Ras orchestrates exit from the cell cycle and light-chain recombination during early B cell development.

Signals through the pre-B cell antigen receptor (pre-BCR) and interleukin 7 receptor (IL-7R) coordinate pre-B cell population expansion with subsequent recombination of the locus encoding immunoglobulin kappa-chain (Igk). Although many 'downstream' effectors of each receptor are known, how they integrate to mediate development has remained unclear. Here we report that pre-BCR-mediated activation of the Ras-MEK-Erk signaling pathway silenced transcription of Ccnd3 (encoding cyclin D3) and coordinated exit from the cell cycle with induction of the transcription factor E2A and the initiation of Igk recombination. IL-7R-mediated activation of the transcription factor STAT5 opposed this pathway by promoting Ccnd3 expression and concomitantly inhibiting Igk transcription by binding to the Igk intronic enhancer and preventing E2A recruitment. Our data show how pre-BCR signaling poises pre-B cells to undergo differentiation after escape from IL-7R signaling.

Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis.

Precise cell-cycle control is critical for plant development and responses to pathogen invasion. Two homologous cyclin-dependent kinase inhibitor genes, SIAMESE (SIM) and SIM-RELATED 1 (SMR1), were recently shown to regulate Arabidopsis (Arabidopsis thaliana) defense based on phenotypes conferred by a sim smr1 double mutant. However, whether these two genes play differential roles in cell-cycle and defense control is unknown. In this report, we show that while acting synergistically to promote endoreplication, SIM and SMR1 play different roles in affecting the ploidy of trichome and leaf cells, respectively. In addition, we found that the smr1-1 mutant, but not sim-1, was more susceptible to a virulent Pseudomonas syringae strain, and this susceptibility could be rescued by activating salicylic acid (SA)-mediated defense. Consistent with these results, smr1-1 partially suppressed the dwarfism, high SA levels, and cell death phenotypes in acd6-1, a mutant used to gauge the change of defense levels. Thus, SMR1 functions partly through SA in defense control. The differential roles of SIM and SMR1 are due to differences in temporal and spatial expression of these two genes in Arabidopsis tissues and in response to P. syringae infection. In addition, flow-cytometry analysis of plants with altered SA signaling revealed that SA is necessary, but not sufficient, to change cell-cycle progression. We further found that a mutant with three CYCD3 genes disrupted also compromised disease resistance to P. syringae. Together, this study reveals differential roles of two homologous cyclin-dependent kinase inhibitors in regulating cell-cycle progression and innate immunity in Arabidopsis and provides insights into the importance of cell-cycle control during host-pathogen interactions.

[Gateway Reflex, a regulator of the inflammation feedback loop by regional neural activation].

Inflammation is observed in many diseases and disorders. We discovered a key machinery of inflammation, the inflammation amplifier, which is induced by the simultaneous activation of NFκB and STAT3 followed by the hyper-activation of NFκB in non-immune cells, including endothelial cells and fibroblasts. Since that discovery, we found the Gateway Reflex, which describes regional neural activations that enhance the inflammation amplifier to create a gateway for immune cells to bypass the blood-brain barrier. In addition, we have identified over 1,000 positive regulators and over 500 targets of the inflammation amplifier, which include a significant numbers of human disease-associated genes. In parallel, we performed a comprehensive analysis of human disease samples and found that the inflammation amplifier was activated during the development of chronic inflammation. Thus, we concluded that the inflammation amplifier is associated with various human diseases and disorders, including autoimmune diseases, metabolic syndromes, neurodegenerative diseases, and other inflammatory diseases. We are now attempting drug discovery for inflammatory diseases and disorders based on the inflammation amplifier and Gateway Reflex. In this review, we discuss the Gateway Reflex as an example for the neuro-immune interaction in vivo.

Deficiency of the cyclin kinase inhibitor p21(WAF-1/CIP-1) promotes apoptosis of activated/memory T cells and inhibits spontaneous systemic autoimmunity.

