Ubiquitin-proteasome Degradation Pathway Research Articles

A coordinated action of Bax, PUMA, and p53 promotes MG132-induced mitochondria activation and apoptosis in colon cancer cells

Targeting the ubiquitin-proteasome degradation pathway has become a promising approach for cancer therapy. Previous studies have shown that proteasome inhibition leads to apoptosis in various cancer cells. The mechanism by which apoptosis occurs are not fully understood and can be cell type and/or inhibitor specific. In this study, we investigated the mechanism of mitochondrial activation by proteasome inhibitors in colon cancer cells. We found that Bax activation and mitochondria translocation were required for apoptosis induced by multiple proteasome inhibitors. In contrast, reactive oxygen species did not seem to be induced by MG132 or bortezomib and antioxidants had no effects on MG132-induced apoptosis. In contrast, treatment with MG132 or bortezomib induced a significant accumulation of p53 and PUMA. Genetic deletion of either p53 or PUMA led to a marked suppression of apoptosis induced by these inhibitors, accompanied with reduced Bax activation and cytochrome c release. Consistently, inhibition of translation by cycloheximide could also effectively abolish the accumulation of p53 and PUMA and suppress MG132-induced Bax activation and apoptosis. These findings thus strongly indicate the critical involvement of p53-, PUMA-, and Bax-mediated mitochondrial activation in proteasome inhibitor-induced apoptosis in colon cancer cells.

Mass Spectrometric Characterization of the Affinity-Purified Human 26S Proteasome Complex

The 26S proteasome is a multisubunit complex responsible for degradation of ubiquitinated substrates, which plays a critical role in regulating various biological processes. To fully understand the function and regulation of the proteasome complex, an important step is to elucidate its subunit composition and posttranslational modifications. Toward this goal, a new affinity purification strategy has been developed using a derivative of the HB tag for rapid isolation of the human 26S proteasome complex for subsequent proteomic analysis. The purification of the complex is achieved from stable 293 cell lines expressing a HB-tagged proteasome subunit and by high-affinity streptavidin binding with TEV cleavage elution. The complete composition of the 26S proteasome complex, including recently assigned new subunits, is identified by LC-MS/MS. In addition, all known proteasome activator proteins and components involved in the ubiquitin-proteasome degradation pathway are identified. Aside from the subunit composition, the N-terminal modification and phosphorylation of the proteasome subunits have been characterized. Twelve novel phosphorylation sites from eight subunits have been identified, and N-terminal modifications are determined for 25 subunits, 12 of which have not been previously reported in mammals. We also observe different N-terminal processing of subunit Rpn2, which results in identification of two different N-termini of the protein. This work presents the first comprehensive characterization of the human 26S proteasome complex by affinity purification and tandem mass spectrometry. The detailed proteomic profiling obtained here is significant to future studies aiming at a complete understanding of the structure-function relationship of the human 26S proteasome complex.

High-Throughput Bioluminescence Screening of Ubiquitin-Proteasome Pathway Inhibitors from Chemical and Natural Sources

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.

Proteomic analysis of steroid-triggered autophagic programmed cell death during Drosophila development

Two morphological forms of programmed cell death, apoptosis and autophagic cell death, remove unneeded or damaged cells during animal development. Although the mechanisms that regulate apoptosis are well studied, little is known about autophagic cell death. A shotgun proteome analysis of purified dying larval salivary glands in Drosophila was used to identify proteins that are expressed during autophagic programmed cell death. A total of 5661 proteins were identified from stages before and after the onset of cell death. Analyses of these data enabled us to identify proteins from a number of interesting categories including regulators of transcription, the apoptosis, autophagy, lysosomal, and ubiquitin proteasome degradation pathways, and proteins involved in growth control. Several of the identified proteins, including the serine/threonine kinase warts (Wts), were not detected using whole-genome DNA microarrays, providing support for the importance of such high-throughput proteomic technology. Wts regulates cell-cycle arrest and apoptosis, and significantly, mutations in wts prevent destruction of salivary glands.

