Cell Cycle Regulatory Proteins P27 Research Articles

Proliferation and differentiation characteristics of neural stem cells during course of cerebral cortical histogenesis.

Recent advancements in the research field of stem cell biology have enabled the realization of regenerative medicine in various systems of the body, including the central nervous system. However, fundamental knowledge regarding how neural stem cells divide and generate young neurons in mammals, especially in vivo, is still inadequate. In this article, we shall summarize the concept of cell cycle/division of neural stem cells that generate projection neurons in the murine cerebral cortex. We shall also review the molecular mechanisms that modulate the critical parameters related to the cell cycle regulatory mechanisms, with special reference to the cell cycle regulatory protein p27(Kip1) , an inhibitor of progression of the cell cycle at the G1 phase. A better understanding of the mechanisms controlling cell cycle progression is expected to contribute to the development of novel strategies to increase the efficiency of neural cell/tissue production, both in vivo and in vitro.

P53 and HIC1 in Brain Cancer Gliomas

Hypermethylated in Cancer 1 (HIC1) is a novel tumor suppressor gene (TSG) which acts as a transcriptional enhancer and growth obliterator, identified by Baylin et al. more than 10 years back. HIC1 is found to be methylated in varieties of human cancers as seen in ductal carcinoma of the breast wherein promoter methylation and allelic loss of 17p13.3 chromosomal locus has been reported without any significant mutation in p53 gene. To determine the targets, Zhang et al. reported that Ephrin-A1 is one potential target for the TSG role of HIC1 in breast cancer cells. The region 17p13.3 consists of Variable Number of Tandem Repeats (VNTR) microsatellite markers such as YNZ22/D17S5/D17S30 and show intermittent DNA hypermethylation in neural cancer, colon cancer and lung cancer. Epigenetic silencing also is very commonly observed in liver cancer, breast cancer, gastric cancer, prostate cancer and is also seen in human male nonseminomatous germ cell tumors. The central coding domain of HIC1 exhibits elevated and eminent level of methylation in acute myeloid leukemia. Our laboratory had showed that 17p13.3 was preferentially affected in high grade primary human gliomas and the same was associated with increased p53 immunopositivity and proliferation within a particular tumor grade. Both HIC1 and p53 are catalogued as tumor repressors, HIC1 practically comply with p53 in cells cultures in laboratory as well as in genetically modified transgenic animals to repress/supress tumors and have binding sites on their promoters. Since P53 has binding site onto the promoter of HIC1 it transcriptionally up-regulates its expression. In corroboration, our result did showed an increased expression of HIC1 with wild type p53 containing U87MG cells whereas less or no expression with mutated cell lines (U373MG) or null p53 (SaOS-2) (Sanjay et al.). The ambiguity was previously reported that wild type P53 continuously being formed and degraded by ubiquitination and proteasomal mediated pathway in absence of stress signal. On contrary, mutant forms of P53 are usually stable and escape degradation from ubiquitination pathway. We observed almost no expression of p53 in U87MG and SaOS-2 cells whereas U373MG showed high level of mutated p53. Conventionally HIC1 is a tumour suppressor gene (TSG), hence the over expression of this gene leads to arrest of cell cycle. Since HIC1 plays the role of TSG, the activities like cell cycle regulation and p53 dependency is not known. We have done a research on the activity of HIC1 in cell cycle as well as during glioma cell line (U87MG) proliferation, that had the wild type p53 gene in both serum containing and serum deprived medium. Reduction in number of cells and cell proliferation was revealed during HIC1 knocking as compared to control siRNA of cells without any treatment with serum containing medium and serum free medium. Flow cytometry and FacsDiva software verified the arrest of the cell cycle at the G2-M phase without any significant increase in the number of cell death i.e., cell apoptosis in both the medium. An increase in the expression of p53 gene associated with the knockdown of the HIC1 was seen. Moreover, we observed an increase in p21, p27 along with p53 gene when compared to the controls. The expression level of another cell cycle regulatory protein p27 found to be very significantly high comparing to controls. This could be the cause of arresting the cell cycle even after knocking down a tumor suppressor gene like HIC1, which was quite unexpected and beyond the convention. The results revealed the significance of HIC1 in the normal progression of cell cycle and also its activity the at molecular level, affecting the homeostasis of p53 gene as well as a number of genes, which are directly or indirectly related to p53, taking part in the cell cycle process.