A characteristic feature of systemic lupus erythematosus is the accumulation of activated/memory T and B cells. These G0/G1-arrested cells express high levels of cyclin-dependent kinase inhibitors such as p21, are resistant to proliferation and apoptosis, and produce large amounts of proinflammatory cytokines. Herein, we show that ablation of p21 in lupus-prone mice allows these cells to reenter the cell cycle and undergo apoptosis, leading to autoimmune disease reduction. Absence of p21 resulted in enhanced Fas/FasL-mediated activation-induced T cell death, increased activation of procaspases 8 and 3, and loss of mitochondrial transmembrane potential. Increased apoptosis was also associated with p53 up-regulation and a modest shift in the ratio of Bax/Bcl-2 toward the proapoptotic Bax. Proliferation and apoptosis of B cells were also increased in p21-/- lupus mice. Thus, modulation of the cell cycle pathway may be a novel approach to reduce apoptosis-resistant pathogenic lymphocytes and to ameliorate systemic autoimmunity.

Roles of the tumor suppressor p53 and the cyclin-dependent kinase inhibitor p21WAF1/CIP1 in receptor-mediated apoptosis of WEHI 231 B lymphoma cells.

Treatment of WEHI 231 immature B lymphoma cells with an antibody against their surface immunoglobulin M (anti-IgM) induces apoptosis and has been studied extensively as a model of self-induced B cell tolerance. Since the tumor suppressor protein p53 has been implicated in apoptosis in a large number of cell types and has been found to be mutated in a variety of B cell tumors, here we sought to determine whether p53 and the p53 target gene cyclin-dependent kinase inhibitor p21(WAF1/CIP1) were involved in anti-IgM-induced cell death. Anti-IgM treatment of WEHI 231 cells increased expression of p53 and p21 protein levels. Ectopic expression of wild-type p53 in WEHI 231 cells induced both p21 expression and apoptosis. Ectopic expression of p21 similarly induced apoptosis. Rescue of WEHI 231 cells from apoptosis by costimulation with CD40 ligand ablated the increase in p21 expression. Lastly, a significant decrease in anti-IgM-mediated apoptosis was seen upon downregulation of endogenous p53 activity by expression of a dominant-negative p53 protein or upon microinjection of an antisense p21 expression vector or antibody. Taken together, the above data demonstrate important roles for p53 and p21 proteins in receptor-mediated apoptosis of WEHI 231 B cells.

p35, the non-cyclin activator of Cdk5, protects podocytes against apoptosis in vitro and in vivo.

Cyclin-dependent kinase-5 is widely expressed and predominantly regulated by the non-cyclin activator p35. Since we recently showed that expression of p35 in the kidney is restricted to podocytes, we examined here its function in mice in which p35 was genetically deleted. The mice did not exhibit kidney abnormalities during glomerular development or during adult life. Conditionally immortalized cultured podocytes, derived from these null mice, did not have any change in their morphology, differentiation, or proliferation. However, when these cultured podocytes were exposed to UV-C irradiation, serum depletion, puromycin aminonucleoside, or transforming growth factor-beta-1, they showed increased apoptosis compared to those from wild-type mice. Levels of Bcl-2 were decreased in these null podocytes but increased after transduction with human p35. Restoration of p35 or the ectopic expression of Bcl-2 reduced the susceptibility of p35-null podocytes to apoptosis. Experimental glomerulonephritis, characterized by podocyte apoptosis and subsequent crescent formation, was utilized to test these findings in vivo. Podocyte apoptosis was significantly increased in diseased p35-null compared with wild-type mice, accompanied by increased glomerulosclerosis and decreased renal function. Our study shows that p35 does not affect glomerulogenesis but controls podocyte survival following injury, in part, by regulating Bcl-2 expression.

Foxo3-/- mice demonstrate reduced numbers of pre-B and recirculating B cells but normal splenic B cell sub-population distribution.