JAK kinases control IL-5 receptor ubiquitination, degradation, and internalization

IL-5, IL-3, and GM-CSF are related hematopoietic cytokines, which regulate the function of myeloid cells and are mediators of the allergic inflammatory response. These cytokines signal through heteromeric receptors containing a specific alpha chain and a shared signaling chain, betac. Previous studies demonstrated that the ubiquitin (Ub) proteasome degradation pathway was involved in signal termination of the betac-sharing receptors. In this study, the upstream molecular events leading to proteasome degradation of the IL-5 receptor (IL-5R) were examined. By using biochemical and flow cytometric methods, we show that JAK kinase activity is required for betac ubiquitination and proteasome degradation but only partially required for IL-5R internalization. Furthermore, we demonstrate the direct ubiquitination of the betac cytoplasmic domain and identify lysine residues 566 and 603 as sites of betac ubiquitination. Lastly, we show that ubiquitination of the betac cytoplasmic domain begins at the plasma membrane, increases after receptor internalization, and is degraded by the proteasome after IL-5R internalization. We propose an updated working model of IL-5R down-regulation, whereby IL-5 ligation of its receptor activates JAK2/1 kinases, resulting in betac tyrosine phosphorylation, ubiquitination, and IL-5R internalization. Once inside the cell, proteasomes degrade the betac cytoplasmic domain, and the truncated receptor complex is terminally degraded in the lysosomes. These data establish a critical role for JAK kinases and the Ub/proteasome degradation pathway in IL-5R down-regulation.

Ubiquitination and Proteasome-Mediated Degradation of BRCA1 and BARD1 during Steroidogenesis in Human Ovarian Granulosa Cells

Germ-line mutations in BRCA1 predispose women to early-onset, familial breast and ovarian cancers. However, BRCA1 expression is not restricted to breast and ovarian epithelial cells. For example, ovarian BRCA1 expression is enriched in ovarian granulosa cells, which are responsible for ovarian estrogen production in premenopausal women. Furthermore, recent tissue culture and animal studies suggest a functional role of BRCA1 in ovarian granulosa cells. Although levels of BRCA1 are known to fluctuate significantly during folliculogenesis and steroidogenesis, the mechanism by which BRCA1 expression is regulated in granulosa cells remains to be elucidated. Here we show that the ubiquitin-proteasome degradation pathway plays a significant role in the coordinated protein stability of BRCA1 and its partner BARD1 in ovarian granulosa cells. Our work identifies the amino-terminal RING domain-containing region of BRCA1 as the degron sequence that is both necessary and sufficient for polyubiquitination and proteasome-mediated protein degradation. Interestingly, mutations in the RING domain that abolish the ubiquitin E3 ligase activity of BRCA1 do not affect its own ubiquitination or degradation in ovarian granulosa cells. The proteasome-mediated degradation of BRCA1 and BARD1 also occurs during the cAMP-dependent steroidogenic process. Thus, the dynamic changes of BRCA1/BARD1 protein stability in ovarian granulosa cells provide an excellent paradigm for investigating the regulation of this protein complex under physiological conditions.

Regulation of the Ets-1 transcription factor by sumoylation and ubiquitinylation

Sumoylation and ubiquitinylation reversibly regulate the activity of transcription factors through covalent attachment to lysine residues of target proteins. We examined whether the Ets-1 transcription factor is modified by sumoylation and/or ubiquitinylation. Among four potential SUMO motifs in Ets-1, we identified lysines 15 and 227 within the LK(15)YE and IK(227)QE motifs, as being the sumoylation acceptor sites. Using transfection of Ets-1 wildtype (WT) or its sumoylation deficient version (Ets-1 K15R/K227R), as well as WT or mutant proteins of the SUMO pathway, we further demonstrated that the E2 SUMO-conjugating enzyme Ubc9 and a E3 SUMO ligase, PIASy, can enhance Ets-1 sumoylation, while a SUMO protease, SENP1, can desumoylate Ets-1. We also found that Ets-1 is modified by K48-linked polyubiquitinylation independently of the sumoylation acceptor sites and is degraded through the 26S proteasome pathway, while sumoylation of Ets-1 does not affect its stability. Finally, sumoylation of Ets-1 leads to reduced transactivation and we demonstrated that previously identified critical lysine residues in Synergistic Control motifs are the sumoylation acceptor sites of Ets-1. These data show that Ets-1 can be modified by sumoylation and/or ubiquitinylation, with sumoylation repressing transcriptional activity of Ets-1 and having no clear antagonistic action on the ubiquitin-proteasome degradation pathway.

