Apoptosis in hematopoietic cells (FL5.12) caused by interleukin-3 withdrawal: relationship to caspase activity and the loss of glutathione.
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The mechanism of cell death caused by cytokine deprivation remains largely unknown. FL5.12 cells (a murine prolymphocytic cell line), following interleukin-3 (IL-3) withdrawal, undergo a decrease in intracellular glutathione (GSH) that precedes the onset of apoptosis. In the present study, the induction of apoptosis following IL-3 withdrawal or GSH depletion with DL-buthionine-[S,R,]-sulfoximine (BSO) was examined. Both conditions caused time-dependent increases in phosphatidylserine externalization, acridine orange and ethidium bromide staining, decreases in mitochondrial membrane potential, processing and activation of caspase-3 and proteolysis of the endogenous caspase substrate poly(adenosine diphosphate ribose)polymerase (PARP). Apoptosis induced by IL-3 deprivation but not BSO also caused lamin B1 cleavage, suggesting activation of caspase-6. Despite a more profound depletion of GSH after BSO than withdrawal of IL-3, the extent of apoptosis was somewhat lower. Benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone (z-VAD.fmk) blocked this caspase activity and prevented cell death after BSO exposure but not after IL-3 deprivation. Following IL-3 withdrawal, the caspase inhibitors z-VAD.fmk and boc-asp(OMe)fluoromethylketone (boc-asp.fmk) prevented the cleavage and activation of caspase-3, the breakdown of lamin B1 and partially mitigated PARP degradation. However, the externalization of phosphatidylserine, the fall in mitochondrial membrane potential and subsequent apoptotic cell death still occurred. These results suggest that IL-3 withdrawal may mediate cell death by a mechanism independent of both caspase activation and the accompanying loss of GSH. (+info)
Apoptosis and cell-cycle arrest in human and murine tumor cells are initiated by isoprenoids.
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Diverse classes of phytochemicals initiate biological responses that effectively lower cancer risk. One class of phytochemicals, broadly defined as pure and mixed isoprenoids, encompasses an estimated 22,000 individual components. A representative mixed isoprenoid, gamma-tocotrienol, suppresses the growth of murine B16(F10) melanoma cells, and with greater potency, the growth of human breast adenocarcinoma (MCF-7) and human leukemic (HL-60) cells. beta-Ionone, a pure isoprenoid, suppresses the growth of B16 cells and with greater potency, the growth of MCF-7, HL-60 and human colon adenocarcinoma (Caco-2) cells. Results obtained with diverse cell lines differing in ras and p53 status showed that the isoprenoid-mediated suppression of growth is independent of mutated ras and p53 functions. beta-Ionone suppressed the growth of human colon fibroblasts (CCD-18Co) but only when present at three-fold the concentration required to suppress the growth of Caco-2 cells. The isoprenoids initiated apoptosis and, concomitantly arrested cells in the G1 phase of the cell cycle. Both suppress 3-hydroxy-3-methylglutaryl CoA reductase activity. beta-Ionone and lovastatin interfered with the posttranslational processing of lamin B, an activity essential to assembly of daughter nuclei. This interference, we postulate, renders neosynthesized DNA available to the endonuclease activities leading to apoptotic cell death. Lovastatin-imposed mevalonate starvation suppressed the glycosylation and translocation of growth factor receptors to the cell surface. As a consequence, cells were arrested in the G1 phase of the cell cycle. This rationale may apply to the isoprenoid-mediated G1-phase arrest of tumor cells. The additive and potentially synergistic actions of these isoprenoids in the suppression of tumor cell proliferation and initiation of apoptosis coupled with the mass action of the diverse isoprenoid constituents of plant products may explain, in part, the impact of fruit, vegetable and grain consumption on cancer risk. (+info)
Strong association of autoantibodies to human nuclear lamin B1 with lupus anticoagulant antibodies in systemic lupus erythematosus.