Pre-Clinical Investigation of Targeted Therapies for Double Hit Diffuse Large B-Cell Lymphoma (DH-DLBCL)

Background: Molecular studies divide DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal lymphoma (PML). Florescence in situ hybridization (FISH) studies identified another subgroup of DLBCL, classified as DH-DLBCL, with a poor clinical outcome harboring concurrent gene rearrangements of the c-MYC, BCL2 and/or BCL6 proto-oncogenes, resulting in the over-expression of c-Myc, Bcl2 and Bcl6 proteins. DH-DLBCL has inferior response rates (RR) to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS). DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, resulting in rapid cellular proliferation and resistance to apoptotic stimuli. In ABC-DLBCL, anti-apoptotic factor MCL-1 is implicated in poor prognosis leading to resistance to standard chemotherapy. C-MYC transcriptionally upregulates Mcl1. Translocation of c-MYC in DH-DLBCL may contribute to the aggressive phenotype and chemotherapy resistance via the MCL-1 pathway. We hypothesize that dual inhibition of both anti-apoptotic proteins BCL2 and MCL1 is an effective strategy in inducing lymphoma cell death in DH-DLBCL.Materials & Methods: At the pre-clinical level, we studied novel therapeutic strategies targeting key-regulatory pathways abnormally enhanced by the dual BCL2/BCL6 and c-MYC gene rearrangements or amplification using DH lymphoma cell lines (Val, DOHH-2, ROS-50). We evaluated the therapeutic value of three novel agents targeting BCL2 (ABT199), c-MYC (JQ-1), and cell cycle regulatory proteins (p21) and other BCL2 family members affecting ABT199 activity (carfilzomib (CFZ), an irreversible proteasome inhibitor) in a panel of DH-DLBCL cell lines. We retrospectively evaluated our single institution outcome experience over the last 15 years in DH-DLBCL patients. Using the lymphoma translational database that includes 611 DLBCL patients, we identified 30 patients (M=17/F=13) with aberrations in c-MYC and BCL2 or BCL6 by FISH. Demographic characteristics and clinical data were collected and analyzed.Results: In vitro, ABT199, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining ABT199 with CFZ and to a lesser degree with JQ-1. Subsequently, we studied the outcome of DHL patients treated at our institute. Mean age at diagnosis was 63 years, mostly Caucasians (N=28). Using the Han's algorithm, 21 of the DH-DLBCL were GCB, 8 were non-GCB, and 1 unclassifiable. 22 patients had rearrangement of c-MYC and BCL2/ or BCL-6 by FISH and 8 had gain/amplification/aneuploidy at these loci. At diagnosis, 96% (n=29) patients presented with stage III/IV, 67% (n=20) with High-intermediate/High IPI score, and 77% (n=23) had extra nodal disease. Front-line chemo-immunotherapy included 1) standard doxorubicin-containing regimens: R-CHOP (N=11) or R-EPOCH (N=8), or 2) intensified regimens: R-DHAC (N=3) and R+HyperCVAD/R+HDMTXCytarabine (N=8). Prophylaxis IT chemotherapy was administered to 21 (70%) patients. The complete remission (CR) rate was 67% (n=20) and 37% (n=11) patients underwent HDC-ASCS (N=9)/allo-BMT (N=2) in first (N=9) or second-remission (N=2). The use of intensified regimens was associated with a non-statistically significant improvement is PFS and OS. Similarly, an improvement in OS was observed among patients receiving HDC-ASCT/allo-BMT as consolidation. As previously noted, CNS prophylaxis was associated with an improvement in OS. Mcl-1 and other Bcl-2 family members expression levels are been determined by IHC. These results highlight the poor clinical outcome of this patient population and the need for alternative therapeutic options.Conclusion: Our data suggests that ABT199 exhibited strong synergistic activity with CFZ. Dual targeting of BCL2 and c-MYC pathways results in synergistic activity in DHL cell lines. Of interest, this pharmacological interaction could be related to the effects of proteasome inhibition on MCL1 and p21 levels in lymphoma cells, further enhancing the activity of ABT199. Using combination therapy to inhibit c-MYC and the proteasome and in turn decreasing MCL1 will render ABT-199 more effective and be a more potent combination in causing apoptosis and lymphoma cell death. Further pre-clinical work is ongoing. DisclosuresNo relevant conflicts of interest to declare.

Novel analogs targeting histone deacetylase suppress aggressive thyroid cancer cell growth and induce re-differentiation.

