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[Cancer Research 63, 3840-3844, July 1, 2003]
© 2003 American Association for Cancer Research

Tumor Biology

p21Waf1/Cip1 Dysfunction in Neuroblastoma

A Novel Mechanism of Attenuating G0-G1 Cell Cycle Arrest1

Pamela P. McKenzie, Mary K. Danks, Richard W. Kriwacki and Linda C. Harris2

Departments of Molecular Pharmacology [P. P. M., M. K. D., L. C. H.] and Structural Biology [R. W. K.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In normal cells in which DNA has been damaged, p53 induces the expression of p21Waf1/Cip1; p21, in turn, binds to cyclin-dependent kinase 2 (cdk2) and inhibits its function. Inhibition of cdk2 results in cell cycle arrest in G0-G1. Although p53 is transcriptionally active and induces p21 expression in neuroblastoma (NB) cells, the G0-G1 checkpoint is attenuated. Here we report that the mechanism that mediates this defect in NB cells is the inability of p21 to bind to, or inhibit the activity of cdk2. However, when recombinant p21 protein was added to NB cell extracts in vitro, the protein inhibited the activity of cdk2. This finding suggests that endogenous p21 protein in NB cells is inactive and may be bound either to a protein complex or in a conformation that precludes its binding to cdk2. The dysfunction of p21 in NB cells represents a novel mechanism by which the G0-G1 cell cycle checkpoint can be inactivated. This mechanism may be important in regulating the growth of NB and potentially other types of tumors. Cdk inhibitors currently being developed for clinical use may be useful therapy for tumors such as NB in which endogenous cdk inhibitors are defective.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NBs3 are the most common extracranial solid tumors in children (1) . Mutations of the p53 gene in NBs are rare (2) , and at the time of diagnosis, a high proportion of NBs respond well to standard chemotherapy regimens. However, the probability of long-term survival of patients with high-risk disease at the time of diagnosis is only 20–40% (1 , 3) .

NB tumors generally respond to cellular DNA damage caused by chemotherapeutic agents resulting in increased levels of cellular p53 protein. The p53 protein then induces the expression of several proteins involved in apoptosis and cell cycle regulation, including Bax and p21Waf-1/Cip-1. The p21Waf1/Cip1 protein not only acts as an assembly factor for cdk4, cdk6, and cyclin D-type complexes that initiate entry into the S phase of the cell cycle (4 , 5) , but p21 also is a cdk2 inhibitor that initiates G1 arrest in response to DNA damage (6) . By specifically inhibiting the activity of the cdk2-cyclin E complex, p21 prevents the phosphorylation of the cdk2 substrate Rb tumor suppressor protein (pRb) at specific sites; this inhibition of Rb phosphorylation results in the arrest of cells in the G0-G1 phase of the cell cycle (5) .

DNA damage-induced expression of p53 and p21 cannot induce G1 arrest in most NB cell lines (7, 8, 9, 10) . Because a defective G0-G1 checkpoint can result in enhanced chemosensitivity (11) , the attenuated arrest in NBs could enhance their chemotherapeutic response. Here we examined the function of p21 in NB cells to elucidate its role in the regulation of the cell cycle of NB and thus to understand how p21 fails to induce G1 arrest in NB cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
The NB-1643 line (wild-type p53, amplified MYCN) was obtained from the Pediatric Oncology Group (12) . SJNB-1 (wild-type p53, no MYCN amplification but elevated MycN expression), SJNB-4 (mutant p53, MYCN amplification), and Rh30 (mutant p53, no MYCN amplification) cell lines were obtained from Dr. Peter Houghton (St. Jude Children’s Research Hospital, Memphis, TN). The NBL-S line (wild-type p53, no MYCN amplification but elevated MycN expression) was obtained from Dr. Garrett Brodeur (The Children’s Hospital of Philadelphia, Philadelphia, PA), and SK-N-SH (wild-type p53, no MYCN amplification) was obtained from the American Type Culture Collection (Manassas, VA). These cell lines have been described elsewhere (8 , 13 , 14) .

