The apoptotic action of the retinoid CD437/AHPN: Diverse effects,common basis

Retinoids, such as all-TRANS-retinoic acid (RA), have found applications in several different types of (cancer) therapies. The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437 or AHPN), an RA receptor (RAR)gamma agonist, not only induces RARgamma-dependent differentiation, but in contrast to RA, it also induces RARgamma-independent apoptosis in many tumor cells. This observation makes this and similar new retinoids very interesting from a clinical perspective. Several genes have been associated with CD437/AHPN-mediated apoptosis, but the multiple activities of this compound and the apparent cell-type-specific responses to treatment have made it difficult to discern a common biochemical basis for the mechanism of its apoptotic action. In this brief review, we present a model which links all CD437/AHPN-associated apoptotic effects. CD437/AHPN rapidly induces DNA adduct formation through an as-yet unknown reaction which is independent of cell type. This is followed by a cell-type-specific, largely p53-independent DNA damage response which can result in engagement of multiple cell death pathways and activation of caspases as a common endpoint. At the same time, the RARgamma-dependent pathway leads to regulation of differentiation-associated, cell-type-specific genes. CD437/AHPN, with its simultaneous differentiation and apoptosis-inducing activities, is a good prototype for new drugs which may be clinically more efficacious than those with a single activity.     

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.     

Molecular Determinants of AHPN (CD437)-Induced Growth Arrest and Apoptosis in Human Lung Cancer Cell Lines

6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN or CD437), originally identified as a retinoic acid receptor γ-selective retinoid, was previously shown to induce growth inhibition and apoptosis in human breast cancer cells. In this study, we investigated the role of AHPN/CD437 and its mechanism of action in human lung cancer cell lines. Our results demonstrated that AHPN/CD437 effectively inhibited lung cancer cell growth by inducing G₀/G₁ arrest and apoptosis, a process that is accompanied by rapid induction of c-Jun, nur77, and p21^WAF1/CIP1. In addition, we found that expression of p53 and Bcl-2 was differentially regulated by AHPN/CD437 in different lung cancer cell lines and may play a role in regulating AHPN/CD437-induced apoptotic process. On constitutive expression of the c-JunAla(63,73) protein, a dominant-negative inhibitor of c-Jun, in A549 cells, nur77 expression and apoptosis induction by AHPN/CD437 were impaired, whereas p21^WAF1/CIP1 induction and G₀/G₁ arrest were not affected. Furthermore, overexpression of antisense nur77 RNA in A549 and H460 lung cancer cell lines largely inhibited AHPN/CD437-induced apoptosis. Thus, expression of c-Jun and nur77 plays a critical role in AHPN/CD437-induced apoptosis. Together, our results reveal a novel pathway for retinoid-induced apoptosis and suggest that AHPN/CD437 or analogs may have a better therapeutic efficacy against lung cancer.     

Apoptosis induced by MNNG in human TK6 lymphoblastoid cells is p53 and Fas/CD95/Apo-1 related

Agents inducing O(6)-methylguanine (O(6)MeG) in DNA, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), are not only highly mutagenic and carcinogenic but also cytotoxic because of the induction of apoptosis. In CHO fibroblasts, apoptosis triggered by O(6)MeG requires cell proliferation and MutSalpha-dependent mismatch repair and is related to the induction of DNA double-strand breaks (DSBs). Furthermore, it is mediated by Bcl-2 degradation and does not require p53 for which the cells were mutated [Cancer Res. 60 (2000) 5815]. Here we studied cytotoxicity and apoptosis induced by MNNG in a pair of human lymphoblastoid cells expressing wild-type p53 (TK6) and mutant p53 (WTK1) and show that TK6 cells are more sensitive than WTK1 cells to cell killing (determined by a metabolic assay) and apoptosis. Apoptosis was a late response observed <24h after treatment and was related to accumulation of p53 and upregulation of Fas/CD95/Apo-1 receptor as well as Bax. The data indicate that MNNG induces apoptosis in lymphoblastoid cells by activating the p53-dependent Fas receptor-driven pathway. This is in contrast to CHO fibroblasts in which, in response to O(6)MeG, the mitochondrial damage pathway becomes activated.     