To develop novel therapies for aggressive thyroid cancers, we have synthesized a collection of histone deacetylase (HDAC) inhibitor analogs named AB1 to AB13, which have different linkers between a metal chelating group and a hydrophobic cap. The purpose of this study was to screen out the most effective compounds and evaluate the therapeutic efficacy. AB2, AB3 and AB10 demonstrated the lowest half-maximal inhibitory concentration (IC50) values in one metastatic follicular and two anaplastic thyroid cancer cell lines. Treatment with each of the three ABs resulted in an increase in apoptosis markers, including cleaved poly adenosine diphosphate ribose polymerase (PARP) and cleaved caspase 3. Additionally, the expression of cell-cycle regulatory proteins p21(WAF1) and p27(Kip1) increased with the treatment of ABs while cyclin D1 decreased. Furthermore, AB2, AB3 and AB10 were able to induce thyrocyte-specific genes in the three thyroid cancer cell lines indicated by increased expression levels of sodium iodide symporter, paired box gene 8, thyroid transcription factor 1 (TTF1), TTF2 and thyroid-stimulating hormone receptors. AB2, AB3 and AB10 suppress thyroid cancer cell growth via cell-cycle arrest and apoptosis. They also induce cell re-differentiation, which could make aggressive cancer cells more susceptible to radioactive iodine therapy.

Pyrrolidine dithiocarbamate inhibits enterovirus 71 replication by down-regulating ubiquitin–proteasome system

Enterovirus 71 (EV71) is the main causative pathogen of hand, foot, and mouth disease (HFMD). The severe neurological complications caused by EV71 infection and the lack of effective therapeutic medicine underline the importance of searching for antiviral substances. Pyrrolidine dithiocarbamate (PDTC), an antioxidant, has been reported to inhibit the replication of coxsackievirus B (CVB) through dysregulating ubiquitin–proteasome system (UPS). In this study, we demonstrated that PDTC exerted potent antiviral effect on EV71. Viral RNA synthesis, viral protein expression, and the production of viral progeny were significantly reduced by the treatment of PDTC in Vero cells infected with EV71. Similar to the previous report about the inhibitory effect of PDTC on UPS, we found that PDTC treatment led to decreased levels of polyubiquitinated proteins in EV71-infected cells. The inhibitory effect of PDTC on UPS was further confirmed by the increased accumulation of cell cycle regulatory proteins p21 and p53, which are normally degraded through UPS, while the expression levels of both proteins remained unchanged. We also showed that PDTC had no impact on the activity of proteasome. Thus, we demonstrated that the down-regulation of PDTC on UPS was the result of its inhibition on ubiquitination. More importantly, this study provides evidence that the inhibition on UPS was required for the antiviral activity of PDTC, since MG132, a potent proteasome inhibitor, significantly inhibited the cytopathic effect and viral protein synthesis in EV71-infected cells. We also found that the antioxidant property of PDTC did not contribute to its antiviral effect, since N-acetyl-l-cysteine, a potent antioxidant, could not inhibit viral replication. In addition, CPE and viral protein synthesis were not inhibited in the cells pretreated with PDTC 2h before viral infection and then cultured in the media with no PDTC supplement, while the antioxidant effect of PDTC was retained. PDTC also showed significant inhibition on apoptosis induced by EV71 infection when it was applied at the early stage of viral infection. Our results collectively suggest that PDTC could be a potential anti-EV71 compound which possesses both antiviral and anti-apoptotic capacity.

