Role of the histone deacetylase inhibitor valproic acid in high-fat diet-induced hypertension via inhibition of HDAC1/angiotensin II axis.

BACKGROUND: Obesity is known as an epidemic worldwide because of consumption of westernized high-fat diets and one of the major risk factors of hypertension. Histone deacetylases (HDACs) control gene expression by regulating histone/non-histone protein deacetylation. HDAC inhibitors exert anti-cancer and anti-inflammatory effects and play a protective role in cardiovascular diseases. In the present study, we tested the effect of an FDA-approved pan-HDAC inhibitor valproic acid (VPA) on high-fat diet (HFD)-induced hypertension in mice. Furthermore, we examined the mechanism of VPA-induced prevention of hypertension. METHODS: Nine-week-old male C57BL/6 mice were fed either a normal diet (ND) or HFD. When the HFD group reached a pre-hypertensive phase (130-140 mm Hg systolic blood pressure), VPA was administered for 6 days (300 mg kg-1 per day). Body weights and blood pressure (BP), expression of renin-angiotensin system (RAS) components and HDAC1 were determined. The direct role of HDAC1 in the expression of RAS components was investigated using gene silencing. RESULTS: HFD accelerated the increase in body weight from 22.4±1.3 to 31.9±3.0 compared to in the ND group from 22.7±0.9 to 26.0±1.7 (P=0.0134 ND vs HFD), systolic BP from 118.5±5.7 to 145.0±3.0 (P<0.001), and diastolic BP from 91.0±13.6 to 121.0±5.0 (P=0.006); BP was not altered in the ND group. HFD increased RAS components and HDAC1 in the kidneys as well as leptin in the plasma. VPA administration prevented the progression of hypertension and inhibited the increase in expression of HDAC1 and RAS components. VPA did not affect plasma leptin level. Knockdown of HDAC1 in MDCK cells decreased the expression of angiotensinogen and type 1 angiotensin II receptor. CONCLUSIONS: VPA prevented HFD-induced hypertension by downregulating angiotensin II and its receptor via inhibition of HDAC1, offering a novel therapeutic option for HFD-induced hypertension.

Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression.

Although ABT-737, a small-molecule Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic agent, ABT-737-induced apoptosis is often blocked in several types of cancer cells with elevated expression of Mcl-1. Cafestol, one of the major compounds in coffee beans, has been reported to have anti-carcinogenic activity and tumor cell growth-inhibitory activity, and we examined whether cafestol could overcome resistance against ABT-737 in Mcl-1-overexpressed human renal carcinoma Caki cells. ABT-737 alone had no effect on apoptosis, but cafestol markedly enhanced ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells, human glioma U251MG cells, and human breast carcinoma MDA-MB231 cells. By contrast, co-treatment with ABT-737 and cafestol did not induce apoptosis in normal human skin fibroblast. Furthermore, combined treatment with cafestol and ABT-737 markedly reduced tumor growth compared with either drug alone in xenograft models. We found that cafestol inhibited Mcl-1 protein expression, which is important for ABT-737 resistance, through promotion of protein degradation. Moreover, cafestol increased Bim expression, and siRNA-mediated suppression of Bim expression reduced the apoptosis induced by cafestol plus ABT-737. Taken together, cafestol may be effectively used to enhance ABT-737 sensitivity in cancer therapy via downregulation of Mcl-1 expression and upregulation of Bim expression.

Long-term subjective tongue function after partial glossectomy.

There have been limited studies of subjective tongue function over long-term follow-up in spite of swallowing and articulation disorders are common complications of glossectomy. To assess long-term subjective swallowing and articulation function after partial glossectomy. A total of 63 patients with the mobile tongue cancer who underwent partial glossectomy without reconstruction were interviewed to score their swallowing and articulation function on a 100-point scale. The relation of this subjective scoring to the perioperative data was subjected to multivariate analysis. The mean patient age was 53·4 (19-81) years, and the mean follow-up duration was 78·9 (14-277) months. Mean swallowing and articulation function score was 87·7 ± 6·1 and 88·6 ± 5·4. Age, follow-up duration, T stage and resection volume were significantly correlated with swallowing function (P = 0·026, 0·029, 0·016, 0·002, respectively); follow-up duration was correlated with articulation function (P = 0·039). Patients who undergo partial glossectomy without reconstruction generally demonstrate good function on long-term follow-up. Subjective dysfunction was correlated with larger resection volume, older age and shorter follow-up duration.