Phosphatase Activation by Epidermal Growth Factor Family Ligands Regulates Extracellular Regulated Kinase Signaling in Undifferentiated Hen Granulosa Cells

Previous work has demonstrated that epidermal growth factor family ligands, signaling through the MAPK/ERK pathway, prevent hen granulosa cell differentiation, in vitro, even in the presence of factors that promote differentiation (e.g. TGFbeta and FSH). The working hypothesis is that a release from tonic inhibitory ERK signaling is prerequisite for the initiation of hen granulosa cell differentiation. Initial results demonstrate that the ERK signaling pathway is desensitized after treatment with TGFalpha or betacellulin. Thus, studies were conducted to evaluate a role for MAPK phosphatases in the termination of ERK signaling in undifferentiated granulosa cells. Subsequent to ligand-induced translocation of ERK to the nucleus, de novo transcription and translation of one or more protein tyrosine or dual-specificity phosphatases results in dephosphorylation and localization of inactivated ERK within the nucleus. RT-PCR amplification reveals expression of the MAPK-selective phosphatases (MKP), MKP-1, -3, and dual-specificity phosphatase 5, in granulosa cells. TGFalpha induces expression (within 3 h) of mRNA encoding the ERK-selective nuclear phosphatase, dual-specificity phosphatase 5, and subsequently (by 20 h) induces mRNA encoding the cytoplasmic phosphatase, MKP-3. Increased expression of phosphatases is associated with the intracellular localization and dephosphorylation of ERK and is inhibited by the selective ERK inhibitor, U0126. In turn, regulation of phosphatase activity occurs via the ubiquitin-proteasome degradation pathway because treatment of cells with the proteasome inhibitor, Z-LLF-CHO, markedly promotes ERK dephosphorylation. These data provide direct evidence for ERK-mediated negative feedback due to regulation of phosphatase activity in undifferentiated granulosa cells.

Novel function of POSH, a JNK scaffold, as an E3 ubiquitin ligase for the Hrs stability on early endosomes

POSH (plenty of SH3s) acts as a scaffold that links activated Rac1 and downstream c-Jun N-terminal kinase (JNK) signaling modules. However, it is unknown whether it's functional domain-mediated roles including the interesting RING-finger domain or its cellular function. Here, we provide evidence that subcellular localization of POSH is regulated by a particular domain of the protein and POSH was colocalized with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) on early endosomes via interaction of Hrs with POSH's two rear SH3 domains. Moreover, the RING domain of POSH specifically regulates the stability of Hrs, but not of JNK1, via a ubiquitin-proteasomal degradation pathway. Finally, we demonstrate that JNK1 does not interact with Hrs under the conditions of POSH interacted with Hrs, but instead reduces the POSH-catalyzed ubiquitination of Hrs and their reciprocal interaction. Together, these data suggest that POSH has a distinct role as a specific E3 ubiquitin ligase for Hrs on early endosomes, and there exists a relationship between its separate activities as a scaffold and as an E3.

A phase II trial of PS-341 (bortezomib) followed by the addition of doxorubicin at progression in incurable adenoid cystic carcinoma of the head and neck: An Eastern Cooperative Oncology Group study

