagent according to the protocol from the manufacturer. After 36 h, the cells were infected with Ad.hPNPaseold-35 at a multiplicity of infection (m.o.i.) of 50 or 100 pfu/cell; 6 h later, the cells were trypsinized and counted and 103 cells were plated in 6-cm dishes. Colonies were counted after 3 weeks. Colony formation assays using hPNPaseold-35-HA in HO-1 cells were performed as described (32).

RNA Isolation and Northern Blot Analysis—Total RNA was extracted from the cells using Qiagen RNeasy mini kit (Qiagen) according to the protocol from the manufacturer, and Northern blotting was performed as described (33). The cDNA probes used were a 400-bp fragment from human c-myc, a 500-bp fragment from hPNPaseold-35, a 500-bp fragment from human GADD34, full-length human c-jun, and full-length human GAPDH.

In Vitro Translation and in Vitro mRNA Degradation Assays—In vitro translation was performed using the TNT-coupled Reticulocyte Lysate Systems (Promega, Madison, WI) using the plasmids pcDNA3.1 as a control, GADD153 expression plasmid, and hPNPaseold-35-HA according to the protocol from the manufacturer. Five µg of total RNA from HO-1 cells were incubated with 5 µl of each in vitro translated protein at 37 °C from 0.5 to 3 h. The RNA was repurified using the Qiagen RNeasy mini kit, and Northern blotting was performed (33).

Western Blot Analysis—Western blotting was performed as previously described (33). Briefly, cells were harvested in radioimmune precipitation assay buffer containing protease inhibitor mixture (Roche Molecular Biochemicals, Mannheim, Germany), 1 mM Na3VO4, and 50 mM NaF and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was used as total cell lysate. Thirty µg of total cell lysate were used for SDS-PAGE and transferred to a nitrocellulose membrane. The primary antibodies included: Myc (1:200; mouse monoclonal), Max (1: 200; rabbit polyclonal), Mad1 (1:200; rabbit polyclonal), p16 (1:200; rabbit polyclonal), p21 (1:200; rabbit polyclonal), p27 (1:200; rabbit polyclonal), p53 (1:200; mouse monoclonal), cyclin E (1:200, rabbit polyclonal), and E2F1 (1:200, rabbit polyclonal) (all from Santa Cruz Biotechnology, Santa Cruz, CA); Rb (1:500, mouse monoclonal) and cyclin A (1:500, mouse monoclonal) (from BD Biosciences); Bcl-2, Bcl-xL, and Bax (1:1000; rabbit polyclonal; kindly provided by Dr. John C. Reed); anti-HA (1:3000; mouse monoclonal; Covance Research Products, Inc., Berkeley, CA); and EF1{alpha} (1:1000; mouse monoclonal; Upstate Biotechnology, Inc., Waltham, MA).

[3H]Thymidine Incorporation Assay—HO-1 cells were plated at a density of 5 x 104 cells in each well of a 12-well plate. The next day the cells were infected with Ad.hPNPaseold-35 at an m.o.i. of 25 or 50 pfu/cell. After 4 days the cells were incubated with 10 µCi/ml [3H]thymidine for 12 h. The cells were washed with phosphate-buffered saline and incubated with 2 ml of ice-cold 10% trichloroacetic acid at 4 °C for 30 min. Trichloroacetic acid-precipitated materials were collected by centrifugation and solubilized with 1 ml of 2% SDS, and 100-µl aliquots were counted in a liquid scintillation counter.

Cell Cycle Analysis— Cells were harvested, washed in phosphate-buffered saline, and fixed overnight at –20 °C in 70% ethanol. The cells were treated with RNase A (1 mg/ml) at 37 °C for 30 min and then with propidium iodide (50 µg/ml). Cell cycle was analyzed using a FACScan flow cytometer, and data were analyzed using CellQuest software (Becton Dickinson, San Jose, CA).

Telomerase Assay—HO-1 cells were infected with either Ad.vec or Ad.hPNPaseold-35 for 1–4 days or untreated or treated with fibroblast IFN-{beta} (2000 units/ml) plus MEZ (10 ng/ml) for 1–4 days, and telomerase assays were performed as described previously (34). Briefly, protein concentrations of cell extracts were determined, and equal amounts of protein were used for the elongation process in which telomerase added telomeric repeats (TTAGGG) to the 3' end of the biotin-labeled primer. These elongation products were amplified by PCR, and the PCR products were denatured and hybridized to digoxigenin-labeled detection probes, specific for the telomeric repeats. The resulting products were immobilized via the biotin label to a streptavidin-coated microtiter plate. Immobilized amplicons were detected with an antibody against digoxigenin that is conjugated to horseradish peroxidase and the sensitive peroxidase substrate 3,3',5,5'-tetramethylbenzidine. The telomerase activity was quantified by measuring the absorbance of the samples at 450 nm (with a reference wavelength of 690 nm) using a microtiter plate reader.

Statistical Analysis—Statistical analysis was performed using oneway analysis of variance, followed by Fisher's protected least significant difference analysis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies demonstrated that infection with Ad.hPNPaseold-35 inhibited colony formation in HO-1 melanoma cells (8). The present studies were conducted to comprehend the molecular mechanism underlying the growth-arresting property of Ad.hPNPaseold-35. Different melanoma cell lines and SV40 T-Ag immortalized primary human melanocytes (FM-516-SV) were infected with Ad.hPNPaseold-35, and the growth of the cells was monitored by standard MTT assays. As shown in Fig. 1, infection with Ad.hPNPaseold-35 resulted in significant growth inhibition in all of the cells. The growth-inhibitory effect became significant from 4 days after infection, and, in certain cell lines (WM278 and MeWo), infection with Ad.hPNPaseold-35 completely inhibited cell growth. In addition, Ad.hPNPaseold-35 infection inhibited the growth of other cell types, including breast, prostate, colon and pancreatic carcinomas, glioblastoma multiforme, fibrosarcoma, and osteosarcoma, irrespective of their p53 or Rb status (data not shown).



