UNIPROT:P10415 - FACTA Search

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Query: UNIPROT:P10415 (Bcl-2)
33,771 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The response to therapy of leukemic cells is largely determined by their capacity of proliferation and apoptosis in presence of the administered drugs. We describe here the main markers used in flow cytometry (FCM) and involved in the assessment of cell cycle parameters: single labeling by Propidium Iodide (PI) and double labeling anti-Bromodeoxyuridine (BrdUrd)/PI which, both in vitro and in vivo, gives cell percentages in the different cell cycle phases. The markers of cell cycle progression can be divided into proliferation markers such as PCNA (proliferating cell nuclear antigen) or Ki-67 and cell cycle progression markers. The latter, which are the core of the cell cycle machinery, are molecules recently characterized (Cyclins, CDKs (cell dependent kinases), CDIs (cyclin-dependent kinase inhibitors)) and their cell expression can be analyzed using FCM. FCM is also one of the best means to detect and quantitate apoptotic cells. Several techniques are described: Nuclear labeling using Hoechst 33342: mitochondrial labeling using DiOC6(3): detection of DNA fragmentation using 1) labeling of fixed and permeabilized cells with a DNA marker or 2) labeling of the free 3' DNA ends using incorporation of labeled deoxynucleotides; detection in apoptotic cells (Bcl-2, Fas, phospholipids...). At last, we analyzed flow cytometry methods to study the cell resistance to Ara-C and anthracyclins. In combination with cell kinetic studies and detection of apoptotic cells, they should increase the efficiency of the acute leukemia treatment.
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PMID:Flow cytometry study of cell cycle, apoptosis and drug resistance in acute leukemia. 903 Sep 62

Aging is associated with lymphopenia and progressive decline in T cell functions; however, the mechanisms underlying these defects are unclear. We analyzed the expression of genes promoting apoptosis (fas/fasL1 and bax) and those inhibiting apoptosis (bcl-2 and bcl-xL) in lymphocytes from aging and young subjects at the protein level, using flow cytometry/Western blotting, and at the mRNA level, using quantitative PCR. Susceptibility of T cell subsets to undergo anti-Fas-induced apoptosis was analyzed by propidium iodide staining, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay, DNA fragmentation assay, and staining with Hoechst 33342 dye. An increased expression of Fas and Fas ligand and a decreased expression of Bcl-2 were observed in both CD4+ and CD8+ T cells from aging as compared with young controls. Increased Fas and decreased Bcl-2 expression were also found in memory cells of both CD4+ and CD8+ T cell subsets from aging. Bax expression was increased in lymphocytes from aging at both the protein and mRNA level. No significant difference was observed in Bcl-xL expression between aging and young; however, the ratio of Bax:Bcl-xL was increased in aging. An increased proportion of CD4+ and CD8+ T cell subsets from aging underwent apoptosis following anti-Fas Ab treatment as compared with CD4+ and CD8+ T cell subsets from young controls. These data suggest that increased apoptosis may be one of the mechanisms responsible for lymphopenia and T cell deficiency associated with human aging.
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PMID:Increased apoptosis of T cell subsets in aging humans: altered expression of Fas (CD95), Fas ligand, Bcl-2, and Bax. 946 19

