VRK1 Signaling Pathway in the Context of the Proliferation Phenotype in Head and Neck Squamous Cell Carcinoma

Published OnlineFirst March 3, 2006; DOI: 10.1158/1541-7786.MCR-05-0212 Published Online First on March 3, 2006

Claudio R. Santos,1 Marıá Rodrı´guez-Pinilla,2 Francisco M. Vega,1 Jose´L. Rodrı´guez-Peralto,3 Sandra Blanco,1 Ana Sevilla,1 Alberto Valbuena,1 Teresa Hernańdez,1 Andre´J. van Wijnen,4 Fengzhi Li,5 Enrique de Alava,1 Montserrat Sańchez-Ce´spedes,2 and Pedro A. Lazo1

1Instituto de Biologıá Molecular y Celular del Cańcer, Consejo Superior de Investigaciones Cientı´ficas, Universidad de Salamanca, Spain; 2Programa de Patologıá Molecular, Centro Nacional de Investigaciones Oncolo´gicas; 3Departamento de Patologıá, Hospital Universitario 12 de Octubre, Madrid, Spain; 4Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts; and 5Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, State University of New York at Buffalo, Buffalo, New York

Abstract positively with VRK1. VRK1 increases the activity of The vaccinia-related kinase (VRK) proteins are a new both the CDK2 and SURVIVIN gene promoters. The family with three members in the human kinome. expression of VRK1 was analyzed in the context of The VRK1 protein phosphorylates several transcription regulators of the G1-S transition. VRK1 protein levels factors and has been postulated to be involved in increase in response to E2F1 and are reduced by regulation of cell proliferation. In normal squamous retinoblastoma and p16. These data suggest that VRK1 epithelium, VRK1 is expressed in the proliferation area. might play a role in cell cycle regulation and is likely to Because VRK1 can stabilize p53, the expression of the represent the beginning of a new control mechanism VRK1 protein was analyzed in the context of the p53 of cell cycle, particularly late in the G1-S phase. pathway and the proliferation phenotype in a series of 73 (Mol Cancer Res 2006;4(3):177–85) head and neck squamous cell carcinomas. VRK1 protein level positively correlated with p53 response proteins, particularly hdm2 and p21. The VRK1 protein also Introduction correlated positively with several proteins associated Head and neck squamous cell carcinoma (HNSCC) with proliferation, such as cyclin-dependent kinase 2 constitutes the sixth most common type of cancer worldwide. (CDK2), CDK6, cdc2, cyclins B1 and A, topoisomerase II, In the United States, it accounts for f3% of new cases and 2% survivin, and Ki67. The level of VRK1 protein behaves of the deaths annually. Pathogenically, the development of these like a proliferation marker in this series of head and neck tumors has been associated with the mutagenic role of tobacco squamous cell carcinomas. To identify a possible carcinogens (1), which can induce specific mutations in regulatory role for VRK1 and because it regulates gene different genes, including p53 (2-5). The strong association transcription, the promoters of two genes were studied, with carcinogens suggests that pathways related with the CDK2 and SURVIVIN, whose proteins correlated cellular response to genotoxic damage might be implicated in HNSCC; in addition, there might be a genetic susceptibility affecting genes that are implicated in DNA repair processes or metabolism of carcinogens (6). There are also ethnic differences Received 10/19/05; revised 1/31/06; accepted 2/9/06. Grant support: Fondo de Investigacioń Sanitaria grant FIS02/0585 (P.A. Lazo); in its incidence, with the Black population presenting higher Ministerio de Educacioń y Ciencia grant SAF2004-02900 (P.A. Lazo); Junta de rates, whereas the Hispanic and Asian populations have lower Castilla y Leoń grants SAN/SA-01/04, SAN/SA-04/05, and CSI05A05 (P.A. rates (1). A role for certain types of human papillomaviruses Lazo); Fundacioń de Investigacioń Medica MM (P.A. Lazo); Ministerio de Ciencia y Tecnologıá grant SAF2002-01595 (M. Sańchez-Ce´spedes); Comunidad de has also been suggested (7, 8). Madrid grant CAM 08.1/0032/2003 (M. Sańchez-Ce´spedes); and Fundacioń The vaccinia-related kinase 1 (VRK1) protein belongs to a Cientı´fica de la Asociacioń Espanõla contra el Cańcer (F.M. Vega), Fundaçaõ para a Cieˆncia e a Tecnologia Portugal (C.R. Santos), Ministerio de Educacioń y Ciencia new family of serine/threonine kinases in the human kinome (S. Blanco and A. Valbuena), and Consejo Superior de Investigaciones Cientificas (9, 10). This family has three members in mammals, but predoctoral fellowships (A. Sevilla). Drosophila and Caenorhabditis elegans have only one homo- 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 logue gene. The inactivation of the C. elegans homologue causes accordance with 18 U.S.C. Section 1734 solely to indicate this fact. embryonic lethality (11). Inactivation of the distant homologue in Requests for reprints: Pedro A. Lazo, Instituto de Biologıá Molecular y Celular yeast, both Schizosaccharomyces pombe and Saccharomyces del Cańcer, Centro de Investigacioń del Cańcer, Consejo Superior de Inves- tigaciones Cientificas, Universidad de Salamanca, Campus Miguel de Unamuno, cerevisiae, has been shown to be implicated in the response to E-37007 Salamanca, Spain. Phone: 34-923-294-804; Fax: 34-923-294-795. DNA damage (12, 13). VRK1 is highly expressed in human E-mail: [email protected] Copyright D 2006 American Association for Cancer Research. tumor cell lines (14) and murine embryos (15), suggesting it doi:10.1158/1541-7786.MCR-05-0212 might be associated with cell proliferation. This association was

Mol Cancer Res 2006;4(3). March 2006 177 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Published OnlineFirst March 3, 2006; DOI: 10.1158/1541-7786.MCR-05-0212

