Meenakshi Upreti

Cyclin-Dependent Kinase 1-Mediated Bcl-xL/Bcl-2 Phosphorylation Acts as a Functional Link Coupling Mitotic Arrest and Apoptosis

ABSTRACT Despite detailed knowledge of the components of the spindle assembly checkpoint, a molecular explanation of how cells die after prolonged spindle checkpoint activation, and thus how microtubule inhibitors and other antimitotic drugs ultimately elicit their lethal effects, has yet to emerge. Mitotically arrested cells typically display extensive phosphorylation of two key antiapoptotic proteins, Bcl-xL and Bcl-2, and evidence suggests that phosphorylation disables their antiapoptotic activity. However, the responsible kinase has remained elusive. In this report, evidence is presented that cyclin-dependent kinase 1 (CDK1)/cyclin B catalyzes mitotic-arrest-induced Bcl-xL/Bcl-2 phosphorylation. Furthermore, we show that CDK1 transiently and incompletely phosphorylates these proteins during normal mitosis. When mitosis is prolonged in the absence of microtubule inhibition, Bcl-xL and Bcl-2 become highly phosphorylated. Transient overexpression of nondegradable cyclin B1 caused apoptotic death, which was blocked by a phosphodefective Bcl-xL mutant but not by a phosphomimetic Bcl-xL mutant, confirming Bcl-xL as a key target of proapoptotic CDK1 signaling. These findings suggest a model whereby a switch in the duration of CDK1 activation, from transient during mitosis to sustained during mitotic arrest, dramatically increases the extent of Bcl-xL/Bcl-2 phosphorylation, resulting in inactivation of their antiapoptotic function. Thus, phosphorylation of antiapoptotic Bcl-2 proteins acts as a sensor for CDK1 signal duration and as a functional link coupling mitotic arrest to apoptosis.

Identification of the major phosphorylation site in Bcl-xL induced by microtubule inhibitors and analysis of its functional significance.

Vinblastine and other microtubule inhibitors used as antimitotic cancer drugs characteristically promote the phosphorylation of the key anti-apoptotic protein, Bcl-xL. However, putative sites of phosphorylation have been inferred based on potential recognition by JNK, and no direct biochemical analysis has been performed. In this study we used protein purification and mass spectrometry to identify Ser-62 as a single major site in vivo. Site-directed mutagenesis confirmed Ser-62 to be the site of Bcl-xL phosphorylation induced by several microtubule inhibitors tested. Vinblastine-treated cells overexpressing a Ser-62 → Ala mutant showed highly significantly reduced apoptosis compared with cells expressing wild-type Bcl-xL. Co-immunoprecipitation revealed that phosphorylation caused wild-type Bcl-xL to release bound Bax, whereas phospho-defective Bcl-xL retained the ability to bind Bax. In contrast, phospho-mimic (Ser-62 → Asp) Bcl-xL exhibited a reduced capacity to bind Bax. Functional tests were performed by transiently co-transfecting Bax in the context of different Bcl-xL mutants. Co-expression of wild-type or phospho-defective Bcl-xL counteracted the adverse effects of Bax expression on cell viability, whereas phospho-mimic Bcl-xL failed to provide the same level of protection against Bax. These studies suggest that Bcl-xL phosphorylation induced by microtubule inhibitors plays a key pro-apoptotic role at least in part by disabling the ability of Bcl-xL to bind Bax.

Key role for Bak activation and Bak-Bax interaction in the apoptotic response to vinblastine

Microtubule inhibitors such as vinblastine cause mitotic arrest and subsequent apoptosis through the intrinsic mitochondrial pathway. However, although Bcl-2 family proteins have been implicated as distal mediators, their precise role is largely unknown. In this study, we investigated the role of Bak in vinblastine-induced apoptosis. Bak was mainly monomeric in untreated KB-3 cells, and multimers corresponding to dimer, trimer, and higher oligomers were observed after vinblastine treatment. The oligomeric Bak species were strongly diminished in cells stably overexpressing Bcl-xL. Immunoprecipitation with a conformation-dependent Bak antibody revealed that vinblastine induced Bak activation. Reciprocal immunoprecipitations indicated that vinblastine induced the interaction of active Bak with active Bax. Furthermore, Bcl-xL overexpression prevented Bak and Bax interaction and strongly inhibited apoptosis, whereas Bcl-2 overexpression did not prevent Bak-Bax interaction and only weakly inhibited apoptosis. The relative contributions of Bak and Bax were investigated using fibroblasts deficient in one or both of these proteins; double knockouts were highly resistant compared with single knockouts, with vinblastine sensitivities in the order of Bak+/Bax+ > Bak+/Bax- > Bak-/Bax+ > Bak-/Bax-. These results highlight Bak as a key mediator of vinblastine-induced apoptosis and show for the first time activation and oligomerization of Bak by an antimitotic agent. In addition, our results suggest that the interaction of the activated forms of Bak and Bax represents a key distal step in the apoptotic response to this important chemotherapeutic drug. [Mol Cancer Ther 2008;7(7):2224–32]

Vinblastine-induced apoptosis is mediated by discrete alterations in subcellular location, oligomeric structure, and activation status of specific Bcl-2 family members.

