[Epidural block associated with improved long-term survival after surgery for colorectal cancer: A retrospective cohort study with propensity score matching].

OBJECTIVE: To investigate the effect of epidural anesthesia on the long-term prognosis of patients after selective colorectal cancer resection surgery. METHODS: This was a retrospective cohort study and approved by local institution review board. Patients who underwent selective colorectal cancer resection surgery from August 2011 to December 2012 in Peking University First Hospital were enrolled. The patients were divided into general anesthesia (GA) group and combined epidural-general anesthesia (EGA) group according to anesthesia type. Primary outcome was patient's long-term survival status. Secondary outcome included the overall incidence of in-hospital complications and length of postoperative in-hospital stay. Propensity score was used to match cases between the two groups based on the probability of receiving EGA. Survival was analyzed by Kaplan-Meier analysis and compared by Log-rank test between the two groups. Multivariate Cox regression analysis was used to investigate the relationship between epidural anesthesia and other variables with long-term survival status. RESULTS: A total of 264 patients were entered into final analysis, including 166 cases in GA group and 98 cases in EGA group. Mean age of the patients was (63.3±12.1) years and mean survival time was 47.2 (95%CI 45.7-48.7) months. Before the propensity score match, the mortality in EGA group was 16.9% (28/166) and 9.2% (9/98) in GA group. But comparison between the two groups had no statistical significance (P=0.091). After the propensity score match, 87 paired cases were matched and analyzed. The risk of long-term mortality in EGA group was lower than that of GA group by Kaplan-Meier analysis (5.7% vs.16.1%, HR=0.344, 95%CI 0.124-0.955, P=0.041). Mean survival time of EGA group was longer than that of GA group (50.3 months vs. 42.9 months, P=0.032). Multivariate Cox regression ana-lysis showed that EGA, in comparison with GA, was related with lower risk of long-term mortality (HR=0.326, 95%CI 0.117-0.909, P=0.032). Age (HR=1.042, 95%CI 1.001-1.085, P=0.046) and preoperative lymph node metastasis (HR=2.924, 95%CI 1.162-7.356, P=0.023) were also related with increased risk of long-term mortality. CONCLUSION: Present study found that perioperative use of epidural anesthesia and analgesia was associated with improvement of the patient's long-term survival. Well-designed studies are needed to verify this hypothesis.

[Clinical characteristics of patients with multiple myeloma harboring 6q deletion].

Objective: To study the clinical and cytogenetic characteristics of patients with multiple myeloma harboring 6q deletion, with the aim to determine the impact of 6q deletion on survival. Methods: This study included the retrospective analysis of 382 newly diagnosed patients with multiple myeloma in our hospital from 2014 to 2017 and compared the clinical and cytogenetic characteristics between patients with and without 6q deletion. The log-rank test and the Cox proportional hazards regression model were used to analyze prognostic factors for progression-free survival (PFS) and overall survival (OS) . Results: Compared to those without 6q, the patients with 6q deletion were older (median age, 63 vs 58 years, P=0.039) , had higher incidence of t (4; 14) (30.4% vs 16.4% , P=0.020) , and higher proportion of complex karyotypes (22.2% vs 5.3% , P=0.001) . Univariate survival analysis using the log-rank test revealed that 6q deletion was associated with shorter PFS. However, by the Cox multivariate proportional hazards regression model, 6q deletion was not an independent prognostic factor and its effect on survival was affected by age, t (4; 14) , and other risk factors. Conclusions: 6q deletion was common in elderly patients with multiple myeloma and was often accompanied by t (4;14) and complex karyotypes. However, 6q deletion was not an independent prognostic factor for multiple myeloma.

The mRNA Distribution of Cancer Stem Cell Marker CD90/Thy-1 Is Comparable in Hepatocellular Carcinoma of Eastern and Western Populations.