B cell antigen receptor (BCR) cross-linking promotes proliferation and survival of mature B cells. Phosphoinositide-3-kinase-mediated down-regulation of pro-apoptotic and anti-mitogenic genes such as the Foxo family of transcription factors is an important component of this process. Previously, we demonstrated that BCR signaling decreases expression of transcripts for Foxo1, Foxo3 and Foxo4. We now show that BCR-induced down-regulation of Foxo3 and Foxo4 mRNA expression occurs via distinct mechanisms from those established for Foxo1. While Foxo1, Foxo3 and Foxo4 bind the same DNA sequence, the differential control of their expression upon B cell activation suggests that they may have unique functions in the B lineage. To begin to address this issue, we evaluated B cell development and function in Foxo3-/- mice. No effect of Foxo3 deficiency was observed with respect to the following parameters in the splenic B cell compartment: sub-population distribution, proliferation, in vitro differentiation and expression of the Foxo target genes cyclin G2 and B cell translocation gene 1. However, Foxo3-/- mice demonstrated increased basal levels of IgG2a, IgG3 and IgA. A significant reduction in pre-B cell numbers was also observed in Foxo3-/- bone marrow. Finally, recirculating B cells in the bone marrow and peripheral blood were decreased in Foxo3-/- mice, perhaps due to lower than normal expression of receptor for sphingosine-1 phosphate, which mediates egress from lymphoid organs. Thus, Foxo3 makes a unique contribution to B cell development, B cell localization and control of Ig levels.

Two unique HLA-A*0201 restricted peptides derived from cyclin E as immunotherapeutic targets in leukemia.

Immunotherapy targeting leukemia-associated antigens has shown promising results. Because of the heterogeneity of leukemia, vaccines with a single peptide have elicited only a limited immune response. Targeting several peptides together elicited peptide-specific cytotoxic T lymphocytes (CTLs) in leukemia patients, and this was associated with clinical responses. Thus, the discovery of novel antigens is essential. In the current study, we investigated cyclin E as a novel target for immunotherapy. Cyclin E1 and cyclin E2 were found to be highly expressed in hematologic malignancies, according to reverse transcription polymerase chain reaction and western blot analysis. We identified two HLA-A*0201 binding nonameric peptides, CCNE1M from cyclin E1 and CCNE2L from cyclin E2, which both elicited the peptide-specific CTLs. The peptide-specific CTLs specifically kill leukemia cells. Furthermore, CCNE1M and CCNE2L CTLs were increased in leukemia patients who underwent allogeneic hematopoietic stem cell transplantation, and this was associated with desired clinical outcomes. Our findings suggest that cyclin E1 and cyclin E2 are potential targets for immunotherapy in leukemia.

Molecular characterization of mammalian cylicin, a basic protein of the sperm head cytoskeleton.

The cytoskeletal calyx structure surrounding part of the nucleus of the mammalian sperm head contains two major kinds of basic proteins, i.e., the approximately 60-kD calicin and a group of very basic (IEP > pH 10) polypeptides ranging in size from approximately 58 to approximately 100 kD ("multiple band proteins," MBPs). We have produced MBP-specific mAbs and have isolated a bovine and a human cDNA clone encoding one of these proteins, termed "cylicin" (from the Greek word c eta kv lambda l zeta for cup or beaker). Bovine cylicin I of a calculated molecular weight of 74,788 contains a high proportion (29%) of positively charged amino acids, resulting in an IEP of 10.55, numerous KKD tripeptides, and is characterized by an organization of the central part of the molecule in nine repeating units of maximally 41 amino acids each of which according to prediction analysis should tend to form an alpha helix. The identity of the polypeptide has been proven by direct amino acid sequencing of > 14 different fragments and by experiments using antibodies raised against a partial cDNA-derived protein segment produced in E. coli. By Northern blot analysis we have identified the 2.4-kb cylicin I mRNA only in testis. The unusual cytoskeletal protein cylicin is compared with other proteins and its possible architectural role during spermiogenesis is discussed.

Early events in DNA replication require cyclin E and are blocked by p21CIP1.

Using immunodepletion of cyclin E and the inhibitor protein p21WAF/CIP1, we demonstrate that the cyclin E protein, in association with Cdk2, is required for the elongation phase of replication on single-stranded substrates. Although cyclin E/Cdk2 is likely to be the major target by which p21 inhibits the initiation of sperm DNA replication, p21 can inhibit single-stranded replication through a mechanism dependent on PCNA. While the cyclin E/Cdk2 complex appears to have a role in the initiation of DNA replication, another Cdk kinase, possibly cyclin A/Cdk, may be involved in a later step controlling the switch from initiation to elongation. The provision of a large maternal pool of cyclin E protein shows that regulators of replication are constitutively present, which explains the lack of a protein synthesis requirement for replication in the early embryonic cell cycle.

