UMLS:C0029463 - FACTA Search

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Query: UMLS:C0029463 (osteosarcoma)
16,637 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Poly(ADP-ribose) polymerase (PARP), which is catalytically activated by DNA strand breaks, has been implicated in apoptosis, or programmed cell death. A protease (CPP32) responsible for the cleavage of PARP and necessary for apoptosis was recently purified and characterized. The coordinated sequence of events related to PARP activation and cleavage in apoptosis has now been examined in individual cells. Apoptosis was studied in a human osteosarcoma cell line that undergoes a slow (8 to 10 days), spontaneous, and reproducible death program in culture. Changes in the abundance of intact PARP, poly(ADP-ribose) (PAR), and a proteolytic cleavage product of PARP that contains the DNA-binding domain were examined during apoptosis in the context of individual, whole cells by immunofluorescence with specific antibodies. The synthesis of PAR from NAD increased early, within 2 days of cell plating for apoptosis, prior to the appearance of internucleosomal DNA cleavage and before the cells become irreversibly committed to apoptosis, since replating yields viable, nonapoptotic cells. Strong expression of full-length PARP was also detected, by immunofluorescence as well as by Western analysis, during this same time period. However, after approximately 4 days in culture, the abundance of both full-length PARP and PAR decreased markedly. After 6 days, a proteolytic cleavage product containing the DNA-binding domain of PARP was detected immunocytochemically and confirmed by Western analysis, both in the nuclei and in the cytoplasm of cells. A recombinant peptide spanning the DNA-binding domain of PARP was expressed, purified, and biotinylated, and was then used as a probe for DNA strand breaks. Fluorescence microscopy with this probe revealed extensive DNA fragmentation during the later stages of apoptosis. This is the first report, using individual, intact cells, demonstrating that poly(ADP-ribosyl)ation of nuclear proteins occurs prior to the commitment to apoptosis, that inactivation and cleavage of PARP begin shortly thereafter, and that very little PAR per se is present during the later stages of apoptosis, despite the presence of a very large number of DNA strand breaks. These results suggest a negative regulatory role for PARP during apoptosis, which in turn may reflect the requirement for adequate NAD and ATP during the later stages of programmed cell death.
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PMID:Intact cell evidence for the early synthesis, and subsequent late apopain-mediated suppression, of poly(ADP-ribose) during apoptosis. 916 7

A transient burst of poly(ADP-ribosyl)ation of nuclear proteins occurs early, prior to commitment to death, in human osteosarcoma cells undergoing apoptosis, followed by caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP). The generality of this early burst of poly(ADP-ribosyl)ation has now been investigated with human HL-60 cells, mouse 3T3-L1, and immortalized fibroblasts derived from wild-type mice. The effects of eliminating this early transient modification of nuclear proteins by depletion of PARP protein either by antisense RNA expression or by gene disruption on various morphological and biochemical markers of apoptosis were then examined. Marked caspase-3-like PARP cleavage activity, proteolytic processing of CPP32 to its active form, internucleosomal DNA fragmentation, and nuclear morphological changes associated with apoptosis were induced in control 3T3-L1 cells treated for 24 h with anti-Fas and cycloheximide but not in PARP-depleted 3T3-L1 antisense cells exposed to these inducers. Similar results were obtained with control and PARP-depleted human Jurkat T cells. Whereas immortalized PARP +/+ fibroblasts showed the early burst of poly(ADP-ribosyl)ation and a rapid apoptotic response when exposed to anti-Fas and cycloheximide, PARP -/- fibroblasts exhibited neither the early poly (ADP-ribosyl)ation nor any of the biochemical or morphological changes characteristic of apoptosis when similarly treated. Stable transfection of PARP -/- fibroblasts with wild-type PARP rendered the cells sensitive to Fas-mediated apoptosis. These results suggest that PARP and poly(ADP-ribosyl)ation may trigger key steps in the apoptotic program. Subsequent degradation of PARP by caspase-3-like proteases may prevent depletion of NAD and ATP or release certain nuclear proteins from poly(ADP-ribosyl)ation-induced inhibition, both of which might be required for late stages of apoptosis.
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PMID:Transient poly(ADP-ribosyl)ation of nuclear proteins and role of poly(ADP-ribose) polymerase in the early stages of apoptosis. 959 11

