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J. Biol. Chem., Vol. 279, Issue 12, 11639-11648, March 19, 2004
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From the Institute of Immunology, Second Military Medical University, Shanghai 200433, People's Republic of China
Received for publication, July 9, 2003 , and in revised form, November 25, 2003.
| ABSTRACT |
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| INTRODUCTION |
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Transcription regulator cyclin/CDK pairs are associated with the initiation, elongation, and processing of RNA transcripts. During this process cyclins interact with the large subunit of RNA polymerase II, phosphorylating the C-terminal domain (CTD) via their partner CDKs. It is widely accepted that transcription and RNA processing are closely related. Capping enzymes, polyadenylation factors, and splicing factors assemble at the CTD of RNA polymerase II. Hyperphosphorylated RNA polymerase II (RNAPIIo) stimulates RNA splicing, and the CTD has been observed to target some splicing factors to RNA transcription sites in vivo (8). Although there is no direct evidence to support a critical role for transcription regulator cyclins in regulation of cell cycle progression or cellular apoptosis, some results have implied that one transcription regulator cyclin, cyclin C, may be important for the regulation of cell proliferation in cooperation with c-Myc. Cyclin C has been reported to regulate both cell cycling and cell adhesion, suggesting that this cyclin may play some part in regulating both the G1/S and G2/M phases of the cell cycle (9). It thus appears that some transcription regulator cyclins may also be involved in cell cycle progression or apoptosis, RNA transcription, and DNA repair, although the precise role of the phenomena remains to be elucidated.
In addition to small nuclear ribonucleoprotein particles and spliceosome-associated proteins, SR proteins compose another group of splicing factors. SR proteins have a characteristic C-terminal Arg/Ser-rich repeat of variable length (RS domain) and RNA-binding domains in the N terminus and constitute a family of highly conserved nuclear phosphoproteins that can function as both essential and alternative pre-mRNA splicing factors (10, 11). SR-related proteins, proteins that are structurally and functionally distinct from the SR family, have also been identified. These proteins generally contain RS domains but not RNA-binding domains, are associated with the transcriptional machinery or mRNA 3'-end formation, and have been implicated in export. Cyclin L1 (originally named cyclin L) is the first known example of a cyclin containing an RS domain in addition to a typical "cyclin box" (12).
We report here a novel candidate cyclin, human cyclin L2 (CCNL2), first identified by large scale random sequencing of a cDNA library of human bone marrow stromal cells (BMSCs). Human cyclin L2 is also a cyclin box and RS repeat region-containing protein and localizes within the nuclear speckles, co-localizing with splicing factors SC-35 and 9G8. We demonstrate that human cyclin L2 associates with RNAPIIo, CDK p110 PITSLRE kinase (CDK11), SC-35, and 9G8. In addition, recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Furthermore, overexpression of exogenous cyclin L2 can suppress the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis, possibly by up-regulating expression of p53 and Bax and down-regulating Bcl-2. These results suggest that cyclin L2 may be a functional cyclin that associates with transcriptional regulation and participates in regulating the pre-mRNA splicing process. The mechanism by which cyclin L2 suppresses the growth and induces apoptosis of tumor cells requires further investigation.
| EXPERIMENTAL PROCEDURES |
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Molecular Cloning of Full-length Human Cyclin L2 and Its Mouse HomologueThe main EST of human cyclin L2 was directly isolated from a human BMSC cDNA library by random sequencing as described previously (14). Briefly, BMSCs were generated from the bone marrow aspirates of an adult patient. Red blood cells were depleted, and the monocytes were plated in 6-well plates (1 x 106 cells/well) in RPMI 1640 supplemented with 10% fetal calf serum, 10% horse serum, 50 µM 2-mercaptoethanol, 10 mM HEPES, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. After 24 h, floating cells were removed, and an adherent stromal cell layer was established. On the 14th day, stromal cells were activated with 10 µg/ml lipopolysaccharide (Sigma) and 100 ng/ml phorbol 12-myristate 13-acetate (Sigma) for 8 h and then collected for cDNA library construction. A plasmid cDNA library of pCMV·SPORT6.0 vector (Invitrogen) was constructed using the Superscript plasmid system (Invitrogen) for cDNA synthesis. Plasmid cloning was performed according to the manufacturer's instructions. Random sequencing was performed to identify a clone, HNA6E9, which contained an insert sharing homology to the known transcription regulator cyclin proteins, in particular cyclin L1 (CCNL1). This clone was hence designated as human cyclin L2 (CCNL2) as defined by HUGO, in accordance with the nomenclature of cyclin family members.
