Inducible degradation of checkpoint kinase 2 links to cisplatin-induced resistance in ovarian cancer cells

Checkpoint kinase 2 (Chk2) is one of the critical kinases governing the cell cycle checkpoint, DNA damage repair, and cell apoptosis in response to DNA damaging signals. In the present report, we demonstrate that Chk2 kinase is degraded at the protein level in response to cisplatin through ubiquitin-proteasome pathway. This degradation was independent of the Thr68 phosphorylation, ATM kinase, and BRCA1 tumor suppressor. Examination of Chk2 protein revealed a decreased expression of Chk2 protein in cisplatin-resistant ovarian cancer cell lines, suggesting that degradation or decreased expression of Chk2 is partially responsible for chemo-resistance. Site-directed mutation of the putative destruction box in the Chk2 protein did not affect the Chk2 degradation induced by cisplatin. Therefore, these results are the first to indicate a novel mechanism of regulating Chk2 in cisplatin-induced resistance of cancer cells.     

Oxidative Stress Responses Involve Oxidation of a Conserved Ubiquitin Pathway Enzyme

Although it is vital that cells detect and respond to oxidative stress to allow adaptation and repair damage, the underlying sensing and signaling mechanisms that control these responses are unclear. Protein ubiquitinylation plays an important role in controlling many biological processes, including cell division. In Saccharomyces cerevisiae, ubiquitinylation involves a single E1 enzyme, Uba1, with multiple E2s and E3s providing substrate specificity. For instance, the conserved E2 Cdc34 ubiquitinylates many substrates, including the cyclin-dependent kinase inhibitor Sic1, targeting it for degradation to allow cell cycle progression. Here we reveal that, in contrast to other ubiquitin pathway E2 enzymes, Cdc34 is particularly sensitive to oxidative inactivation, through sequestration of the catalytic cysteine in a disulfide complex with Uba1, by levels of oxidant that do not reduce global ubiquitinylation of proteins. This Cdc34 oxidation is associated with (i) reduced levels of Cdc34-ubiquitin thioester forms, (ii) increased stability of at least one Cdc34 substrate, Sic1, and (iii) Sic1-dependent delay in cell cycle progression. Together, these data reveal that the differential sensitivity of a ubiquitin pathway E2 enzyme to oxidation is utilized as a stress-sensing mechanism to respond to oxidative stress.     

Ubiquitin-conjugating Enzyme Cdc34 and Ubiquitin Ligase Skp1-Cullin-F-box Ligase (SCF) Interact through Multiple Conformations

In the ubiquitin-proteasome system, protein substrates are degraded via covalent modification by a polyubiquitin chain. The polyubiquitin chain must be assembled rapidly in cells, because a chain of at least four ubiquitins is required to signal for degradation, and chain-editing enzymes in the cell may cleave premature polyubiquitin chains before achieving this critical length. The ubiquitin-conjugating enzyme Cdc34 and ubiquitin ligase SCF are capable of building polyubiquitin chains onto protein substrates both rapidly and processively; this may be explained at least in part by the atypically fast rate of Cdc34 and SCF association. This rapid association has been attributed to electrostatic interactions between the acidic C-terminal tail of Cdc34 and a feature on SCF called the basic canyon. However, the structural aspects of the Cdc34-SCF interaction and how they permit rapid complex formation remain elusive. Here, we use protein cross-linking to demonstrate that the Cdc34-SCF interaction occurs in multiple conformations, where several residues from the Cdc34 acidic tail are capable of contacting a broad region of the SCF basic canyon. Similar patterns of cross-linking are also observed between Cdc34 and the Cul1 paralog Cul2, implicating the same mechanism for the Cdc34-SCF interaction in other members of the cullin-RING ubiquitin ligases. We discuss how these results can explain the rapid association of Cdc34 and SCF.     

Signaling and function of caspase and c-jun N-terminal kinase in cisplatin-induced apoptosis

Caspases and c-Jun N-terminal kinase (JNK) are activated in tumor cells during induction of apoptosis. We investigated the signaling cascade and function of these enzymes in cisplatin-induced apoptosis. Treatment of Jurkat T-cells with cisplatin induced cell death with DNA fragmentation and activation of caspase and JNK. Bcl-2 overexpression suppressed activation of both enzymes, whereas p35 and CrmA inhibited only the DEVDase (caspase-3-like) activity, indicating that the activation of these enzymes may be differentially regulated. Cisplatin induced apoptosis with the cytochrome c release and caspase-3 activation in both wild-type and caspase-8-deficient JB-6 cells, while the Fas antibody induced these apoptotic events only in wild-type cells. This indicates that caspase-8 activation is required for Fas-mediated apoptosis, but not cisplatin-induced cell death. On the other hand, cisplatin induced the JNK activation in both the wild-type and JB-6 cells, and the caspase-3 inhibitor Z-DEVD-fmk did not inhibit this activation. The JNK overexpression resulted in a higher JNK activity, AP-1 DNA binding activity, and metallothionein expression than the empty vector-transfected cells following cisplatin treatment. It also partially protected the cells from cisplatin-induced apoptosis by decreasing DEVDase activity. These data suggest that the cisplatin-induced apoptotic signal is initiated by the caspase-8-independent cytochrome c release, and the JNK activation protects cells from cisplatin-induced apoptosis via the metallothionein expression.     

