A specific anti-cyclin D1 intrabody represses breast cancer cell proliferation by interrupting the cyclin D1-CDK4 interaction.

BACKGROUND: Cyclin D1 overexpression may contribute to development of various cancers, including breast cancer, and thus may serve as a key cancer diagnostic marker and therapeutic target. In our previous study, we generated a cyclin D1-specific single-chain variable fragment antibody (ADκ) from a human semi-synthetic single-chain variable fragment library. ADκ specifically interacted with recombinant and endogenous cyclin D1 proteins through an unknown molecular basis to inhibit HepG2 cell growth and proliferation. RESULTS: Here, using phage display and in silico protein structure modeling methods combined with cyclin D1 mutational analysis, key residues that bind to ADκ were identified. Notably, residue K112 within the cyclin box was required for cyclin D1-ADκ binding. In order to elucidate the molecular mechanism underlying ADκ anti-tumor effects, a cyclin D1-specific nuclear localization signal-containing intrabody (NLS-ADκ) was constructed. When expressed within cells, NLS-ADκ interacted specifically with cyclin D1 to significantly inhibit cell proliferation, induce G1-phase arrest, and trigger apoptosis of MCF-7 and MDA-MB-231 breast cancer cells. Moreover, the NLS-ADκ-cyclin D1 interaction blocked binding of cyclin D1 to CDK4 and inhibited RB protein phosphorylation, resulting in altered expression of downstream cell proliferation-related target genes. CONCLUSION: We identified amino acid residues in cyclin D1 that may play key roles in the ADκ-cyclin D1 interaction. A nuclear localization antibody against cyclin D1 (NLS-ADκ) was constructed and successfully expressed in breast cancer cells. NLS-ADκ exerted tumor suppressor effects via blocking the binding of CDK4 to cyclin D1 and inhibiting phosphorylation of RB. The results presented here demonstrate anti-tumor potential of intrabody-based cyclin D1-targeted breast cancer therapy.

Bioinformatics and in-silico findings reveal medical features and pharmacological targets of biochanin A against colorectal cancer and COVID-19.

Severe mortality due to the COVID-19 pandemic resulted from the lack of effective treatment. Although COVID-19 vaccines are available, their side effects have become a challenge for clinical use in patients with chronic diseases, especially cancer patients. In the current report, we applied network pharmacology and systematic bioinformatics to explore the use of biochanin A in patients with colorectal cancer (CRC) and COVID-19 infection. Using the network pharmacology approach, we identified two clusters of genes involved in immune response (IL1A, IL2, and IL6R) and cell proliferation (CCND1, PPARG, and EGFR) mediated by biochanin A in CRC/COVID-19 condition. The functional analysis of these two gene clusters further illustrated the effects of biochanin A on interleukin-6 production and cytokine-cytokine receptor interaction in CRC/COVID-19 pathology. In addition, pathway analysis demonstrated the control of PI3K-Akt and JAK-STAT signaling pathways by biochanin A in the treatment of CRC/COVID-19. The findings of this study provide a therapeutic option for combination therapy against COVID-19 infection in CRC patients.

Cyclin D1b induces changes in the macrophage phenotype resulting in promotion of tumor metastasis.

In breast cancer, tumor-associated macrophages with activated phenotypes promote tumor invasion and metastasis. The more aggressive mesenchymal-like breast cancer cells have a selective advantage, skewing macrophages toward the more immunosuppressive subtype. However, the mechanism underlying this shift is poorly understood. Cyclin D1b is a highly oncogenic variant of cyclin D1. Our previous study showed that non-metastatic epithelial-like breast cancer cells were highly metastatic in vivo when cyclin D1b was overexpressed. The present study determined whether cyclin D1b contributed to the interaction between breast cancer cells and macrophages. The results showed that cyclin D1b promoted the invasion of breast cancer cells in vitro. Specifically, through overexpression of cyclin D1b, breast cancer cells regulated the differentiation of macrophages into a more immunosuppressive M2 phenotype. Notably, tumor cells overexpressing cyclin D1b activated macrophages and induced migration of breast cancer cells. Further investigations indicated that SDF-1 mediated macrophage activation through breast cancer cells overexpressing cyclin D1b. These results revealed a previously unknown link between aggressive breast cancer cells and Tumor-associated macrophages, and highlighted the importance of cyclin D1b activity in the breast cancer microenvironment.

