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[Cancer Research 64, 8328-8333, November 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

24-Hour Oscillation of Mouse Methionine Aminopeptidase2, a Regulator of Tumor Progression, Is Regulated by Clock Gene Proteins

Hiroo Nakagawa1, Satoru Koyanagi2, Takako Takiguchi1, Yukako Kuramoto2, Shinji Soeda2, Hiroshi Shimeno2, Shun Higuchi1 and Shigehiro Ohdo1

1 Clinical Pharmacokinetics, Division of Clinical Pharmacy, Department of Medico-Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan, and 2 Department of Biochemistry, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Methionine aminopeptidase2 (MetAP2) plays an important role in the growth of endothelial cells during the tumor angiogenesis stage. Recently, we have clarified that mouse methionine aminopeptidases (mMetAPs) show a 24-hour rhythm in implanted tumor masses. In the present study, we investigated the mechanism underlying the 24-hour rhythm of mMetAP2 activity in tumor-bearing mice under a light-dark (lights on from 7 a.m. to 7 p.m.) cycle. The 5' flanking region of mMetAP2 included eight E-boxes. The transcription of the mMetAP2 promoter was enhanced by the mCLOCK:mBMAL1 heterodimer, and its activation was inhibited by mPER2 or mCRY1. Deletion and mutation of the E-boxes in the region indicated that the E-box nearest to the initiation start site played an important role in the transcriptional regulation by clock genes. In sarcoma180-bearing mice, the pattern of binding of mCLOCK and mBMAL1 to the E-box and transcription of the mMetAP2 promoter showed a 24-hour rhythm with higher levels from the mid-light to early dark phase. The pattern of mMetAP2 transcription was closely associated with that of mMetAP2 mRNA expression in three types of tumor-bearing mice. mMetAP2 protein expression varied with higher levels from the late-dark to early light phase. The rhythmicity of the protein expression was synchronous with that of the activity of mMetAPs but out of phase with that of the mMetAP2 mRNA expression. These results suggest that the 24-hour rhythm of mMetAP2 activity is regulated by the transcription of clock genes within the clock feedback loops.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The methione aminopeptidases (MetAPs) are a class of proteases that selectively remove methionine from the NH2 terminus of newly synthesized proteins (1) . Protein synthesis is initiated with methionine in eukaryotes. MetAPs remove the initiator methionine of nascent polypeptide chains and play an important role in cell cycle initiation in eukaryotes (2 , 3) . MetAPs have been identified in all mammalian tissues and cells examined (4 , 5) . Down-regulation of human MetAP2 expression by an antisense oligonucleotide leads to the inhibition of endothelial cell proliferation (6) . Antisense of MetAP2 also induces apoptosis in human mesothelioma cells and rat hepatoma cells (7 , 8) . These results suggest that MetAP2 plays an important role in the growth of not only endothelial cells during the tumor angiogenesis stage but of tumor cells in mammals.

One approach to increasing the efficacy of pharmacotherapy is to administer the drugs at the time of day when they are most effective and/or best tolerated. A chronopharmacological strategy can enhance the effects of drugs and/or attenuate their toxicity (9, 10, 11) . Daily variations in biological functions, such as gene expression and protein synthesis, are thought to be important factors affecting the efficacy of drugs. MetAP2 has been examined as a physiologic target for the potent angiogenesis inhibitor TNP-470, a synthetic analogue of fumagillin, which acts by directly inhibiting endothelial cell proliferation (12) . Recently, we clarified that the antitumor effect of TNP-470 is more potent in mice injected with the drug during the early light phase than in those injected with the drug during the early dark phase (9) . In a nondrugged state, mouse methionine aminopeptidase (mMetAP) activity in tumor masses showed a significant 24-hour rhythm. The dosing time-dependent antitumor effect of TNP-470 is closely related to the 24-hour rhythm of mMetAP activity in tumor masses. However, the mechanism underlying the 24-hour rhythm of the enzyme activity has not been clarified.

The central circadian pacemaker in mammals is located in the hypothalamic suprachiasmatic nucleus (13) . The core circadian oscillator is composed of interacting positive and negative transcription–translation feedback loops. CLOCK and BMAL1 induce Period (Per) and Cryptochrome (Cry) gene expression (14) . PER and CRY proteins in turn act as negative components of feedback loops by suppressing CLOCK:BMAL1 heterodimer-mediated transcription through CACGTG E-box enhancer elements (15 , 16) . Recent studies have discovered circadian oscillations in the transcription of various genes in both peripheral tissues and cultured cells (17, 18, 19) . The transcriptional machinery of the core circadian clockwork also regulates clock-controlled output rhythms. Namely, the CLOCK:BMAL1 heterodimer acts through an E-box enhancer to activate the transcription of vasopressin, albumin D-element-binding protein, and prokineticin2 mRNAs, thereby showing a specific circadian output function (20, 21, 22) . Several factors can cause alterations to the clock function leading to illness and altered homeostatic regulation (10 , 11) . The functional significance of the mammalian peripheral clocks is still unknown.

