Cyclin G2 Is Up-regulated during Growth Inhibition and B Cell Antigen Receptor-mediated Cell Cycle Arrest
1997; Elsevier BV; Volume: 272; Issue: 19 Linguagem: Inglês
10.1074/jbc.272.19.12650
ISSN1083-351X
AutoresMary C. Horne, Karen Donaldson, Gay Lynn Goolsby, David Tran, Michael Mulheisen, Johannes Hell, Alan F. Wahl,
Tópico(s)Cancer Research and Treatments
ResumoHuman cyclin G2 together with its closest homolog cyclin G1 defines a novel family of cyclins (Horne, M. C., Goolsby, G. L., Donaldson, K. L., Tran, D., Neubauer, M., and Wahl, A. F. (1996)J. Biol. Chem. 271, 6050–6061). Cyclin G2 is highly expressed in the immune system where immunologic tolerance subjects self-reactive lymphocytes to negative selection and clonal deletion via apoptosis. Here we investigated the effect of growth inhibitory signals on cyclin G2 mRNA abundance in different maturation stage-specific murine B cell lines. Upon treatment of wild-type and p53 null B cell lines with the negative growth factor, transforming growth factor β1, or the growth inhibitory corticosteroid dexamethasone, cyclin G2 mRNA levels were increased in a time-dependent manner 5–14-fold over control cell levels. Unstimulated immature B cell lines (WEHI-231 and CH31) and unstimulated or IgM B cell receptor (BCR) -stimulated mature B cell lines (BAL-17 and CH12) rapidly proliferate and express low levels of cyclin G2 mRNA. In contrast, BCR-stimulated immature B cell lines undergo growth arrest and coincidentally exhibit an ∼10-fold increase in cyclin G2 transcripts and a decrease in cyclin D2 message. Costimulation of WEHI-231 and CH31 cells with calcium ionophores and protein kinase C agonists partially mimics anti-IgM stimulation and elicits a strong up-regulation of cyclin G2 mRNA and down-regulation of cyclin D2 mRNA. Signaling mutants of WEHI-231 that are deficient in the phosphoinositide signaling pathway and consequently resistant to the BCR stimulus-induced growth arrest did not display a significant increase in cyclin G2 or decrease in cyclin D2 mRNAs when challenged with anti-IgM antibodies. The two polyclonal activators lipopolysaccharide and soluble gp39, which inhibit the growth arrest response of immature B cells, suppressed cyclin G2 mRNA expression induced by BCR stimulation. These results suggest that in murine B cells responding to growth inhibitory stimuli cyclin G2 may be a key negative regulator of cell cycle progression. Human cyclin G2 together with its closest homolog cyclin G1 defines a novel family of cyclins (Horne, M. C., Goolsby, G. L., Donaldson, K. L., Tran, D., Neubauer, M., and Wahl, A. F. (1996)J. Biol. Chem. 271, 6050–6061). Cyclin G2 is highly expressed in the immune system where immunologic tolerance subjects self-reactive lymphocytes to negative selection and clonal deletion via apoptosis. Here we investigated the effect of growth inhibitory signals on cyclin G2 mRNA abundance in different maturation stage-specific murine B cell lines. Upon treatment of wild-type and p53 null B cell lines with the negative growth factor, transforming growth factor β1, or the growth inhibitory corticosteroid dexamethasone, cyclin G2 mRNA levels were increased in a time-dependent manner 5–14-fold over control cell levels. Unstimulated immature B cell lines (WEHI-231 and CH31) and unstimulated or IgM B cell receptor (BCR) -stimulated mature B cell lines (BAL-17 and CH12) rapidly proliferate and express low levels of cyclin G2 mRNA. In contrast, BCR-stimulated immature B cell lines undergo growth arrest and coincidentally exhibit an ∼10-fold increase in cyclin G2 transcripts and a decrease in cyclin D2 message. Costimulation of WEHI-231 and CH31 cells with calcium ionophores and protein kinase C agonists partially mimics anti-IgM stimulation and elicits a strong up-regulation of cyclin G2 mRNA and down-regulation of cyclin D2 mRNA. Signaling mutants of WEHI-231 that are deficient in the phosphoinositide signaling pathway and consequently resistant to the BCR stimulus-induced growth arrest did not display a significant increase in cyclin G2 or decrease in cyclin D2 mRNAs when challenged with anti-IgM antibodies. The two polyclonal activators lipopolysaccharide and soluble gp39, which inhibit the growth arrest response of immature B cells, suppressed cyclin G2 mRNA expression induced by BCR stimulation. These results