To gain a broader insight into the role of Bcl-2 proteins in apoptosis induced after mitotic arrest, we investigated the subcellular location, oligomeric structure, and protein interactions of Bax, Bcl-2, and Bcl-xL in vinblastine-treated KB-3 cells. Vinblastine induced the translocation of Bax from the cytosol to the mitochondria, which was accompanied by conformational activation and oligomerization of Bax. Bcl-2 was located in the mitochondria, underwent multisite phosphorylation after vinblastine treatment, and was strictly monomeric under all conditions. In contrast, in control cells, Bcl-xL existed in both monomeric (30 kDa) and oligomeric (150 kDa) forms. Treatment with agents that induced Bcl-xL phosphorylation (microtubule inhibitors) caused loss of the 150-kDa form, but this species was unaffected by apoptotic stimuli that did not stimulate phosphorylation. Vinblastine also promoted Bax activation and Bax oligomerization in HCT116 colon cancer cells. Both wild-type and Bax-deficient HCT116 cells expressed the 150-kDa form of Bcl-xL, which was depleted similarly in both cell lines upon vinblastine treatment. Co-immunoprecipitation studies revealed that in untreated KB-3 cells inactive cytosolic Bax interacted with Bcl-xL, whereas in vinblastine-treated cells, activated mitochondrial Bax did not interact with Bcl-xL. Interaction of Bcl-2 with Bax was not observed under any condition. Overexpression of Bcl-xL inhibited vinblastine-induced Bax activation and Bax dimerization and in parallel inhibited apoptosis. The results indicate that vinblastine-induced apoptosis requires translocation, activation, and oligomerization of Bax and is associated with specific changes in the oligomeric properties of Bcl-xL, which occur independently of Bax.

Radiation induced oxidative stress: I. Studies in Ehrlich solid tumor in mice.

Understanding the response of tumors to ionizing radiation might potentially lead to improvement in tumor control and patient morbidity. Since the antioxidant status is likely to be linked to radioresponse, its modulation needs to be examined. Therefore, Swiss albino male mice (7–8 weeks old) with Ehrlich solid tumors were irradiated with different doses of gamma rays (0–9 Gy) at a dose rate of 0.0153 Gy/s; and enzymes involved in antioxidant functions were determined in the tumors. Radiation effects in terms of oxidative damage, LDH, nitric oxide and DNA fragmentation were also examined.In tumors, the specific activity of SOD was increased with dose but declined 6 Gy onwards. GST, DTD and GSH showed an almost progressive increase. These enhanced activities might have resulted from the increased protein expression. This possibility was supported by the Western Blot analysis for GST protein. These changes might be closely linked to the radiation-induced oxidative stress as reflected by the enhanced levels of peroxidative damage, DNA fragmentation, LDH activity and nitric oxide levels. These findings may have relevance to radiation therapy of cancer as the elevated antioxidant status of irradiated tumors is likely to limit the effectiveness of radiation dose and adversely affect the therapeutic gain.

Induction of Apoptosis by Vinblastine via c-Jun Autoamplification and p53-Independent Down-Regulation of p21WAF1/CIP1

Vinblastine treatment in all cell lines examined causes a robust increase in c-Jun protein expression and phosphorylation and a corresponding increase in activator protein-1 (AP-1) transcriptional activity. We show in KB-3 carcinoma cells that this is due to a strong autoamplification loop involving the proximal AP-1 site in the c-Jun promoter, resulting in highly increased c-Jun mRNA and c-Jun protein. Inhibitors of RNA transcription and protein translation blocked both vinblastine-induced c-Jun expression and apoptotic cell death, suggesting that apoptosis is dependent, at least in part, on transcription/translation. Small interfering RNA (siRNA) to c-Jun was used to interrupt the amplification cycle and was found to be highly effective, reducing vinblastine-induced c-Jun expression at both the mRNA and protein levels by 90%. Apoptosis and caspase-3 activation were significantly inhibited in c-Jun siRNA-treated cells. To uncover potential mechanisms of c-Jun-mediated cell death and protection by c-Jun siRNA, candidate target genes were examined. Chromatin immunoprecipitation revealed preferential association of c-Jun with the p21 (cyclin-dependent kinase inhibitor) gene promoter after vinblastine treatment. In KB-3 cells, which have compromised p53 function, and in p53-null cells but not in p53 wild-type cells, vinblastine caused down-regulation of p21 expression concomitant with increased c-Jun expression, suggesting a role for c-Jun in negative regulation of the p21 promoter independent of p53. These results provide strong evidence that c-Jun induction in response to vinblastine plays a proapoptotic role in part via down-regulation of p21, promoting cycling and subsequent cell death of mitotically impaired cells.