Antibodies.
Monoclonal antibodies against cdk4 or cdk2 that were used for Western blots were obtained from Oncogene Research Products (Cambridge, MA); those against Rb protein were obtained from PharMingen (San Diego, CA); and those against ß-tubulin were from ICN Biomedicals, Inc. (Aurora, OH). Affinity-purified rabbit antibodies specific for p21 (C-19) and monoclonal antibodies specific for cyclin E (HE12), cdk2 (D-12), and p21 (187) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Agarose-conjugated antibodies used in the IP assays, i.e., anti-p21 (C-19), anti-cdk2 (M2), anti-cdk4 (H-22), and anti-cyclin E (HE111), were also obtained from Santa Cruz Biotechnology.

Adenovirus Vectors and Transduction.
The adenovirus vector Ad.p21 containing the p21Waf1/Cip1 cDNA was obtained from Dr. Wafik el-Diery (University of Pennsylvania School of Medicine, Philadelphia, PA), and the Ad.p53 (Av1p53) vector containing wild-type p53 cDNA was obtained from Genetic Therapy, Inc. (a Novartis Company; Gaithersburg, MD; Ref. 15 ). Dr. Prem Seth (Human Gene Therapy Research Institute, Des Moines, IA) provided the Ad.p16 vector, which contained the p16Ink4a cDNA, and Dr. Janet Houghton (St. Jude) provided the control adenovirus vector (Ad.VC). NB and Rh30 cells were transduced as described previously (9) .

Transfection and Luciferase Assays.
FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN) was used according to the manufacturer’s instructions. The Firefly Luciferase Reporter System (Promega Corporation, Madison, WI) was used with the plasmid vector pGL2-Control as the positive control, and pGL2-Basic as the negative control. The (E2F-1)3-luc E2F-responsive promoter reporter plasmid was provided by Dr. Srikumar Chellappan (Columbia Presbyterian Medical Center, New York, NY). Luciferase activity was measured by using the Promega Luciferase Assay system according to the manufacturer’s instructions.

Western Blot Analysis.
Cell extracts were prepared and Western blot analyses were carried out as described previously (9) .

IP Studies.
The IP studies were carried out as described previously (16) with the following modifications: cell extracts were prepared in 20 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, and 0.1% Tween 20, containing freshly added protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 10 mM sodium fluoride) and phosphatase inhibitor (1 mM sodium orthovanadate). In addition, cellular protein (400 µg) was diluted to 0.5 ml with 20 mM HEPES (pH 7.5); 150 mM NaCl, and 0.1% Tween 20, containing freshly added phosphatase and protease inhibitors (1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 mM sodium fluoride).

Cyclin E-associated Cdk2 Kinase Assay.
Protein extracts (100 µg) were incubated overnight with antibodies against cyclin E and protein G-conjugated agarose beads. The assays were then carried out as described previously (17 , 18) . Gels were exposed to phosphoimage screens and were analyzed by using a Molecular Dynamics PhosphoImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).

Cell Cycle Analysis.
After adenoviral transduction, cells were resuspended (1 x 106 cells/ml) in a solution of propidium iodide, and their DNA content was analyzed as described previously (9) .

In Vitro Assay of cdk2 in Complex with Recombinant p21B Protein.
His-tagged recombinant p21B protein, which contains amino acid residues 9 through 84 of p21Waf1/Cip1, was purified as described previously (19) . The cell extracts were incubated with p21B protein (final concentration, 200 nM) and DTT (final concentration, 2 mM) for 1 h before IP. For IP analysis, the p21B containing cell extract was divided equally, agarose-conjugated p21 antibodies were added to an aliquot, and anti-His tag antibodies were added to the remaining aliquot. The IPs incubated for an additional 1 h at room temperature while rotating. Each aliquot was analyzed by Western blot analysis for the presence of cdk2 within the complex.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Absence of Mutations in p21 in NB Cells.
G1 arrest is attenuated in wild-type p53 expressing NB cell lines NB-1643 and SK-N-SH that have experienced DNA damage (9) , a finding that suggested that p21 is not functional in these cells. Therefore, we performed reverse transcription-PCR analysis and sequenced the coding region of the p21cDNA from the NB-1643 and SK-N-SH cell lines. The p21 cDNA sequences in all of the NB cell lines examined were identical to the wild-type sequence (data not shown).

