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. 2002 May;4(3):268–274. doi: 10.1038/sj.neo.7900235

Cyclin K as a Direct Transcriptional Target of the p53 Tumor Suppressor1

Toshiki Mori 1, Yoshio Anazawa 1, Kuniko Matsui 1, Seisuke Fukuda 1, Yusuke Nakamura 1, Hirofumi Arakawa 1
PMCID: PMC1531701  PMID: 11988847

Abstract

Cyclin K, a newly recognized member of the “transcription” cyclin family, may play a dual role by regulating CDK and transcription. Using cDNA microarray technology, we found that cyclin K mRNA was dramatically increased in U373MG, a glioblastoma cell line deficient in wild-type p53, in the presence of exogenous p53. An electrophoretic mobility-shift assay showed that a potential p53-binding site (p53BS) in intron 1 of the cyclin K gene could indeed bind to p53 protein. Moreover, a heterologous reporter assay revealed that the p53BS possessed p53-dependent transcriptional activity. Colony-formation assays indicated that overexpression of cyclin K suppressed growth of T98G, U373MG and SW480 cells. The results suggested that cyclin K may play a role in regulating the cell cycle or apoptosis after being targeted for transcription by p53.

Keywords: cyclin K, cell cycle, p53, p53 target gene, cDNA microarray

Introduction

The tumor suppressor gene p53 encodes a transcription factor that binds to specific DNA sequences and activates transcription of its target genes [1]. Numerous p53 targets have already been discovered by our group and others. Of those, p21/WAF1 and BAX are believed to be the most important because they mediate two major functions of p53, cell cycle arrest and apoptosis, respectively [2–4]. Given that hundreds of possible p53-binding sequences may be present in the human genome [5], we assume that the tumor-suppressive role of p53 probably reflects physiological activities of numerous and widely diverse target genes. Hence, identification of additional p53 targets may continually yield clues to the pathways involved in tumorigenesis and clarify mechanisms that protect cells from a variety of cellular stresses.

Cyclin K was first identified in a yeast screen based on its ability to restore cell cycle progression and rescue Saccharomyces cerevisiae cells from lethality resulting from deletion of G1 cyclin [6]. Cyclin K was also isolated in a two-hybrid screen using human CDK9 as bait, and biochemical analysis confirmed that cyclin K can activate CDK9 activity by forming a stable protein complex with CDK9 [7]. The CDK9-cyclin K complex phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII), a reaction that is considered to be one of the most important steps in transcription of many genes [8]. However, the function of cyclin K remains to be elucidated in detail.

Until now, our efforts to isolate p53-target genes have involved two different methods. One method was differential display, using a cell line in which expression of an exogenous wild-type p53 gene can be regulated under the control of the lactose operon [9]. In this manner we isolated five novel p53 targets, TP53TG1 [9]; TP53TG3 [10]; fractalkine, which is a chemokine regulating migration and targeting cytotoxic T cell and NK cell [11]; p53R2, which is commonly involved in p53-dependent DNA repair in response to diverse agents of DNA damage [12]; and p53DINP1, which mediates the p53-dependent apoptotic pathway [13]. The other method was a yeast enhancer-trap system that allowed direct cloning of functional p53-binding sequences from human genomic DNA [5]. By isolating and sequencing cosmid clones containing every p53-binding site (p53BS) obtained in that way, we identified four novel p53-target genes in the genomic regions surrounding the binding sequences: GML [14], P2XM [15], BAI1 [16] and p53AIP1 [17]. GML contributes to the sensitivity of cells to anticancer drugs; BAI1 inhibits angiogenesis in gliomas, and the p53AIP1 product is a pivotal mediator of p53-induced apoptosis.

As a third approach to isolation of additional p53-target genes we have been applying cDNA-microarray technology. That effort has yielded evidence that cyclin K is yet another direct transcriptional target of p53.

Materials and Methods

Cell Lines

Human cancer cell lines U373MG (glioblastoma), and SW480 (colon carcinoma) cells were purchased from the American Type Culture Collection (Manassas, VA). T98G (glioblastoma) was purchased from the Human Science Research Resource Bank (Tokyo, Japan). All cells were cultured under conditions recommended by their respective depositors.