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further supported by experiments where VRK1 inactivation with at the protein level with regard to the p53 pathway and other small interfering RNA resulted in a block of cell division (16). proteins related with the proliferation phenotype, as well as VRK1 is expressed at very high level in the retina neurons, and identified the role of VRK1 as a regulatory factor of genes its expression drops dramatically the first day after birth (17). In implicated in cell cycle control, such as cyclin-dependent chronic myelogenous leukemia, the expression of VRK1 can kinase 2 (CDK2) and survivin. It is postulated that VRK1 plays differentiate responders from nonresponders cases to treatment a role in both G1-S and G2-M progression of the cell cycle. with imatinib (18). In B cells, analysis by quantitative mass spectrometry indicates that it is down-regulated when Myc Results expression is induced (19). VRK1 is also regulated in the Expression of VRK1 in Normal Squamous Epithelium response to peroxisome proliferators in murine hepatocytes (20). The expression of human VRK1 was determined in normal VRK1 expression is also activated by E2F and inhibited by p16 epithelium. The determination was done in a tonsil that has a and nonphosphorylated retinoblastoma (Rb; ref. 21). VRK1 has a well-characterized squamous epithelium. VRK1 was detected serine/threonine kinase activity and phosphorylates several as present particularly near the basal layer where cellular transcription factors, including human p53 (22), and can also proliferation takes place, and as the epithelial cells differentiate, cooperate with the c-Jun NH2-terminal kinase pathway by the signal for VRK1 is lost (Fig. 1, top). VRK1 was also phosphorylation of c-Jun (23) and ATF2 (24). All these proteins detected in many lymphocytes within the follicles. The same phosphorylated by VRK1 have been associated with cellular sample was analyzed for the expression of the Ki67 antigen, a responses to stress (25-27). VRK1 contributes to p53 stability by typical proliferation marker; this antigen is also present in the 18 two mechanisms, one of them dependent on Thr phosphory- same compartment as VRK1, although fewer cells were stained lation, and also seems to be implicated in the control of normal (Fig. 1, bottom). We conclude from these observations that both proliferation in the absence of cellular stress (16). The loss of VRK1 and Ki67 seem to be located within the proliferation area VRK1 also affects the endocytic transport with a phenotype of squamous epithelia. similar to that induced by silencing of mitogen-activated protein kinase (28). The other VRK members are not well known. VRK2 VRK1/p53 Pathway in Head and Neck Tumors is located in the cytoplasm and is membrane bound; it is The substrates thus far identified regarding the enzymatic catalytically active, but nothing is known regarding its substrates activity of VRK1 are transcription factors (23, 24) and include (29). VRK3 is catalytically inactive and probably functions as a p53 (16, 22). Therefore, it was decided to study if a correlation scaffold protein (29). Among the sensor mechanisms of cellular response to DNA damage is the p53 molecule that has been dubbed as the guardian of the genome (30). The p53 reaction to cellular damage triggers several types of responses, ranging from stopping the cell cycle to induction of apoptosis (31, 32). Thus, the p53 pathway plays a central role in cancer biology (32, 33), being at the center of all processes implicated in the cellular response to genotoxic stress (25). The p53 protein contributes to proliferation by its participation in the checkpoints that controls the cell cycle (34-36). In the control of these processes, phosphorylation of p53 is a major regulatory mechanism, and several kinases phosphor- ylating p53 have been implicated in cancer and in its therapeutic responses, such as ATM, ATR, CHK1, CHK2, and c-Jun NH2- terminal kinase among others (37-40). The p53 protein also can modulate in some circumstances cellular sensitivity to radiotherapy (41) and chemotherapeutic drugs (42-44). VRK1, a new regulator of p53, phosphorylates p53 in Thr18 (22, 45), resulting in its stabilization and favoring its interaction with the transcriptional coactivator p300 (16). Phosphorylated Thr18 affects the interaction of p53 with hdm2 (46, 47), and it is acquiring more relevance lately (48, 49), particularly because phosphorylation in other better known residues, such as Ser15 or Ser20, seems to be dispensable for p53 activity (49-51). The phosphorylation of p53 Thr18 has been associated with cellular senescence (52) and with the response to Taxol (53). These observations led to the proposal that VRK1 might be a component of a novel mechanism that controls basal p53 levels during FIGURE 1. Expression of human VRK1 in normal squamous epithelium. A biopsy from a human tonsil was stained for immunohistochemical normal proliferation, or suboptimal stress situations, and thus per- detection of the VRK1 (top) and Ki67 (bottom) proteins. In the section, the mits the cell to respond when situations of severe stress arise (16). normal squamous epithelium and part of the follicle with lymphocytes are clearly identified. The staining in the epithelium is located in the zone of In this work, we have studied in a series of squamous head proliferation. VRK1 was detected with a rabbit polyclonal antibody (VE1). and neck carcinomas the correlation between VRK1 expression The Ki67 antigen was detected with MIB1 antibody.

Mol Cancer Res 2006;4(3). March 2006 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Published OnlineFirst March 3, 2006; DOI: 10.1158/1541-7786.MCR-05-0212