The full-length sequence of human cyclin L2 was isolated from Raji human leukemia cells, which express high levels of human cyclin L2, with the PCR primers 5'-ATGAATTCGAAGGGGAGTCGGCGGCACAAAATGG-3' (sense) and 5'-CAGGATCCCACCTCCGATGCCTGCTGTGCCCAGGGTGGTC-3' (antisense) and Advantage polymerase (Clontech). The PCR product was cloned in-frame into the myc-6His-tagged expression vector pcDNA3.1/Myc-His(-)B (Invitrogen) and then sequenced. The positive clone was denoted as pCCNL2-wt. The full-length sequence of human cyclin L2 is available in the GenBankTM data base under accession number AY037150 [GenBank] .
Specific primers 5'-ATGAATTCGAGTCGGCGGCACAAAATG-3' (sense) and 5'-TGCCCACTGTCTCATCAATCAAAT-3' (antisense) were used to amplify the mouse homologue of human cyclin L2, designated as mouse cyclin L2, from the murine melanoma line B16 by RT-PCR. DNA sequencing showed that it encoded a putative protein sharing high homology with human cyclin L2. The full-length sequence of mouse cyclin L2 is available in the GenBankTM data base under accession number AY337018 [GenBank] .
RT-PCR and Northern Blot AnalysisTotal cellular RNA was isolated using Trizol reagent (Invitrogen), and first strand cDNA was prepared using the Superscript II system with an oligo(dT)15 primer (Invitrogen). cDNA synthesis was checked by PCR with human
-actin primers as a positive control. Human adult multiple tissue cDNA panels were purchased from Clontech. RT-PCR was performed with primers specific for human cyclin L2 5'-CAGGCTGTACTCCGGGGTGCTCATCA-3' (sense) and 5'-CACGTGTCACCGGTCCCTCAGTTCCATTTC-3' (antisense) and human cyclin L1 5'-GGACGACGACCACGACGAC-3' (sense) and 5'-CGGAATCTGAAGTGCTCTAGC-3' (antisense). The reaction was subjected to denaturing (95 °C for 30 s), annealing (55 °C for 30 s), and extension (72 °C for 30 s) for 30 cycles, and PCR products were confirmed by DNA sequencing.
Human adult multiple tissue Northern blots (Clontech) were used to detect human cyclin L2 mRNA expression. A 324-bp XhoI fragment spanning positions 11811504 of human cyclin L2 cDNA was prepared as a probe and labeled by random priming with [
-32P]dCTP (Amersham Biosciences). Molecular hybridization was performed in ExpressHyb hybridization solution (Clontech) according to the manufacturer's instructions. After stringent washing the filters were exposed for autoradiography at -80 °C.
RT-PCR analysis of apoptosis-related factors p53, Bax, and Bcl-2 was performed as described above. All PCR products were resolved on 1.8% agarose gels. The sequence of all primers is available on request.
Expression of GST Fusion Protein and Generation of Polyclonal AntibodyThe coding sequence of cyclin L2 cDNA was cloned in-frame into pGEX-4T3 (Amersham Biosciences) for GST fusion protein expression. GST-CCNL2 fusion protein expressed in Escherichia coli BL21 (DE3) (Stratagene) was affinity-purified with glutathione-Sepharose 4B (Amersham Biosciences) and eluted in 10 mM reduced glutathione. Antibody to recombinant human cyclin L2 (anti-CCNL2) was raised in rabbits against purified GST-CCNL2 fusion protein.
Mammalian Expression Vector Construction and Cell Transfection The construction of pCCNL2-wt containing the full-length coding region of human cyclin L2 tagged by myc-6His was described above. Domain-negative mutants of human cyclin L2, including pCCNL2-
CL (deletion of both cyclin domains) and pCCNL2-
RS (deletion of RS region), were generated by PCR mutations. Full-length cyclin L2 was cloned into pcDNA3.1/Myc-His(-)B vector in a negative direction to give the antisense construct pCCNL2-AS. The myc-6His tag was replaced by a FLAG sequence in the pCCNL2-wt vector to generate pCCNL2-FLAG.
pCCNL2-wt, pCCNL2-
CL, pCCNL2-
RS, pCCNL2-AS, or pcDNA3.1/Myc-His(-)B mock vectors were transfected using liposome-mediated LipofectAMINE reagent (Invitrogen) into human hepatocellular carcinoma SMMC 7721 cells according to the manufacturer's instructions and screened under 500 µg/ml G418 (Calbiochem) for
35 weeks. Cell clones of stably transected cells were obtained by the limited dilution method.
