Reactive oxygen species and mitochondrial sensitivity to oxidative stress determine induction of cancer cell death by p21

p21(Waf1/Cip1/Sdi1) is a cyclin-dependent kinase inhibitor that mediates cell cycle arrest. Prolonged p21 up-regulation induces a senescent phenotype in normal and cancer cells, accompanied by an increase in intracellular reactive oxygen species (ROS). However, it has been shown recently that p21 expression can also lead to cell death in certain models. The mechanisms involved in this process are not fully understood. Here, we describe an induction of apoptosis by p21 in sarcoma cell lines that is p53-independent and can be ameliorated with antioxidants. Similar levels of p21 and ROS caused senescence in the absence of significant death in other cancer cell lines, suggesting a cell-specific response. We also found that cells undergoing p21-dependent cell death had higher sensitivity to oxidants and a specific pattern of mitochondrial polarization changes. Consistent with this, apoptosis could be blocked with targeted expression of catalase in the mitochondria of these cells. We propose that the balance between cancer cell death and arrest after p21 up-regulation depends on the specific effects of p21-induced ROS on the mitochondria. This suggests that selective up-regulation of p21 in cancer cells could be a successful therapeutic intervention for sarcomas and tumors with lower resistance to mitochondrial oxidative damage, regardless of p53 status.     

Cyclooxygenase-independent induction of p21WAF-1/cip1, apoptosis and differentiation by L-745,337, a selective PGH synthase-2 inhibitor, and salicylate in HT-29 cells

In order to dissect out cyclooxygenase-dependent from cyclooxygenase-independent mechanisms in the antiproliferative effects of selective prostaglandin H synthase (PGHS)-2 inhibitors, we compared the effects of L-745,337 (a highly selective PGHS-2 inhibitor) with sodium salicylate (a weak PGHS inhibitor) on prostanoid production, induction of the cyclin-dependent kinase inhibitor p21^WAF-1/cip1, mutant p53 (m273-p53) levels, apoptosis and differentiation in human colon adenocarcinoma HT-29 cells. L-745,337 dose-dependently suppressed the cyclooxygenase activity of HT-29 cells (IC₅₀: 0.24 M). Four-day treatment with L-745,337 caused a concentration-dependent inhibition of cell growth (IC₅₀: 0.9 mM) associated with the induction of p21^WAF-1/cip1 and an increase in the proportion of apoptotic nuclei (EC₅₀: 0.1 and 0.34 mM, respectively) while reducing the levels of m273-p53 (IC₅₀: 0.2 mM). Sodium salicylate, at the concentration of 10 mM that did not affect prostanoid formation, caused a 60% reduction of cell growth associated with a 3-fold induction of p21^WAF-1/cip1 and a 60% increase in the proportion of apoptotic nuclei. Ultrastructural analysis showed that L-745,337 (0.5 mM) and sodium salicylate (10 mM) caused the induction of a differentiated phenotype. We conclude that high concentrations of L-745,337 and sodium salicylate inhibit colon cancer cell growth by a mechanism unrelated to cyclooxygenase inhibition that may involve p53-independent induction of the tumor suppressor p21^WAF-1/cip1.     

Adriamycin (ADM) induced apoptosis in Transitional Cell Cancer (TCC) cell lines accompanied by p21 WAF1/CIP1 induction

Genotoxic stimuli, including anticancer drugs, induce apoptosis in cancer cells through increase of p53, p21WAF1/CIP1 , at least in part. Bcl-2 and Bax modify this pathway or directly regulated by p53. Here we studied Adriamycin (ADM)-induced apoptosis in four human bladder cancer cell lines in respect of p53, p21WAF1/CIP1 and Bcl-2 family proteins. After ADM, treatment bladder cancer cells underwent dose-dependent cell death with typical morphologic features of apoptosis. Among four cell lines RT4 with wt p53, low ratio of Bcl-2 to Bax and induction of p21WAF1/CIP1 after ADM treatment, was the most sensitive to induction of apoptosis. Thus, p53, p21WAF1/CIP1 , Bcl-2 and Bax status might determine susceptibility of bladder cancer cells to ADM induced apoptosis.     