Elucidation of molecular events mediating induction of apoptosis by synthetic retinoids using a CD437-resistant ovarian carcinoma cell line

Retinoids have great promise in the area of cancer therapy and chemoprevention. Although some tumor cells are sensitive to the growth inhibitory effect of all-trans-retinoic acid (ATRA), many ovarian tumor cells are not. 6-((1-Admantyl)-4-hydroxyphenyl)-2-naphthalenecarboxylic acid (CD437) is a conformationally restricted synthetic retinoid that induces growth arrest and apoptosis in both ATRA-sensitive and ATRA-resistant ovarian tumor cell lines. To better understand the mechanism by which CD437 induces apoptosis in ovarian tumor cell lines, we prepared a cell line, CA-CD437R, from the ATRA-sensitive ovarian cell line, CA-OV-3, which was resistant to CD437. We found that the CD437-resistant cell line was also resistant to the induction of apoptosis by tumor necrosis factor-alpha but not resistant to the induction of apoptosis by another synthetic retinoid, fenretinide N-(4-hydroxyphenyl)retinamide. We also show that this cell line remains ATRA-sensitive and exhibits no deficiencies in RAR function. Analysis of this CD437-resistant cell line suggests that the pathway for induction of apoptosis by CD437 is similar to the pathway utilized by tumor necrosis factor-alpha and different from the pathway induced by the synthetic retinoid, fenretinide N-(4-hydroxyphenyl)retinamide. The CA-CD437R cell line is a valuable tool, permitting us to further elucidate the molecular events that mediate apoptosis induced by CD437 and other synthetic retinoids. Results of experiments utilizing this cell line suggest that the alteration responsible for resistance of CA-CD437R cells to CD437 induced event maps after the activation of p38 and TR3 expression, prior to mitochondrial depolarization, subsequent release of cytochrome c and activation of caspase-9 and caspase-3.     

Induction of apoptosis and stress response in ovarian carcinoma cell lines treated with ST1926, an atypical retinoid

To understand the molecular mechanisms mediating apoptosis induction by a novel atypical retinoid, ST1926, the cellular response to drug treatment was investigated in IGROV-1 ovarian carcinoma cells carrying wild-type p53 and a cisplatin-resistant p53 mutant subline (IGROV-1/Pt1). Despite a similar extent of drug-induced DNA strand breaks, the level of apoptosis was substantially higher in p53 wild-type cells. p53 activation and early upregulation of p53-target genes were consistent with p53-dependent apoptosis in IGROV-1 cells. Stress-activated protein kinases were activated in both cell lines in response to ST1926. This event and activation of AP-1 were more pronounced in IGROV-1/Pt1 cells, in which the modulation of DNA repair-associated genes suggests an increased ability to repair DNA damage. Inhibition of JNK or p38 stimulated ST1926-induced apoptosis only in IGROV-1 cells, whereas inhibition of ERKs enhanced apoptosis in both the cell lines. Such a pattern of cellular response and modulation of genes implicated in DNA damage response supports that the genotoxic stress is a critical event mediating drug-induced apoptosis. The results are consistent with apoptosis induction through p53-dependent and -independent pathways, regulated by MAP kinases, which likely play a protective role.     

Induction of apoptosis and mitosis inhibition by degraded DNA lipotransfection mimicking genotoxic drug effects

Genotoxic damage induces cell cycle arrest and/or apoptosis by activation of p53 oncosuppressor protein. A number of anticancer drugs are genotoxic and their damaging effect upon cells is mediated by this mechanism. Microinjection of defined DNA species directly into nucleus has been reported previously to activate p53 and inhibit cell cycle. Here, we demonstrate that simple addition of heterogeneous degraded DNA to cultured cells (Rat-1 fibroblasts) in combination with lipotransfecting agent DOTAP leads to apoptosis induction and mitosis inhibition by a molecular mechanism which mimics that of the cellular response to genotoxic anticancer agents. Indeed, both cellular effects induced by lipotransfected degraded DNA (essentially, heterogeneous small DNA fragments) are associated to p53 activation and modulated by two apoptosis-related genes, such as bcl-2 and c-myc, which also modulate the apoptotic threshold to anticancer agents. Here we raise the hypothesis of exogenous DNA segment lipotransfection as possible new tool for anticancer therapy.     