Immunoprecipitation and Western BlotCells were lysed in cell lysis buffer (Cell Signaling). Anti-Myc immunoprecipitations were performed using mouse anti-Myc monoclonal antibody (Invitrogen) cross-linked to protein G-Sepharose beads (Santa Cruz Biotechnology) using dimethyl pimelimidate. Immunoprecipitation products or cell extracts were then separated by 12% SDS-PAGE, transferred onto nitrocellulose membranes, and subjected to Western blot analysis. Relevant proteins were visualized using anti-PITSLRE kinase (Santa Cruz Biotechnology), anti-RNAPIIo (Covance), anti-hypophosphorylated RNA polymerase II (RNAPIIa) (Biodesign), anti-splicing factors SC-35 (Santa Cruz Biotechnology) and 9G8 (Santa Cruz Biotechnology), anti-53 (Santa Cruz Biotechnology), anti-Bcl-2 (Cell Signaling), or anti-Bax (Cell Signaling) as primary antibodies. Blots were then probed with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology), and reactive protein bands were visualized with LumiGlo reagents (Cell Signaling).
Fluorescence Confocal Microscopy AnalysispCCNL2-FLAG-transfected SMMC 7721 cells growing on glass coverslips placed in 6-well plates were fixed, incubated with mouse biotin-labeled anti-FLAG antibody (Sigma), and then stained with Avidin-Texas Red (BD Pharmingen). Endogenous splicing factors SC-35 and 9G8 were detected with above-mentioned primary antibodies and fluorescent isothiocyanate-coupled anti-goat IgG antibody as secondary antibody. Stained cells on glass coverslips were washed briefly in phosphate-buffered saline and then observed by fluorescence confocal microscopy (Carl Zeiss).
In Vitro Splicing AssaysPrecursor RNA was transcribed from the plasmid pCI-pA102 (kindly gifted by Prof. Stein Saeboe-Larssen, University of Oslo, Norway) containing a
-globin precursor coding sequence (15), in a transcription reaction with T7 Cap-Scribe (Roche Applied Science) and [
-32P]UTP (Amersham Biosciences) as described by the manufacturer. The labeled RNA was run on a 6% denaturing polyacrylamide gel. The gel was briefly exposed to film, and the band corresponding to the full-length RNA was excised from the gel. RNA was eluted from the gel slice in elution buffer (0.5 M NH4 acetate, 2 mM EDTA, 0.2% SDS) overnight at 37 °C. The eluted sample was precipitated with ethanol and stored as dry pellets at -80 °C. Splicing reactions were performed as described (16) and contained 80 mM potassium acetate, 4 mM magnesium acetate, 20 mM creatine phosphate, 1 mM freshly prepared ATP, 1 unit/ml RNasin (Takara), 2 ng of labeled
-globin RNA precursor, 50 µg of HeLa nuclear extract (Promega), or 25 µg of HeLa nuclear extract plus 250 ng of GST-CCNL2 fusion protein in a total volume of 20 µl. Reactions were incubated at 30 °C for 3 h, extracted with phenol/chloroform, and precipitated with ethanol. The reaction products were run on 6% denaturing polyacrylamide gels and were visualized by autoradiography.
In Vitro Analysis for Cell GrowthCell proliferation of transiently or stably transfected SMMC 7721 cells was measured by MTT dye reduction assay (17). Briefly, cells were seeded into 96-well plates, and on the day of harvest, 100 µl of spent medium was replaced with an equal volume of fresh medium containing 10% MTT 5 mg/ml stock. Plates were incubated at 37 °C for 4 h; then 100 µl of Me2SO (Sigma) was added to each well, and plates were shaken at room temperature for 10 min. Cellular viability was determined by measuring the absorbance of the converted dye at a wavelength of 570 nm.