Interaction between human cyclin A and adenovirus E1A-associated p107 protein.

The products of the adenovirus early region 1A (E1A) gene are potent oncoproteins when tested in standard transformation and immortalization assays. Many of the changes induced by E1A may be due to its interaction with cellular proteins. Four of these cellular proteins are the retinoblastoma protein (pRB), p107, cyclin A, and p33cdk2. The pRB and p107 proteins are structurally related and have several characteristics in common, including that they both bind to the SV40 large T oncoprotein as well as to E1A. Cyclin A and p33cdk2 are thought to function in the control of the cell cycle. They bind to one another, forming a kinase that closely resembles the cell cycle-regulating complexes containing p34cdc2. Cyclin A is now shown to bind to p107 in the absence of E1A. The association of p107 with cyclin A suggests a direct link between cell cycle control and the function of p107.

Measurement of changes in Cdk2 and cyclin o-associated kinase activity in apoptosis.

Many cell cycle regulatory proteins have been shown to be able to regulate cell death. Activation of Cdk2 has been shown to be necessary for apoptosis of quiescent cells such as thymocytes, neurons, and endothelial cells. This activation is stimulus-specific because it occurs in glucocorticoid and DNA damage but not in CD95-induced apoptosis in thymocytes. Apoptotic Cdk2 activation in lymphoid cells is controlled by a recently identified protein, cyclin O, and its activity is modulated by p53 and members of the Bcl-2 protein family. In this chapter, we describe methods for measuring changes in Cdk2 activity during apoptosis. In addition, we also show the details of the generation of an antibody able to immunoprecipitate the cyclin O complexes from apoptotic cells in native conditions and its use to measure the kinase activity associated with this proapoptotic cyclin.

Cell cycle arrest effects of large-dose FTY720 on lymphocytes in mouse skin transplantation models.

To probe into the mechanism of immunosuppression of FTY720, the authors study the cell cycle arrest effects of large-dose FTY720 on lymphocytes in mouse skin transplantation models and the expression of cell cycle-related factors in cell cycle regulation system in mouse skin allograft models using flow cytometry, Immunohistochemical staining, and reverse transcriptase-polymerase chain reaction (RT-PCR). FTY720 could prolong survival days of graft in mouse skin transplantation models obviously (p < 0.01). In thymus gland and lymph node, compared with the control group, cell percentage in G0-G1 increases and cell percentage in G2-M and S decreases in the treated group (p < 0.01). Expression of P16 increases and expression of cyclin D1 decreases in the treated group (p < 0.05). In thymus gland, it is shown by semiquantitative RT-PCR that the quantity of mRNA of P16 increases in the treated group (p < 0.01), and the quantity of mRNA of cyclin D1 decreases in the treated group (p < 0.01). FTY720 is a kind of effective immunosuppressant. FTY720 could induce cell cycle arrest in thymus gland and lymph node and change the expression of protein and the transcription of mRNA of cell cycle-related factor.

Cyclin D1 is dispensable for G1 control in retinoblastoma gene-deficient cells independently of cdk4 activity.

To elucidate the regulator-versus-target relationship in the cyclin D1/cdk4/retinoblastoma protein (pRB) pathway, we examined fibroblasts from RB-1 gene-deficient and RB-1 wild-type littermate mouse embryos (ME) and in human tumor cell lines that differed in the status of the RB-1 gene. The RB+/+ and RB-/- ME fibroblasts expressed similar protein levels of D-type cyclins, cdk4, and cdk6, showed analogous spectra and abundance of cellular proteins complexed with cdk4 and/or cyclins D1 and D2, and exhibited comparable associated kinase activities. Of the two human cell lines established from the same sarcoma biopsy, the RB-positive SKUT1B cells contained cdk4 that was mainly associated with D-type cyclins, contrary to a predominant cdk4-p16INK4 complex in the RB-deficient SKUT1A cells. Antibody-mediated neutralization of cyclin D1 arrested the RB-positive ME and SKUT1B cells in G1, whereas this cyclin appeared dispensable in the RB-deficient ME and SKUT1A cells. Lack of requirement for cyclin D1 therefore correlated with absence of functional pRB, regardless of whether active cyclin D1/cdk4 holoenzyme was present in the cells under study. Consistent with a potential role of cyclin D/cdk4 in phosphorylation of pRB, monoclonal anti-cyclin D1 antibodies supporting the associated kinase activity failed to significantly affect proliferation of RB-positive cells, whereas the antibody DCS-6, unable to coprecipitate cdk4, efficiently inhibited G1 progression and prevented pRB phosphorylation in vivo. These data provide evidence for an upstream control function of cyclin D1/cdk4, and a downstream role for pRB, in the order of events regulating transition through late G1 phase of the mammalian cell division cycle.

