Synchronization of mammalian cells by Lovastatin

Keyomarsi, Khandan

doi:10.1007/bf00122161

Cited by 15 publications

(28 citation statements)

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“…Lovastatin is an inhibitor of HMG COA reductase which is the rate limiting enzyme of the cholesterol biosynthesis pathway (Alberts et al, 1980). Though lovastatin has been primarily prescribed for patients with high cholesterol levels it has also been used as an effective agent in cell synchronization for both tumor and normal cells Keyomarsi, 1996). The inhibition of the cholesterol biosynthesis pathway by lovastatin not only blocks mevalonate synthesis (the product of HMG COA reductase) but also prevents the farnesylation and geranylgeranylation (intermediate products of the cholesterol pathway) of several signal transduction proteins such as Ras, Rap and many G proteins, thereby preventing their proper intracellular localization and function (Goldstein and Brown, 1990;).…”

Section: Discussionmentioning

confidence: 99%

“…All other chemicals used were reagent grade. Before addition to cultures, lovastatin was converted from its inactive lactone prodrug form to its active dihydroxy-open acid as described previously (Keyomarsi et ai, 1991;Keyomarsi, 1996). The culture conditions for 76N, 70N normal cell strains, MCF-10A immortalized cell line, and MCF-7, ZR75T, MDA-MB-157, Hs578T, T47D, and MDA-MB-231 breast cancer cell lines were described previously .…”

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confidence: 99%

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Cyclin E, A Potential Prognostic Marker in Breast Cancer

Keyomarsi¹

1998

Self Cite

105

143

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PuMic reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining fS^adta heeded, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for Teducing this burden to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302, and to the Office of Management and Budget,, Washington, DC 20503 AGENCY USE ONLY (Leave blank) REPORT DATE April 2000 REPORT TYPE AND DATES COVEREDFinal (1 Oct 94 -30 Mar 00) TITLE AND SUBTITLE Cyclin E, a Potential Prognostic Marker in Breast Cancer AUTHOR(S)Khandan Keyomarsi, Ph.D. Cyclin E, a G1 cyclin essential for S phase entry with a profound role in oncogenesis, is overexpressed and present in lower molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. Alteration and overexpression of cyclin E are linked to poor patient outcome. We have established the use of cyclin E as a prognostic marker for breast cancer. Tumor specimens from 400 breast cancer patients were examined; changes of cyclin E expression were compared with seven other established tumor markers to patient outcome. Altered expression of cyclin E was observed in 91% of all breast cancers with poor prognosis where patients either died of breast cancer or were still with cancer at the last contact date. Similarly in 93% of all breast cancer patients where cyclin E was either not altered, or its alteration was minimal, patients had a favorable prognosis. We have also deciphered the mechanism of deregulation of cyclin E in breast cancer cells giving rise to the LMW forms of cyclin E. Our studies show that the LMW forms of cyclin E detected at a much higher level in tumor cells, arise from post-translational action of a protease. We have been able to generate and knockout the tumor specific LMW pattern of cyclin E by transient transfection of FLAG-tagged cyclin E constructs harboring these mutations in a breast cancer cell line. We have identified a novel elastase-type consensus sequence in the amino-terminus of cyclin E responsible for generating these tumor specific LMW isoforms. Lastly, studies using the drug Lovastatin demonstrated that this highly prescribed drug used to lower cholesterol levels is also capable of inducing all four cyclin dependent kinase inhibitors (CKIs) in a cell specific manner via cell cycle independent mechanism, through the inhibition of the proteasome. We have also devised a novel treatment strategy that protects normal cells against the toxic effects of chemotherapeutic agents, by reversibly arresting the normal cells in the G1 phase of the cell cycle. Where copyrighted material is quoted, permission has been obtained to use such material. FUNDING NUMBERS DAMD17-94-J-4081 PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)H...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Cyclin E, A Potential Prognostic Marker in Breast Cancer

Keyomarsi¹

1998

Self Cite

105

143

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“…To compare the cell-sorting approach with commonly used synchronization methods, we generated LC-MS data from HeLa cells synchronized in S phase using the double thymidine block (DTB) technique (Whitfield et al, 2002), and in G 1 phase by lovastatin (Keyomarsi, 1996). Although HeLa cells are among the easiest to synchronize, perfect synchrony is never attained, and in this case 20% of DTB cells were not in S phase, while 22% of lovastatin-treated cells were not in G 1 (Figure S1K).…”

Section: Cell Sorting Allows Metabolism Measurements In Pure G 1 and Sg 2 M Subpopulationsmentioning

confidence: 99%

“…In mammalian cells, data obtained using synchronization methods indicate that central metabolic processes like glycolysis (Colombo et al, 2011), glycogen utilization (Favaro et al, 2012), glutamine catabolism (Ahn et al, 2017), and polyamine synthesis (Bettuzzi et al, 1999) are cyclic. However, synchronization methods are problematic in that they block cell-cycle progression by inhibiting metabolic processes such as thymidine or mevalonate synthesis (Whitfield et al, 2002;Keyomarsi 1996), and hence may disturb metabolism. Moreover, most mammalian cells are difficult to synchronize (Cooper, 2004), and consequently most data have been gathered from a few transformed cell lines that are amenable to synchronization, including HeLa and mouse 3T3 cells.…”