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OBJECTIVE: To determine the frequency and clinical significance of high titers of IgG autoantibodies to nuclear lamin B1 in a large number of unselected and well-characterized systemic lupus erythematosus (SLE) patients, disease controls, and normal healthy controls. METHODS: A cross-sectional study of anti-lamin B1 autoantibodies, as measured by enzyme-linked immunosorbent assay using human recombinant lamin B1 autoantigen, was performed on serum samples obtained at first evaluation of 238 consecutive French Canadian adults: 61 healthy control subjects, 20 patients with osteoarthritis, 22 with ankylosing spondylitis, 11 with autoimmune hepatitis, 30 with rheumatoid arthritis, and 94 with SLE. SLE patients were studied for 57 disease manifestations. A case-control study was performed to analyze the relationship between anti-lamin B1 status and thrombotic manifestations between SLE onset and last followup. RESULTS: High titers of anti-lamin B1 were strikingly restricted to a subset of 8 SLE patients (8.5%). The mean anti-lamin B1 titer was higher in this subset than in the other SLE patients or any control group (P<0.001). By univariate analysis and stepwise multiple logistic regression, the most striking association of anti-lamin B1 was with lupus anticoagulant (LAC) antibodies (P = 0.00001). Although LAC were significantly associated with thrombosis in our SLE patients, anti-lamin B1 was not. The frequency of thrombosis in SLE patients expressing both LAC and anti-lamin B1 was similar to that in patients without LAC (P = 1.0). However, patients expressing LAC without anti-lamin B1 had a greater frequency of thrombosis (P = 0.018). CONCLUSION: High titers of IgG anti-lamin B1 autoantibodies are highly specific for a subset of SLE patients whose clinical characteristics include the presence of LAC and other laboratory manifestations of the antiphospholipid syndrome. The presence of LAC without anti-lamin B1 may define a subset of SLE patients at greater risk for thrombosis. (+info)
Translational control of nuclear lamin B1 mRNA during oogenesis and early development of Xenopus.
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Cytoplasmic polyadenylation of specific mRNAs is commonly correlated with their translational activation during development. A canonical nuclear polyadenylation element AAUAAA (NPE) and cytoplasmic polyadenylation element(s) (CPE) are necessary and sufficient for polyadenylation during egg maturation. We have characterized cis-acting sequences of Xenopus nuclear lamin B1 mRNA that mediate translational regulation. By injection of synthetic RNAs into oocytes we show that the two CPE-like elements found in the 3'-untranslated region of B1 mRNA act as translational repressors in oocytes. The same CPEs in conjunction with the NPE confer transient polyadenylation and translational activation during egg maturation. Poly(A) length determination of the endogenous lamin B1 mRNA reveals a gradual increase of poly(A) tail length in early development up to mid-blastula, and a shortening of poly(A) tails during gastrulation and neurulation. The same kinetic and extent of polyadenylation and poly(A) tail shortening is observed with synthetic RNAs injected into fertilized eggs. Polyadenylation and translational activation of these RNAs is independent of the two CPEs and a NPE during early development. While translational regulation of lamin B1 mRNA functions in parts via established mechanisms, the pattern of polyadenylation and deadenylation during early development points to a novel mode of translational regulation. (+info)
Decreased and aberrant nuclear lamin expression in gastrointestinal tract neoplasms.
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BACKGROUND: Altered expression of lamins A/C and B1, constituent proteins of the nuclear lamina, may occur during differentiation and has also been reported in primary lung cancer. AIMS: To examine the expression of these proteins in gastrointestinal neoplasms. PATIENTS: Archival human paraffin wax blocks and frozen tissue from patients undergoing surgical resection or endoscopic biopsy. METHODS: Immunohistochemistry and western blotting using polyclonal antisera against A type lamins and lamin B1. RESULTS: The expression of lamin A/C was reduced and was frequently undetectable by immunohistochemistry in all primary colon carcinomas and adenomas, and in 7/8 primary gastric cancers. Lamin B1 expression was reduced in all colon cancers, 16/18 colonic adenomas, and 6/8 gastric cancers. Aberrant, cytoplasmic labelling with both antibodies occurred in some colonic cancers and around one third of colonic adenomas. Cytoplasmic lamin A/C expression was detected in 3/8 gastric cancers. Lamin expression was reduced in gastric dysplasia, but not intestinal metaplasia, atrophy, or chronic gastritis. Lamin expression was low in carcinomas of oesophagus, prostate, breast, and uterus, but not pancreas. CONCLUSIONS: Reduced expression of nuclear lamins, sometimes together with aberrant, cytoplasmic immunoreactivity is common in gastrointestinal neoplasms. Altered lamin expression may be a biomarker of malignancy in the gastrointestinal tract. (+info)
The apoptotic effects and synergistic interaction of sodium butyrate and MG132 in human retinoblastoma Y79 cells.
