PTEN Expression Is Decreased in Pediatric Burkitt Lymphoma Tissues but Does Not Correlate with AKT Activation.

Abstract Abstract 2664 Background: We have recently shown that a subset of pediatric Burkitt lymphoma (pBL) show gain of 13q31, containing the microRNA (miRNA) MIR-17–92 locus, as well as associated increased expression of miRNA from that locus. PTEN, a tumor suppressor phosphatase that negatively regulates the PI3K/AKT/mTOR pro-survival pathway, is negatively regulated by MIR-17–92. Therefore, we hypothesized that PTEN protein and mRNA expression would be decreased in pBL cases with 13q31 locus gain and high expression of MIR-17–92. Because PTEN negatively regulates the PI3K/AKT/mTOR pathway, we also assessed AKT activation in pBL tissues. Methods: We assessed PTEN protein expression by immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded (FFPE) pBL tissues from a cohort of patients (n=25) where MIR-17–92 expression levels and status of the 13q31 locus have been previously determined (Schiffman/Miles, Brit J Haematol 155:477, 2011). From FFPE tissues, we assayed PTEN mRNA levels by quantitative RT-PCR. The results of protein and mRNA expression were then correlated with presence or absence of a 13q31 gain and the expression level of MIR17. To evaluate AKT activation in pBL, we assayed for phospho-(p)-AKT by IHC. In addition, PTEN, AKT, and p-AKT protein levels were evaluated by western blotting in Raji and Ramos BL cell lines. Results: PTEN protein was weakly expressed in 5/25 cases (20%) of pBL. PTEN protein showed moderate nuclear and cytoplasmic staining in a subset of paracortical and germinal center lymphocytes in reactive lymph node control tissues, and vascular endothelial cells provided internal positive control in tumor tissue. pBL tissues showed decreased relative PTEN mRNA expression compared to reactive lymph node controls (1.56 vs. 2.61, p<3E-07). There was no significant difference in PTEN mRNA levels between PTEN protein positive and negative cases (1.27 vs. 1.61, p=0.22); cases with or without 13q31gain (1.49 vs. 1.57, p=0.60); or cases with high or low expression of MIR17. p-AKT was detected by IHC in 1/25 cases (4%). The BL cell lines Raji and Ramos showed very low levels of PTEN protein but no detectable p-AKT; total AKT was readily detectable. Discussion: pBL shows decreased PTEN mRNA, and most cases are negative for PTEN protein expression. Unexpectedly, higher MIR-17–92 expression level or 13q31 gain did not show an inverse relationship with PTEN expression at the protein or mRNA level. Despite decreased PTEN expression, p-AKT was detected in only 1/25 pBL cases and was not detected in BL cell lines. These findings suggest that decreased PTEN expression does not lead to AKT activation in pBL. Ongoing studies will investigate the potential of alternate PTEN targets and/or alternate targets for MIR-17–92 in pBL. Disclosures: No relevant conflicts of interest to declare.

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Characterization of immunoglobulin mrna expression in burkitt lymphoma cell lines

International Journal of Cancer ◽

10.1002/ijc.2910430534 ◽

1989 ◽

Vol 43 (5) ◽

pp. 930-935 ◽

Cited By ~ 6

Author(s):

Roberto Anker ◽

James Caldwell ◽

Jane Brokaw ◽

Brian A. Pollok

Keyword(s):

Mrna Expression ◽

Cell Lines ◽

Burkitt Lymphoma ◽

Lymphoma Cell ◽

Burkitt Lymphoma Cell

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Synaptotagmin 13 Is Highly Expressed in Estrogen Receptor-Positive Breast Cancer

Current Oncology ◽

10.3390/curroncol28050346 ◽

2021 ◽

Vol 28 (5) ◽

pp. 4080-4092

Author(s):

Takahiro Ichikawa ◽

Masahiro Shibata ◽

Takahiro Inaishi ◽

Ikumi Soeda ◽

Mitsuro Kanda ◽

...