Involvement of Cyclin-Dependent Kinase Activities in CD437-Induced Apoptosis

A novel synthetic retinoid, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), is a selective ligand of the RARgamma nuclear receptor. We examined the in vitro effects of CD437 and found that CD437 induces S phase arrest within 24 to 48 h, followed by cell death, in the p53-negative Hep3B and the p53-positive HepG2 human hepatoma cell lines. Based on observations of cellular and nuclear fragmentation, chromatin condensation, and DNA fragmentation, the CD437-mediated cell-killing effect appears to be due to apoptosis. On morphological examination, a number of CD437-treated cells were found to have increased 5- to 10-fold in size and persisted as single giant cells without cell division, while the remainder underwent nuclear division (multiple nuclei) but were unable to complete cytokinesis, and finally all died by apoptosis. In HepG2 cells that possessed wild-type p53, CD437-induced S phase arrest and apoptosis were accompanied by the up-regulation of cyclin A, cyclin B, p53, p21(CIP1/Waf1), Bad, and Bcl-Xs proteins and by a decrease in Bcl-2 protein levels. In Hep3B cells, CD437-mediated S phase arrest and apoptosis were also associated with a concomitant up-regulation of cyclin A, cyclin B, Bad, and Bcl-Xs. However, Hep3B cells did not express p53 or Bcl-2 messages. Olomoucine and roscovitine, the potent p34(cdc2) and CDK2 inhibitors, effectively blocked CD437-mediated cyclin A- and B-dependent kinase activation and prevented CD437-induced cell death. Furthermore, antisense oligonucleotide complementary to cyclin A and B mRNA significantly rescued CD437-induced apoptosis. These findings suggest that activation of cyclin A- and B-dependent kinases is a critical determinant of apoptotic death mediated by CD437.     

LYG-202 inhibits the proliferation of human colorectal carcinoma HCT-116 cells through induction of G1/S cell cycle arrest and apoptosis via p53 and p21(WAF1/Cip1) expression

We recently established that LYG-202, a new flavonoid with a piperazine substitution, exerts an anti-tumor effect in vivo and in vitro. In the present study, we demonstrate that LYG-202 induces G1/S phase arrest and apoptosis in human colorectal carcinoma HCT-116 cells. Data showed that the blockade of the cell cycle was associated with increased p21(WAF1/Cip1) and Rb levels and reduced expression of cyclin D1, cyclin E, and CDK4. Moreover, PARP cleavage, activation of caspase-3, caspase-8, and caspase-9, and an increased ratio of Bax/Bcl-2 were detected in LYG-202-induced apoptosis. Additionally, activation of p53 resulted in the up-regulation of its downstream targets PUMA and p21(WAF1/Cip1), as well as the down-regulation of its negative regulator MDM2, suggesting that the p53 pathway may play a crucial role in LYG-202-induced cell cycle arrest and apoptosis. Furthermore, siRNA knockdown of p53 attenuated the G1 cell cycle arrest and apoptosis induced by LYG-202, as the effects of LYG-202 on up-regulation of p21(WAF1/Cip1) and down-regulation of Bcl-2 and pro-caspase-3 were partly inhibited in p53 siRNA transfected cells compared with control siRNA transfected cells. Collectively, these data indicate that LYG-202 exerts its anti-tumor potency by activating the p53-p21 pathway for G1/S cell cycle arrest and apoptosis in colorectal cancer cells.     

Cyclooxygenase-2 overexpression inhibits platelet-derived growth factor-induced mesangial cell proliferation through induction of the tumor suppressor gene p53 and the cyclin-dependent kinase inhibitors p21waf-1/cip-1 and p27kip-1.

Cyclooxygenase-2 (COX-2) is an inducible enzyme and serves as a source of paracrine prostaglandin E2 (PGE2) formation in many tissues. In glomerular immune injury COX-2 formation is up-regulated in association with increased mesangial cell growth. To examine whether COX-2 exerts growth modulating effects on glomerular cells, we established two separate COX-2-overexpressing mesangial cell lines (COX-2+) and assessed their proliferative response to the potent mesangial cell growth-promoting factor, platelet-derived growth factor (PDGF). PDGF increased proliferation in mock-transfected cells. In contrast, PDGF did not induce proliferation in COX-2+ cells. Our results also showed that the tumor suppressor protein p53 and the cyclin-dependent kinase inhibitors p21(cip-1) and p27(kip-1) were up-regulated in COX-2+ cells de novo as well as under PDGF-stimulated conditions. To study whether COX-2 products are required for these effects, COX-2+ cells were treated with indomethacin (1 microg/ml) or NS-398 (3 microm). Unexpectedly, both COX inhibitors had no significant effect on cell proliferation, not on the protein levels of p53, p21(cip-1), or p27(kip-1). To evaluate the role of p21(cip-1) and p27(kip-1), COX-2 was overexpressed in mesangial cells derived from p21(cip-1) (p21-/- COX-2+) and p27(kip-1) (p27-/- COX-2+) null mice. In contrast to the wild type COX-2+ cells, p21-/- COX-2+ and p27-/- COX-2+ cells proliferated in response to PDGF. These data suggest that COX-2 inhibits mesangial cell proliferation by a novel mechanism that is independent of prostaglandin synthesis, but involves p53, p21(cip-1), and p27(kip-1).     