Epidemiology of hepatocellular carcinoma (HCC) showed a correlation between incidence and geographical-relevant risk factors. This study aims to compare the distributions of cancer stem cells (CSC) in two distant populations in Asia and Europe. We analyzed 52 and 43 selected HCC patients undergoing hepatectomy in Ho Chi Minh City (Vietnam) and Trieste (Italy). Each patient sample consisted of HCC, peri-HCC, and non-tumoral (distal) tissue. Demographic data were recorded together with clinical findings. The protocol for the collection of tissue samples and RNA was standardized in both laboratories and gene expression analysis was performed in a single laboratory with identical PCR conditions. Baseline data showed comparable laboratory findings between the two cohorts. mRNA distribution showed a comparable pattern of all CSC markers analyzed with the expression of CD90 progressively increasing from distal and peri-HCC to be highest in HCC (p < 0.001), confirmed by immunofluorescence data. CD90 mRNA distribution was related to HBV-related HCC and a tumor diameter less than 5 cm. Patients with high tumoral CD90 mRNA had a shorter time (p < 0.05) to tumor recurrence compared to patients with lower CD90. This comparative study showed that CD90 mRNA expressions are comparable between Eastern and Western HCC cases.

Has-miR-17 increases the malignancy of gastric lymphoma by HSP60/TNFR2 pathway.

The purpose of this study was to investigate the expression and mechanism of miR-17 in gastric lym-phoma. miR-17mimics, miR-17 inhibitors and negative controls were transfected into human gastric lymphoma cell line cyp6d. The proliferation, invasion and apoptosis of cyp6d cells were detected by CCK-8, Transwell and TUNEL methods, respectively. The expression and clinicopathological features of miR-17 in gastric lymphoma were analyzed by real-time quantitative PCR. The target gene of miR-17 was predicted by targetscan 7.2, and the expression of miR-17 related protein was detected by Western blot. The results showed that the expression of miR-17 in gastric lymphoma was significantly higher than that in normal tissues (P < 0.05), which was closely related to lymph node metastasis, tumor size and distant metastasis (P < 0.05). The high expression of miR-17 significantly promoted the proliferation and invasion of cyp6d cells and inhibited apoptosis (P < 0.05). The high expression of miR-17 can regu¬late the expression of HSP60 and TNFR2. It has been found that miR-17 can promote the development of gastric lymphoma by regulating HSP60/TNFR2 pathway, which is a potential molecular target for the diagnosis and treatment of gastric lymphoma.

Differential capacity of CD90+ cells in autophagy activation following chemotherapy in hepatocellular carcinoma.

INTRODUCTION AND OBJECTIVES: Analysis of cancer biomarkers is an important tool in developing targeted-therapy and in modulating chemoresistance. Here, we analyze the relevance of CD90, a marker of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) and its correlation with autophagy. MATERIALS AND METHODS: For in vivo study, 86 specimens were collected from 43 patients undergoing liver resections. In each patient, HCC nodule (HCC) and surrounding non-tumor (SNT) were collected. For in vitro study, HCC cells JHH6 subpopulations expressing CD90+ and CD90- were isolated using magnetic-sorter and confirmed by flow-cytometry. Upon doxorubicin treatment, autophagy turn-over was analyzed by RTqPCR for mRNA expression, Western blot for protein expression, and autophagosome staining for autophagy-flux. Cytotoxicity test was performed by MTT assay. Gene and protein analysis were performed in clinical samples together with immunohistostaining. RESULTS: CD90 mRNA expression was higher in HCC than in SNT for 8-fold (p < 0.001). LC3-II protein was up-regulated in the HCC in comparison with the SNT (p < 0.05). In vitro model showed that CD90+ and CD90- cells had diverse expressions of autophagy-related genes. Upon doxorubicin treatment, autophagy was activated in both cells by increasing LC3-II protein expression, autophagic vacuoles, and dysregulation of autophagy-related mRNAs. A differential autophagic capacity was noticed between two subpopulations and it was correlated with cellular toxicity assay. CONCLUSIONS: We demonstrated the relevance of differential autophagy capacity of CD90+ cells in HCC. Autophagy was involved in cancer-defense mechanism against doxorubicin. Cancer promoting function of autophagy in CD90+ cells was also related to cancer environment.

On the mechanical behavior of austenitic stainless steel with nano/ultrafine grains and comparison with micrometer austenitic grains counterpart and their biological functions.

