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Human CLK2 Links Cell Cycle Progression, Apoptosis, and Telomere Length Regulation

2003; Elsevier BV; Volume: 278; Issue: 24 Linguagem: Inglês

10.1074/jbc.m300286200

1083-351X

Ning Jiang, Claire Bénard, Hania Kébir, Eric A. Shoubridge, Siegfried Hekimi,

DNA Repair Mechanisms

Mutations in the clk-2 gene of the nematode Caenorhabditis elegans affect organismal features such as development, behavior, reproduction, and aging as well as cellular features such as the cell cycle, apoptosis, the DNA replication checkpoint, and telomere length. clk-2 encodes a novel protein (CLK-2) with a unique homologue in each of the sequenced eukaryotic genomes. We have studied the human homologue of CLK-2 (hCLK2) to determine whether it affects the same set of cellular features as CLK-2. We find that overexpression of hCLK2 decreases cell cycle length and that inhibition of hCLK2 expression arrests the cell cycle reversibly. Overexpression of hCLK2, however, renders the cell hypersensitive to apoptosis triggered by oxidative stress or DNA replication block and gradually increases telomere length. The evolutionary conservation of the pattern of cellular functions affected by CLK-2 suggests that the function of hCLK2 in humans might also affect the same organismal features as in worms, including life span. Surprisingly, we find that hCLK2 is present in all cellular compartments and exists as a membrane-associated as well as a soluble form. Mutations in the clk-2 gene of the nematode Caenorhabditis elegans affect organismal features such as development, behavior, reproduction, and aging as well as cellular features such as the cell cycle, apoptosis, the DNA replication checkpoint, and telomere length. clk-2 encodes a novel protein (CLK-2) with a unique homologue in each of the sequenced eukaryotic genomes. We have studied the human homologue of CLK-2 (hCLK2) to determine whether it affects the same set of cellular features as CLK-2. We find that overexpression of hCLK2 decreases cell cycle length and that inhibition of hCLK2 expression arrests the cell cycle reversibly. Overexpression of hCLK2, however, renders the cell hypersensitive to apoptosis triggered by oxidative stress or DNA replication block and gradually increases telomere length. The evolutionary conservation of the pattern of cellular functions affected by CLK-2 suggests that the function of hCLK2 in humans might also affect the same organismal features as in worms, including life span. Surprisingly, we find that hCLK2 is present in all cellular compartments and exists as a membrane-associated as well as a soluble form. Identifying and studying the processes and the genes that are involved in determining the rate of aging is a challenging area of modern genetics. In particular, it would be of interest to determine whether the activity of specific genes limits human life span. Several epidemiological studies of centenarians are being carried out with this goal in mind under the hypothesis that there might be a genetic basis for the exceptional life span of very long lived individuals (1Perls T. Terry D.F. Silver M. Shea M. Bowen J. Joyce E. Ridge S.B. Fretts R. Daly M. Brewster S. Puca A. Kunkel L. Results Probl. Cell Differ. 2000; 29: 1-20Crossref PubMed Scopus (10) Google Scholar). However, given the pervasive evolutionary conservation of physiological processes among organisms, a practical approach to find genes that might be involved in human aging is to first investigate the genetic basis of aging in lower organisms. The nematode genetic model system, Caenorhabditis elegans, is being extensively used to this end, and a number of genes that have been identified in this organism for their effect on aging are now also being studied in vertebrates (2Hekimi S. Burgess J. Bussiere F. Meng Y. Benard C. Trends Genet. 