Taking the findings described above into consideration, we hypothesized that the transcription of mMetAP2 mRNA may be regulated by clock gene proteins in tumor masses. The purpose of the present study was to investigate the mechanism underlying the 24-hour rhythm of mMetAP2 activity in implanted tumor masses: (1) we cloned the 5' flanking region of the mMetAP2 gene and identified an E-box in the arrangement, (2) we investigated whether the transcription of mMetAP2 mRNA was enhanced by the mCLOCK:mBMAL1 heterodimer and the activation was inhibited by mPER2 or mCRY1 in sarcoma180 tumor masses using the luciferase reporter assay, (3) we identified the E-boxes that were enhanced by the clock gene heterodimer after deletion and mutation of the E-box in the 5' flanking region of mMetAP2, (4) we observed when the mCLOCK:mBMAL1 heterodimer bound to the 5' flanking region and the transcription of mMetAP2 mRNA occurred in Sarcoma180-bearing mice, and (5) we determined the 24-hour rhythm of mMetAP2 mRNA and protein expression in three types of tumor (Sarcoma180, Lewis lung carcinoma, and B16 melanoma)-bearing mice.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Cells.
Male ICR mice (5 weeks old) were purchased from Charles River Japan, Inc. (Kanagawa, Japan). They were housed in a light-controlled room (lights on from 7 a.m. to 7 p.m.) at room temperature (24°C ± 1°C) and a relative humidity of 60% ± 10% with food and water ad libitum. All mice were adapted to the light/dark cycle for 2 weeks before the experiments began. During the dark period, a dim red light was used to aid in treatment of the mice. Two murine tumor cells lines (Sarcoma180 and B16 melanoma) were commercially obtained from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan). Lewis lung carcinoma cells were supplied by the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). The tumor cells were maintained in Dulbecco’s Modified Eagle’s Medium, supplemented with 10% heated-inactivated fetal bovine serum, 0.5% penicillin, and 0.5% streptomycin at 37°C in a humidified 5% CO2 atmosphere. A 50-µL volume of 1.5 ± 106 viable tumor cells was inoculated into the right hind footpads of each mouse.

Experimental Design.
To observe the existence of E-boxes in the 5' flanking region of the mMetAP2 gene, the unknown arrangement was amplified by PCR from Sarcoma180 genomic DNA. To investigate how the rhythmic variation of mMetAP2 mRNA expression occurs in tumor masses, the transcriptional activity of clock gene proteins in the cloning of the 5' flanking region of mMetAP2 was measured using a luciferase reporter assay system. To identify which E-box in the mMetAP2 promoter region is enhanced by the clock gene, deletion of the promoter region and mutation of the E-box were performed. To observe when the mCLOCK:mBMAL1 heterodimer binds the mMetAP2 promoter region and transcription occurs in the implanted Sarcoma180 masses, formaldehyde-cross–linked chromatin immunoprecipitation and semiquantitative PCR analysis were performed. The tumor masses were removed at the zeitgeber times ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22 on day 14 after the implantation of each mass of tumor cells. To determine the 24-hour rhythm of mMetAP2 mRNA and protein expression, tumor masses (Sarcama180, Lewis lung carcinoma, and B16 melanoma) were removed from six tumor-bearing mice at the zeitgeber times described above. mMetAP2 mRNA or its protein levels were measured by reverse transcription-PCR (RT-PCR) or Western blot analysis.

Cloning of the 5' Flanking Region of mMetAP2 and Construction of mMetAP2 Promoter Reporter.
The 5' flanking region of the mMetAP2 gene was amplified by PCR from mouse tumor cell Sarcoma180 DNA (DNA Databank of Japan accession no. AB181293). PCR was performed using the forward primer: 5'-GAT CAA TAA TGA ATG CTT-3' and reverse primer: 5'-AGG GCC CGC TCC TTC TTC A-3'. These primers were designed based on the arrangement of the 5' flanking region of rat MetAP2 (GenBank accession no. U37710). The PCR products were purified and ligated into the TOPO-XL vector (TA cloning kit, Invitrogen, Carlsbad, CA). A 1212-bp (–1272 to –60) 5' flanking region of mMetAP2 was isolated from the TOPO-XL vector using HindIII and XhoI, and this region was ligated into the Luciferase pGL3 basic vector (Promega, Madison, WI).