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FIG. 1.
Infection with Ad.hPNPaseold-35 inhibits viability in melanoma cells. Different melanoma cell lines and FM-516 immortal melanocytes were either uninfected (diamond) or infected with Ad.vec (square) or Ad.hPNPaseold-35 (triangle) at an m.o.i. of 100 pfu/cell. Cell viability was monitored by standard MTT assay for 8 days. The data represent mean ± S.D. of three independent experiments each performed in octuplicate.

 

To investigate the mechanism of Ad.hPNPaseold-35-mediated growth inhibition, cell cycle analysis was performed following Ad.hPNPaseold-35 infection in HO-1 cells. When the cells were infected with Ad.hPNPaseold-35 at a high m.o.i. of 100 pfu/cell for 4 days, there was a significant increase in sub-G0 population of cells indicating apoptosis and a decrease in the S phase indicating inhibition of DNA synthesis (Fig. 2, A and B). When the kinetics of killing was slowed down by infecting cells at a low m.o.i. of 25 pfu/cell, there was an initial significant increase in cells in the G1 phase of the cell cycle (Fig. 2, C and D) at 3 and 6 days after infection. This increase was also accompanied by a marked decrease in the S phase. At later time points, the cells infected with Ad.hPNPaseold-35, but not control or Ad.vec-infected cells, started to die by apoptosis (Fig. 2D). The kinetics of cell death was very slow when HO-1 cells were infected with Ad.hPNPaseold-35 at a low m.o.i. It is worth noting that at 25 pfu/cell ~90% of the cells are infected with adenovirus (data not shown). From these observations it might be inferred that infection with Ad.hPNPaseold-35 induces cell cycle arrest at the G1 phase that ultimately culminates in apoptosis.



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FIG. 2.
Infection with Ad.hPN Paseold-35 induces G1 arrest, decreases S phase, and induces apoptosis in HO-1 human melanoma cells. A, HO-1 cells were either uninfected or infected with Ad.vec or Ad.hPNPaseold-35 at an m.o.i. of 100 pfu/cell and cell cycle was monitored by flow cytometry 4 days after infection. B, percentage of cell population in different phases of the cell cycle following the infection protocol as in panel A. White, gray, and black bars represent control, Ad.vec-infected and Ad.hPNPaseold-35-infected cells, respectively. C, cell cycle analysis by flow cytometry of HO-1 cells either uninfected or infected with Ad.vec or Ad.hPNPaseold-35 at an m.o.i. of 25 pfu/cell 3 days after infection. D, percentage of cell population in different phases of the cell cycle following the infection protocol as in panel B at 3, 6 and 8 days after infection. White, gray, and black bars represent control, Ad.vec-infected and Ad.hPNPaseold-35-infected cells, respectively.

 

The inhibition of DNA synthesis following Ad.hPNPaseold-35 infection was confirmed using a [3H]thymidine incorporation assay. As shown in Fig. 3A, infection with Ad.vec did not have an impact on DNA synthesis. Infection with Ad.hPNPaseold-35 reduced DNA synthesis by ~40% at an m.o.i. of 25 pfu/cell and by ~75% at 50 pfu/cell 4 days after infection.



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FIG. 3.
Infection with Ad.hPNPaseold-35 inhibits [3H]thymidine incorporation and telomerase activity. A, HO-1 cells were either uninfected or infected with Ad.vec (m.o.i. 100 pfu/cell) or Ad.hPNPase old-35 at an m.o.i. of either 25 or 50 pfu/cell. 3H thymidine incorporation assay was performed 4 days after infection. The data represent mean ± S.D. of two independent experiments each performed in triplicates. B, HO-1 cells were either untreated (filled diamonds) or treated with IFN-{beta} + MEZ (filled squares) for the indicated time periods and telomerase activity was measured as described under "Materials and Methods." The data represent mean ± S.D. of two independent experiments. C, HO-1 cells were infected as in A except at an m.o.i. of 100 pfu/cell. Telomerase activity was measured as described under "Materials and Methods." Open circles and open squares represent Ad.vec- and Ad.hPNPaseold-35-infected groups, respectively. The data represent mean ± S.D. of two independent experiments.

 

Telomerase activity is decreased in both terminal differentiation and senescence. As shown in Fig. 3B, telomerase activity decreased in a time-dependent manner to ~50% when HO-1 cells were treated with IFN-{beta} + MEZ for up to 4 days. This treatment protocol results in the induction of irreversible growth arrest and terminal differentiation in HO-1 melanoma cells (2, 5). Based on these findings, telomerase activity was also determined following Ad.hPNPaseold-35 infection. As shown in Fig. 3C, infection with Ad.hPNPaseold-35 at an m.o.i. of 100 pfu/cell, but not with Ad.vec, inhibited telomerase activity by almost 60% at day 4 after infection.

One of the factors that facilitate entry into the S phase of the cell cycle is Myc. During terminal differentiation of melanoma cells, c-myc mRNA expression is down-regulated (35). The expression level of c-myc mRNA following Ad.hPNPaseold-35 infection was, therefore, determined by Northern blot analysis. The expression of c-myc mRNA began decreasing 2 days after Ad.hPNPaseold-35 infection but not in uninfected or Ad.vec-infected cells even at 4 days after infection (Fig. 4A). This decrease correlates with the expression of hPNPaseold-35 mRNA that was also detected 2 days after infection. It should be noted that, under basal condition, hPNPaseold-35 mRNA is undetectable in HO-1 cells. The expression of the GAPDH housekeeping gene remained unchanged following Ad.hPNPaseold-35 infection.



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FIG. 4.
Effect of Ad.hPNPaseold-35 on Myc, Mad1, Max, and EF1{alpha} expression and cell growth. A, HO-1 cells were either uninfected or infected with either Ad.vec or Ad.hPNPaseold-35 at an m.o.i. of 100 pfu/cell. Samples were collected on day 4 after infection in case of control and Ad.vec-infected cells. Expression of the mRNAs indicated was analyzed by Northern blot analysis. B, HO-1 cells were treated as in panel A. Expression of the proteins indicated was analyzed by Western blot analysis. C, HEK-293 cells were transfected with either empty vector or p290-myc (2, 3). The cells were harvested after 48 h and the expression of Myc was analyzed by Western blot analysis. D, HO-1 cells were transfected with either empty vector or p290-myc (2, 3). The next day the cells were either uninfected or infected with Ad.vec (m.o.i. 100 pfu/cell) or Ad.hPNPaseold-35 at an m.o.i. of either 50 or 100 pfu/cell. 6 h later, the cells were trypsinized, counted and 103 cells were plated per 6-cm dish. Colony numbers (>50 cells) were determined after 3 weeks. E, graphical representation of the colony numbers as described in panel D. The data represent mean ± S.D. of two independent experiments each done in quintuplicate.