TGF-beta1 is a multifunctional regulatory peptide (25 kDa) inducing growth arrest and apoptosis in many normal and neoplastic cells. In the present study, the involvement of proapoptotic (bax) and antiapoptotic (bcl-2) genes in the molecular mechanism of TGF-beta1-induced apoptosis of L1210 leukemic cells was investigated. Bax transcript was measured using the RT-PCR method with GAPDH as a "housekeeping gene" control, whereas Bcl-2 protein was determined using flow cytometry (FITC-conjugated monoclonal anti-Bcl-2 antibody and FITC-conjugated mouse anti-IgG1 antibody as a negative control). Apoptosis was evaluated using fluorescence microscopy and flow cytometry after cell staining with DAPI and sulforhodamine or propidium iodide and Hoechst 33342. ROS generation was assessed by flow cytometry using the oxidation-sensitive fluorescent marker C-DCDHF-DA. The response of L1210 leukemic cells to TGF-beta1 was two-directional: 1) partial arrest of the cell cycle at G1-S transition, and 2) induction of apoptotic cell death. TGF-beta1 increased the number of leukemic cells with typical morphological features of apoptosis: cell shrinkage, condensation of chromatin, pyknosis and fragmentation of nuclei, followed by secondary necrosis. DNA cleavage led to a decrease of the nuclear DNA content and the appearance of a hypodiploid peak sub-G1 in the DNA histogram. The extraction of low-molecular weight DNA fragments from apoptotic cells showed that TGF-beta1-induced apoptosis concerned first of all the cells from G1 phase. Two phases of intracellular ROS generation in TGF-beta1-treated cultures were observed: the first (rapid, 60 min after TGF-beta1 administration), and the second (slow, occurring between 24 and 48h of experiment, respectively). The increase of apoptotic cell number in TGF-beta1-treated cultures (2% FCS/RPMI 1640) was associated with the decrease of cell number expressing bcl-2, and with a significant drop of Bcl-2 level in the remaining cells after 24 h. The dose-dependent relationship between TGF-beta1 concentration and Bcl-2 level was nonlinear and described by power series regression. The lowest Bcl-2 level was noted at 1 ng/ml of TGF-beta1 concentration. The increase of Bax mRNA:GAPDH mRNA ratio was observed 3h after TGF-beta1 (1 ng/ml) administration to both the maintenance (2% FCS/RPMI) and growth promoting (10% FCS/RPMI) medium. Regardless of TGF-beta1 treatment, the quantity of Bax transcript was dependent on FCS concentration, being higher in the growth promoting medium. The results of this study indicate that bax may function as a primary response gene and together with lowered Bcl-2 level may determine the induction of apoptotic process in L1210 leukemic cells exposed to TGF-beta1.
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PMID:Expression of bcl-2 and bax in TGF-beta 1-induced apoptosis of L1210 leukemic cells. 962 23

We examined effects of the anti-epidermal growth factor receptor monoclonal antibody C225 on proliferation, cell cycle phase distribution, apoptosis, and radiosensitivity in squamous cell carcinoma (SCC) cell lines derived from head and neck cancer patients. Exposure to C225 in culture inhibits SCC proliferation in a time-dependent manner, and the degree of growth inhibition, compared to controls, ranges from 20 to 75%. Flow cytometry analysis demonstrates that C225 treatment induces accumulation of cells in G1, which is accompanied by a 2-3-fold decrease in the percentage of cells in S phase. C225 exposure also induces apoptosis in SCC populations, as demonstrated by flow cytometry analysis using dual stainings of merocyanine 540 and Hoechst 33342. Western blot analysis indicates that C225 exposure induces accumulation of hypophosphorylated retinoblastoma protein and increases expression of p27KIP1. An increase in Bax expression and concurrent decrease in Bcl-2 expression are observed when SCC cells are exposed to C225. Examination of C225 effects on radiation response in SCCs demonstrates enhancement in radiosensitivity and amplification of radiation-induced apoptosis. These effects are observed in both single-dose and fractionated radiation experiments. C225 represents a promising growth-inhibitory agent that can influence cellular proliferation, apoptosis, and radiosensitivity in SCCs of the head and neck.
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PMID:Epidermal growth factor receptor blockade with C225 modulates proliferation, apoptosis, and radiosensitivity in squamous cell carcinomas of the head and neck. 1021 3

Spinal cord injury is accompanied by an initial inflammatory reaction followed by secondary injury that is caused, in part, by apoptosis. Recruitment of leukocytes from the blood compartment to the site of inflammation in the injured spinal cord has been attributed to locally generated chemotactic agents (cytokines and chemokines). In addition to upregulation in the message levels of a number of chemokines, we have found up-regulation in the message levels of several chemokine receptors following spinal cord contusion injury. To reduce the inflammatory response after spinal cord injury, we have blocked the interaction of chemokine receptors with their ligands using the vMIPII chemokine antagonist. Using a rat model of spinal cord contusion injury, we show that continuous infusion of the antagonist for up to 7 days results in a decrease in infiltrating hematogenous cells at the site of injury. Histological evaluation ofthe tissue showed fewer activated macrophages at the site of injury. Concomitantly, reduced neuronal loss and gliosis were observed in the antagonist infused spinal cord. In addition, increased expression of Bcl-2 gene, an endogenous inhibitor of apoptosis, was seen in the antagonist infused spinal cord at 7 days post injury. Morphologically, staining with the bisbenzamide dye Hoechst 33342 showed significantly more apoptotic bodies in the vehicle compared to antagonist infused spinal cord. Our data suggest that chemokine antagonist infusion post-injury results in limiting the inflammatory response following spinal cord contusion injury, thereby attenuating neuronal loss, possibly due to decreased apoptosis. These findings support the contention that disrupting chemokine interactions with their receptors may be an effective approach in reducing the secondary damage after spinal cord injury.
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PMID:Chemokine antagonist infusion attenuates cellular infiltration following spinal cord contusion injury in rat. 1065 86