Rsf-1, a chromatin remodeling protein, induces DNA damage and promotes genomic instability

Rsf-1 (HBXAP) has been reported as an amplified gene in human cancer, including the highly aggressive ovarian serous carcinoma. Rsf-1 protein interacts with SNF2H to form an ISWI chromatin remodeling complex, RSF. In this study, we investigated the functional role of Rsf-1 by observing phenotypes after expressing it in nontransformed cells. Acute expression of Rsf-1 resulted in DNA damage as evidenced by DNA strand breaks, nuclear γH2AX foci, and activation of the ATM-CHK2-p53-p21 pathway, leading to growth arrest and apoptosis. Deletion mutation and gene knockdown assays revealed that formation of a functional RSF complex with SNF2H was required for Rsf-1 to trigger DNA damage response (DDR). Gene knock-out of TP53 alleles, TP53 mutation, or treatment with an ATM inhibitor abolished up-regulation of p53 and p21 and prevented Rsf-1-induced growth arrest. Chronic induction of Rsf-1 expression resulted in chromosomal aberration and clonal selection for cells with c-myc amplification and CDKN2A/B deletion. Co-culture assays indicated Rsf-1-induced DDR as a selecting barrier that favored outgrowth of cell clones with a TP53 mutation. The above findings suggest that increased Rsf-1 expression and thus excessive RSF activity, which occurs in tumors harboring Rsf-1 amplification, can induce chromosomal instability likely through DDR.     

Hypoxia induces p53-dependent transactivation and Fas/CD95-dependent apoptosis

p53 triggers apoptosis in response to cellular stress. We analyzed p53-dependent gene and protein expression in response to hypoxia using wild-type p53-carrying or p53 null HCT116 colon carcinoma cells. Hypoxia induced p53 protein levels and p53-dependent apoptosis in these cells. cDNA microarray analysis revealed that only a limited number of genes were regulated by p53 upon hypoxia. Most classical p53 target genes were not upregulated. However, we found that Fas/CD95 was significantly induced in response to hypoxia in a p53-dependent manner, along with several novel p53 target genes including ANXA1, DDIT3/GADD153 (CHOP), SEL1L and SMURF1. Disruption of Fas/CD95 signalling using anti-Fas-blocking antibody or a caspase 8 inhibitor abrogated p53-induced apoptosis in response to hypoxia. We conclude that hypoxia triggers a p53-dependent gene expression pattern distinct from that induced by other stress agents and that Fas/CD95 is a critical regulator of p53-dependent apoptosis upon hypoxia.     

Cutting edge: a novel mechanism for rescue of B cells from CD95/Fas-mediated apoptosis.

CD95-induced apoptosis contributes to the maintenance of homeostasis in both B and T lymphocyte-mediated immunity. B cells increase CD95 expression in response to activation signals and become susceptible to CD95-induced apoptosis. Protection from CD95-mediated death signals can be induced in mature B cells by signals delivered through the B cell Ag receptor. In this paper we demonstrate for the first time that rescue from apoptosis can occur independently of de novo protein synthesis. This rescue from apoptosis prevents activation of caspase 8, the apical caspase in the CD95 death pathway, and CD95-FADD (Fas-associated death domain containing protein) association does not occur normally. Thus B cell activation signals can biochemically modify proximal elements of the CD95 death pathway and regulate the sensitivity of cells to apoptosis induction at an early stage in programmed cell death.     

Caspase inhibition in apoptotic T cells triggers necrotic cell death depending on the cell type and the proapoptotic stimulus