 

Down-regulation of Myc protein by different stimuli is usually accompanied by up-regulation of Mad1, the transcriptional repressor belonging to the Max family of transcription factors (9). In this context, the expressions of Myc, its heterodimer partner Max, and Mad1 were determined by Western blot analysis following Ad.hPNPaseold-35 infection. As anticipated from Northern blot analysis, Myc expression started decreasing 2 days after Ad.hPNPaseold-35 infection but not in uninfected or Ad.vec-infected cells at 4 days after infection (Fig. 4B). This down-regulation was accompanied by up-regulation of Mad1 protein. The level of Max protein remained unchanged, indicating that infection with Ad.hPNPaseold-35 switches the Myc-Max transcriptional activator to Mad1-Max transcriptional repressor. The expression level of the EF1{alpha} housekeeping gene did not change under any condition.

We next addressed whether c-myc overexpression could protect HO-1 cells from Ad.hPNPaseold-35-mediated cell death. At first we determined whether the Myc expression plasmid generates the appropriate protein. For this assay, HEK-293 cells were used because transfection efficiency in these cells is very high, permitting easy detection of expressed protein by Western blot analysis. As shown in Fig. 4C, transfection of p290-myc (2, 3) in HEK-293 cells resulted in significant overexpression of Myc in comparison with the cells transfected with empty vector. For protection assays, HO-1 cells were transfected with p290-myc (2, 3), infected with Ad.hPNPaseold-35, and the growth of the cells were analyzed by colony formation assays. Overexpression of Myc provided partial but significant protection against Ad.hPNPaseold-35-mediated cell death (Fig. 4, D and E) consistent with the possibility that one pathway by which Ad.hPNPaseold-35 induces growth suppression and cell death is by down-regulation of Myc.

hPNPaseold-35 is a 3',5'-exoribonuclease, prompting us to determine whether it can directly degrade c-myc mRNA. For this analysis a C-terminal HA-tagged hPNPaseold-35-expressing construct (hPNPaseold-35-HA) was created. The authenticity of the construct was first confirmed by transfecting it into HEK-293 cells. As shown in Fig. 5A, Western blot analysis using anti-HA antibody detected a single protein of ~90 kDa in size only in the hPNPaseold-35-HA-transfected cells. To check whether this construct has functional similarity to Ad.hPNPaseold-35, HO-1 cells were transfected with hPNPaseold-35-HA and cell growth was monitored by colony formation assay. As shown in Fig. 5B, overexpression of hPNPaseold-35-HA reduced colony-forming ability by ~45% indicating that hPNPaseold-35-HA also has growth-suppressing properties. After establishing that hPNPaseold-35-HA generates functional protein, this construct was used to prepare in vitro translated hPNPaseOLD-35. The effect of hPNPaseOLD-35 protein on c-myc mRNA was investigated by in vitro mRNA degradation assays. As shown in Fig. 5C, incubation with in vitro translated hPNPaseOLD-35 resulted in degradation of c-myc mRNA. This effect was specific for c-myc because the mRNAs for the GADPH housekeeping gene, cell growth regulatory gene c-jun, and apoptosis-inducing gene GADD34 were not degraded. This effect was also specific for hPNPaseOLD-35 because incubation with in vitro translated GADD153, a transcription factor, did not result in mRNA degradation.



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FIG. 5.
hPNPaseold-35 degrades c-myc mRNA in vitro. A, HEK-293 cells were transfected with either empty vector or hPNPaseold-35-HA. The cells were harvested after 48 h and the expression of hPNPaseOLD-35-HA was analyzed by Western blot analysis using anti-HA antibody. B, HO-1 cells were transfected with either empty vector or hPNPaseold-35-HA. Colony formation assays were performed as described under "Materials and Methods." The columns are graphical representations of the plating efficiency. The data represent mean ± S.D. of two independent experiments each done in quintuplicate. C, in vitro degradation assay was performed as described under "Materials and Methods." The expression of c-myc, GAPDH, GADD34 and c-jun mRNAs were detected by Northern blot analysis.

 

Because Ad.hPNPaseold-35 infection reduces the S phase of the cell cycle, the expression level of the regulators of G1 to S transition was checked. This checkpoint is guarded by CDK inhibitors. Ad.hPNPaseold-35 infection resulted in progressive up-regulation of p27KIP1, and the level of p21CIP1/WAF-1/MDA-6 was down-regulated (Fig. 6). The expression of p16INK4A could not be detected in these cells, which is a result of the fact that a majority of melanomas have genomic abnormalities in the p16INK4A gene. The expression level of p53 did not change upon Ad.hPNPaseold-35 infection. There was a significant decrease in the levels of cyclin A, cyclin E, hyperphosphorylated form of Rb, and E2F1 following Ad.hPNPaseold-35 infection when compared with control or Ad.vec-infected cells at 4 days after infection. The expression level of the EF1{alpha} housekeeping gene did not change under any condition.



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FIG. 6.
Expression profiles of regulators of G1 checkpoint following Ad.hPNPaseold-35 infection. Infection protocol was performed as described in Fig. 4A. Expression of the indicated proteins were detected by Western blot analysis.