The expression of apoptosis-related proteins: TGF-beta1 (local inductor), TGF-beta-receptor, Bax (promoter), Bcl-2 (inhibitor) and CPP-32 (executor of apoptosis); the subcellular distribution of Bax; as well as the number and morphology of apoptotic cells in low-, moderate-, and high-involuted mammary glands of sow (four to six days after weaning) were investigated. The immunohistochemical study demonstrated a statistically significant increase in the integrated optical density (IOD) of lobuloalveolar mammary tissue labelling with anti-Bax antibody from low- through moderate-, to high-involuted glands. The immunoelectron microscopy revealed that Bax was localised in the cytosol, on the membranes of mitochondrium and rough endoplasmic reticulum, in nuclear envelope pores, and over heterochromatin of mammary epithelial cells. The increase in Bax/Bcl-2 ratio (2.3, 2.6 and 5.6 for low-, moderate-, and high-involuted glands, respectively) indicated the increasing susceptibility of mammary epithelial cells to apoptosis in the course of involution. The highest Bax/Bcl-2 ratio in high-involuted glands coincided with the highest expression of CPP-32 (caspase 3), TGF-beta1 and TGF-beta1 receptor. The number of apoptotic cells (simultaneous TUNEL and Hoechst 33342 staining) was 2.7, 3.4 and 3.8% for low-, moderate-, and high-involuted glands, respectively. The ultrastructural evaluation showed characteristic morphological features of apoptosis such as: margination and condensation of chromatin; pyknosis and fragmentation of the nucleus; and formation of apoptotic bodies. Phagocytosis of apoptotic cells by macrophages was also documented. The results of the present study suggest the involvement of Bax/Bcl-2 check-point in the regulation, CPP-32 in the execution, but TGF-beta1 in the induction of apoptosis of mammary epithelial cells in the involuting mammary gland of sow.
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PMID:Expression of apoptosis-related proteins in involuting mammary gland of sow. 1129 Apr 45

Renal Cell Carcinomas (RCCs) exhibit strong resistance to the most chemotherapeutic treatments probably due to the expression of various multidrug resistance (MDR) genes. Overexpression of P-glycoprotein (Pgp) is established as one such factor, but other mechanisms such as at-MDR, characterized by attenuated DNA-topoisomerase II (topoII) activity, may be functional as well. In addition, regulating proteins involved in apoptosis can exhibit multidrug resistant features. However, prevention of apoptosis as a mechanism of MDR has not yet been assessed in RCC, nor has the cytotoxicity of a variety of chemotherapeutic agents known to trigger apoptotic or necrotic cell death been tested in RCC in a systematic fashion. Using immunohistochemistry and Western blotting, Bcl-2 and Bax expression was determined in a panel of multidrug resistant RCC lines featuring Pgp and/or at-MDR. The results were related to apoptotic activity and kind of cell death in these cell lines, demonstrated by incubation with Hoechst 33342 and propidium iodide after treatment with various cytotoxic agents and quantitated by MTT. In the drug resistant sublines, some decreased Bax and strongly increased Bcl-2 expression was seen by immunohistochemistry indicating prevention of apoptosis as a distinct feature of MDR in RCC. This was confirmed by Western blotting. Sublines revealed significant resistance for all drugs, except for CC-313 and DiMIQ. However, these drugs induced necrotic cell death, in contrast to all other drugs tested, which induced apoptotic cell death. We conclude that, in chemoselected RCC sublines, multidrug resistance appears to be functional due to inhibition of apoptosis, apart from the MDR1 and at-MDR resistance mechanisms. CC-313 and DiMIQ are very potent cytotoxic agents in RCC, probably because they do not kill by induction of apoptosis.
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PMID:Inhibition of apoptotic proteins causes multidrug resistance in renal carcinoma cells. 1184 68

Apoptosis and necrosis represent two distinct types of cell death. Apoptosis possesses unique morphologic and biochemical features which distinguish this mechanism of programmed cell death from necrosis. Extrinsic apoptotic cell death is receptor-linked and initiates apoptosis by activating caspase 8. Intrinsic apoptotic cell death is mediated by the release of cytochrome c from mitochondrial and initiates apoptosis by activating caspase 3. Cancer chemotherapy utilizes apoptosis to eliminate tumor cells. Agents which bind to the minor groove of DNA, like camptothecin and Hoechst 33342, inhibit topoisomerase I, RNA polymerase II, DNA polymerase and initiate intrinsic apoptotic cell death. Hoechst 33342-induced apoptosis is associated with disruption of TATA box binding protein/TATA box complexes, replication protein A/single-stranded DNA complexes, topoisomerase I/DNA cleavable complexes and with an increased intracellular concentration of E2F-1 transcription factor and nitric oxide concentration. Nitric oxide and transcription factor activation or respression also regulate the two apoptotic pathways. Some human diseases are associated with excess or deficient rates of apoptosis, and therapeutic strategies to regulate the rate of apoptosis include inhibition or activation of caspases, mRNA antisense to reduce anti-apoptotic factors like Bcl-2 and survivin and recombinant TRAIL to activate pro-apoptotic receptors, DR4 and DR5.
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PMID:Apoptosis: biochemical aspects and clinical implications. 1241 95

