Calcium pools, calcium entry, and cell growth

The Ca2+ pump and Ca2+ release functions of intracellular Ca2+ pools have been well characterized. However, the nature and identity of Ca2+ pools as well as the physiological implications of Ca2+ levels within them, have remained elusive. Ca2+ pools appear to be contained within the endoplasmic reticulum (ER); however, ER is a heterogeneous and widely distributed organelle, with numerous other functions than Ca2+ regulation. Studies described here center on trying to determine more about subcellular distribution of Ca2+ pools, the levels of Ca2+ within Ca2+ pools, and how these intraluminal Ca2+ levels may be physiologically related to ER function. Experiments utilizing in situ high resolution subcellular morphological analysis of ER loaded with ratiometric fluroescent Ca2+ dyes, indicate a wide distribution of inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools within cells, and large changes in the levels of Ca2+ within pools following InsP3-mediated Ca2+ release. Such changes in Ca2+ may be of great significance to the translation, translocation, and folding of proteins in ER, in particular with respect to the function of the now numerously described luminal Ca2+-sensitive chaperonin proteins. Studies have also focussed on the physiological role of pool Ca2+ changes with respect to cell growth. Emptying of pools using Ca2+ pump blockers can result in cells entering a stable quiescent G0-like growth state. After treatment with the irreversible pump blocker, thapsigargin, cells remain in this state until they are stimulated with essential fatty acids whereupon new pump protein is synthesized, functional Ca2+ pools return, and cells reenter the cell cycle. During the Ca2+ pool-depleted growth-arrested state, cells express a Ca2+ influx channel that is distinct from the store-operated Ca2+ influx channels activated after short-term depletion of Ca2+ pools. Overall, these studies indicate that significant changes in intraluminal ER Ca2+ do occur and that such changes appear linked to alteration of essential ER functions as well as to the cell cycle-state and the growth of cells.

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Mutations at sites involved in Suc1 binding inactivate Cdc2

Molecular and Cellular Biology ◽

10.1128/mcb.11.12.6177-6184.1991 ◽

1991 ◽

Vol 11 (12) ◽

pp. 6177-6184

Author(s):

B Ducommun ◽

P Brambilla ◽

G Draetta

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Specific Interaction ◽

Physiological Role ◽

Negative Effects ◽

Alanine Scanning Mutagenesis ◽

Cycle Progression ◽

Mutant Proteins ◽

Additional Protein

suc1+ encodes an essential cell cycle regulator of the fission yeast Schizosaccharomyces pombe. Its product, a 13-kDa protein, interacts with the Cdc2 protein kinase. Both positive and negative effects on cell cycle progression have been attributed to Suc1. To date, the exact mechanisms and the physiological role of the interaction between Suc1 and Cdc2 remain unclear. Here we have studied the molecular basis of this association. We show that Cdc2 can bind Suc1 or its mammalian homolog directly in the absence of any additional protein component. Using an alanine scanning mutagenesis method, we analyzed the interaction between Cdc2 and Suc1. We show that the integrity of several domains on the Cdc2 protein, including sites directly involved in catalytic activity, is required for binding to Suc1. Furthermore, Cdc2 mutant proteins unable to bind Suc1 (but able to bind cyclins) are nonfunctional when overexpressed in S. pombe, indicating that a specific interaction with Suc1 is required for Cdc2 function.

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RHBDL4 protects against endoplasmic reticulum stress by regulating the morphology and distribution of ER sheets

10.1101/2021.06.15.448480 ◽

2021 ◽

Author(s):

Viorica Liebe Lastun ◽

Matthew Freeman

Keyword(s):

Cell Cycle ◽

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Er Stress ◽

Liver Steatosis ◽

Physiological Role ◽

Cell Types ◽

Rhomboid Protease ◽

Rapid Changes

In metazoans, the architecture of the endoplasmic reticulum (ER) differs between cell types, and undergoes major changes through the cell cycle and according to physiological needs. Although much is known about how the different ER morphologies are generated and maintained, especially the ER tubules, how context dependent changes in ER shape and distribution are regulated and the factors involved are less characterized. Here, we show that RHBDL4, an ER-resident rhomboid protease, modulates the shape and distribution of the ER, especially under conditions that require rapid changes in the ER sheet distribution, including ER stress. RHBDL4 interacts with CLIMP-63, a protein involved in ER sheet stabilisation, and with the cytoskeleton. Mice lacking RHBDL4 are sensitive to ER stress and develop liver steatosis, a phenotype associated with unresolved ER stress. Our data introduce a new physiological role of RHBDL4 and also imply that this function does not require its enzymatic activity.

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Adaptation to calcium deprivation in the rat: effects of parathyroidectomy.