 

The next question that arose was the biological significance of the growth arrest mediated by hPNPaseold-35. Because hPNPaseold-35 is induced during terminal differentiation by IFN-{beta} and MEZ, we employed an antisense approach to determine the role of hPNPaseold-35 in the growth arrest associated with treatment with IFN-{beta}, MEZ, or IFN-{beta} + MEZ. Ad.hPNPaseold-35AS was constructed and evaluated for activity. HO-1 cells were transfected with hPNPaseold-35-HA, followed by infection with Ad.hPNPaseold-35AS. The expression of hPNPaseOLD-35-HA was detected in the cell lysates by Western blot analysis using anti-HA antibody. As shown in Fig. 7A, hPNPaseOLD-35-HA could be detected in the cells following hPNPaseold-35-HA transfection (lane 2). However, infection with Ad.hPNPaseold-35AS at an m.o.i. of 50 or 100 pfu/cell resulted in marked reduction of hPNPaseOLD-35-HA (lanes 3 and 4), suggesting that Ad.hPNPaseold-35AS could effectively inhibit the expression of hPNPaseOLD-35. We generated a cell line in the HO-1 background that stably overexpresses hPNPaseold-35AS (HO-1-hPNPaseold-35AS). HO-1-pREP4 and HO-1-hPNPaseold-35AS cells were infected with Ad.hPNPaseold-35AS at an m.o.i. of 5, 25, and 100 pfu/cell, and after 24 h the cells were treated with either IFN-{beta} or MEZ, alone or in combination. Cell viability was assayed after 5 days. It should be noted that HO-1 cells are relatively refractory to IFN-{beta} and a high concentration of IFN-{beta} (2000 units/ml) is required to induce growth inhibition (2, 5). Infection with Ad.hPNPaseold-35AS provided small but significant protection against IFN-{beta}-induced growth inhibition both in HO-1-pREP4 and in HO-1-hPNPaseold-35AS in a dose-dependent manner (Fig. 7, B and D). As shown in Fig. 7B, treatment with IFN-{beta} reduced cell viability by ~75% in HO-1-pREP4. In HO-1-pREP4 infected with Ad.hPNPaseold-35AS at an m.o.i. of 100 pfu/cell and in HO-1-hPNPaseold-35AS, cell viability was reduced by ~55% by IFN-{beta} (Fig. 7B, columns 4 and 5, respectively). When HO-1-hPNPaseold-35AS cells were infected with Ad.hPNPaseold-35AS at an m.o.i. of 100 pfu/cell, the cell viability was reduced by only ~45% (Fig. 7B, column 8). A combination of IFN-{beta} and MEZ treatment reduced cell viability of HO-1-pREP4 by ~94% (Fig. 7D, column 1). This inhibitory effect was partially reversed in HO-1-pREP4 cells infected with Ad.hPNPaseold-35AS at an m.o.i. of 100 pfu/cell, and in HO-1-hPNPaseold-35AS cells, the reduction in cell viability was ~87% (Fig. 7D, columns 4 and 5, respectively). In HO-1-hPNPaseold-35AS cells infected with Ad.hPNPaseold-35AS at an m.o.i. of 100 pfu/cell, the reduction in cell viability was ~80% (Fig. 7D, column 8). The antisense approach did not provide protection against MEZ-induced growth arrest (Fig. 7C), even at an m.o.i. of 100 pfu/cell. Because of nonspecific cytotoxicities of adenovirus infection, an m.o.i. higher than 100 pfu/cell was not employed in these studies. These results indicate that, as a predominantly type I interferon-inducible gene, hPNPaseold-35 might be involved in mediating growth arrest induced by IFN-{beta}. The observation that infection of Ad.hPNPaseold-35AS in HO-1-hPNPaseold-35AS cells provided superior protection indicates that the dosage of antisense molecules might determine the relative level of protection from growth inhibition.



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FIG. 7.
Antisense inhibition of hPNPaseold-35 provides protection against IFN-{beta}-mediated growth inhibition. A, HO-1 cells were transfected with 5 µg each of either pcDNA3.1 or hPNPaseold-35-HA. The cells were infected with either Ad.vec or Ad.hPNPaseold-35AS at an m.o.i. of 50 or 100 pfu/cell 6 h later. The expression of hPNPaseOLD-35-HA was analyzed in cell lysates by Western blot analysis using Anti-HA antibody 48 h after infection. B, HO-1-pREP4 and HO-1-hPNPaseold-35AS cells were infected with either Ad.vec or Ad.hPNPaseold-35AS at an m.o.i. of 5, 25 and 100 pfu/cell. 24 h later, the cells were treated with IFN-{beta} (2000 units/ml) for 5 days and cell viability was assayed by MTT assay. C, HO-1-pREP4 and HO-1-hPNPaseold-35AS cells were infected as in Fig. 7B and were treated with MEZ (10 ng/ml). Cell viability was assayed after 5 days. D, HO-1-pREP4 and HO-1-hPNPaseold-35AS cells were infected as in Fig. 7B and were treated with IFN-{beta} + MEZ. Cell viability was assayed after 5 days. For panels B, C, and D the column numbers represent the legends at the bottom of the figure and the data represent mean ± S.D. of two independent experiments each performed in octuplicate.

 

Because Ad.hPNPaseold-35 infection induces apoptosis in HO-1 cells, the effect of Ad.hPNPaseold-35 infection on the expression levels of pro- and anti-apoptotic genes were examined. Infection with Ad.hPNPaseold-35 resulted in down-regulation of the anti-apoptotic protein Bcl-xL (Fig. 8A). The expression levels of the anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax remained unchanged. Stable HO-1 cell lines expressing either Bcl-2 or Bcl-xL were generated, and these cell lines were infected with Ad.hPNPaseold-35. As shown in Fig. 8B, overexpression of Bcl-xL, but not Bcl-2, provided partial protection against Ad.hPNPaseold-35-induced cell death.



