Measurement of Cdk1/Cyclin B Kinase Activity by Specific Antibodies and Western Blotting.

Quantitative measurement of enzyme activity is a valuable approach to study how cells function. We present a method to measure the activity of the enzyme Cdk1/cyclin B. This enzyme is required by all eukaryotic cells to enter mitosis. Therefore, a biochemical assay to measure Cdk1/cyclin B activity can be used to identify cell populations that are in mitosis or to detect inhibitors of Cdk1/Cyclin B in vitro. A key distinction of the method presented here, compared to others, is that it uses a recombinant protein, a specific antibody, and a western blot apparatus, which makes the technique available to cell and molecular biology laboratories who do not wish to use radioisotopes, which are commonly required for other protein kinase assays.

Membrane attack by complement: the assembly and biology of terminal complement complexes.

Complement system activation plays an important role in both innate and acquired immunity. Activation of the complement and the subsequent formation of C5b-9 channels (the membrane attack complex) on the cell membranes lead to cell death. However, when the number of channels assembled on the surface of nucleated cells is limited, sublytic C5b-9 can induce cell cycle progression by activating signal transduction pathways and transcription factors and inhibiting apoptosis. This induction by C5b-9 is dependent upon the activation of the phosphatidylinositol 3-kinase/Akt/FOXO1 and ERK1 pathways in a Gi protein-dependent manner. C5b-9 induces sequential activation of CDK4 and CDK2, enabling the G1/S-phase transition and cellular proliferation. In addition, it induces RGC-32, a novel gene that plays a role in cell cycle activation by interacting with Akt and the cyclin B1-CDC2 complex. C5b-9 also inhibits apoptosis by inducing the phosphorylation of Bad and blocking the activation of FLIP, caspase-8, and Bid cleavage. Thus, sublytic C5b-9 plays an important role in cell activation, proliferation, and differentiation, thereby contributing to the maintenance of cell and tissue homeostasis.

Immunity against cyclin B1 tumor antigen delays development of spontaneous cyclin B1-positive tumors in p53 (-/-) mice.

We previously identified cyclin B1-specific T cells and antibodies in cancer patients with cyclin B1-positive (+) tumors and also in some healthy individuals. We also demonstrated that these responses may be important in cancer immunosurveillance by showing that vaccination against cyclin B1 prevents growth of transplantable cyclin B1(+) tumors in mice. Constitutive overexpression of cyclin B1 was determined to correlate with the lack of p53 function. This allowed us to use p53(-/-) mice as a model that better approximates human disease. These p53(-/-) mice spontaneously develop cyclin B1(+) tumors. At 5-6 weeks of age, when the mice were still healthy with no evidence of tumor, they received the cyclin B1 vaccine and were then observed for tumor growth. We demonstrate that cyclin B1 vaccination delays spontaneous cyclin B1(+) tumor growth and increases median survival of tumor-bearing p53(-/-) mice.

PPARalpha ligands cause lymphocyte depletion and cell cycle block and this is associated with augmented TRB3 and reduced Cyclin B1 expression.