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This study deals with the apoptotic effect exerted on human retinoblastoma Y79 cells by both sodium butyrate and an inhibitor of 26S proteasome [z-Leu-Leu-Leu-CHO (MG132)] and their synergistic effect. Exposure to sodium butyrate (1-4 mM) induced an accumulation of cells in the G2-M phase that was already visible after 24 h of treatment, when morphological and biochemical signs of apoptosis appeared only in a small number of cells (5-10%). Thereafter, the apoptotic effects increased progressively with slow kinetics, reaching a maximum after 72 h of exposure, when they concerned a large fraction of cells (>75% with 4 mM sodium butyrate). Sodium butyrate stimulated the conversion of procaspase-3 into caspase-3 and also induced the cleavage of poly-(ADP-ribose) polymerase and lamin B, two hallmarks of apoptosis. All of the apoptotic signals were suppressed by benzyloxy carbonyl-Val-Ala-Asp-fluoromethylketone (a general inhibitor of caspase activities), whereas acetyl-Asp-Glu-Val-Asp aldehyde, a specific inhibitor of caspase-3 activity, only induced a partial reversion of the apoptotic effects. Sodium butyrate also decreased the Bcl-2 level, whereas it increased the Bax level and stimulated the release of cytochrome c from the mitochondria, an event that was most likely responsible for the activation of caspase-3. Finally, sodium butyrate activated 26S proteasome, the major extralysosomal degradative machinery, which is responsible for the degradation of short-lived proteins. Consequently, the levels of p53, N-myc, and IkappaBalpha (factors that play regulatory roles in apoptosis) diminished, whereas the nuclear level of nuclear factor kappaB concomitantly increased. Treatment of Y79 cells with MG132 induced apoptosis with more rapid kinetics than with sodium butyrate. The effects appeared after 8 h of incubation, reaching a maximum at 24 h, and they were accompanied by increased levels of N-myc, p53, and IkappaBalpha. MG132 also favored the release of cytochrome c from the mitochondria and increased the activity of caspase-3. When Y79 cells were exposed to combinations of sodium butyrate and MG132, the latter compound suppressed the decreasing effect induced by sodium butyrate on the levels of p53, N-myc, and IkappaBalpha and the increasing effect on the nuclear level of nuclear factor kappaB. Moreover, an increase in the level of Bax and an enhancement in the release of cytochrome c from the mitochondria were observed. Clear synergistic effects concerning the activation of both caspase-3 and apoptosis were induced by a combination of suboptimal doses of sodium butyrate and MG132. The results support the conclusion that MG132 potentiates the apoptotic effect of sodium butyrate by suppressing its stimulatory effect on 26S proteasome activity. Synergistic interactions between butyrate and inhibitors of proteasome could represent a new important tool in tumor therapy and, in particular, the treatment of retinoblastoma. (+info)
Colocalization of intranuclear lamin foci with RNA splicing factors.
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The lamins form a fibrous network underlying the inner nuclear membrane termed the nuclear lamina. In order to gain insights into the role of lamins in nuclear organization, we have characterized a monoclonal antibody (LA-2H10) raised against recombinant rat lamin A that labels nuclei in a speckled pattern in all cells of unsynchronized populations of HeLa and rat F-111 fibroblast cells, unlike the typical nuclear periphery staining by another monoclonal antibody to lamin A, LA-2B3. In immunolocalization studies the lamin A speckles or foci were found to colocalize with the RNA splicing factors SC-35 and U5-116 kD, but not with p80 coilin found in coiled bodies. Lamin B1 was also associated with these foci. These foci dispersed when cells entered mitosis and reformed during anaphase. The differential reactivity of LA-2H10 and LA-2B3 was retained after nuclei were extracted with detergents, nucleases and salt to disrupt interactions of lamins with chromatin and other nuclear proteins. Using deletion fragments of recombinant lamin A, the epitope recognized by LA-2H10 was located between amino acids 171 and 246. Our findings are consistent with a structural role for lamins in supporting nuclear compartments containing proteins involved in RNA splicing. (+info)
Start sites of bidirectional DNA synthesis at the human lamin B2 origin.
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The initiation sites of bidirectional synthesis at the DNA replication origin located at the 3' end of the human lamin B2 gene were investigated. RNA-primed nascent DNA molecules were subjected to second-strand synthesis with appropriate primers, amplified by ligation-mediated polymerase chain reaction, and size fractionated. Evidence for precise start sites was obtained. Exploration of close to 1 kilobase, coupled to inhibition of Okazaki fragment synthesis, demonstrates that the leading strands initiate at precise nucleotides on either helix, overlapping by three base pairs, within the area bound to a protein complex possibly analogous to the prereplicative complex of yeast. (+info)