Conventional coarse-grained (CG) biomedical austenitic stainless steel with grain size in the micrometer range was subjected to a novel phase reversion concept involving severe cold deformation, followed by annealing, when the cold deformed martensite reverts to austenite with grain size in the nanometer/ultrafine (NG/UFG) regime (~200-400 nm). The mechanical behavior of CG and NG/UFG steels was studied via load-controlled and displacement-controlled experiments using a nanoindentation technique with the aim to simulate micromotion. The plastic zone associated with the indentation-induced deformed region was characterized by post-mortem electron microscopy of the deformed region to elucidate the deformation mechanism. Nanoscale twinning was the deformation mechanism in steel with grain size in the NG/UFG regime, and contributed to the ductility of high strength steel. In contrast, strain-induced martensite contributed to the ductility of low strength CG steel with micrometer grain size. Interestingly, besides the differences in the mechanical behavior, the biological functions of the two steels were remarkably different. Higher cell attachment, proliferation and higher expression level of prominent proteins, fibronection, actin and vinculin were favored by a surface with grain size in the nanometer regime and was in striking contrast with the surface with micrometer grain size. This behavior is attributed to the differences in the fraction of grain boundaries that are high energy two-dimensional defects. The study advances our understanding of the mechanical behavior of biomaterials and their cellular functions.

Heredity and cardiometabolic risk: naturally occurring polymorphisms in the human neuropeptide Y(2) receptor promoter disrupt multiple transcriptional response motifs.

OBJECTIVES: The neuropeptide Y(2) G-protein-coupled receptor (NPY2R) relays signals from PYY or neuropeptide Y toward satiety and control of body mass. Targeted ablation of the NPY2R locus in mice yields obesity, and studies of NPY2R promoter genetic variation in more than 10,000 human participants indicate its involvement in control of obesity and BMI. Here we searched for genetic variation across the human NPY2R locus and probed its functional effects, especially in the proximal promoter. METHODS AND RESULTS: Twin pair studies indicated substantial heritability for multiple cardiometabolic traits, including BMI, SBP, DBP, and PYY, an endogenous agonist at NPY2R. Systematic polymorphism discovery by resequencing across NPY2R uncovered 21 genetic variants, 10 of which were common [minor allele frequency (MAF) >5%], creating one to two linkage disequilibrium blocks in multiple biogeographic ancestries. In vivo, NPY2R haplotypes were associated with both BMI (P = 3.75E-04) and PYY (P = 4.01E-06). Computational approaches revealed that proximal promoter variants G-1606A, C-599T, and A-224G disrupt predicted IRF1 (A>G), FOXI1 (T>C), and SNAI1 (A>G) response elements. In neuroendocrine cells transfected with NPY2R promoter/luciferase reporter plasmids, all three variants and their resulting haplotypes influenced transcription (G-1606A, P < 2.97E-06; C-599T, P < 1.17E-06; A-224G, P < 2.04E-06), and transcription was differentially augmented or impaired by coexpression of either the cognate full-length transcription factors or their specific siRNAs at each site. Endogenous expression of transcripts for NPY2R, IRF1, and SNAI1 was documented in neuroendocrine cells, and the NPY2R mRNA was differentially expressed in two neuroendocrine tissues (adrenal gland, brainstem) of a rodent model of hypertension and the metabolic syndrome, the spontaneously hypertensive rat. CONCLUSION: We conclude that common genetic variation in the proximal NPY2R promoter influences transcription factor binding so as to alter gene expression in neuroendocrine cells, and consequently cardiometabolic traits in humans. These results unveil a novel control point, whereby cis-acting genetic variation contributes to control of complex cardiometabolic traits, and point to new transcriptional strategies for intervention into neuropeptide actions and their cardiometabolic consequences.

Additional effects of bisphenol A and paraben on the induction of calbindin-D(9K) and progesterone receptor via an estrogen receptor pathway in rat pituitary GH3 cells.