Colony Forming AssayStably transfected SMMC 7721 or parental SMMC 7721 cells (5 x 104/ml) were mixed with 1 ml of MethoCult methylcellulose-based medium (StemCell, British Columbia, Canada) and plated in 6-well plates according to the manufacturer's instructions. After 710 days of incubation at 37 °C in a humidified atmosphere containing 5% CO2 in air, colonies (>50 cells) were counted using an inverted microscope.
Apoptosis AssayTransiently transfected SMMC 7721 cells were washed, resuspended in staining buffer, and examined by ApoAlert Annexin V Apoptosis kit (BD Biosciences) and PI according to manufacturer's instruction. Stained cells were analyzed by FACS (FACS-calibur, BD Biosciences).
In Vivo Gene Transfer and Evaluation of Tumor Growthnu/nu mice were subcutaneously inoculated with 3 x 106 SMMC 7721 cells. When the tumors had grown to
2.5 mm in diameter, mice were randomly divided into three groups of five tumor-bearing mice. In vivo electroporation was performed to deliver naked DNA into pre-established tumors (18, 19). Briefly, 20 µg of plasmid DNA in 30 µl of saline was injected into the tumors of tumor-bearing mice. One minute after injection, the site where the plasmid was injected was sandwiched in an electrode (BTX 533 2-needle array electrode) with poles 5 mm in diameter. Three electrical pulses of 20-ms duration at a voltage of 600 V were delivered using an ECM 830 Square Wave Electroporation System (BTX). Following this procedure, tumor growth of tumor-bearing mice was monitored every other day. The tumor volume was calculated as follows: V = 0.4 (a x b2) (V = volume, a = maximum tumor diameter, b = diameter at 90° to a) (19).
On the 32nd day following treatment, mice were killed. The tissues in the inoculated site of the tumor-bearing mice were dissected, fixed in 4% paraformaldehyde, and then embedded in paraffin. Deparaffinized 5-µm-thick specimens were subjected to hematoxylin and eosin staining and immunohistochemical ABC staining, respectively.
| RESULTS |
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-folds. The predicted three-dimensional structure of human cyclin L2 generated by Swiss model did indeed indicate that there was an obvious
-helix in the N terminus (data not shown) of human cyclin L2, which was in agreement with the common structural feature of the cyclin family. In addition to the highly conserved cyclin box region in the N terminus, there was also a conserved "serine/arginine-rich (RS)" repeat region in the C terminus of the human cyclin L2 protein (Fig. 1A), similar to that of cyclin L1 and some splicing factor proteins known as SR protein. Although two transmembrane regions (amino acids 1024 and 224228) were predicted, no obvious signal peptide was found, suggesting that human cyclin L2 may be a non-secretory intracellular protein. A significant number of nuclear location signals indicated that human cyclin L2 might be a nuclear protein (Fig. 1A). The strong hydrophilicity in the C terminus of the human cyclin L2 was also consistent with features of cyclin L1 and cyclin K, both of which are important members of the transcriptional regulation cyclin family. Identification of an Isoform and Mouse Homologue of Human Cyclin L2During the cloning and sequencing of human cyclin L2 cDNA, a novel isoform was found in Raji cells. This new isoform of human cyclin L2 was designated as human cyclin L2s (CCNL2s) and submitted to NCBI GenBankTM (accession number AY116620 [GenBank] ). The sequence of human cyclin L2s was identical to the 5' region of human cyclin L2. The coding region cDNA of cyclin L2s was 684 bp in length, encoding a theoretical protein of 227 amino acids. There were two conservative cyclin domains in putative cyclin L2s protein, lacking the RS repeat region.
A mouse homologue of human cyclin L2 was identified from mouse melanoma B16 cells and designated as mouse cyclin L2. The mouse cyclin L2 cDNA was 1617 bp in length, encoding a putative protein of 518 amino acids with a calculated molecular mass of 58.0 kDa and an isoelectric point of 10.21. Mouse cyclin L2 protein showed 90% identity to and 93% similarity with human cyclin L2. There were also two cyclin domains and one SUA7 domain in mouse cyclin L2 protein, highly consistent with human cyclin L2. A BLAST search of the NCBI genome data base indicated that the mouse cyclin L2 gene is located in the low terminus of mouse chromosome 4. A previous report (20) identified a 224-amino acid protein, mouse ANIA-6B, that was the same as N terminus of mouse cyclin L2. Based on the protein and genome structural analysis of human and mouse cyclin L2, mouse ANIA-6B might be the alternatively spliced isoform of mouse cyclin L2. The sequence of mouse cyclin L2 is available in the GenBankTM data base under accession number AY337018 [GenBank] .