Identification of Herpesvirus Proteins That Contribute to G 1 /S Arrest

Lytic infection by herpesviruses induces cell cycle arrest at the G1/S transition. This appears to be a function of multiple herpesvirus proteins, but only a minority of herpesvirus proteins have been examined for cell cycle effects. To gain a more comprehensive understanding of the viral proteins that contribute to G1/S arrest, we screened a library of over 200 proteins from herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus (EBV) for effects on the G1/S interface, using HeLa fluorescent, ubiquitination-based cell cycle indicator (Fucci) cells in which G1/S can be detected colorimetrically. Proteins from each virus were identified that induce accumulation of G1/S cells, predominantly tegument, early, and capsid proteins. The identification of several capsid proteins in this screen suggests that incoming viral capsids may function to modulate cellular processes. The cell cycle effects of selected EBV proteins were further verified and examined for effects on p53 and p21 as regulators of the G1/S transition. Two EBV replication proteins (BORF2 and BMRF1) were found to induce p53 but not p21, while a previously uncharacterized tegument protein (BGLF2) was found to induce p21 protein levels in a p53-independent manner. Proteomic analyses of BGLF2-interacting proteins identified interactions with the NIMA-related protein kinase (NEK9) and GEM-interacting protein (GMIP). Silencing of either NEK9 or GMIP induced p21 without affecting p53 and abrogated the ability of BGLF2 to further induce p21. Collectively, these results suggest multiple viral proteins contribute to G1/S arrest, including BGLF2, which induces p21 levels likely by interfering with the functions of NEK9 and GMIP. Most people are infected with multiple herpesviruses, whose proteins alter the infected cells in several ways. During lytic infection, the viral proteins block cell proliferation just before the cellular DNA replicates. We used a novel screening method to identify proteins from three different herpesviruses that contribute to this block. Several of the proteins we identified had previously unknown functions or were structural components of the virion. Subsets of these proteins from Epstein-Barr virus were studied for their effects on the cell cycle regulatory proteins p53 and p21, thereby identifying two proteins that induce p53 and one that induces p21 (BGLF2). We identified interactions of BGLF2 with two human proteins, both of which regulate p21, suggesting that BGLF2 induces p21 by interfering with the functions of these two host proteins. Our study indicates that multiple herpesvirus proteins contribute to the cell proliferation block, including components of the incoming virions.

Acetazolamide triggers death inducing autophagy in T‐47D breast cancer cells

The inhibitory effects of acetazolamide on the growth and proliferation of epithelial breast cancer cells (T-47D) were investigated. Analysis of morphological changes indicated little apoptosis in T-47D cells incubated with acetazolamide, according to data from flow cytometry, DNA laddering, and expression of AIF. However, an increase in caspase-3 activity was detected in cells. This was concomitant with an increase in DFF45/DFF40 ratio leading to inhibition of caspase-3 activity, DNA fragmentation and progression of apoptosis. Flow cytometry also confirmed that acetazolamide had no significant effect on cell cycle progression. These results are consistent with lack of change in the expression of cell cycle regulatory proteins p21, p27, cdc2 and cyclinD1. Increased expression of ATG5, p53 and DRAM, along with an increase in BCLN1/Bcl-2 ratio, indicated that acetazolamide inhibited the proliferation of T-47D cells by inducing autophagy. Increased expression of PTEN, along with decreased expression of Akt1, also showed that acetazolamide treatment resulted in death inducing autophagy. Collectively the results indicate that autophagy is an adequate mechanism mediating the anti-cancer effects of acetazolamide in T-47D cells through engagement of p53/DRAM pathway and attenuation of Akt survival signalling.

Dual Involvement of Growth Arrest-Specific Gene 6 in the Early Phase of Human IgA Nephropathy

BackgroundGas6 is a growth factor that causes proliferation of mesangial cells in the development of glomerulonephritis. Gas6 can bind to three kinds of receptors; Axl, Dtk, and Mer. However, their expression and functions are not entirely clear in the different glomerular cell types. Meanwhile, representative cell cycle regulatory protein p27 has been reported to be expressed in podocytes in normal glomeruli with decreased expression in proliferating glomeruli, which inversely correlated with mesangial proliferation in human IgA nephropathy (IgAN).MethodsThe aim of this study is to clarify Gas6 involvement in the progression of IgAN. Expression of Gas6/Axl/Dtk was examined in 31 biopsy proven IgAN cases. We compared the expression levels with histological severity or clinical data. Moreover, we investigated the expression of Gas6 and its receptors in cultured podocytes.ResultsIn 28 of 31 cases, Gas6 was upregulated mainly in podocytes. In the other 3 cases, Gas6 expression was induced in endothelial and mesangial cells, which was similar to animal nephritis models. Among 28 podocyte type cases, the expression level of Gas6 correlated with the mesangial hypercellularity score of IgAN Oxford classification and urine protein excretion. It also inversely correlated with p27 expression in glomeruli. As for the receptors, Axl was mainly expressed in endothelial and mesangial cells, while Dtk was expressed in podocytes. In vitro, Dtk was expressed in cultured murine podocytes, and the expression of p27 was decreased by Gas6 stimulation.ConclusionsGas6 was uniquely upregulated in either endothelial/mesangial cells or podocytes in IgAN. The expression pattern can be used as a marker to classify IgAN. Gas6 has a possibility to be involved in not only mesangial proliferation via Axl, but also podocyte injury via Dtk in IgAN.