ß-Lapachone induces programmed necrosis through the RIP1-PARP-AIF-dependent pathway in human hepatocellular carcinoma SK-Hep1 cells.

ß-Lapachone activates multiple cell death mechanisms including apoptosis, autophagy and necrotic cell death in cancer cells. In this study, we investigated ß-lapachone-induced cell death and the underlying mechanisms in human hepatocellular carcinoma SK-Hep1 cells. ß-Lapachone markedly induced cell death without caspase activation. ß-Lapachone increased PI uptake and HMGB-1 release to extracellular space, which are markers of necrotic cell death. Necrostatin-1 (a RIP1 kinase inhibitor) markedly inhibited ß-lapachone-induced cell death and HMGB-1 release. In addition, ß-lapachone activated poly (ADP-ribosyl) polymerase-1(PARP-1) and promoted AIF release, and DPQ (a PARP-1 specific inhibitor) or AIF siRNA blocked ß-lapachone-induced cell death. Furthermore, necrostatin-1 blocked PARP-1 activation and cytosolic AIF translocation. We also found that ß-lapachone-induced reactive oxygen species (ROS) production has an important role in the activation of the RIP1-PARP1-AIF pathway. Finally, ß-lapachone-induced cell death was inhibited by dicoumarol (a NQO-1 inhibitor), and NQO1 expression was correlated with sensitivity to ß-lapachone. Taken together, our results demonstrate that ß-lapachone induces programmed necrosis through the NQO1-dependent ROS-mediated RIP1-PARP1-AIF pathway.

Antipsychotic agent thioridazine sensitizes renal carcinoma Caki cells to TRAIL-induced apoptosis through reactive oxygen species-mediated inhibition of Akt signaling and downregulation of Mcl-1 and c-FLIP(L).

Thioridazine has been known as an antipsychotic agent, but it also has anticancer activity. However, the effect of thioridazine on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitization has not yet been studied. Here, we investigated the ability of thioridazine to sensitize TRAIL-mediated apoptosis. Combined treatment with thioridazine and TRAIL markedly induced apoptosis in various human carcinoma cells, including renal carcinoma (Caki, ACHN, and A498), breast carcinoma (MDA-MB231), and glioma (U251MG) cells, but not in normal mouse kidney cells (TMCK-1) and human normal mesangial cells. We found that thioridazine downregulated c-FLIP(L) and Mcl-1 expression at the post-translational level via an increase in proteasome activity. The overexpression of c-FLIP(L) and Mcl-1 overcame thioridazine plus TRAIL-induced apoptosis. We further observed that thioridazine inhibited the Akt signaling pathway. In contrast, although other phosphatidylinositol-3-kinase/Akt inhibitors (LY294002 and wortmannin) sensitized TRAIL-mediated apoptosis, c-FLIP(L) and Mcl-1 expressions were not altered. Furthermore, thioridazine increased the production of reactive oxygen species (ROS) in Caki cells, and ROS scavengers (N-acetylcysteine, glutathione ethyl ester, and trolox) inhibited thioridazine plus TRAIL-induced apoptosis, as well as Akt inhibition and the downregulation of c-FLIP(L) and Mcl-1. Collectively, our study demonstrates that thioridazine enhances TRAIL-mediated apoptosis via the ROS-mediated inhibition of Akt signaling and the downregulation of c-FLIP(L) and Mcl-1 at the post-translational level.

Comparison of techniques for double-lumen endobronchial intubation: 90° or 180° rotation during advancement through the glottis.

BACKGROUND: During endobronchial intubation with a double-lumen endobronchial tube (DLT), the DLT is conventionally rotated through 90° when the bronchial tip is just past the vocal cords. This study was performed to investigate if rotation of the DLT through 180° decreases postoperative hoarseness, sore throat, or vocal cord injuries. METHODS: Patients (n=164) undergoing thoracic surgery were randomized into two groups. Just after the bronchial tip passed the glottis, left-sided DLTs were rotated 90° (Group 90, n=84) or 180° (Group 180, n=80) counterclockwise and advanced. In the Group 180, DLTs were re-rotated 90° clockwise after the tracheal tip passed the glottis. Resistance during the advance of DLTs was assessed. Hoarseness and sore throat were evaluated for three postoperative days. Vocal cords were examined on the first postoperative day. RESULTS: In nine patients allocated to Group 90, the DLT could not be advanced past the glottis because of severe resistance. There was less resistance to advancement of the DLT in Group 180 compared with Group 90 (P<0.001). The incidence of hoarseness was comparable between the two groups. Sore throat and vocal cord injuries occurred less frequently in Group 180 compared with Group 90 (20 vs 40%, P=0.008; 19 vs 47%, P=0.032). CONCLUSIONS: Rotation of a DLT through 180° facilitated its passage through the glottis and reduced the incidence of postoperative sore throat and vocal cord injuries.