suggest that in murine B cells responding to growth inhibitory stimuli cyclin G2 may be a key negative regulator of cell cycle progression. Proliferation signals promote the coordinated progression of a cell through the cell division cycle. In eukaryotes this process is controlled by the sequential formation, activation, and inhibition of cyclin-cyclin-dependent kinase (CDK) 1The abbreviations used are: CDK, cyclin-dependent kinase; CDKI, cyclin-dependent kinase inhibitor; ORF, open reading frame; PCD, programmed cell death; BCR, B cell receptor; LPS, lipopolysaccharide; Ca2+i, cytoplasmic calcium; DAG, diacylglycerol; PdBu, phorbol 12, 13-dibutyrate; gp, glycoprotein; TGF-β, transforming growth factor β1; PKC, protein kinase C; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. complexes (1Pines J. Adv. Cancer Res. 1995; 66: 181-212Crossref PubMed Google Scholar). Active cyclin-CDK complexes phosphorylate specific targets such as the tumor suppressor RB, various transcription factors, DNA polymerase α, and cytoskeletal proteins (2Nigg E.A. Curr. Opin. Cell Biol. 1993; 5: 187-193Crossref PubMed Scopus (220) Google Scholar) and thus trigger progression through the cell cycle. 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Chem. 1996; 271: 6050-6061Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The mRNAs for human cyclins G1 and G2 are strongly expressed in tissues rich in terminally differentiated cells (cardiac and skeletal muscle for cyclin G1 and cerebellum for cyclin G2) and tissues populated with cells subjected to PCD (spleen and thymus). Murine cyclin G1 mRNA is expressed independently of p53 in a number of tissues of p53 null mice (e.g. brain, heart muscle, and stomach) and can be up-regulated in a p53 null murine B cell line by TGF-β treatment (6Horne M.C. Goolsby G.L. Donaldson K.L. Tran D. Neubauer M. Wahl A.F. J. Biol. Chem. 1996; 271: 6050-6061Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). While cyclin G1 mRNA is constitutively expressed and encodes a protein with no prototypic "destruction box" involved in ubiquitin-dependent degradation (42Glotzer M. Murray A.W. Kirschner M.W. 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We cloned the cDNA encoding the murine form of cyclin G2 and investigated its expression pattern in rodent tissues and various murine cell lines. Our results indicate that cyclin G2 transcripts are present at high levels in murine B cells treated with agents causing growth inhibition or growth arrest but not in cells receiving a positive costimulus that promotes cell cycle progression. In contrast, we found that transcripts for cyclin D2, the D-type G1-phase cyclin associated with proliferation in B cells (44Tanguay D.A. Chiles T.C. J. Immunol. 1996; 156: 539-548PubMed Google Scholar), are down-regulated during G1-phase growth arrest. The up-regulation of cyclin G2 mRNA during cell cycle arrest and its expression in terminally differentiated tissues suggest that this cyclin, and perhaps the related cyclins G1 and I, may function in specific contexts as negative coordinators of cell cycle progression. Phorbol 12,13-dibutyrate (PdBu), ionomycin (calcium salt), and propidium iodide were obtained from Calbiochem. Lipopolysaccharide Escherichia coli serotype 0111:B4 (LPS), porcine transforming growth factor β1 (TGF-β), dexamethasone and 5-bromo 2′-deoxyuridine (BrDu) were purchased from Sigma. Fluorescein isothiocyanate-conjugated goat anti-BrDu antibodies were obtained from Becton-Dickinson (Mountain View, CA.) and μ chain-specific F(ab′)2 goat anti-IgM antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). COS cell supernatants containing soluble gp39 was the generous gift of Dr. Diane Hollenbaugh (Bristol-Myers Squibb) (45Kiener P.A. Moran-Davis P. Rankin B.M. Wahl A.F. Aruffo A. Hollenbaugh D. J. Immunol. 1995; 155: 4917-4925Crossref PubMed Google Scholar). The murine B cell lines WEHI-231 (46Lanier L.L. Warner N.L. J. Immunol. 1981; 126: 626-631PubMed Google Scholar) and BAL-17 (47Kim K.J. Kanellopoulos-Langevin C. Merwin R.M. Sachs D.H. Asofsky R. J. 