Deletion of mMetAP2 Promoter Region.
The deletion was used to clone the mMetAP2 promoter. A 782-bp (–842 to –60) and 422-bp (–482 to –60) 5' flanking region were produced using the forward primers: 5'-CTA GGTACC (Kpn I) ATC CTA GTA AGA GCA CCA TT-3' and 5'-CTA GGTACC (Kpn I) CTA TTG TCT GTA GGA CTG CA-3' and reverse primer: 5'-CAC GCTAGC (Nhe I) AGG GCC CGC TCC TTC TTC A-3'. Because these primers had a restriction site, PCR products were ligated to PGL3basic vector.

Mutation of E-Boxes in the mMetAP2 Promoter Region.
Two E-boxes in the 422-bp (–482 to –60) 5' flanking region of the mMetAP2 gene were mutated at bp –163 to –168 (CAAGTG to GAATTC), –200 to –205 (CAGTTG to GAATTC), or both using a QuickChange Site-directed Mutagenesis kit (Stratagene, La, Jolla, CA).

Cell Transfection and Luciferase Assay.
On the day before transfection, the cells were seeded (1 x 105/well) into six-well plates containing Dulbecco’s Modified Eagle’s Medium, supplemented with 10% fetal bovine serum. Cells were transfected with 100 ng of reporter construct and 1 µg (total) of expression construct using Lipofectamine Plus (Invitrogen), according to the manufacturer’s instructions. To correct for variations in transfection efficiency, 0.5 ng of pRL-SV40 (Promega) was cotransfected in all experiments. The total amount of DNA per well was adjusted by adding pcDNA3.1 vector (Invitrogen). At 48 hours post-transfection, the cells were disrupted with 200 µL of passive lysis buffer (Promega). Luciferase activity was determined using a Dual Luciferase Reporter Assay System (Promega). For each sample, the measured luciferase activity was corrected for transfection efficiency by dividing the firefly luciferase activity (expressed from the reporter construct) by the Renilla luciferase activity (expressed from pRL-SV40). The level of transcription activity in individual experiments was normalized to the corresponding control (set at 1-fold). Expression constructs were made as follows: the coding regions of mClock, mBmal1, mPer2, and mCry1 were obtained by RT-PCR and used after conformation of their sequences. All coding regions were ligated into the pcDNA3.1 vector.

Formaldehyde-Cross–Linked Chromatin Immunoprecipitation Assays.
Tumor masses removed from Sarcoma180-bearing mice were cross-linked with formaldehyde for 20 minutes. Cross-linked samples were sonicated on ice, and nuclear fractions were obtained by centrifugation at 10,000 x g for 10 minutes. Supernatants were diluted with 10 volumes of lysis buffer [50 mmol/L HEPES (pH 7.4), 140 mmol/L NaCl/1 mmol/L/EDTA/10% glycerol/0.5% NP40, and 0.25% Triton X-100] and incubated with antibodies against mCLOCK, mBMAL1, acetyl histone H3 (Upstate, Lake Placid, NY), and RNA polymerase II (Santa Cruz Biotechnology, Santa Cruz, CA). Chromatin/antibody complexes were extracted using a protein G agarose kit (Roche Diagnostics, Mannheim, Germany). DNA was isolated using the GeneElute Mammalian Genomic DNA kit (Sigma-Chemical Co., Steinheim, Germany) and subjected to PCR using the following primers for the E-box: forward: 5'-TTA GAT GTC CCT CAA CAG AA-3' and reverse: 5'-AGG GCT CAC TCC TTC TTC A-3' and for acetyl histone H3 or polymerase II: forward: 5'-TTA GAT GTC CCT CAA CAG AA-3' and reverse: 5'-GGA TCC AGG TCG CGA TTC-3'.