PPARalpha ligands are medications used clinically to prevent cardiovascular events, however studies have shown that these agents are also anti-inflammatory. Our previous studies have shown that PPARalpha ligands induce lymphocyte depletion. PPARalpha ligands also potently upregulate TRB3, a protein that has been associated with cell cycle arrest. Therefore the following studies were undertaken to determine the mechanisms associated with lymphocyte depletion. Our studies demonstrate that WY14,643, a PPARalpha ligand, decreases the amount of lymphocytes recovered after stimulation and reduces cellular divisions. Cells treated with WY14,643 also accumulate in the G2/S phase of the cell cycle. TRB3 has been shown to inhibit the phosphorylation of AKT/Protein Kinase B, and reduced activation of AKT has been associated with decreased cellular divisions and survival. However in lymphocytes, TRB3 did not reduce the phosphorylation of AKT, and WY14,643 treatment was associated with enhanced activation of AKT. Drosophila tribbles (TRB3 homolog) causes G2 arrest by decreasing the expression of a Cdc25c homolog. Lymphocytes stimulated and treated with WY14,643 have reduced expression of Cdc25c, however this is not associated with enhanced expression of phosphorylated-Cdc2 which induces G2 arrest. Instead we observed that WY14,643 consistently reduces the protein and mRNA expression of Cyclin B1. Moreover, TRB3 inhibits activation of a Cyclin B1 promoter construct. In summary, we propose that PPARalpha ligands may reduce cellular number by augmenting TRB3 expression, which in turn induces cell cycle arrest by reducing the expression of Cyclin B1. Reduced cellular divisions and cell cycle arrest may be responsible for some of the immunomodulatory effects of these agents that have been consistently observed in human trials.

Healthy individuals have T-cell and antibody responses to the tumor antigen cyclin B1 that when elicited in mice protect from cancer.

We previously identified the aberrantly expressed cell cycle regulator cyclin B1 as a tumor antigen recognized by antibodies and T cells from patients with breast, lung, and head and neck cancers. Ordinarily expressed only transiently in the G2/M stage of the cell cycle in normal cells, cyclin B1 is constitutively expressed at high levels in the cytoplasm of these and many other tumor types, leading to its recognition by the cancer patient's immune system. We report here an unexpected observation that cyclin B1-specific antibody and memory CD4 and CD8 T cells are also found in many healthy individuals who have no history of cancer. Moreover, young as well as older healthy people have these responses suggesting that events other than cancer, which occur either early in life or throughout life, may lead to aberrant cyclin B1 expression and anti-cyclin B1 immunity. The role, if any, of immunity to this tumor-associated antigen is not known. We wanted to determine specifically whether immunity to cyclin B1 might be important in the immunosurveillance of cyclin B1+ tumors. We therefore tested in mice the effectiveness of vaccine-elicited anti-cyclin B1 immunity against a cyclin B1+ mouse tumor that was chosen based on our published observation that cyclin B1 overexpression is associated with the lack of p53 function. We found that cyclin B1 DNA prime-protein boost vaccine protected mice from a challenge with a tumor cell line that was established from a tumor arising in the p53(-/-) mouse that spontaneously overexpresses cyclin B1.

CD8 T-cell responses against cyclin B1 in breast cancer patients with tumors overexpressing p53.

PURPOSE: This study aimed to examine CD8 T-cell reactivity in breast cancer patients against cyclin B1-derived peptides restricted by the human leukocyte antigen (HLA)-A2 molecule. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cells from 36 breast cancer patients were analyzed by enzyme-linked immunosorbent spot (ELISPOT) for the presence of T cells recognizing the cyclin B1-derived peptides CB9 (AKYLMELTM) and CB-P4 (AKYLMELCC), in addition to modified versions of CB9, CB9L2 (ALYLMELTM) and CB9M2 (AMYLMELTM), both of which display higher affinity to HLA-A2. RESULTS: Twelve patients harbored a memory CD8 T-cell response against at least one of the peptides; strongest reactivity was detected against the CB9L2 peptide. Because the level of cyclin B1 has been shown to be influenced by the level of p53, which in turn is elevated in cancer cells because of point mutation, we analyzed the level of p53 protein in biopsies from the patients by immune histochemistry. Combined data showed that anti-cyclin B1 reactivity was predominantly detected in patients with tumors characterized by elevated expression of p53. Interestingly, no reactivity was detected against six peptides derived from the p53 protein. CONCLUSIONS: Our data support the notion of cyclin B1 as a prominent target for immunologic recognition in cancer patients harboring p53-mutated cancer cells. Because mutation of p53 is one of the most frequent genetic alterations in human cancers, this suggests that immunotherapy based on targeting of cyclin B1 is broadly applicable in a large proportion of cancer patients.

