Parameters of Apoptotic Pathways Altered by Polyamine Depletion

Figure 5 indicates the apoptotic pathways delineated in IEC-6 cells and the references to the effects of polyamines on the specific components of those pathways. Yuan et al. (26) found that polyamine depletion prevented the release of cytochrome c from mitochondria after camptothecin. This was accompanied by increased Bcl-2 and Bcl-XL and decreased Bax and ¿-Bid in the cells incubated with DFMO. In addition to inhibiting caspase 3 activation, DFMO significantly reduces the activation of caspase 6, 8, and 9 in response to TNF-a/CHX (27). Thus, polyamine depletion inhibits apoptosis by decreasing both the extrinsic and intrinsic pathways. The remaining proapoptotic step shown in Fig. 5 involves JNK, which is activated by TNF-a through the TNFR1

Tnf Induced Apoptotic Pathway
Fig. 5. Apoptotic pathways in IEC-6 cells activated by TNF-a/CHX and by damage induced by camptothecin or y-irradiation. Reference numbers refer to articles demonstrating changes in a particular pathway component and the effect of polyamine depletion on it.

receptor and TRAF2. TNF-a also activates JNK in IEC-6 cells, and that activation is dependent on polyamines (27). Polyamine depletion inhibits JNK activation, and inhibition of JNK with the specific inhibitor, SP-600125, prevented apoptosis, caspase 9 activation, and cytochrome c release. The mechanism by which JNK activates apoptosis is not clear; however, the results indicate that JNK acts upstream from cytochrome c release, possibly through the Bcl-2 family proteins.

Polyamine depletion causes the sustained activation of ERK in response to TNF-a/CHX, and the ERKs mediate a strong antiapoptotic response (18). Pretreatment of polyamine-depleted cells with the membrane permeable MEK 1/2 inhibitor, U-0126, inhibited TNF-a-induced phosphorylation of ERK and increased DNA fragmentation, JNK activity, and caspase 3 activity. IEC-6 cells expressing constitutively active MEK1 were protected from apoptosis and had decreased JNK activity in response to TNF-a. Conversely, dominant-negative MEK1 cells had high basal levels of JNK activity, cytochrome c release, and spontaneous apoptosis. Depletion of polyamines in dominantnegative MEK1 cells did not prevent JNK activation, cytochrome c release, or apoptosis in response to TNF-a/CHX on camptothecin (18).

Polyamine depletion rapidly activates NF-kB (28). Within 1 h of administering DFMO, p65 rapidly redistributes from the cytoplasm to the nucleus of IEC-6 cells, and the degradation of IkBa is complete by 5 h. These times correlated with a 50% drop in putrescine levels at 1 h and a total absence of putrescine by 3 h. All of these early changes were prevented by the addition of putrescine to the cells incubated with DFMO (28). Li et al. (29) also reported that polyamine depletion led to the activation of NF-kB from 4 to 8 d after constant exposure to DFMO. They showed that inhibition of NF-KB-binding activity by MG-132 or sulfasalazine prevented the increased apoptosis to staurosporine in polyamine-depleted cells and the inhibition of apoptosis in the same cells in response to TNF-a/CHX. Wang and his coworkers followed this report by showing that activation of NF-kB by polyamine depletion led to the synthesis of c-IAP2 and XIAP, as well as the inhibition of caspase 3 activity (30). They did not attempt to reconcile these findings with their data indicating that NF-kB activation enhanced staurosporine-induced apoptosis (29). This observation that DFMO increased staurosporine-induced apoptosis in IEC-6 cells remains the only report of enhanced apoptosis in IECs following depletion of polyamines. The mechanism leading to this effect is also unknown.

TNF-a-induced activation of NF-kB requires Akt, a serine/threonine protein kinase originally identified as the oncogene transduced by the acute transforming retrovirus Akt (31). After cytokine stimulation, Akt translocates to the inner surface of the plasma membrane, where it is phosphorylated and activated by PI3K (Fig. 5). Although Akt inhibits apoptosis by preventing the release of cytochrome c, the mechanism is not clear. Glycogen synthase kinase-3p (GSK-3P), an important downstream target of Akt, is inhibited by Akt phosphorylation. Increased activity of GSK-3P has been shown to induce apoptosis in several cell types (32). Zhang et al. (33) found that inhibition of Akt increases apoptosis in polyamine depleted IEC-6 cells, and suggested that Akt suppresses caspase 3 activity through a process involving the phosphorylation of GSK-3P. Bhattacharya et al. (34) found that the inhibition of GSK-3P had no effect on apoptosis induced by TNF-a in IEC-6 cells. They went on to show that inhibition of PI3-kinase and overexpressing of DN-Akt significantly increased apoptosis in polyamine-depleted cells. Constitutive activation of Akt by polyamine depletion was prevented in DN-Akt cells, thereby reversing the protective effect of polyamine depletion. Inhibitors of PI3 kinase and expression of DN-Akt prevented Akt activation and the subsequent translocation of NF-kB to the nucleus (34). CA-Akt expression increased the resistance to TNF-a-induced apoptosis and increased the activation of NF-kB. Polyamine depletion of cells transfected with DN-Akt prevented spontaneous and TNF-a-induced IkBa phosphorylation. Prevention of NF-kB activation in cells transfected with DN-IkBa increased spontaneous apoptosis and restored it in polyamine-depleted cells. Thus protection from apoptosis by Akt in polyamine-deficient cells is dependent on the activation of the transcription factor, NF-kB, and is independent of GSK-3p. Akt regulates the mitochondrial pathway, preventing the activation of caspase 9 and, hence, caspase 3 via the actions of NF-kB (34).

Signal transducers and activators of transcription (STAT) proteins are a family of latent transcription factors that undergo ligand-dependent phosphorylation and activation.

STAT3 activates the acute phase response genes and binds to the SIE (sis-inducible element) found in the c-fos promoter that is regulated by PDGF platelet-derived growth factor (PDGF). Pfeffer et al. (35) found that DFMO rapidly induced STAT3 activation as determined by its tyrosine phosphorylation, translocation from the cytoplasm to the nucleus, and presence in SIE-dependent DNA-protein complexes. Activation of STAT3 resulted in the activation of a STAT3-dependent reporter construct. Additional phosphorylation of STAT3 at serine 727 is thought to increase its transcriptional activity and is mediated by the mitogen-activated protein kinases pathway. STAT3 is constitu-tively activated in a wide variety of primary tumors and induces cell survival in association with survivin expression in gastric cancer cells (36). STAT3 signaling has been reported to mediate the survival of intestine epithelial cells transfected with oncogenic Ras. Thus, the role of STAT3 as a mediator of cell survival is well established.

Bhattacharya et al. (37) determined that polyamine depletion induced STAT3 phosphorylation at both Tyr-705 and Ser-727, resulting in localization to the cell periphery and nucleus, respectively. Sustained phosphorylation of STAT3 at both residues occurred after polyamine-depleted cells were exposed to TNF-a. Inhibition of STAT3 increased the sensitivity of polyamine-depleted cells to apoptosis, and the protective effect of polyamine depletion was eliminated in cells expressing DN-STAT3. Additional evidence was obtained, indicating that DFMO increased both the transcription and translation of antiapoptotic proteins, Bcl-2, Mcl-1, and BIRC3. The levels of these proteins were significantly reduced in cells expressing DN-STAT3. Therefore, the activation of STAT3 and its subsequent effects as a transcription factor appear to be an important part of the mechanism that protects polyamine-depleted cells from apop-tosis. This protection may be mediated by the expression of antiapoptotic proteins Bcl-2, Mcl-1, and BIRC3 (37).

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