Quantitative Reverse Transcription-PCR Analysis of mMetAP2 mRNA.
Total RNA was extracted from tumor-bearing mice using TRIzol reagent (Invitrogen). The cDNA of mouse mMetAP2 (GenBank accession no. AF434712) and mouse glyceraldehyde-3-phosphate dehydrogenase (mGAPDH: M32599) were synthesized and amplified with a superscript one-step RT-PCR system (Invitrogen). PCRs were performed for 30 cycles with mMetAP2 and mGAPDH in a single tube. The mMetAP2 primers used were forward: 5'-CAG TAT GAT GAC ATC TG-3' and reverse: 5'-CTT GGA AGC CTT ATT GGC A-3', yielding a 467-bp product. The mGAPDH primers used were forward: 5'-GAC CTC AAC TAC ATG GTC TAC A-3' and reverse: 5'-ACT CCA CGA CAT ACT CAG CAC-3', yielding a 178-bp product. To evaluate the quantitative reliability of RT-PCR, we performed a kinetic analysis of the amplified products to ensure that signals were derived only from the exponential phase of the amplification. From each sample after the first 27 cycles of amplification, we drew a 5-µL aliquot for electrophoresis and submitted the tubes for one more cycle of PCR. This procedure was repeated for a total of 32 cycles. The PCR products were run on 3% agarose gel. The gel was photographed with Polaroid-type film after staining with ethidium bromide, and the intensity of ethidium bromide fluorescence in each band was assessed using NIH Image software. The ratio of the amplified target to the amplified competitor (calculated by dividing the value of mMetAP2 by that of the internal control mGAPDH) was compared. To calculate relative RNA values while equalizing differences in the peak of the rhythm in the controls, values were normalized so that the peak value equaled 1.

Western Blot Analysis of mMetAP2 Proteins.
Tumor masses and healthy tissues were homogenated with 500 µL of 10 mmol/L HEPES-KOH (pH 7.5), 50 mmol/L KCl, 3 mmol/L Mg(OAc)2, 0.3 mmol/L EDTA, 10% glycerol, 0.5% Triton-100, 100 µmol/L phenylmethylsulfonylfluoride, and 7 mmol/L 2-mercaptoethanol. After the removal of insoluble materials by centrifugation at 12,000 x g for 10 minutes, the resulting supernatants were used for the experiments. Lysates containing 60 µg of total protein were mixed with an equal volume of 2x sample buffer [0.125 mol/L Tris-HCl (pH 6.8), 10% 2-mercaptoethanol, 4% SDS, 10% sucrose, and 0.004% bromphenol blue] and boiled at 95°C for 5 minutes. Protein concentrations in the tumor masses and tissue lysates were determined by Lowry’s method (BCA Protein Assay kit, Pierce, Rockford, IL). The lysate samples were separated on 15% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were reacted with antibody against mMetAP2 (Zymed Laboratories, Inc., San Francisco, CA) and mGAPDH (Santa Cruz Biotechnology). The immunocomplexes were additionally reacted with peroxidase-conjugated secondary antibodies and made visible with 4-chloro naphthol as a peroxidase substrate. Immunoblot analysis with mMetAP2 or GAPDH polyclonal antibody revealed a single band of Mr 67,000 or 38,000.

Statistical Analysis.
ANOVA and Bonferroni’s test were used for multiple comparisons. A 5% level of probability was considered to be significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of the 5' Flanking Region of the mMetAP2 Gene.
This is the first demonstration of cloning of the 5' flanking region of MetAP2 in the mouse (Fig. 1)Citation . The sequence for the 5' flanking region of the gene was analyzed. Consequently, although an E-box with the sequence CACGTG and an especially high affinity for clock gene proteins was not observed within 1272 bp of the mMetAP2 promoter from the transcription start site, eight E-boxes with a CANNTG sequence were observed.



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  		Fig. 1. Nucleotide sequence of the 5' flanking region of the mMetAP2 gene. A 1212 (–1272 to –60)-bp fragment from the 5' flanking region containing the putative core promoter is shown. The known transcription start site is designated as +1. Underlines, E-boxes (CANNTG); white boxes, forward and reverse primers.

 
Activation of the mMetAP2 Promoter Region by Clock Gene Proteins.
We performed a transient transcription assay using the Bp 1212-LUC (–1272 to –60) 5' flanking region of the mMetAP2 gene luciferase reporter plasmids to elucidate whether mMetAP2 mRNA levels are controlled by clock gene proteins (Fig. 2)Citation . Cotransfection of mCLOCK+mBMAL1 with 1 µg of total plasmid increased mPer1 promoter activity, a positive control, by 4.7-fold (Fig. 2A)Citation . Cotransfection of the mCLOCK:mBMAL1 heterodimer increased mMetAP2 promoter activity by 3-fold (Fig. 2B)Citation . Coexpression of the mMetAP2 promoter with mBMAL1 or mCLOCK had little effect on promoter activity. Next, the suppressive effect of mPER2 or mCRY1 on the mMetAP2 transcription enhanced by the mCLOCK:mBMAL1 heterodimer was investigated. Both proteins suppressed mMetAP2 promoter activity in a dose-dependent manner. Cotransfection of mPER2 partially suppressed the induction of mMetAP2 by the mCLOCK:mBMAL1 heterodimer, whereas cotransfection of mCRY1 abolished the induction.