Role of reactive oxygen species and p53 in chromium(VI)-induced apoptosis

Apoptosis is a programmed cell death mechanism to control cell number in tissues and to eliminate individual cells that may lead to disease states. The present study investigates chromium(VI) (Cr(VI))-induced apoptosis and the role of reactive oxygen species (ROS) and p53 in this response. Treatment of human lung epithelial cells (A549) with Cr(VI) caused apoptosis as measured by DNA fragmentation, mitochondria damage, and cell morphology. Cr(VI)-induced apoptosis is contributed to ROS generation, resulting from cellular reduction of Cr(VI) as measured by flow cytometric analysis of the stained cells, oxygen consumption, and electron spin resonance spin trapping. Scavengers of ROS, such as catalase, aspirin, and N-acetyl-L-cysteine, decreased Cr(VI)-induced apoptosis, whereas NADPH and glutathione reductase, enhancers of Cr(VI)-induced ROS generation, increased it. p53 is activated by Cr(VI), mostly by ROS-mediated free radical reactions. Cr(VI)-induced ROS generation occurred within a few minutes after Cr(VI) treatment of the cells, whereas p53 induction took at least 5 h. The level of Cr(VI)-induced apoptosis was similar in both p53-positive cells and p53-negative cells independent of p53 status in the early stage (0-3 h) of Cr(VI) treatment. However, at the later stage (3-24 h), the level of the apoptosis is higher in p53-positive cells than in p53-negative cells. These results suggest that ROS generated through Cr(VI) reduction is responsible to the early stage of apoptosis, whereas p53 contributes to the late stage of apoptosis and is responsible for the enhancement of Cr(VI)-induced apoptosis at this stage.     

p73beta is regulated by protein kinase Cdelta catalytic fragment generated in the apoptotic response to DNA damage

Protein kinase C (PKC) delta is cleaved by caspase-3 to a kinase-active catalytic fragment (PKCdeltaCF) in the apoptotic response of cells to DNA damage. Expression of PKCdeltaCF contributes to the induction of apoptosis by mechanisms that are presently unknown. Here we demonstrate that PKCdeltaCF associates with p73beta, a structural and functional homologue of the p53 tumor suppressor. The results show that PKCdeltaCF phosphorylates the p73beta transactivation and DNA-binding domains. One PKCdeltaCF-phosphorylation site has been mapped to Ser-289 in the p73beta DNA-binding domain. PKCdeltaCF-mediated phosphorylation of p73beta is associated with accumulation of p73beta and induction of p73beta-mediated transactivation. By contrast, PKCdeltaCF-induced activation of p73beta is attenuated by mutating Ser-289 to Ala (S289A). The results also demonstrate that PKCdeltaCF stimulates p73beta-mediated apoptosis and that this response is attenuated with the p73beta(S289A) mutant. These findings demonstrate that cleavage of PKCdelta to PKCdeltaCF induces apoptosis by a mechanism in part dependent on PKCdeltaCF-mediated phosphorylation of the p73beta Ser-289 site.     

Pharmacological activation of a novel p53-dependent S-phase checkpoint involving CHK-1

We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1.     

c-Myc can induce DNA damage,increase reactive oxygen species,and mitigate p53 function: a mechanism for oncogene-induced genetic instability

Oncogene overexpression activates p53 by a mechanism posited to involve uncharacterized hyperproliferative signals. We determined whether such signals produce metabolic perturbations that generate DNA damage, a known p53 inducer. Biochemical, cytological, cell cycle, and global gene expression analyses revealed that brief c-Myc activation can induce DNA damage prior to S phase in normal human fibroblasts. Damage correlated with induction of reactive oxygen species (ROS) without induction of apoptosis. Deregulated c-Myc partially disabled the p53-mediated DNA damage response, enabling cells with damaged genomes to enter the cycle, resulting in poor clonogenic survival. An antioxidant reduced ROS, decreased DNA damage and p53 activation, and improved survival. We propose that oncogene activation can induce DNA damage and override damage controls, thereby accelerating tumor progression via genetic instability.     