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Table 1. Correlations of VRK1 with p53, hdm2, and p21 in sion and cellular proliferation phenotype were analyzed in these Head and Neck Tumors with Wild-type p53 HNSCC cases. The markers determined by immunohistochemistry in addition to VRK1 were bcl2, p21, p53, p27, p16, Antigen levels (score) VRK1 (0) VRK1 (1) VRK1 (2) Totals hdm2, cdk2, cdk6, cdc2, cyclin A, cyclin B1 (nuclear and A. cytosolic), cyclin D1, cyclin D3, topoisomerase II a, survivin, p53 (À) 10 (71%) 30 (85%) 8 (53%) 48 chk2, Ki67, Rb, and phosphorylated Rb (Rb-P). All these p53 (+) 4 (29%) 5 (15%) 7 (47%) 16 markers were quantified and analyzed with respect to VRK1 Totals 14 35 15 64 B. protein level. Nine of them presented a positive correlation with hdm2 (À) 12 (86%) 24 (68%) 9 (60%) 45 VRK1 that reached statistical significance (Table 2). Interest- hdm2 (+) 2 (14%) 11 (32%) 6 (40%) 19 Totals 14 35 15 64 ingly, the correlation was associated with several markers C. related with the proliferation and cell cycle progression p21 (À) 8 (57%) 21 (60%) 5 (33%) 34 phenotype. No statistically significant correlation was found p21 (+) 6 (53%) 14 (40%) 10 (67%) 30 Totals 14 35 15 64 with the remaining proteins studied, although it is likely that with a larger sample, some of them might also be significant. Antigen levels (score) p53 (0) p53 (1) p53 (2) p53 (3) Totals Interestingly, it did not correlate with cyclins D1 and D3 that D. are required early in the G1 phase. hdm2 (À) 24 (89%) 7 (77%) 8 (66%) 6 (37%) 45 The staining of some of these antigens that positively hdm2 (+) 3 (11%) 2 (13%) 4 (33%) 10 (63%) 19 correlated with VRK1 is shown in Fig. 3. The Rb-P protein Totals 27 9 12 16 64 (P < 0.09), an important protein for cell cycle progression, CDK2, and survivin staining are shown (Fig. 3), because they were selected for further study to try to establish a link between could be detected between the levels of VRK1 protein and them and VRK1. those of p53 (Table 1A) and p53 response proteins, such as hdm2 or p21. For these four proteins, a panel of 64 HNSCCs VRK1 Activates the CDK2 Gene Promoter was available for study in a tissue array. Different levels of Next, the potential connection between VRK1 levels and VRK1 protein could be detected by immunohistochemistry in other proteins that positively correlated in this series of HNSCC different tumors, ranging from almost no expression to very was determined. Based on the tumor expression data, and the high level expression (Fig. 2). As shown in Table 1, the fact that VRK1 can activate transcription factors, it was decided immunostaining for hdm2 and p21 was grouped into two categories and compared with the levels of VRK protein. As can be observed, as the level of VRK1 increased, so was the level of the p53 targets, such as hdm2 and p21, indicating an augmented activity of p53-dependent transcriptional regulation. The correlation reached statistical significance between VRK1 and Hdm2 (P < 0.02; Table 1B) but not between p21 (P < 0.6) and p53 (P < 0.2; Table 1C), although a positive correlation trend was clearly detectable with these two proteins. In the positive case shown in Fig. 1, p53 is functional because the proteins hdm2 and p21 coded by p53-dependent genes are also increased. However, and although there seemed to be an association between levels of p53 and VRK1 (Table 1A), it is possible that determination of p53 gene status, instead of only protein immunostaining, would lead to more definitive results. This was not possible in the present study because there was no DNA available to determine if p53 was mutated and thus stabilized. The inverse correlation between p53 and hdm2 is also detectable (Table 1D). No correlation between VRK1 levels and survival could be detected in this series of patients (data not shown), probably due to the advanced stage of the disease at the time of diagnosis.

Relation between Proteins Implicated in the Proliferation Phenotype and VRK1 The proliferation phenotype is a hallmark of the cancer FIGURE 2. VRK1/p53/hdm2 pathway in head and neck squamous cell carcinomas. Immunohistochemical detection of the VRK1 protein and its phenotype resulting from alterations in growth regulation (54). relation with p53 and p53 response proteins hdm2 and p21. Negative (left) The p53 protein is one of the main regulatory proteins of the and positive (right) cases. The four proteins were analyzed in the same cell cycle, participating in several checkpoints. Because p53 is a tumor present in a tissue microarray to show the range and variation of the signal. In the positive case, p53 appears to be functional because the target of VRK1 and because of the role of p53 in cell cycle levels of two proteins (p53 and hdm2) expressed from p53-dependent regulation, several proteins associated with cell cycle progres- genes are also high.

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Table 2. Positive Correlation of VRK1 with Proteins Impli- cells from apoptosis, and is usually an indicator of bad cated in the Proliferation Phenotype in HNSCC prognosis (62). These biological effects are consistent with the role of VRK1 as a regulator of p53. Because there was a Protein P (P < 0.05) positive correlation between VRK1 and survivin protein levels hdm2 0.02 in these biopsies, it was determined if VRK1 could also be CDK2 0.01 implicated in SURVIVIN gene regulation. To study this effect, CDK6 0.009 Cos1 cells were transfected with plasmid pCEFL-HA-VRK1 CB1N (cyclin B1 nuclear) 0.02 Topoisomerase II 0.01 and several constructs of the SURVIVIN gene promoter with a Survivin 0.01 luciferase reporter. In these experiments, VRK1 induced an up- Cdc2 0.001 regulation of SURVIVIN gene expression (Fig. 5A). The Ki67 0.03 Cyclin A 0.008 response element in the SURVIVIN gene promoter is located between residues À1430 and À649. The survivin promoter has NOTE: The statistical analysis is described in Materials and Methods. Only the a basal activity level, and its magnitude was increased f3-fold proteins with P < 0.05 are shown. in the presence of VRK1, an effect similar to that detected with the CDK2 promoter. The SURVIVIN gene promoter also to test if VRK1 could be an activator of genes coding for increased its transcriptional activity in a dose-dependent proteins with which VRK1 was detected as having a positive manner with respect to VRK1 (Fig. 5B). correlation in these HNSCC cases. For this aim, it was tested if VRK1 expression could affect the transcriptional activity of the E2F1 Activates VRK1 Expression CDK2 and SURVIVIN gene promoters. The association Up to now, all the evidence points to a role of VRK1 in between VRK1 and the CDK2 and/or survivin proteins might proliferation. Therefore, it was attempted to determine in what be due to two different possibilities. One is that because VRK1 phase of the cell cycle VRK1 might be implicated. The can activate transcription factors, both CDK2 and SURVIVIN transcription factor E2F is one of the main inducers of cell cycle genes are located in the response pathway of VRK1 activation progression through the G1-S phase. Earlier experiments using and thus the observation of their common overexpression. microarray expression analysis have been shown that expres- Alternatively, the three protein (VRK1, CDK2, and survivin) sion of E2F1 increases the level of VRK1 message (21). E2F is expression levels might be a common component of the same activated by release from the Rb-E2F complex when Rb is phenotype and respond perhaps to another common signal. phosphorylated by cyclin D/CDK complexes. In addition, in To attempt to discriminate between the two possibilities, the this series of HNSCC cases, there was a positive correlation effect of VRK1 overexpression on the human CDK2 gene between Rb-P protein and VRK1 (P < 0.09). Based on these promoter activity was first determined. The human CDK2 gene two lines of evidence, it was decided to determine if there was a plays a role mainly late during G1-S and G2-M phases of the sequential connection between the release of free E2F1 by its cell cycle (55-57). The CDK2 gene promoter was analyzed in overexpression and the level of VRK1 protein. For this aim, Cos1 cells that were transfected with a plasmid pCEFL-HA- U2OS cells (p16À/À), which have low levels of endogenous VRK1 expressing VRK1 and different constructs of the CDK2 E2F1, were transfected with a plasmid expressing E2F1, or a gene promoter linked to the luciferase reporter gene (58). The combination of plasmids expressing the cell cycle inhibitors CDK2 promoter has some activity by itself but was further activated by VRK1 (Fig. 4A), and an additional increase in activity was identified as mediated by an element located between nucleotides À68 and À440 in the promoter sequence. This effect is a potentiation of the response already observed in the absence of VRK1 (Fig. 4A). When the À2400 promoter region was included, the increase in activity was not detected when VRK1 was overexpressed, suggesting that the VRK1 role in the regulation of the CDK2 gene promoter is complex. To further confirm the effect of VRK1 on the À68 to À440 region of the CDK2 gene promoter, a dose response experiment was done. Cos1 cells were transfected with the CDK2 luciferase construct containing from the À683 position in combination with increasing amounts of the plasmid expressing VRK1. As shown in Fig. 4B, as the amount of VRK1 was increased, so did the activity of this CDK2 promoter region, thus confirming the existence of a VRK1 response element in this promoter.