NB Cells Fail To Undergo a G0-G1 Arrest in Response to Adenoviral-mediated Overexpression of p21.
Because the p21 coding sequence of the NB cell lines contained no mutations, we hypothesized that the level of p21 induced in these cells after DNA damage is insufficient to mediate G0-G1 arrest. To test this hypothesis, we used the adenovirus vector Ad.p21 (MOI: 5, 10, or 20 pfu/cell) to increase the intracellular level of p21 in NB cells. We then analyzed p21 expression and the cell cycle of the cultured cells.

For this study, we chose the NB cell line NB-1643 and a control rhabdomyosarcoma cell line, Rh30. NB-1643 cells are typical NB cells in that expression of endogenous p21 is induced after DNA damage, but no concomitant increase in the number of cells in G0-G1 occurs (9) . In contrast, the number of Rh30 cells arrested in G0-G1 typically increases in response to an increased level of p21 protein (9) . The MOI chosen were sufficient to transduce 100% of the cells of both cell types as determined by transduction with Ad.LacZ adenoviral vector and staining the cells for ß-galactosidase. Ad.p21 at a MOI of 10 resulted in a low level of cell death of <5% for each cell line, as determined by the proportion of cells in the sub-G1 fraction after cell cycle analysis by flow cytometry (data not shown). The level of cellular p21 increased in the NB-1643 and Rh30 cell lines after exposure to Ad.p21 (Fig. 1A)Citation , but the percentage of only Rh30 cells in G1 arrest substantially increased (Fig. 1B)Citation . Therefore, the defect in the G0-G1 arrest pathway in NB cells appeared to be downstream of the induction of p21 expression.



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  		Fig. 1. The overexpression of p21 does not inhibit the progression of NB-1643 cells through the cell cycle. A, NB-1643 and Rh30 cells were exposed to increasing amounts of the adenovirus Ad.p21, which contained p21 cDNA (MOI: 5, 10, and 20 pfu). The level of p21 expression was determined by Western blot analysis, which was performed after the cells had undergone one cell doubling. Equal amounts of protein were loaded on the gel, and ß-tubulin was used as a loading control. B, After transduction with increasing MOI of Ad.p21, Rh30 cells (squares) showed a relative increase in the proportion of cells accumulated in G0-G1 arrest; a minimal effect was observed in the NB cells (circles).

 
Inability of p21 To Inhibit cdk2 Activity in NB Cells.
When functional, p21 binds to cyclin E-cdk2 complexes to inhibit the kinase activity of cdk2. After exposure to Ad.p21, the Rh30 cells demonstrated a 60% reduction in relative cyclin-E-dependent cdk2 activity as determined by the amount of phosphorylated HH1 (Fig. 2A)Citation . In contrast, no reduction in HH1 phosphorylation was observed in Ad.p21-treated NB-1643 cells. This finding suggested that the defect in the p21 pathway involves the inability of p21 to inhibit cdk2 activity. To confirm this theory, we also analyzed cdk2 activity by evaluating the phosphorylation of another cdk2 substrate, Rb. Cells were exposed to empty adenoviral vector (Ad.VC), Ad.p21, Ad.p53, or IR, and the phosphorylation status of pRb was evaluated by Western blot analysis. pRb was expressed only in Ad.p21- or Ad.p53-treated Rh30 control cells (Fig. 2B)Citation , demonstrating that cdk activity in these cells was inhibited. In contrast, empty vector treatment, Ad.p53, Ad.p21, and IR exposure did not alter the level of phosphorylation of Rb in NB cells; only ppRb was detected in NB-1643 cells (Fig. 2B)Citation . This second method of analysis demonstrated that cdk activity was not inhibited by p21 under these experimental conditions and suggest that proteins in the p21 pathway that are affected by the phosphorylation status of Rb, such as the E2F transcription factors, may be deregulated in NB cells. The slight decrease in Rb expression after exposure to either IR or the adenoviral vectors likely results from down-regulation of Rb expression in the proportion of cells that are undergoing apoptosis (20 , 21) .