Phosphorylation of Tip60 by GSK-3 determines the induction of PUMA and apoptosis by p53

Activation of p53 by DNA damage results in either cell-cycle arrest, allowing DNA repair and cell survival, or induction of apoptosis. As these opposite outcomes are both mediated by p53 stabilization, additional mechanisms to determine this decision must exist. Here, we show that glycogen synthase kinase-3 (GSK-3) is required for the p53-mediated induction of the proapoptotic BH3 only-protein PUMA, an essential mediator of p53-induced apoptosis. Inhibition of GSK-3 protected from cell death induced by DNA damage and promoted increased long-term cell survival. We demonstrate that GSK-3 phosphorylates serine 86 of the p53-acetyltransferase Tip60. A Tip60(S86A) mutant was less active to induce p53 K120 acetylation, histone 4 acetylation, and expression of PUMA. Our data suggest that GSK-3 mediated Tip60S86 phosphorylation provides a link between PI3K signaling and the choice for or against apoptosis induction by p53.     

The nuclear death domain protein p84N5 activates a G2/M cell cycle checkpoint prior to the onset of apoptosis

In contrast to extracellular signals, the mechanisms utilized to transduce nuclear apoptotic signals are not well understood. Characterizing these mechanisms is important for predicting how tumors will respond to genotoxic radiation or chemotherapy. The retinoblastoma (Rb) tumor suppressor protein can regulate apoptosis triggered by DNA damage through an unknown mechanism. The nuclear death domain-containing protein p84N5 can induce apoptosis that is inhibited by association with Rb. The pattern of caspase and NF-kappaB activation during p84N5-induced apoptosis is similar to p53-independent cellular responses to DNA damage. One hallmark of this response is the activation of a G(2)/M cell cycle checkpoint. In this report, we characterize the effects of p84N5 on the cell cycle. Expression of p84N5 induces changes in cell cycle distribution and kinetics that are consistent with the activation of a G(2)/M cell cycle checkpoint. Like the radiation-induced checkpoint, caffeine blocks p84N5-induced G(2)/M arrest but not subsequent apoptotic cell death. The p84N5-induced checkpoint is functional in ataxia telangiectasia-mutated kinase-deficient cells. We conclude that p84N5 induces an ataxia telangiectasia-mutated kinase (ATM)-independent, caffeine-sensitive G(2)/M cell cycle arrest prior to the onset of apoptosis. This conclusion is consistent with the hypotheses that p84N5 functions in an Rb-regulated cellular response that is similar to that triggered by DNA damage.     

E2F1 and E2F3 activate ATM through distinct mechanisms to promote E1A-induced apoptosis

Deregulation of the Rb-E2F pathway occurs in many cancers and results in aberrant cell proliferation as well as an increased propensity to undergo apoptosis. In most cases, apoptosis in response to Rb inactivation involves the activation of p53 but the molecular details of the signaling pathway connecting Rb loss to p53 are poorly understood. Here we demonstrate that the E1A oncoprotein, which binds and inhibits Rb family members, induces the accumulation and phosphorylation of p53 through the DNA damage-responsive ATM kinase. As a result, E1A-induced apoptosis is significantly impaired in cells lacking ATM. In contrast, inactivation of ARF, which is widely believed to activate p53 in response to oncogenic stress, has no effect on p53 induction and only a modest effect on apoptosis in response to E1A. Both E2F1 and E2F3 contribute to ATM-dependent phosphorylation of p53 and apoptosis in cells expressing E1A. However, deregulated E2F3 activity is implicated in the DNA damage caused by E1A while E2F1 stimulates ATM- and NBS1-dependent p53 phosphorylation and apoptosis through a mechanism that does not involve DNA damage.     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV-hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.Key words: UVB irradiation, p53, DNA damage, DNA damage responses, apoptosis, senescence