Chemoprevention by Hippophae rhamnoides: Effects on Tumorigenesis, Phase II and Antioxidant Enzymes, and IRF-1 Transcription Factor

Abstract: Fruits or berries of Hippophae rhamnoides (sea buckthorn), a rich source of vitamins A, C, and E, carotenes, flavonoids, and microelements such as sulfur, selenium, zinc, and copper, are edible and have been shown to protect from atopic dermatitis, hepatic injury, cardiac disease, ulcer, and atherosclerosis. However, its mechanism of action is not clear. We show that Hippophae inhibits benzo(a)pyrene-induced forestomach and DMBA-induced skin papillomagenesis in mouse. This decrease in carcinogenesis may be attributed to the concomitant induction of phase II enzymes such as glutathione S-transferase and DT-diaphorase and antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase in the mouse liver. This was accompanied by a remarkable induction of the transcription factor interferon regulatory factor-1 in the Hippophae-treated liver. Our results strongly suggest that Hippophae fruit is able to decrease carcinogen-induced forestomach and skin tumorigenesis, which might involve up-regulation of phase II and antioxidant enzymes as well as DNA-binding activity of IRF-1, a known antioncogenic transcription factor causing growth suppression and apoptosis induction for its anticancer effect.

Regulation of Bax by c-Jun NH2-terminal kinase and Bcl-xL in vinblastine-induced apoptosis

Microtubule inhibitors, such as vinblastine, are widely used in cancer chemotherapy. Vinblastine exerts its antitumor effect by inducing apoptosis. In KB-3 cells, we have shown previously that vinblastine activates c-Jun NH(2)-terminal protein kinase (JNK) and causes Bax mitochondrial translocation and activation. In this study, we sought to test the hypothesis that JNK and Bcl-xL act as positive and negative regulators, respectively, of Bax translocation. The JNK inhibitor SP600125 inhibited vinblastine-induced JNK activation and in concert inhibited Bax mitochondrial translocation, Bax oligomerization, and Bax activation. Furthermore, the JNK inhibitor blocked vinblastine-induced apoptosis. The ability of vinblastine to induce Bax translocation and the inhibitory effect of SP600125 were confirmed in cells stably expressing GFP-Bax. However, if transiently overexpressed, Bax localized to the mitochondria, and this was associated with loss of viability and subsequent cell death. If Bcl-xL was co-expressed with Bax, the cells readily tolerated Bax overexpression. Indeed, physical interaction between Bcl-xL and Bax but not Bak was demonstrated by co-immunoprecipitation. These findings provide novel insight into the role of Bax and its regulation in vinblastine-induced apoptosis.

Synthesis and Anticancer Activity of Sclerophytin-Inspired Hydroisobenzofurans

Three structurally related sets of hydroisobenzofuran analogs of sclerophytin A were prepared in three or four steps from (S)-(+)-carvone via an aldol-cycloaldol sequence. The most potent members of each set of analogs exhibited IC(50)s of 1-3 microM in growth inhibitory assays against KB3 cells. The NCI 60-cell line 5-dose assay for analog 6h revealed a GI(50)=0.148 microM and LC(50)=9.36 microM for the RPMI-8226 leukemia cell line, and a GI(50)=0.552 microM and LC(50)=26.8 microM for the HOP-92 non-small cell lung cancer cell line.

Correlation Statistics for cDNA Microarray Image Analysis

In this paper, correlation of the pixels comprising a microarray spot is investigated. Subsequently, correlation statistics, namely, Pearson correlation and Spearman rank correlation, are used to segment the foreground and background intensity of microarray spots. The performance of correlation-based segmentation is compared to clustering-based (PAM, k-means) and seeded-region growing techniques (SPOT). It is shown that correlation-based segmentation is useful in flagging poorly hybridized spots, thus minimizing false-positives. The present study also raises the intriguing question of whether a change in correlation can be an indicator of differential gene expression