p63cdc13, a B-type cyclin, is associated with both the nucleolar and chromatin domains of the fission yeast nucleus.

The cellular distribution of the fission yeast mitotic cyclin B, p63cdc13, was investigated by a combination of indirect immunofluorescence light microscopy, immunogold electron microscopy, and nuclear isolation and fractionation. Immunofluorescence microscopy of wild-type cells and the cold-sensitive mutant dis2.11 with a monospecific anti-p63cdc13 antiserum was consistent with the association of a major subpopulation of fission yeast M-phase protein kinase with the nucleolus. Immunogold electron microscopy of freeze-substituted wild-type cells identified two nuclear populations of p63cdc13, one associated with the nucleolus, the other with the chromatin domain. To investigate the cell cycle regulation of nuclear labeling, the mutant cdc25.22 was synchronized through mitosis by temperature arrest and release. Immunogold labeling of cells arrested at G2M revealed gold particles present abundantly over the nucleolus and less densely over the chromatin region of the nucleus. Small vesicles around the nucleus were also labeled by anti-p63cdc13, but few gold particles were detected over the cytoplasm. Labeling of all cell compartments declined to zero through mitosis. Cell fractionation confirmed that p63cdc13 was substantially enriched in both isolated nuclei and in a fraction containing small vesicles and organelles. p63cdc13 was not extracted from nuclei by treatment with RNase A, Nonidet P40 (NP-40), Triton X-100, and 0.1 M NaCl, although partial solubilization was observed with DNase I and 1 M NaCl. A known nucleolar protein NOP1, partitioned in a similar manner to p63cdc13, as did p34cdc2, the other subunit of the M-phase protein kinase. We conclude that a major subpopulation of the fission yeast mitotic cyclin B is targeted to structural elements of the nucleus and nucleolus.

Cyclin D(1) and D(3) expression in vestibular schwannomas.

OBJECTIVES: The G1 regulators of the cell cycle, cyclin D(1) and D(3), have been implicated in the regulation of Schwann cell proliferation and differentiation. The purpose of this study is to evaluate cyclin D(1) and D(3) protein expression and the corresponding clinical characteristics of vestibular schwannomas. STUDY DESIGN AND METHODS: Tissue sections of 15 sporadic vestibular schwannomas were prepared. Immunohistochemical analysis of the vestibular schwannomas was performed with anticyclin D(1) and anticyclin D(3) antibodies. The immunoreactivity was evaluated in comparison with adjacent vestibular nerves. Tissue sections of breast carcinoma and prostate carcinoma were used as positive controls for cyclin D(1) and D(3) staining, respectively. Patient demographics, tumor characteristics, and cyclin D expression were reviewed, and statistical analysis was performed. RESULTS: While the breast carcinoma control expressed abundant cyclin D(1) protein, none of the 15 vestibular schwannomas showed detectable cyclin D(1) staining. In contrast, seven of 15 vestibular schwannomas stained positive for the cyclin D(3) protein. Cyclin D(3) staining was taken up in the nucleus of schwannoma tumor cells in greater proportion than Schwann cells of adjacent vestibular nerve. Although sample size was small, no significant difference in the average age of presentation, tumor size, and male to female ratios for the cyclin D(3)(+) or cyclin D(3)(-) groups was found. CONCLUSION: The Cyclin D(1) protein does not appear to play a prominent role in promoting cell cycle progression in vestibular schwannomas. In contrast, cyclin D(3) expression was seen in nearly half of the tumors examined, suggesting that it may have a growth-promoting role in some schwannomas. Further studies are needed to define its cellular mechanism.