Keyword(s):

Breast Cancer ◽

Estrogen Receptor ◽

Mrna Expression ◽

Cell Lines ◽

Mrna Levels ◽

Pcr Array ◽

Array Analysis ◽

Expression Levels ◽

Er Positive ◽

Mrna Expression Levels

Background: Accumulating evidence indicates tumor-promoting roles of synaptotagmin 13 (SYT13) in several cancers; however, no studies have investigated its expression in breast cancer (BC). This study aimed to clarify the significance of SYT13 in BC. Methods: SYT13 mRNA expression levels were evaluated in BC cell lines. Polymerase chain reaction (PCR) array analysis was conducted to determine the correlation between expression levels of SYT13 and other tumor-associated genes. Then, the association of SYT13 expression levels in the clinical BC specimens with patients’ clinicopathological factors was evaluated. These findings were subsequently validated using The Cancer Genome Atlas (TCGA) database. Results: Among 13 BC cell lines, estrogen receptor (ER)-positive cells showed higher SYT13 mRNA levels than ER-negative cells. PCR array analysis revealed positive correlations between SYT13 and several oncogenes predominantly expressed in ER-positive BC, such as estrogen receptor 1, AKT serine/threonine kinase 1, and cyclin-dependent kinases 4. In 165 patients, ER-positive specimens exhibited higher SYT13 mRNA expression levels than ER-negative specimens. The TCGA database analysis confirmed that patients with ER-positive BC expressed higher SYT13 levels than ER-negative patients. Conclusion: This study suggests that SYT13 is highly expressed in ER-positive BC cells and clinical specimens, and there is a positive association of SYT13 with the ER signaling pathways.

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Implication of HIF-1α but Not HIF-2α in the Hypoxic Response of Human Hematopoietic Progenitors.

Blood ◽

10.1182/blood.v104.11.4154.4154 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4154-4154

Author(s):

Yanyan Zhang ◽

Adlen Foudi ◽

Magali Berthebaud ◽

Dorothee Buet ◽

Peggy Jarrier ◽

...

Keyword(s):

Mrna Expression ◽

Cell Lines ◽

Hematopoietic Cells ◽

Receptor Expression ◽

Metabolic Adaptation ◽

Protein Accumulation ◽

Mrna Levels ◽

Hematopoietic Progenitors ◽

Cd34 Cells ◽

Cxcr4 Mrna

Abstract Maturing hematopoietic cells are exposed to hypoxia as they develop and migrate within the bone marrow microenvironment. Previous studies using non hematopoietic cell lines and monocytes showed that CXCR4 is strongly induced by hypoxia but little is known on the regulation of CXCR4 by hypoxia in the other hematopoietic cells and during hematopoietic development. We analyzed the expression and regulation of hypoxia-inducible transcription factor-1a (HIF1a) and 2a (HIF2a), the master regulators of metabolic adaptation to hypoxia, during hematopoiesis. Real time quantitative RT-PCR showed that HIF-1a mRNA was present on all the non hematopoietic and hematopoietic cells lines including HL-60, HEL, TF1, K562, KG1, U937, Jurkat and Mo7e. In contrast, HIF-2a mRNA expression was variable among the cell lines and was detected only at very low level in some cells such as KG1, Jurkat and HEL. Hypoxia exposure rapidly induced VEGF mRNA expression in the cells that expressed HIF-1a mRNA and exhibited HIF-1a protein accumulation. Interestingly, CXCR4 induction was observed only in the cells that exhibit significant expression of HIF-2a mRNA and HIF-2a protein accumulation. A strong correlation between HIF-2a mRNA levels and the induction of CXCR4 mRNA expression by hypoxia was found. Human CD34+ cells also expressed high levels of HIF-1a mRNA, whereas HIF-2a mRNA was barely detected. Interestingly, as observed for several myeloid cell lines, CD34+ cells exhibited a strong induction of VEGF expression in response to hypoxia and hypoxia mimetic agents cobalt chloride and desferrrioxamine whereas CXCR4 receptor expression was not induced suggesting that CXCR4 mRNA induction is related to the expression of HIF-2a. Altogether these data indicated that the hypoxic responses of human hematopoietic progenitors are independent of HIF-2a. Moreover, they establish that CXCR4 regulation by hypoxia is linked to HIF-2a protein expression.