5573 Background: Adenoid cystic carcinoma (ACC) is usually resistant to chemotherapy. Bortezomib (B) is a highly selective inhibitor of the 26S proteasome which is central for the ubiquitin-proteasome degradation pathway. Inhibition of NF-κB activity achieved by B may be important for inhibition of the growth of ACC. Preclinical studies have shown synergy between B and doxorubicin (D). Methods: Eligibility criteria included incurable ACC, any number of prior therapies but without an anthracycline, unidimensionally measurable disease, ECOG performance status 0–2, ejection fraction within normal limits (WNL), absolute neutrophil count >1500/μL, platelets >100,000/μL, and total bilirubin WNL. Patients (pts) with stable disease (SD) for 9 months or more were excluded. Pts received B 1.3 mg/m2 IV push on days 1,4,8, and 11, every 21 days, until progression. Doxorubicin 20 mg/m2 IV on days 1 and 8 was added at the time of progression. Response assessment was performed after every 2 cycles using RECIST. Immunohistochemistry for biomarkers affected by the ubiquitin-proteasome pathway on baseline tumor and measurement of serum inflammatory and angiogenic cytokines before and in response to therapy were performed. Results: 25 pts were enrolled; 18 were females; median age was 56 years (26–73); distant metastatic sites: lung (21), liver (4), bone (2). Best response to single-agent B: 16 pts (64%) had SD, 7 progressed, and 2 were unevaluable (1 with symptomatic deterioration). The median progression-free survival was 8.5 months. In 4 evaluable pts who received B plus D, 1 partial response and 2 SD were observed. 1 pt died 7 months after starting treatment without documented progression. Worst grade (G) 3/4 toxicities with B alone in 22 evaluable pts were: G3 neutropenia 5%, G4 neutropenia 5%, G3 thrombocytopenia 5%, G3 sensory neuropathy 14%; G3 generalized weakness 5%, G3 fatigue 9%, G3 anorexia 5%, G3 diarrhea 5%, G3 hyponatremia 5%, G3 dyspnea 5%. Conclusions: B was well tolerated and resulted in disease stabilization in a high percentage of pts but no objective responses. Updated response data with the addition of D will be available. No significant financial relationships to disclose.

Bortezomib (VELCADE®) in metastatic breast cancer: pharmacodynamics, biological effects, and prediction of clinical benefits

Bortezomib (VELCADE) is a potent inhibitor of the 26S proteasome with broad antitumor activity. We performed a phase II study of bortezomib to evaluate its clinical effects in patients with metastatic breast cancer. Twelve patients with metastatic breast cancer were treated with bortezomib (VELCADE) at a dosage of 1.5 mg/m(2) administered biweekly for 2 weeks with 1 week of rest in a 21-day cycle. The primary objective was clinical response rate. Toxicity and pharmacodynamics data were also obtained. No objective responses were observed. One patient had stable disease, and 11 others experienced disease progression. The median survival time was 4.3 months (range, 0.9-37 months). The most common grade 3 or 4 toxicities included fatigue (58%; n = 7) and skin rash (33%; n = 4). The mean inhibition of specific chymotryptic activity was 53.1% (+/- 13.33%). A statistically significant reduction in the plasma interleukin-6 level was seen (P = 0.0354). Bortezomib was well tolerated but showed limited clinical activity against metastatic breast cancer when used as a single agent. The future development of this agent for the treatment of breast cancer should be guided by in vivo models that optimize activity in combination with other antitumor agents.

ERADication of ion channels destined for the plasma membrane. Focus on “Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of β-cell ATP-sensitive potassium channels”

surface expression of ion channels and receptors on the plasma membrane is of critical importance in the control of cellular signaling and communication. Each cell contains a menagerie of plasma membrane proteins, many of them oligomeric ion channels, receptors, and cell adhesion molecules. Proper

Autosomal Dominant Inclusion Body Myopathy, Paget Disease of Bone, and Frontotemporal Dementia

Autosomal dominant proximal limb girdle or inclusion body myopathy, associated with Paget disease of bone and frontotemporal dementia (IBMPFD) is a recently described disorder that maps to chromosome 9p21.1-p12. We refined the critical locus and identified the gene as the Valosin Containing Protein (VCP) gene, a member of the AAA-ATPase superfamily using a candidate gene approach. Six missense mutations were found to co-segregate with affected individuals only, two of these representing mutation hot spots. We report the clinical and molecular findings in 99 individuals in 13 families. VCP is associated with a variety of cellular activities, including the control of cell cycle, membrane fusion, and the ubiquitin-proteasome degradation pathway. Previous studies have associated VCP mutants in cell lines with vacuole formation and aggregate formation. Identification of VCP as the gene causing IBMPFD has important implications for understanding the pathogenesis of neurodegenerative disorders.