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  		Fig. 2. Transcriptional regulation of the mMetAP2 promoter region by clock gene proteins. A, left, the transcriptional regulation of mPer1-LUC reporter activity by clock genes in Sarcoma180 cells. The amounts of transfected mCLOCK and mBMAL1 plasmids (total 0.25–1 µg) are listed at the bottom of each bar. B, right, the Bp1212-LUC (–1272 to –60) 5' flanking region of the mMetAP2 gene activity by clock genes in Sarcoma180 cells. The presence (+) or absence (–) of plasmids (0.5 µg of mCLOCK or mBMAL1) is denoted. All values are shown as fold increases compared with the control. Each value is the mean with SE of three experiments.

 
Activation of the Deleted mMetAP2 Promoter Region by Clock Genes.
In addition to the mMetAP2 promoter vector, the Bp 1212-LUC (–1272 to –60) 5' flanking region of the mMetAP2 gene, we made shorter constructs, such as Bp 782-LUC (–842 to –60), which included five E-boxes, and Bp 422-LUC (–482 to –60), which included two E-boxes, in the 5' flanking region (Fig. 3)Citation . To investigate which E-box is important for the transcription of mMetAP2 mRNA, a luciferase assay was performed using each promoter vector. These reporter vectors retained 2.3- to 3.9-fold greater levels of activity than the control (pGL3 basic vector intact).



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  		Fig. 3. Transcriptional regulation of the deleted mMetAP2 promoter region by the mCLOCK:mBMAL1 heterodimer. A schematic representation of the mMetAP2 promoter reporters is shown on the left, with the names of the reporters listed in the middle and their corresponding relative activities shown on the right. The amounts of the transfected mCLOCK and mBMAL1 plasmids are 0.5 µg each. All values are shown as fold increases compared with each control. White boxes, the location of the E-box (CANNTG) in the promoter region. Each value is the mean with SE of three experiments.

 
Activation of the Mutated E-Box in the mMetAP2 Promoter Region by Clock Genes.
As shown in Fig. 3Citation , each of the two E-boxes contained in the Bp 422-LUC (–482 to –60) 5' flanking region of the mMetAP2 gene is important for the transcription of mMetAP2 mRNA. We constructed a mutation in the two E-boxes and identified which E-box is important. Consequently, transcription activity decreased with mutation in the E-box nearest to the transcription start site (Fig. 4)Citation .



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  		Fig. 4. Transcriptional regulation of the mutated E-box in the mMetAP2 promoter region by mCLOCK:mBMAL1. A schematic representation of the mutated mMetAP2 promoter reporters is shown on the left, with the names of the reporters listed in the middle and their corresponding relative activities shown on the right. The amounts of the transfected mCLOCK and mBMAL1 plasmids are 0.5 µg each. All values are shown as fold increases compared with each control. White boxes, the location of the E-box (CANNTG) in the promoter region; white X boxes, the mutated E-box. Each value is the mean with SE of three experiments.

 
Contrasting Rhythms in Transcription Factor Binding and Chromatin Remodeling for the mMetAP2 Gene.
We examined the binding of mCLOCK and mBMAL1 to the mMetAP2 E-box in tumor mass nuclear extracts from Sarcoma180-bearing mice using chromatin immunoprecipitation with antibodies against clock gene proteins. Fig. 5ACitation shows a schematic representation of the mCLOCK:mBMAL1 heterodimer-binding, acetyl histone (AcH3), and RNA polymerase II-binding regions. A significant 24-hour rhythm was demonstrated for the binding of mCLOCK and mBMAL1 with higher levels of binding from the mid-light to early dark phase and low levels from the mid-dark to early light phase (Fig. 5BCitation ; P < 0.01, respectively). Then, we examined the amount of AcH3 in the mMetAP2 promoter region to assay for rhythmic changes in chromatin structure using the same method. There was a significant 24-hour rhythm with higher levels from the mid-light to early dark phase and lower levels from the mid-dark to early light phase (Fig. 5CCitation ; P < 0.01). We also examined the binding of polymerase II to the mMetAP2 promoter to assay for dynamic changes in coactivator assembly necessary for transcriptional activity. There was also a significant 24-hour rhythm in the binding of polymerase II with higher levels of binding from the mid-light to early dark phase and lower levels from the mid-dark to early light phase (Fig. 5CCitation ; P < 0.01).



