Zoya N. Demidenko

Rapamycin increases oxidative stress response gene expression in adult stem cells Abstract Balancing quiescence with proliferation is of paramount importance for adult stem cells in order to avoid hyperproliferation and cell depletion. In some models, stem cell exhaustion may be reversed with the drug rapamycin, which was shown can suppress cellular senescence  in vitro  and extend lifespan in animals. We hypothesized that rapamycin increases the expression of oxidative stress response genes in adult stem cells, and that these gene activities diminish with age. To test our hypothesis, we exposed mice to rapamycin and then examined the transcriptome of their spermatogonial stem cells (SSCs). Gene expression microarray analysis revealed that numerous oxidative stress response genes were upregulated upon rapamycin treatment, including superoxide dismutase 1, glutathione reductase, and delta-aminolevulinate dehydratase. When we examined the expression of these genes in 55-week-old wild typ

Stress Response of Glioblastoma Cells Mediated by miR-17-5p Targeting PTEN and the Passenger Strand miR-17-3p Targeting MDM2 ABSTRACT Tumor development not only destroys the homeostasis of local tissues but also the whole body, and thus the tumor cells have to face the body’s defense system, a shortage of nutrition and oxygen, and chemotherapeutic drug treatment. In response to these stresses, tumor cells often alter gene expression and microRNA levels to facilitate survival. We have demonstrated that glioblastoma cells deprived of nutrition or treated with chemotherapeutics drugs expressed increased levels of miR-17. Ectopic transfection of miR-17 prolonged glioblastoma cell survival when the cells were deprived with nutrition or treated with chemotherapeutic drugs. Expression of miR-17 also promoted cell motility, invasion, and tube-like structure formation. We found that these phenotypes were the results of miR-17 targeting PTEN. As a consequence, HIF1α and VEGF were up-regulated. E

Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer ABSTRACT We previously reported MELK (maternal embryonic leucine zipper kinase) as a novel therapeutic target for breast cancer. MELK was also reported to be highly upregulated in multiple types of human cancer. It was implied to play indispensable roles in cancer cell survival and indicated its involvement in the maintenance of tumor-initiating cells. We conducted a high-throughput screening of a compound library followed by structure-activity relationship studies, and successfully obtained a highly potent MELK inhibitor OTSSP167 with IC 50  of 0.41 nM. OTSSP167 inhibited the phosphorylation of PSMA1 (proteasome subunit alpha type 1) and DBNL (drebrin-like), which we identified as novel MELK substrates and are important for stem-cell characteristics and invasiveness. The compound suppressed mammosphere formation of breast cancer cells and exhibited significant t

A combination of temsirolimus, an allosteric mTOR inhibitor, with clofarabine as a new therapeutic option for patients with acute myeloid leukemia ABSTRACT Signaling through the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream effectors, Akt and mechanistic target of rapamycin (mTOR), is aberrantly activated in acute myeloid leukemia (AML) patients, where it contributes to leukemic cell proliferation, survival, and drug-resistance. Thus, inhibiting mTOR signaling in AML blasts could enhance their sensitivity to cytotoxic agents. Preclinical data also suggest that allosteric mTOR inhibition with rapamycin impaired leukemia initiating cells (LICs) function. In this study, we assessed the therapeutic potential of a combination consisting of temsirolimus [an allosteric mTOR complex  1  (mTORC 1 ) inhibitor] with clofarabine, a nucleoside analogue with potent inhibitory effects on both ribonucleotide reductase and DNA polymerase. The drug combination (CLO-TOR) displayed syner

Small-molecule inhibition of BRD4 as a new potent approach to eliminate leukemic stem- and progenitor cells in acute myeloid leukemia (AML) ABSTRACT Acute myeloid leukemia (AML) is a life-threatening stem cell disease characterized by uncontrolled proliferation and accumulation of myeloblasts. Using an advanced RNAi screen-approach in an AML mouse model we have recently identified the epigenetic ‘reader’ BRD4 as a promising target in AML. In the current study, we asked whether inhibition of BRD4 by a small-molecule inhibitor, JQ1, leads to growth-inhibition and apoptosis in primary human AML stem- and progenitor cells. Primary cell samples were obtained from 37 patients with freshly diagnosed AML (n=23) or refractory AML (n=14). BRD4 was found to be expressed at the mRNA and protein level in unfractionated AML cells as well as in highly enriched CD34 + /CD38 -  and CD34 + /CD38 +  stem- and progenitor cells in all patients examined. In unfractionated leukemic cells, submicromolar conce

Down-regulation of Sox7 is associated with aberrant activation of Wnt/β-catenin signaling in endometrial cancer ABSTRACT Although the mortality rate of endometrial cancer is comparatively low in gynecologic malignancies, a rising trend of this cancer has been observed for the past decade. The understanding of the molecular mechanism will favor for the clinical management of this disease. Aberrant activation of Wnt/β-catenin signaling pathway plays a major role in the pathogenesis of endometrioid adenocarcinoma including this cancer type. In this study, we reported that Sox7, one of Sox transcriptional factors, was frequently underexpressed in endometrial cancer and importantly, it was associated with dysregulation of the Wnt/β-catenin signaling activity. Immunohistochemical and quantitative RT-PCR analyses showed that Sox7 was underexpressed and was associated with high-grade tumor ( P =0.021), increased expressions of β-catenin ( P =0.038) and its downstream targets; CyclinD1 ( P <