In vivo and in vitro demonstration of herb-drug interference in human breast cancer cells treated with tamoxifen and trastuzumab

In recent trends, patients with breast cancer seek integrative medical treatment when receiving either hormonal (tamoxifen [Tam]) or target (trastuzumab) therapy. Our previous in vitro studies demonstrated that the Chinese medicine Si-Wu-Tang (SWT) stimulates MCF-7 cell growth via activation of estrogen receptor α and human epidermal growth factor receptor 2 (HER2) signaling. The present study demonstrates herb-drug interference with cell proliferation in tumor-bearing mice treated with SWT and Tam in vivo and with proliferation capacity in breast cancer cells treated with SWT and trastuzumab in vitro. To assess in vivo SWT + Tam interference, we randomly separated female MCF-7-implanted athymic nude mice into five groups, namely, vehicle (n = 11), estradiol (n = 8), SWT (n = 8), Tam (n = 11), and SWT + Tam (n = 8). All mice were killed after 21 days of treatment. Body weight, uterine weight, tumor volume, and tumor weight were measured. To assess in vitro SWT-trastuzumab interference, we cotreated BT-474 and SK-BR-3 breast cancer cells with SWT and trastuzumab. This was followed by (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays and cell cycle analysis to measure cell proliferation and by Western blot analysis to analyze protein expression in growth-related signal pathways. SWT reversed Tam-induced antiproliferative effects on tumor weight and tumor volume and increased estrogen receptor α and N-cadherin expression in the SWT + Tam-treated group compared with the Tam-treated group. Furthermore, SWT reversed trastuzumab-induced antiproliferative activity in HER2 cell lines (SK-BR-3 and BT-474) through increased phosphorylation of the cell cycle regulatory protein p27(Kip1) and possibly of the antiapoptosis protein P38. Based on the in vivo and in vitro demonstration of herb-drug interference in breast cancer cells, we conclude that physicians should pay more attention to such interference when treating patients with receptor-positive (estrogen receptor-positive, progesterone receptor-positive, or HER2) breast cancers.

Bisdemethoxycurcumin suppresses MCF-7 cells proliferation by inducing ROS accumulation and modulating senescence-related pathways

BackgroundBisdemethoxycurcumin (BDMC) is a natural derivative of curcumin present in the phenolic components extracted from the dried rhizome of Curcuma longa L. BDMC demonstrated potential chemotherapeutic activities but the underlying mechanisms have not been fully clarified. In the present study, the role of reactive oxidative species (ROS) in the anti-cancer effects of BDMC was investigated. MethodsMCF-7 cells were exposed to BDMC, and then the cell proliferation, colony formation ability and cell cycle profile were analyzed. Cellular ROS level was determined by flow cytometry and fluorescent microscope observation using specific fluorescent probes. Mitochondrial membrane potential (ψm) was assessed using JC-1. In addition, effects of BDMC on senescence-related molecules were analyzed by western blot assay. ResultsBDMC significantly inhibited MCF-7 breast cancer cell proliferation, while a rapid rise of the intracellular ROS level accompanied with a reduction of Δψm were observed. In addition, BDMC activated the pro-apoptotic protein p53 and its downstream effector p21 as well as the cell cycle regulatory proteins p16 and its downstream effector retinoblastoma protein (Rb). All of these BDMC-induced effects were counteracted with the pre-incubation of the antioxidant N-acetylcysteine (NAC). ConclusionsThese results suggested that BDMC-induced ROS accumulation may contribute to its inhibitory effect on MCF-7 cell viability through regulation of p53/p21 and p16/Rb pathways.

Fancd2 and p21 function independently in maintaining the size of hematopoietic stem and progenitor cell pool in mice

Fanconi anemia patients suffer from progressive bone marrow failure. An overactive p53 response to DNA damage contributes to the progressive elimination of Fanconi anemia hematopoietic stem and progenitor cells (HSPC), and hence presents a potential target for therapeutic intervention. To investigate whether the cell cycle regulatory protein p21 is the primary mediator of the p53-dependent stem cell loss, p21/Fancd2 double-knockout mice were generated. Surprisingly double mutant mice displayed even more severe loss of HSPCs than Fancd2−/− single mutants. p21 deletion did not rescue the abnormal cell cycle profile and had no impact on the long-term repopulating potential of Fancd2−/− bone marrow cells. Collectively, our data indicate that p21 has an indispensable role in maintaining a normal HSPC pool and suggest that other p53-targeted factors, not p21, mediate the progressive elimination of HSPC in Fanconi anemia.

Abstract 3322: GW4716 selectively targets multiple p53 mutant cancer models.