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  		Fig. 5. Contrasting rhythms in transcription factor binding and chromatin remodeling for the mMetAP2 gene. A, schematic representation of the 5' flanking region of the mMetAP2 gene. White boxes, the promoter sequence of the E-box (CANNTG). Dotted line arrows, PCR amplification region for E-box or acetyl histone H3 (AcH3), polymeraseII (PolII). B, 24-hour rhythms in mCLOCK and mBMAL1 binding to the promoter E-box. Representative PCR of the binding patterns are shown, along with the input DNA control. The graph shows relative PCR values (P < 0.01, respectively; ANOVA). For ChIP PCR abundance, the mean value at the peak time (Anti-CLOCK and BMAL1; ZT10) was set to 1. Each value is the mean with SE of six experiments. C, 24-hour rhythms of AcH3 and RNA PolII binding to the mMetAP2 promoter region. Representative PCR of the binding patterns is shown. The graph shows relative PCR values (P < 0.01, respectively; ANOVA). For ChIP PCR abundance, the mean value at the peak time (Anti-AcH3; ZT10, Anti-PolII; ZT6) was set to 1. Each value is the mean with SE of six experiments. Horizontal bar at top of B and C, light and dark cycles.

 
24-Hour Rhythm of mMetAP2 mRNA and mMetAP2 Protein Expression in Tumor-Bearing Mice.
There was a significant 24-hour rhythm of mMetAP2 mRNA expression with higher levels from the mid-light to early dark phase and lower levels from the late-dark to early light phase in all of the tumor (Sarcama180, Lewis lung carcinoma, and B16 melanoma)-bearing mice. (Fig. 6ACitation ; P < 0.01, respectively). This result indicates that the 24-hour rhythm of mMetAP2 mRNA expression corresponded to that of mMetAP2 transcription in Sarcoma180 masses (Fig. 5CCitation and Fig. 6ACitation ). We also determined the 24-hour rhythm of mMetAP2 protein production in implanted tumor masses. In the three different types of tumor masses, the abundance of mMetAP2 protein varied with higher levels from the late-dark to early light phase and lower levels from the late-light to early dark phase (Fig. 6B)Citation . This result indicates that the peak of mMetAP2 protein occurs ~12 hours after the peak of mMetAP2 mRNA. On the other hand, the 24-hour rhythm of mMetAP2 protein expression corresponded to that of mMetAP activity.



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  		Fig. 6. Twenty-four-hour rhythm of mMetAP2 mRNA and protein in three different types of tumor (Sarcoma180, Lewis lung carcinoma, and B16 melanoma)-bearing mice. A, 24-hour rhythm in relative mRNA levels of mMetAP2. Tumor masses were removed from six tumor-bearing mice at ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22 on day 14 after tumor implantation. Relative mRNA levels are based on a mean value at the peak time (Sarcoma180 and Lewis lung carcinoma; ZT10, B16 melanoma; ZT6) of 1. Each value is the mean with SE of six mice. Horizontal bar at bottom, light and dark cycles. GAPDH mRNA was used as an internal control for transcripts, the expression of which was constant throughout the day. B, representative Western blots for the 24-hour rhythm of mMetAP2 protein expression in tumor-bearing mice. The extracts were measured by Western blot analysis with mMetAP2 polyclonal antibody. Horizontal bar at top, light and dark cycles. GAPDH protein was used as an internal control for transcripts, the expression of which was constant throughout the day. This examination was performed three times.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the purpose of the present study was to investigate the mechanism underlying the 24-hour rhythm of mMetAP2 activity, the arrangement of the 5' flanking region of the mMetAP2 gene has not been clarified as yet in the mouse. Therefore, cloning of the 5' flanking region of mMetAP2 was performed in the mouse. Comparison of the arrangement of the 5' flanking region of the gene with the known rat sequence revealed ~99% homology between the mouse and rat (23) . The arrangement and position of eight E-boxes in the 5' flanking region of mMetAP2 were also conserved between the mouse and rat. Recently, we demonstrated the mechanism underlying the 24-hour rhythm of vascular endothelial growth factor expression in hypoxic tumor cells (24) . The negative limbs of the feedback loop, PER and CRY protein, inhibit the hypoxia-induced activation of vascular endothelial growth factor transcription. On the other hand, mMetAP2 mRNA expression may not be enhanced by hypoxia, because there was no hypoxia-responsive element in the 5' flanking region of the mMetAP2 gene.