Novel function of POSH, a JNK scaffold, as an E3 ubiquitin ligase for the Hrs stability on early endosomes

POSH ( plenty of SH3s) acts as a scaffold that links activated Rac1 and downstream c-Jun N-terminal kinase (JNK) signaling modules. However, it is unknown whether it's functional domain-mediated roles including the interesting RING-finger domain or its cellular function. Here, we provide evidence that subcellular localization of POSH is regulated by a particular domain of the protein and POSH was colocalized with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) on early endosomes via interaction of Hrs with POSH's two rear SH3 domains. Moreover, the RING domain of POSH specifically regulates the stability of Hrs, but not of JNK1, via a ubiquitin–proteasomal degradation pathway. Finally, we demonstrate that JNK1 does not interact with Hrs under the conditions of POSH interacted with Hrs, but instead reduces the POSH-catalyzed ubiquitination of Hrs and their reciprocal interaction. Together, these data suggest that POSH has a distinct role as a specific E3 ubiquitin ligase for Hrs on early endosomes, and there exists a relationship between its separate activities as a scaffold and as an E3.

Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of β-cell ATP-sensitive potassium channels

ATP-sensitive potassium (K(ATP)) channels of pancreatic beta-cells mediate glucose-induced insulin secretion by linking glucose metabolism to membrane excitability. The number of plasma membrane K(ATP) channels determines the sensitivity of beta-cells to glucose stimulation. The K(ATP) channel is formed in the endoplasmic reticulum (ER) on coassembly of four inwardly rectifying potassium channel Kir6.2 subunits and four sulfonylurea receptor 1 (SUR1) subunits. Little is known about the cellular events that govern the channel's biogenesis efficiency and expression. Recent studies have implicated the ubiquitin-proteasome pathway in modulating surface expression of several ion channels. In this work, we investigated whether the ubiquitin-proteasome pathway plays a role in the biogenesis efficiency and surface expression of K(ATP) channels. We provide evidence that, when expressed in COS cells, both Kir6.2 and SUR1 undergo ER-associated degradation via the ubiquitin-proteasome system. Moreover, treatment of cells with proteasome inhibitors MG132 or lactacystin leads to increased surface expression of K(ATP) channels by increasing the efficiency of channel biogenesis. Importantly, inhibition of proteasome function in a pancreatic beta-cell line, INS-1, that express endogenous K(ATP) channels also results in increased channel number at the cell surface, as assessed by surface biotinylation and whole cell patch-clamp recordings. Our results support a role of the ubiquitin-proteasome pathway in the biogenesis efficiency and surface expression of beta-cell K(ATP) channels.

HSP25 Inhibits Protein Kinase Cδ-mediated Cell Death through Direct Interaction

Heat shock protein 25 (HSP25) interferes negatively with apoptosis through several pathways that involve its direct interaction with cytochrome c or Akt. Here we show that HSP25 inhibits protein kinase C (PKC) delta-mediated cell death through direct interaction. HSP25 binds to kinase-active PKCdelta to inhibit its kinase activity and translocation to the membrane, which results in reduced cell death. Deletion constructs of HSP25 and PKCdelta identified amino acids 90-103 of HSP25 and the C-terminal V5 region of PKCdelta as binding sites. In addition, the interaction between HSP25 and PKCdelta induced HSP25 phosphorylation at Ser-15 and Ser-86, and these phosphorylations permitted HSP25 release from PKCdelta. Based on these observations, we propose that after PKCdelta activation, HSP25 binds to the exposed V5 region of PKCdelta. This novel function of HSP25 accounts for its cytoprotective properties via the inhibition of PKCdelta and the enhancement of HSP25 phosphorylation.