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CHARACTERIZATION OF COLLAGEN (IV) MRNA IN CELL LINES OF BREAST CANCER

Jurnal Teknologi ◽

10.11113/jt.v77.6752 ◽

2015 ◽

Vol 77 (25) ◽

Author(s):

Afzan Mat Yusof ◽

Mardhiah Mohammad ◽

Sharifah Norbaizura Syed Bahrom ◽

Syahirah Kaja Mohideen ◽

Ridhwan Roshdi ◽

...

Keyword(s):

Breast Cancer ◽

Mrna Expression ◽

Cancer Cells ◽

Cell Lines ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Cancer Cell Lines ◽

Collagen Iv ◽

Breast Cancer Cell Lines ◽

Mrna Levels

Breast cancer incidence rate has increased in the 5 recent years with 14% increases in mortality. The structural change in the collagen chain has led to alterations in the cancer cells. Various biological processes, such as differentiation or gene expression, are regulated through extracelullar matrix (ECM)[1]. The restructuring of the collagenous architecture in the hypoxic microenvironment may influence the invasive growth of the cancer cells. With the increased stress within the cell, the invasion of cancer cells into the ECM was triggered. This cell lines model would enable the exploration of the relationship between the extracellular matrices component and the tumor proliferation. The aim of this study is to characterize the collagen (IV) mRNA expression in the breast cancer cell. Breast cancer (MCF7) cell lines were cultured and harvested upon confluent. The RNA was extracted from the cell lines and then the cDNA were synthesized. The collagen (IV) mRNA levels in breast cancer cell lines were measured using real time PCR and GAPDH was used as an internal control. The level of COL4A2 (IV) mRNA expression was higher compared with COL4A1 (IV) mRNA. The level of COL4A5 (IV) mRNA was reduced significantly in breast cancer cells lines. Overall, the expression of COL4A1-A6 (IV) was reduced. The reduced amount of collagen (IV) in breast cancer cell lines suggested that the collagen was restructured and this has triggered the tumor invasion into the ECM.

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Contribution of constitutive activation of Akt to the pathogenesis of mantle cell lymphoma (MCL) independent of PTEN protein expression

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.8101 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 8101-8101

Author(s):

C. Economopoulou ◽

A. Psyrri ◽

E. Liakata ◽

S. Papageorgiou ◽

P. Economopoulou ◽

...

Keyword(s):

Protein Expression ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Pten Protein ◽

Akt Activation ◽

Constitutive Activation ◽

Protein Levels ◽

Phosphorylated Akt ◽

Mantle Cell

8101 Background: To determine whether the PI3K/Akt signaling pathway is involved in the pathogenesis of mantle cell lymphoma (MCL), we investigated the phosphorylation status of Akt in primary MCL cases and cell lines. We also sought to determine whether loss of the phosphatase PTEN is the mechanism of Akt activation in MCL. Methods: We evaluated the protein levels of Akt, phosphorylated Akt and PTEN in 15 primary MCL cases and 2 cell lines by immunoblotting Results: Akt was phosphorylated in 3 of 15 MCL cases and in 2 of 2 mantle cell lymphoma cell lines. PTEN protein was expressed in 15 of 15 primary mantle cell lymphoma cases and in 2 of 2 MCL cell lines. Conclusions: We conclude that constitutive activation of the PI3K/Akt pathway contributes to the pathogenesis of MCL. Loss of PTEN protein expression is not the responsible mechanism for Akt activation. Alternative mechanisms of Akt activation are being evaluated to identify markers predictive for response to PI3K/Akt inhibitors in MCL. No significant financial relationships to disclose.

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Association of Asparagine Synthetase Expression and Sensitivity to L-Asparaginase in Cell Lines and Primary Pediatric Acute Lymphoblastic Leukemia Samples.