There are concerns about the combined estrogenic effects of chemicals since mixtures of these chemicals exist in our environment. This study investigated potential additional interactions between bisphenol A (BPA) and isobutylparaben (IBP), which are major xenoestrogens used in the manufacture of plastics, cosmetics, drugs, and other products. The combined effects of these two chemicals were analyzed by measuring the expression of calbindin-D(9k) (CaBP-9k) in rat pituitary cancer GH3 cells. GH3 cells were treated with single and combination doses of both chemicals (BPA single doses: 10(-7), 10(-6) and 10(-5) M; IBP single doses: 10(-7), 10(-6) and 10(-5) M, and each of the BPA and IBP doses combined). Prior to treatment, cells were temporarily transfected with a plasmid containing an ERE-luciferase reporter gene. Luciferase activity was measured as an indicator of ER activation by 17ß-estradiol (E2), BPA, and IBP. BPA (10(-5) M) combined with IBP (10(-7) M and 10(-6) M) induced a significant increase in the luciferase activity. Twenty-four hours after treatment, dose-dependent effects were observed in both single and combined dose groups, and several combination doses induced significant increases in the expression of CaBP-9k and progesterone receptor (PR) at both transcriptional and translational levels. Pre-treatment with ICI 182,780, a pure estrogen antagonist, significantly reversed BPA- and IBP-induced CaBP-9k and PR upregulation in GH3 cells. Taken together, these results indicate that BPA and IBP may have additionally increased estrogenic potency via an estrogen receptor-mediated pathway.

Nutritional and Hormonal Induction of Fatty Liver Syndrome and Effects of Dietary Lipotropic Factors in Egg-type Male Chicks.

This experiment was conducted with male chicks to investigate the influence of hormones and nutrients on the development of fatty liver syndrome (FLS) as well as the effects of dietary lipotropic factors on hepatic fat accumulation and lipogenic enzyme gene expression. A total of two-hundred sixteen 4-wk-old Hy-Line male chicks were divided into six groups and fed an experimental diet (T1, low-energy diet with low levels of lipotropic factors; T2, high-energy diet with low levels of lipotropic factors; T3 and T5, low-energy diet with high levels of lipotropic factors; T4 and T6, high-energy diet with high levels of lipotropic factors) for six weeks. The chicks in T5 and T6 groups were treated with intramuscular injections of estradiol benzoate for three days prior to biopsy and clinical analysis of FLS. Chicks treated with estrogen had significantly greater liver weights than untreated chicks. The abdominal fat contents were increased in chicks consuming high-energy diets as compared to those consuming low-energy diets. Treatment with estrogen significantly increased the concentrations of serum cholesterol, triacylglycerol and phospholipid (p<0.05). The hepatic triacylglycerol levels were tenfold higher in the estrogen treated chicks than in the untreated chicks. There were no significant differences in malondialdehyde levels between the treatment groups. Estrogen treatment dramatically increased the levels of fatty acid synthetase, acetyl-CoA carboxylase and ApoB mRNA. The results indicated that treatment with exogenous estrogen in growing male chicks induced hepatic fat accumulation, which might be partially due to increased lipogenic enzyme gene expression.

Synergistic effects of parabens on the induction of calbindin-D(9k) gene expression act via a progesterone receptor-mediated pathway in GH3 cells.

Although the endocrine-disrupting bioactivity of parabens is weakly estrogenic (parabens are xenoestrogens), their combined synergistic effect is unknown. The aim of this study was to investigate the effects of methyl paraben (MP), ethyl paraben (EP), propyl paraben (PP), isopropyl paraben (IPP), butyl paraben (BP), and isobutyl paraben (IBP), either alone or in combination (MP + EP + PP + BP; PP + IPP; and BP + IBP) on the induction of the estrogenic biomarker gene, calbindin-D(9k) (CaBP-9k), in rat pituitary lactosomatotrophic GH3 cells. The expression of CaBP-9k mRNA and protein was analyzed using real-time PCR and Western blot analysis, respectively. After 24 h of treatment, a significant increase in CaBP-9k expression was observed. This was dependent upon the length of the paraben alkyl chains (shortest in MP and longest in IBP). Interestingly, the synergistic effects of these paraben combinations were observed at a dose (10(-5) M) of these parabens, which induced the highest expression of CaBP-9k mRNA and protein. To investigate the involvement of estrogen receptors (ERs) and progesterone receptors (PRs), through which parabens exert their effects, the expression levels of ERα and PR-B were also examined. The expression of ERα mRNA and protein fluctuated after paraben treatment in GH3 cells, which was not significant. However, the expression level of ERα gene was induced when cotreated with 17ß-estradiol (E2) and ICI 182, 780 (estrogen receptor antagonist). The different combinations of parabens induced the expression of the PR-B gene, which was abolished by cotreatment with ICI 182,780. The expression patterns of CaBP-9k and PR-B genes appeared to be similar in response to paraben treatments. This implied that CaBP-9k expression in GH3 cells may be induced by parabens via a PR-mediated pathway. Taken together, these results suggest that exposure to multiple parabens at low concentrations may increase their synergistic estrogenic activities in GH3 cells through a PR-mediated pathway.