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The resulting polymerase chain reaction fragments were purified by agarose gel electrophoresis using the GeneClean II® DNA purification kit (Bio 101, La Jolla, CA) and used to screen a λ Zap II murine thymus cDNA library (Stratagene, La Jolla, CA). Cross-species screening of the murine cDNA library with human cyclin G2 cDNA probes was done at low stringency with the hybridization buffer containing 30% formamide and filters hybridized and washed at 37 °C. Development of the filters with alkaline phosphatase-conjugated anti-digoxigenin antibodies and the Lumi-Phos™ 530 reagent (Boehringer Mannheim) was done according to manufacturer's protocol. The isolated phagemid DNA was amplified, extracted, and purified for sequence analysis following the manufacturer's recommended methods and standard techniques (50Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). DNA sequences were determined using the Sequenase™ (version 2.0) system following procedures recommended by the manufacturer (United States Biochemical Corp.). cDNA fragments present in the Lambda Zap® II phagemid (Stratagene) cloning vectors were sequenced from reactions primed with either a vector-specific oligonucleotide or oligonucleotides homologous to the cloned fragment's internal sequences. [α33P]dATP or [α32P]dATP (at 800 Ci/mmol) was used to radioactively label the DNA fragments. Nucleotide sequences were read from scanned gels with the aid of BioImage® sequence analysis software. The computer-aided editing and alignment of DNA sequences was accomplished using Genetics Computer Group (GCG) (Madison, WI) sequence analysis software. Additional nucleotide and cDNA-derived peptide sequence comparisons were performed using the BLAST program. Final alignments were performed using the GCG Pileup and Pretty programs. Murine lymphocytes were separated into progressive stages of the cell cycle by centrifugal elutriation, and the cell cycle position of elutriated fractions was determined as described previously (6Horne M.C. Goolsby G.L. Donaldson K.L. Tran D. Neubauer M. Wahl A.F. J. Biol. Chem. 1996; 271: 6050-6061Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Unelutriated cell populations stimulated with different reagents were examined using dual-parameter flow cytometric analysis of total DNA content and newly incorporated BrDu. Briefly, following a 20–30-min pulse of a culture with 10 μm BrDu, ∼2.0 × 106 cells were sedimented by centrifugation, washed in phosphate-buffered saline and fixed with ice-cold 70% ethanol, and stored at 4 °C until staining and analysis could be performed. The permeabilization and staining with propidium iodide and fluorescein isothiocyanate-conjugated anti-BrDu antibodies were done following protocols supplied by Becton-Dickinson. Flow cytometry was performed utilizing either a FACScan and Lysis II software (Becton-Dickinson Instruments, San Jose, CA) or Coulter EPICS Profile II Analyzer with Multigraph and MultiCycle software (Coulter Electronics, Miami, FL). Total RNA was isolated from murine tissues and cells utilizing TRIzol® reagent (Life Technologies, Inc.). The glyoxal denaturation of total RNA and electrophoresis in glyoxal-agarose was done following a standard protocol (50Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). After electrophoresis, the relative amount and quality of the RNA was controlled by short wave UV fluorescent shadowing of the ribosomal RNAs on a F-254 TLC plate. The fractionated RNAs were transferred and fixed to MagnaGraph® nylon membranes (MSI, Westboro, MA) followed by removal of residual glyoxal as described (50Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Membranes were routinely stained with methylene blue to control for RNA transfer efficiency as described (50Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The [α32P]dTTP and [α32P]dCTP radioactive labeling of DNA fragments was done using polymerase chain reaction generated and GeneClean II®-isolated DNA fragments as templates and reagents obtained from the Life Technologies, Inc. random priming kit. Hybridization of DNA probes to Northern blots was done according to the methods described by the manufacturer of the nylon membrane and standard protocols (50Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). PhosphorImaging (Molecular Dynamics) of the washed Northern blot filters was routinely obtained immediately before autoradiography. All Northern blot experiments were performed at least twice with reproducible results. Hybridization screening of a murine thymic cDNA library with an internal cDNA fragment of the human cyclin G2 ORF (6Horne M.C. Goolsby G.L. Donaldson K.L. Tran D. Neubauer M. Wahl A.F. J. Biol. Chem. 1996; 271: 6050-6061Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) at low stringency identified 15 independent overlapping partial cDNA clones encompassing the full murine cyclin G2 ORF. One clone comprised nearly the full ORF lacking only the first two nucleotides of the translation initiation codon. In contrast to the human cDNA fragments of cyclin G2 cloned from a Jurkat lambda ZapII cDNA library (6Horne M.C. Goolsby G.L. Donaldson K.L. Tran D. Neubauer M. Wahl A.F. J. Biol. 