Blood ◽

10.1182/blood.v114.22.2738.2738 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2738-2738

Author(s):

Ivana Hermanova ◽

Jan Trka ◽

Julia Starkova

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Amino Acid ◽

Mrna Expression ◽

Cell Line ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Asparagine Synthetase ◽

Leukemic Cells ◽

Mrna Levels ◽

Basal Expression

Abstract Abstract 2738 Poster Board II-714 L-Asparaginase (L-Asp) is an important component in the combined chemotherapy for childhood acute lymphoblastic leukemia (ALL). Administration of L-Asp leads to depletion of plasmatic asparagine and consequently causes loss of intracellular asparagine. As a non-essential amino acid, asparagine is synthesized from aspartate and glutamine by asparagine synthetase (ASNS). Primary ALL cells are believed to have low ASNS expression and therefore to be sensitive to asparagine depletion. Although increased ASNS level was shown to be connected with L-Asp resistance the exact relationship between ASNS expression and L-Asp sensitivity is not clear. We and others have previously shown TEL/AML1[+] ALL blasts express more ASNS mRNA than TEL/AML[-] do although primary TEL/AML[+] cell are in vitro more sensitive to treatment with L-Asp. Hutson et al (1997) showed that amino acid deprivation led to increased expression of ASNS on mRNA and protein level as well as to increased biological activity. On the other hand, Nan Su et al described negative correlation between L-Asp sensitivity and ASNS protein rather than mRNA levels. Therefore, in our studies we concentrated on protein expression of ASNS in patients' samples. So far, there has been no reproducible published data on ASNS protein detection by Western blot in primary patients' samples. Despite using 3 different antibodies and precise optimization we were not able to detect ASNS protein in patients' samples in contrast to cell lines. Transcripts' levels confirmed significantly lower (2 log) expression of ASNS in patients' leukemic cells compared to leukemic cell lines. Therefore, for further studies on gene and protein relation we had to rely on cell lines as a model. We detected ASNS gene expression and ASNS protein content in four ALL cell lines: REH (TEL/AML1[+]), UOCB6 (TEL/AML1[+]), NALM6 (TEL/PDGFRB[+]) and RS4;11 (MLL/AF4[+]). ASNS mRNA levels were in accord with sensitivity to L-Asp. UOCB6 as the most resistant cell line (IC50=0.04U/ml) had the highest expression of ASNS (normalized ASNS, nASNS=4.946), then NALM6 (IC50=0.01U/ml; nASNS=1.8), REH (IC50=0.6.10−4; nASNS=1.176) and RS4;11 (IC50<0.3.10−4; nASNS=0.024). ASNS protein levels significantly differed through passages in REH cells, likely due to rapid turnover. For the remaining three cell lines L-Asp sensitivity correlated also with protein content. We have previously shown that different basal expression levels do not affect short-term dynamics of ASNS expression after L-Asp administration. Here we were interested to see the changes of sensitivity to L-Asp using gradient silencing of ASNS by RNAi in two cell lines with different basal expression: REH cell line with intermediate ASNS mRNA expression and RS4;11 cell line with very low mRNA expression. Gradient silencing revealed that L-Asp sensitivity correlated with ASNS expression till 50% decrease; further silencing did not potentiate the effect. The same response was seen in both cell lines despite different basal ASNS expression and sensitivity to L-Asp. The ASNS is glutamine dependent enzyme therefore we also studied expression of glutamate dehydrogenase (GDH), an enzyme necessary for glutamine synthesis. We found significantly lower GDH mRNA expression in primary TEL/AML1[+] blasts in comparison with TEL/AML[-] blasts (p=0.019), which might lead to deficiency of glutamine in these cells and consequently higher sensitivity to L-Asp. Accordingly, silencing of ASNS in REH tended to increase GDH expression levels. Our data confirm that generally, both ASNS mRNA and protein expression inversely correlate with the sensitivity to L-Asp in the cell lines. However, it may be misleading to draw conclusions for the patients' cells directly from the results obtained in cell line models. The expression patterns of ASNS in primary leukemic cells differ even from those of genotypically identical cell lines. The control of basal levels of ASNS in leukemic cells remains to be elucidated. Our results implicate an important role of GDH and glutamine metabolic pathway in the regulation of ASNS activity. This work was supported by MSM0021620813 and GAUK 7835. Disclosures: No relevant conflicts of interest to declare.

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Fluconazole inhibits human adrenocortical steroidogenesis in vitro

Journal of Endocrinology ◽

10.1530/joe-12-0310 ◽

2012 ◽

Vol 215 (3) ◽

pp. 403-412 ◽

Cited By ~ 29

Author(s):

R van der Pas ◽

L J Hofland ◽

J Hofland ◽

A E Taylor ◽

W Arlt ◽

...