Regulation and molecular mechanisms of calcium transport genes: do they play a role in calcium transport in the uterine endometrium?

Maintenance of calcium (Ca) balance in the uterus is critically important for many physiological functions, including smooth muscle contraction during embryo implantation. Ca transport genes, i.e., transient receptor potential cation channel subfamily V members 5/6 (TRPV5/6), calbindins, plasma membrane Ca(2+)-ATPase 1 (PMCA1), and NCX1/NCKX3, may play roles in the uterus for Ca transport and reproductive function. Although these Ca transport genes may have a role in Ca metabolism, their role(s) and molecular mechanisms require further elucidation. In this review, we highlight the expression and regulation of Ca transport genes in the uterus to clarify their potential role(s). Since Ca transport genes are abundantly expressed in reproductive tissues in a distinct manner, they may be involved in specific uterine functions including fetal implantation, Ca homeostasis, and endometrial cell production.

Changes in hepatic lipid parameters and hepatic messenger ribonucleic acid expression following estradiol administration in laying hens (Gallus domesticus).

Fatty liver hemorrhagic syndrome (FLHS) is characterized by increased hepatic triacylglycerol content associated with liver hemorrhages and results in a sudden decline in egg production. Genetic, environmental, nutritional, and hormonal factors have all been implicated in the etiology of FLHS, but the exact cause of FLHS is still unknown. Estrogens have been implicated in the development of excess fat content of the liver and in the etiology of FLHS. This study investigated estradiol (E(2)) administration in hens and its effect on lipid metabolism. Hy-Line Brown laying hens were intramuscularly injected with E(2) on a daily basis for 3 wk. The dosages were 0, 0.5, and 1.0 mg/kg of BW, with corn oil injections used as a control. Egg production and quality were measured among the groups, with no significant difference seen in egg production. Liver weights of hens treated with E(2) were greater than those of control hens, but the increase was not statistically significant. Serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase activities and E(2) plasma concentrations increased in a dose-dependent manner, with plasma concentration of E(2) increasing from 6,900 to 19,000 pg/mL. No significant differences in free cholesterol or phospholipids were observed, but there was a significant increase in hepatic triacylglycerol levels. Injection with E(2) showed an increased expression of mRNA for peroxisome proliferator-activated receptor γ (23-fold), but not for peroxisome proliferator-activated receptor α. A statistically significant increase was seen for fatty acid synthase, apolipoprotein B, and adenosine triphosphate citrate lyase, but not for acetyl coenzyme A carboxylase, apolipoprotein VLDL-II, microsomal triglyceride transport protein, or malic enzyme. For proteins involved in the oxidation of E(2), only cytochrome P450 3A37 showed a statistically significant increase. The present results suggest that E(2) upregulates the synthesis of fatty acids and triacylglycerols and the accumulation of hepatic lipids by increasing mRNA expression related to lipid metabolism, and that excess E(2) in the blood leads to activation of E(2) catabolic metabolism (cytochrome P450 3A37)-related mRNA expression.

Cell surface expression and turnover of the alpha-subunit of the epithelial sodium channel.

The renal epithelial cell line A6, derived from Xenopus laevis, expresses epithelial Na(+) channels (ENaCs) and serves as a model system to study hormonal regulation and turnover of ENaCs. Our previous studies suggest that the alpha-subunit of Xenopus ENaC (alpha-xENaC) is detectable as 150- and 180-kDa polypeptides, putative immature and mature alpha-subunit heterodimers. The 150- and 180-kDa alpha-xENaC were present in distinct fractions after sedimentation of A6 cell lysate through a sucrose density gradient. Two anti-alpha-xENaC antibodies directed against distinct domains demonstrated that only 180-kDa alpha-xENaC was expressed at the apical cell surface. The half-life of cell surface-expressed alpha-xENaC was 24-30 h, suggesting that once ENaC matures and is expressed at the plasma membrane, its turnover is similar to that reported for mature cystic fibrosis transmembrane conductance regulator. No significant changes in apical surface expression of alpha-xENaC were observed after treatment of A6 cells with aldosterone for 24 h, despite a 5.3-fold increase in short-circuit current. This lack of change in surface expression is consistent with previous observations in A6 cells and suggests that aldosterone regulates ENaC gating and increases channel open probability.