Correlation between cyclin B1 immunostaining in cervical biopsies and HPV detection by PCR.

In recent years, experimental data on the roles of viruses and cyclin B1 in G2/M arrest has accumulated. The aim of our study was to immunohistochemically examine a series of cervical squamous intraepithelial lesions for the presence of cyclin B1 and test for a possible correlation with morphologic findings or human papillomavirus (HPV) types detected by polymerase chain reaction. One hundred and three biopsies and/or conization specimens were examined, including high-grade lesions (25), low-grade lesions (52), biopsies showing nondiagnostic atypia (15), and biopsies without histopathologic alterations (11). Three patterns of immunopositivity were recognized, according to the level of epithelium exhibiting positively stained cells. Immunoreactivity for cyclin B1 above the basal/parabasal cells correlated with HPV presence. Cyclin B1-stained cells may represent "prekoilocytes," whose eventual progression to koilocytes would depend on several parameters related to the intricacies of HPV infection. In cases of nondiagnostic atypia immunoreactivity, cyclin B1 could be considered as a surrogate test.

Cyclin B1 is commonly expressed in the cytoplasm of primary human acute myelogenous leukemia cells and serves as a leukemia-associated antigen associated with autoantibody response in a subset of patients.

OBJECTIVES: Aberrant expression of cyclin B1, a cell cycle regulator, is related to prognosis in various human malignancies. Additionally, cytoplasmic expression of cyclin B1 in epithelial malignancies is associated with a specific T-cell response and presumably also a humoral immune response. We therefore investigated (i) whether a similar expression pattern could be detected in native human acute myelogenous leukemia (AML) cells and (ii) whether cyclin B1 specific antibodies could be detected in AML. METHODS: AML cell expression of cyclin B1 was analyzed by flow cytometry and confocal microscopy. Humoral immune response in AML patient sera against cyclin B1 was analyzed by ELISA. RESULTS: AML cell expression of cyclin B1 was detected for all 42 patients; but the percentage of cyclin B1 positive cells showed a wide variation between patients. Confocal microscopy demonstrated that 32/42 (76%) patient samples showed abnormal cytoplasmic expression. Furthermore, the cytoplasmic expression was maintained after 14 d of in vitro culture and differentiation of the AML cells towards a dendritic cell phenotype. Cyclin B1 specific serum antibodies could be detected for seven of 65 patients with untreated AML. CONCLUSIONS: Our studies demonstrate that primary human AML cells show aberrant cytoplasmic expression of cyclin B1 for a majority of patients and a specific humoral immune response was also detected for a subset of patients with untreated leukemia.

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.

Cross-priming of cyclin B1, MUC-1 and survivin-specific CD8+ T cells by dendritic cells loaded with killed allogeneic breast cancer cells.

INTRODUCTION: The ability of dendritic cells (DCs) to take up whole tumor cells and process their antigens for presentation to T cells ('cross-priming') is an important mechanism for induction of tumor specific immunity. METHODS: In vitro generated DCs were loaded with killed allogeneic breast cancer cells and offered to autologous naïve CD8+ T cells in 2-week and/or 3-week cultures. CD8+ T cell differentiation was measured by their capacity to secrete effector cytokines (interferon-gamma) and kill breast cancer cells. Specificity was measured using peptides derived from defined breast cancer antigens. RESULTS: We found that DCs loaded with killed breast cancer cells can prime naïve CD8+ T cells to differentiate into effector cytotoxic T lymphocytes (CTLs). Importantly, these CTLs primed by DCs loaded with killed HLA-A*0201- breast cancer cells can kill HLA-A*0201+ breast cancer cells. Among the tumor specific CTLs, we found that CTLs specific for HLA-A2 restricted peptides derived from three well known shared breast tumor antigens, namely cyclin B1, MUC-1 and survivin. CONCLUSION: This ability of DCs loaded with killed allogeneic breast cancer cells to elicit multiantigen specific immunity supports their use as vaccines in patients with breast cancer.

