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.

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.

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.

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.

Identification of HLA ligands and T-cell epitopes for immunotherapy of lung cancer.

INTRODUCTION: Lung cancer is the most common cancer worldwide. Every year, as many people die of lung cancer as of breast, colon and rectum cancers combined. Because most patients are being diagnosed in advanced, not resectable stages and therefore have a poor prognosis, there is an urgent need for alternative therapies. Since it has been demonstrated that a high number of tumor- and stromal-infiltrating cytotoxic T cells (CTLs) is associated with an increased disease-specific survival in lung cancer patients, it can be assumed that immunotherapy, e.g. peptide vaccines that are able to induce a CTL response against the tumor, might be a promising approach. METHODS: We analyzed surgically resected lung cancer tissues with respect to HLA class I- and II-presented peptides and gene expression profiles, aiming at the identification of (novel) tumor antigens. In addition, we tested the ability of HLA ligands derived from such antigens to generate a CTL response in healthy donors. RESULTS: Among 170 HLA ligands characterized, we were able to identify several potential targets for specific CTL recognition and to generate CD8+ T cells which were specific for peptides derived from cyclin D1 or protein-kinase, DNA-activated, catalytic polypeptide and lysed tumor cells loaded with peptide. CONCLUSIONS: This is the first molecular analysis of HLA class I and II ligands ex vivo from human lung cancer tissues which reveals known and novel tumor antigens able to elicit a CTL response.

Requirement of epithelial integrin-linked kinase for facilitation of Citrobacter rodentium-induced colitis.

BACKGROUND: Integrin-linked kinase (ILK) is a serine-threonine kinase that transduces extracellular matrix-related cues into intracellular signals, with fundamental roles in cell motility, development and cancer. Recently ILK been shown to have an important role in bacterial epithelial cell attachment, through ILK-bacterial OspE binding. Here we report on the role of epithelial derived ILK in response to Citrobacter rodentium infection. METHODS: C. rodentium was administered to both control and intestinal epithelial cell ILK knockout mice. Histological inflammatory scores were assessed, and cytokines measured by ELISA as well as RT-PCR, in mouse colons. Bacterial colonization was determined by plating homogenates onto MacConkey agar, and immunofluorescence microscopy performed using anti-LPS and anti-Tir antibodies. RESULTS: ILK-ko mice exhibited reduced weight loss at 15 days post-infection (p < 0.01) and demonstrated reduced histological inflammatory scores (p < 0.01), reduced CCL2 and pro-inflammatory cytokines. This was not due to reduced colonization, but was associated with an altered pattern of C. rodentium bacterial migration. Attenuated fibronectin expression was found in the ILK-ko mice. C. rodentium exposure was shown to increase ILK expression in cell lines, and in murine epithelium in vivo. In ILK-ko mice reduced activation of ser473Akt and reduced crypt proliferation, together with reduced cyclin D1 expression were observed. CONCLUSIONS: ILK influences the host response to C. rodentium -induced infection, independently of reduced colonization in the ILK knockout mice. The reduced inflammation and dramatically attenuated hyperplastic cryptal response to infection in this group, are at least in part the result of, the reduction in CCL2 and cyclin D1 expression respectively.

Leptin signaling maintains B-cell homeostasis via induction of Bcl-2 and Cyclin D1.

Regulation of apoptosis and cell cycle progression plays an essential role in the maintenance of B-cell homeostasis, because a fine balance of survival and expansion is critical for preventing lymphocytic disorders. Although remarkable progress in understanding B-cell development has been achieved, much less is known concerning niches that are critical to the maintenance of B-cell homeostasis. Leptin has recently been recognized to be important for modulating the immune responses, but it has remained unclear how leptin signaling influences B-cell physiology. A variety of lymphocytic malignancies have been reported to be linked to leptin, and therefore it is necessary to elucidate the mechanisms involved. Here we demonstrate that leptin promotes B-cell homeostasis by inhibiting apoptosis and by inducing cell cycle entry through the activation of expressions of B-cell CLL/lymphoma 2 (Bcl-2) and cyclin D1. We further show that leptin can induce Bcl-2 and cyclin D1 expression by two pathways, including the direct activation of their promoters and suppression of microRNAs (miRNAs) that target their putative 3'untranslated regions. Amplification of these leptin-modulated miRNAs inhibited B lymphoma cell growth. These findings provide insights into mechanisms for leptin regulation of the humoral immune system and suggest new therapeutic strategies for leptin receptor expressing malignancies.

Apurinic/apyrimidinic endonuclease 1 is a key modulator of keratinocyte inflammatory responses.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) functions in both DNA repair and redox signaling, making it an attractive emerging therapeutic target. However, the role of APE1 in cutaneous inflammatory responses is largely unknown. In this study, we report that APE1 is a key upstream regulator in TLR2-dependent keratinocyte inflammatory responses. We found that nuclear expression of APE1 in epidermal layers was markedly up-regulated in psoriatic skin. APE1 was essential for the transcriptional activation and nuclear translocation of hypoxia-inducible factor-1alpha and NF-kappaB, both of which are crucial for inflammatory signaling in keratinocytes. Moreover, APE1 played a crucial role in the expression of TLR2-mediated inflammatory mediators, including TNF-alpha, CXCL8, and LL-37, in HaCaT cells and human primary keratinocytes. Silencing of APE1 attenuated cyclin D1/cyclin-dependent kinase 4 expression and phosphorylation of ERK1/2 and Akt, thereby affecting keratinocyte proliferation. Importantly, TLR2-induced generation of reactive oxygen species contributed to the nuclear translocation and expression of APE1, suggesting an autoregulatory circuit in which the subcellular localization of APE1 is associated with the production of APE1 per se through reactive oxygen species-dependent signaling. Taken together, these findings establish a role for APE1 as a master regulator of TLR2-dependent inflammatory responses in human keratinocytes.