VRK1 Activates the SURVIVIN Gene Promoter Survivin is a protein required for cell cycle progression (59), and in some tumors, such as meningiomas, it also varies in FIGURE 3. Immunohistochemical detection of the expression of some proliferation markers Rb, CDK2, and survivin that positively correlates with correlation with the Ki67 antigen (60). Survivin participates in a VRK1 in HNSCC. Example of negative (À) and positive (+) cases for the mitotic arrest checkpoint regulated by p53 (61), protects the antigens to illustrate the difference between cases.

VRK1 Pathway in Head and Neck Carcinomas 181

Discussion Different types of evidence suggested that VRK1 is likely to play a role in cell proliferation. In this work, a positive correlation has been found between VRK1 and a large number of proliferation-related proteins, suggesting that this VRK1 correlation is consistent with its implication in different aspects of this phenotype and cell cycle regulation in the context of HNSCC. The advanced stage of the tumors analyzed is better suited to find a correlation with phenotypic aspects of the dominant tumor cell population than with their clinical characteristics. It has been postulated that the role of VRK1 in normal proliferation is to maintain the p53 molecule in a readiness state that will permit cell cycle progression under nonstress situation (16). Regarding the p53 pathway, the accumulation of some p53 response proteins, such as hdm2, was expected, because the phosphorylation of p53 in Thr18 by VRK1 partially stabilizes a transcriptionally active p53 molecule that can not interact with hdm2; consequently, hdm2 expression was increased as a result of the transcription potential of p53 (16). However, the correlation with the level of p53 is less clear, although a positive trend was observed. This may be due in part to the occurrence of p53 mutations that might behave differently (63-65); in these patients, the mutational status of p53 could not be determined; and also to the existence of an autoregulatory loop between VRK1 and p53.6 However, in most cases of this series where p53 correlated with VRK1, it also correlated with p21 and hdm2, which suggests that at least in this group, p53 must be wild type because it seemed to be functional. The correlation with several proliferation markers, such as some cdks and cyclins, is consistent with a role in the context of cell cycle regulation, notwithstanding other functions that remain to be identified. Several of the proteins that positively correlated with VRK1 in this series were already known to be prognostic markers in HNSCC; these include cdc2 (66), cyclin A (67), and survivin (68), among others (69). Another protein, Ki67, is a generally accepted proliferation marker and is widely FIGURE 4. VRK1 activates the human CDK2 gene promoter. A. Cos1 used for this purpose in tumor analysis (70). However, in this cells line were transfected with 1 Ag of luciferase constructs containing several fragments of the CDK2 gene promoter in the pGL2 plasmid and series, VRK1 could not be used for a prognostic correlation the pRL-tk-luciferase as internal control with and without 2 Ag of pCEFL- because of the already advanced stage of the tumors. HA-VRK1. The dual luciferase was determined as described in Materials The expression of cdk2 and cdk6 clearly indicates a potential and Methods. The specific promoter-luciferase activity was normalized with Renilla luciferase used as internal control and shown as a relative role associated with cell cycle progression. The positive value. Columns, mean of three independent experiments determined each correlation with some cdk, such as cdk2, cdk6, or cdc2, indicates by triplicate; bars, SD. As controls for the detection system, the plasmids that VRK1 might be required for a function that is necessary at pGL2-basic and pGL2-control (SV40 enhancer/promoter) were used. B. VRK1 dose response of the CDK2 gene promoter construct up to the the same time that these proteins, probably late in G1 to the G2-M 683 nucleotide. Cos1 cells were transfected with 1 Ag of pCDK2(-683)- À phase. CDK6 phosphorylates Rb in the G1-S transition, thus luc and the indicated amount of plasmid pCEFL-HA-VRK1. This experiment was done three times, and the activity was determined in releasing the transcription factor E2F1, and increased CDK2 triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001. activity is a consequence of this activation and cell cycle progression. This role is further supported by the positive correlation with phosphorylated Rb and with the accumulation of p16 and Rb as negative control. The level of endogenous VRK1 nuclear cyclin B1 required for progression through the G2-M protein was determined with a specific monoclonal antibody phase of the cell cycle. In this context, survivin also promotes (1F6). As shown in Fig. 6, the E2F1 protein induces a 2-fold progression by relieving the inhibition of cdk2 complexes (59). increase in VRK1 protein, and the inhibitory combination of However, this may not be the only role for VRK1 within correct p16 plus Rb, used as a negative control, somewhat reduces the cell cycle progression. The positive correlation with markers, endogenous VRK1 basal level. These data suggest that the such as survivin, a protein required for stable checkpoint E2F1 protein required for G1-S transition increases the level of VRK1, an observation consistent with the potential role of VRK1 as a control mechanism during cell cycle and proliferation. 6 Unpublished results.