2001; 17: 712-718Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 3Tissenbaum H.A. Guarente L. Dev. Cell. 2002; 2: 9-19Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The clk-2 mutants of C. elegans display a pleiotropic phenotype (reviewed in Ref. 4Benard C. Hekimi S. Mech. Ageing Dev. 2002; 123: 869-880Crossref PubMed Scopus (5) Google Scholar) that includes a slowing down of numerous physiological processes, including embryonic and postembryonic development, behavioral rates, and reproduction (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). clk-2 mutants also show an increase in life span that is particularly dramatic in combination with mutations in other genes, such as clk-1 and daf-2 (2Hekimi S. Burgess J. Bussiere F. Meng Y. Benard C. Trends Genet. 2001; 17: 712-718Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 6Lakowski B. Hekimi S. Science. 1996; 272: 1010-1013Crossref PubMed Scopus (425) Google Scholar). The clk-2 mutations are temperature-sensitive (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar, 7Ahmed S. Alpi A. Hengartner M.O. Gartner A. Curr. Biol. 2001; 11: 1934-1944Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and at 25 °C produce a lethal embryonic phenotype resulting in differentiated but highly disorganized embryos (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). This is likely to be the null phenotype, since it is also produced by RNA interference at all temperatures. Extensive temperature shift experiments have demonstrated that clk-2 is required for embryonic development only during a narrow time window in which oocyte maturation, fertilization, the completion of meiosis, and the initiation of embryonic development occurs (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). However, these events as well as subsequent embryonic development appear to proceed entirely normally until the 100-cell stage, after which aberrant development becomes apparent. Surprisingly, all clk-2 phenotypes, including the phenotypes observed in adults that are ∼1000 times larger than the eggs produced by the mother, are rescued by a maternal effect; i.e. homozygous mutant animals, issued from a heterozygous mother, appear wild type. This maternal rescue effect suggests that the presence of maternally provided clk-2 product might induce a self-maintained epigenetic state, although the possibility that the maternally provided clk-2 product can still function efficiently after extreme dilution cannot be excluded. A number of cellular phenotypes of clk-2 mutants have also been identified in addition to the organismal phenotypes described above (7Ahmed S. Alpi A. Hengartner M.O. Gartner A. Curr. Biol. 2001; 11: 1934-1944Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 8Gartner A. Milstein S. Ahmed S. Hodgkin J. Hengartner M.O. Mol. Cell. 2000; 5: 435-443Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). For example, the germ lines of clk-2 mutants do not respond normally to ionizing radiation. In the wild type, irradiation leads to cell cycle arrest in the mitotic phase of the germ line and to apoptotic cell death in the meiotic phase of the germ line. Both of these responses are abolished in clk-2 mutants. In addition, clk-2 mutants fail to respond with cell cycle arrest to treatment with hydroxyurea (HU), 1The abbreviations used are: HU, hydroxyurea; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; GST, glutathione S-transferase. a drug that blocks DNA replication, suggesting a defect in the S-phase replication checkpoint. Taken together, these cellular phenotypes suggest that clk-2 mutants are defective in important aspects of the normal cellular response to DNA damage. The C. elegans clk-2 gene encodes a protein of 877 amino acids that is similar to Saccharomyces cerevisiae Tel2p and has a unique homologue in every eukaryotic genome sequenced to date (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). Yeast cells carrying the hypomorphic tel2-1 mutation grow slowly and have short telomeres (9Runge K.W. Zakian V.A. Mol. Cell. Biol. 1996; 16: 3094-3105Crossref PubMed Scopus (85) Google Scholar). The telomeres shorten gradually in the tel2 cells, reaching their shortest lengths only after ∼150 generations. In addition to affecting the length of telomeres, Tel2p has also been shown to be involved in the telomere position effect, contributing to silencing of subtelomeric regions. Mutations in other genes, such as tel1, that also affect telomere length do not result in abnormal telomere position effect, indicating that the telomere position effect defect in tel2 mutants is not a simple consequence of the altered telomere length (10Zakian V.A. Annu. Rev. Genet. 