The peroxisome proliferator-activated receptor agonist troglitazone (TRO) was used for treatment of non-insulin-dependent diabetes until its removal from the market because of its severe hepatotoxicity. However, the mechanism for its hepatotoxicity is still poorly understood. In this study, we investigated whether TRO caused cell death by altering signaling pathways associated with cell damage and survival in human hepatoma cells. Our data reveal that TRO caused time- and concentration-dependent apoptosis of HepG2 and Chang liver human hepatoma cells, as evidenced by DNA fragmentation and staining with Hoechst 33342. In contrast, 50 or 100 microM rosiglitazone, a structural analog of TRO, did not cause apoptosis in these hepatoma cells. TRO activated both c-Jun N-terminal protein kinase (JNK) and p38 kinase about 5-fold between 0.5 and 8 h before they returned to control levels at 16 h in HepG2 cells. In contrast, TRO failed to activate the extracellular signal-regulated kinase. Furthermore, TRO increased the levels of proapoptotic proteins, Bad, Bax, release of cytochrome c, and cleavage of Bid in a time-dependent manner. The antiapoptotic Bcl-2 protein level decreased in hepatoma cells treated with TRO. Pretreatment of hepatoma cells with a selective JNK inhibitor, anthra[1,9-cd]pyrazol-6(2H)-one (SP600125), significantly reduced the rate of TRO-induced cell death, whereas 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), an inhibitor of p38 kinase, had little effect on apoptosis. Pretreatment with SP600125 also prevented JNK activation and c-Jun phosphorylation. In addition, rosiglitazone, which is not as toxic to hepatoma cells as TRO, did not stimulate JNK activity. Transfection of cDNA for the dominant-negative mutant JNK-KR (Lys-->Arg) or SEK1-KR (Lys-->Arg), an immediate upstream kinase of JNK, significantly reduced TRO-induced JNK activation and cell death rate. Furthermore, SP600125 pretreatment effectively prevented the TRO-mediated changes in Bad, Bax, Bid cleavage, and cytochrome c release. These data strongly suggest that hepatotoxic TRO causes apoptosis by activating the JNK-dependent cell death pathway accompanied by increased Bid cleavage and elevation of proapoptotic proteins.
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PMID:Critical role of c-Jun N-terminal protein kinase activation in troglitazone-induced apoptosis of human HepG2 hepatoma cells. 1252 12

Angelica sinensis (Oliv.) Diels, a traditional Chinese medicine, has been widely prescribed in treatment of gynecological diseases. Bio-based assays for extracts of Angelica sinensis showed that the acetone extract (AE-AS) had dose-dependently antiproliferative effect on A549, HT29, DBTRG-05MG and J5 human cancer cells. The IC50 values of AE-AS on mentioned cancer cells ranged from 35 to 50 microg/ml after 24 h of treatment. After 72 h of exposure, AE-AS (40 microg/ml) significantly reduced A549 cell proliferation to 24 +/- 3.2% of control. In A549 cells, the cell cycle analysis showed that AE-AS induced a significant increase in the number of cells in G0/G1, with a concomitant decrease in the number of cells in S phase. AE-AS-induced chromatin changes and apoptosis of A549 cells were confirmed by Hoechst 33342 DNA staining and annexin V staining. A549 cells treated with AE-AS caused activation of caspase-9 and -3, and AE-AS-induced apoptosis could be inhibited by the broad-spectrum caspase inhibitor, z-VAD-fmk. The Western blot indicated the AE-AS-triggered apoptosis is mediated via suppression of Bcl-2 oncoprotein expression rather than p53 or Bax. Besides, AE-AS decreased the levels of cdk4 protein was observed. These results indicate that the AE-AS could induce G1/S arrest and activate the mechanism of apoptosis in human cancer cells. Extracts obtained from different methods of fractionation might possess distinct bioactivity. These results prompted us to further evaluate the in vivo anticancer effects and elucidate the chemical composition profile of AE-AS.
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PMID:Acetone extract of Angelica sinensis inhibits proliferation of human cancer cells via inducing cell cycle arrest and apoptosis. 1526 63

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