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FIG. 8.
Effect of Ad.hPNPaseold-35 on the expression of pro- and anti-apoptotic molecules. A, infection protocol was performed as described in Fig. 4A. Expressions of the indicated proteins were detected by Western blot analysis. B, HO-1 (white column), HO-1-bcl-2 (gray column) and HO-1-bcl-xL (black column) cells were either uninfected or infected with Ad.vec or Ad.hPNPaseold-35 at an m.o.i. of 100 pfu/cell. Cell viability was determined by MTT assay 5 days after infection. The data represent mean ± S.D. of three independent experiments each performed in octuplicate.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular mechanism by which Ad.hPNPaseold-35 induces growth arrest has striking resemblance to that observed during terminal differentiation and senescence. The Max network of transcription factors has been implicated in mediating cell cycle arrest during differentiation. Myc levels rapidly diminish during terminal differentiation of many cell types, and enforced expression of Myc inhibits or modulates terminal differentiation of myoblasts, erythroleukemia cells, adipocytes, B lymphoid cells, and myeloid cells among others (3639). Inhibition of Myc is able to block mitogenic signals and drive cells toward terminal differentiation. Stable transfection of multiple copies of the Myc DNA-binding sequence, resulting in sequestration and squelching of endogenous Myc, accelerates proliferative senescence in rat embryo fibroblasts (40). During the transition from an undifferentiated to a differentiated state, there is a switch from Myc-Max to Mad-Max complexes that function as transcriptional repressors. Overexpression of the Mad family transcription factors Mad1 and Mxi1 arrests cells in the G1 phase of the cell cycle and promotes differentiation of some cell types (4143). There is an intimate relationship between members of the Max network and the CDK inhibitors. The expression of both Mad1 and p27KIP1 are up-regulated during differentiation (4447). Terminally differentiated melanocytes induced by cAMP display elevated levels of p27KIP1 (48). Moreover, Myc is down-regulated during differentiation as levels of p27KIP1 increase. It has been demonstrated that Mad1 and p27KIP1 cooperate to promote terminal differentiation of granulocytes and to inhibit Myc expression and cyclin E-CDK2 activity (49). Overexpression of Mad1 in human melanoma cells results in a reduced proliferation rate, increased G0/G1 cell cycle accumulation, and active melanin synthesis (50). Our observation that Ad.hPNPaseold-35 infection results in down-regulation of Myc and up-regulation of Mad1 and that overexpression of Myc can protect from Ad.hPNPaseold-35-induced cell death suggests the involvement of the Max network in this process. More importantly, we observed that telomerase activity decreases following Ad.hPNPaseold-35 infection, which is also observed during terminal differentiation and senescence (51). During the induction of terminal differentiation in HO-1 cells by IFN-{beta} + MEZ treatment, telomerase activity was also decreased (Fig. 3B). It has been shown that human telomerase catalytic subunit (hTERT) is transcriptionally regulated by Myc-Max (52). Thus, down-regulation of Myc can partially explain the decreased telomerase activity following Ad.hPNPaseold-35 infection.

There are several models of growth arrest in which down-regulation of Myc is associated with up-regulation of p27KIP1. Retinoic acid-induced G1 arrest of human myeloid cells results in sequential down-regulation of Myc and cyclin E and upregulation of p27KIP1 (53). When deprived of adhesion, mammary epithelial cells arrest in the G1 phase of the cell cycle (54). This arrest is associated with down-regulation of Myc and up-regulation of p27KIP1 and overexpression of Myc in nonadherent cells reverses the inhibition of cell cycle progression. One interesting finding of the present study is that, although the level of p27KIP1 increased, that of p21CIP1/WAF-1/MDA-6 decreased in response to Ad.hPNPaseold-35 infection in HO-1 cells. Although both of these CDK inhibitors are involved in G1 arrest, the association of Myc with p27KIP1 is more profound than that with p21CIP1/WAF-1/MDA-6. In addition to being a transcriptional repressor for p27KIP1, Myc also drives the synthesis of a putative p27KIP1-sequestering protein, which renders the hyperphosphorylated p27KIP1 available for ubiquitination (55). Accordingly, it is likely that the down-regulation of Myc protein could prevent the synthesis of this putative protein, making p27KIP1 unavailable for ubiquitination. p27KIP1 is a recognized target for SCF, the ubiquitin ligase, and one of the critical components of SCF is Cul1, a Myc target gene (56). Thus, down-regulation of Myc results in increased p27KIP1 by multiple pathways. Two models of senescence-like growth arrest, one because of iron chelation in hepatocytes, the other because of inhibition of phosphoinositide 3-kinase pathway in mouse embryo fibroblasts, are also associated with up-regulation of p27KIP1 and down-regulation of p21CIP1/WAF-1/MDA-6 (57, 58). In both cases, the levels of p53 and p16INK4A were also decreased. We could not detect p16INK4A in HO-1 cells because the majority of melanomas are associated with genetic abnormalities in the p16INK4A locus. By Western blot analysis we could not detect any change in p53 protein level following Ad.hPNPaseold-35 infection. Thus, we could not attribute the down-regulation of p21CIP1/WAF-1/MDA-6, which lies downstream of p53, as a result of down-regulation of p53. Interestingly, we observed that both p21CIP1/WAF-1/MDA-6 and p53 were down-regulated at the mRNA level after Ad.hPNPaseold-35 infection (data not shown). A detailed study monitoring the half-life of p53 mRNA and protein, its phosphorylation status, and transcription-inducing activity following Ad.hPNPaseold-35 infection might be required to address the issue of whether there is any association between p53 and subsequent p21CIP1/WAF-1/MDA-6 down-regulation.

In the present study we observed that Ad.hPNPaseold-35 infection induces initial cell cycle arrest but eventually the cells die by apoptosis. Inhibition of c-myc in M14 human melanoma cells by an inducible antisense approach resulted in G1 cell cycle arrest, which was associated with increased levels of p27KIP1 and decreased levels of p21CIP1/WAF-1/MDA-6 (59). These cells eventually became apoptotic, and it was shown that upregulation of p27KIP1 was directly responsible for promoting apoptosis. It has been shown that, in addition to inhibiting cell cycle progression, overexpression of p27KIP1 alone could induce apoptosis (60, 61). Overexpression of Bcl-2 can protect HeLa cells from p27KIP1-induced apoptosis (61). Ad.hPNPaseold-35 infection did not alter the level of Bcl-2 protein in HO-1 cells; however, there was a significant reduction in Bcl-xL protein level and overexpression of Bcl-xL in these cells could protect HO-1 cells from Ad.hPNPaseold-35-mediated cell death. Bcl-2 and Bcl-xL are functionally similar molecules, and they are often functionally interchangeable, depending upon the cell type. In fact, in HO-1 melanoma cells, it has been shown previously that apoptosis-inducing agents modulate the level of Bcl-xL protein at a higher level than that of Bcl-2 protein (30).