The Korean version of the University of Washington Quality of Life Questionnaire for Patients with head and neck cancer, and its use in an initial validation study of 56 patients.

The University of Washington Quality of Life (UW-QOL) questionnaire is often used to assess health-related quality of life (HRQOL) of head and neck cancer patients. The aim of this study was to translate the UW-QOL version 4 into the Korean language and to carry out an initial validation study. A recognized methodology for translation of questionnaires was used. The validation study used the final Korean version between March and September 2009. Adult patients were recruited, with a confirmed diagnosis of head and neck cancer, therapy completed and disease-free for at least 1 year. The UW-QOL was successfully translated into Korean. 56 patients completed Korean versions of UW-QOL, the Beck Depression Inventory and the World Health Organization Quality of Life-BREF and various expected correlations were confirmed first between the two UW-QOL subscales (Spearman 0.54 p<0.001) and then of these subscales with the other concurrent measures. Lower (worse) UW-QOL scores were seen for later stage patients in all 12 domains. The Korean version of UW-QOL is ready for use in the assessment of HRQOL for Korean patients. Validation work needs to be continued to further establish psychometric properties of the questionnaire for use in this population.

Kinematic analysis of hyolaryngeal complex movement in patients with dysphagia development after pneumonectomy.

PURPOSE: Aim of the study was to investigate the swallowing kinematics of patients with dysphagia which developed after pneumonectomy. METHODS: We investigated the swallowing kinematics of patients with dysphagia development after pneumonectomy and compared them with age- and gender-matched normal controls. The following swallowing parameters were compared: (1) maximum anterior and superior displacement (mm) of the hyoid bone; (2) maximum anterior and superior displacement (mm) of the larynx; (3) maximum epiglottic rotation angle ( degrees ); and (4) pharyngeal delay time (PDT) (sec) using videofluoroscopy. RESULTS: Significant differences were found in the maximum superior displacement of the hyoid bone ( P = 0.028) and larynx ( P = 0.001). Pharyngeal delay time showed a significant difference between the two groups ( P = 0.001). When we dichotomized patients to vocal cord palsy and non-palsy subgroups, no significant difference was found in all parameters. CONCLUSION: Our results indicate that dysphagia development after pneumonectomy is characterized by reduced hyolaryngeal elevation during swallowing and delay of the pharyngeal swallowing reflex. Further study must be done to reveal the exact mechanism of this phenomenon.

Endoscopically assisted reduction of nasal bone fractures.

The standard treatment for a nasal bone fracture is closed reduction within 10 days. After that time, callus and fibrous connective tissue will limit a precise reduction. This study evaluated endoscopically assisted reduction for the treatment of nasal bone fractures in patients who miss the optimal operating time. Fifteen patients underwent endoscopically assisted correction of nasal bone fractures. The surgery was performed with the patients under general anesthesia. An intercartilaginous incision was made. The depressed bony fragments were repositioned under endoscopic visualization. In all cases, good anatomic reduction was obtained, the postoperative course was uneventful, with no complications, and the patients were satisfied with the shape of their noses. Endoscopy appears to be the best tool for visualizing intraoperative repositioning control, enabling the surgeon to confirm a fracture site with callus and to perform an accurate reduction. Endoscopically assisted reduction provides an alternative option in the treatment of patients outside the optimal temporal window for surgery.

Class III beta-tubulin, but not ERCC1, is a strong predictive and prognostic marker in locally advanced head and neck squamous cell carcinoma.

