Stereospecific alignment of the X and Y elements is required for major histocompatibility complex class II DRA promoter function.

The regulatory mechanisms controlling expression of the major histocompatibility complex (MHC) class II genes involve several cis-acting DNA elements, including the X and Y boxes. These two elements are conserved within all murine and human class II genes and are required for accurate and efficient transcription from MHC class II promoters. Interestingly, the distance between the X and Y elements is also evolutionarily conserved at 18 to 20 bp. To investigate the function of the invariant spacing in the human MHC class II gene, HLA-DRA, we constructed a series of spacing mutants which alters the distance between the X and Y elements by integral and half-integral turns of the DNA helix. Transient transfection of the spacing constructs into Raji cells revealed that inserting integral turns of the DNA helix (+20 and +10 bp) did not reduce promoter activity, while inserting or deleting half-integral turns of the DNA helix (+15, +5, and -5 bp) drastically reduced promoter activity. The loss of promoter function in these half-integral turn constructs was due neither to the inability of the X and Y elements to bind proteins nor to improper binding of the X- and Y-box-binding proteins. These data indicate that the X and Y elements must be aligned on the same side of the DNA helix to ensure normal function. This requirement for stereospecific alignment strongly suggests that the X- and Y-box-binding proteins either interact directly or are components of a larger transcription complex which assembles on one face of the DNA double helix.

Upstream stimulatory factor regulates expression of the cell cycle-dependent cyclin B1 gene promoter.

Progression through the somatic cell cycle requires the temporal regulation of cyclin gene expression and cyclin protein turnover. One of the best-characterized examples of this regulation is seen for the B-type cyclins. These cyclins and their catalytic component, cdc2, have been shown to mediate both the entry into and maintenance of mitosis. The cyclin B1 gene has been shown to be expressed between the late S and G2 phases of the cell cycle, while the protein is degraded specifically at interphase via ubiquitination. To understand the molecular basis for transcriptional regulation of the cyclin B1 gene, we cloned the human cyclin B1 gene promoter region. Using a chloramphenicol acetyltransferase reporter system and both stable and transient assays, we have shown that the cyclin B1 gene promoter (extending to -3800 bp relative to the cap site) can confer G2-enhanced promoter activity. Further analysis revealed that an upstream stimulatory factor (USF)-binding site and its cognate transcription factor(s) are critical for expression from the cyclin B1 promoter in cycling HeLa cells. Interestingly, USF DNA-binding activity appears to be regulated in a G2-specific fashion, supporting the idea that USF may play some role in cyclin B1 gene activation. These studies suggest an important link between USF and the cyclin B1 gene, which in part explains how maturation promoting factor complex formation is regulated.

Mechanism of c-myc regulation by c-Myb in different cell lineages.

Activation of the murine c-myc promoter by murine c-Myb protein was examined in several cell lines by using a transient expression system in which Myb expression vectors activate the c-myc promoter linked to a chloramphenicol acetyltransferase reporter gene or a genomic beta-globin gene. S1 nuclease protection analyses confirmed that the induction of c-myc by c-Myb was transcriptional and affected both P1 and P2 start sites in a murine T-cell line, EL4, and a myelomonocytic line, WEHI-3. Mutational analyses of the c-myc promoter revealed that two distinct regions could confer Myb responsiveness in two T-cell lines, a distal site upstream of P1 and a proximal site within the first noncoding exon. In contrast, only the proximal site was required for other cell lineages examined. Five separate Myb-binding sites were located in this proximal site and found to be important for c-Myb trans activation. DNA binding was necessary for c-myc activation, as shown by the loss of function associated with mutation of Myb's DNA-binding domain and by trans-dominant repressor activity of the DNA binding, trans-activation-defective mutant. The involvement of additional protein factors was addressed by inhibiting protein synthesis with cycloheximide in a conditional expression system in which the activity of presynthesized Myb was under the control of estrogen. These experiments indicate that de novo synthesis of additional proteins was not necessary for c-myc trans activation. Together these data reveal two cell lineage-dependent pathways by which c-Myb regulates c-myc; however, both pathways are mechanistically indistinguishable in that direct DNA binding by Myb is required for activating c-myc whereas neither de novo protein synthesis nor other labile proteins are necessary.

Characterization of human E4BP4, a phosphorylated bZIP factor.