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  		Fig. 2. The function of cdk2 is not inhibited in p21Waf1/Cip1-expressing NB-1643 cells. A, densitometric analysis of HH1 phosphorylation is depicted as the relative level in cyclin E-dependent cdk2 kinase activity. HH1 phosphorylation was determined in NB-1643 and Rh30 cells before (solid bar) and after Ad.p21 (MOI, 10 pfu) treatment (open bars). The activity for the control untreated cell extract was set to 1. The figure shows the mean decrease ± SE in kinase activity relative to this control after p21 expression. Data from three experiments are combined. B, Western blot analysis of the phosphorylation status of the tumor suppressor RB protein (pRb) was determined after treatment with IR (10 Gy), control adenovirus vector (Ad.VC), or an adenovirus expressing either p21 (Ad.p21; MOI, 10 pfu) or wild-type p53 (Ad.p53; MOI, 10 pfu). C, the relative level of E2F-responsive promoter luciferase activity substantially decreased in Rh30 cells after exposure to Ad.p21 (MOI, 10 pfu); no change in activation of E2F was observed in the NB-1643 cells. Results presented as the mean ± SE are representative of at least three independent experiments.

 
Rb sequesters E2F proteins, thereby inhibiting transcriptional activation of E2F-responsive promoters of S-phase-specific genes (22) . Therefore, we used a luciferase reporter assay to evaluate the activation of an E2F-responsive promoter in extracts of Ad.p21-exposed Rh30 and NB-1643 cells. Normal sequestration of E2F by pRb in Rh30 cells was indicated by the 79% reduction in luciferase activity of these cell extracts. In contrast, the overexpression of p21 in NB-1643 cells did not affect luciferase activity, consistent with the activation of the E2F-responsive promoter in the presence of ppRb (Fig. 2C)Citation .

Absence of Binding of p21 to cdk2 in NB Cell Lines.
The inability of p21 to suppress cdk2 activity in NB cells suggested that p21 does not bind to active cdk2 in these cells. Therefore, we performed a series of sequential IPs with antibodies, first against p21 and then against cyclin E, to determine whether p21 binds to cdk2-cyclin E complexes. Most of the p21 in Ad.p21-treated Rh30 cells was immunoprecipitated in complex with cdk2 and cyclin E (Fig. 3ACitation , Lanes 4 and 6). This finding was consistent with p21 acting to reduce cdk2 kinase activity resulting in a decreased proportion of ppRb in Rh30 cells exposed to Ad.p21. In extracts of Ad.p21-treated NB-1643 cells, very little cdk2-cyclin E coprecipitated with p21 (Fig. 3ACitation , Lanes 3 and 4) suggesting that p21 could not bind to this complex. Even when equivalent levels of p21 were expressed in the two cell lines, efficient binding of cdk2 to p21 was only observed in Rh30 cell extract (data not shown). The cyclin E that was observed to coprecipitate with p21 likely occurred by the direct binding of p21 to cyclin E in the absence of cdk2 (23) . A lack of p21-cdk2 binding was observed in analyses of three other NB cell lines: SJNB-1, NBL-S (Fig. 3B)Citation , and SK-N-SH (data not shown). The sequential IP studies using anti-cyclin-E antibodies resulted in coprecipitation of cdk2 and cyclin E in the NB cell extract (Fig. 3A)Citation . The presence of these complexes, which lacked p21, supports the hypothesis that although cdk2 is active in these cells (Fig. 2A)Citation , it is unable to bind to p21.



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  		Fig. 3. IP analyses of NB cells revealed the absence of p21 in complex with cdk2. A, extracts from untreated or Ad.p21-treated NB-1643 and Rh30 cells were sequentially subjected to IP analyses with antibodies, first against p21 and then cyclin E. Western blot analysis demonstrated that p21 did not coprecipitate with cdk2 in NB cells. B, IP analysis of additional Ad.p21-treated (MOI, 10 pfu) NB cells from SJNB-1 and NBL-S NB lines demonstrated that the cdk2 present in the cell extracts did not coprecipitate in complex with p21.