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1977.232.3.e336 ◽

1977 ◽

Vol 232 (3) ◽

pp. E336

Author(s):

J T Pento ◽

L C Waite ◽

P J Tracy ◽

A D Kenny

Keyword(s):

Adaptive Response ◽

Calcium Transport ◽

Parathyroid Tissue ◽

Adaptation Period ◽

Short Term ◽

High Calcium

The role of parathyroid hormone (PTH) in the adaptive response in gut calcium transport to calcium deprivation has been studied in the rat using both the in vitro everted duodenal sac and the in situ ligated duodenal segment technique. Intact or parathyroidectomized (PTX) young rats were placed on a low calcium (0.01%) diet for 7-, 14-, or 21-day adaptation periods and compared with control rats maintained on a high calcium (1.5%) diet. Prior PTX (3 days before the start of the adaptation period) abolished the adaptive response (enhanced calcium transport) induced by calcium deprivation for a 7-day adaptation period, but did not abolish a response after a 21-day period. A 14-day adaptation period gave equivocal results. It is concluded that PTH appears to be necessary for short-term (7-day) adaptation, but not for long-term (21-day) adaptation to calcium deprivation. However, if accessory parathyroid tissue is present, the data could be interpreted differently: the essentiality of PTH for the adaptive response might be independent of the length of the adaptation period. The data also contribute to a possible resolution of the controversy concerning the involvement of PTH in the regulation of intestinal calcium transport in the rat.

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PP2ARts1 is a master regulator of pathways that control cell size

The Journal of Cell Biology ◽

10.1083/jcb.201309119 ◽

2014 ◽

Vol 204 (3) ◽

pp. 359-376 ◽

Cited By ~ 47

Author(s):

Jessica Zapata ◽

Noah Dephoure ◽

Tracy MacDonough ◽

Yaxin Yu ◽

Emily J. Parnell ◽

...

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Cell Size ◽

Control Cell ◽

Size Control ◽

Regulatory Subunit ◽

Delay Cell ◽

Cell Cycle Entry ◽

G1 Cyclin

Cell size checkpoints ensure that passage through G1 and mitosis occurs only when sufficient growth has occurred. The mechanisms by which these checkpoints work are largely unknown. PP2A associated with the Rts1 regulatory subunit (PP2ARts1) is required for cell size control in budding yeast, but the relevant targets are unknown. In this paper, we used quantitative proteome-wide mass spectrometry to identify proteins controlled by PP2ARts1. This revealed that PP2ARts1 controls the two key checkpoint pathways thought to regulate the cell cycle in response to cell growth. To investigate the role of PP2ARts1 in these pathways, we focused on the Ace2 transcription factor, which is thought to delay cell cycle entry by repressing transcription of the G1 cyclin CLN3. Diverse experiments suggest that PP2ARts1 promotes cell cycle entry by inhibiting the repressor functions of Ace2. We hypothesize that control of Ace2 by PP2ARts1 plays a role in mechanisms that link G1 cyclin accumulation to cell growth.

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Rational targeted therapies to overcome microenvironment-dependent expansion of mantle cell lymphoma

Blood ◽

10.1182/blood-2016-06-720490 ◽

2016 ◽

Vol 128 (24) ◽

pp. 2808-2818 ◽

Cited By ~ 39

Author(s):

David Chiron ◽

Céline Bellanger ◽

Antonin Papin ◽

Benoit Tessoulin ◽

Christelle Dousset ◽

...

Keyword(s):

Cell Cycle ◽

Drug Resistance ◽

Cell Cycle Progression ◽

Cell Lymphoma ◽

Cycle Progression ◽

Key Points ◽

Mantle Cell ◽

Molecular Profiles

Key Points CD40L plus cytokines induces cell-cycle progression and loss of mitochondrial priming, leading to drug resistance in MCL. CD40L plus cytokines mimics in situ molecular profiles and allows the development of new approaches by integrating the role of the microenvironment.

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Data Supporting a New Physiological Role for Brain Apelin in the Regulation of Hypothalamic Oxytocin Neurons in Lactating Rats

Endocrinology ◽

10.1210/en.2011-0206 ◽

2011 ◽

Vol 152 (9) ◽

pp. 3492-3503 ◽

Cited By ~ 14

Author(s):

Laurence Bodineau ◽

Christopher Taveau ◽

Hong-Hanh Lê Quan Sang ◽

Guillaume Osterstock ◽

Isabelle Queguiner ◽

...

Keyword(s):

Direct Action ◽

Physiological Role ◽

Double Immunofluorescence ◽

Endogenous Ligand ◽

Electrophysiological Measurements ◽

Apelin Receptor ◽

Immunofluorescence Labeling ◽

G Protein Coupled

Apelin is a bioactive peptide identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ in 1998. The present data show that apelin modulates the activity of magnocellular and parvocellular oxytocin (OXY) neurons in the lactating rat. A combination of in situ hybridization and immunohistochemistry demonstrated the presence of apelin receptor mRNA in hypothalamic OXY neurons. Double immunofluorescence labeling then revealed the colocalization of apelin with OXY in about 20% of the hypothalamic OXY-positive neurons. Intracerebroventricular apelin administration inhibited the activity of magnocellular and parvocellular OXY neurons, as shown by measuring the c-fos expression in OXY neurons or by direct electrophysiological measurements of the electrical activity of these neurons. This effect was correlated with a decrease in the amount of milk ejected. Thus, apelin inhibits the activity of OXY neurons through a direct action on apelin receptors expressed by these neurons in an autocrine and paracrine manner. In conclusion, these findings highlight the inhibitory role of apelin as an autocrine/paracrine peptide acting on OXY neurons during breastfeeding.