In this report we described the isolation of transcription factor E4BP4 by lambda gt11 expression cloning using a probe containing the CRE/ATF-like sequence located between -2764 bp and -2753 bp in the upstream regulatory region for the human IL-1 beta gene. DNaseI protection, gel mobility shift analysis, and cotransfection studies were performed to investigate the binding and functional properties of E4BP4 using IL-1 beta promoter sequences. By DNaseI footprinting, a protection pattern was generated over the CRE/ATF-like site and the flanking sequences by bacterially produced E4BP4. Competition experiment by gel shift assay indicated that E4BP4 bound specifically to CRE/ATF-like site, not NF kappa B-like site. In cotransfection studies, E4BP4 repressed promoter activity and this repression was mediated through the CRE/ATF-like site. Mutational analysis of E4BP4 suggested that the DNA binding as well as repression activities required leucine heptad repeat domain. Analysis of E4BP4 produced in Escherichia coli and Sf9 cells infected with recombinant baculovirus indicated that baculovirus produced protein showed enhanced binding to the CRE/ATF-like site compared to the E. coli-produced protein. Analysis of posttranslational modifications indicated that E4BP4 produced in Sf9 cells was phosphorylated and this phosphorylation was important for the DNA binding activity of E4BP4.

Transcriptional regulation of the HLA-DRA gene.

Class II Major Histocompatibility Complex (MHC) products are essential initiators of cellular immune responses. Their cell surface expression demonstrates aspects of tissue-restricted, inducible, differentiation-dependent, and coordinate regulation indicative of complex molecular controls mediated primarily at the level of transcription. This article reviews our current understanding of transcriptional regulation of one such human class II MHC gene, DRA. Considerable effort has been exerted toward characterizing the cis-acting regulatory sequences and trans-acting DNA binding proteins responsible for regulating DRA transcription. Recent application of novel molecular techniques has resulted in the cloning and characterization of some of these regulatory proteins. A model is presented herein which accommodates the current knowledge of DRA gene regulation.

Syntheses and biological activities of a novel group of steroidal derived inhibitors for human Cdc25A protein phosphatase.

Silica gel supported pyrolysis of an azido-homo-oxa steroid led to rearrangement, presumably by a mechanism similar to that of solution phase Schmidt fragmentation, to produce a group of novel inhibitors for the oncogenic cell cycle regulator Cdc25A phosphatase. Cyano-containing acid 17, one of the best inhibitors in this group, inhibited the activity of Cdc25A protein phosphatase reversibly and noncompetitively with an IC(50) value of 2.2 microM. Structure-activity relationships revealed that a phosphate surrogate such as a carboxyl or a xanthate group is required for inhibitory activity, and a hydrophobic alkyl chain, such as the cholesteryl side chain, contributes greatly to the potency. Without the cyano group, acid 26 and xanthate 27 were found to be more selective over Cdc25A (IC(50) = 5.1 microM and 1.1 microM, respectively) than toward CD45 (IC(50) > 100 microM, in each case), a receptor protein tyrosine phosphatase. Several of these inhibitors showed antiproliferative activities in the NCI 60-human tumor cell line screen. These steroidal derived Cdc25 inhibitors provide unique leads for the development of dual-specificity protein phosphatase inhibitors.

The admid system: generation of recombinant adenoviruses by Tn7-mediated transposition in E. coli.

A new system has been developed for generating recombinant adenoviruses by Tn7-mediated transposition in E. coli. Low copy number E. coli plasmids containing a full-length adenoviral genome with lacZattTn7 replacing E1 have been constructed. The adenovirus plasmid or admid, as well as high copy number progenitors, were stably maintained in E. coli strain DH10B. Several transfer vectors containing a mammalian expression cassette flanked by Tn7R and Tn7L were used as donors to transpose the mini-Tn7 into the E1 region of the adenoviral genome. Transposed recombinant admids are readily identified by their beta-galactosidase phenotype. Transfection of admid DNA into producer cells resulted in the efficient production of infectious adenovirus. This easy-to-use, efficient system generates pure, clonal stocks of recombinant adenovirus without successive rounds of plaque purification.

Histone deacetylase inhibitors promote apoptosis and differential cell cycle arrest in anaplastic thyroid cancer cells.