BACKGROUND: Recent researches revealed that class III beta-tubulin (TUBB3) is a prognostic marker in various tumors and role of TUBB3 in head and neck squamous cell carcinoma (HNSCC) is not defined yet. We analyzed the significance of TUBB3 expression along with p53 and ERCC1 in locally advanced HNSCC patients receiving cisplatin-based induction chemotherapy. MATERIALS AND METHODS: Retrospective review of medical records at Seoul National University Hospital between 1998 and 2007 was carried out. Immunohistochemical stain of TUBB3, p53, and ERCC1 was done in paraffin-embedded tumor tissue. We assessed response to treatment, progression-free survival (PFS), overall survival (OS), and cancer-specific survival (CSS). RESULTS: Eighty-five patients with oropharyngeal, hypopharyngeal, and laryngeal cancers received induction chemotherapy with 5-fluorouracil (5-FU) and cisplatin (n = 55), or 5-FU, cisplatin, and docetaxel (Taxotere) (n = 30). Eighty-three received definitive treatment after induction chemotherapy, where 62 received radiotherapy and 21 received surgery. TUBB3-positive patients showed lower response rate than TUBB3-negative patients (69% versus 88%, P = 0.039). Shorter median PFS was observed in TUBB3-positive group (12 versus 47 months, P = 0.001). Shorter median OS was observed in TUBB-positive group not reaching statistical significance (30 versus 59 months, P = 0.072). TUBB3 status significantly influenced CSS (35 months versus not reached, P = 0.017). Positive p53 status was related to poorer OS and CSS. ERCC1 showed no influence on chemotherapy response, PFS, OS, and CSS. CONCLUSION: TUBB3 is a predictive and prognostic marker along with well-known p53 in HNSCC patients receiving cisplatin-based induction chemotherapy. Clinical impact of ERCC1 is not evident in this setting.

NAG-1 up-regulation mediated by EGR-1 and p53 is critical for quercetin-induced apoptosis in HCT116 colon carcinoma cells.

Quercetin, a flavonoid molecule ubiquitously present in nature, has multiple effects on cancer cells, including the inhibition of cell proliferation and migration. However, the responsible molecular mechanisms are not fully understood. We found that quercetin induces the expression of NAG-1 (Non-steroidal anti-inflammatory drug activated gene-1), a TGF-beta superfamily protein, during quercetin-induced apoptosis of HCT116 human colon carcinoma cells. Reporter assays using the luciferase constructs containing NAG-1 promoter region demonstrate that early growth response-1 (EGR-1) and p53 are required for quercetin-mediated activation of the NAG-1 promoter. Overexpression of NAG-1 enhanced the apoptotic effect of quercetin, but suppression of quercetin-induced NAG-1 expression by NAG-1 siRNA attenuated quercetin-induced apoptosis in HCT116 cells. Taken together, the present study demonstrates for the first time that quercetin induces apoptosis via NAG-1, providing a mechanistic basis for the apoptotic effect of quercetin in colon carcinoma cells.

Bcl-2 attenuates anticancer agents-induced apoptosis by sustained activation of Akt/protein kinase B in U937 cells.

Aberrant overexpression of antiapoptotic members of the Bcl-2 protein family contributes to resistance to anticancer therapeutic drugs. Thus, this protein represent attractive target for novel anticancer agents. In the present study, we determined the effect of the anti-apoptosis protein Bcl-2 on caspase-3 activation, PLC-gamma1 degradation and Akt activation during the various anticancer agents-induced apoptosis. Treatment with chrysin for 12 h produced morphological features of apoptosis in U937 cells, which was associated with caspase-3 activation and PLC-gamma1 degradation. Induction of apoptosis was also accompanied by down-regulation of XIAP and inactivation of Akt. Chrysin-induced caspase-3 activation, PLC-gamma1 degradation and apoptosis were significantly attenuated in Bcl-2 overexpressing U937/Bcl-2 cells. Ectopic expression of Bcl-2 appeared to inhibit ceramide-, and Akt specific inhibitor (SH-6)-induced apoptosis by sustained Akt activation. Thus, our findings imply that some of the biological functions of Bcl-2 may be attributed to their ability to inhibit anticancer agents-induced apoptosis through the sustained Akt activation.

Bcl-2 overexpression attenuates resveratrol-induced apoptosis in U937 cells by inhibition of caspase-3 activity.

Resveratrol has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. In the present study, we determined the effect of high intracellular levels of the anti-apoptosis protein Bcl-2 on caspase-3 activation, PLC-gamma1 degradation and cytochrome c release during resveratrol-induced apoptosis. For this, we used U937/vector and U937/Bcl-2 cells, which were generated by transfection of the cDNA of the Bcl-2 gene. As compared with U937/vector, U937/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment with 60 or 100 microM resveratrol for 24 h produced morphological features of apoptosis and DNA fragmentation in U937/vector cells, respectively. This was associated with caspase-3 activation and PLC-gamma1 degradation. In contrast, resveratrol-induced caspase-3 activation and PLC-gamma1 degradation and apoptosis were significantly inhibited in U937/Bcl-2 cells. Bcl-2 overexpressing cells exhibited less cytochrome c release and sustained expression levels of the IAP proteins during resveratrol-induced apoptosis. In addition, these findings indicate that Bcl-2 inhibits resveratrol-induced apoptosis by a mechanism that interferes with cytochrome c release and activity of caspase-3 that is involved in the execution of apoptosis.