Cyclin D1: potential utility as marker for Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a rare disorder of unknown etiopathogenesis. Diagnosis is based on the identification of CD1a positive histiocytic infiltrate. Activation of the mitogen-activated-protein-kinase (MAPK) is constantly observed in LCH and therefore downstream markers such as cyclin D1 may be a useful marker for LCH. The aim of this study was to investigate the expression of cyclin D1 in LCH. We assessed the immunohistochemical expression of cyclin D1 (clone SP4-R) in series of 16 cases of confirmed LCH. Expression of Cyclin D1 was scored as weak, moderate, and strong nuclear staining and results were interpreted by two pathologists. The percentage of positivity was assessed. The mean age of patients was 13.7 years old with a male to female ratio of 1:3. The most common involved site was bone (n = 9; 56,3%), followed by lymph node (n = 5; 31,2%) and skin (n = 2; 12,5%). All cases showed nuclear staining for cyclin D1 with variable intensity. It was assessed moderate in 43,8% (n = 7) and strong in 56,2% (n = 9). The percentage of positive cells was >50% in 13 cases and <50% in 3 cases. Our results have shown that all cases of Langerhans cell histiocytosis from various sites express cyclin D1. This finding may be attributed to MAPK pathway activation that has been described in LCH. Otherwise, cyclin D1 is not significantly expressed in reactive Langerhans cell proliferations. Therefore, cyclin D1 immunohistochemistry may be useful as a diagnostic marker and in excluding non-neoplastic mimics of LCH.

Leukotriene B4 induces proliferation of rat pulmonary arterial smooth muscle cells via modulating GSK-3ß/ß-catenin pathway.

Leukotriene B4 (LTB4) has been found to contribute to pulmonary arterial smooth muscle cells (PASMCs) proliferation and pulmonary arterial remodeling therefore the development of pulmonary arterial hypertension (PAH). Yet, the underlying molecular mechanisms remain poorly understood. The present study aims to address this issue. Our results demonstrate that LTB4 dose- and time-dependently induced proliferation of primary cultured rat PASMCs, this was accompanied with the activation of phosphatidylinositol-3-kinase/Akt (PI3K/Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathways, and consequent inactivation of glycogen synthase kinase-3ß (GSK-3ß), up-regulation of ß-catenin and induction of cyclin D1 expression. The presence of PI3K inhibitor (LY294002) or MEK inhibitor (U0126) or prior silencing of ß-catenin with siRNA suppressed LTB4-induced cyclin D1 up-regulation and PASMCs proliferation. In addition, inactivation or lack of GSK-3ß up-regulated ß-catenin and cyclin D1 in PASMCs. Taken together, our study indicates that activation of PI3K/Akt and ERK1/2 pathways mediates LTB4-induced PASMCs proliferation by modulating GSK-3ß/ß-catenin/cyclin D1 axis and suggests that targeting this pathway might have potential value in alleviating vascular remodeling and benefit PAH.

Changes in apoptosis, proliferation and T lymphocyte subtype on thymic cells of SPF chickens infected with reticuloendotheliosis virus.

Reticuloendotheliosis virus (REV), an avian retrovirus is able to infect a variety of birds and can cause immunosuppression. The aim of this study was to investigate the relationship of thymic lymphocytes apoptosis, proliferation and T cell subtype with immunosuppression. In this study, a hundred and twenty one-day old SPF chickens were randomly divided into control groups (group C) and a REV infection groups (group I). The chickens of group I received intraperitoneal injections of REV with 104.62/0.1 ml TCID50. On day 14, 21, 28 and 35 post-inoculation, the chickens of C group and I group were sacrificed by cardiac puncture blood collection, and the thymic lymphocytes was sterile collected. The proliferation ability of lymphocytes was tested by Cell Counting Kit-8. Flow cytometry was performed to detect apoptosis, cell cycle stage and the change in T cell subtype. The RNA genome copy numbers of REV virus were detected using real-time PCR. Real-time PCR and western blotting were performed to analyze the expression of CyclinD1 and Bcl-2. Our results showed that REV genome copy number steadily declined, the proliferation potential of thymic lymphocytes was inhibited, lymphocytes apoptosed, the ratio of CD4+/CD8+ decreased and the expression of CyclinD1 and Bcl-2 were firstly inhibited, then rapidly recovered. Thus, immunosuppression lead by REV is closely related to the change of T cell subtype, apoptosis, and proliferation of thymic lymphocytes.