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.

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.

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.

Cyclin D1-specific cytotoxic T lymphocytes are present in the repertoire of cancer patients: implications for cancer immunotherapy.

PURPOSE: Cyclin D1, a key cell cycle regulator, is overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for cancer immunotherapy. Nevertheless, it had previously been suggested that efficient T cells recognizing cyclin D1-derived epitopes are absent from the repertoire because of thymic deletion. We attempted to induce autologous CTL from healthy donors and patients with cyclin D1-overexpressing tumors using a highly efficient T-cell expansion system based on CD40-activated B cells as antigen-presenting cells. EXPERIMENTAL DESIGN: Cyclin D1-derived, HLA-A*0201-restricted epitopes were predicted by multiple computer algorithms, screened in HLA-A2-binding assays, and used for T-cell stimulation. The generated CTL lines and clones were analyzed by IFN-gamma enzyme-linked immunosorbent spot assay or cytolysis assay. RESULTS: After screening, at least two naturally processed and presented HLA-A*0201-binding cyclin D1 epitopes were identified. CTL specific for these epitopes could be successfully generated from HLA-A2(+) donors. T cells efficiently recognized target cells pulsed with the cognate peptide and cyclin D1-expressing tumor cell lines in an HLA-A*0201-restricted manner. More importantly, HLA-A*0201-matched, primary cyclin D1(+) tumor cells were efficiently recognized by cyclin D1-specific CTL. These CTL could be generated from patients with mantle cell lymphoma and cyclin D1(+) colon cancer. CONCLUSIONS: These results underscore that cyclin D1 needs to be considered as a target for broad-based antitumor immunotherapy.

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.

CXCL12/CXCR4 signaling promotes human thymic dendritic cell survival regulating the Bcl-2/Bax ratio.

CXCL12, a member of the chemokine CXC subfamily, and its physiologic receptor CXCR4 are essential for the development of various organs during embryonic development and are also involved in the control of cell survival, proliferation and migration in adult tissues. In the human thymus, CXCL12 is produced by epithelial cells located in the subcapsular and medullary regions and CXCR4 is expressed in different thymocyte subpopulations. Several results have demonstrated that CXCL12/CXCR4 signaling participates in different intrathymic processes including the control of human precursor cell survival and proliferation, and the exit of mature thymocytes to the periphery. In this study, we show that CXCL12 is also produced by human thymic dendritic cells (DCs), most of which express CXCR4 receptor. The addition of exogenous CXCL12 significantly inhibited the serum depletion-induced apoptosis in thymic DCs, and the treatment with neutralizing antibodies against CXCL12 or CXCR4 decreased their survival. The survival-promoting effect of CXCL12 was mediated by the up-regulation of Bcl-2 protein expression and the concomitant down-regulation of Bax protein expression. The higher viability of thymic DCs also enhanced their allostimulatory capacity. Taken together, the results suggest a new function of CXCL12 in the human thymus controlling the survival and functionality of thymic DCs.

[Immunohistochemical analysis of nuclear factor, p38, and cyclin D1 proteins in premalignant lesions and carcinomas of the colorectal mucosa].

BACKGROUND/AIMS: Nuclear factor-kappaB p65 (NF-kappaB p65), nuclear factor-kappaB1 p50 (NF-kappaB p50) have been shown to play a role in cell proliferation, apoptosis, cytokine production, and oncogenesis. Recently, p38 mitogen-activated protein kinase (MAPK)/ NF-kappaB/ cyclin D1 signaling pathway has been shown to play an important part in the pathogenesis of human cancers. This study was designed to investigate the expression of NF-kappaB p65, NF-kappaB p50, p38 MAPKalpha, and cyclin D1 proteins in premalignant lesions of colon and colorectal adenocarcinoma. METHODS: Paraffin sections of 20 normal mucosa, 20 low-grade tubular adenoma, 20 high-grade tubular adenoma and 64 adenocarcinoma tissues were analysed immunohistochemically for the expression of NF-kappaB p65, NF-kappaB p50, p38 MAPKalpha, and cyclin D1 proteins. RESULTS: The expression of NF-kappaB p65, NF-kappaB p50, and p38 MAPKalpha proteins were significantly higher in adenocarcinoma tissue in comparison with that in normal mucosa, low-grade tubular adenoma, and high-grade tubular adenoma tissues. Expression of NF-kappaB p50 was more frequent in poorly differentiated histologic grade, presence of nodal metastasis, and advanced stage. Expression of p38 MAPKalpha protein was higher in advanced tumor stage, presence of nodal metastasis and advanced stage. Synchronous expression of NF-kappaB p65, NF-kappaB p50, p38 MAPKalpha, and cyclin D1 proteins were significantly higher in adenocarcinoma tissue. CONCLUSIONS: With the increased expression of NF-kappaB p65, NF-kappaB p50, and p38 MAPKalpha proteins, p38 MAPK/ NF-kappaB/ cyclin D1 signaling pathway may play a role in the pathogenesis of colorectal carcinoma.

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.

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.

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.

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.