Neoplastic transformation and tumorigenesis associated with overexpression of phospholipase D isozymes in cultured murine fibroblasts.

Phospholipase D (PLD) has been suggested to play an important role in a variety of cellular functions. PLD activity has been shown to be significantly elevated in many tumours and transformed cells, suggesting the possibility that PLD might be involved in tumorigenesis. In this study, we have established stable cell lines overexpressing PLD1 and PLD2 from fibroblast cells. These cells, but not control cells, showed altered growth properties and anchorage-independent growth in soft agar. Both PLD1 and PLD2 also induced an up-regulation of the activity of matrix metalloprotease-9 as detected by zymograms. Furthermore, both PLD1 and PLD2 transformants, but not vector-transfectants, induced undifferentiated sarcoma when transplanted into nude mice. Both PLD1- and PLD2-mediated cell cycle distributions in stable cell lines revealed an increased fraction of cells in the S phase compared with control cells. Interestingly, the level of cyclin D3 protein, known as an activator of G(1) to S phase transition in the cell cycle, was aberrantly high in cells overexpressing PLD1 and PLD2 compared with control cells. These results suggest that overexpression of PLD isozymes may play an important role in neoplastic transformation.

Phorbol myristate acetate inhibits okadaic acid-induced apoptosis and downregulation of X-linked inhibitor of apoptosis in U937 cells.

Okadaic acid is a specific inhibitor of serine/threonine protein phosphatase 1 (PP-1) and 2A (PP-2A). The phosphorylation and dephosphorylation at the serine/threonine residues on proteins play important roles in regulating gene expression, cell cycle progression, and apoptosis. In this study, phosphatase inhibitor okadaic acid induces apoptosis in U937 cells via a mechanism that appears to involve caspase 3 activation, but not modulation of Bcl-2, Bax, and Bcl-X(L) expression levels. Treatment with 20 or 40 nM okadaic acid for 24 h produced DNA fragmentation in U937 cells. This was associated with caspase 3 activation and PLC-gamma1 degradation. Okadaic acid-induced caspase 3 activation and PLC-gamma1 degradation and apoptosis were dose-dependent with a maximal effect at a concentration of 40 nM. Moreover, PMA (phorbol myristate acetate), PKC (protein kinase C) activator, protected U937 cells from okadaic acid-induced apoptosis, abrogated okadaic acid-induced caspase 3 activation, and specifically inhibited downregulation of XIAP (X-linked inhibitor of apoptosis) by okadaic acid. PMA cotreated U937 cells exhibited less cytochrome c release and sustained expression levels of the IAP (inhibitor of apoptosis) proteins during okadaic acid-induced apoptosis. In addition, these findings indicate that PMA inhibits okadaic acid-induced apoptosis by a mechanism that interferes with cytochrome c release and activity of caspase 3 that is involved in the execution of apoptosis.

Involvement of ERK and protein tyrosine phosphatase signaling pathways in EGCG-induced cyclooxygenase-2 expression in Raw 264.7 cells.

Prostaglandins play regulatory roles in a variety of physiological and pathological processes in immune response and inflammation. Epigallocatechin-3-gallate (EGCG) is known to potent antitumor agent with antioxidant property. We first investigated the effect of EGCG on the production of prostaglandin E(2) (PGE(2)) and the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of PGE(2), using macrophage cell line, Raw264.7. Our results showed that COX-2 expression and PGE(2) production are upregulated by EGCG treatment and that this induction of COX-2 is regulated in part at the transcriptional level. In addition, we demonstrated the signal transduction pathway of mitogen-activated protein kinase (MAP kinase) in EGCG-mediated COX-2 expression. The MEK inhibitor (PD098059) prevented EGCG-induced COX-2 expression, whereas sodium orthovanadate (protein-tyrosine phosphatase inhibitor) significantly enhanced COX-2 expression and PGE(2) production. These results suggest that EGCG mediated COX-2 expression and PGE(2) production is associated with the activation of both the ERK and protein-tyrosine phosphatase signaling pathways.

Arsenic trioxide induces G2/M growth arrest and apoptosis after caspase-3 activation and bcl-2 phosphorylation in promonocytic U937 cells.