EXPRESSION OF BIOLOGICAL MARKERS INDUCED BY IONIZING RADIATION AT THE LATE PERIOD AFTER EXPOSURE IN A WIDE RANGE OF DOSES. / EKSPRESIIa RADIATsIINO-INDUKOVANYKh BIOLOGIChNYKh MARKERIV U VIDDALENOMU PERIODI PISLIa OPROMINENNIa U ShYROKOMU INTERVALI DOZ.

OBJECTIVE: To study radiation induced biological markers of the late period after exposure. SUBJECTS AND METHODS: A study was performed in 235 Chornobyl accident male clean-up workers exposed in 1986-1987 (doses of external exposure: (M ± SD: 419.48 ± 654.60; range 0.10-3,500 mSv); age 58,34 ± 6,57 years. Controlgroup included 45 non-exposed subjects (mean age: 50.60 ± 5.37 (M ± SD). Gene expression was performed by RT-PCR on 7900HT Analyzer using TLDA for BCL2, CDKN2A, CLSTN2, GSTM1, IFNG, IL1B, MCF2L, SERPINB9, STAT3, TERF1, TERF2,TERT, TNF, TP53, CCND1 genes. Relative telomere length (RTL) was analysed by flow-FISH; immune cell subsets,γ-H2AÐ¥ and CyclinD1 expression by flow cytometry. RESULTS: A statistically significant and dose-dependent decrease in expression of the BCL2, SERPINB9, CDKN2A, andSTAT3 genes was demonstrated in parallel to a dose-dependent overexpression of MCF2L and upregulation of TP53 (upto 100 mSv). IL1B expression was the highest in exposed to doses from 0.1 to 100 mSv with a negative correlationbetween at IL1B expression and CD19+3-, CD3-HLA-DR+, CD4+8- cell counts and CD4+/CD8+ ratio. Hyperexpression ofTNF gene in doses above 100 mSv to 1,000 mSv was shown, and in higher doses a combination of TNF downregula-tion with increase in IFNG gene expression were demonstrated with correlations with numbers of CD3+16+56+ andCD25+ lymphocytes and inhibition of expression CLSTN2. An increased expression of γ-H2AÐ¥ and Cyclin D1 corre-lated to radiation dose, telomere shortening to age and concommittant pathology. CONCLUSIONS: Cellular immunity, gene expression, telomere length, intracellular protein parameters are shown to beamong perspective biological markers at a late period after radiation exposure.

Role of calcium-sensing receptor, Galectin-3, Cyclin D1, and Ki-67 immunohistochemistry to favor in the diagnosis of parathyroid carcinoma.

BACKGROUND: As histopathological findings of parathyroid carcinoma are not certain, the diagnosis of tumors with degenerative changes may be difficult. In these cases, immunohistochemical markers are beneficial. We aimed to research the acceptability of calcium-sensing receptor (CaSR), Galactin-3, Cyclin D1, and Ki-67 as helpful markers in parathyroid tumors in cases which are difficult to diagnose. MATERIALS AND METHODS: Those cases who had been diagnosed with atypical parathyroid adenoma and parathyroid carcinoma between 2010 and 2015 were reevaluated. Immunohistochemical markers were applied to this cases. RESULTS: About 21 cases were parathyroid adenoma, 14 were atypical adenoma, and 10 cases were parathyroid carcinoma. According to the immunohistochemical results, global loss of CaSR staining was seen in 50% (5/10) of the patients with carcinoma while there was no loss of staining in those with parathyroid adenoma (P = 0,001). Global loss of CaSR staining was found in only one out of 14 cases with atypical adenoma. The expression of Galactin-3 was found to be positive in 40% (4/10) of carcinoma cases, 71.4% (10/14) of those with atypical adenoma, and 14.3% (3/21) of those with adenoma (P = 0,002). Cyclin D1 expression was determined to be positive in 70% (7/10) of patients with carcinoma, 71.4% (10/14) of atypical adenoma cases, and 23.8% (5/21) of those with adenoma. The Ki-67 proliferation index was seen to be above 5% in 50% (5/10) of carcinoma cases and 35,7% (5/14) of those with atypical adenoma. CONCLUSION: In these studies, it has been emphasized that the global loss of CaSR staining was used as a negative marker in the diagnosis of carcinoma. In this study, we have also confirmed that the global loss of CaSR staining is a useful marker to determine potential increased malignancy.