Abstract The TP53 gene is the most commonly mutated gene in all cancers, with over 50% of human cancers harboring an inactivating mutation. Most frequently when p53 is mutated, the mutation occurs as a single nucleotide substitution within the DNA-binding domain. The selective advantage for retaining mutant p53 varies between cancer cells. Mutant p53 can have both gain-of-function and loss-of-function properties. Given the high frequency of p53 mutations in human cancers, understanding mutant p53 function and identifying novel therapies is a clinically relevant and important area of research. We have identified a novel pharmacological approach that selectively targets p53 mutant cancer cells across multiple cancer models using the acyl hydrazone GW4716. We have found cancer cells harboring a mutation in p53, treated with GW4716 for 24h, exhibit significantly greater cell death measured by DNA fragmentation (subG1) than p53 wild-type cells in both breast cancer and glioblastoma models. In contrast, GW4716 has little/no effect on normal (non-transformed) cells Furthermore, the mode of cell death induced by GW4716 differs between p53 wild-type and mutant cells. p53 mutants undergo a G2/M arrest, exhibit characteristics of mitotic catastrophe and ultimately apoptose in response to GW4716. MAPK signaling proteins (p38 and MK2) involved in G2/M checkpoint control, progression through mitosis, and cell death are induced by GW4716 treatment in p53 mutant cells. In contrast, p53 wild-type cells exhibit an induction of cell cycle regulatory proteins (p53 and p21) and the p53-specific autophagy marker DRAM, with a concomitant inhibition of p62 (SQSTM1). This leads us to hypothesize that the cytotoxic effects of GW4716 treatment occur in a p53-dependent manner, and the mode of cell death caused by GW4716 differs depending on p53 status. We hypothesize the loss of functional p53 leads to aberrant mitosis and ultimately apoptosis. We also hypothesize that p53 wild-type cells, which are less sensitive to GW4716, undergo autophagy. With the increasing emphasis on personalized medicine, these data suggest a clinically relevant role for GW4716 in the treatment of multiple p53 mutant cancers. Citation Format: Mary Heckler, Christopher Albanese, Rebecca Riggins. GW4716 selectively targets multiple p53 mutant cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3322. doi:10.1158/1538-7445.AM2013-3322 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Tetrandrine triggers apoptosis and cell cycle arrest in human renal cell carcinoma cells

Tetrandrine is a cytotoxic compound capable of exerting remarkable antitumor activity against many cancer cells in vitro and in vivo. However, little is known about its effect on human renal cell carcinoma (RCC). In the present study, using RCC 786-O, 769-P and ACHN cell lines as the model system, we demonstrated the anticancer effect of tetrandrine against RCC and clarified its underlying mechanisms. Tetrandrine treatment showed growth inhibitory effects on RCC cells in a time- and dose-dependent manner. Additionally, flow cytometric studies revealed that tetrandrine was capable of inducing G1 cell cycle arrest and apoptosis in RCC cells. Mechanically, activation of caspase-8, caspase-9, and caspase-3 and increasing expression of cell cycle regulatory protein p21(WAF1/CIP1) and p27(KIP1) were observed in tetrandrine-treated RCC cells. This study provides the first evidence that tetrandrine triggered apoptosis and cell cycle arrest in RCC 786-O, 769-P and ACHN cells in vitro; these events are associated with caspase cascade activation and upregulation of p21 and p27. Our results thus provide rational evidence supporting the application of tetrandrine as a novel therapeutic agent against RCC in the clinical setting.

P27 as a Prognostic Factor of Early Cervical Carcinoma

The aim of this study was to clarify whether the evaluation of cell-cycle regulatory protein p27 can serve as a prognostic factor in patients with International Federation of Gynecology and Obstetrics (FIGO) stage IB cervical carcinoma. A retrospective study was performed on 130 surgically treated patients with FIGO stage IB cervical carcinoma with at least a 5-year follow-up. The expression of p27 was investigated independently by 2 experienced pathologists using immunohistochemistry. The prognostic significance of established prognostic factors and p27 expression were analyzed using univariate and multivariate analyses. In a univariate analysis, lymph node status, tumor diameter, Gynecologic Oncology Group (GOG) score, lymph vascular space invasion, and p27 expression were significant prognostic factors for overall survival (OS). We found a correlation between p27 expression and lymph node status, tumor diameter, invasion, and GOG score. The p27 expression was a statistically significant prognostic factor for OS in a univariate analysis (log-rank test, P = 0.03). In a multivariate analysis, only lymph node status and tumor diameter were statistically significant prognostic factors for OS. This study demonstrated that a low p27 expression is associated with lymph node metastasis, deep stromal invasion, tumor diameter more than 20 mm, and high GOG score and had a prognostic influence on OS in a univariate analysis in a series of 130 women with FIGO stage IB cervical carcinoma. Lymph node status and the diameter of the tumor were the only statistically significant prognostic factors in multivariate analysis.