Expression Pattern of Human Cyclin L2 mRNAThe expression of human cyclin L2 mRNA was detected by RT-PCR and Northern blot analysis. RT-PCR detected expression of human cyclin L2 in all human hematopoietic cell lines and solid tumor cell lines examined (Fig. 2, A and B), including U-937 (histiocytic lymphoma), NB4 and HL-60 (promyelocytic leukemia), KG-1 (acute myelogenous leukemia), K-562 (chronic myelogenous leukemia), MOLT-4, Jurkat, and Hut 78 (T lymphoblastic leukemia), Daudi (B lymphoma) and NAMALWA (Burkitt's lymphoma), Reh (acute lymphocytic leukemia), HeLa (cervix epithelioid carcinoma), U251 (glioma), CaoV-3 (ovary adenocarcinoma), PC-3 (prostate adenocarcinoma), HT-29 (colon adenocarcinoma), SMMC 7721 (hepatocellular carcinoma), and MCF-7 (breast adenocarcinoma). Relatively higher levels of human cyclin L2 mRNA expression were observed in K-562, Reh, CaoV-3, U251, and HeLa cells.
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We also examined the expression of human cyclin L1 in the above cell and tissue templates. Interestingly, the results revealed differential expression of human cyclin L2 and L1. Unlike cyclin L2, cyclin L1 expression was more restricted in cultured cell lines. Cyclin L1 was not detected in K-562, U-937, and NAMALWA cells and in other solid tumor cells except for CaoV-3. Its expression was much lower than that of cyclin L2. On human multiple tissue cDNA panels, the expression patterns of both cyclins were also dissimilar (Fig. 2C). The differential distribution of closely related cyclins belonging to the same class may reflect a functional diversity, resulting from the existence of a finely tuned mechanism of the cell cycle and transcriptional regulation by different members of the multiple cyclin family.
Nuclear Cytolocalization of Human Cyclin L2Because human cyclin L2 contained nuclear localization signals and was predicted to be a nuclear protein, we examined the subcellular localization of FLAG-tagged cyclin L2 to determine whether it localized within the nucleus. pCCNL2-FLAG was co-transfected into SMMC 7721 cells with pEGFP-Nuc (encoding a nuclear-localized form of GFP with nuclear location signals). The expression of CCNL2-FLAG protein was assayed by Western blot analysis with anti-FLAG antibody, and an
59-kDa protein was detected in the crude extract of pCCNL2-FLAG-transfected SMMC 7721 cells (data not shown), consistent with the calculated molecular weight of cyclin L2. In transfected SMMC 7721 cells, we observed both generalized nucleoplasmic and punctated nuclear staining (a "speckled" pattern of expression) of CCNL2-FLAG protein (Fig. 3B), which preferentially co-localized with the nuclear GFP signal (Fig. 3C). The result confirmed the structure prediction that cyclin L2 is a typical nuclear protein.
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Human Cyclin L2 Associates with RNA Polymerase II, Splicing Factors SC-35, 9G8, and CDK p110 PITSLREThe cell nucleus (particularly the nuclear speckles) is believed to contain compartments enriched for nuclear proteins involved in transcriptional regulation and mRNA processing. Human cyclin C, H, K, and the closest homologue of cyclin L2, cyclin L1, have been proposed to play roles in the regulation of basal transcription through their association with and activation of CDKs that phosphorylate the CTD of the large subunit of RNA polymerase II. To explore the possibility that human cyclin L2 may associate with RNA polymerase II or other CDKs and splicing factors and to delineate the domain(s) of cyclin L2 required for these interactions, several domain-negative constructs tagged by myc-6His were overexpressed in SMMC 7721 cells and immunoprecipitated with anti-Myc antibody cross-linked with protein G-Sepharose beads. As shown in Fig. 4A, anti-Myc antibody could detect expression of Myc-tagged CCNL2-wt (59 kDa), CCNL2-
CL (39 kDa, both cyclin domains deleted) and CCNL2-
RS (51 kDa, RS repeat region deleted). We then examined the association of cyclin L2 with RNA polymerases II and other CDKs and splicing factors.