Role of 14-3-3η as a Positive Regulator of the Glucocorticoid Receptor Transcriptional Activation

The glucocorticoid receptor (GR), a member of the nuclear receptor superfamily, mediates the effects of glucocorticoids. It is known that 14-3-3 family proteins interact with GR and regulate its transcriptional activity. They also bind to several molecules and influence many cellular events by altering their subcellular localization and/or acting as a chaperone. Recently, it has been proposed that ligand-activated degradation of GR occurs via the ubiquitin-proteasomal degradation pathway and that inhibition of proteasomal activity induces up-regulation of GR and enhances the transcriptional activity of GR. To examine the function of 14-3-3eta in the glucocorticoid-dependent signal pathway, we studied the regulatory role of 14-3-3eta in ligand-induced GR transcriptional activation. 14-3-3eta Enhanced the transcriptional activity of GR, and the levels of GR were higher in cells transfected with the 14-3-3eta expression vector in response to glucocorticoid. The GR level increased in both cytosol and nucleus, and endogenous GR was also elevated by 14-3-3eta in HeLa cells. 14-3-3eta Inhibited ligand-induced down-regulation of GR. Proteasomal inhibition did not induce any synergistic effect on the 14-3-3eta-induced increase in GR in response to glucocorticoid, and inhibition of translation did not block elevation of GR by 14-3-3eta, indicating that 14-3-3eta induces stabilization of GR. These results suggest that 14-3-3eta functions as a positive regulator in the glucocorticoid signal pathway by blocking the degradation of GR and inducing an elevation of GR, thus enhancing the transcriptional activity of GR.

Quantitative Proteomic Profiling of Primary Cutaneous CD30+ T-Cell Lymphoma Progression.

Primary cutaneous CD30+ T-cell lymphoproliferative disorders (LPD) represent a complex spectrum of related conditions that are not very well understood. Many may progress to more aggressive or systemic lymphomas. The factors involved in the progression of these LPDs are largely unknown. Advances in robust high throughput proteomics techniques permit elucidation of the molecular mechanisms underlying disease development and progression. In this study we applied a quantitative proteomics methodology involving isotope coded affinity tag reagents (ICAT™), 3-dimensional liquid chromatography (3-D LC), and tandem mass spectrometry (MS/MS) to evaluate the differential protein expression patterns of two unique T-cell lymphoma derived cell lines. The Mac-1 and Mac-2A are clonally related cell lines derived from the same cutaneous T-cell lymphoma (CTCL) at relatively indolent versus aggressive stages of tumor progression, respectively. Equivalent quantities of total cell lysates obtained from the two cell lines were labeled with cleavable ICAT™ and dialyzed prior to digestion with proteinase K. The labeled peptides were then subjected to strong cation-exchange (SCX) chromatography followed by further purification of each offline fraction using avidin affinity chromatography. These cysteine enriched ICAT™ labeled peptides were analyzed by reverse phase nanospray LC-MS/MS. Differential expression of 490 unique proteins was determined. Of these, a total of 59 proteins showed a 1.5 fold or greater increase in abundance while 86 proteins exhibited a similar decrease in the clinically aggressive lymphoma cells as compared to the more indolent form. Functional classification of the differentially expressed proteins revealed increased expression of those involved in cell signaling (neurotrophic tyrosine kinase receptor, ATP binding cassette transporter 1, SH3 protein interacting with NCK), transcription (HFK1, general transcription factor IIIC), and cell adhesion (ICAM, protocadherin) in the advanced lymphoma. There was also decreased expression in the advanced lymphoma of proteins which were involved in apoptosis (caspase 8), cell signaling (STAT4, LIM kinase), cell cycle regulation (CDC10 homolog, ankyrin 3), cell adhesion (laminin, periostin), and ion transport (voltage-gated potassium channel). Overexpression of MALT1/paracaspase that leads to activation of the NF-kappaB pathway was observed in the advanced lymphoma. Proteins such as Smad4 which is involved in regulation of the TGF-beta signaling pathway and ubiquitin specific protease of the ubiquitin-proteasomal degradation pathway that in turn regulates Smads were differentially expressed. Some of the proteins identified by MS were selected for confirmation by Western blot analysis. This study implicates deregulation of multiple signal transduction pathways during progression of CTCL and provides novel insights into the underlying molecular pathogenetic mechanisms involved.