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cycle regulation, further supports a role for VRK1 in regulation of genes implicated in cell proliferation. Each gene, CDK2 and SURVIVIN, must have a in their upstream regulatory region a sequence recognized by a transcription factor regulated by VRK1. The activation of the human SURVIVIN gene promoter by VRK1 locates the presence of a functional element between nucleotides À1430 and À649, an active region initially detected in HeLa cells (74). In Cos1 cells, the same response region has been detected, but in the presence of VRK1, there is a significant increase in transcriptional activity. The human CDK2 gene promoter has a response element located between À440 and À68, which is important for basal expression in NIH3T3 cells (58). This element is also functional in Cos1 cells, but this region also increases its transcriptional activity in the presence of VRK1. Microarrays studies have shown that VRK1 gene expression is up-regulated by activation of the Rb pathway (21) and thus provided additional evidence for a role in proliferation. This has been experimentally confirmed in this report because overexpression of E2F1, a general transcription factor released by activated (phosphorylated) Rb and required for G1-S progression, was able to induce an increase in the level of VRK1 protein. Members of the E2F family have predictive value for lymph node metastasis in HNSCC (75). These data places VRK1 expression late in the G1-S transition of the cell cycle, because after activation of Rb, the E2F1 transcription factor up-regulates VRK1, which is consistent with the lack of correlation between early cyclins D1 and D2 and VRK1. The implication of E2F1 has also been reported to be required for the expression of the cyclin A gene (76, 77); overexpression of cyclin A correlated positively with VRK1 in this study. Furthermore, E2F1 induces phosphorylation and accumulation of p53 (78), an observation consistent with the effects of VRK1 on p53 (16, 22). All the evidence points to a role for VRK1 required throughout the progression of the cell cycle and perhaps with FIGURE 5. VRK1 activates the human SURVIVIN promoter. A. The Cos1 cell line was transfected with 0.8 Ag luciferase constructs containing more than one role. If that is the case, it is likely to be placed at the SURVIVIN promoter in the pGL2 plasmid and the pRL-tk-luciferase as some point high in the pathway so that more than one control internal control with and without 2 Ag of pCEFL-HA-VRK1. The dual luciferase was determined as described in Materials and Methods. The point might be affected. This generic role for VRK1, or the specific promoter-luciferase activity was normalized with Renilla luciferase existence of other proteins with such a role, must exist in higher used as internal control and shown as a relative value. As controls for the eukaryotes because other proteins, such as cdk2 or cdk4, seem detection system, the plasmids pGL2-basic and pGL2-control (SV40 enhancer/promoter) were used. Columns, means from three experiments to be dispensable in mammalian cell cycle progression as determined in triplicate; bars, SD. B. VRK1 dose response of the shown in the corresponding knock-out mice (79). SURVIVIN promoter using the construct p-2480-SURV-Luc and the HNSCCs have a poor prognosis and are very resistant to indicated amount of plasmid pCEFL-HA-VRK1. This experiment was done three times, and the activity was determined in triplicate. *, P < 0.05; radiotherapy and chemotherapy. Because of that, survival has **, P < 0.01; ***, P < 0.001. not improved significantly in the last 20 years. In this context, the identification of new pathways that may be of potential use activation (71) that also participates in apoptosis protection and as diagnostic marker or therapeutic target represents a major correct cell division (72), might indicate additional effects for aim of cancer research. In this context, the characterization of VRK1. It is interesting to note that survivin has been associated the VRK1 pathway might be an important new step in the better with the response to Taxol (73), which is able to induce the knowledge and future control of HNSCC. unique phosphorylation of p53 in Thr18 (53), a phosphorylation that can be done by VRK1 (16, 22). Because VRK1 can regulate transcription factors, it is very Materials and Methods likely that it will modulate some of the genes whose proteins Samples have been found associated with proliferation and perhaps The 73 cases of HNSCCs were diagnosed and obtained with provide a clue to what phase of the cell cycle VRK1 might written informed consent between 1998 and 2000 in several participate. The activation by VRK1 of the gene promoters of Spanish hospitals according to the institutional guidelines and two of the proteins with which it correlates positively, CDK2 ethics review committees. All tumors were in stages III and IV and SURVIVIN, which are important for different aspects of cell according the WHO classification. The median age of the

VRK1 Pathway in Head and Neck Carcinomas 183

patients was 56 years; the median overall survival for the cohort Antibodies was 37.7 months; the median to progression was 29.1 months. The antibody specific for human VRK1 was a rabbit The characteristics of these cases have already been reported polyclonal (VE1) previously described and prepared with a (67). Tumor biopsies were taken at diagnosis before initiation of GST-VRK1 fusion protein (16). The proteins p53, survivin, treatment. Biopsies were fixed in formalin and embedded in cdk2, cdk6, cyclin B1, topoisomerase II, cdc2, ki67, Rb-P, and paraffin. These biopsies were used to prepare a tissue array made cyclin Awere detected with the following antibodies. CDK2 was with a tissue arrayer (Beecher Instruments, Silver Spring, MD) detected with antibody clone 8D4 (Neomarkers, Freemont, CA). as previously reported (67). The biopsies were examined by two The p53 was detected with clone DO-7 (Novocastra, Newcastle pathologists who selected tumor areas, avoiding those with upon Tyne, United Kingdom); cdc2 with antibody 1 (Transduc- necrosis, inflammation, and keratinization. To assess reproduc- tion Laboratory, Lexington, KY); CDK6 with antibody BD (BD ibility from each tumor, two separate areas were taken from the PharMingen, San Diego, CA); cyclin A with antibody GEG cylinders (0.6-mm diameter). Sections (3 Am thick) were cut and (Novocastra); hdm2 with clone 1F2 (Oncogene, La Jolla, CA); transferred to positively charged surface glass slides. Sections and Ki67 with clone M1B1 (DAKO, Glostrup, Denmark). The were dried for 16 hours at 56jC followed by dewaxing and processing of the biopsies, the dilutions used for primary rehydration through a graded ethanol series and washed with antibodies, and the secondary antibodies for detection have all PBS. To retrieve antigens, the slides were treated in a pressure been described (67). The cutoff criteria has been previously cooker for 2 minutes in 10 mmol/L citrate buffer (pH 6.5). The reported (67). Briefly, hdm2 immunostaining was scored as tissue array slides were then used for immunochemical analysis. negative when <5% of tumor cells presented immunoreactivity. Mesenchymal and endothelial cells were used as the internal positive control. For p21, staining was scored as negative when <10% of tumor cells showed immunoreactivity. Nuclear immunostaining of some lymphoid and granulocytic cells was taken as the internal positive control. For p53, immunostaining was scored as negative when <10% of tumor cells showed nuclear staining. In the case of topoisomerase, <5% was considered negative, between 5% and 30% was moderate (level 1), and >30% was high expression (level 2). For VRK1 and survivin, the criteria was 0 when the number of positive cells were below 5%, level 1 when between 5% and 50%, and level 2 with >50% of the population (68, 80).

Statistical Analysis The statistical analysis and criteria for quantification has been previously described (67). Briefly, the frequencies were compared either by the Fisher’s exact test or the m2 contingency test using the SPSS program version 10.0.5 (SPSS, Inc., Chicago, IL), and differences with a P < 0.05 were considered as statistically significant.