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Cyclins, together with their CDK partners, regulate the orderly progression of the cell cycle. We investigated whether human cyclin L2 could possibly interact with known CDKs. Because the CDK p110 PITSLRE (also called CDK11) has been identified as the CDK partner for human cyclin L1 (12) and mouse cyclin ANIA-6A (20), the mouse homologue of human cyclin L1, the association of human cyclin L2 and PITSLRE was assayed by immunoprecipitation. As shown in Fig. 4B, human cyclin L2 could also associate with p110 PITSLRE, suggesting that p110 PITSLRE might be the CDK partner of human cyclin L2. It remains to be determined whether cyclin L2 can partner with other CDKs.
The results also showed that association with RNAPIIo and p110 PITSLRE required the presence of the N-terminal cyclin domains of human cyclin L2, because in cells overexpressing CCNL2-
CL, a truncated protein lacking both cyclin domains, no association of cyclin L2 with RNAPIIo or p110 PITSLRE was detected. In CCNL2-
RS-expressing cells, the levels of RNAPIIo and p110 PITSLRE precipitated were approximately the same as those with CCNL2-wt, suggesting that RS repeats might not be necessary for RNAPIIo and p110 PITSLRE interaction. Notably, the interactions with SC-35 and 9G8 in CCNL2-
CL-expressing cells were similar to those with CCNL2-wt, whereas in CCNL2-
RS-expressing cells, interactions decreased. Although this indicates a requirement for only the RS repeat region, the possibility cannot be not excluded that indirect interactions between cyclin L2 with SC-35 and 9G8 may occur, perhaps linked by p110 PITSLRE via the cyclin domains of cyclin L2.
Together with the nuclear distribution, the findings indicate that human cyclin L2 might play an active role in the cell nucleus, with involvement in the RNA processing complex via association with hyperphosphorylated RNA polymerase II, the splicing factors SC-35 and 9G8, and the CDK p110 PITSLRE.
Human Cyclin L2 Stimulates Pre-mRNA SplicingBecause cyclin L2 both co-localizes and associates with the splicing factors SC-35 and 9G8, does it contribute to splicing function? We performed in vitro splicing assays using HeLa cell nuclear extract (50 µg), assaying the effect of anti-CCNL2 polyclonal sera on in vitro splicing of a
-globin RNA precursor. As shown in Fig. 5, the presence of preimmune sera or anti-GST antibody did not affect splicing, whereas anti-CCNL2 sera remarkably inhibited splicing activity, suggesting that human cyclin L2 is involved in pre-mRNA splicing. We also performed in vitro splicing assays using sub-optimal amounts of HeLa cell nuclear extract (25 µg), with the addition of GST or GST-CCNL2 recombinant protein. In the absence of additional protein, or when GST was added to the reaction, the splicing activity was greatly reduced compared with that obtained with 50 µg of HeLa cell nuclear extract. Addition of recombinant GST-CCNL2 protein (250 ng) strongly stimulated splicing activity (Fig. 5). Human cyclin L2 thus appears to play a positive role in pre-mRNA splicing.
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Proliferation of SMMC 7721 cells transfected with different cyclin L2 constructs was analyzed by MTT assay. As shown in Fig. 6A, the proliferation of pCCNL2-wt and pCCNL2-
RS transiently transfected SMMC 7721 cells was inhibited compared with that of mock vector-transfected or parental SMMC 7721 cells (p < 0.05), although transfection of pCCNL2-AS or pCCNL2-
CL had minor effects on cell proliferation. The viability of stably transfected SMMC 7721 cells was further analyzed by colony formation assay. The colony formation rate of CCNL2-wt-transfected SMMC 7721 cells was significantly reduced compared with that of pCCNL2-AS-transfected, mock vector-transfected, and parental SMMC 7721 cells (p < 0.05, Fig. 6B). The results suggested that overexpression of human cyclin L2 might inhibit in vitro growth of human hepatocellular carcinoma.