 
Sequestration of p21 by cdk4.
We hypothesized that cdk4, or another cellular protein, could sequester p21 in NB cells and prevent it from forming a complex with cdk2. To block the binding of p21 to cdk4 complexes, NB-1643 and Rh30 control cells were treated with Ad.p16 for 24 h before they were treated with Ad.p21. Because p16Ink4a preferentially binds cdk4 and cdk6 and specifically inhibits their activity, but not that of cdk2 (reviewed by Sherr and Roberts, Ref. 5 ), we hypothesized that if p16 prevents p21 from binding to cdk4, then p21 would be available to bind to cdk2, inhibit its activity, and mediate G0-G1 arrest.

Cdk4 immunoprecipitates from both Ad.p21-treated Rh30 and NB-1634 cells contained a proportion of p21 bound to cdk4 (Fig. 4A)Citation . However, p21 immunoprecipitates from the control Rh30 cells contained both cdk4 and cdk2 (Fig. 4BCitation , Lane 3), a finding that suggests that in Rh30 cells, p21 inhibits cdk2 activity and, consequently, pRb phosphorylation. The lack of p21 binding to cdk2 in NB-1643 cell extracts suggested that cdk4 may be inhibiting their interaction. Therefore, we hypothesized that if p16 could block the p21 binding sites on cdk4, p21 would then be free to bind cdk2. In Rh30 cells, p16 blocked a proportion of p21 from binding to cdk4 complexes; however, a corresponding increase in cdk2-p21 complexes was not observed (Figs. 4BCitation , Lane 4). These results suggest that the cdk2-p21 binding was maximal in the Rh30 cells and that release of p21 from cdk4 could not bind additional cdk2 protein.



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  		Fig. 4. Overexpression of p16 blocks p21 from binding to cdk4 complexes in NB cells but does not facilitate the formation of p21-cdk2 complexes. Cells were either untreated (Lane 1) or exposed to Ad.p16 (Lane 2), Ad.p21 (Lane 3), or Ad.p16 and then Ad.p21 24 h later (Lane 4). Cells were harvested after one cell doubling, and cell extracts were divided and immunoprecipitated with antibodies against cdk4 (A) or p21 (B). Western blot analyses of both sets of IPs were done to show the presence of p21 and cdk4. The same Western blot of p21 immunoprecipitates was then reprobed with anti-cdk2 antibodies (B). Western blots of cell extracts were probed with anti-pRb antibodies (C). After exposure to Ad.p16, Ad.p21, or both, NB cells expressed predominantly ppRb, whereas pRb was predominant in extracts of the control cells (C).

 
The p21 immunoprecipitates of the NB cells exposed to Ad.p21 contained more cdk4 than cdk2 (Figs. 4BCitation , Lane 3). When NB-1643 cells were pretreated with Ad.p16, the amount of cdk4-p21 complexes that formed decreased (Fig. 4BCitation , Lane 4) a finding that suggests that p16 blocked a proportion of p21 from binding cdk4. After treatment with both Ad.p16 and Ad.p21, NB-1634 cells showed a minimal increase in p21-cdk2 binding (Fig. 4BCitation , Lane 4); however, ppRb was still present in these cells (Fig. 4CCitation , Lane 4), and the percentage of cells in G0-G1 remained unchanged (data not shown). It is unknown why the intensity of the pRb band does not increase on reduction of the hyperphosphorylated form in the Rh30 cell extracts on p21 and p16 expression. However, this same observation has been made by others (17 , 24 , 25) . Because binding of p21 to cdk2 inhibits kinase activity, these results suggest that in NB cells, an insufficient number of p21-cdk2 complexes formed to elicit a biological response. The elevated level of p21 expression after Ad.p16 treatment alone was probably caused by adenovirus-mediated induction of wild-type p53 (26) .

Recombinant p21B Protein Can Bind To and Inhibit cdk2 Activity in NB Extracts.
Despite the increased availability of intracellular p21 that resulted from exposure to Ad.p21, alone or in combination with Ad.p16, only a small amount bound cdk2 and cyclin E-cdk2 activity persisted in NB cells. This finding supports our hypothesis that the binding of p21 to cdk2 is impaired. The sequence analysis of the cdk2 cDNA in NB-1643 cells revealed no mutations (data not shown). To eliminate the possibility that the protein-binding defect in NB cells is caused by a defect in the cdk2-binding site on p21, we performed an in vitro cdk2-binding assay in NB extracts. For these experiments we used p21B, a recombinant histidine (His)-tagged truncated p21 protein that includes the cdk inhibitory domain and has previously been shown to inhibit cdk2 kinase activity in vitro (19) .