Polymer Cancerostatics Targeted with an Antibody Fragment Bound via a Coiled Coil Motif: In Vivo Therapeutic Efficacy against Murine BCL1 Leukemia.

A BCL1 leukemia-cell-targeted polymer-drug conjugate with a narrow molecular weight distribution consisting of an N-(2-hydroxypropyl)methacrylamide copolymer carrier and the anticancer drug pirarubicin is prepared by controlled radical copolymerization followed by metal-free click chemistry. A targeting recombinant single chain antibody fragment (scFv) derived from a B1 monoclonal antibody is attached noncovalently to the polymer carrier via a coiled coil interaction between two complementary peptides. Two pairs of coiled coil forming peptides (abbreviated KEK/EKE and KSK/ESE) are used as linkers between the polymer-pirarubicin conjugate and the targeting protein. The targeted polymer conjugate with the coiled coil linker KSK/ESE exhibits 4× better cell binding activity and 2× higher cytotoxicity in vitro compared with the other conjugate. Treatment of mice with established BCL1 leukemia using the scFv-targeted polymer conjugate leads to a markedly prolonged survival time of the experimental animals compared with the treatment using the free drug and the nontargeted polymer-pirarubicin conjugate.

Impairment of Hematopoietic Precursor Cell Activation during the Granulopoietic Response to Bacteremia in Mice with Chronic-Plus-Binge Alcohol Administration.

Alcohol abuse impairs immune defense. To study the effect of chronic-plus-binge alcohol exposure on the granulopoietic response, acute alcohol intoxication (intraperitoneal injection of 5 g alcohol/kg body weight) was introduced to mice chronically fed on the Lieber-DeCarli low-fat liquid alcohol diet for 5 weeks. Bacteremia was induced by intravenous injection of Escherichia coli Bacteremia caused a remarkable increase in marrow lin- c-kit+ Sca-1+ cells. Activation of cell proliferation supported the increase in marrow lin- c-kit+ Sca-1+ cells. Alcohol administration inhibited this activation of lin- c-kit+ Sca-1+ cells. The bone marrow of pair-fed control mice receiving intraperitoneal saline stored a large number of mature granulocytes expressing a high level of Gr1 (Gr1hi cells). The proportion of Gr1hi cells and the total number of Gr1+ cells were markedly reduced in the bone marrow, along with an increase in the ratio of Gr1+ granulocytes in peripheral white blood cells following bacteremia. E. coli infection stimulated proliferation of granulopoietic precursor cells, resulting in a marked increase in the ratio of immature Gr1lo cells in the bone marrow. Alcohol administration itself triggered marrow release of Gr1+ cells, resulting in reduction of the marrow granulocyte reserve with an elevation of granulocytes in the circulation. Alcohol also impaired activation of granulopoietic precursor proliferation following bacteremia. Alcohol disrupted lipopolysaccharide (LPS)-TLR4-ERK1/2-cyclin D1 signaling and inhibited upregulation of Sca-1 and C/EBPß expression by lineage-negative marrow cells in response to bacteremia. These results indicate that chronic-plus-binge alcohol exposure inhibits the granulopoietic response by disrupting key cell signaling for hematopoietic precursor cell activation and commitment to granulocyte lineage development.

Effects of metal ions on the structure and activity of a human anti-cyclin D1 single-chain variable fragment AD5.

Cyclin D1 has become a potential target for anti-tumor therapy. Recently, a novel human anti­cyclin D1 single­chain variable fragment (AD5) was identified, which demonstrated specific binding activity to cyclin D1 and exhibited anti­tumor effects. However, the detailed characteristics of AD5 remain unclear. In the present study, the structure and activity of AD5 in the presence of copper II (Cu2+) or iron III (Fe3+) metal ions was investigated by fluorescence spectroscopy, synchronous fluorescence and enzyme­linked immunosorbent assay. Cu2+ and Fe3+ were able to bind to AD5 and quench the fluorescence intensity of AD5 primarily by static quenching, which slightly altered the conformation of AD5 at temperatures of 293, 298 and 303 K; however, these temperatures demonstrated different effects on the activity of AD5. These results may be of value for the clinical application of anti-cyclin D1 single chain antibodies in the future.

The involvement and possible mechanism of pro-inflammatory tumor necrosis factor alpha (TNF-α) in thoracic ossification of the ligamentum flavum.

