Elimination of Cyclin D1 in Vertebrate Cells Leads to an Altered Cell Cycle Phenotype, Which Is Rescued by Overexpression of Murine Cyclins D1, D2, or D3 but Not by a Mutant Cyclin D1
1997; Elsevier BV; Volume: 272; Issue: 16 Linguagem: Inglês
10.1074/jbc.272.16.10859
ISSN1083-351X
AutoresJill M. Lahti, Haimin Li, Vincent J. Kidd,
Tópico(s)Cell death mechanisms and regulation
ResumoDT40 lymphoma B-cells normally express cyclins D1 and D2 but not D3. When cyclin D1 expression was extinguished in these cells by gene knockout, specific alterations in their ability to transit the cell cycle were observed. These changes are exemplified by a delay of approximately 2 h in their progression through a normal 14-h cell cycle. This delay results in an increase in the number of cells in the G2/M phase population, most likely due to triggering of checkpoints in G2/M, inability to enter G1 normally, and/or alterations of crucial event(s) in early G1. The defect(s) in the cell cycle of these D1 "knockout" cells can be rescued by overexpression of any normal mouse D-type cyclin but not by a mutant mouse cyclin D1 protein that lacks the LXCXE motif at its amino terminus. These data suggest that the cell cycle alterations observed in the D1−/− cells are a direct effect of the absence of the cyclin D1 protein and support the hypothesis that the D-type cyclins have separate, but overlapping, functions. Elimination of cyclin D1 also resulted in enhanced sensitivity to radiation, resulting in a significant increase in apoptotic cells. Expression of any normal murine D-type cyclin in the D1−/− cells reversed this phenotype. Intriguingly, expression of the mutant cyclin D1 in the D1 −/− cells partially restored resistance to radiation-induced apoptosis. Thus, there may be distinct differences in cyclin D1 complexes and/or its target(s) in proliferating and apoptotic DT40 lymphoma B-cells. DT40 lymphoma B-cells normally express cyclins D1 and D2 but not D3. When cyclin D1 expression was extinguished in these cells by gene knockout, specific alterations in their ability to transit the cell cycle were observed. These changes are exemplified by a delay of approximately 2 h in their progression through a normal 14-h cell cycle. This delay results in an increase in the number of cells in the G2/M phase population, most likely due to triggering of checkpoints in G2/M, inability to enter G1 normally, and/or alterations of crucial event(s) in early G1. The defect(s) in the cell cycle of these D1 "knockout" cells can be rescued by overexpression of any normal mouse D-type cyclin but not by a mutant mouse cyclin D1 protein that lacks the LXCXE motif at its amino terminus. These data suggest that the cell cycle alterations observed in the D1−/− cells are a direct effect of the absence of the cyclin D1 protein and support the hypothesis that the D-type cyclins have separate, but overlapping, functions. Elimination of cyclin D1 also resulted in enhanced sensitivity to radiation, resulting in a significant increase in apoptotic cells. Expression of any normal murine D-type cyclin in the D1−/− cells reversed this phenotype. Intriguingly, expression of the mutant cyclin D1 in the D1 −/− cells partially restored resistance to radiation-induced apoptosis. Thus, there may be distinct differences in cyclin D1 complexes and/or its target(s) in proliferating and apoptotic DT40 lymphoma B-cells. Regulation of the vertebrate cell cycle requires the periodic formation, activation, and inactivation of unique protein kinase complexes that consist of cyclin (regulatory) and cyclin-dependent kinase (CDK 1The abbreviations used are: CDK and cdk, cyclin-dependent protein kinases; pRb, retinoblastoma protein; cki, cyclin-dependent protein kinase inhibitors; p34cdc2, p34 cell division control-2 protein kinase;Ggccnd1, Gallus gallus cyclin D1 gene;CCND1, human cyclin D1 gene; neo, neomycin; hyg, hygromycin; RSV, Rous sarcoma virus; kb, kilobase pair(s); IP, immunoprecipitate; LXCXE, leucine-X-cysteine-X-glutamic acid; IVTT,in vitro transcribe and translate; BudR, bromodeoxyuridine; MI, mitotic index. 