A photodynamic pathway to apoptosis and necrosis induced by dimethyl tetrahydroxyhelianthrone and hypericin in leukaemic cells: possible relevance to photodynamic therapy.
(9/10114)
The mechanism of cell death induction by dimethyl tetrahydroxyhelianthrone (DTHe), a new second-generation photodynamic sensitizer, is analysed in human leukaemic cell lines in comparison with the structurally related hypericin. DTHe has a broad range of light spectrum absorption that enables effective utilization of polychromatic light. Photosensitization of HL-60 cells with low doses of DTHe (0.65 microM DTHe and 7.2 J cm(-2) light energy) induced rapid apoptosis of > or =90% of the cells. At doses > or =2 microM, dying cells assumed morphological necrosis with perinucleolar condensation of chromatin in HL-60 and K-562 cell lines. Although nuclear fragmentation that is characteristic to apoptosis was prevented, DNA digestion to oligonucleosomes proceeded unhindered. Such incomplete apoptosis was more prevalent with the related analogue hypericin throughout most doses of photosensitization. Despite hypericin being a stronger photosensitizer, DTHe exhibited advantageous phototoxic properties to tumour cells, initiating apoptosis at concentrations about threefold lower than hypericin. Photosensitization of the cells induced dissociation of the nuclear envelope, releasing lamins into the cytosol. DTHe also differed from hypericin in effects exerted on the nuclear lamina, causing release of an 86-kDa lamin protein into the cytosol that was unique to DTHe. Within the nucleus, nuclear envelope lamin B underwent covalent polymerization, which did not affect apoptotic nuclear fragmentation at low doses of DTHe. At higher doses, polymerization may have been extensive enough to prevent nuclear collapse. Hut-78, CD4+ cells were resistant to the photodynamically activated apoptotic pathway. Beyond the tolerated levels of photodynamic damage, these cells died exclusively via necrosis. Hut-78 cells overexpress Bcl-X(L) as well as a truncated Bcl-X(L)tr isoform that could contribute to the observed resistance to apoptosis. (+info)
Microsatellite instability, Epstein-Barr virus, mutation of type II transforming growth factor beta receptor and BAX in gastric carcinomas in Hong Kong Chinese.
(10/10114)
Microsatellite instability (MI), the phenotypic manifestation of mismatch repair failure, is found in a proportion of gastric carcinomas. Little is known of the links between MI and Epstein-Barr virus (EBV) status and clinicopathological elements. Examination of genes mutated through the MI mechanism could also be expected to reveal important information on the carcinogenic pathway. Seventy-nine gastric carcinomas (61 EBV negative, 18 EBV positive) from local Hong Kong Chinese population, an intermediate-incidence area, were examined. Eight microsatellite loci, inclusive of the A10 tract of type II transforming growth factor beta receptor (TbetaR-II), were used to evaluate the MI status. MI in the BAX and insulin-like growth factor II receptor (IGF-IIR) genes were also examined. High-level MI (>40% unstable loci) was detected in ten cases (12.7%) and low-level MI (1-40% unstable loci) in three (3.8%). High-level MI was detected in two EBV-associated cases (11%) and the incidence was similar for the EBV-negative cases (13%). The high-level MIs were significantly associated with intestinal-type tumours (P = 0.03) and a more prominent lymphoid infiltrate (P = 0.04). Similar associations were noted in the EBV-positive carcinomas. The high-level MIs were more commonly located in the antrum, whereas the EBV-associated carcinomas were mostly located in body. Thirteen cardia cases were negative for both high-level MI and EBV. All patients aged below 55 were MI negative (P = 0.049). Of the high-level MIs, 80% had mutation in TbetaR-II, 40% in BAX and 0% in IGF-IIR. Of low-level MIs, 33% also had TbetaR-II mutation. These mutations were absent in the MI-negative cases. Of three lymphoepithelioma-like carcinomas, two cases were EBV positive and MI negative, one case was EBV negative but with high-level MI. In conclusion, high-level MIs were present regardless of the EBV status, and were found in a particular clinicopathological subset of gastric carcinoma patient. Inactivation of important growth regulatory genes observed in these carcinomas confirms the importance of MI in carcinogenesis. (+info)
Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression.