Keyword(s):

Mrna Expression ◽

Cell Lines ◽

Primary Cultures ◽

Cell Number ◽

Mrna Levels ◽

Steroidogenic Enzymes ◽

Maximum Inhibition ◽

H295r Cells ◽

17 Hydroxyprogesterone

The antifungal agent ketoconazole is often used to suppress cortisol production in patients with Cushing's syndrome (CS). However, ketoconazole has serious side effects and is hepatotoxic. Here, the in vitro effects of ketoconazole and fluconazole, which might be less toxic, on human adrenocortical steroidogenesis were compared. The effects on steroidogenesis were examined in primary cultures of nine human adrenocortical tissues and two human adrenocortical carcinoma cell lines. Moreover, the effects on mRNA expression levels of steroidogenic enzymes and cell growth were assessed. Ketoconazole significantly inhibited 11-deoxycortisol (H295R cells; maximum inhibition 99%; EC50 0.73 μM) and cortisol production (HAC15 cells; 81%; EC50 0.26 μM and primary cultures (mean EC50 0.75 μM)). In cultures of normal adrenal cells, ketoconazole increased pregnenolone, progesterone, and deoxycorticosterone levels, while concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, DHEA, and androstenedione decreased. Fluconazole also inhibited 11-deoxycortisol production in H295R cells (47%; only at 1 mM) and cortisol production in HAC15 cells (maximum inhibition 55%; EC50 35 μM) and primary cultures (mean EC50 67.7 μM). In the cultures of normal adrenals, fluconazole suppressed corticosterone, 17-hydroxypregnenolone, and androstenedione levels, whereas concentrations of progesterone, deoxycorticosterone, and 11-deoxycortisol increased. Fluconazole (1 mM) slightly increased STAR mRNA expression in both cell lines. Neither compound affected mRNA levels of other steroidogenic enzymes or cell number. In conclusion, by inhibiting 11β-hydroxylase and 17-hydroxylase activity, pharmacological concentrations of fluconazole dose dependently inhibit cortisol production in human adrenocortical cells in vitro. Although fluconazole seems less potent than ketoconazole, it might become an alternative for ketoconazole to control hypercortisolism in CS. Furthermore, patients receiving fluconazole because of mycosis might be at risk for developing adrenocortical insufficiency.

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Specific and reliable detection of Myosin 1C isoform A by RTqPCR in prostate cancer cells

PeerJ ◽

10.7717/peerj.5970 ◽

2018 ◽

Vol 6 ◽

pp. e5970 ◽

Cited By ~ 2

Author(s):

Aleena A. Saidova ◽

Daria M. Potashnikova ◽

Anna V. Tvorogova ◽

Ivan V. Maly ◽

Wilma A. Hofmann ◽

...

Keyword(s):

Prostate Cancer ◽

Mrna Expression ◽

Cancer Cells ◽

Cell Lines ◽

Reference Genes ◽

Critical Issue ◽

Cell Detection ◽

Mrna Levels ◽

Prostate Cell ◽

Prostate Cell Lines

Background Prostate cancer (PC) diagnostics and treatment often present a challenging task due to cancer subtype heterogeneity and differential disease progression in patient subgroups. Hence, the critical issue is finding a reliable and sensitive diagnostic and prognostic PC marker, especially for cases of biopsies with low percentages of cancer cells. Isoform A of myosin 1C was shown to be expressed in PC cells and responsible for their invasive properties, however, its feasibility for diagnostic purposes remains to be elucidated. Methods To verify the role of myosin 1C isoform A mRNA expression as a putative prostate cancer marker we performed RT qPCR normalized by three reference genes (GAPDH, YWHAZ, HPRT1) on PC3, RWPE-1, LNCaP and 22Rv1 cell lines. Myosin 1C isoform A detection specificity was confirmed by immunofluorescence staining, cancer and non-cancer prostate cell lines were immunophenotyped by flow cytometry. Results Median normalized mRNA expression level of myosin 1C isoform A in PC cells (PC3 and 22Rv1) is two orders of magnitude higher compared to RWPE-1 cells, which functionally correspond to benign prostate cells. Myosin 1C isoform A expression allows PC cell detection even at a dilution ratio of 1:1000 cancer to non-cancer cells. At the protein level, the mean fluorescence intensity of myosin 1C isoform A staining in PC3 nuclei was only twice as high as in RWPE-1, while the immunophenotypes of both cell lines were similar (CD44+/CD90-/CD133-/CD57-/CD24+-). Conclusions We report a distinct difference in myosin 1C isoform A mRNA levels in malignant (PC3) and benign (RWPE-1) prostate cell lines and suggest a combination of three reference genes for accurate data normalization. For the first time we provide an immunophenotype comparison of RWPE-1 and PC3 cells and demonstrate that RT qPCR analysis of MYO 1C A using appropriate reference genes is sufficient for PC detection even in low-abundance cancer specimens.