Proteasome inhibition leads to significant reduction of Bcr-Abl expression and subsequent induction of apoptosis in K562 human chronic myelogenous leukemia cells.

The chimeric oncogene bcr-abl is detected in virtually every case of chronic myelogenous leukemia. It has been shown that cells (such as K562) expressing Bcr-Abl/p210, a protein tyrosine kinase, not only undergo cellular transformation but also demonstrate multiple drug resistance. Recent studies also demonstrate that the proteasome is involved in the survival signaling pathway(s). In the current study, we tested the hypothesis that the proteasome might play a role in regulating Bcr-Abl function. We have demonstrated by using a variety of inhibitors that inhibition of the proteasome, but not of the cysteine protease, activity is able to activate the apoptotic cell death program in K562 cells. Proteasome inhibition-induced apoptosis is demonstrated by condensation and fragmentation of nuclei, appearance of an apoptotic population with sub-G1 DNA content, the internucleosomal fragmentation of DNA, and cleavage of poly(ADP-ribose) polymerase, and can be blocked by a specific caspase-3-like tetrapeptide inhibitor. Western blot analysis with specific antibodies to c-Abl and Bcr proteins show that treatment of K562 cells with a proteasome inhibitor results in significant reduction of Bcr-Abl protein expression, which occurs several hours before the onset of apoptotic execution. Levels of c-Abl/p145 and Bcr/p160 proteins, however, remain essentially unaltered at that time. Furthermore, reduced Bcr-Abl expression is reflected in significantly attenuated Bcr-Abl-mediated protein tyrosine phosphorylation. Taken together, these results indicate that proteasome inhibition is sufficient to inactivate Bcr-Abl function and subsequently activate the apoptotic death program in cells that are resistant to apoptosis induced by chemotherapy.

Bcl-2- and CrmA-inhibitable dephosphorylation and cleavage of retinoblastoma protein during etoposide-induced apoptosis.

Cell numbers are regulated by a balance between proliferation and apoptosis (programmed cell death). Recent evidence suggests that proteins regulating cell proliferation also mediate apoptosis. Therefore, cellular fate might be determined by cross talk between regulators of cell cycle progression and apoptosis. Previously, we had found that during DNA damage-induced apoptosis, retinoblastoma protein (RB), an important G1/S regulator and tumor suppressor, became dephosphorylated and then immediately cleaved into p48 and p68 fragments. Here, we report that expression of the Bcl-2 oncoprotein, an inhibitor of caspases (interleukin 1 -converting enzyme-like proteases), blocked RB dephosphorylation, RB cleavage and apoptosis in etoposide-treated human Jurkat T cells. In addition, expression of the cowpox virus CrmA protein, a direct inhibitor of caspases, also inhibited both RB changes and apoptosis. Taken together, our findings demonstrate important roles for caspases in the processes of etoposide-induced RB dephosphorylation, RB proteolysis and apoptosis.

p53-independent dephosphorylation and cleavage of retinoblastoma protein during tamoxifen-induced apoptosis in human breast carcinoma cells.

We have investigated several molecular events that occur during the process of tamoxifen-induced apoptosis in human breast carcinoma cells. We show that the treatment of either MCF-7 (containing wild-type p53) or MDA-MB-231 cells (containing mutant p53) with tamoxifen resulted in apoptotic nuclear changes and an increase in the pre-G1 apoptotic population. This was accompanied by activation of the caspase enzymes, as evidenced by specific cleavage of poly(ADP-ribose) polymerase and retinoblastoma (RB) protein. The RB protein was cleaved at both an interior and carboxyl terminus cleavage site. In addition, dephosphorylation of RB was found at an early stage of tamoxifen-induced apoptosis in both cell lines. However, neither induction of p53 in MCF-7 cells nor induction of p21 in either cell line was detected, suggesting that tamoxifen-induced RB dephosphorylation and apoptosis are independent of the p53/p21 pathway. We also observed an increase in levels of the pro-apoptotic Bax protein, the inhibitory cytokine TGF-beta1 and the transcription factor c-Myc in tamoxifen-treated MDA-MB-231 cells, suggesting the possible involvement of these proteins during apoptosis in this system.