Antisense oligonucleotides selected by hybridisation to scanning arrays are effective reagents in vivo.

Transcripts representing mRNAs of three Xenopus cyclins, B1, B4 and B5, were hybridised to arrays of oligonucleotides scanning the first 120 nt of the coding region to assess the ability of the immobilised oligonucleotides to form heteroduplexes with their targets. Oligonucleotides that produced high heteroduplex yield and others that showed little annealing were assayed for their effect on translation of endogenous cyclin mRNAs in Xenopus egg extracts and their ability to promote cleavage of cyclin mRNAs in oocytes by RNase H. Excellent correlation was found between antisense potency and affinity of oligonucleotides for the cyclin transcripts as measured by the array, despite the complexity of the cellular environment.

The immunohistochemical outline of p27kip1, cyclin B1 and cyclin D3 in pleomorphic adenomas.

AIM: Pleomorphic adenomas of salivary glands are benign lesions which may sometimes relapse even after complete surgical removal. This risk has led to the search for methods to provide predictive data on the biological behaviour of such neoplasia. The authors intend to evaluate the degree of cellular aggression of these tumours by finding prognostic data using the antigens involved in cellular proliferative activity. Therefore they have chosen for this study: p27kip1, cyclin B1 and Cyclin D3. METHODS: Seventeen mixed tumours, 2 of them relapsed, underwent the direct immunohystochemical PAP technique for the determination of antigens p27kip1, cyclins B1 and D3 of the tissue. RESULTS: The results obtained show that the verification of these markers may reveal a potential risk of biological deviation and that their expression is independent of the degree of cellularity in neoplasias. CONCLUSIONS: On the basis of the results, the conclusion is drawn that there is no relation between the expressivity of the mentioned antigens and histological characters of pleomorphic adenomas.

The cdk2 kinase is required for the G1-to-S transition in mammalian cells.

In the cell cycle of fission and budding yeast, the p34cdc2/CDC28 kinase is required for both the G1-to-S and G2-to-M phase transitions. In vertebrates, the homologous p34cdc2 kinase is required for G2-to-M phase transitions but appears to be dispensable for DNA synthesis. We have investigated the function of a related kinase, p33cdk2, using serum-stimulated quiescent human fibroblasts. While the p33cdk2 protein was expressed at constant levels throughout the cell cycle, p33cdk2 kinase activity was first detected a few hours prior to the onset of DNA synthesis. Microinjection of anti-p33cdk2 antibodies blocked cells from entering S phase. Pre-adsorption of these antibodies with cdk2 protein abrogated their blocking effect suggesting that the G1 arrest caused by these antibodies is cdk2-specific. These results indicate that p33cdk2 is required for the G1-to-S phase transition in mammalian cells. We also show evidence to suggest that the cyclin E/p33cdk2 complex is likely to be required for entry into S phase since the timing of the cyclin E-associated kinase activity was coincident with that of p33cdk2 and preclearing of either component abolished the majority of the histone H1 kinase activity present in the lysates harvested from the late G1.

Cyclin C/CDK8 is a novel CTD kinase associated with RNA polymerase II.

A number of cyclin/kinase complexes have been identified in mammalian cells that are essential for controlled cell proliferation. Cyclin C was isolated by virtue of its ability to rescue the triple CLN mutation in yeast; however, until now its function has remained unclear. Cyclin C associates with a novel cyclin dependent kinase, CDK8, and we demonstrate that this complex is associated with kinase activity towards the carboxy-terminal domain (CTD) of RNA polymerase II. We have identified at least two distinct cyclin C/CDK8 containing complexes within the cell, a larger complex over 500 kD in size, that also contains the largest subunit of RNA polymerase II, and a smaller 170 kD species. Both of these cyclin C complexes retain potent CTD kinase activity. We further demonstrate that the cyclin C/CDK8 complex associates with the large subunit of RNA polymerase II in vivo, implicating a potential role for cyclin C/CDK8 in regulating its activities.