Heterozygosity for the proteasomal Psmc1 ATPase is insufficient to cause neuropathology in mouse brain, but causes cell cycle defects in mouse embryonic fibroblasts

The ubiquitin proteasome system (UPS) is a fundamental cellular pathway, degrading most unwanted intracellular soluble proteins. Dysfunction of the UPS has been associated with normal aging as well as various age-related pathological conditions, including chronic human neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, leading to a significant interest in the involvement of this degradative system in neurones. We previously reported that the 26S proteasome was essential for neuronal homeostasis and survival in mouse brains following conditional genetic homozygous knockout of a key subunit of the multi-meric 26S proteasome (19S ATPase Psmc1). Here, we investigated the effects of Psmc1 heterozygosity in the mouse brain and primary mouse embryonic fibroblasts. Neuropathologically and biochemically, Psmc1 heterozygous (Psmc1+/−) knockout mice were indistinguishable from wild-type mice. However, we report a novel age-related accumulation of intraneuronal lysine 48-specific polyubiquitin-positive granular staining in both wild-type and heterozygous Psmc1 knockout mouse brain. In Psmc1+/− MEFs, we found a significant decrease in PSMC1 levels, altered 26S proteasome assembly and a notable G2/M cell cycle arrest that was not associated with an increase in the cell cycle regulatory protein p21. The disturbance in cell cycle progression may be responsible for the growth inhibitory effects in Psmc1+/− MEFs.

Disorder-function relationships for the cell cycle regulatory proteins p21 and p27

The classic structure-function paradigm has been challenged by a recently identified class of proteins: intrinsically disordered proteins (IDPs). Despite their lack of stable secondary or tertiary structure, IDPs are prevalent in all forms of life and perform myriad cellular functions, including signaling and regulation. Importantly, disruption of IDP homeostasis is associated with numerous human diseases, including cancer and neurodegeneration. Despite wide recognition of IDPs, the molecular mechanisms underlying their functions are not fully understood. Here we review the structural features and disorder-function relationships for p21 and p27, two cyclin-dependent kinase (Cdk) regulators involved in controlling cell division and fate. Studies of p21 bound to Cdk2/cyclin A revealed that a helix stretching mechanism mediates binding promiscuity. Further, investigations of Tyr88-phosphorylated p27 identified a signaling conduit that controls cell division and is disrupted in certain cancers. These mechanisms rely upon a balance between nascent structure in the free state, induced folding upon binding, and persistent flexibility within functional complexes. Although these disorder-function relationships are likely to be recapitulated in other IDPs, it is also likely that the vocabulary of their mechanisms is much more extensive than is currently understood. Further study of the physical properties of IDPs and elucidation of their links with function are needed to fully understand the mechanistic language of IDPs.

Effect of aspirin administration on reversal of tumor-induced suppression of myelopoiesis in T-cell lymphoma bearing host

The present investigation studied the effect of aspirin administration in tumor-bearing hosts on bone marrow cellularity and myelopoiesis. Aspirin administration to mice bearing a transplantable T-cell lymphoma, designated as Dalton's lymphoma (DL), augmented proliferation of bone marrow cells (BMC). BMC of aspirin-administered tumor-bearing mice were found to be predominantly in the S phase of cell cycle releasing them from G0/G1 arrest. Aspirin-exposed BMC also showed an altered expression of survival and cell cycle regulatory proteins p53, bcl2, caspase-activated deoxyribonuclease (CAD), cyclin B1 and cyclin D. Moreover, the BMC of aspirin-administered tumor-bearing mice showed an augmented colony-forming ability and differentiation in the macrophage lineage with an activated phenotype of bone marrow-derived macrophages (BMDM) with respect to macrophage-mediated tumoricidal activity and production of nitric oxide, IL-1β, TNFα and VEGF. On the other hand aspirin administration to normal mice showed little effect on bone marrow cellularity and myeloid differentiation. In this model, aspirin had a myelopoetic action in tumor-bearing host.