Recombinant p21B was added to Ad.p21-treated NB-1643 cell extract, which was then divided equally. IP analyses were carried out by using either agarose-conjugated anti-p21 or anti-His tag antibodies. Results from Western blot analyses confirmed the presence of cdk2 in the extracts before they were subjected to IP analysis (Fig. 5A)Citation . Recombinant p21B was detected in samples immunoprecipitated with anti-His tag antibodies (Fig. 5B)Citation . The cdk2 formed an in vitro complex with p21B (Fig. 5CCitation , bottom panel) that resulted in a 79% reduction in cdk2 activity, as indicated by the decreased amount of phosphorylated HH1 (Fig. 5D)Citation . This reduction represents an additional 64% decrease compared with that seen in NB cells treated with Ad.p21 alone (Fig. 2A)Citation . Similar results were obtained in NB cells that were not treated with Ad.p21 (data not shown). Addition of p21B to Ad.p21-treated Rh30 extracts had no additional effect on the cdk2 kinase activity (Fig. 5D)Citation .



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  		Fig. 5. Recombinant purified p21B binds to cdk2 in Ad.p21-treated NB-1643 cells. A, Western blot analysis of NB cell extracts in the absence (-) or presence (+) of 200 nM recombinant His-tagged p21B protein revealed that the expression of cdk2 in these cells was not affected by p21B treatment. B, IP of extracts containing p21B demonstrate the presence of adenovirus-generated p21 (anti-p21 IP) and recombinant p21 (anti-his IP). C, IP analysis using anti-p21 and anti-His antibodies revealed that cdk2 was in complex with p21B protein in NB cells. D, the assay of cyclin E-associated cdk2 activity in NB-1643 and Rh30 cell extracts in the absence or presence of p21B recombinant protein. Cdk2 activity decreased in the presence of p21B. No change was observed in the Rh30 cell extracts.

 
Because recombinant p21B bound to cdk2 and inhibited its activity in NB cell extracts, this finding suggests that the cdk2 in NB cells is normal but that the endogenously translated p21 does not function appropriately. We concluded that the inability of p21 to bind to and inhibit cyclin-E-dependent cdk2 activity is a mechanism for the defective G1-arrest phenotype observed in NB cells.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The G1 checkpoint of the cell cycle is defective in most NB cell lines (7 , 9 , 10) ; therefore, NB cells progress through the cell cycle unregulated even after DNA damage, resulting in genetic instability and rapidly growing tumors.

In normal cells that sustain DNA damage, p21Waf1/Cip1, a member of the Cip/Kip family of cdk inhibitors, induces G0-G1 cell cycle arrest (6) . Although once considered to be a broad inhibitor of all cyclins and cdks, p21 family members are now believed to function as assembly factors for D-type cyclins and cdk4/6 complexes but as inhibitors of cdk2. Specifically, the p21-cyclin D-cdk4/6 complex is catalytically active (4) ; however, p21 and p27 inhibit the kinase activity of the cyclin E-cdk2 complex and prevent the entry of the cell into S phase (27, 28, 29) . The accumulation of active cyclin D-cdk4/6 complexes during early G1 initiates the phosphorylation of pRb, which is completed by cyclin E-cdk2 complexes during mid-to-late G1 phase. The subsequent release of members of the E2F family of transcription factors by ppRb facilitates the entry of the cell into S phase (30 , 31) .

Ad.p21 treatment of control Rh30 cells resulted in a decrease in cdk2 kinase activity, predominantly pRb, a decrease in E2F activity, and a G0-G1 arrest, as would be expected in cells in which p21 function is intact. The lack of a G0-G1 arrest observed in NB-1643 cells was associated with the inability of p21 to inhibit cdk2 activity, alter the phosphorylation status of pRb, or decrease E2F transcriptional activity. The defect in p21 function in NB cells was likely attributable to an insufficient amount of the p21 protein in complex with cdk2.