CD95 (Fas/Apo-1) triggers apoptotic cell death via a caspase-dependent pathway. Inhibition of caspase activation blocks proapoptotic signaling and thus, prevents execution of apoptosis. Besides induction of apoptotic cell death, CD95 has been reported to trigger necrotic cell death in susceptible cells. In this study, we investigated the interplay between apoptotic and necrotic cell death signaling in T cells. Using the agonistic CD95 antibody, 7C11, we found that caspase inhibition mediated by the pancaspase inhibitor, zVAD-fmk, prevented CD95-triggered cell death in Jurkat T cells but not in A3.01 T cells, although typical hallmarks of apoptosis, such as DNA fragmentation or caspase activation were blocked. Moreover, the caspase-independent cell death in A3.01 cells exhibited typical signs of necrosis as detected by a rapid loss of cell membrane integrity and could be prevented by treatment with the radical scavenger butylated hydroxyanisole (BHA). Similar to CD95-induced cell death, apoptosis triggered by the DNA topoisomerase inhibitors, camptothecin or etoposide was shifted to necrosis when capsase activation was inhibited. In contrast to this, ZVAD was fully protective when apoptosis was triggered by the serpase inhibitor, Nalpha-tosyl-phenyl-chloromethyl ketone (TPCK). TPCK was not protective when administered to anti-CD95/ZVAD-treated A3.01 cells, indicating that TPCK does not possess anti-necrotic activity but fails to activate the necrotic death pathway. Our findings show (a) that caspase inhibition does not always protect apoptotic T cells from dying but merely activates a caspase-independent mode of cell death that results in necrosis and (b) that the caspase-inhibitor-induced shift from apoptotic to necrotic cell death is dependent on the cell type and the proapoptotic stimulus.     

Environmental chemical-induced pro/pre-B cell apoptosis: analysis of c-Myc,p27Kip1, and p21WAF1 reveals a death pathway distinct from clonal deletion

Polycyclic aromatic hydrocarbons (PAH) are common environmental pollutants that suppress the immune system in part by inducing pro/pre-B cell apoptosis. The PAH-induced death signaling pathway resembles the signaling cascade activated during clonal deletion and modeled by B cell receptor cross-linking or by dexamethasone exposure of immature surface Ig(+) B cells in that apoptosis is mediated by NF-kappa B down-regulation. Because a PAH-induced, clonally nonrestricted deletion of B cells would have important implications for B cell repertoire development, the nature of the PAH-induced intracellular death signal was studied further. Particular emphasis was placed on the roles of growth arrest and c-Myc, p27(Kip1), and p21(WAF1) expression, because all of these elements contribute to clonal deletion. As in clonal deletion models, and as predicted by the down-regulation of NF-kappa B, PAH-induced death of pro/pre-B cells was at least partially dependent on c-Myc down-regulation. Furthermore, whereas dexamethasone induced a G(0)/G(1) cell cycle arrest, PAH had no effect on pro/pre-B cell growth, indicating that growth arrest and apoptosis occur by separable signaling pathways in this early phase of B cell development. Finally, in contrast to clonal deletion, PAH-induced pro/pre-B cell death was not dependent on p27(Kip1) or p21(WAF1) up-regulation but did coincide with p53 induction. These results distinguish the PAH-induced apoptosis pathway from that activated during clonal deletion and indicate that signaling cascades leading to growth arrest and/or apoptosis in pro/pre-B cells differ from those active at later B cell developmental stages.     

Induction of p21(CIP1/Waf1) and activation of p34(cdc2) involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells

The biological activity of retinoic acid (RA) was examined in human hepatoma Hep3B cells. Under serum-deprived conditions, RA induced S/M-phase elevation and mitotic index increase within 24 h, followed by apoptosis. This RA-induced apoptosis was accompanied by p53-independent up-regulation of endogenous p21(CIPI/Waf1) and Bax proteins, as well as activation of p34(cdc2) kinase, and increase of Rb2 protein level and phosphorylation pattern. In addition, RA had no effect on the levels of Bcl-XL; Bcl-XS; cyclins A, B, D1, D3, or E; or Rb1 expression but markedly down-modulated Cdk2 kinase activity and reduced Cdk4 expression. RA also slightly delayed p27(Kip1) expression. Olomoucine, a potent p34(cdc2) and Cdk2 inhibitor, effectively blocked RA-mediated p34(cdc2) kinase activation and prevented RA-induced apoptosis. Furthermore, antisense oligonucleotide complementary to p21(CIP2/Waf1) and p34(cdc2) mRNA significantly rescued RA-induced apoptosis. Our data indicate that p21(CIP2/Waf1) overexpression may not be the only regulatory factor necessary for RA-induced apoptosis in human hepatoma Hep3B cells. RA treatment leads to Rb2 hyperphosphorylation, and p34(cdc2) kinase activation is coincident with an aberrant mitotic progression, followed by appearance of abnormal nucleus. This aberrant cell cycle progression appeared requisite for RA-induced cell death. These findings suggest that inappropriate regulation of the cell cycle regulators p21(CIP2/Waf1) and p34(cdc2) is coupled with induction of Bax and involved in cell death with apoptosis when Hep3B cells are exposed to RA.