Bortezomib reverses the proliferative and antiapoptotic effect of neuropeptides on prostate cancer cells

Neuropeptides are important signal initiators in advanced prostate cancer, partially acting through activation of nuclear factor kappa B. Central to nuclear factor kappa B regulation is the ubiquitin-proteasome system, pharmacological inhibition of which has been proposed as an anticancer strategy. We investigated the putative role of the proteasome inhibitor bortezomib in neuropeptides signaling effects on prostate cancer cells. Human prostate cancer cell lines, LNCaP and PC-3, were used to examine cell proliferation, levels of proapoptotic (caspase-3, Bad) and cell cycle regulatory proteins (p53, p27, p21), as well as total and phosphorylated Akt and p44/42 mitogen-activated protein kinase proteins. Furthermore, 20S proteasome activity, subcellular localization of nuclear factor kappa B and transcription of nuclear factor kappa B target genes, interleukin-8 and vascular endothelial growth factor, were assessed. Neuropeptides (endothelin-1, bombesin) increased cell proliferation, whereas bortezomib decreased proliferation and induced apoptosis, an effect maintained after cotreatment with neuropeptides. Bad, p53, p21 and p27 were downregulated by neuropeptides in PC-3, and these effects were reversed with the addition of bortezomib. Neuropeptides increased proteasomal activity and nuclear factor kappa B levels in PC-3, and these effects were prevented by bortezomib. Interleukin-8 and vascular endothelial growth factor transcripts were induced after neuropeptides treatment, but downregulated by bortezomib. These results coincided with the ability of bortezomib to reduce mitogen-activated protein kinase signaling in both cell lines. These findings are consistent with bortezomib-mediated abrogation of neuropeptides-induced proliferative and antiapoptotic signaling. Thus, the effect of the drug on the neuropeptides axis needs to be further investigated, as neuropeptide action in prostate cancer might entail involvement of the proteasome.

Oncogenic Viruses and Tumor Glucose Metabolism: Like Kids in a Candy Store

Oncogenic viruses represent a significant public health burden in light of the multitude of malignancies that result from chronic or spontaneous viral infection and transformation. Although many of the molecular signaling pathways that underlie virus-mediated cellular transformation are known, the impact of these viruses on metabolic signaling and phenotype within proliferating tumor cells is less well understood. Whether the interaction of oncogenic viruses with metabolic signaling pathways involves enhanced glucose uptake and glycolysis (both hallmark features of transformed cells) or dysregulation of molecular pathways that regulate oxidative stress, viruses are adept at facilitating tumor expansion. Through their effects on cell proliferation pathways, such as the PI3K and MAPK pathways, the cell cycle regulatory proteins p53 and ATM, and the cell stress response proteins HIF-1α and AMPK, viruses exert control over critical metabolic signaling cascades. Additionally, oncogenic viruses modulate the tumor metabolomic profile through direct and indirect interactions with glucose transporters, such as GLUT1, and specific glycolytic enzymes, including pyruvate kinase, glucose 6-phosphate dehydrogenase, and hexokinase. Through these pathways, oncogenic viruses alter the phenotypic characteristics and energy-use methods of transformed cells; therefore, it may be possible to develop novel antiglycolytic therapies to target these dysregulated pathways in virus-derived malignancies.

Recovery of the Cell Cycle Inhibition in CCl(4)-Induced Cirrhosis by the Adenosine Derivative IFC-305.

Introduction. Cirrhosis is a chronic degenerative illness characterized by changes in normal liver architecture, failure of hepatic function, and impairment of proliferative activity. The aim of this study is to know how IFC-305 compound induces proliferation of the liver during reversion of cirrhosis. Methods. Once cirrhosis has been installed by CCl4 treatment for 10 weeks in male Wistar rats, they were divided into four groups: two received saline and two received the compound; all were euthanized at 5 and 10 weeks of treatment. Liver homogenate, mitochondria, and nucleus were used to measure cyclins, CDKs, and cell cycle regulatory proteins PCNA, pRb, p53, E2F, p21, p27, HGF, liver ATP, and mitochondrial function. Results. Diminution and small changes were observed in the studied proteins in the cirrhotic animals without treatment. The IFC-305-treated rats showed a clear increase in most of the proteins studied mainly in PCNA and CDK6, and a marked increased in ATP and mitochondrial function. Discussion/Conclusion. IFC-305 induces a recovery of the cell cycle inhibition promoting recovery of DNA damage through the action of PCNA and p53. The increase in energy and preservation of mitochondrial function contribute to recovering the proliferative function.