Thoracic ossification of the ligamentum flavum (TOLF) is characterized by ectopic bone formation in the ligamentum flavum and is considered to be a leading cause of thoracic spinal canal stenosis and myelopathy. However, the underlying etiology is not well understood. An iTRAQ proteomics was used to reveal the involvement of inflammation factors in TOLF. TNF-α is a pro-inflammatory cytokine implicated in the pathogenesis of many human diseases. Protein profiling analysis showed that the protein level of TNF-α increased in the ossified ligamentum flavum of TOLF, which was confirmed by western blot. The effects of TNF-α on primary ligamentum flavum cells was examined. Cell proliferation assay demonstrated that primary cells from the ossified ligamentum flavum of TOLF grew faster than the control. Flow cytometry assay indicated that the proportions of cells in S phase of cell cycle of primary cells increased after TNF-α stimulation. To address the effect of TNF-α on gene expression, primary cells were derived from ligamentum flavum of TOLF patients. Culture cells were stimulated by TNF-α. RNA was isolated and analyzed by quantitative RT-PCR. G1/S-specific proteins cyclin D1 and c-Myc were upregulated after TNF-α stimulation. On the other hand, osteoblast differentiation related genes such as Bmp2 and Osterix (Osx) were upregulated in the presence of TNF-α. TNF-α activated Osx expression in a dose-dependent manner. Interestingly, a specific mitogen-activated protein kinase ERK inhibitor U0126, but not JNK kinase inhibitor SP600125, abrogated TNF-α activation of Osx expression. This suggests that TNF-α activates Osx expression through the mitogen-activated protein kinase ERK pathway. Taken together, we provide the evidence to support that TNF-α involves in TOLF probably through regulating cell proliferation via cyclin D1 and c-Myc, and promoting osteoblast differentiation via Osx.

A cyclin D1-positive diffuse large B-cell lymphoma of germinal center B-cell-like subtype in the right tonsil: A rare case report.

INTRODUCTION: Cyclin D1-positive tumor cells are commonly found in mantle cell lymphoma but they are very rare in diffuse large B-cell lymphoma. CLINICAL FINDINGS/PATIENT CONCERNS: Here we present a rare case of cyclin D1-positive diffuse large B-cell lymphoma in the right tonsil of a 50-year-old man. Computed tomographic imaging detected a mass, about 2.5 cm × 1.8 cm in size, in the left side of the oropharynx. DIAGNOSES: Microscopically, the tumor cells were located under the pharyngeal mucosa and diffusely arranged. The tumor cells were large, with marked nuclear atypia. On performing immunohistochemistry, the tumor cells showed diffuse positive staining for CD10, CD20, cyclin D1, and Pax-5, and negative staining for CD3, CD15, CD30, CD56, and CK. Bcl-6 and Mum-1 expression were observed in 60% and 80% of tumor cells, respectively. The tumor Ki67 index was about 60%. Based on these findings, The tumor was diagnosed as a rare cyclin D1-positive diffuse large B-cell lymphoma rather than a mantle cell lymphoma. CONCLUSION: Cyclin D1-positive large B-cell lymphoma is rare, but as large B-cell lymphoma is a common type of lymphoma, cyclin D1-positive large B-cell lymphoma should be considered a major possibility during differential diagnosis, including in the tonsils.

Accelerated cell cycle progression of human regulatory T cell-like cell line caused by continuous exposure to asbestos fibers.

Asbestos exposure causes malignant tumors such as lung cancer and malignant mesothelioma. Based on our hypothesis in which continuous exposure to asbestos of immune cells cause reduction of antitumor immunity, the decrease of natural killer cell killing activity with reduction of NKp46 activating receptor expression, inhibition of cytotoxic T cell clonal expansion, reduced CXCR3 chemokine receptor expression and production of interferon-γ production in CD4+ T cells were reported using cell line models, freshly isolated peripheral blood immune cells from health donors as well as asbestos exposed patients such as pleural plaque and mesothelioma. In addition to these findings, regulatory T cells (Treg) showed enhanced function through cell-cell contact and increased secretion of typical soluble factors, interleukin (IL)-10 and transforming growth factor (TGF)-ß, in a cell line model using the MT-2 human polyclonal T cells and its sublines exposed continuously to asbestos fibers. Since these sublines showed a remarkable reduction of FoxO1 transcription factor, which regulates various cell cycle regulators in asbestos-exposed sublines, the cell cycle progression in these sublines was examined and compared with that of the original MT-2 cells. Results showed that cyclin D1 expression was markedly enhanced, and various cyclin-dependent kinase-inhibitors were reduced with increased S phases in the sublines. Furthermore, the increase of cyclin D1 expression was regulated by FoxO1. The overall findings indicate that antitumor immunity in asbestos-exposed individuals may be reduced in Treg through changes in the function and volume of Treg.