Little information exists concerning the response of anaplastic thyroid carcinoma (ATC) cells to histone deacetylase inhibitors (HDAIs). In this study, the cellular response to the histone deacetylase inhibitors, sodium butyrate and trichostatin A, was analyzed in cell lines derived from primary anaplastic thyroid carcinomas. HDAIs repress the growth (proliferation) of ATC cell lines, independent of p53 status, through the induction of apoptosis and differential cell cycle arrest (arrested in G1 and G2/M). Apoptosis increases in response to drug treatment and is associated with the appearance of the cleaved form of the caspase substrate, poly-(ADP-ribose) polymerase (PARP). Cell cycle arrest is associated with the reduced expression of cyclins A and B, the increased expression of the cyclin-dependent kinase inhibitors, p21(Cip1/WAF1) and p27Kip1, the reduced phosphorylation of the retinoblastoma protein (pRb), and a reduction in cdk2 and cdk1-associated kinase activities. In ATC cells overexpressing cyclin E, drug treatment failed to replicate these events. These results suggest that growth inhibition of ATC cells by HDAIs is due to the promotion of apoptosis through the activation of the caspase cascade and the induction of cell cycle arrest via a reduction in cdk2- and cdk1-associated kinase activities.

Role of self carriers in the immune response and tolerance. XII. Effect of epitope density and antigen-presenting cell phenotype on the presentation of hapten-modified self for the induction of immunity or tolerance in vitro.

The data presented in the accompanying paper (J. P. Cogswell, R. P. Phipps, and D. W. Scott, Cell. Immunol. 114, 55-70, 1988) indicate that certain macrophage-like and lymphoid dendritic-like (P388AD.2) tumor lines which express major histocompatibility encoded class II (Ia) antigens and produce interleukin 1 (IL-1) are uniquely able to present hapten-modified self (HMS) in an immunogenic fashion in vivo. In the current study, the relationship between phenotype and function has been confirmed utilizing a completely in vitro system. This investigation revealed that B-cell priming required T cells restricted to P388AD.2's I-A antigens. In addition, exogenous IL-1 reconstituted the response of an IL-1-deficient tumor (P388AD.2-ILd), although it had no effect on the other nonimmunogenic Ia+ tumor lines. Unlike the in vivo system, effective B-cell tolerance was induced when P388AD.2 was modified with high concentrations (10 mM) of hapten or when highly haptenated tumor was added to 0.1 mM TNBS-modified P388AD.2. These results suggest that positive regulation of in vitro immune responses to HMS is dependent upon the phenotype of the accessory cell carrier (with lymphoid dendritic-like cells being unusually potent), while negative regulation is associated with high epitope density. This system now allows the dissection of the properties of different accessory cells and the signals required for B-cell priming or tolerance induction.

The W element is a positive regulator of HLA-DRA transcription in various DR+ cell types.

The W and P elements are cis-acting transcriptional regulators of the human class II MHC gene HLA-DRA necessary for maximal promoter activity in DR+ B cells. Proteins that bind specifically to W may mediate promoter activity from a DNA segment containing W and P (W/P). In this report, we demonstrate that a -143 to -123 bp region that lacks P is sufficient to mediate the W elements function and to bind the W proteins W-B1 and W-B2 in the Raji B-lymphoblastoid cell line. In contrast to previous reports, we find that W/P is not a B cell-specific element; rather that its promoter activity parallels active transcription of the endogenous DRA gene. In addition, we show that tissue-restricted regulation by W/P is not correlated with the ubiquitous (W-B1) and lymphoid-specific (W-B2) distribution of the W binding proteins. We suggest that mechanisms in addition to binding to W may account for W/P-dependent transcriptional activation in DR+ cells.

Role of self carriers in the immune response and tolerance. X. A lymphoid dendritic-like tumor, P388AD.2, acts as a novel immunogenic carrier for hapten.

Hapten-modified spleen cells, peritoneal exudate cells, and certain lymphoid tumors preferentially induce specific tolerance after i.v. administration. In contrast to these tolerogenic carrier cells, we found that a haptenated lymphoid dendritic-like tumor, P388AD.2, acts as a potent immunogen after i.v. injection. The immunogenicity of P388AD.2 was analyzed by measuring the specific augmentation of plaque-forming cell (PFC) responses when spleen cells from mice previously injected with haptenated tumor cells were challenged in vitro with thymus-independent antigens. Optimal immunization was found to be dependent on cell dose and hapten concentrations. Further studies indicated that P388AD.2 elicited a response which was T cell-dependent and which involved both the so-called Lyb-3,5,7- and Lyb-3,5,7+ B cell populations. Injection of haptenated tumor into different mouse strains suggested that H-2 compatibility was required to prime B cells in vivo, although significant augmentation could also be achieved in allogeneic C57B1/6J mice. The enhanced PFC responses elicited in H-2b mice could not be explained by allo-recognition of class I or II MHC determinants. In toto, these results suggest that P388AD.2 acts as a unique accessory cell for the presentation of hapten-modified self.