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Hayakawa T. Nojima H. Okayama H. Oncogene. 1993; 8: 2113-2118PubMed Google Scholar), which is homologous to the epidermal growth factor and polyoma virus middle T antigen autophosphorylation sites (6Horne M.C. Goolsby G.L. Donaldson K.L. Tran D. Neubauer M. Wahl A.F. J. Biol. Chem. 1996; 271: 6050-6061Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 51Tamura K. Kanaoka Y. Jinno S. Nagata A. Ogiso Y. Shimizu K. Hayakawa T. Nojima H. Okayama H. Oncogene. 1993; 8: 2113-2118PubMed Google Scholar, 52Kavanaugh W.M. Turck C.W. Williams L.T. Science. 1995; 268: 1177-1179Crossref PubMed Scopus (225) Google Scholar). The murine cyclin G2 and murine cyclin A proteins share 47% similarity and 26% identity. Our analysis indicates that human cyclin I, a cyclin of unknown function highly expressed in brain and skeletal muscle (7Nakamura T. Sanokawa R. Sasaki Y.F. Ayusawa D. Oishi M. Mori N. Exp. Cell Res. 1995; 221: 534-542Crossref PubMed Scopus (74) Google Scholar), is more related to full-length human cyclins G1 and G2 (52% similarity and 30% identity for both) than to cyclin A (46% similarity and 24% identity). Crystallography of cyclin A has defined a new structural motif consisting of two tandem repeats of a five-helix bundle referred to as the "cyclin fold" (10Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1225) Google Scholar, 53Brown N.R. Noble M.E. Endicott J.A. Garman E.F. Wakatsuki S. Mitchell E. Rasmussen B. Hunt T. Johnson L.N. Structure. 1995; 3: 1235-1247Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 54Russo A.A. Jeffrey P.D. Patten A.K. Massague J. Pavletich N.P. Nature. 1996; 382: 325-331Crossref PubMed Scopus (805) Google Scholar). The first repeat spans the cyclin box (α1–α5, see Fig. 1) immediately followed by the second (α1′–α5′). The amino acid identity between the cyclin G family and cyclin A is highest in the cyclin box region, yet it extends to the amino- and carboxyl-terminal regions suggesting that the G family of cyclins possess a cyclin fold structure similar to cyclin A (Fig. 1). The amino-terminal region of cyclin A contributes important residues for CDK binding and structural integrity of cyclin A and is likely to have a similar function for cyclins G1, G2, and I. Cyclin A residues Arg-211 and Asp-240 form a buried salt bridge connecting helix 1 with helix 2 and are essential for cyclin A-CDK activity (9Lees E.M. Harlow E. Mol. Cell. Biol. 1993; 13: 1194-1201Crossref PubMed Scopus (124) Google Scholar, 10Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1225) Google Scholar, 53Brown N.R. Noble M.E. Endicott J.A. Garman E.F. Wakatsuki S. Mitchell E. Rasmussen B. Hunt T. Johnson L.N. Structure. 1995; 3: 1235-1247Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 55Stewart E. Kobayashi H. Harrison D. Hunt T. EMBO J. 1994; 13: 584-594Crossref PubMed Scopus (93) Google Scholar). The equivalent residues in the sequences of cyclins G1, G2, and I are conserved, as they are among most cyclins (3Hunt T. Semin. Cell Biol. 1991; 2: 213-222PubMed Google Scholar). Amino acids at position 266 (lysine) and 295 (glutamate) in cyclin A are crucial CDK contact residues (10Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1225) Google Scholar, 53Brown N.R. Noble M.E. Endicott J.A. Garman E.F. Wakatsuki S. Mitchell E. Rasmussen B. Hunt T. Johnson L.N. Structure. 1995; 3: 1235-1247Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar) and are maintained in cyclins G1 and G2 and human cyclin I, although the residues equivalent to the cyclin A 266 lysine in murine and human cyclin G2 have been conservatively exchanged with an arginine (Fig. 1). A
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