Construction and Infection of Recombinant Adenovirus

Replication-deficient recombinant viruses Ad-p53 and Ad-LacZ, containing wild-type p53 and LacZ genes, respectively, were generated and purified as described previously [17]. U373MG cells were infected by the viral solutions at a multiplicity of infection of 80 and incubated at 37°C until the time of harvest.

RNA preparation

Total RNAs were isolated from transfected cells with Trizol reagent (Life Technologies, Rockville, MD) following the manufacturer's instructions, on a time course of 0, 6, 12, 24, and 48 hours after infection. Poly(A)+ RNAs were purified with mRNA purification kits (Amersham Pharmacia Biotech, Uppsala, Sweden).

cDNA Microarray

The cDNA microarray was fabricated essentially as described elsewhere [18] In brief, we amplified human cDNAs, including ESTs of interest, based on sequences in the UniGene database (National Center for Biotechnology Information, Bethesda, MD) using gene-specific sets of oligonucleotide primers. 9216 independent PCR products ranging in size from 0.2 to 1 kb were purified and robotically spotted onto type 7 glass slides (Amersham Pharmacia Biotech) by means of the Microarray Spotter Generation III (Amersham). Aliquots of poly(A)+ RNA (1 µg) were reverse transcribed and second-strand synthesis was performed. Double-stranded cDNAs were then amplified once with the Ampliscribe T7 Transcription Kit (Epicentre Technologies, Madison, WI). Finally, 2.5-µg aliquots of amplified RNA from U373MG cells infected with Ad-LacZ or Ad-p53 were labeled with Cy3 or Cy5, respectively, using a cDNA synthesis system (Life Technologies). Arrays were hybridized with equal amounts of Cy3- and Cy5-labeled DNA probes, then washed and scanned by the ArrayScanner according to the supplier's protocol (Amersham Pharmacia Biotech). Signal intensities of Cy3 and Cy5 from individual spots were quantified and analyzed by substituting backgrounds using ArrayVision software (Imaging Research, Ontario, Canada).

Northern Blot Hybridization

A 2-µg aliquot of each poly(A)+ RNA was separated by electrophoresis on a 1% agarose gel containing 6% formalin and transferred onto a nylon membrane. Membranes blotted with poly(A)+ RNA from various human tissues were purchased from Clontech, Palo Alto, CA. Prehybridization and hybridization were carried out in solutions containing 50% formamide, 5x Denhardt's solution, 6x SSC, and 1% salmon sperm DNA. The probe, a 344-bp cDNA fragment carrying coding sequences of cyclin K, was labeled with [α32P]dCTP using a random primer labeling kit (Megaprime, Amersham Pharmacia Biotech) according to the manufacturer's instructions. The blots were hybridized with the radioactive probes at 42°C for 16 hours, washed with 0.1 x SSC/0.1%SDS at 55°C, and exposed to Kodak X-Omat films (Eastman Kodak, Rochester, NY) at -80°C for 24 hours (cyclin K) or 1 hour (β-actin).

cDNA Library Screening

We constructed a cDNA library using poly(A)+ RNA obtained from U373MG cells infected with Ad-p53 for 48 hours and screened 1x106 independent colonies of this library with the same probe that was used for Northern blotting.

Electrophoretic Mobility-Shift Assay (EMSA)

Oligonucleotides representing the candidate sequence (p53BS) were synthesized and annealed (p53BS sense, 5′-AAACTAGCTTGCAGACATGCTG-3′; p53BS antisense, 5′-CAGCATGTCTGCAAGCTAGTTT-3′). Nuclear extracts from H1299 cells infected with Ad-p53 were incubated for 30 minutes at RT with 2.0 µg of sonicated salmon sperm DNA, EMSA buffer (0.5x TBE, 20 mM HEPES (pH 7.5), 0.1 M NaCl, 1.5 mM MgCl2, 10 mM DTT, 20% glycerol, 0.1% NP-40, 1 mM PMSF, 10 mg/ml pepstatin, 10 mg/ml leupeptin), the [γ33P]ATP-labeled double-strand oligomer, and, in some cases, with monoclonal anti-p53 antibodies PAb421 (Oncogene Science, Cambridge, MA) and/or PAb1801 (Santa Cruz Biotechnology, Santa Cruz, CA). After incubation, the samples were electrophoresed in native 4% polyacrylamide gels using 0.5x TBE. The gels were dried and exposed for autoradiography at -80°C for 3 hours.