Inhibition of Proteasome by MG-132 Treatment Causes Extra Phragmoplast Formation and Cortical Microtubule Disorganization during M/G1 Transition in Synchronized Tobacco Cells

The 26S proteasome plays essential roles in cell cycle progression in various types of cell. We previously reported that the inhibition of 26S proteasome activities by a proteasome inhibitor, MG-132, exclusively caused cell cycle arrest in synchronized tobacco BY-2 cells. Here we report a further observation of 26S proteasome involvement during M/G1 transition utilizing a transgenetic BY-2 cell line that stably expresses a GFP-alpha-tubulin fusion protein (BY-GT16). Interestingly, MG-132 treatment caused the arrest of cell cycle progression prior to entering the G1 phase. Indeed, phragmoplast-like structures were formed and cortical microtubules were not organized after the collapse of the original phragmoplasts. Additionally, actin microfilaments showed irregular rearrangements when further incubated with MG-132 and as the phragmoplast-like structures developed. Since these phragmoplast-like structures had a similar configuration and ability to form cell plates to that of the original phragmoplasts, we designated these phragmoplast-like structures as extra phragmoplasts. Furthermore, we showed that a tobacco kinesin-related polypeptide of 125 kDa (TKRP125) localized in the extra phragmoplasts and that its protein level remained unchanged during MG-132 treatment. We propose that TKRP125 might be one of the possible targets of the ubiquitin-proteasome degradation pathway during M/G1 transition.

Phase I trial of bortezomib (PS-341) in combination with docetaxel in patients with advanced solid tumors

3052 Background: Bortezomib (PS-341), a first-in-class dipeptidyl boronic acid, is an inhibitor of the 26S proteasome, an ATP-dependent multicatalytic protease central to the ubiquitin-proteasome degradation pathway. The principal mode of bortezomib detoxification is via deboronization involving cytochrome P4503A (CYP3A), which also metabolizes docetaxel. A phase I study combining bortezomib and docetaxel is being performed to determine the MTD/toxicities and pharmacokinetics (PK) of this combination in refractory solid tumor pts. Methods: Pts received escalating doses of weekly docetaxel (d1 and d8) and twice-weekly bortezomib (d2, d5, d9, and d12) in 3-wk cycles. Two pts were enrolled at each dose level, with cohort expansion to 6pts for DLT (occurring in cycles 1 or 2). Dose level 1 (DL1), 2 (DL2), and 3 (DL3) consisted of docetaxel/bortezomib 25/0.8 mg/m2, 25/1.0 mg/m2, and 30/1.0 mg/m2, respectively. CYP3A activity, determined using the erythromycin breath test (ERMBT), and docetaxel PK were evaluated during weeks 1 (docetaxel alone) and 5 (docetaxel + bortezomib). Results: Thirteen pts (10 M/3 F; mean (range) age, 62 (40–76) years; ECOG PS, 0–1) received a total of 31 cycles. DL2 was expanded for DLT which occurred in 2 of 6 pts (febrile neutropenia [1 pt] and thrombocytopenia [1pt]), thus exceeding the MTD. DL1 was then expanded to 6 pts where no DLTs occurred; one additional patient will be enrolled to replace a pt not evaluable for toxicity to define the MTD at DL1. One pt received 2 cycles at DL3 with dose-reduction to DL2; gr 3 thrombocytopenia occurred at cycle 3. Both episodes of gr3 thrombocytopenia were transient (<7d) without clinical sequelae. The ERMBT parameter, 14C-exhaled/h (%), and docetaxel clearance did not change between weeks 1 and 5; mean (SD) values were 1.99 (0.84)% vs 2.31 (0.81)% (P=.44) and 29.2 (13.5) L/h vs 27.0 (14.6) (P = .49), respectively. Conclusions: The MTD of docetaxel given weekly for 2 of 3 weeks with bortezomib twice-weekly (4 doses) has been defined. Bortezomib treatment did not alter CYP3A activity and docetaxel CL demonstrating lack of a drug interaction with this drug combination. Supported by U01 CA70095 and Aventis. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Aventis (Speaker's Bureau) Aventis