A murine erythroleukemic cell line (1-2-3) which expresses only the temperature-sensitive mutant p53 gene (Ala-to-Val substitution at codon 135) was established. These cells showed typical characteristics of apoptosis, when they were cultured at 32 degrees C. In this process, p53 recovered the wild-type p53 function and the expression of the p21 (waf1/cip1/sdi1), cyclin G1 and gadd45 genes was increased. However, no significant changes were detected in the expression of the mdm2, bcl-2, bax, fas and fasl genes, suggesting the existence of other genes associated with apoptosis. Genes up-regulated by p53 were screened by the mRNA differential display method. One of the up-regulated genes was identified as the elongation factor 1 alpha (EF-1 alpha) gene. EF-1 alpha is also a microtubule-severing protein. Upon the temperature-shift, the cells developed the morphology and the localization of alpha-tubulin similar to those of the cells treated with vincristine, a drug that affects microtubules. The microtubule-severing associated with up-regulation of EF-1 alpha by p53 may be a cause of the cell death. On the other hand, the function of cyclin G1 is not so clear despite the fact that 1-2-3 cells showed a significant increase of the cyclin G1 gene during the early stage of apoptosis. The yeast two-hybrid system was used to identify cyclin G1-associated proteins. One is a cytochrome c (Cyt c) oxidase subunit II (COXII). Cyclin G1 and COXII were co-immunoprecipitated from an extract of human osteosarcoma cell line that expressed high levels of cyclin G1. COX activity was also increased by temperature-shift in this cell line. The pattern of changes in COX activity was closely reflected by the expression of the cyclin G1 gene. Cyclin G1 and COXII associate physically with each other in vivo and that activation of COXII by binding to cyclin G1 upregulated by p53 may be associated with apoptosis. These two new pathways, p53-EF-1 alpha-microtubule-severing (-distortion of cytoskeleton) and p53-cyclin G1-COXII (-CytC, ATP-caspase-3 activation), may cooperate to induce apoptosis in this cell line.
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PMID:The mechanisms of death of an erythroleukemic cell line by p53: involvement of the microtubule and mitochondria. 1019 36

Spontaneous apoptosis in human osteosarcoma cells was observed to be associated with a marked increase in the intracellular abundance of p53. Immunoprecipitation and immunoblot analysis revealed that, together with a variety of other nuclear proteins, p53 undergoes extensive poly(ADP-ribosyl)ation early during the apoptotic program in these cells. Subsequent degradation of poly(ADP-ribose) (PAR), attached to p53 presumably by PAR glycohydrolase, the only reported enzyme to degrade PAR, was apparent concomitant with the onset of proteolytic processing and activation of caspase-3, caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP), and internucleosomal DNA fragmentation during the later stages of cell death. The decrease in PAR covalently bound to p53 also coincided with the marked induction of expression of the p53-responsive genes bax and Fas. These results suggest that poly(ADP-ribosyl)ation may play a role in the regulation of p53 function and implies a regulatory role for PARP and/or PAR early in apoptosis.
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PMID:Poly(ADP-ribosyl)ation of p53 during apoptosis in human osteosarcoma cells. 1023 7

We have focused on the roles of PARP and poly(ADP-ribosyl)ation early in apoptosis, as well as during the early stages of differentiation-linked DNA replication. In both nuclear processes, a transient burst of PAR synthesis and PARP expression occurs early, prior to internucleosomal DNA cleavage before commitment to apoptosis as well as at the round of DNA replication prior to the onset of terminal differentiation. In intact human osteosarcoma cells undergoing spontaneous apoptosis, both PARP and PAR decreased after this early peak, concomitant with the inactivation and cleavage of PARP by caspase-3 and the onset of substantial DNA and nuclear fragmentation. Whereas 3T3-L1, osteosarcoma cells, and immortalized PARP +/+ fibroblasts exhibited this early burst of PAR synthesis during Fas-mediated apoptosis, neither PARP-depleted 3T3-L1 PARP-antisense cells nor PARP -/- fibroblasts showed this response. Consequently, whereas control cells progressed into apoptosis, as indicated by induction of caspase-3-like PARP-cleavage activity, PARP-antisense cells and PARP -/- fibroblasts did not, indicating a requirement for PARP and poly(ADP-ribosyl)ation of nuclear proteins at an early reversible stage of apoptosis. In parallel experiments, a transient increase in PARP expression and activity were also noted in 3T3-L1 preadipocytes 24 h after induction of differentiation, a stage at which approximately 95% of the cells were in S-phase, but not in PARP-depleted antisense cells, which were consequently unable to complete the round of DNA replication required for differentiation. PARP, a component of the multiprotein DNA replication complex (MRC) that catalyzes viral DNA replication in vitro, poly(ADP-ribosyl)ates 15 of approximately 40 MRC proteins, including DNA pol alpha, DNA topo I, and PCNA. Depletion of endogenous PARP by antisense RNA expression in 3T3-L1 cells results in MRCs devoid of any DNA pol alpha and DNA pol delta activities. Surprisingly, there was no new expression of PCNA and DNA pol alpha, as well as the transcription factor E2F-1 in PARP-antisense cells during entry into S-phase, suggesting that PARP may play a role in the expression of these proteins, perhaps by interacting with a site in the promoters for these genes.
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PMID:Involvement of PARP and poly(ADP-ribosyl)ation in the early stages of apoptosis and DNA replication. 1033 50