Cyclin B1 and other cyclins as tumor antigens in immunosurveillance and immunotherapy of cancer.

Uncontrolled cell division is an indispensable event in tumor progression, and numerous molecules involved in this process have been the focus of intense investigation in tumor biology. Cyclins, molecules that orchestrate normal cell cycle progression, are abnormally overexpressed in various human cancers. We review evidence that the immune system recognizes some abnormally expressed cyclins as tumor antigens, such as cyclin B1, and we analyze the potential of cyclins D, E, and A to serve a similar function in cancer immunosurveillance.

T cell-dependent antibody responses against aberrantly expressed cyclin B1 protein in patients with cancer and premalignant disease.

PURPOSE: Cyclin B1-derived peptides were shown by us to be targets of tumor-specific CD8(+) T cells in patients with breast and head and neck cancer. We obtained further evidence of cyclin B1 immunogenicity and its potential to serve as a tumor-specific antigen by analyzing its ability to elicit T cell-dependent humoral immune responses in vivo in patients with different types of tumors. EXPERIMENTAL DESIGN: Recombinant cyclin B1 protein from two different sources was purified and used as antigen in ELISA assays to test sera from patients with breast, pancreatic, colon, and lung cancer for the presence of anti-cyclin B1 antibody. We also analyzed patients with benign lung disease, premalignant disease, and a known history of heavy smoking. RESULTS AND CONCLUSIONS: Cyclin B1 elicits helper T cell-dependent antibody responses in vivo. Tumors with higher level of cyclin B1 expression elicit higher anti-cyclin B1 antibody levels. Antibodies in patients with breast and colon cancer are primarily of the IgG isotype whereas patients with pancreatic and lung cancer have in addition anti-cyclin B1 IgA. Cyclin B1-specific IgG was also detected in long-term smokers and in patients with preneoplastic lung disease. Immune responses to aberrantly expressed cyclin B1 in tumors and premalignant lesions should be further explored as diagnostic and prognostic markers, in addition to their immunotherapeutic potential.

Evaluation of anticyclin B1 serum antibody as a diagnostic and prognostic biomarker for lung cancer.

The primary aims of this study were to establish a high-throughput assay and analytic procedures that would allow us to assess with accuracy and reproducibility the presence of anti-cyclin B1 antibodies in a large cohort of subjects at-risk for development of lung cancer. The overall goal is to identify factors significantly associated with immune responses against this lung tumor antigen and to evaluate the relationship between anticyclin B1 antibodies and lung cancer risk in a prospective cohort. Successful cancer treatment relies on early detection. The immune system could respond to tumor antigens arising from altered host factors and provide an early readout for cancer presence. This study examines immune responses to the tumor antigen cyclin B1. Lung cancer patients have antibodies against cyclin B1. Unscheduled overexpression of this cell cycle regulator occurs early in tumorigenesis and might be detected by the immune system long before clinical diagnosis of cancer. Antibodies recognizing cyclin B1 were measured using semiautomated ELISAs in sera samples from subjects without detectable lung cancer enrolled in the Pittsburgh Lung Screening Study (PLuSS), a longitudinal study of long-term smokers over age 50. Factors significantly associated with antibody presence were identified using linear regression analysis. We have established a highly reproducible, semiautomated ELISA-based assay for the high-throughput assessment of serum antibody titers. Using this technology and a small subset of PluSS subjects, cyclin B1 antibody levels were found to be high in a small proportion of subjects tested. Regression analysis identified gender as well as age in women smokers to be significant determinants of cyclin B1 antibody levels, association not seen in male subjects. This work represents an important first step toward defining the importance of the presence of anticyclin B1 antibodies in an at-risk population and assessing the predictive value of serum cyclin B1 antibody as relates to lung cancer risk.