3',5' processing or degradation of RNA controls many cellular events including the maturation of 5.8 S rRNA, the processing of many small RNAs, and the turnover of different types of mRNAs (6265). In eubacteria and chloroplasts, 3',5' RNA decay occurs through a multiprotein complex called the degradosome (66, 67). The bacterial degradosome contains several proteins, including the endoribonuclease RNase E, the exoribonuclease PNPase, enolase, and an RNA helicase (RhlB) (68, 69). The chloroplast degradosome contains only PNPase and no other proteins (67). In eukaryotic cells this processing is performed by the exosome which consists of at least 11 proteins, all of which either possess 3',5'-exoribonuclease activity or are predicted to contain exoribonuclease or RNA binding activity based on their homology with prokaryotic proteins such as RNase PH, RNase R, RNase D, and the RNA-binding domain of the S1 ribosomal protein (63, 70). The yeast exosome does not contain PNPase; however, the structural model based on the protein-protein interactions of the exosome subunits reveal that it resembles the structure of bacterial PNPase, which form homotrimers (71). With the isolation of the human exosome components, a similar structural model for human exosome has also been proposed (72). Although this model excludes the involvement of hPNPaseold-35 in the human exosome, it might be possible that hPNPaseold-35 has evolved to regulate a more specific function as is documented by its involvement in growth arrest during terminal differentiation and senescence. Another possible specialized function of hPNPaseold-35 could be its involvement in an alternative IFN-stimulated RNA-decay pathway that might include hPNPaseold-35, mda-5, a putative RNA helicase having growth suppressive property and mda-E-63, a gene having an RNase II motif (32, 73). Our observation that antisense inhibition of hPNPaseold-35 rescues the cells from IFN-{beta}-mediated growth inhibition confirms its biological role in mediating interferon action.

Terminal differentiation and senescence are two of the important ways by which the human body protects itself from the lethal effects of carcinogenesis. The observation that hPNPaseold-35 lies at the crossroads of these two important physiological processes and induces growth arrest supports a potential tumor suppressor role for this molecule. Further rigorous in vivo studies are required to confirm this possibility and to establish the functional properties of hPNPaseold-35. These studies will also indicate whether this gene and its encoded protein can be used for therapeutic applications. Experiments in fibroblasts and primary human melanocytes are also necessary to address the question of whether overexpression of hPNPaseold-35 can induce a true senescence-like phenotype and also whether specific differentiation markers are altered during this process. These studies should be very enlightening and may provide new insights into the processes of terminal differentiation and senescence and the functional relationships between these alternative cellular fates.


    FOOTNOTES
 
* This work was supported in part by Grants CA35675 and CA97318 from the NCI, National Institutes of Health, by the Samuel Waxman Cancer Research Foundation, and by the Chernow Endowment (all to P. B. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** Supported by National Institutes of Health Grant NS34746 and the Ataxia-Telangiectasia Children's Project. Back

¶¶ Michael and Stella Chernow Urological Cancer Research Scientist. To whom correspondence should be addressed: Dept. of Pathology, College of Physicians and Surgeons, Columbia University, 630 W. 168th St., P & S Box 23, BB-1505, New York, NY 10032. Fax: 212-305-8177; E-mail: pbf1{at}columbia.edu.

1 The abbreviations used are: IFN, interferon; MEZ, mezerein; m.o.i., multiplicity of infection; pfu, plaque-forming unit(s); AS, antisense; HA, hemagglutinin; CDK, cyclin-dependent kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Rb, retinoblastoma; GAPDH, glyceradehyde-3-phosphate dehydrogenase. Back