Urinary leukotriene and Bcl I polymorphism of glucocorticoid receptor gene in preschoolers with recurrent wheezing and high risk of asthma

BACKGROUND: Urinary leukotriene (LTE4) is an important marker of airway inflammation presence. A relationship between single nucleotide polymorphism in the glucocorticoid receptor (GCR) gene promoter (Bcl I polymorphism), development of asthma and sensitivity to glucocorticoids has been hypothesised. OBJECTIVE: To explore the possible association between the Bcl I polymorphism and baseline levels of urinary LTE4 in preschoolers with recurrent wheezing episodes. We prospectively enrolled and classified 86 preschoolers based on the risk of developing asthma (by the Asthma Predictive Index [API]). METHODS: At admission standardised questionnaires for demographics and respiratory illness characteristics were completed. The Bcl I polymorphism of the GCR was determined by a PCR-RFLP assay from blood samples, and urinary leukotriene was assessed from urine samples by an enzyme immunoassay. RESULTS: We enrolled 86 preschoolers (46 with positive API and 40 with negative API). There were no statistical differences in demographic, respiratory illnesses and wheezing episodes characteristics between both groups. Also, the prevalence of Bcl I polymorphism was similar between positive vs. negative API groups (34.8% vs. 38.9% for homozygote GG, 56.5% vs. 52.8% for heterozygote GC, 8.7% vs. 8.3% for homozygote CC, respectively, p = 0.94). However, urinary LTE4 (median [IQR]) was higher in preschoolers with positive than negative API (7.18 [5.57-8.96 pg/ml] vs. 6.42 [3.96-8.07 pg/ml], p = 0.02, respectively). CONCLUSIONS: In our population, wheezing preschoolers with positive API exhibit higher levels of urinary LTE4 than those with negative API; but there were no differences in Bcl I polymorphism of the GCR

No disponible

Bone marrow and splenic histology in hairy cell leukaemia.

Hairy cell leukaemia is a rare chronic neoplastic B-cell lymphoproliferation that characteristically involves blood, bone marrow and spleen with liver, lymph node and skin less commonly involved. Histologically, the cells have a characteristic appearance with pale/clear cytoplasm and round or reniform nuclei. In the spleen, the infiltrate involves the red pulp and is frequently associated with areas of haemorrhage (blood lakes). The cells stain for B-cell related antigens as well as with antibodies against tartrate-resistant acid phosphatase, DBA44 (CD72), CD11c, CD25, CD103, CD123, cyclin D1 and annexin A1. Mutation of BRAF -V600E is present and antibody to the mutant protein can be used as a specific marker. Bone marrow biopsy is essential in the initial assessment of disease as the bone marrow may be inaspirable or unrepresentative of degree of marrow infiltration as a result of the tumour associated fibrosis preventing aspiration of the tumour cell component. Bone marrow biopsy is important in the assessment of therapy response but in this context staining for CD11c and Annexin A1 is not helpful as they are also markers of myeloid lineage and identification of low level infiltration may be obscured. In this context staining for CD20 may be used in conjunction with morphological assessment and staining of serial sections for cyclin D1 and DBA44 to identify subtle residual infiltration. Staining for CD79a and CD19 is not recommended as these antibodies will identify plasma cells and can lead to over-estimation of disease. Staining for CD20 should not be used in patients following with anti-CD20 based treatments. Down regulation of cyclin D1 and CD25 has been reported in patients following BRAF inhibitor therapy and assessment of these antigens should not be used in this context. Histologically, hairy cell leukaemia needs to be distinguished from other B-cell lymphoproliferations associated with splenomegaly including splenic marginal zone lymphoma, splenic diffuse red pulp small B-cell lymphoma and hairy cell leukaemia variant. This can be done by assessment of the spleen but as this is now rarely performed in this disorder distinction is almost always possible by a combination of morphological and immunophenotypic studies on bone marrow trephine biopsy, which can be supplemented by assessment of BRAF-V600E mutation assessment in borderline cases.