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RS-overexpressing SMMC 7721 cells were much greater than that among pCCNL2-AS, pCCNL2-
CL, mock vector-transfected or parental SMMC 7721 cells. Human cyclin L2 may thus be involved in the induction of cell apoptosis, with conserved cyclin domains playing a vital role in this biological activity. In order to determine whether overexpression of cyclin L2 in human hepatocellular carcinoma could mediate therapeutic effects in vivo, human hepatocellular carcinoma-bearing nude mice were used as a model. Purified human cyclin L2 expression plasmid was delivered into the tumors by in vivo electroporation. Immunohistochemical analysis of hepatocellular carcinoma tissue confirmed high expression of Myc-tagged human cyclin L2 protein in tissues from tumor-bearing nude mice intratumorally transfected with pCCNL2-wt (Fig. 7A, panel a), indicating that in vivo electroporation can effectively mediate human cyclin L2 gene transfer into tumors. We next investigated whether intratumoral gene transfer of human cyclin L2 could inhibit the growth of parental SMMC 7721 cells in nude mice. We found that, compared with mock DNA, intratumoral gene transfer of human cyclin L2 could significantly inhibit the growth of SMMC 7721 tumors in nude mice (Fig. 7B). These findings indicate that intratumoral transfer of the human cyclin L2 gene can inhibit tumor growth significantly in vivo, consistent with the results obtained in vitro.
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Overexpression of Human Cyclin L2 Up-regulates the Expression of p53 and Bax and Down-regulates the Expression of Bcl-2Overexpression of human cyclin L2 induced the apoptosis of SMMC 7721 cells in vitro. Cellular apoptosis involves complex molecular cascades, and dysfunction of a variety of genes may lead to the onset and progression of apoptosis. We analyzed the expression of the apoptosis-related protein p53, anti-apoptotic protein Bcl-2, and pro-apoptotic protein Bax by Western blot and RT-PCR analysis. As shown in Fig. 8A, transient expression of CCNL2-wt and CCNL2-
RS induced an increase in p53 and Bax protein expression (3- and 4-fold, respectively) in SMMC 7721 cells 72 h post-transfection compared with pCCNL2-AS-, pCCNL2-
CL-, mock vector-transfected, or parental SMMC 7721 cells. Significantly up-regulated p53 and Bax mRNA expression was also detected by RT-PCR (Fig. 8B). Conversely, Bcl-2 expression was down-regulated in SMMC 7721 cells overexpressing human cyclin L2. These results demonstrate that overexpression of human cyclin L2 modulates the expression of critical apoptotic factors, leading to cell apoptosis. It would be interesting to determine whether other apoptotic factors may also have a relationship with apoptosis induced by overexpression of human cyclin L2.
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| DISCUSSION |
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The various aspects of RNA elongation and processing, including capping, splicing, and polyadenylation of nascent transcripts, are tightly coupled by protein factors physically associating with the CTD of RNA polymerase II (32). Human cyclin L2 possesses RS-repeat domains, a characteristic feature of SR protein/spliceosome components, and associates and co-localizes with the known splicing factors SC-35 and 9G8. RS domains have been discovered in several factors associated with transcription components, including the SR-like CTD-associated factors (33) and in a 3'-end cleavage factor, cleavage factor-I 68-kDa subunit (34), suggesting that communication between splicing and both transcription and 3'-end processing might in some cases be mediated by interactions involving RS domain proteins (35, 36). We have confirmed the stimulatory effect of cyclin L2 in pre-mRNA splicing. This activity may be mediated by interaction through its cyclin domains with RNA polymerase II and CDK p110 PITSLRE, or direct or indirect interaction via cyclin domain/RS repeat region with SC-35 and 9G8, as an RNA elongation/processing unit, in the nuclear speckles. Of note, while this manuscript was in revision, de Graaf et al. (37) reported cyclin L1 and cyclin L2 as interaction partners and substrates of DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1A), a nuclear speckle-locating protein kinase with transcription factors and splicing factors as its substrates. Because human cyclin L2 and cyclin L1 are expressed differentially at different levels in different cells and tissues, it is possible that these two closely related cyclins contribute to tissue-specific regulation of splicing.
The PITSLRE protein kinases are parts of the large family of p34cdc2-related kinases whose functions appear to be linked with progression of the cell cycle and the signal transduction of apoptosis and oncogenesis. PITSLRE CDK also interacts selectively with RNAPIIo but not with RNAPIIa, and PITSLRE proteins also contain many serine/arginine dipeptide repeats, characteristic of spliceosome components. The p110 PITSLRE has been shown to participate in a signaling pathway that may regulate the processing of RNA transcription. It interacts with the RNA-binding protein RNPS1, an activator of mRNA splicing, RNA polymerase II, and multiple transcriptional elongation factors, regulating some aspects of RNA splicing and/or transcription in proliferating cells (3840). Due to its identification as a partner for human cyclin L1 (12) and its mouse homologue cyclin ANIA-6A (20), it is referred to as CDK11. We show that human cyclin L2 can associate with p110 PITSLRE in a cyclin domain-dependent manner, suggesting that it is also likely to be a partner CDK for cyclin L2.