In the present study, a significant 24-hour rhythm was demonstrated for mMetAP2 mRNA expression in implanted tumor masses. The clock genes control downstream events by regulating the expression of clock-controlled output genes. The CLOCK:BMAL1 heterodimer acts through an E-box enhancer element of the output genes to activate transcription, and its activation is inhibited by PER or CRY protein (14 , 15) . The expression of clock genes in implanted cells is subordinated to the dominance exerted by the central clock of the host animal (25) . The rhythmic patterns of clock gene expression in tumor masses are similar to those reported in healthy tissues, such as liver and skeletal muscle (15 , 26, 27, 28) . In addition, the 24-hour rhythm of mCRY1 protein expression was antiphase to that of mMetAP2 mRNA expression in the present study (9 , 24) . Therefore, the transcription of mMetAP2 mRNA may be regulated by clock gene proteins in tumor masses. Monitoring clock gene protein levels is not enough to estimate whether mMetAP2 mRNA is increased by the mCLOCK:mBMAL1 heterodimer in vivo, e.g., mPer and mCry mRNA expression are enhanced by clock gene proteins of the mCLOCK:mBMAL1 heterodimer (15 , 16) . mPer2 mRNA shows a peak at ZT14, and the protein shows a peak at ZT18 (24 , 26) . On the other hand, mCry1 mRNA shows a peak at ZT22 as does the protein. Namely, the transcriptional pattern of mPer2 and mCry1 mRNA enhanced by the mCLOCK:mBMAL1 heterodimer differed between the two genes. Thus, whether mMetAP2 mRNA is increased by the mCLOCK:mBMAL1 heterodimer is not clear. Therefore, we performed the luciferase reporter assay in vitro.

We performed a transient transcription assay using luciferase reporter plasmids of the mMetAP2 promoter. The mCLCOK:mBMAL1 heterodimer increased mMetAP2 promoter activity, and mPer2 or mCRY1 suppressed the transcription of mMetAP2 mediated by the mCLOCK:mBMAL1 heterodimer. mCRY protein was more suppressive than mPER2 protein. The results suggest that mCRY1 acts as a major suppressive regulator of the 24-hour rhythm of mMetAP2 mRNA expression in tumor cells. Furthermore, we investigated which E-box participates in the transcription of mMetAP2 mRNA among the eight E-boxes in the mMetAP2 promoter region. Deletion of the first six E-boxes increased the mMetAP2 promoter activity from 3- to 4-fold relative to the intact PGL3 basic, and most, if not all, of this activity was from the proximal E-box. The results suggest that the E-box nearest to the initiation start site plays an important role in the transcriptional regulation by clock genes. In the case of the mPer1 promoter region, the E-box closest to the initiation start site also shows greater transcriptional activity than any other E-boxes (14) . The transcription of mMetAP2 mRNA using a luciferase reporter assay involves interacting positive and negative transcriptional translation feedback loops, but the experimental result for cultured cells may not necessarily reflect the situation in the body.

The chromatin immunoprecipitation assay can detect a time-dependent change in the amount of clock gene protein combining with the promoter domain, which includes the E-box, although a conventional gel shift assay cannot (29) . Therefore, a chromatin immunoprecipitation assay was performed to investigate whether mCLOCK and mBMAL1 directly combine with the E-box in tumor-bearing mice and the binding of the mCLOCK:mBMAL1 heterodimer mediating the rhythmic drive of mMetAP2 transcription is regulated by time-dependent changes in chromatin structure. Consequently, mCLOCK and mBMAL directly combined with the E-box in the mMetAP2 promoter region. The 24-hour rhythm of the binding of mCLOCK:mBMAL1 showed the same waveform as that of mMetAP2 mRNA expression. Although mCLOCK shows a rhythm of small amplitude, mBMAL1 shows a rhythm of large amplitude (21 , 27 , 30) . Therefore, the rhythm in the binding of mCLOCK:mBMAL1 to the mMetAP2 promoter may be caused by the large change in the mBMAL1 protein level in addition to the small change in the mCLOCK protein level. Similar findings are demonstrated for the transcription of Per1 or Cry1 by mCLOCK:mBMAL1 (26 , 29) . The mCLOCK:mBMAL1 heterodimer exerts its transcriptional activity by forming a transcriptional coactivator complex with p300 histone acetyltansferase (29) . mCRY1 protein disrupts the transcriptional coactivator complex, p300, thereby reducing histone acetyltransferase activity and altering chromatin structure to decrease mCLOCK:mBMAL1 transcriptional activation. In the case of mMetAP2, the rhythmic changes in the amount of AcH3 and polymerase II binding were similar to those in the mMetAP2 mRNA expression. These results suggest that transcription of mMetAP2 mRNA is controlled by clock gene proteins. In addition, the transcriptional regulation is accompanied by expression of AcH3 and the binding of polymerase II.