Hyper sensitive protein detection by Tandem-HTRF reveals Cyclin D1 dynamics in adult mouse.

We present here a novel method for the semi-quantitative detection of low abundance proteins in solution that is both fast and simple. It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target. This novel approach we termed "Tandem-HTRF", can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format. Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge. Thus our method, Tandem-HTRF offers a promising way to investigate subtle variations in the dynamics of sparse proteins from limited biological material.

A novel vaccine for mantle cell lymphoma based on targeting cyclin D1 to dendritic cells via CD40.

BACKGROUND: Mantle cell lymphoma (MCL) is a distinct clinical pathologic subtype of B cell non-Hodgkin's lymphoma often associated with poor prognosis. New therapeutic approaches based on boosting anti-tumor immunity are needed. MCL is associated with overexpression of cyclin D1 thus rendering this molecule an interesting target for immunotherapy. METHODS: We show here a novel strategy for the development of recombinant vaccines carrying cyclin D1 cancer antigens that can be targeted to dendritic cells (DCs) via CD40. RESULTS: Healthy individuals and MCL patients have a broad repertoire of cyclin D1-specific CD4(+) and CD8(+) T cells. Cyclin D1-specific T cells secrete IFN-γ. DCs loaded with whole tumor cells or with selected peptides can elicit cyclin D1-specific CD8(+) T cells that kill MCL tumor cells. We developed a recombinant vaccine based on targeting cyclin D1 antigen to human DCs via an anti-CD40 mAb. Targeting monocyte-derived human DCs in vitro with anti-CD40-cyclin D1 fusion protein expanded a broad repertoire of cyclin D1-specific CD4(+) and CD8(+) T cells. CONCLUSIONS: This study demonstrated that cyclin D1 represents a good target for immunotherapy and targeting cyclin D1 to DCs provides a new strategy for mantle cell lymphoma vaccine.

A crucial role for the homeodomain transcription factor Hhex in lymphopoiesis.

The hematopoietically expressed homeobox gene, Hhex, is a transcription factor that is important for development of definitive hematopoietic stem cells (HSCs) and B cells, and that causes T-cell leukemia when overexpressed. Here, we have used an Hhex inducible knockout mouse model to study the role of Hhex in adult hematopoiesis. We found that loss of Hhex was tolerated in HSCs and myeloid lineages, but resulted in a progressive loss of B lymphocytes in the circulation. This was accompanied by a complete loss of B-cell progenitors in the bone marrow and of transitional B-cell subsets in the spleen. In addition, transplantation and in vitro culture experiments demonstrated an almost complete failure of Hhex-null HSCs to contribute to lymphoid lineages beyond the common lymphoid precursor stage, including T cells, B cells, NK cells, and dendritic cells. Gene expression analysis of Hhex-deleted progenitors demonstrated deregulated expression of a number of cell cycle regulators. Overexpression of one of these, cyclin D1, could rescue the B-cell developmental potential of Hhex-null lymphoid precursors. Thus, Hhex is a key regulator of early lymphoid development, functioning, at least in part, via regulation of the cell cycle.

Ellagic acid normalizes mitochondrial outer membrane permeabilization and attenuates inflammation-mediated cell proliferation in experimental liver cancer.

Despite great advances in our understanding of the molecular causes of liver cancer, significant gaps still remain in our knowledge of the disease pathogenesis and development of effective strategies for early diagnosis and treatment. The present study was conducted to evaluate the chemopreventive activity of ellagic acid (EA) against experimental liver cancer in rats. This is the first report that implies a possible role of EA in controlling liver cancer through activation of mitochondrial outer membrane permeability via activating proteins such as Bax, bcl-2, cyt-C, and caspase-9, which play important roles in apoptosis. Downregulation of NF-κB, cyclin D1, cyclin E1, matrix metalloproteinases (MMP)-2, MMP-9, and proliferating cell nuclear antigen (PCNA) were noted in EA-treated experimental rats and controlled inflammation mediated liver cancer when compared to the diethylnitrosamine (DEN)-induced group. Transmission electron microscopy (TEM) analysis of the livers of experimental rats demonstrated that EA treatment renovated its internal architecture. Overall, these results demonstrate the value of molecular approaches in identifying the potential role of EA as an effective chemopreventive agent.