(11/10114)
Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and permeability factor that is potently angiogenic in vivo. We report here studies that suggest that VEGF potentiates angiogenesis in vivo and prolongs the survival of human dermal microvascular endothelial cells (HDMECs) in vitro by inducing expression of the anti-apoptotic protein Bcl-2. Growth-factor-enriched and serum-deficient cultures of HDMECs grown on collagen type I gels with VEGF exhibited a 4-fold and a 1.6-fold reduction, respectively, in the proportion of apoptotic cells. Enhanced HDMEC survival was associated with a dose-dependent increase in Bcl-2 expression and a decrease in the expression of the processed forms of the cysteine protease caspase-3. Cultures of HDMECs transduced with and overexpressing Bcl-2 and deprived of growth factors showed enhanced protection from apoptosis and exhibited a twofold increase in cell number and a fourfold increase in the number of capillary-like sprouts. HDMECs overexpressing Bcl-2 when incorporated into polylactic acid sponges and implanted into SCID mice exhibited a sustained fivefold increase in the number of microvessels and a fourfold decrease in the number of apoptotic cells when examined 7 and 14 days later. These results suggest that the angiogenic activity attributed to VEGF may be due in part to its ability to enhance endothelial cell survival by inducing expression of Bcl-2. (+info)
Kinetics of neuroendocrine differentiation in an androgen-dependent human prostate xenograft model.
(12/10114)
It was previously shown in the PC-295 xenograft that the number of chromogranin A (CgA)-positive neuroendocrine (NE) cells increased after androgen withdrawal. NE cells did not proliferate and differentiated from G0-phase-arrested cells. Here we further characterized NE differentiation, androgen receptor status, and apoptosis-associated Bcl-2 expression in the PC-295 model after androgen withdrawal to assess the origin of NE cells. PC-295 tumor volumes decreased by 50% in 4 days. Intraperitoneal bromodeoxyuridine (BrdU) incorporation and MIB-1 labeling decreased to 0%, and the apoptosis was maximal at day 4. Androgen receptor expression and prostate-specific antigen (PSA) serum levels decreased rapidly within 2 days. The number of NE cells increased 6-fold at day 4 and 30-fold at day 7. Five and ten percent of the CgA-positive cells were BrdU positive after continuous BrdU labeling for 2 and 4 days, respectively. However, no MIB-1 expression was observed in CgA-positive cells. NE cells expressed the regulated secretory pathway marker secretogranin III but were negative for androgen receptor and Bcl-2. Bcl-2 expression did increase in the non-NE tumor cells. In conclusion, androgen withdrawal leads to a rapid PC-295 tumor regression and a proliferation-independent induction of NE differentiation. The strictly androgen-independent NE cells that were still present after 21 days differentiated mainly from G0-phase-arrested cells. (+info)
Control of apoptosis in Epstein Barr virus-positive nasopharyngeal carcinoma cells: opposite effects of CD95 and CD40 stimulation.
(13/10114)
The expression and function of CD95 and CD40 were investigated in malignant cells from EBV-positive undifferentiated nasopharyngeal carcinomas (NPCs). Large amounts of CD95 and CD40 expression were detected in 15 of 16 EBV-positive NPC specimens. In contrast, CD95 was not detected in two biopsies from patients with EBV-negative differentiated NPCs. We tested whether the CD95 apoptotic pathway was functional in NPC cells by treating two EBV-positive NPC tumor lines in vitro with a CD95 agonist. In both cases, NPC cells were extremely susceptible to CD95-mediated apoptosis, despite strong constitutive expression of Bcl-x. Combined CD40 and CD95 stimulation was used to investigate the possible anti-apoptotic activity mediated by CD40. The CD40 receptor was activated by incubating NPC cells with murine L cells producing CD154, the CD40 ligand. This treatment resulted in a strong inhibition of CD95-related cytotoxicity. Such an anti-apoptotic effect of CD40 is well known for B lymphocytes, but has not previously been reported for epithelial cells. These data suggest that NPC tumor-infiltrating lymphocytes, which often produce the CD40 ligand in situ, may increase the survival of malignant cells, thereby enhancing tumor growth in patients. (+info)
Role of cytokine signaling molecules in erythroid differentiation of mouse fetal liver hematopoietic cells: functional analysis of signaling molecules by retrovirus-mediated expression.