Role of self carriers in the immune response and tolerance. XI. Correlation of Ia expression and interleukin-1 production with delivery of immunogenic signals in vivo by hapten-modified accessory cell-like tumor lines.

It was recently demonstrated that a lymphoid dendritic-like tumor, P388AD.2, presented hapten-modified self (HMS) in an immunogenic fashion even after injection via the normally "tolerogenic" intravenous (iv) route. To determine whether this property was unique to the P388AD.2 line, other hapten-modified tumors were administered iv and the result of their presentation was measured by changes in the number of splenic plaque-forming cells (PFC) following in vitro challenge with thymic-independent antigens. Of the six tumors tested, two (P388 and J774.5R) primed for augmented PFC responses, while four others (P388NA.10, P388D1, WEHI-231, and 70Z/3) did not. When these tumors were compared for Ia expression and production of interleukin-1 (IL-1), it was discovered that (1) all of the immunogenic tumors were Ia+ and IL-1 producing (IL-1+), although not all Ia+,IL-1+ tumors could elicit augmented PFC responses; (2) none of the tumors that were deficient in either Ia expression or IL-1 production could prime B-cell responses in vivo; and (3) the ability to augment PFC responses was proportional to the density of Ia on the immunogenic tumors. These results demonstrated that P388AD.2 was not the only tumor line capable of presenting HMS iv as an immunogen, and that the accessory cell phenotype is critical for the induction of an immunogenic response in vivo.

X-box-binding proteins positively and negatively regulate transcription of the HLA-DRA gene through interaction with discrete upstream W and V elements.

Previous reports have identified that the class II box, consisting of the positive regulatory X and Y boxes, is important for expression of all class II major histocompatibility genes. In this paper, we identify additional sequences upstream from the class II box that regulate constitutive transcription of a human class II gene, HLA-DRA, in the B-lymphoblastoid cell line Raji. Using 5' promoter deletions, substitution mutants, and nuclease S1 protection assays, we mapped a positive element, called W, between -135 and -117 base pairs and a negative element, called V, from -193 to -179 base pairs. Sequence comparisons revealed that W and V share homology with the HLA-DRA X box situated downstream. Gel-mobility-shift assays confirmed that the Raji nuclear proteins that bound to W and V elements were competed with by an HLA-DRA X-box oligonucleotide. These results suggest that X-box-binding proteins mediate both positive and negative effects on transcription by means of interaction with multiple elements (W, V, and X) within the same HLA-DRA gene.

p53 regulates a G2 checkpoint through cyclin B1.

The p53 tumor suppressor controls multiple cell cycle checkpoints regulating the mammalian response to DNA damage. To identify the mechanism by which p53 regulates G2, we have derived a human ovarian cell that undergoes p53-dependent G2 arrest at 32 degrees C. We have found that p53 prevents G2/M transition by decreasing intracellular levels of cyclin B1 protein and attenuating the activity of the cyclin B1 promoter. Cyclin B1 is the regulatory subunit of the cdc2 kinase and is a protein required for mitotic initiation. The ability of p53 to control mitotic initiation by regulating intracellular cyclin B1 levels suggests that the cyclin B-dependent G2 checkpoint has a role in preventing neoplastic transformation.

Lymphoma models for B-cell activation and tolerance. II. Growth inhibition by anti-mu of WEHI-231 and the selection and properties of resistant mutants.