    ACKNOWLEDGMENTS
 
We thank Dr. Riccardo Dalla-Favera for providing p290-myc (2, 3) and Dr. John C. Reed for providing the Bcl-2 and Bcl-xL expression constructs and anti-Bcl-2, Bcl-xL, and Bax antibodies.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Fisher, P. B., and Grant, S. (1985) Pharmacol. Ther. 27, 143–166[CrossRef][Medline] [Order article via Infotrieve]
  2. Fisher, P. B., Prignoli, D. R., Hermo, H., Jr., Weinstein, I. B., and Pestka, S. (1985) J. Interferon Res. 5, 11–22[Medline] [Order article via Infotrieve]
  3. Graham, G. M., Guarini, L., Moulton, T. A., Datta, S., Ferrone, S., Giacomini, P., Kerbel, R. S., and Fisher, P. B. (1991) Cancer Immunol. Immunother. 32, 382–390[Medline] [Order article via Infotrieve]
  4. Guarini, L., Graham, G. M., Jiang, H., Ferrone, S., Zucker, S., and Fisher, P. B. (1992) Pigment Cell Res. Suppl. 2, 123–131
  5. Jiang, H., Su, Z. Z., Boyd, J., and Fisher, P. B. (1993) Mol. Cell Differ. 1, 41–66
  6. Hayflick, L. (1976) N. Engl. J. Med. 295, 1302–1308[Medline] [Order article via Infotrieve]
  7. Serrano, M., and Blasco, M. A. (2001) Curr. Opin. Cell Biol. 13, 748–753[CrossRef][Medline] [Order article via Infotrieve]
  8. Leszczyniecka, M., Kang, D.-C., Sarkar, D., Su, Z. Z., Holmes, M., Valerie, K., and Fisher, P. B. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 16636–16641[Abstract/Free Full Text]
  9. Grandori, C., Cowley, S. M., James, L. P., and Eisenman, R. N. (2000) Annu. Rev. Cell Dev. Biol. 16, 653–699[CrossRef][Medline] [Order article via Infotrieve]
  10. Ferre-D'Amare, A. R., Pognonec, P., Roeder, R. G., and Burley, S. K. (1994) EMBO J. 13, 180–189[Abstract]
  11. Murre, C., McCaw, P. S., Vaessin, H., Caudy, M., Jan, L. Y., Jan, Y. N., Cabrera, C. V., Buskin, J. N., Hauschka, S. D., Lassar, A. B., et al. (1989) Cell 58, 537–544[Medline] [Order article via Infotrieve]
  12. Blackwood, E. M., and Eisenman, R. N. (1991) Science 251, 1211–1217[Medline] [Order article via Infotrieve]
  13. Blackwood, E. M., Luscher, B., and Eisenman, R. N. (1992) Genes Dev. 6, 71–80[Abstract]
  14. Ayer, D. E., and Eisenman, R. N. (1993) Genes Dev. 7, 2110–2119[Abstract]
  15. Zervos, A. S., Gyuris, J., and Brent, R. (1993) Cell 72, 223–232[Medline] [Order article via Infotrieve]
  16. Hurlin, P. J., Queva, C., Koskinen, P. J., Steingrimsson, E., Ayer, D. E., Copeland, N. G., Jenkins, N. A., and Eisenman, R. N. (1995) EMBO J. 14, 5646–5659[Abstract]
  17. Eilers, M., Picard, D., Yamamoto, K. R., and Bishop, J. M. (1989) Nature 340, 66–68[CrossRef][Medline] [Order article via Infotrieve]
  18. Karn, J., Watson, J. V., Lowe, A. D., Green, S. M., and Vedeckis, W. (1989) Oncogene 4, 773–787[Medline] [Order article via Infotrieve]
  19. Bartek, J., and Lukas, J. (2001) Curr. Opin. Cell Biol. 13, 738–747[CrossRef][Medline] [Order article via Infotrieve]
  20. Baldin, V., Lukas, J., Marcote, M. J., Pagano, M., and Draetta, G. (1993) Genes Dev. 7, 812–821[Abstract]
  21. Resnitzky, D., Gossen, M., Bujard, H., and Reed, S. I. (1994) Mol. Cell. Biol. 14, 1669–1679[Abstract]
  22. Bouchard, C., Thieke, K., Maier, A., Saffrich, R., Hanley-Hyde, J., Ansorge, W., Reed, S., Sicinski, P., Bartek, J., and Eilers, M. (1999) EMBO J. 18, 5321–5333[Abstract/Free Full Text]
  23. Perez-Roger, I., Solomon, D. L., Sewing, A., and Land, H. (1997) Oncogene 14, 2373–2381[CrossRef][Medline] [Order article via Infotrieve]
  24. Hermeking, H., Rago, C., Schuhmacher, M., Li, Q., Barrett, J. F., Obaya, A. J., O'Connell, B. C., Mateyak, M. K., Tam, W., Kohlhuber, F., Dang, C. V., Sedivy, J. M., Eick, D., Vogelstein, B., and Kinzler, K. W. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2229–2234[Abstract/Free Full Text]
  25. Coller, H. A., Grandori, C., Tamayo, P., Colbert, T., Lander, E. S., Eisenman, R. N., and Golub, T. R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3260–3265[Abstract/Free Full Text]
  26. Pusch, O., Bernaschek, G., Eilers, M., and Hengstschlager, M. (1997) Oncogene 15, 649–656[CrossRef][Medline] [Order article via Infotrieve]
  27. Sears, R., Ohtani, K., and Nevins, J. R. (1997) Mol. Cell. Biol. 17, 5227–5235[Abstract]
  28. Yang, W., Shen, J., Wu, M., Arsura, M., FitzGerald, M., Suldan, Z., Kim, D. W., Hofmann, C. S., Pianetti, S., Romieu-Mourez, R., Freedman, L. P., and Sonenshein, G. E. (2001) Oncogene 20, 1688–1702[CrossRef][Medline] [Order article via Infotrieve]
  29. Claassen, G. F., and Hann, S. R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9498–9503[Abstract/Free Full Text]
  30. Lebedeva, I. V., Su, Z. Z., Chang, Y., Kitada, S., Reed, J. C., and Fisher, P. B. (2002) Oncogene 21, 708–718[CrossRef][Medline] [Order article via Infotrieve]
  31. Valerie, K. (1999) in Biopharmaceutical Drug Design and Development (Wu-Pong, S., and Rojanasakul, Y., eds) pp. 69–142, Humana Press, Totowa, NJ
  32. Kang, D. C., Gopalkrishnan, R. V., Wu, Q., Jankowsky, E., Pyle, A. M., and Fisher, P. B. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 637–642[Abstract/Free Full Text]
  33. Sarkar, D., Su, Z. Z., Lebedeva, I. V., Sauane, M., Gopalkrishnan, R. V., Valerie, K., Dent, P., and Fisher, P. B. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10054–10059[Abstract/Free Full Text]
  34. Wood, L. D., Halvorsen, T. L., Dhar, S., Baur, J. A., Pandita, R. K., Wright, W. E., Hande, M. P., Calaf, G., Hei, T. K., Levine, F., Shay, J. W., Wang, J. J., and Pandita, T. K. (2001) Oncogene 20, 278–288[CrossRef][Medline] [Order article via Infotrieve]
  35. Jiang, H., Lin, J., Young, S. M., Goldstein, N. I., Waxman, S., Davila, V., Chellappan, S. P., and Fisher, P. B. (1995) Oncogene 11, 1179–1189[Medline] [Order article via Infotrieve]