Plasmids, Transfections, Transcription Reporter Assays, and Immunoblots Plasmid pCEFL-HA-VRK1 has already been described (16). pX-p16, pCMV-E2F1, and pCMV-Rb were from M. Malum- bres (Centro Nacional de Investigaciones Oncolo´gicas, Madrid, Spain). Plasmid p-68-CDK2-Luc, p-440-CDK2-Luc, p-683- CDK2-Luc, and p-2400-CDK2-Luc have been previously described (58). The survivin gene promoter constructs pLuc- FIGURE 6. Effect of overexpression of E2F1 on VRK1 protein level in U2OS cells (p16À/À). A. Immunoblot detection of the effects of E2F1 or the 1430c, pLuc-649c, pLuc-441c, pLuc-230c, and pLuc-42c were combination of p16 and Rb on VRK1 levels. This Western blot is a previously reported (74). U2OS (p16À/À) and Cos1 cells grown representative experiment. B. Quantification of the effects relative of E2F1 or the combination of p16 and Rb on VRK1 levels. Columns, mean of three in DMEM supplemented with 10% FCS. experiments; bars, SD. *, P < 0.01. The cells were transfected with the For transfections of Cos1, 300,0000 cells were plated in indicated amount of plasmids, pCMV-E2F1 or the combination of pX-p16 P35 dishes. The cells were transfected with 0.8 Agof and pCMV-Rb, expressing the proteins indicated in the graph, and the VRK1 protein was determined with a specific monoclonal antibody (clone pSurvivin-luc constructs and 50 ng of pRL-tk-luc as internal 1F6) 48 hours after transfection in whole-cell lysates. The signal was control, or 1 Ag of pCDK-luc constructs and 20 ng of pRL- quantified in the linear response range and was normalized with respect to TK-luc using 6 AL of the JetPEI reagent (Polytransfection, the h-actin level. As control for proteins that inhibit cell proliferation, a transfection was done using a combination of p16 and Rb expression Illkirch, France) according to manufacturer’s instructions. plasmids (in these transfections, equal amounts of each plasmid were Luciferase activity was determined with a Dual luciferase used, and the amount indicated corresponds to the total specific plasmid DNA used). Immunoblot from a representative experiment. The details of system from Promega (Madison, WI) following the manu- the experiment are described in Materials and Methods. facturer’s instructions.