1The abbreviations used are: CDK and cdk, cyclin-dependent protein kinases; pRb, retinoblastoma protein; cki, cyclin-dependent protein kinase inhibitors; p34cdc2, p34 cell division control-2 protein kinase;Ggccnd1, Gallus gallus cyclin D1 gene;CCND1, human cyclin D1 gene; neo, neomycin; hyg, hygromycin; RSV, Rous sarcoma virus; kb, kilobase pair(s); IP, immunoprecipitate; LXCXE, leucine-X-cysteine-X-glutamic acid; IVTT,in vitro transcribe and translate; BudR, bromodeoxyuridine; MI, mitotic index.; catalytic) subunits. 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These agents have no apparent effect on cell cycle once cells have passed the restriction point late in G1 phase of the cell cycle. Finally, it has been shown that ectopic expression of cyclin D1 inhibits MyoD and myogenin-mediated skeletal muscle differentiation indirectly (48Skapek S.X. Rhee J. Kim P.S. Novitch B.G. Lassar A.B. Mol. Cell. Biol. 1996; 16: 7043-7053Crossref PubMed Scopus (110) Google Scholar). To determine the role of cyclin D1 in muscle differentiation, experiments were designed to determine whether MyoD·myogenin and/or pRb were targets of cyclin D1·CDK phosphorylation. Mutation of CDK phosphorylation sites in myogenin had no effect on the ability of cyclin D1 to inhibit differentiation. Ectopic MyoD expression in fibroblasts can induce muscle-specific gene expression, as long as wild-type pRb is also expressed (48Skapek S.X. Rhee J. Kim P.S. Novitch B.G. Lassar A.B. Mol. Cell. Biol. 1996; 16: 7043-7053Crossref PubMed Scopus (110) Google Scholar). 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DT40 cells provide a unique reagent to easily and quickly examine the necessity for each of these cyclins individually and collectively, as well as interactions between the various cyclins and other cell cycle proteins. These lymphoma B-cells normally express cyclin D1 and D2 but not cyclin D3. Elimination of cyclin D1 in DT40 cells is not lethal; however, ablation of cyclin D1 gene expression results in marked alterations in the ability of these cells to transit the cell cycle which, in turn, affects their normal growth rate. Furthermore, loss of cyclin D1 did not result in a compensatory increase in the levels of cyclin D2 in these cells. Overexpression of normal mouse cyclin D1, D2, or D3 in the D1 −/− cells restored normal growth and cell cycle progression. However, a mutant murine cyclin D1 lacking the LXCXE motif (the so-called retinoblastoma protein (pRb)-binding motif that enhances D-type cyclin interaction with pRb) did not rescue the D1 −/− phenotype (44Ewen M.E. Sluss H.K. 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Finally, expression of either murine cyclin D1, D2, or D3 in the DT40 D1−/− cells resulted in decreased sensitivity to radiation-induced apoptosis, indistinguishable to what was observed in the parental DT40 cell line. Expression of the mutant cyclin D1 partially rescued cells from radiation-induced apoptosis, suggesting that the mechanism(s) involving cyclin D1 in this pathway is distinct from its role in normal cell cycle progression. Thus, in avian B-lymphoma cells cyclin D1 appears to fully suppress apoptosis, whereas a mutant cyclin D1 partially rescued these cells, suggesting that the targets of cyclin D1·CDK activity in apoptotic cells may be distinct from those in proliferating cells. Agarose was purchased from FMC Bioproducts (Rockland, ME). Restriction and modification enzymes were obtained from New England Biolabs (Beverly, MA) and Boehringer Mannheim. Urea and cell culture reagents, including chicken serum, were purchased from Life Technologies, Inc. Sequenase and DNA sequencing reagents were purchased from U. S. Biochemical Corp. All radioisotopes were purchased from DuPont NEN. All chemicals were purchased from either Sigma or Fisher. The chicken cosmid genomic library was obtained from Clonetech (Palo Alto, CA). The PrimeIt labeling kit and Duralose membranes were purchased from Stratagene (La Jolla, CA). Selection drugs were purchased from Life Technologies, Inc., Sigma, or Boehringer Mannheim. Fluorescein-conjugated goat anti-mouse and goat anti-rabbit antibodies were obtained from Boehringer Mannheim or Southern Biotechnology. Rabbit polyclonal anti-cyclin D1 antibody was kindly provided by Dr. Charles Sherr (see Ref. 2Nurse P. Nature. 