Heterologous-Reporter Assay

Heterologous-reporter vectors designed to possess the 500-bp segment corresponding to intron 1 of the cyclin K gene were constructed to contain the p53BS (intron 1-wt). Using specifically designed oligonucleotides (sense, 5′-AAAACGCGTCTGGCTTGACAAAAGTGGTTC-3′; antisense, 5′-AAACTCGAGTAGCGACTCTTTAACTTCTAC-3′), the segment was generated by PCR and ligated between the MluI and XhoI sites of the pGL3-promoter vector (Promega, Madison, WI). Then two nucleotide-exchanged mutations (the 4th and 16th nucleotides of p53BS) were inserted (intron 1-mt). In brief, the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used twice, according to the manufacturer's instructions, using two sets of oligonucleotides: mt (4th) BS sense, 5′-GATAAGAAAACTATCTTGCAGACATGCTG-3′ and mt (4th) BS antisense, 5′-CAGCATGTCTGCAAGATAGTTTTCTTATC-3′ and mt (16th) BS sense, 5′-GCTTGCAGACATTCTGCAACGCAGCAGAC-3′, and mt (16th) BS antisense, 5′-GTCTGCTGCGTTGCAGAATGTCTGCAAGC-3′. The constructs of all plasmids were confirmed by sequencing.

H1299 cells were plated in six-well culture plates (1x105 cells) 24 hours before cotransfection of 1 µg of pGL3-BS, pGL3-mtBS, or pGL3-promoter vector with wild-type or mutant-type p53 expression vectors (wt-p53 or mt-p53) in combination with 1 µg of pRL-TK vector (Promega), using 6 µl of FuGENE 6 transfection reagent according to the manufacturer's optimized methodology (Roche, Basel, Switzerland). Forty eight hours after transfection, the cells were lysed in 500 µl of a passive lysis buffer (Promega). Cell lysates were forwarded directly to the luciferase assay. The dual luciferase system (Promega) was applied for the sequential measurement of firefly and Renilla luciferase activities with specific substrates of beetle luciferin and coelenterazine, respectively. Quantification of both luciferase activities and calculation of relative ratios were carried out manually with a luminometer.

Construction of Expression Vectors (pcDNA-cyclin K-S and pcDNA-cyclin K-AS)

Plasmids (pcDNA-cyclin K-S) designed to express cyclin K were constructed by cloning the entire coding region of cyclin K cDNA into the pcDNA3.1(+) expression vector (Invitrogen, San Diego, CA), which carries a CMV promoter and a gene conferring neomycin resistance. The same fragment was inserted into the pcDNA3.1(-) vector for construction of the antisense expression vector as a control (pcDNA-cyclin K-AS). The constructs were confirmed by sequencing.

Colony-Formation Assay

T98G, U373MG, and SW480 cells (5x105 each) were plated in 10-cm culture dishes 24 hours before transfection. Eight micrograms of expression vector (pcDNA-cyclin K-S or pcDNA-cyclin K-AS) were transfected using 24 µl of FuGENE 6 transfection reagent (Roche); 48 hours later the harvested cells were diluted and replated on 10-cm dishes. The transfected cells were allowed to grow in the presence of 0.8 mg/ml of G-418 (geneticin, Life Technologies) for 2 weeks, after which the colonies formed from each cell were fixed with 10% formaldehyde, stained with crystal violet, and counted.

Results

To identify additional genes that are transcriptional targets of p53, we applied cDNA-microarray technology using as probes cDNAs reverse transcribed from mRNAs of p53-deficient U373MG cells infected by either Ad-p53 or Ad-LacZ. Duplicate microarrays containing 9216 human cDNAs were hybridized with a mixture of Cy5-labeled cDNA probes corresponding to Adp53-infected cells and Cy3-labeled cDNA probes corresponding to AdLacZ-infected cells. Dozens of genes showed increased signal intensities of Cy5, in a time-dependent manner. Among them, one EST (UniGene accession number Hs.12186) revealed striking induction by p53. To clone the full-length cDNA of this gene, we constructed a cDNA library using poly(A)+ RNA obtained from U373MG cells infected with Ad-p53, and screened 106 independent colonies of the cDNA library using a 400-bp DNA fragment of the EST as a probe. As a result, 20 independent clones were isolated and sequenced. A BLAST search of the public database indicated that DNA sequences of this gene were identical to parts of cyclin K (GenBank accession number AF060515). Comparison of the cDNA with genomic DNA (GenBank accession number AL110504) indicated that the archived cDNA sequence lacked the first exon. Northern blot analysis showed that the 2.8-kb mRNA of cyclin K was rapidly induced in U373MG after infection with Ad-p53 (Figure 1A). Multi-tissue Northern blots revealed ubiquitous expression of cyclin K (data not shown).