An early transient burst of poly(ADP-ribosyl)ation of nuclear proteins was recently shown to be required for apoptosis to proceed in various cell lines (Simbulan-Rosenthal, C., Rosenthal, D., Iyer, S., Boulares, H., and Smulson, M. (1998) J. Biol. Chem. 273, 13703-13712) followed by cleavage of poly(ADP-ribose) polymerase (PARP), catalyzed by caspase-3. This inactivation of PARP has been proposed to prevent depletion of NAD (a PARP substrate) and ATP, which are thought to be required for later events in apoptosis. The role of PARP cleavage in apoptosis has now been investigated in human osteosarcoma cells and PARP -/- fibroblasts stably transfected with a vector encoding a caspase-3-resistant PARP mutant. Expression of this mutant PARP increased the rate of staurosporine and tumor necrosis factor-alpha-induced apoptosis, at least in part by reducing the time interval required for the onset of caspase-3 activation and internucleosomal DNA fragmentation, as well as the generation of 50-kilobase pair DNA breaks, thought to be associated with early chromatin unfolding. Overexpression of wild-type PARP in osteosarcoma cells also accelerated the apoptotic process, although not to the same extent as that apparent in cells expressing the mutant PARP. These effects of the mutant and wild-type enzymes might be due to the early and transient poly(ADP-ribose) synthesis in response to DNA breaks, and the accompanying depletion of NAD apparent in the transfected cells. The accelerated NAD depletion did not seem to interfere with the later stages of apoptosis. These results indicate that PARP activation and subsequent cleavage have active and complex roles in apoptosis.
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PMID:Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells. 1043 58

We examined the effects of flavopiridol (FP), a cyclin-dependent kinase inhibitor, on doxorubicin (DOX)-induced cell killing in an osteosarcoma cell line (SaOs-2) that lacks functional retinoblastoma protein (pRb). The IC50 value for DOX was 7-fold lower when combined with a low dose (100 nM) FP in pRb-deficient SaOs-2 cells than in the absence of FP. In contrast, the IC50 value for DOX was not decreased in the presence of 100 nM FP in pRb-restored SaOs-2 cells. Consistent with this, FP enhanced DOX-induced activation of caspase-3, which correlates with apoptosis, in pRb-deficient cells but not in pRb-restored cells. Additional studies showed that FP decreased DOX-induced cell accumulation in S phase in retinoblastoma-restored cells but not in pRb-deficient cells. An increased expression of p21 and inhibition of cyclin-dependent kinase 2 kinase activity by FP was also observed in pRb-deficient cells but not in retinoblastoma-restored SaOs-2 cells. We conclude that pRb plays a key role in determining whether FP selectively sensitizes DOX-induced cell killing in human sarcoma cells. Because lack of functional pRb is a common abnormality in human cancers, the combination of FP with DOX in tumors lacking pRb would be worthy of further investigation.
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PMID:Selective sensitization of retinoblastoma protein-deficient sarcoma cells to doxorubicin by flavopiridol-mediated inhibition of cyclin-dependent kinase 2 kinase activity. 1128 34

The oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a multifunctional molecule that induces monocytic differentiation of human myeloid leukemia cells and inhibits proliferation of diverse human tumor cell lines. The present studies on human osteosarcoma cells demonstrate that CDDO induces mitochondrial cytochrome c release, caspase-3 activation, and internucleosomal DNA fragmentation. Overexpression of the caspase-8 inhibitor CrmA blocked CDDO-induced cytochrome c release and apoptosis. By contrast, overexpression of the antiapoptotic Bcl-x(L) protein blocked CDDO-induced cytochrome c release, but only partly inhibited caspase-3 activation and apoptosis. In concert with these findings, we demonstrate that CDDO: 1) activates caspase-8 and thereby caspase-3 by a cytochrome c-independent mechanism and 2) induces cytochrome c release by caspase-8-dependent cleavage of Bid. The results also demonstrate that treatment of osteosarcoma cells with CDDO induces differentiation, as assessed by alkaline phosphatase activity and osteocalcin production, and that this response is abrogated in cells that overexpress CrmA. These findings demonstrate that CDDO induces both osteoblastic differentiation and apoptosis by caspase-8-dependent mechanisms.
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PMID:The novel triterpenoid CDDO induces apoptosis and differentiation of human osteosarcoma cells by a caspase-8 dependent mechanism. 1130 92

This paper studies the cytotoxic effect induced by the topoisomerase I inhibitor camptothecin in human osteosarcoma Saos-2 cells, which lack p53 and contain a non-functional form of the product of the retinoblastoma gene, pRb. Cytotoxicity induced by camptothecin was dose- and time-dependent; the treatment with 100 nM camptothecin reduced cell viability by 50% at 32 h and by 75% at 72 h of exposure. The cytotoxic effect was caused by apoptosis, as ascertained by morphological evidence, acridine orange-ethidium bromide staining and flow cytometric analysis. Apoptosis was accompanied by both the activation of caspase-3 and the fragmentation of poly(ADP-ribose) polymerase. Treatment with camptothecin caused a threefold increase in the activity of c-Jun N-terminal kinase (JNK) and an eightfold increase in the level of phosphorylated c-Jun. The introduction of the RB gene into Saos-2 cells reduced the rate of cell growth. Moreover, stable clones of transfected cells were resistant to camptothecin. Exposure to 100 nM camptothecin for 72 h reduced the viability of transfected cells by only 10%; moreover, very modest effects were observed on the activity of JNK as well as on the level of phosphorylated c-Jun. The results reported in this paper support the conclusion that the expression of wild-type pRb in Saos-2 cells exerts an anti-apoptotic influence through the control of JNK activity.
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PMID:pRb suppresses camptothecin-induced apoptosis in human osteosarcoma Saos-2 cells by inhibiting c-Jun N-terminal kinase. 1141 38

Several endonucleases are implicated in the internucleosomal DNA fragmentation associated with apoptosis. The human Ca2+- and Mg2+-dependent endonuclease DNAS1L3 is inhibited by poly(ADP-ribosyl)ation in vitro, and its activation during apoptosis shows a time course similar to that of the cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). The role of the cleavage and consequent inactivation of PARP-1 by caspase-3 in the activation of DNAS1L3 has now been investigated further both in vitro and in vivo. In an in vitro system based on purified recombinant proteins and NAD, caspase-3 prevented the inhibition of DNAS1L3 endonuclease activity by wild-type PARP-1 but not that induced by a caspase-3-resistant PARP-1 mutant. The induction by etoposide of apoptosis in human osteosarcoma cells (which were shown not to express endogenous DNAS1L3) was accompanied by internucleosomal DNA fragmentation only after transfection of the cells with a plasmid encoding DNAS1L3. This DNA fragmentation in etoposide-treated cells was blocked by 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, an inhibitor of intracellular Ca2+ release. Expression of the endonuclease subunit of DNA fragmentation factor (DFF40) and cleavage of its inhibitor, DFF45, were not sufficient to cause internucleosomal DNA fragmentation in osteosarcoma cells during etoposide-induced apoptosis. Coexpression of caspase-3-resistant PARP-1 mutant with DNAS1L3 in osteosarcoma cells blocked etoposide-induced internucleosomal DNA fragmentation and resulted in persistent poly(ADP-ribosyl)ation of DNAS1L3; it did not, however, prevent the activation of caspase-3 and the consequent cleavage of endogenous PARP-1. These results indicate that PARP-1 cleavage during apoptosis is not simply required to prevent excessive depletion of NAD and ATP but is also necessary to release DNAS1L3 from poly(ADP-ribosyl)ation-mediated inhibition.
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PMID:Regulation of DNAS1L3 endonuclease activity by poly(ADP-ribosyl)ation during etoposide-induced apoptosis. Role of poly(ADP-ribose) polymerase-1 cleavage in endonuclease activation. 1169 7

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