Arsenic trioxide has recently been shown to inhibit growth and induce apoptosis in acute promyelocytic leukemia (APL), but little is known about the molecular mechanisms mediating these effects. Here we demonstrate that treatment of promonocytic U937 cells with arsenic trioxide leads to G2/M arrest which was associated with a dramatic increase in the levels of cyclin B and cyclin B-dependent kinase and apoptosis. We further show that apoptosis occurs after bcl-2 phosphorylation and caspase-3 activation followed by cleavage of PARP and PLC-gamma1 degradation and DNA fragmentation. The arsenic trioxide-induced apoptosis could be blocked by the protein synthesis inhibitor cycloheximide. In addition, pretreatment of U937 cells with the DNA polymerase inhibitor aphidicolin also blocked apoptosis, but did not cause the arrest of cells in the G2/M phase. The findings suggest that arsenic trioxide exerts its growth-inhibitory effects by modulating expression and/or activity of several key G2/M regulatory proteins. Furthermore, arsenic trioxide-mediated G2/M arrest correlates with the onset of apoptosis.

Immune response induced by immunization with Hepatitis B virus core DNA isolated from chronic active hepatitis patients.

There are many mutations in the gene encoding Hepatitis B virus (HBV) core antigen of chronic active hepatitis patients, and such mutations are most likely to be related to the severity of disease. Here, we constructed plasmids containing wild-type and deletion type of HBV core gene (HBc) to develop an experimental DNA vaccine and to compare immunogenicity of two types of HBc vaccine. Twenty-nine wild-types and seven deletion types of HBc were detected in sera of 32 Korean patients with chronic active hepatitis. Four wild-types (W1, W2, W4, W6) and two deletion types (D3, D4) of HBc were cloned into the pcDNA3 vector. Intramuscular immunization with wild-type HBc efficiently increased serum anti-HBc antibody response in a dose-dependent manner. Anti-HBc antibody response in mice injected with W6 increased 14 days after immunization, and peaked after 30 days and was maintained at least up to 50 days. W6 immunization induced a specific cytotoxic T lymphocyte response to W6-transfected 3LL (3LL-W6), and reduced the sizes of tumor mass of mice challenged with 3LL-W6 or 3LL transfected with D4. However, intramuscular immunization with D3 and D4 did not show antibody response at all. D3 and D4 have 157 bp (from 331 to 491 bp) and 122 bp (from 327 to 448 bp) gene deletion, respectively, and these encode class II MHC-restricted T-cell epitope. Altogether, these results suggest that mutant virus that has deleted HBc gene may evade immune systems due to loss of T-cell epitope.

Low levels of cyclin D and nonfunctional Rb protein affect cdk6 association with cyclin-dependent kinase inhibitor p27(Kip1).

p27(Kip1) associates with cyclin/cdk complexes and inhibiting cdk activity, and overexpression of p27(Kip1) induces G1 arrest. We found that p27(Kip1) overexpression inhibits cdk2 kinase activity, but not cdk6 kinase activity in HeLa cells. The amount of p27(Kip1) associated with cdk2 was significantly higher than that associated with cdk6. cdk6 complexes contained detectable amounts of p27(Kip1) in all human cell lines examined, except in HeLa cells where p27(Kip1) preferentially associated with cdk2. It appears that in HeLa cells overexpressed p27(Kip1) fails to inhibit cdk6 kinase activity because of low binding affinity of cdk6 to p27(Kip1). The low binding affinity is due to a low level of the cdk6/cyclin D complexes. Functional inactivation of pRb has an effect on p27(Kip1) association with cdk6/cyclin D complexes.

The levels of MDM2 protein are decreased by a proteasome-mediated proteolysis prior to caspase-3-dependent pRb and PARP cleavages.

MDM2 is a substrate of caspase-3 in p53-mediated apoptosis. In addition, MDM2 mediates its own ubiquitination in a RING finger-dependent manner. Thus, we investigated whether MDM2 is degraded through a ubiquitin-dependent proteasome pathway in the absence of p53. When HL-60 cells, p53 null, were treated with etoposide, MDM2 was markedly decreased prior to caspase-3-dependent retinoblastoma tumor suppressor protein (pRb) and poly (ADP- ribose) polymerase (PARP) cleavages. Moreover, down-regulation of MDM2 level was not coupled with its mRNA down-regulation. However, the level of MDM2 was partially restored by proteasome inhibitors such as LLnL and lactacystin, even in the presence of etoposide. Our results suggest that, in the p53 null status, MDM2 protein level is decreased by proteasome-mediated proteolysis prior to caspase-3-dependent PARP and pRb cleavages.