Specific changes in the posttranslational regulation of nucleolin in lymphocytes from patients infected with human immunodeficiency virus.

Lymphocytes isolated from human immunodeficiency virus (HIV)-infected patients have dysregulated cell-cycle control, consisting of increased activation of the cyclin B1/p34 cdc2 complex and abnormal nucleolar structure. To better characterize the molecular features of the HIV-associated cell-cycle perturbations, we performed a detailed analysis of the posttranslational regulation of nucleolin, a key structural protein in the nucleolus. We found that, in concanavalin A-stimulated lymphocytes from HIV-infected patients, the inappropriate activation of the cyclin B1/p34 cdc2 kinase complex is temporally associated with increased threonine phosphorylation, augmented fragmentation, and prominent extranuclear and cell-surface localization of nucleolin. Importantly, increased lymphocyte apoptosis is observed at the time of cell-surface localization of nucleolin. These results may delineate a direct molecular link between abnormal activation of cyclin B1/p34 cdc2 and the changes in the nucleolar structure, thus providing a better molecular definition of HIV-associated cell-cycle dysregulation.

Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer's disease.

Cell cycle events play a major role in the loss of neurons in advanced Alzheimer's disease (AD). It is currently unknown, however, whether the same is true for the neuronal losses in early disease stages. To explore this issue we analyzed brain autopsy material from individuals clinically categorized with mild cognitive impairment (MCI), many if not most of whom will progress to AD. Immunocytochemistry for three cell cycle-related proteins, proliferating cell nuclear antigen, cyclin D, and cyclin B, was performed on sections from hippocampus, basal nucleus of Meynert, and entorhinal cortex. The results obtained from MCI cases were compared with material from individuals diagnosed with AD and those without cognitive impairment. In both hippocampus and basal nucleus, there was a significant percentage of cell cycle immunopositive neurons in the MCI cases. These percentages were similar to those found in the AD cases but significantly higher than non-cognitively impaired controls. In entorhinal cortex, the density of cell cycle-positive neurons was greater in MCI than in AD. However, we observed large variations in the percentages of immunopositive neurons from individual to individual. These findings lend support to the hypothesis that both the mechanism of cell loss (a cell cycle-induced death) and the rate of cell loss (a slow atrophy over several months) are identical at all stages of the AD disease process. The implication of the findings for human clinical trials is discussed.

Immune recognition of cyclin B1 as a tumor antigen is a result of its overexpression in human tumors that is caused by non-functional p53.

Cyclin B1, which plays a key role in the control of cell cycle progression from G(2) through M phase, was recently identified by us as a tumor antigen recognized by human T-cells. To understand what makes this normal molecule antigenic, we compared its expression in malignant versus normal cells. Immunohistology showed overexpression of cyclin B1 protein in tumors compared to surrounding normal tissue and localization in the cytoplasm rather than the nucleus. Cyclin B1 is overexpressed at protein and mRNA level in many tumor cell lines including breast, lung, colorectal carcinoma, lymphoma and leukemia. While overexpressed in tumor cells at all stages of the cell cycle, its expression still peaks at G(2)/M phase, as it does in normal cells. We compared cyclin B1 expression in two cell clones derived from the same colorectal tumor cell line, one wild type for p53 (HCT116p53(+/+)) and one with deleted p53 (HCT116p53(-/-)). HCT116p53(+/+) cells had undetectable (normal) level of cyclin B1 protein, while HCT116p53(-/-) cells showed overexpression. When reconstituted with p53, HCT116p53(-/-) cells reverted to normal cyclin B1 expression. We conclude that p53 plays an important role in cyclin B1 regulation and that tumors with mutated p53 will be good candidates for cyclin B1 based immunotherapy.

Identification of cyclin B1 as a shared human epithelial tumor-associated antigen recognized by T cells.