184 Santos et al.

For transfections, 500,000 U2OS cells were plated in P35 16. Vega FM, Sevilla A, Lazo PA. p53 stabilization and accumulation induced by human vaccinia-related kinase 1. Mol Cell Biol 2004;24:10366 – 80. dishes. After 24 hours, cells were transfected with the 17. Dorrell MI, Aguilar E, Weber C, Friedlander M. Global gene expression indicated amounts of pX-p16, pCMV-E2F1, or pCMV-Rb analysis of the developing postnatal mouse retina. Invest Ophthalmol Vis using 10 AL of the JetPEI reagent from Polytransfection Sci 2004;45:1009 – 19. according to manufacturer’s instructions. Forty-eight hours 18. McLean LA, Gathmann I, Capdeville R, Polymeropoulos MH, Dressman M. after transfection, cells were washed twice with ice-cold PBS; Pharmacogenomic analysis of cytogenetic response in chronic myeloid leukemia patients treated with imatinib. Clin Cancer Res 2004;10:155 – 65. harvested in radioimmunoprecipitation assay buffer [150 19. Shiio Y, Eisenman RN, Yi EC, Donohoe S, Goodlett DR, Aebersold R. mmol/L NaCl, 1.5 mmol/L MgCl2, 10 mmol/L NaF, 10% Quantitative proteomic analysis of chromatin-associated factors. J Am Soc Mass glycerol, 4 mmol/L EDTA, 1% Triton X-100, 0.1% SDS, 1% Spectrom 2003;14:696 – 703. deoxycholate, 50 mmol/L HEPES (pH 7.4)], plus 1 mmol/L 20. Tien ES, Gray JP, Peters JM, Vanden Heuvel JP. Comprehensive gene expression analysis of peroxisome proliferator-treated immortalized hepatocytes: Na3VO4,10Ag/mL leupeptin, 10 Ag/ml aprotinin, and 1 identification of peroxisome proliferator-activated receptor {alpha}-dependent mmol/L phenylmethylsulfonyl fluoride; incubated on ice for growth regulatory genes. Cancer Res 2003;63:5767 – 80. 20 minutes; and precleared by centrifugation at 13,200 rpm 21. Vernell R, Helin K, Muller H. Identification of target genes of the p16INK4A- for 20 minutes at 4 jC in an Eppendorf 5415D minifuge. pRB-E2F pathway. J Biol Chem 2003;278:46124 – 37. Thirty micrograms of the extracts were analyzed by SDS- 22. Lopez-Borges S, Lazo PA. The human vaccinia-related kinase 1 (VRK1) phosphorylates threonine-18 within the mdm-2 binding site of the p53 tumour PAGE under denaturing conditions and transferred onto suppressor protein. Oncogene 2000;19:3656 – 64. Immobilon-P membranes (Millipore, Bedford, MA) for 1 23. Sevilla A, Santos CR, Barcia R, Vega FM, Lazo PA. c-Jun phosphorylation hour at 90 V. The membranes were blocked with 5% skimmed by the human vaccinia-related kinase 1 (VRK1) and its cooperation with the milk in TBS-T and then incubated with the specific antibody. N-terminal kinase of c-Jun (JNK). Oncogene 2004;23:8950 – 8. As secondary antibody, a sheep/anti-mouse/horseradish per- 24. Sevilla A, Santos CR, Vega FM, Lazo PA. Human vaccinia-related kinase 1 (VRK1) activates the ATF2 transcriptional activity by novel phosphorylation on oxidase (Amersham Biosciences, Little Chalfont, United Thr-73 and Ser-62 and cooperates with JNK. J Biol Chem 2004;279:27458 – 65. Kingdom) was used at a 1:5,000 dilution. The chemilumines- 25. Wahl GM, Carr AM, The evolution of diverse biological responses to DNA cence in the blots was detected using the with the enhanced damage: insights from yeast and p53. Nat Cell Biol 2001;3:E277 – 86. chemiluminescence reagent from Amersham Biosciences. 26. van Dam H, Wilhelm D, Herr I, Steffen A, Herrlich P, Angel P. ATF-2 is preferentially activated by stress-activated protein kinases to mediate c-jun induction in response to genotoxic agents. EMBO J 1995;14:1798 – 811. References 27. Sugden PH, Clerk A. Cell stresses regulate the phosphorylation state of c-Jun 1. Diaz EM, Sturges EM, Laramore GE, Sabichi AL, Clayman G. Neoplasms of and ATF2 in ventricular myocytes. Biochem Soc Trans 1997;25:221S. the head and neck. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Cancer medicine, Vol. 2. Hamilton (Ontario, Canada): American Cancer Society- 28. Pelkmans L, Fava E, Grabner H, et al. Genome-wide analysis of human BC Dekker, Inc.; 2003. pp. 1325 – 72. kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature 2005; 436:78 – 86. 2. Koch WM, Brennan JA, Zahurak M, et al. p53 mutation and locoregional treatment failure in head and neck squamous cell carcinoma. J Natl Cancer Inst 29. Nichols RJ, Traktman P. Characterization of three paralogous members of 1996;88:1580 – 6. the mammalian vaccinia related kinase family. J Biol Chem 2004;279:7934 – 46. 3. Brennan JA, Boyle JO, Koch WM, et al. Association between cigarette 30. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000; smoking and mutation of the p53 gene in squamous-cell carcinoma of the head 408:307 – 10. and neck. N Engl J Med 1995;332:712 – 7. 31. Levine A. p53, the cellular gatekeeper for growth and division. Cell 1997; 4. Olshan AF, Weissler MC, Pei H, Conway K. p53 mutations in head and neck 88:323 – 31. cancer: new data and evaluation of mutational spectra. Cancer Epidemiol 32. Prives C, Hall PA. The p53 pathway. J Pathol 1999;187:112 – 26. Biomarkers Prev 1997;6:499 – 504. 33. Bargonetti J, Manfredi JJ. Multiple roles of the tumor suppressor p53. Curr 5. Blons H, Laurent-Puig P. TP53 and head and neck neoplasms. Hum Mutat Opin Oncol 2002;14:86 – 91. 2003;21:252 – 7. 34. Bunz F, Dutriaux A, Lengauer C, et al. Requirement for p53 and p21 to 6. Crowe DL, Hacia JG, Hsieh CL, Sinha UK, Rice H. Molecular pathology of sustain G2 arrest after DNA damage. Science 1998;282:1497 – 501. head and neck cancer. Histol Histopathol 2002;17:909 – 14. 35. Taylor WR, Stark GR. Regulation of the G2/M transition by p53. Oncogene 7. Herrero R. Chapter 7: human papillomavirus and cancer of the upper 2001;20:1803 – 15. aerodigestive tract. J Natl Cancer Inst Monogr 2003;31:47 – 51. 36. Chehab NH, Malikzay A, Appel M, Halazonetis TD. Chk2/hCds1 functions 8. Gillison ML. Human papillomavirus-associated head and neck cancer is a as a DNA damage checkpoint in G(1) by stabilizing p53. Genes Dev 2000; distinct epidemiologic, clinical, and molecular entity. Semin Oncol 2004;31: 14:278 – 88. 744 – 54. 37. Okorokov AL. p53 in a crosstalk between DNA repair and cell cycle 9. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein checkpoints. Cell Cycle 2003;2:233 – 5. kinase complement of the human genome. Science 2002;298:1912 – 34. 38. Oren M. Decision making by p53: life, death and cancer. Cell Death Differ 10. Lazo PA, Vega FM, Sevilla A. Vaccinia-related kinase-1. Afcs Nature 2003;10:431 – 42. Molecule Page, doi:10.1038/: mp a003025.003001, 2005. 39. Bode AM, Dong Z. Post-translational modification of p53 in tumorigenesis. 11. Kamath RS, Fraser AG, Dong Y, et al. Systematic functional analysis of the Nat Rev Cancer 2004;4:793 – 805. Caenorhabditis elegans genome using RNAi. Nature 2003;421:231 – 7. 40. Meek DW. The p53 response to DNA damage. DNA Repair (Amst) 2004;3: 12. Hoekstra MF, Liskay RM, Ou AC, DeMaggio AJ, Burbee DG, Heffron F. 1049 – 56. HRR25, a putative protein kinase from budding yeast: association with repair of 41. Fei P, El-Deiry WS. P53 and radiation responses. Oncogene 2003;22: damaged DNA. Science 1991;253:1031 – 4. 5774 – 83. 13. Dhillon N, Hoekstra MF. Characterization of two protein kinases from 42. Liem AA, Appleyard MV, O’Neill MA, Hupp TR, Chamberlain MP, Schizosaccharomyces pombe involved in the regulation of DNA repair. EMBO J Thompson AM. Doxorubicin and vinorelbine act independently via p53 1994;13:2777 – 88. expression and p38 activation respectively in breast cancer cell lines. Br J 14. Nezu J, Oku A, Jones MH, Shimane M. Identification of two novel human Cancer 2003;88:1281 – 4. putative serine/threonine kinases, VRK1 and VRK2, with structural similarity to 43. Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of vaccinia virus B1R kinase. Genomics 1997;45:327 – 31. resistance. Oncogene 2003;22:7265 – 79. 15. Vega FM, Gonzalo P, Gaspar ML, Lazo PA. Expression of the VRK 44. Sullivan A, Syed N, Gasco M, et al. Polymorphism in wild-type p53 (vaccinia-related kinase) gene family of p53 regulators in murine hematopoietic modulates response to chemotherapy in vitro and in vivo. Oncogene 2004; development. FEBS Lett 2003;544:176 – 80. 23:3328 – 37.