1996; 30: 141-172Crossref PubMed Scopus (170) Google Scholar). In contrast to the viable tel2–1 mutants, cells that fully lack Tel2p die rapidly with an abnormal cell morphology, which suggests that Tel2p also has telomere-independent functions in yeast (9Runge K.W. Zakian V.A. Mol. Cell. Biol. 1996; 16: 3094-3105Crossref PubMed Scopus (85) Google Scholar). Worm clk-2 mutants also have altered telomere length. In contrast to the phenotype in yeast, clk-2(qm37) mutants have lengthened telomeres, and transgenic expression of clk-2 shortens some telomeres (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). Although yeast Tel2p can bind single- and double-stranded DNA and RNA under some in vitro conditions (11Kota R.S. Runge K.W. Nucleic Acids Res. 1998; 26: 1528-1535Crossref PubMed Scopus (29) Google Scholar, 12Kota R.S. Runge K.W. Chromosoma. 1999; 108: 278-290Crossref PubMed Scopus (22) Google Scholar), a functional worm CLK-2::GFP fusion protein accumulates predominantly in the cytoplasm (5Benard C. McCright B. Zhang Y. Felkai S. Lakowski B. Hekimi S. Development. 2001; 128: 4045-4055Crossref PubMed Google Scholar). These findings, together with the broad pleiotropy observed mostly in worms, but also in yeast, suggest that clk-2 and tel2 mutations affect telomere length indirectly. Cell Culture—All cells were grown in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (plus nonessential medium amino acids for HT-1080 and SK-HEP-1) at 37 °C in an atmosphere of 5% CO2 and 95% air. Construction of the Plasmid pLXSH-hclk2 and Establishment of a Stable Cell Line Overexpressing hCLK2—A cDNA clone hk02952 (insert size 4337 bp), containing the full-length hclk2 cDNA sequence, as well as parts of intronic sequences (1929–2171, 2288–2456, and 2812–3434) was obtained from Kazusa DNA Research Institute, Japan. Using this cDNA as a template, two fragments that exclude the intron sequences, Δhclk2-A (from bp 256 to 1929) and Δhclk2-B (from bp 1929 to 3434) were generated by PCR and cloned into a pcDNA3.1/V5/His/TOPO vector (Invitrogen) to produce pcDNA3.1-Δhclk2-A and pcDNA3.1-Δhclk2-B. A BamHI-EcoRV fragment from pcDNA3.1-Δhclk2-A(–) was subcloned into the BamHI–HpaI site of pLXSH (13Miller A.D. Buttimore C. Mol. Cell. Biol. 1986; 6: 2895-2902Crossref PubMed Scopus (1144) Google Scholar) to produce pLXSH-Δhclk2-A. A BamHI fragment from pcDNA3.1-Δhclk2-A(–) was inserted into the BamHI site of pLXSH-Δhclk2-A to produce pLXSH-hclk2. Stable virus-producing cell lines were generated using procedures described previously (14Miller A.D. Miller D.G. Garcia J.V. Lynch C.M. Methods Enzymol. 1993; 217: 581-599Crossref PubMed Scopus (378) Google Scholar). Briefly, the retroviral constructs were used to transfect GP + E86 ecotropic packaging cells (15Markowitz D. Goff S. Bank A. J. Virol. 1988; 62: 1120-1124Crossref PubMed Google Scholar), and viruses thus produced were used to infect the amphotropic packaging cell line PA317. Selection was performed 48 h after infection in 400 units/ml hygromycin B and continued until colonies were visible. The colonies were pooled and expanded to establish the virus-producing cell lines. Target cells (see Table I) were transduced with the retrovirus as described (16Lochmuller H. Johns T. Shoubridge E.A. Exp. Cell Res. 1999; 248: 186-193Crossref PubMed Scopus (83) Google Scholar) and selected in hygromycin at the concentrations indicated. All surviving cells were kept together as a pool, which constitutes the SK-HEP-1-overexpressing hCLK2 cell line.Table ICell lines infectedName (ATCC No)Tissue derivationHygromycinunits/mlC2C12 (CRL-1772)Mouse myoblast400Rat1-R12 (CRL-2210)Rat fibroblast200A549 (CCL-18S)Human lung carcinoma900SK-N-ASHuman neuroblastoma400SK-HEP-1Human liver adenocarcinoma400HT-1080Human fibrosarcoma400293Human kidney carcinoma400MCH58Human fibroblast100 Open table in a new tab Construction of Plasmid pTRE2-hclk2 and Establishment of a Double Stable Tet-off HT-1080 Line with Inducible hCLK2—The hclk2 full-length cDNA sequence containing engineered NotI and EcoRV sites was generated by PCR and inserted into the NotI–EcoRV site of pTRE2 (Clontech, Palo Alto, CA) to produce plasmid pTRE2-hclk2. The plasmid DNA was transfected into premade Tet-off HT-1080 cells (Clontech) using the superfect reagent (Qiagen). Cells were selected in 400 units/ml hygromycin 48 h after infection, and selection was continued until colonies were visible. 