Figure 1.

Figure 1

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Transcriptional activation of cyclin K by p53 and a potential p53BS in the cyclin K gene. (A) Northern blot analysis of cyclin K mRNA expression in the U373MG glioblastoma cell line at the indicated times after infection with either Adp53 or AdLacZ. Hybridization of the same blot with a β-Actin probe quantifies the amount of mRNA loaded in each lane. (B) Genomic structure and a p53BS of cyclin K. The cyclin K gene consists of 11 exons that span a 35-kb genomic region, and a potential p53 BS was found in intron 1 (arrow). The consensus sequence for p53 binding is indicated as (R) =purine, (Y) =pyrimidine, and (W) =A or T. Of the 20 base pairs of p53BS (with a two-base spacer), 19 match the consensus sequence.

To determine whether cyclin K is a direct target of p53, we searched for a p53-binding sequence(s) within its 35-kb genomic sequence (AL110504). A potential p53-binding sequence (p53BS) was found in intron 1. Nineteen of the 20 nucleotides of this p53BS matched the consensus p53-binding sequence proposed by el-Deiry et al. [19] (Figure 1B).

We performed an EMSA using a nuclear extract purified from H1299 cells infected by Ad-p53, after synthesizing oligonucleotides corresponding to the p53BS. As shown in Figure 2A, this sequence bound to a protein contained in the nuclear extract (lane 1), and the band was super-shifted in the presence of mouse monoclonal anti-p53 antibody Pab421, indicating that p53BS bound to the p53 protein (lane 2). This evidence was further clarified by specific competition with self-DNA but not with nonspecific DNA (lanes 3 and 4), and also by the fact the band was supersuper-shifted by addition of another mouse monoclonal anti-p53 antibody, Pab1801 (lane 5).

Figure 2.

Figure 2

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Cyclin K as a direct target of p53. (A) EMSA. Anti-p53 antibodies Pab421 and Pab1801 were present in the lanes designated “+”. Interaction between p53 protein and DNA was inhibited by unlabeled oligonucleotides corresponding to the binding site of the cyclin K gene (competitor DNA [self]), but not by nonspecific oligonucleotides (competitor DNA [nonspecific]). (B) Luciferase assay. 500-bp fragments corresponding to intron 1 of the cyclin K gene, containing the possible p53-binding sequence without or with point mutations, were cloned into Luciferase reporter vector (wt-BS or mt-BS). These two vectors were cotransfected into H1299 cells along with wt-p53, mt-p53 (R175H or C277R), or pcDNA3.1 vector (mock). Luciferase activity was indicated relative to the activity of reporter vector without the p53-binding sequence.

To examine p53-dependent transcriptional activity of p53BS, we performed a heterologous reporter assay using a plasmid containing 500-bp fragments encompassing p53BS or p53mtBS (intron 1-wt or intron 1-mt) in intron 1 of the cyclin K gene. As shown in Figure 2B, luciferase activity of pGL3-BS (intron 1-wt) was enhanced more than 10-fold by cotransfection with wt-p53 vector than by cotransfection with either mt-p53 vector or mock vector. Moreover, the mutated sequence (intron 1-mt) apparently inhibited the wild-type p53-dependent enhancement of luciferase activity. Taken together, these results indicated that cyclin K is a direct target for p53.

We further examined whether endogenous p53 could induce cyclin K expression in response to cellular stresses causing DNA damage, such as gamma irradiation, adriamycin treatment, and UV irradiation. As shown in Figure 3, the expression of cyclin K was induced by three different stresses in MCF7 p53+/+ cells. However, the expression was not induced at all by any stress in H1299 p53-/- cells (Figure 3). The result indicated that cyclin K is transcriptionally activated in response to diverse cellular stresses in a p53-dependent manner.