Our work here also revealed that overexpression of human cyclin L2 suppressed the growth of hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, and induced cellular apoptosis in vitro. Cyclin L2 is ubiquitously expressed throughout the body, and in a variety of human cell lines, including SMMC 7721 cells. Under normal conditions, tumor cells such as SMMC 7721 cells may have anti-apoptotic mechanisms in place which antagonize the apoptosis-inducing effects of cyclin L2. However, when cyclin L2 is overexpressed, the apoptosis-inducing effects of cyclin L2 may counterattack these anti-apoptotic mechanisms, resulting in an imbalance between apoptosis and cell survival and cell cycle regulation, if any. Previous studies have implicated other cyclins in apoptosis and growth inhibition of tumor cells. A potential p53-binding site is present in intron 1 of the cyclin K gene, and colony formation assays indicate that overexpression of cyclin K suppresses growth of the glioblastoma cell lines U373MG and T98G and colon carcinoma SW480 cells (41). At high levels, cyclin G1, a p53-responsive gene induced in alternative reading frame-arrested cells, also induces a G1-phase arrest in mammalian cells that coincides with p53 activation. Interestingly, growth inhibition by cyclin G1 does not require p53 but instead exhibits partial retinoblastoma protein dependence (42). A recent study by cDNA microarray and RT-PCR indicates that cyclin L1 is commonly overexpressed in well differentiated but not in poorly differentiated primary head and neck tumors, compared with corresponding correlative normal tissues (43). Other RS repeat-containing proteins such as Hs-BTF (44) can also act as apoptosis-promoting proteins that interact with Bcl-2-related proteins. To gain insight into the mechanism of cyclin L2-induced apoptosis in SMMC 7721 cells, we assessed the expression of the apoptosis-related proteins p53, Bax, and Bcl-2. In our study, following transient expression of cyclin L2 in SMMC 7721 cells, p53 and Bax expression was observed to increase significantly, and down-regulation of Bcl-2 expression could also be observed. Previous studies have demonstrated that overexpression of p53 can transactivate Bax expression and inhibit Bcl-2 expression, resulting in apoptotic cell death (45). Combined with other observations, including its association with RNAPII, CDK p110 PITSLRE, and splicing factors, and its stimulatory effect in pre-mRNA splicing, our results suggest that overexpression of cyclin L2 might regulate the RNA transcription and processing of certain apoptosis-related factors, possibly through a p53-dependent increase in Bax and reduction in Bcl-2 pathway signaling, leading to tumor cell growth inhibition.
In conclusion, we report here the cloning of human and mouse cyclin L2, the association of human cyclin L2 with p110 PITSLRE and the splicing factors SC-35 and 9G8, and the potential involvement of human cyclin L2 in the stimulation of pre-mRNA splicing. Growth inhibition and apoptosis-inducing effects of human cyclin L2 on tumor cells was also observed both in vitro and in vivo. Further experiments are required to elucidate the roles of the novel molecule, human cyclin L2, in transcriptional regulation and cell growth suppression and the related mechanisms.
| FOOTNOTES |
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* This work was supported by National Natural Science Foundation of China 30121002, the National Key Basic Research Program of China 2001CB510002, and the National High Biotechnology Development Program of China 2002BA711A01. 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. ![]()
Both authors contributed equally to this work. ![]()
To whom correspondence should be addressed: Institute of Immunology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People's Republic of China. Fax: 86-21-6538-2502; E-mail: caoxt{at}public3.sta.net.cn.
1 The abbreviations used are: CDK, cyclin-dependent kinase; CTD, C-terminal domain; RNAPIIo, hyperphosphorylated RNA polymerase II; RNAPIIa, hypophosphorylated RNA polymerase IIa; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GST, glutathione S-transferase; GFP, green fluorescent protein; BMSCs, bone marrow stromal cells; RT, reverse transcriptase; PI, propidium iodide. ![]()
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