30 colonies were picked, and immunoblot analysis using anti-hCLK2 antibodies (see below) showed that five clones (numbers 3, 6, 11, 19, and 21) overexpressed hCLK2 in an inducible manner. Cells were grown in the presence of doxycycline (1 μg/ml) to turn off hCLK2 expression and grown in the absence of doxycycline to turn on hCLK2 expression. Immunoblot analysis showed that the level of expression of hCLK2 in the Tet-off cell line (clone 21) was dependent on the dosage of doxycycline. Knocking Down the Expression of hCLK2 by Sequence-specific Small Interfering RNA (siRNA)—siRNA oligonucleotides were synthesized by Dharmacon Research (Lafayette, Colorado). siRNA duplex selection and transfection were performed as described (17Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8185) Google Scholar). The sequence of the siRNA for targeting endogenous hclk2 was as follows: sense siRNA, 5′-GCGGUAUCUCGGUGAGAUGdT-3′; antisense siRNA, 5′-CAUCUCACCGAGAUACCGCdT-3′. For each well of a six-well plate, 240 pmol of siRNA duplex was used. Cells were exposed to the siRNA treatment on day 1, and they were passaged 1:4 on day 4. Preparation of Antibodies Directed against hCLK-2 Protein—Two separate antigens were used to develop anti-hCLK2 polyclonal antibodies. The first antigen was generated as follows. A PCR fragment corresponding to bases 1516–1929 of the hclk2 clone hk02952, encoding amino acids 414–551 of hCLK2, was cloned into the pGEX-3X expression vector (Amersham Biosciences). A GST-hCLK2-(414–551) protein of the expected size (∼46 kDa) was expressed in DH10b bacteria and purified by affinity chromatography on a GST slurry. This recombinant protein was injected into two rabbits (2779 and 2780) to obtain polyclonal antibodies. To generate the second antigen, a PCR fragment corresponding to bases 279–1519 of the hclk2 clone hk02952, encoding amino acids 2–415 of hCLK2, was cloned into the pGEX-3X expression vector. A GST-hCLK2-(2–415) protein of the expected size (∼78 kDa) was expressed in DH10b bacteria and was purified from bacterial inclusion bodies. This recombinant protein was injected into two rabbits (2838 and 2839) to obtain polyclonal antibodies. All four sera specifically react to hCLK2 by the following criteria. The terminal bleed of each rabbit recognizes the corresponding bacterial antigen, in vitro translated hCLK2, a band at the expected size of ∼100 kDa in cell extracts, and a strong band of the same size in cells overexpressing hCLK2 (see Table I). This ∼100-kDa band is not detected by any of the preimmune sera. Moreover, this band disappears upon preabsorbtion of the antibody with the corresponding purified GST-hCLK2 protein but not upon preabsorbtion with other unrelated bacterially expressed proteins, including GST fusions. Also, the intensity of this band is drastically reduced in hclk-2-siRNA-treated cells as compared with controls. The serum from rabbit 2780 gave the strongest reaction and was used for immunoblot analyses throughout this study. Immunoblot Analysis—Cultured cells were trypsinized and pelleted and then resuspended in 5× volumes of extraction buffer (500 mm NaCl, 20 mm Tris, pH 8.0, 1% Nonidet P-40, 1 mm dithiothreitol, and protease inhibitors (Roche Applied Science)). The resuspended cells were submitted to five freeze-thaw cycles (frozen in liquid nitrogen and thawed at 37 °C). Cell debris were removed by centrifugation, and the quantity of protein was measured (Bio-Rad protein assay). 