The mode of cisplatin-induced cell death in CYP2E1-overexpressing HepG2 cells: modulation by ERK, ROS, glutathione, and thioredoxin

In a previous study, E47 HepG2 cells that overexpress human CYP2E1 were shown to be more sensitive to cisplatin than C34 cells that do not express CYP2E1. In this study, we found that this sensitivity was due to an earlier activation of ERK in the E47 cells compared to the C34 cells. Glutathione depletion by L-buthionine sulfoximine (BSO) enhanced cisplatin cytotoxicity via increasing production of reactive oxygen species (ROS) and activation of ERK. In contrast, elevation of glutathione by glutathione ethyl ester (GSHE) decreased cisplatin/BSO cytotoxicity by decreasing ROS production and ERK activation. Inhibition of ERK activation by U0126 protected against cisplatin/BSO cytotoxicity via inhibiting ROS production but not restoring intracellular glutathione content. Examination of the mode of cell death showed that U0126 inhibited cisplatin-induced necrosis but not apoptosis. Cisplatin-induced apoptosis was caspases-dependent; BSO switched cisplatin-induced apoptosis to necrosis via decreasing activity of caspases, and GSHE switched cisplatin/BSO-induced necrosis back to apoptosis through maintaining activity of caspases. Similar to GSHE, U0126 partially switched cisplatin/BSO induced necrosis to apoptosis via restoring activity of caspases. Cisplatin lowered levels of thioredoxin, especially in the presence of BSO. Although U0126 failed in restoring intracellular glutathione levels, it restored thioredoxin levels, which maintain the activity of the caspases. These results suggest that thioredoxin can replace glutathione to promote the active thiol redox state necessary for caspase activity, and thus glutathione and thioredoxin regulate the mode of cisplatin toxicity in E47 cells via redox regulation of caspase activity.     

Important amino acid properties for determining the transition state structures of two-state protein mutants

In this study, the role of V12-Rac1 in the cisplatin-induced apoptosis was investigated. Cisplatin-induced apoptosis is associated with cytochrome c release, which can be inhibited by V12-Rac1 expression. The analysis of mitogen-activated protein kinase activity indicated that V12-Rac1 expression led to a decrease in p38 activity after exposure to cisplatin but not c-jun N-terminal kinase and extracellular signal-regulated kinase. Using pharmacological inhibitors, it was found that only p38 is a critical mediator in the cisplatin-induced apoptosis of NIH3T3 cells. This suggests that V12-Rac1 can stimulate the anti-apoptotic signaling pathway in response to cisplatin, and that decreased p38 activity caused by V12-Rac1 expression in cisplatin-treated NIH3T3 cells is crucial for V12-Rac1-dependent cell survival.     

ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apoptosis

Cisplatin is one of the most effective anti-cancer drugs; however, the use of cisplatin is limited by its toxicity in normal tissues, particularly injury of the kidneys. The mechanisms underlying the therapeutic effects of cisplatin in cancers and side effects in normal tissues are largely unclear. Recent work has suggested a role for p53 in cisplatin-induced renal cell apoptosis and kidney injury; however, the signaling pathway leading to p53 activation and renal apoptosis is unknown. Here we demonstrate an early DNA damage response during cisplatin treatment of renal cells and tissues. Importantly, in the DNA damage response, we demonstrate a critical role for ATR, but not ATM (ataxia telangiectasia mutated) or DNA-PK (DNA-dependent protein kinase), in cisplatin-induced p53 activation and apoptosis. We show that ATR is specifically activated during cisplatin treatment and co-localizes with H2AX, forming nuclear foci at the site of DNA damage. Blockade of ATR with a dominant-negative mutant inhibits cisplatin-induced p53 activation and renal cell apoptosis. Consistently, cisplatin-induced p53 activation and apoptosis are suppressed in ATR-deficient fibroblasts. Downstream of ATR, both Chk1 and Chk2 are phosphorylated during cisplatin treatment in an ATR-dependent manner. Interestingly, following phosphorylation, Chk1 is degraded via the proteosomal pathway, whereas Chk2 is activated. Inhibition of Chk2 by a dominant-negative mutant or gene deficiency attenuates cisplatin-induced p53 activation and apoptosis. In vivo in C57BL/6 mice, ATR and Chk2 are activated in renal tissues following cisplatin treatment. Together, the results suggest an important role for the DNA damage response mediated by ATR-Chk2 in p53 activation and renal cell apoptosis during cisplatin nephrotoxicity.     

Inhibition of extracellular signal-regulated protein kinase or c-Jun N-terminal protein kinase cascade, differentially activated by cisplatin, sensitizes human ovarian cancer cell line.

We have studied the roles of c-Jun N-terminal protein kinase (JNK) and extracellular signal-regulated protein kinase (ERK) cascade in both the cisplatin-resistant Caov-3 and the cisplatin-sensitive A2780 human ovarian cancer cell lines. Treatment of both cells with cisplatin but not transplatin isomer activates JNK and ERK. Activation of JNK by cisplatin occurred at 30 min, reached a plateau at 3 h, and declined thereafter, whereas activation of ERK by cisplatin showed a biphasic pattern, indicating the different time frame. Activation of JNK by cisplatin was maximal at 1000 microM, whereas activation of ERK was maximal at 100 microM and was less at higher concentrations, indicating the different dose dependence. Cisplatin-induced JNK activation was neither extracellular and intracellular Ca(2+)- nor protein kinase C-dependent, whereas cisplatin-induced ERK activation was extracellular and intracellular Ca(2+)- dependent and protein kinase C-dependent. A mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, PD98059, had no effect on the cisplatin-induced JNK activity, suggesting an absence of cross-talk between the ERK and JNK cascades. We further examined the effect of each cascade on the viability following cisplatin treatment. Either exogenous expression of dominant negative c-Jun or the treatment by PD98059 induced sensitivity to cisplatin in both cells. Our findings suggest that cisplatin-induced DNA damage differentially activates JNK and ERK cascades and that inhibition of either of these cascades sensitizes ovarian cancer cells to cisplatin.