VRK1 Pathway in Head and Neck Carcinomas 185

45. Barcia R, Lopez-Borges S, Vega FM, Lazo PA. Kinetic properties of p53 63. Kim E, Deppert W. Transcriptional activities of mutant p53: when mutations phosphorylation by the human vaccinia-related kinase 1. Arch Biochem are more than a loss. J Cell Biochem 2004;93:878 – 86. Biophys 2002;399:1 – 5. 64. Olive KP, Tuveson DA, Ruhe ZC, et al. Mutant p53 gain of function in two 46. Jabbur JR, Zhang W. p53 Antiproliferative function is enhanced by mouse models of Li-Fraumeni syndrome. Cell 2004;119:847 – 60. aspartate substitution at Threonine 18 and Serine 20. Cancer Biol Ther 2002;1: 65. Lang GA, Iwakuma T, Suh YA, et al. Gain of function of a p53 hot 277 – 83. spot mutation in a mouse model of Li-Fraumeni syndrome. Cell 2004;119: 47. Schon O, Friedler A, Bycroft M, Freund S, Fersht A. Molecular 861 – 72. mechanism of the interaction between MDM2 and p53. J Mol Biol 2002; 66. Wada S, Yue L, Furuta I. Prognostic significance of p34cdc2 expression in 323:491 – 501. tongue squamous cell carcinoma. Oral Oncol 2004;40:164 – 9. 48. Bean LJ, Stark GR. Regulation of the accumulation and function of p53 by 67. Rodriguez-Pinilla M, Rodriguez-Peralto JL, Hitt R, et al. Cyclin A as a phosphorylation of two residues within the domain that binds to Mdm2. J Biol predictive factor for chemotherapy response in advanced head and neck cancer. Chem 2002;277:1864 – 71. Clin Cancer Res 2004;10:8486 – 92. 49. Jackson MW, Agarwal MK, Agarwal ML, et al. Limited role of N-terminal 68. Lo Muzio L, Pannone G, Staibano S, et al. Survivin expression in oral phosphoserine residues in the activation of transcription by p53. Oncogene 2004; squamous cell carcinoma. Br J Cancer 2003;89:2244 – 8. 23:4477 – 87. 69. Hunter KD, Parkinson EK, Harrison PR. Profiling early head and neck 50. Sluss HK, Armata H, Gallant J, Jones SN. Phosphorylation of serine 18 cancer. Nat Rev Cancer 2005;5:127 – 35. regulates distinct p53 functions in mice. Mol Cell Biol 2004;24:976 – 84. 70. Brown DC, Gatter KC. Ki67 protein: the immaculate deception? Histopa- 51. Thompson T, Tovar C, Yang H, et al. Phosphorylation of p53 on key serines thology 2002;40:2 – 11. is dispensable for transcriptional activation and apoptosis. J Biol Chem 2004; 279:53015 – 22. 71. Carvalho A, Carmena M, Sambade C, Earnshaw WC, Wheatley SP. Survivin is required for stable checkpoint activation in Taxol-treated HeLa cells. J Cell Sci 52. Ferbeyre G, de Stanchina E, Lin AW, et al. Oncogenic ras and p53 cooperate 2003;116:2987 – 98. to induce cellular senescence. Mol Cell Biol 2002;22:3497 – 508. 72. Altieri DC. Survivin in apoptosis control and cell cycle regulation in cancer. 53. Stewart ZA, Tang LJ, Pietenpol JA. Increased p53 phosphorylation after Prog Cell Cycle Res 2003;5:447 – 52. microtubule disruption is mediated in a microtubule inhibitor- and cell-specific manner. Oncogene 2001;20:113 – 24. 73. Ling X, Bernacki RJ, Brattain MG, Li F. Induction of survivin expression by 54. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57 – 70. Taxol (paclitaxel) is an early event, which is independent of Taxol-mediated G2/M arrest. J Biol Chem 2004;279:15196 – 203. 55. Malumbres M, Barbacid M. To cycle or not to cycle: a critical decision in cancer. Nat Rev Cancer 2001;1:222 – 31. 74. Li F, Altieri DC. Transcriptional analysis of human survivin gene expression. Biochem J 1999;344 Pt 2:305 – 11. 56. Malumbres M, Hunt SL, Sotillo R, et al. Driving the cell cycle to cancer. Adv Exp Med Biol 2003;532:1 – 11. 75. Roepman P, Wessels LF, Kettelarij N, et al. An expression profile for diagnosis of lymph node metastases from primary head and neck squamous cell 57. Aleem E, Berthet C, Kaldis P. Cdk2 as a master of S phase entry: fact or carcinomas. Nat Genet 2005;37:182 – 6. fake? Cell Cycle 2004;3:35 – 7. 76. Henglein B, Chenivesse X, Wang J, Eick D, Brechot C. Structure and cell 58. Xie R-L, Gupta S, Miele A, et al. The tumor suppressor interferon regulatory cycle-regulated transcription of the human cyclin A gene. Proc Nat Acad Sci factor 1 interferes with SP1 activation to repress the human CDK2 promoter. U S A 1994;91:5490 – 4. J Biol Chem 2003;278:26589 – 96. 77. Schulze A, Zerfass K, Spitkovsky D, et al. Cell cycle regulation of the cyclin 59. Suzuki A, Hayashida M, Ito T, et al. Survivin initiates cell cycle entry by the A gene promoter is mediated by a variant E2F site. Proc Natl Acad Sci U S A competitive interaction with Cdk4/p16(INK4a) and Cdk2/cyclin E complex 1995;92:11264 – 8. activation. Oncogene 2000;19:3225 – 34. 78. Rogoff HA, Pickering MT, Debatis ME, Jones S, Kowalik TF. E2F1 induces 60. Kayaselcuk F, Zorludemir S, Bal N, Erdogan B, Erdogan S, Erman T. The phosphorylation of p53 that is coincident with p53 accumulation and apoptosis. expression of survivin and Ki-67 in meningiomas: correlation with grade and Mol Cell Biol 2002;22:5308 – 18. clinical outcome. J Neurooncol 2004;67:209 – 14. 79. Malumbres M, Sotillo R, Santamaria D, et al. Mammalian cells cycle 61. Beltrami E, Plescia J, Wilkinson JC, Duckett CS, Altieri DC. Acute ablation without the D-type cyclin-dependent kinases Cdk4 and Cdk6. Cell 2004;118: of survivin uncovers p53-dependent mitotic checkpoint functions and control of 493 – 504. mitochondrial apoptosis. J Biol Chem 2004;279:2077 – 84. 80. Weinman EC, Roche PC, Kasperbauer JL, et al. Characterization of antigen 62. Schimmer AD. Inhibitor of apoptosis proteins: translating basic knowledge processing machinery and Survivin expression in tonsillar squamous cell into clinical practice. Cancer Res 2004;64:7183 – 90. carcinoma. Cancer 2003;97:2203 – 11.

VRK1 Signaling Pathway in the Context of the Proliferation Phenotype in Head and Neck Squamous Cell Carcinoma

Mol Cancer Res Published OnlineFirst March 3, 2006.

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