Rapamycin: Killing two birds with one stone Inhibition of TOR signaling leads to extended longevity in both invertebrate and invertebrate species by modulating a number of downstream molecular pathways [1,2]. Drugs that inhibit the TOR pathway can serve as powerful tools to translate the effects of the TOR pathway on lifespan in simpler invertebrate model systems to more complex systems like mammals. One of the most promising drugs to slow aging is rapamycin, an inhibitor of TOR, which was previously shown to extend lifespan in mice when administered late in life [3]. As aging is one of the biggest risk factors for cancer, one of the outstanding questions is whether drugs that slow aging will also slow age-related increases in cancer incidence. A recent study describes how treatment of mice initiated early in life can not only retard aging but also slow age-related increase in cancer. Work by Anisimov and colleagues examines the impact on health and lifespan of Rapamycin treated inbred

Accumulation of hypoxia-inducible factor-1 is limited by transcription-dependent depletion Abstract In the presence of oxygen and iron, hypoxia-inducible factor (HIF-1alpha) is rapidly degraded via the prolyl hydroxylases (PHD)/VHL pathways. Given striking similarities between p53 and HIF-1alpha regulation, we previously suggested that HIF-1 transcriptionally initiates its own degradation and therefore inhibitors of transcription must induce HIF-1alpha. Under normoxia, while inducing p53, inhibitors of transcription did not induce HIF-1alpha. Under hypoxia or low iron (DFX), inhibitors of transcription dramatically super-induced HIF-1alpha. Removal of inhibitors resulted in outburst of the HIF-1-dependent transcription followed by depletion of HIF-1alpha. Although hypoxia/DFX induced PHD3, we excluded the PHD/VHL pathway in the regulation of HIF-1alpha under hypoxia/DFX. The transcription-dependent degradation of HIF-1alpha under hypoxia occurs via the proteasome and is accelerated by

Cytostatic Activity of Paclitaxel in Coronary Artery Smooth Muscle Cells is Mediated Through Transient Mitotic Arrest Followed by Permanent Post-Mitotic Arrest: Comparison with Cancer Cells Abstract The anti-cancer agent paclitaxel (PTX) is an effective anti-restenosis agent on drug eluting stents, primarily due to growth inhibition of coronary artery smooth muscle cells (CASMC) across a wide dose range. In this study, we compared the effects of PTX on CASMC to apoptotic-prone HL60 leukemia cells and apoptotic-reluctant A549 lung cancer cells to assess cell survival mechanisms. In comparison to HL60 and A549 cells, CASMC had a shorter mitotic arrest and a lower mitotic index. While CASMC and A549 cells did not become apoptotic and displayed a multi-nucleated phenotype, HL60 cells showed prolonged mitotic arrest followed by apoptosis. CASMC exiting mitosis were arrested in G1 as MN tetraploid cells, with decreased levels of cyclin B1 and PCNA. CASMC remained metabolically active, becomi

Contact inhibition and high cell density deactivate the mammalian target of rapamycin pathway, thus suppressing the senescence program Significance This work solves longstanding mysteries in the field of contact inhibition (CI), cancer, and aging. As shown here during CI, cells do not undergo senescence, thus resuming proliferation after replating. We found that CI was associated with inhibition of the mammalian target of rapamycin (mTOR) pathway, which is required for the senescence program. In cancer cells, lacking CI, mTOR was still inhibited in high cell density by an alternative mechanism. Our work explains why CI is reversible and how cells can avoid senescence in vivo, allowing the organism to last for decades. Implications for cancer therapy are discussed. Abstract During cell cycle arrest caused by contact inhibition (CI), cells do not undergo senescence, thus resuming proliferation after replating. The mechanism of senescence avoidance during CI is unknown. Recently, it was d

Growth Stimulation Leads to Cellular Senescence When the Cell Cycle Is Blocked Zoya N Demidenko   ,  Mikhail V Blagosklonny Abstract We tested a hypothesis that activation of growth-promoting pathways is required for cellular senescence. In the presence of serum, induction of p21 caused senescence, characterized by beta-Galactosidase staining, cell hypertrophy, increased levels of cyclin D1 and active TOR (target of rapamycin, also known as mTOR). Serum starvation and rapamycin inhibited TOR and prevented the expression of some senescent markers, despite high levels of p21 and cell cycle arrest. In the presence of serum, p21-arrested cells irreversibly lost proliferative potential. In contrast, when cells were arrested by p21 in the absence of serum, they retained the capacity to resume proliferation upon termination of p21 induction. In normal human cells such as WI38 fibroblasts and retinal pigment epithelial (RPE) cells, serum starvation caused quiescence, which was associated with