50 μg of protein were separated on 7.5 or 12% polyacrylamide gels and transferred to nitrocellulose. The membranes were preincubated in blocking solution (TBS-T plus 5% nonfat milk) at room temperature for 1 h and then incubated with the primary antibody at 4 °C overnight at the following concentrations: rabbit anti-hCLK2 antibody (1:500 to 1:1000), mouse anti-α-tubulin antibody (1:10000; Sigma), rabbit anti-actin antibody (1:500; Sigma), mouse anti-cytochrome c (1–2 μg/ml; Molecular Probes, Eugene, OR), and mouse anti-p300 (2 μg/ml; Upstate Biotechnology, Inc., Lake Placid, NY). After 3 × 15 min TBS-T washes, the membranes were incubated in blocking solution at room temperature for 2 h. The membranes were then incubated with donkey anti-rabbit IgG secondary antibody (1:3000; Jackson Immunoresearch Laboratories) or goat anti-mouse IgG (1:20000; Pierce) at room temperature for 1 h, followed by three 15-min TBS-T washes. Finally, the signal was detected by chemiluminescence (Amersham Biosciences). Immunostaining—Cells were transiently transfected with a plasmid, and 24 h later, they were seeded on coverslips. Forty-eight hours later, the coverslips were fixed in 4% paraformaldehyde/PBS for 10 min, permeabilized in acetone for 3 min, and then incubated at room temperature for 1 h with rabbit polyclonal anti-hCLK2 (2780, 2838, 1:100–1000), followed by biotinylated goat anti-rabbit or mouse IgG (1:5000) for 1 h. Finally, the cells were incubated with fluorescein-conjugated streptavidin (10 μg/ml) for 30 min and viewed under a Leitz fluorescence microscope. Similar results were obtained with 2780 and 2838 sera. The pattern observed was not detected by the preimmune sera or the secondary antibody alone. In addition, the observed pattern disappears upon preabsorbtion of the antibody with the corresponding purified GST-hCLK2 protein but not upon preabsorbtion with other unrelated bacterially expressed proteins, including GST fusions. Growth Rate Assay—Cells were seeded in six-well dishes at 1 × 105/well. At the times indicated, the cells were trypsinized and counted with a hemocytometer. Cell Death Assay—Cells were seeded at 1 × 105 in six-well dishes. The next day, the cells were treated by γ-ray (20 grays) and counted 72 h later. A series of different apoptosis-inducing agents was also investigated, and the cells were analyzed at various times following treatment (see Table II). Cell viability was measured by the trypan blue exclusion method, by counting with a hemocytometer.Table IICell death assaysTreatmentWorking concentrationTime of treatmenthEtoposide100 μM24Sodium azide15 μM48Menadione12 μM24Anisomycin2 μM16t-Butyl hydroperoxide40 μM48Staurosporine2 μM24All-trans-retinoic acid4 μM96Hydrogen peroxide0.5 μM24Juglone0.5 μM24Hydroxyurea0.6 mM96Tunicamycin5 μg/ml24 Open table in a new tab Measuring the Length of Telomeres—Genomic DNA from cultured cells was recovered by phenol-chloroform extraction and ethanol precipitation. 10 μg of DNA was digested by HinfI and RsaI (10 units/μg DNA) at 37 °C overnight. The completely digested DNA was separated on 0.7% agarose gel at 23 V for 24 h and transferred by capillary transfer to a positively charged nylon membrane (Amersham Biosciences) overnight. The telomere-specific sequence (5′-TTAGGGTTAGGGTTAGGG-3′) was used as a probe to detect telomeric repeats. The membrane was incubated in prehybridization solution (5× SSC, 5× Denhardt's solution, 0.1% SDS) for 1 h at 50 °C, followed by an overnight incubation in hybridization solution (5× SSC, 0.1% SDS, and 5′-32P-end-labeled probe) at 37 °C. The membrane was then washed in 3× SSC, 0.1% SDS at 42 °C for 3 × 10 min and exposed at room temperature overnight. Preparation of Subcellular Fractions—Subcellular fractionation was performed as described (18Krajewski S. Tanaka S. Takayama S. Schibler M.J. Fenton W. Reed J.C. Cancer Res. 1993; 53: 4701-4714PubMed Google Scholar). From 1 to 10 × 107 cells were washed twice with ice-cold PBS and resuspended in buffer (0.25 m sucrose, 10 mm Tris-HCl, pH 7.5, 1 mm EDTA, protease inhibitors (Roche Applied Sciences)) at a concentration of 2 × 107 cells/ml. Cells were homogenized on ice (10–20 strokes at 1000 rpm; Potter-Elvehjem) until 95% of the cells were lysed based on trypan blue dye uptake. The samples were transferred to 1.5-ml Eppendorf centrifuge tubes (1 ml/tube) and centrifuged at 500 × g for 5 min