One possible explanation for the failure of p21 to bind to cdk2 complexes in NB cells is that another protein(s) is bound to p21 or cdk2, thereby preventing the formation of p21-cdk2 complexes. It is unlikely that cdk2 is directly bound to other proteins because the cyclin E-cdk2 complexes present in NB-1643 cells were functional (Figs. 2ACitation and 3BCitation ). However, p21 may be bound to other proteins, and/or p21 could be in a conformation that precludes its binding to cdk2 but that allows its binding to cdk4. This theory was supported by the observation that overexpression of p16 prevented a proportion of p21 protein from binding to cdk4 complexes, and yet the number of p21-cdk2 complexes that formed was insufficient to prevent cdk2 activity and the hyperphosphorylation of pRb. We hypothesized that another NB protein(s) mediates changes in the conformation of p21 or masks the cdk2-binding site on p21. To address this question, a truncated recombinant p21B protein that inhibits cdk2 kinase activity in vitro (19) was added to NB cell extracts. The recombinant p21B protein bound to and inhibited the cdk2 activity efficiently, irrespective of Ad.p21 treatment (Fig. 5)Citation . These results suggest that either posttranslational modification of endogenous p21 occurs in NB cells or NB cells contain a protein that binds to and inhibits the function of endogenously translated p21, but not that of recombinant p21B.

In summary, the overexpression of p21 in NB cells does not inhibit the kinase activity of cdk2, nor does it induce significant G0-G1 arrest. Furthermore, p21 and cdk2 cannot bind even in cell extracts in vitro. On the basis of these findings, we propose a model of p21 function in NB cells. In normal cells, cyclin D-cdk4 complexes containing p21 are catalytically active, yet the binding of p21 to cyclin E-cdk2 complexes inhibits kinase function. In NB cells, cyclin D-cdk4 complexes containing p21 are also active. However, only a limited amount of p21 binds to cdk2, and the kinase activity of cyclin E-cdk2 remains intact. We speculate that endogenous p21 in NB cells may be in a conformation that precludes its binding to cdk2. An altered conformation might allow the binding of p21 to cdk4 complexes but restrict binding to cdk2.

In conclusion, the inability of p21 in NB cells to bind to cyclin E-cdk2 complexes and inhibit cdk2 activity results in the maintenance of ppRb and continued progression through the cell cycle. The defective G0-G1 cell cycle checkpoint of NB cells may result in the replication of damaged DNA, genetic instability, and rapidly dividing tumor cells. However, novel cancer therapies that are being developed at present for clinical use and that target cdk activity may be useful for the treatment of tumors such as NB, in which endogenous cdk inhibitors are defective.


   ACKNOWLEDGMENTS
 
We thank Queen Rodgers and Limin Xiao for technical assistance, Chris Morton for densitometric analysis, Richard Ashmun and the Flow Cytometry Laboratory at St. Jude for cell cycle analysis, and John Easton for helpful discussions. We also thank the Department of Biomedical Communications, particularly Renee Baxter, Julie Groff, and Linda Rawlinson, for formatting our figures, and the Department of Scientific Editing for editing this manuscript.


   FOOTNOTES
 
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.

1 Supported by NIH Grants CA23099 and CA77541 (to L. C. H.), CA79763 (to M. K. D.), CA82491 (to R. W. K.), and Cancer Center Support Grant CA21765, and by the American Lebanese Syrian Associated Charities (ALSAC). Back

2 To whom requests for reprints should be addressed, at Department of Molecular Pharmacology, Mail Stop 230, St. Jude Children’s Hospital, Memphis, TN 38105. Phone: (901) 495-3440; Fax: (901) 495-4293; E-mail: linda.harris{at}stjude.org Back

3 The abbreviations used are: NB, neuroblastoma; cdk, cyclin-dependent kinase; HH1, histone H1; IP, immunoprecipitation; IR, ionizing radiation; MOI, multiplicity/multiplicities of infection; ppRb, hyperphosphorylated Rb (tumor suppressor protein); pRb, hypophosphorylated Rb (tumor suppressor protein); Rb, retinoblastoma (tumor suppressor protein); pfu, plaque-forming unit(s). Back

Received 10/21/02. Accepted 4/25/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Matthay K. K. Neuroblastoma: a clinical challenge and biological puzzle. CA Cancer J. Clin., 45: 179-192, 1995.[Abstract]
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