RB and apoptotic cell death.

Homeostasis of cell numbers is achieved by balancing the proliferative and death states of cells. Proper regulation in a cell requires an accurate coordination between these two processes. Indeed, dysregulation of cell cycle progression is essential for the initiation of apoptosis. Retinoblastoma protein (RB) is an important tumor suppressor and a cell cycle regulator. Most recent studies suggest that RB also plays a regulatory role in the process of apoptosis. During the onset of apoptosis, the hyperphosphorylated form of RB (p120/hyper) is converted to a hypophosphorylated form (p115/hypo), which is mediated by a specific protein-serine/ threonine phosphatase activity. Accompanied by the internucleosomal fragmentation of DNA, the newly formed p115/hypo/RB is immediately cleaved by a protease that has properties of the caspase family. During apoptosis, RB is also cleaved in its carboxyl terminus by a caspase-3-like activity. By contrast, the unphosphorylated form of RB (p110/unphos) remains uncleaved during apoptosis. Further studies suggest that p110/unphos/RB functions as an inhibitor of apoptosis. Therefore, regulation of the RB proteolytic activities and consequent RB levels is important for the determination of cellular fate.

Novel dipeptidyl proteasome inhibitors overcome Bcl-2 protective function and selectively accumulate the cyclin-dependent kinase inhibitor p27 and induce apoptosis in transformed, but not normal, human fibroblasts.

It has been suggested that overexpression of the Bcl-2 oncoprotein in human cancer cells contributes to their resistance to apoptosis induced by chemotherapy. We report here that a novel dipeptidyl proteasome inhibitor, CEP1612, at low concentrations rapidly induces apoptosis in human Jurkat T cells overexpressing Bcl-2 and also in all human prostate, breast, tongue and brain tumor cell lines we have tested to date, without exception. In contrast, etoposide, a standard anticancer drug, fails to kill these cells when employed under the same conditions. The apoptosis-inducing abilities of CEP1612 and its analogous compounds match precisely their order for inhibition of the proteasome chymotrypsin-like activity. CEP1612-induced apoptosis is p53-independent, inhibitable by a tetrapeptide caspase inhibitor, and associated with accumulation of the cyclin-dependent kinase inhibitors p21 and p27. Furthermore, CEP1612 selectively accumulates p27 and induces apoptosis in simian virus 40-transformed, but not the parental normal, human fibroblasts. Proteasome inhibitors such as those investigated herein might therefore have potential use as novel anticancer drugs.

Characterization of interior cleavage of retinoblastoma protein in apoptosis.

Previously we reported that at the onset of apoptotic execution, retinoblastoma protein (RB) was cleaved in its interior region, resulting in production of two major fragments, p48 and p68, and that the RB interior cleavage was mediated by a caspase-like activity. Here, we further characterized the RB interior cleavage process in human leukemia cells treated with the anticancer agent etoposide. We found that the RB interior cleavage activity was much more sensitive to two specific tetrapeptide caspase inhibitors, YVAD-CMK and DEVD-FMK, than the poly(ADP-ribose) polymerase cleavage activity, suggesting that two distinct caspases are involved in these processes. Several Asp residues are located in amino acids 341-421 of RB protein, and cleavage of any one of these sites by a caspase would generate a p48, which contains the amino terminus, and a p68 fragment, which contains the A/B pocket and the carboxyl terminus. This hypothesis was supported by the fact that the p48 and p68 fragments had selective binding affinity to different RB antibodies and that the p48 was found only in the low-salt-extracted cytoplasmic fraction, while the p68 was only in the nuclear fraction, of the apoptotic cells. However, the nuclear binding partner of the p68 RB fragment is not the transcription factor E2F-1 since a specific E2F-1 antibody coimmunoprecipitated only the unphosphorylated form of RB, but not the p68 fragment. Lastly, we confirmed that RB also underwent dephosphorylation and carboxyl terminal cleavage during apoptosis, as we and others reported previously.