1990; 344: 503-508Crossref PubMed Scopus (2197) Google Scholar), and anti-BudR antibodies were obtained from Boehringer Mannheim. The DNA-specific dye 4′,6-diamidino-2-phenylindole was obtained from Boehringer Mannheim. Oligonucleotides for DNA sequencing were synthesized on an Applied Biosystems 394 DNA/RNA Synthesizer by the Molecular Resources Facility of St. Jude Children's Research Hospital. Cyclin D1 cDNAs were isolated from a UG9 T-cell cDNA library (55Gobel T.W.F. Chen C.H. Lahti J. Kubota T. Kuo C.-L. Aebersold R. Hood L.E. Cooper M.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1094-1098Crossref PubMed Scopus (51) Google Scholar) by low stringency hybridization with a human cyclin D1 cDNA, kindly provided by Dr. Steve Reed (56Lew D.J. Dulic V. Reed S.I. Cell. 1991; 66: 1197-1206Abstract Full Text PDF PubMed Scopus (649) Google Scholar). The nucleotide sequence of these cDNAs was determined as described previously (57Xiang J. Lahti J.M. Grenet J. Easton J. Kidd V.J. J. Biol. Chem. 1994; 269: 15786-15794Abstract Full Text PDF PubMed Google Scholar). Oligonucleotides were spaced approximately 80–100 base pairs apart spanning the entire cDNA. A full-length cyclin D1 cDNA was transcribed and translated in vitro, producing a 36-kDa protein that could be immunoprecipitated with the mouse polyclonal cyclin D1 antibody (see Fig. 4, panel A). The corresponding cyclin D1 (Gallus gallus ccnd1) gene was isolated by screening a Cornish White Rock chicken cosmid library (Stratagene) with the full-length cyclin D1 cDNA as described previously (58Li H. Grenet J. Kidd V.J. Gene (Amst.). 1995; 167: 341-342Crossref PubMed Scopus (8) Google Scholar). A 13-kb BamHI restriction fragment containing exons 1–3 of the cyclin D1 gene was subcloned into the pKS plasmid and either this DNA, or cosmid DNA, was used for double-strand DNA sequence analysis of the gene. The same oligonucleotides used for establishing the cDNA sequence were used to establish the molecular organization (intron/exon boundaries, exons) of the gene by sequencing the DNA strands in both directions. Genomic DNA was isolated as described previously (59Lahti J.M. Valentine M. Xiang J. Joens B. Amann J. Grenet J. Richmond G. Look A.T. Kidd V.J. Nat. Genet. 1994; 7: 370-375Crossref PubMed Scopus (110) Google Scholar). The constructs containing the inserted drug selection cassettes were made by digestion of the 13-kbBamHI subclone that contained exons 1–3 of theGgccnd1 gene, with the enzyme BstI that opened this fragment at a single site in exon 2 (Fig. 2, panel A). This exon encodes a major portion of the cyclin box region. Approximately 5.5 kb of genomic DNA were left intact on the 5′ side of this insertion, and approximately 7.5 kb of genomic DNA remained on the 3′ side. Restriction fragments containing either the neo orhyg gene under the control of a β-actin gene promoter (kindly provided by Dr. C. Thompson) were cloned into thisBstI site by blunt-end ligation using Klenow enzyme. Further details concerning the selectable marker genes can be found elsewhere (60Takeda S. Masteller E.L. Thompson C.B. Buerstedde J.-M. Proc. Natl. Acad. Sci. U. S. 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Insertion of the neo cassette (2 kb in size) generated a 17-kb XbaI hybridizing fragment (Fig. 2, panels Cand D), and insertion of the hyg gene cassette (3 kb in size) generated an 18-kb XbaI hybridizing fragment (Fig. 2, panels C and D). DT40 lymphoma B cells (kindly provided by Dr. C-L. Chen) were grown in suspension cell culture in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1% chicken serum, penicillin, streptomycin, and glutamine. pCycD1-neoand pCycD1-hyg were linearized and transfected into DT40 cells by electroporation (550 V, 25 microfarads). Twenty-four hours after DNA transfection, the appropriate concentration of selection drug (2 mg/ml G418 or 1.5 mg/ml hygromycin) was added to the culture medium, and the cells