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Heterogeneity in Simvastatin-Induced Cytotoxicity in AML Is Related to Differential Ras-Isoprenylation, Rather Than to Blockade of Cholesterol Synthesis.

Blood ◽

10.1182/blood.v114.22.1718.1718 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1718-1718

Author(s):

Karen van der Weide ◽

Patrick M. Korthuis ◽

Folkert Kuipers ◽

Elisabeth G.E. de Vries ◽

Edo Vellenga

Keyword(s):

Mrna Expression ◽

Cell Lines ◽

Cholesterol Synthesis ◽

Mevalonate Pathway ◽

Conflicts Of Interest ◽

Proper Function ◽

Mrna Levels ◽

Sensitive Cell ◽

Synthesis Route

Abstract Abstract 1718 Poster Board I-744 Introduction Cholesterol synthesis is enhanced in blast cells of acute myeloid leukemia (AML) patients, as indicated by high mRNA levels of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoAR) and LDL receptor (LDLR) and increased LDL uptake. In addition, AML cells exposed to chemotherapeutic drugs acutely increase their cholesterol content, which renders these cells less susceptible to chemotherapy. Statins inhibit HMG-CoAR, the rate-limiting enzyme of the mevalonate pathway. As a result, both the cholesterol synthesis route and the isoprenylation route are blocked. The latter route leads to the production of the isoprenoids farnesyl (FPP) and geranylgeranyl pyrophosphate (GGPP). Small GTP-ases Ras and Rho require farnesyl or geranylgeranyl isoprenylation for their proper function, a process which is catalyzed by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase). Simvastatin has shown to be cytotoxic to primary (CD34+) AML cells in vitro, however, these effects are heterogeneous. The aim of the present study was to elucidate the mechanism of simvastatin-induced cytotoxicity and the heterogeneity therein. This could be extensively studied by making use of cell lines that display a strong heterogeneity in responsiveness to simvastatin. Methods In 9 myeloid leukemic cell lines we mimicked or rescued the cytotoxic effects of simvastatin with compounds acting on either the cholesterol synthesis route or the isoprenylation route. Results In the cell lines, we identified 3 subgroups: sensitive (EC50 3-15 μM: NB4, OCI-M3, HL60), medium sensitive (EC50 16-50 μM: THP-1, U937, K562), and insensitive (EC50 60-100 μM: UT7-GM, TF-1, KG1a) to simvastatin, as determined by assessing cytotoxicity using a chemoluminescence assay. In all cell lines, simvastatin-induced cytotoxicity could completely be rescued by mevalonate, a compound directly downstream of HMG-CoAR. This indicates that the mevalonate pathway is indeed the key pathway involved in statin-induced cytotoxicity. To assess the role of the cholesterol synthesis route, mRNA levels of HMG-CoAR and LDLR were determined following simvastatin-treatment. In all cell lines, HMG-CoAR (1.5-7.8 fold) and LDLR (1.5-3.2 fold) mRNA expression were increased. No relationship existed between the basal or induced mRNA expression levels of both genes and simvastatin-induced cytotoxicity. In addition, squalene, a cholesterol precursor, did not rescue simvastatin-induced cell death. The role of the isoprenylation route, which is linked to the GTP-ases Ras and Rho, was assessed by rescue studies: simvastatin-induced cytotoxicity could especially be rescued by GGPP (68%), but also by FPP (42%), in the sensitive cell lines. Rescue was less pronounced in the insensitive cell lines (12% and 26% by GGPP and FPP, respectively), indicating a difference between the cell lines at the level of isoprenylation. This observation was confirmed by the finding that GGTI-298, a GGTase inhibitor, and to a lesser extent tipifarnib, an FTase inhibitor, were more effective in the sensitive cell lines compared to the insensitive cell lines (60% versus 96% viability at 0.2 μM, p=0.005; and 55% versus 78% viability at 5 μM, p=0.001, respectively). Also the ERK inhibitor U0126, acting downstream of Ras, was more effective in the sensitive cell lines when looking at cell viability (48% versus 70%, p=0.01) and pERK inhibition, as determined by Western blot. This is in line with the observed relationship between simvastatin-induced cytotoxicity and inhibition of pERK. Y27632, an inhibitor of ROCK (downstream target of Rho), and LY294,002, an Akt inhibitor (downstream of Ras), were equally cytotoxic in all cell lines. Currently, experiments are ongoing to obtain more detailed information on the mechanism involved by assessing the activity of Ras, GGTase and FTase, and we will confirm our findings in primary AML cells. Conclusion Our data indicate that the Ras-isoprenylation route, rather than the cholesterol synthesis route of the mevalonate pathway is responsible for simvastatin-induced cytotoxicity and the heterogeneous response between AML cells. Supported by a grant of the Dutch Cancer Society Disclosures No relevant conflicts of interest to declare.