(14/10114)
Erythropoietin (EPO) and its cell surface receptor (EPOR) play a central role in proliferation, differentiation, and survival of erythroid progenitors. Signals induced by EPO have been studied extensively by using erythroid as well as nonerythroid cell lines, and various controversial results have been reported as to the role of signaling molecules in erythroid differentiation. Here we describe a novel approach to analyze the EPO signaling by using primary mouse fetal liver hematopoietic cells to avoid possible artifacts due to established cell lines. Our strategy is based on high-titer retrovirus vectors with a bicistronic expression system consisting of an internal ribosome entry site (IRES) and green fluorescent protein (GFP). By placing the cDNA for a signaling molecule in front of IRES-GFP, virus-infected cells can be viably sorted by fluorescence-activated cell sorter, and the effect of expression of the signaling molecule can be assessed. By using this system, expression of cell-survival genes such as Bcl-2 and Bcl-XL was found to enhance erythroid colony formation from colony-forming unit-erythroid (CFU-E) in response to EPO. However, their expression was not sufficient for erythroid colony formation from CFU-E alone, indicating that EPO induces signals for erythroid differentiation. To examine the role of EPOR tyrosine residues in erythroid differentiation, we introduced a chimeric EGFR-EPOR receptor, which has the extracellular domain of the EGF receptor and the intracellular domain of the EPOR, as well as a mutant EGFR-EPOR in which all the cytoplasmic tyrosine residues are replaced with phenylalanine, and found that tyrosine residues of EPOR are essential for erythroid colony formation from CFU-E. We further analyzed the function of the downstream signaling molecules by expressing modified signaling molecules and found that both JAK2/STAT5 and Ras, two major signaling pathways activated by EPOR, are involved in full erythroid differentiation. (+info)
Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.
(15/10114)
We investigated mechanisms of cell death during hypoxia/reoxygenation of cultured kidney cells. During glucose-free hypoxia, cell ATP levels declined steeply resulting in the translocation of Bax from cytosol to mitochondria. Concurrently, there was cytochrome c release and caspase activation. Cells that leaked cytochrome c underwent apoptosis after reoxygenation. ATP depletion induced by a mitochondrial uncoupler resulted in similar alterations even in the presence of oxygen. Moreover, inclusion of glucose during hypoxia prevented protein translocations and reoxygenation injury by maintaining intracellular ATP. Thus, ATP depletion, rather than hypoxia per se, was the cause of protein translocations. Overexpression of Bcl-2 prevented cytochrome c release and reoxygenation injury without ameliorating ATP depletion or Bax translocation. On the other hand, caspase inhibitors did not prevent protein translocations, but inhibited apoptosis during reoxygenation. Nevertheless, they could not confer long-term viability, since mitochondria had been damaged. Omission of glucose during reoxygenation resulted in continued failure of ATP production, and cell death with necrotic morphology. In contrast, cells expressing Bcl-2 had functional mitochondria and remained viable during reoxygenation even without glucose. Therefore, Bax translocation during hypoxia is a molecular trigger for cell death during reoxygenation. If ATP is available during reoxygenation, apoptosis develops; otherwise, death occurs by necrosis. By preserving mitochondrial integrity, BCL-2 prevents both forms of cell death and ensures cell viability. (+info)
Apoptosis during breast carcinoma progression.
(16/10114)
The purpose of this study was to investigate apoptosis, proliferation, and the expression of apoptosis-influencing proteins bcl-2 and bax and estrogen and progesterone receptors during breast carcinoma progression. The material consisted of 53 paired breast carcinoma samples representing primary and recurrent tumors and 24 control samples. The recurrent sample was located either in the breast scar tissue or at a distant metastatic site. Apoptosis was detected both morphologically and by 3' end labeling of fragmented DNA. Cell proliferation was evaluated immunohistochemically by the MIB index. The expressions of bcl-2, bax, and estrogen and progesterone receptors were studied immunohistochemically. There was a significant increase in the extent of apoptosis and proliferation in recurrent tumors compared to the primary lesions (P = 0.015 and P = 0.038, respectively). In primary tumors with an apoptotic index of >0.50%, the survival of the patients was significantly shorter (P = 0.015). In cases with a significant increase in apoptosis or proliferation in the recurrent tumor, the survival of the patients was significantly shorter (P = 0.009 and P = 0.003, respectively). Of the variables analyzed, bcl-2 expression and a positive estrogen receptor status were significantly associated with a low extent of apoptosis (P = 0.010 and P = 0.042, respectively). Their changes were parallel to the changes in apoptosis during tumor progression, although the associations did not reach statistical significance. The results show that increased apoptosis is associated with a worse prognosis in breast carcinoma. A significant increase in apoptosis in recurrent breast carcinoma lesions predicts a worse clinical outcome. (+info)