Section: Introductionmentioning

confidence: 99%

Mapping Metabolic Events in the Cancer Cell Cycle Reveals Arginine Catabolism in the Committed SG2M Phase

et al. 2019

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Background In addition to serving as building blocks for protein synthesis, amino acids also provide energy and precursors that are used by cells through catabolism. Mechanistic target of rapamycin complex 1 (mTORC1) is a central coordinator of cellular metabolism. However, little is known regarding the function of mTORC1 in amino acid catabolism. The aims of this study were to explore the mechanism by which mTORC1 controls the conversion of glutamate to α-ketoglutarate and ornithine to putrescine, and mTORC1 regulates the expression amino acid catabolism-related genes in hepatocyte. Methods HL-7702 hepatocytes were treated with glutamate, ornithine, rapamycin or SC75741, alone or in combination; the plasmids pRNAT-U6.1/Neo-shRaptor and pIRES2-EGFP-Rheb were transfected into HL-7702 cells to silencing Raptor or overexpressing Rheb. The intracellular content of glutamate, oxaloacetate, α-ketoglutaric acid, and aspartic acid, and the intracellular level of aspartate aminotransferase (AST), ornithine decarboxylase (ODC), glutamate dehydrogenase (GDH), and glutamic acid decarboxylase (GAD) were measured by ELISA. The concentrations of intracellular ornithine and putrescine were measured by HPLC. The mRNA level of amino acid catabolism-related genes was detected by qRT-PCR, and the protein level of mTORC1 and NF-κB was investigated by western blot. Results Our data showed that rapamycin inhibits the utilization of glutamate and ornithine in HL-7702 hepatocytes. mTORC1 regulates the expression of AST and ODC through the transcription factor NF-κB in response to glutamate or ornithine. Further, inactivated mTORC1 by Raptor silencing downregulated the expression of AST , ODC , GDH and GAD , while enhanced mTORC1 by Rheb overexpression upregulated NF-κB activation and the indicated genes expression in hepatocytes. Inhibited NF-κB by inhibitor SC75741 decreased the AST , ODC , GDH , and GAD expression. Conclusions 4 Our results demonstrate that mTORC1 regulates amino acid catabolism by inducing the expression of AST , ODC , GDH , and GAD , which is mediated by NF-κB. This finding constitutes a novel mechanism by which amino acid catabolism is regulated in hepatocytes.

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“…It is known that positioning of endolysosomal compartments in the cell affects their membrane composition, maturation and signaling (Cabukusta and Neefjes, 2018; Hu et al, 2015; Hyttinen et al, 2013). To better understand the mechanisms behind dynein clustering and to causally relate dynein clustering to endolysosomal membrane cholesterol levels, we manipulated cholesterol levels with two commonly used drugs: U18666A that increases endolysosomal membrane cholesterol and Lovastatin that decreases cellular and endolysosomal membrane cholesterol levels (Keyomarsi, 1996; Rocha et al, 2009). mCherry-ORP1L-positive endolysosomes in ORP1L KO HeLa cells treated with U18666A indeed had higher membrane cholesterol levels (by 4.5-fold) compared to those in cells treated with lovastatin as measured by filipin intensity (Figure 2-- figure supplement 1A).…”

Section: Resultsmentioning

confidence: 99%

ORP1L regulates dynein clustering on endolysosmal membranes in response to cholesterol levels

Thakur

Relich

Sorokina

et al. 2020

Preprint

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The sub-cellular positioning of endolysosomal compartments is crucial for regulating their downstream function. In particular, the positioning of endolysosomes at two sub-cellular locations: the cell periphery versus the cell peri-nuclear region; impacts autophagy, mTOR (mechanistic target of rapamycin) signaling and other cellular processes. Yet, the mechanisms that regulate the trafficking and positioning of endolysosomes at these two sub-cellular locations are poorly understood. Here, using super-resolution microscopy, we show that the retrograde motor dynein forms nano-clusters on endolysosomal membranes. Surprisingly, dynein nano-clusters are larger on peripherally positioned endolysosomes that contain higher cholesterol levels compared to perinuclearly positioned ones. By perturbing endolysosomal membrane cholesterol levels, we show that dynein clustering directly depends on the amount of cholesterol on the endolysosomal membrane. Finally, we show that the dynein adapter protein ORP1L (Oxysterol Binding Protein Homologue) regulates dynein clustering and endolysosomal positioning through its cholesterol binding domain in response to membrane cholesterol levels.

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Synchronization of mammalian cells by Lovastatin

Cited by 15 publications

References 20 publications

Cyclin E, A Potential Prognostic Marker in Breast Cancer

Cyclin E, A Potential Prognostic Marker in Breast Cancer

Mapping Metabolic Events in the Cancer Cell Cycle Reveals Arginine Catabolism in the Committed SG2M Phase

ORP1L regulates dynein clustering on endolysosmal membranes in response to cholesterol levels

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