to pellet the nuclei. The nuclear pellet was then resuspended in 0.5–2 ml of 1.6 m sucrose containing 50 mm Tris-HCl, pH 7.5, 25 mm KCl, 5 mm MgCl2. After underlayering with 1–2 ml of 2.0–2.3 m sucrose containing the same buffer and centrifugation at 150,000 × g for 60 min, the resulting nuclear pellets were resuspended in 0.1–0.3 ml of 1% Triton X-100-containing buffer (0.15 m NaCl, 10 mm Tris (pH 7.4), 5 mm EDTA, 1% Triton X-100). The supernatant resulting from the initial low speed centrifugation was subjected to centrifugation at 10,000 × g for 15 min at 4 °C to obtain the heavy membrane fraction (a pellet that should include mitochondria, lysosomes, Golgi, and rough endoplasmic reticulum). The supernatant was centrifuged for 60 min at 15,000 × g to obtain the light membrane fraction (a pellet that should include the smooth and rough endoplasmic reticulum) and the cytosolic fraction (supernatant). The heavy membrane and light membrane fractions were resuspended in 1% Triton-containing lysis buffer. An equal amount of protein (50 μg) from each fraction was analyzed by immunoblot. Growth Stimulation by Overexpression of hCLK2 in SK-HEP-1 Cells—To achieve high levels of hCLK2 expression in cultured cells, we used a retroviral vector expressing hCLK2 to infect a panel of cell lines (see "Experimental Procedures") and established stable cell lines, derived from pools of cells infected with the vector expressing hCLK2 or the empty vector control. A high level of hCLK2 expression was detected in all of the established cell lines (Fig. 1A and data not shown). In every case, the cells expressing hCLK2 did not show any morphological alterations compared with controls (data not shown). We found, however, that the growth rate of SK-HEP-1 (19Heffelfinger S.C. Hawkins H.H. Barrish J. Taylor L. Darlington G.J. In Vitro Cell Dev. Biol. 1992; 28A: 136-142Crossref PubMed Scopus (132) Google Scholar) cells overexpressing hCLK2 was increased over the control line (Fig. 2A), indicating that growth rate is sensitive to the level of hCLK2. We then used SK-HEP-1 cells for all subsequent characterization of the function of hCLK2. Other cell lines did not display obvious effects on growth, and their phenotype was not studied further (see "Experimental Procedures").Fig. 2The growth rate of SK-HEP-1 cells is affected by the level of expression of hCLK2.A, SK-HEP-1 cells overexpressing hCLK2 and control cells were plated at a density of 1 × 105/well in a six-well dish. At the indicated time (in days), cells were harvested, and the number of cells were counted using a hemocytometer. B, SK-HEP-1 cells were plated at a density of 1.0 × 105/well in a six-well dish and treated by siRNA or buffer the next day (day 1) at a density of about 1.5 × 105/well. Cell counts were done as above. For both panels, the means and S.E. of triplicate experiments are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Reducing the Level of hCLK2 Expression Causes Reversible Growth Arrest—To investigate the consequences of a loss of function of hclk2, we used the siRNA technique (17Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8185) Google Scholar). SK-HEP-1 cells were treated with either 1) hclk2-specific siRNA, 2) siRNA for luciferase, a gene that is not normally found in human cells, or 3) the same volume of siRNA annealing buffer. The level of hCLK2 and the cell number were determined daily for several days following siRNA treatment (Figs. 1B and 2B). The immunoblots demonstrate that when the cells were treated with hclk2-specific siRNA, the level of hCLK2 was significantly decreased by day 2 and remained low until at least day 6. As expected, neither luciferase siRNA nor siRNA annealing buffer alone resulted in a decrease of the expression of hCLK2. In addition, the expression of actin was not affected by hclk2- specific siRNA, luciferase siRNA, or siRNA annealing buffer alone (Fig. 1B). hclk2 siRNA treatment dramatically slowed cellular growth rate, in contrast to treatment with luciferase siRNA, which had only a minor effect (Fig. 2B). The effect on growth rate lasted until day 7, after which time the cells appeared to recover from the treatment