We reported previously that mMetAP activity in Sarcoma180-bearing mice showed a significant 24-hour rhythm (9) . A higher level of activity was observed from the late-dark to early light phase and a lower level from the late-light to early dark phase in implanted Sarcoma180 masses. Because a specific assay method for only mMetAP2 activity has not been established, it was impossible to separately measure mMetAP2 and the subtype mMetAP1. Consequently, the activity of mMetAPs in the previous study included both mMetAP2 and mMetAP1 activity. Therefore, we measured the protein levels of mMetAP2 in three different types of tumor masses using a specific antibody for mMetAP2. The protein levels of mMetAP2 also showed a 24-hour rhythm associated with the rhythmicity of mMetAP activity in Sarcoma180-bearing mice. On the other hand, the peak of mMetAP2 protein was 12 hours later than the peak of mMetAP2 mRNA in Sarcoma180. Similar findings were confirmed in different cell lines, such as Lewis lung carcinoma and B16 melanoma. Furthermore, CRY2 protein behaves similarly to mMetAP2 protein (26) . mCRY2 mRNA shows a peak at ZT2, and its protein shows a peak at ZT14. The mechanism involved is unclear at present. It is suggested that the synthesis of mMetAP2 protein from its mRNA requires more time than that of other proteins.

Although MetAP2 was initially implicated in the proliferation of endothelial cells during the tumor-angiogenesis stage, it has since been identified in tumor cells and normal cells and may be implicated in the proliferation of both (4 , 12 , 31) . There was a significant 24-hour rhythm of mMetAP2 mRNA expression in all of the tumor (Sarcoma180, Lewis lung carcinoma, and B16melanoma)-bearing mice. In addition, a significant 24-hour rhythm has been demonstrated for mMetAP2 mRNA levels in healthy liver (32) . The rhythmic patterns of clock gene expression in tumor masses are similar to those reported in healthy tissues (15 , 26, 27, 28) . These results suggest that the 24-hour rhythm of mMetAP2 in healthy tissue may also be controlled by the clock feedback loops.

The present study suggests that the 24-hour rhythm of mMetAP2 is controlled by the clock feedback loops. The transcription of the mMetAP2 promoter is enhanced by the clock gene proteins of the mCLOCK:mBMAL1 heterodimer, and mPER2 or mCRY1 inhibits mMetAP2 promoter activation by mCLOCK:mBMAL1 in vitro. In addition, the transcription of mMetAP2 mRNA is generated by the mCLOCK:mBMAL1 heterodimer in vivo. TNP-470, inhibiting MetAP2 activity, has significant clinical advantages as a therapeutic agent for treating tumors, and research into the design of tumor therapies using TNP-470 was reported recently (33) . The present study also supports the notion that choosing the most appropriate time of day for drug administration will aid in establishing rational chronotherapeutics with TNP-470 for the treatment of tumors.


   FOOTNOTES
 
Grant support: Grant-in-Aid for Scientific Research on Priority Areas "Cancer" (14030062, 15025254, and 16023245; S. Ohdo); Grant-in-Aid for Scientific Research (B) (S. Ohdo; 16390042) from the Ministry of Education, Culture, Sports, Science and Technology, Japan; Grant-in-Aid from the Pharmacological Research Foundation, Tokyo (S. Ohdo); Grant-in-Aid from Japan Research Foundation for Clinical Pharmacology (S. Ohdo); Grant-in-Aid for Encouragement of Young Scientists from the Japan Society for the Promotion of Science (S. Koyanagi; 16790194); Grant-in-Aid from Japan Research Foundation for Clinical Pharmacology (S. Koyanagi); and Grant-in-Aid from Takeda Science Foundation (S. Koyanagi).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Shigehiro Ohdo, Clinical Pharmacokinetics, Division of Clinical Pharmacy, Department of Medico-Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Phone: 81-92-642-6658; Fax: 81-92-642-6660; E-mail: ohdo{at}phar.kyushu-u.ac.jp

Received 6/16/04. Revised 8/29/04. Accepted 9/16/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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