  36. Miner, J. H., and Wold, B. J. (1991) Mol. Cell. Biol. 11, 2842–2851[Medline] [Order article via Infotrieve]
  37. Coppola, J. A., and Cole, M. D. (1986) Nature 320, 760–763[Medline] [Order article via Infotrieve]
  38. Birrer, M. J., Raveh, L., Dosaka, H., and Segal, S. (1989) Mol. Cell. Biol. 9, 2734–2737[Medline] [Order article via Infotrieve]
  39. Larsson, L. G., Ivhed, I., Gidlund, M., Pettersson, U., Vennstrom, B., and Nilsson, K. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 2638–2642[Abstract]
  40. Rudolph, C., Halle, J. P., and Adam, G. (1998) Exp. Cell Res. 239, 361–369[CrossRef][Medline] [Order article via Infotrieve]
  41. Chen, J., Willingham, T., Margraf, L. R., Schreiber-Agus, N., DePinho, R. A., and Nisen, P. D. (1995) Nat. Med. 1, 638–643[Medline] [Order article via Infotrieve]
  42. Roussel, M. F., Ashmun, R. A., Sherr, C. J., Eisenman, R. N., and Ayer, D. E. (1996) Mol. Cell. Biol. 16, 2796–2801[Abstract]
  43. Cultraro, C. M., Bino, T., and Segal, S. (1997) Mol. Cell. Biol. 17, 2353–2359[Abstract]
  44. Casaccia-Bonnefil, P., Tikoo, R., Kiyokawa, H., Friedrich, V., Jr., Chao, M. V., and Koff, A. (1997) Genes Dev. 11, 2335–2346[Abstract/Free Full Text]
  45. Hauser, P. J., Agrawal, D., Flanagan, M., and Pledger, W. J. (1997) Cell Growth Differ. 8, 203–211[Abstract]
  46. Levine, E. M., Close, J., Fero, M., Ostrovsky, A., and Reh, T. A. (2000) Dev. Biol. 219, 299–314[CrossRef][Medline] [Order article via Infotrieve]
  47. Hsieh, F. F., Barnett, L. A., Green, W. F., Freedman, K., Matushansky, I., Skoultchi, A. I., and Kelley, L. L. (2000) Blood 96, 2746–2754[Abstract/Free Full Text]
  48. Haddad, M. M., Xu, W., Schwahn, D. J., Liao, F., and Medrano, E. E. (1999) Exp. Cell Res. 253, 561–572[CrossRef][Medline] [Order article via Infotrieve]
  49. McArthur, G. A., Foley, K. P., Fero, M. L., Walkley, C. R., Deans, A. J., Roberts, J. M., and Eisenman, R. N. (2002) Mol. Cell. Biol. 22, 3014–3023[Abstract/Free Full Text]
  50. Ohta, Y., Hamada, Y., Saitoh, N., and Katsuoka, K. (2002) Exp. Dermatol. 11, 439–447[CrossRef][Medline] [Order article via Infotrieve]
  51. Pandita, T. K. (2002) Oncogene 21, 611–618[CrossRef][Medline] [Order article via Infotrieve]
  52. Kyo, S., Takakura, M., Taira, T., Kanaya, T., Itoh, H., Yutsudo, M., Ariga, H., and Inoue, M. (2000) Nucleic Acids Res. 28, 669–677[Abstract/Free Full Text]
  53. Dimberg, A., Bahram, F., Karlberg, I., Larsson, L. G., Nilsson, K., and Oberg, F. (2002) Blood 99, 2199–2206[Abstract/Free Full Text]
  54. Benaud, C. M., and Dickson, R. B. (2001) Oncogene 20, 4554–4567[CrossRef][Medline] [Order article via Infotrieve]
  55. Obaya, A. J., Mateyak, M. K., and Sedivy, J. M. (1999) Oncogene 18, 2934–2941[CrossRef][Medline] [Order article via Infotrieve]
  56. O'Hagan, R. C., Ohh, M., David, G., de Alboran, I. M., Alt, F. W., Kaelin, W. G., Jr., and DePinho, R. A. (2000) Genes Dev. 14, 2185–2191[Abstract/Free Full Text]
  57. Yoon, G., Kim, H. J., Yoon, Y. S., Cho, H., Lim, I. K., and Lee, J. H. (2002) Biochem. J. 366, 613–621[CrossRef][Medline] [Order article via Infotrieve]
  58. Collado, M., Medema, R. H., Garcia-Cao, I., Dubuisson, M. L., Barradas, M., Glassford, J., Rivas, C., Burgering, B. M., Serrano, M., and Lam, E. W. (2000) J. Biol. Chem. 275, 21960–21968[Abstract/Free Full Text]
  59. D'Agnano, I., Valentini, A., Fornari, C., Bucci, B., Starace, G., Felsani, A., and Citro, G. (2001) Oncogene 20, 2814–2825[CrossRef][Medline] [Order article via Infotrieve]
  60. Katayose, Y., Kim, M., Rakkar, A. N., Li, Z., Cowan, K. H., and Seth, P. (1997) Cancer Res. 57, 5441–5445[Abstract]
  61. Wang, X., Gorospe, M., Huang, Y., and Holbrook, N. J. (1997) Oncogene 15, 2991–2997[CrossRef][Medline] [Order article via Infotrieve]
  62. Mitchell, P., Petfalski, E., and Tollervey, D. (1996) Genes Dev. 10, 502–513[Abstract]
  63. Mitchell, P., Petfalski, E., Shevchenko, A., Mann, M., and Tollervey, D. (1997) Cell 91, 457–466[Medline] [Order article via Infotrieve]
  64. Allmang, C., Kufel, J., Chanfreau, G., Mitchell, P., Petfalski, E., and Tollervey, D. (1999) EMBO J. 18, 5399–5410[Abstract/Free Full Text]
  65. Jacobs, J. S., Anderson, A. R., and Parker, R. P. (1998) EMBO J. 17, 1497–1506[Abstract/Free Full Text]
  66. Bessarab, D. A., Kaberdin, V. R., Wei, C. L., Liou, G. G., and Lin-Chao, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3157–3161[Abstract/Free Full Text]
  67. Baginsky, S., Shteiman-Kotler, A., Liveanu, V., Yehudai-Resheff, S., Bellaoui,M., Settlage, R. E., Shabanowitz, J., Hunt, D. F., Schuster, G., and Gruissem, W. (2001) RNA 7, 1464–1475[Abstract/Free Full Text]
  68. Carpousis, A. J., Van Houwe, G., Ehretsmann, C., and Krisch, H. M. (1994) Cell 76, 889–900[Medline] [Order article via Infotrieve]
  69. Py, B., Higgins, C. F., Krisch, H. M., and Carpousis, A. J. (1996) Nature 381, 169–172[CrossRef][Medline] [Order article via Infotrieve]
  70. Allmang, C., Petfalski, E., Podtelejnikov, A., Mann, M., Tollervey, D., and Mitchell, P. (1999) Genes Dev. 13, 2148–2158[Abstract/Free Full Text]
  71. Aloy, P., Ciccarelli, F. D., Leutwein, C., Gavin, A. C., Superti-Furga, G., Bork, P., Bottcher, B., and Russell, R. B. (2002) EMBO Rep. 3, 628–635[Abstract/Free Full Text]
  72. Raijmakers, R., Egberts, W. V., van Venrooij, W. J., and Pruijn, G. J. (2002) J. Mol. Biol. 323, 653–663[CrossRef][Medline] [Order article via Infotrieve]
  73. Jiang, H., Kang, D. C., Alexandre, D., and Fisher, P. B. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 12684–12689[Abstract/Free Full Text]