and resumed growth. No increase in cell death or other obvious changes were observed, indicating that the arrest was not the consequence of major damage to the cells. Treated cells were also sorted by fluorescence-activated cell sorting according to DNA content (data not shown). The arrested cells treated with hclk2 siRNA did not appear to have arrested in any particular phase of the cell cycle. Overexpression of hCLK2 Produces Hypersensitivity to Apoptosis Triggered by Oxidative Stress or DNA Replication Block—Prompted by the findings in the germ line of C. elegans, where clk-2 mutations affect the response to ionizing radiation and to DNA replication block induced by HU, we investigated the response of SK-HEP-1 cells overexpressing hCLK2 to 10 different agents capable of inducing apoptotic cell death as well as to HU and γ-rays. The cells overexpressing hCLK2 did not show any general increase in sensitivity to apoptotic stimuli but were specifically hypersensitive to two methods of increasing oxidative stress: menadione treatment, which leads to intracellular overproduction of superoxide (20Jamieson D.J. Rivers S.L. Stephen D.W. Microbiology. 1994; 140: 3277-3283Crossref PubMed Scopus (118) Google Scholar), and t-butyl hydroperoxide treatment, which leads to the production of the highly toxic hydroxyl radical (21Sano M. Kawabata H. Tomita I. Yoshioka H. Hu M.L. J. Toxicol. Environ. Health. 1994; 43: 339-350Crossref PubMed Scopus (6) Google Scholar) (Fig. 3A). The cells were also hypersensitive to the DNA synthesis inhibitor HU (Fig. 3A). To verify that the cell death observed was indeed apoptotic, we stained the cells using the TUNEL method (22Desjardins L.M. MacManus J.P. Exp. Cell Res. 1995; 216: 380-387Crossref PubMed Scopus (85) Google Scholar), which consists of in situ labeling of the 3′-OH ends of the cleaved DNA typical of apoptotic cells. A significant increase in the number of TUNEL-positive nuclei was observed in cells treated with the compounds that produced increased cell death compared with controls, namely menadione, t-butyl hydroperoxide, and hydroxyurea (Fig. 3B). We have also investigated the response of siRNA-treated SK-HEP-1 cells and found that the cells depleted for hCLK2 did not show any general increase in sensitivity to apoptotic stimuli (data not shown). It is unclear whether a reduction in hCLK2 levels has no effect on the sensitivity of the cells to the agents used or whether the arrest produced by siRNA treatment prevents the detection of any effect. Overexpression of hCLK2 Gradually Lengthens Telomeres—To investigate whether hclk2 affects telomere length in human cells, as it does in S. cerevisiae and in C. elegans, we determined the telomere length of SK-HEP-1 cells overexpressing hCLK2 and of SK-HEP-1 control cells by Southern blot analysis. We examined the telomere length at regular intervals during prolonged culturing (138 population doublings) (Fig. 4). The telomere length of the cells overexpressing hCLK2 gradually grew longer at an average rate of ∼15 bp/population doubling, whereas it remained absolutely stable in the control cells (Fig. 4). Additional population doublings do not appear to increase telomere length further (data not shown). hCLK2 Is Present in Most Compartments of the Cell—To determine the subcellular localization of hCLK2, we used immunocytochemistry to detect native and overexpressed hCLK2 in SK-HEP-1 cells. The level of native hCLK2 appeared to be too low to be detectable by this method with our antisera directed against hCLK2 (see "Experimental Procedures"). However, in cells overexpressing hCLK2, the signal appeared to be everywhere in the cell, filling both the cytoplasm and the nucleus (Fig. 5A). The same distribution was also observed in another overexpressing cell line HT-1080 (Fig. 5B). Controls included immunocytochemistry using the preimmune sera, the secondary antibody alone, and sera preabsorbed with a number of bacterial antigens. We determined the subcellular distribution of hCLK2 by immunocytochemistry following treatment with etoposide and menadione, two apoptotic triggering agents, which result in DNA replication inhibition and o