Figure 3.

Figure 3

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Induction of endogenous cyclin K mRNA by DNA damages by 1.0 µg/ml adriamycin (top), 50 Gy gamma radiation (middle), or 10 J/m2 UV radiation (bottom) in MCF7 cells (p53+/+) and H1299 cells (p53-/-). Expressions of p21WAF1 and β-actin were used as a positive control and a quantity control, respectively, in Northern blot to examine cyclin K expression at the indicated times after exposure to diverse genotoxins.

To test whether cyclin K might regulate the cell cycle or apoptosis, we performed colony-formation assays. T98G and U373MG glioblastoma cell lines and SW480 colorectal cancer cell line were transfected with pcDNA-cyclin K-S (sense), pcDNA-cyclin K-AS (antisense) or pcDNA3.1 (mock). Transfected cells were allowed to grow in medium containing G-418; colonies formed by each cell line were fixed, stained, and counted 2 weeks later. In all three cancer cell lines, overexpression of sense-cyclin K (cyclin K-S) caused the suppression of cell growth (Figure 4). Especially in T98G and U373MG cells, a reduction of more than 50% in colony numbers was observed when cells had been transfected with sense-cyclin K (cyclin K-S) compared to cells transfected with antisense-cyclin K (cyclin K-AS) and mock vector (Figure 4). The results suggested that cyclin K might play a role in suppression of cell growth after being targeted for transcription by p53.

Figure 4.

Figure 4

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Colony-formation assay. Numbers of G-418 resistant colonies grown from each of two glioblastoma cell lines (T98G and U373MG) and one colorectal cancer cell line (SW480) transfected with pcDNA-cyclin K-S (sense), pcDNA-cyclin K-AS (antisense) or pcDNA3.1 (mock). The experiments were repeated at least three times using triplicate samples, and the average scores are shown with error bar.

Discussion

Cell cycle regulation and apoptosis are the most important features for p53's tumor suppression. In fact, overproduction of exogenous p53 in cancer cells causes the remarkable growth suppression through induction of cell cycle arrest or apoptosis. These functions are mediated by several p53-target genes. p21WAF1 and 14-3-3σ are pivotal mediators for cell cycle arrests at G1 and G2 phases, respectively. BAX and p53AIP1 mediate p53-dependent apoptosis. In addition, cyclin G was reported to be a direct target of p53, which might be involved in cell cycle regulation or apoptosis [21,22]. However, except for its sequence similarity to other cyclins, there is no evidence that it regulates cell cycle. In this study, we demonstrated that cyclin K is the second known example, the first being cyclin G, of cyclin genes as a p53 target. Moreover, we showed that ectopic expression of cyclin K induced the growth suppression in several cancer cell lines. Although the mechanism for cyclin K-dependent growth control remains to be elucidated, our results clearly suggest its involvement in cell cycle regulation or apoptosis.

Cyclin K was recently identified in a genetic screen in yeast by virtue of its ability to complement the lethality associated with the deletion of G1 cyclin genes [6]. It can form a stable protein complex with CDK9; in published experiments the specific activity of the CDK9-cyclin K complex was 10 to 15 times greater than that of CDK9 alone [7]. This complex phosphorylates the CTD of RNAPII and functionally substitutes for positive transcription factor b (P-TEFb), comprised of CDK9 and cyclin T, to regulate transcription in vitro [20]. Phosphorylation of the CTD of RNAPII is an important step in transcription, and P-TEFb may facilitate elongation at many genes [8]. Our results and these observations prompt us to speculate that p53 might regulate transcription of many genes indirectly, by activating expression of cyclin K.

Taken together, we propose a model that cyclin K might regulate the transcription of some cell cycle regulators or apoptosis-related factors through phosphorylation of CTD of RNAPII, resulting in cell cycle arrests or apoptosis. Discovery of cyclin K as a p53 target, which is involved in transcriptional regulation of multiple genes, could also provide a reason why p53 can amplify and boost its physiological functions efficiently.

Footnotes

1

This work was supported in part by grant #13216031 from the Ministry of Education, Culture, Sports, Science and Technology to H. A., and in part by “Research for the Future” Program grant #00L01402 from The Japan Society for the Promotion of Science to Y. N.

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