Regulation of the growth of murine B-cell lymphomas has been used as a model for tolerance induction. The inhibition by anti-immunoglobulin reagents of the growth of WEHI-231 and several variant clones has now been studied. The parental line is exquisitely sensitive to growth inhibition by heterologous or monoclonal anti-mu or anti-k reagents and ceases to incorporate thymidine within 24-48 hr of exposure to anti-immunoglobulin reagents. Growth inhibition is initially reversible, but prolonged exposure to anti-mu results in cell death. This inhibition is specific for immunoglobulin light and heavy chains since growth is not inhibited by antibodies directed at either class I or class II histocompatibility antigens. In order to study the mechanism of growth inhibition, we have mutagenized WEHI-231 with ethylmethane sulfonate and cloned the surviving colonies in the presence of anti-mu. Such variants, which have been repeatedly recloned, are able to grow normally in the presence of anti-mu up to 100 micrograms/ml. These "resistant" clones, while expressing amounts of surface IgM similar to that observed on WEHI-231, do not differ markedly in their ability to cap their immunoglobulin receptors compared to the parental line but appear to have lost class II antigens. Cell cycle analysis revealed that anti-mu causes a block in the transition of WEHI-231 from G1 to S phase. The relevance of these processes to models of B-cell tolerance induction are discussed.

Dominant-negative polo-like kinase 1 induces mitotic catastrophe independent of cdc25C function.

Polo-like kinase 1 (PLK1), which has been shown to have a critical role in mitosis, is one possible target for cancer therapeutic intervention. PLK1, at least in Xenopus, starts the mitotic cascade by phosphorylating and activating cdc25C phosphatase. Also, loss of PLK1 function has been shown to induce mitotic catastrophe in a HeLa cervical carcinoma cell line but not in normal Hs68 fibroblasts. We wanted to understand whether the selective mitotic catastrophe in HeLa cells could be extended to other tumor types, and, if so, whether it could be attributable to a tumor-specific loss of dependence on PLK1 for cdc25C activation. When PLK1 function was blocked through adenovirus delivery of a dominant-negative gene, we observed tumor-selective apoptosis in most tumor cell lines. In some lines, dominant-negative PLK1 induced a mitotic catastrophe similar to that published in HeLa cells (K. E. Mundt et al., Biochem. Biophys Res. Commun., 239: 377-385, 1997). Normal human mammary epithelial cells, although arrested in mitosis, appeared to escape the loss of centrosome maturation and mitotic catastrophe seen in tumor lines. Mitotic phosphorylation of cdc25C and activation of cdk1 was blocked by dominant-negative PLK1 in human mammary epithelial cells as well as in the tumor lines regardless of whether they underwent mitotic catastrophe. These data strongly argue that the mitotic catastrophe is not attributable to a lack of dependence for PLK1 in activating cdc25C.

NF-kappa B regulates IL-1 beta transcription through a consensus NF-kappa B binding site and a nonconsensus CRE-like site.

In these studies, we show that NF-kappa B induces transcription from the human pro-IL-1 beta (IL-1 beta) gene. A recombinant plasmid pIL-1(-4000)-CAT, containing 4 kb of the IL-1 beta gene upstream regulatory sequence was transactivated by the p65 subunit of NF-kappa B or by treatment of the cells with a combination of NF-kappa B inducers including LPS, PMA, and dibutyryl cyclic AMP (L+P+C) in U937 cells. Coexpression of p65 with L+P+C treatment led to a synergistic response, whereas coexpression of the I kappa B alpha/MAD-3 protein, in place of p65, blocked L+P+C induction. A series of 5' deletion mutants of the IL-1 beta promoter were used to define two p65 response regions: region I located between -2800 to -2720 bp and region II located between -512 and -133 bp. Electrophoretic mobility shift assays confirmed that NF-kappa B-like proteins could bind to two consensus binding sites in region II. A site-specific mutation in only one of these NF-kappa B sites (-296/-286 bp) caused a specific loss of induction by p65 or L+P+C. A cyclic AMP response element (CRE) site (-2761/-2753 bp) in region I has been shown previously to be critical for L+P+C induction. Mutation of the CRE in an enhancerless test plasmid containing two copies of region I blocked transactivation by p65. Likewise, coexpression of I kappa B alpha inhibited CRE-dependent L+P+C induction of the wild-type counterpart. These data show that NF-kappa B regulates a nonconsensus CRE site in addition to the consensus binding site at -296/-286 bp and suggest that NF-kappa B may play multiple roles in the induction of IL-1 beta transcription.

Cyclin-dependent kinase activation and S-phase induction of the cyclin B1 gene are linked through the CCAAT elements.