were selected for ∼14 days. Single cells, isolated by flow sorting, were expanded into individual clones. Genomic DNA was isolated from multiple single cell-derived clones, digested with the appropriate restriction enzymes and hybridized with the cyclin D1 5′-flanking genomic probe (Fig. 2, panel A) to screen for homologous recombinants. Disruption of one, heterozygote (+/−), or both, homozygote (−/−), cyclin D1 (Ggccnd1) alleles was observed. For all experiments, multiple single cell clones of −/+ and −/− cyclin D1 disruptions were examined by both Southern and Northern blotting. RNA was isolated for Northern blotting, and the blots were hybridized to the cyclin D1 cDNA probe as described previously (57Xiang J. Lahti J.M. Grenet J. Easton J. Kidd V.J. J. Biol. Chem. 1994; 269: 15786-15794Abstract Full Text PDF PubMed Google Scholar,62Lahti J.M. Xiang J. Heath L.S. Campana D. Kidd V.J. Mol. Cell. Biol. 1995; 15: 1-11Crossref PubMed Scopus (191) Google Scholar). Equal loading of RNA samples was verified by reprobing these blots with a human β-actin cDNA probe. The probes were prepared using a random labeling kit (Stratagene) as specified by the manufacturer. The blots were visualized by using a Molecular Dynamics 400A PhosphorImager. Exposure times were as follows: 12 h for the cyclin D1 cDNA, 2 h for the cyclin B2 cDNA, and 20 min for the β-actin cDNA. Cyclin D1 protein expression was examined by immunoprecipitation of [35S]methionine-labeled cell lysates using either mouse polyclonal cyclin D1, D2, or D3 antiserum as described previously by others (28Matsushime H. Ewen M.E. Strom D.K. Kato J.-Y. Hanks S.K. Roussel M.F. Sherr C.J. Cell. 1992; 71: 323-334Abstract Full Text PDF PubMed Scopus (760) Google Scholar). As controls for these experiments, the mouse preimmune sera were used in all experiments, and an appropriate unlabeled cyclin D-GST fusion protein was used as a competitor to demonstrate specificity of the various D-type cyclin immunoprecipitations in DT40 cells (Fig. 4, panel A). Cells were selected in 2 mg/ml G418 or 1.5 mg/ml hygromycin and then maintained in cell culture under continuous drug selection at the same concentrations. For the growth curves, 2 × 105 cells were plated in triplicate and grown in the presence of 10% serum. Each point represents the average of three determinations ± the standard error of the mean. Growth curves were obtained for each of the cell lines for 7 days at 1-day intervals. DT40 cells were synchronized by first using centrifugal elutriation to enrich the G1 phase population of cells. These cells were then blocked at the G1/S boundary using 25 mg/ml aphidicolin for 10 h. These synchronized cells were removed from the drug and then placed into culture media containing BudR, harvested at 2-h intervals, and replicating (BudR +) cells stained and counted. Cytospin slides were prepared and stained with an anti-BudR antibody and a fluorescein labeled secondary antibody according to the protocol from the manufacturer (Boehringer Mannheim). The cells were also stained with 4′,6-diamidino-2-phenylindole, and the number of metaphase cells was determined as well. For the BudR incorporation and mitotic index experiments, two separate clonal D1 −/− cell lines were examined (clones 1 and 3). For cell cycle analysis, asynchronously growing cells were stained with propidium iodide, and their DNA content was determined using the fluorescence activated cell sorter. This analysis was performed as described previously (62Lahti J.M. Xiang J. Heath L.S. Campana D. Kidd V.J. Mol. Cell. Biol. 1995; 15: 1-11Crossref PubMed Scopus (191) Google Scholar) using asynchronously growing cultures by analyzing approximately 50,000 cells from polyclonal, or single cell, populations for each sample. The percentage of cells in each phase of the cell cycle was determined from the histograms using the MODFIT cell cycle analysis program. Rescue of the cyclin D1 −/− DT40 cells was accomplished by transfecting a clonal D1 −/− cell line (clone 1) with either a RSV-mouse CycD1 cDNA, RSV-mouse CycD2, RSV-mouse
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