We eluted peptides from class I molecules of HLA-A2.1(+) breast adenocarcinoma and loaded reverse phase high-performance liquid chromatography (HPLC) fractions onto dendritic cells to prime naive CD8(+) T cells. Fractions that supported growth of tumor-specific cytotoxic T lymphocytes were analyzed by nano-HPLC micro-ESI tandem mass spectrometry. Six HLA-A2.1-binding peptides, four 9-mers (P1-P4) differing in the COOH-terminal residue, and two 10-mers (P5 and P6) with an additional COOH-terminal alanine, were identified in one fraction. Peptide sequences were homologous to cyclin B1. We primed CD8(+) T cells from another HLA-A2.1(+) healthy donor with synthetic peptides and generated P4-specific responses. We also detected memory T cells specific for one or more of these peptides in patients with breast cancer and squamous cell carcinomas of the head and neck (SCCHN). T cells from one patient, restimulated once in vitro, could kill the tumor cell line from which the peptides were derived. Immunohistochemical analysis of tumor lines and tissue sections showed cyclin B1 overexpression and aberrant localization in the cytoplasm instead of the nucleus. Sequencing genomic DNA and cDNA corresponding to P1-P6 region showed that differences in COOH-terminal residues were not due to either DNA mutations or errors in transcription, suggesting a high error rate in translation of cyclin B1 protein in tumors.

Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase.

In vertebrate cells, the nuclear entry of Cdc2-cyclin B1 (MPF) during prophase is thought to be essential for the induction and coordination of M-phase events. Phosphorylation of cyclin B1 is central to its nuclear translocation, but the kinases that are responsible remain unknown. Here we have purified a protein kinase from Xenopus M-phase extracts that phosphorylates a crucial serine residue (S147) in the middle of the nuclear export signal sequence of cyclin B1. We have identified this kinase as Plx1 (ref. 16), a Xenopus homologue of Polo-like kinase (Plk)-1. During cell-cycle progression in HeLa cells, a change in the kinase activity of endogenous Plk1 toward S147 and/or S133 correlates with a kinase activity in the cell extracts. An anti-Plk1 antibody depletes the M-phase extracts of the kinase activity toward S147 and/or S133. An anti-phospho-S147 antibody reacts specifically with cyclin B1 only during G2/M phase. A mutant cyclin B1 in which S133 and S147 are replaced by alanines remains in the cytoplasm, whereas wild-type cyclin B1 accumulates in the nucleus during prophase. Co-expression of constitutively active Plk1 stimulates nuclear entry of cyclin B1. Our results indicate that Plk1 may be involved in targeting MPF to the nucleus during prophase.

Pneumocystis carinii uses a functional cdc13 B-type cyclin complex during its life cycle.

Pneumocystis carinii causes severe pneumonia in immunocompromised patients. Recent studies indicate that P. carinii uses a Cdc2 cyclin-dependent kinase to control its proliferation. To further study the regulation of the life cycle of P. carinii, we characterized the P. carinii B-type cyclin termed Cdc13, whose binding to Cdc2 is necessary for kinase activity. Antibodies to B-type cyclins (Cdc13) specifically immunoprecipitated Cdc2/ Cdc13 complexes with associated kinase activity from P. carinii extracts. To clone P. carinii cdc13, degenerate polymerase chain reaction was undertaken using primers generated from amino-acid motifs conserved in fungal Cdc13 proteins. This amplicon was used to obtain full-length genomic and complementary DNA (cDNA) clones. A specific synthetic peptide antibody generated to P. carinii Cdc13 further demonstrated differential Cdc2/Cdc13 activity over the life cycle of P. carinii, with greater activity in cysts compared with trophic forms of the organism. Finally, P. carinii cdc13 cDNA was used to rescue mutant Schizosaccharomyces pombe strains containing temperature-sensitive deficiencies of endogenous Cdc13 activity, thus verifying function of the P. carinii Cdc13 protein. Therefore, P. carinii contains a Cdc13 cyclin, which is variably active over its life cycle and which promotes fungal proliferation.