Control of cell proliferation is dependent on the regulated expression of the cyclin genes. Induction of cyclin B1 gene expression in S phase has been shown to require sequences within the first 90 bp of the proximal promoter region. In this study, we defined the cell cycle regulatory elements within this region and explored the mechanism by which the cyclin B1 gene is activated. A CDE-like element that is important in S-phase regulation of other genes was not required for correct cell cycle expression of cyclin B1. Instead, two CCAAT boxes were essential for S-phase induction of cyclin B1 gene in both NIH3T3 and HeLa cells. Induction of cyclin B1 by cyclin/cyclin-dependent kinase (cdk) complexes were examined by cotransfection of the reporter along with appropriate expression vectors. Complexes of cdk4 with cyclin D1 or cdk2 with cyclin E or A can activate the cyclin B1 promoter, and activation is uniquely dependent on the CCAAT elements in both normal and heterologous contexts. This transcription factor NF-Y binds to both CCAAT elements. These findings suggest that S phase-specific induction of the cyclin B1 promoter is dependent upon NF-Y binding to the CCAAT elements and is correlated with activation by cyclin-dependent kinases.

NF-kappaB activation provides the potential link between inflammation and hyperplasia in the arthritic joint.

The transcription factor NF-kappaB is a pivotal regulator of inflammatory responses. While the activation of NF-kappaB in the arthritic joint has been associated with rheumatoid arthritis (RA), its significance is poorly understood. Here, we examine the role of NF-kappaB in animal models of RA. We demonstrate that in vitro, NF-kappaB controlled expression of numerous inflammatory molecules in synoviocytes and protected cells against tumor necrosis factor alpha (TNFalpha) and Fas ligand (FasL) cytotoxicity. Similar to that observed in human RA, NF-kappaB was found to be activated in the synovium of rats with streptococcal cell wall (SCW)-induced arthritis. In vivo suppression of NF-kappaB by either proteasomal inhibitors or intraarticular adenoviral gene transfer of super-repressor IkappaBalpha profoundly enhanced apoptosis in the synovium of rats with SCW- and pristane-induced arthritis. This indicated that the activation of NF-kappaB protected the cells in the synovium against apoptosis and thus provided the potential link between inflammation and hyperplasia. Intraarticular administration of NF-kB decoys prevented the recurrence of SCW arthritis in treated joints. Unexpectedly, the severity of arthritis also was inhibited significantly in the contralateral, untreated joints, indicating beneficial systemic effects of local suppression of NF-kappaB. These results establish a mechanism regulating apoptosis in the arthritic joint and indicate the feasibility of therapeutic approaches to RA based on the specific suppression of NF-kappaB.

Plk3 functionally links DNA damage to cell cycle arrest and apoptosis at least in part via the p53 pathway.

Polo-like kinase 3 (Plk3, previously termed Prk) contributes to regulation of M phase of the cell cycle (Ouyang, B., Pan, H., Lu, L., Li, J., Stambrook, P., Li, B., and Dai, W. (1997) J. Biol. Chem. 272, 28646-28651). Plk3 physically interacts with Cdc25C and phosphorylates this protein phosphatase predominantly on serine 216 (Ouyang, B., Li, W., Pan, H., Meadows, J., Hoffmann, I., and Dai, W. (1999) Oncogene 18, 6029-6036), suggesting that the role of Plk3 in mitosis is mediated, at least in part, through direct regulation of Cdc25C. Here we show that ectopic expression of a kinase-active Plk3 (Plk3-A) induced apoptosis. In response to DNA damage, the kinase activity of Plk3 was rapidly increased in an ATM-dependent manner, whereas that of Plk1 was markedly inhibited. Recombinant Plk3 phosphorylated in vitro a glutathione S-transferase fusion protein containing p53, but not glutathione S-transferase alone. Recombinant Plk1 also phosphorylated p53 but on residues that differed from those targeted by Plk3. Co-immunoprecipitation and pull-down assays demonstrated that Plk3 physically interacted with p53 and that this interaction was enhanced upon DNA damage. In vitro kinase assays followed by immunoblotting showed that serine 20 of p53 was a target of Plk3. Furthermore, expression of a kinase-defective Plk3 mutant (Plk3(K52R)) resulted in significant reduction of p53 phosphorylation on serine 20, which was correlated with a decrease in the expression of p21 and with a concomitant increase in cell proliferation. These results strongly suggest that Plk3 functionally links DNA damage to cell cycle arrest and apoptosis via the p53 pathway.