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Mechanisms of high-glucose/insulin-mediated desensitization of acute insulin-stimulated glucose transport and Akt activation

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00644.2007 ◽

2008 ◽

Vol 294 (5) ◽

pp. E870-E881 ◽

Cited By ~ 22

Author(s):

Katherine A. Robinson ◽

Maria G. Buse

Keyword(s):

Insulin Resistance ◽

Glucose Transport ◽

High Glucose ◽

Uncoupling Protein ◽

Specific Inhibitor ◽

Pten Expression ◽

Pten Protein ◽

Akt Activation ◽

Insulin Resistant ◽

Resistant Cells

High-glucose/low-dose insulin-mediated insulin resistance of glucose transport was studied in 3T3-L1 adipocytes. In this model, proximal insulin signaling, including insulin receptor substrate (IRS)-1-bound phosphatidylinositol 3-kinase (PI 3-kinase) activation, is preserved, but insulin-stimulated protein kinase B (Akt) activation is markedly impaired. To assess a difference in acute insulin-stimulated production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5) P3], cells were labeled with [32P]orthophosphate, and glycerophosphoinositides were quantified by HPLC. Although basal PtdIns(3,4,5) P3 was similar, insulin stimulated its production 33.6% more in controls ( P < 0.03) than in insulin-resistant cells. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein, a lipid phosphatase that dephosphorylates PtdIns(3,4,5) P3 in the 3-position, was significantly and specifically increased in insulin-resistant cells. Treatment with rapamycin [a specific inhibitor of mammalian target of rapamycin complex 1 (mTORC1)] inhibited the increased PTEN expression and partially restored insulin-stimulated glucose transport and Akt activation to insulin-resistant cells. Acute insulin markedly stimulated Ser636/639 phosphorylation of IRS-1; this was rapamycin inhibited but was significantly decreased in cells that had been preexposed to insulin, whereas total IRS-1 was unaffected. These findings were essentially paralleled by changes in the activation of p70 S6 kinase and S6-ribosomal protein. Overexpression of uncoupling protein-1 or manganese superoxide dismutase did not prevent the development of insulin-resistant glucose transport and impaired Akt activation in high-glucose/low-insulin-pretreated cells. The insulin resistance associated with glucotoxicity in our model reflects in part decreased availability of PtdIns(3,4,5) P3, which correlates with increased PTEN protein expression. Chronic activation of mTORC1 plays a role in stimulating PTEN expression and possibly in activation or induction of a phosphoprotein phosphatase. No evidence was found for a role for increased mitochondrial superoxide production in this model.

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