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THAP11 Functions as a Tumor Suppressor in Gastric Cancer through Regulating c-Myc Signaling Pathways

BioMed Research International ◽

10.1155/2020/7838924 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11

Author(s):

Jing Zhang ◽

Huahua Zhang ◽

Haiyan Shi ◽

Fenghui Wang ◽

Juan Du ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Cycle ◽

Cell Growth ◽

Cancer Cell Growth ◽

Protein Levels ◽

Cycle Arrest ◽

A Cell ◽

Regulation Mechanisms ◽

Cell Growth Suppression

We aim to investigate the role of THAP11 (thanatos-associated protein11) in gastric cancer and its regulation mechanisms. THAP11 expression was analyzed in 51 pairs of GC tissues and the corresponding paracancerous tissues by qRT-PCR and Western blot. After THAP11 was overexpressed or knocked-down, cell proliferation, cell cycle, and apoptosis were detected in MKN-45 cells. We found that THAP11 was significantly downregulated in GC tissues and GC cell lines. Functionally, THAP11 overexpression markedly inhibited cell growth, induced G1/G0 cell-cycle arrest, and promoted cell apoptosis of MKN-45 cells, while silencing of THAP11 led to increased cell growth, increased DNA synthesis, and inhibited apoptosis. In addition, THAP11 negatively regulated the expression of c-Myc, decreased cyclinD1 protein, and increased p27 and p21 protein levels. We also found cell growth suppression induced by THAP11 was rescued by c-Myc overexpression, further confirming that THAP11 suppresses gastric cancer cell growth via the c-Myc pathway. THAP11 acts as a cell growth suppressor and exerts its role possibly through negatively regulating c-Myc pathway in gastric cancer.

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Insights Into the Role of ASPP2 (Apoptosis Stimulating Protein of p53-2) in Lymphomagenesis.

Blood ◽

10.1182/blood.v114.22.1967.1967 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1967-1967

Author(s):

Kerstin M Kampa ◽

Sandra Mueller ◽

Michael Bonin ◽

Marcus M Schittenhelm ◽

Charles D Lopez

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

T Cell ◽

Cell Growth ◽

Tumor Suppressor ◽

Target Genes ◽

Tumor Formation ◽

Transcriptional Networks ◽

Damage Response

Abstract Abstract 1967 Poster Board I-990 ASPP2 is a member of a family of p53 binding proteins that enhance apoptosis, in part through selective stimulation of p53 transactivation of pro-apoptotic target genes. Low ASPP2 expression is found in many human cancers and has been associated with poor clinical outcome in patients with aggressive lymphoma. Using an ASPP2+/- mouse model, we have previously demonstrated that ASPP2 is a haploinsufficient tumor suppressor and that reduced ASPP2 expression results in attenuated damage-response thresholds (Kampa et al., PNAS 2009). While ASPP2-/- mice are not viable, ASPP2+/- mice have an increased incidence of -irradiation-induced tumors compared to ASPP2+/+ mice.γspontaneous and ASPP2+/- mice develop high-grade thymic T-cell lymphomas after -irradiation. Moreover, primary ASPP2+/- thymocytes have an attenuatedγ -irradiation compared to ASPP2+/+ thymocytes.γapoptotic response after To explore the mechanisms of how attenuated ASPP2 expression could increase thymic lymphomagenesis and attenuate apoptosis, we performed global gene expression profiling on unirradiated, and 5 Gy irradiated ASPP2+/+ and ASPP2+/- thymocytes using an Affymetrix Mouse GeneChip® Array. We found significant differences in gene expression between ASPP2+/+ and ASPP2+/- thymocytes, in both unirradiated and irradiated sets. Using Ingenuity Pathway Analysis software, we found that amongst the highest scoring pathways displaying differences were those associated with cell growth, tumor formation, hematologic malignancies, immune response, cell death and cell cycle regulation. We additionally studied global phosphorylation patterns using 2-dimensional gel electrophoresis, fluorescent phosphoprotein dye Pro-Q Diamond staining, and liquid chromatography tandem mass spectrometry to determine the posttranscriptional mechanisms mediated by attenuated ASPP2 expression. Analysis of the phosphoproteome of ASPP2+/+ and ASPP2+/- mouse embryonic fibroblasts (with and without irradiation) revealed differences in the phosphorylation status of 108 peptides/proteins including those involved in regulating cell cycle checkpoints, T-cell receptor signaling, cell stress response, DNA repair mechanisms, cell growth, translation and transcription. Differential expression of the identified genes and proteins was verified by PCR and Western Blot. Thus, reduced ASPP2 expression affects global transcriptional as well as post-transcriptional networks intimately involved in the development of hematologic disorders–suggesting that ASPP2 function is by far more complex than solely enhancing the expression of pro-apoptotic p53 target genes. Given that ASPP2 is a bona fide tumor suppressor, reduced ASPP2 levels result in global dysregulation of pathways engaged in tumor suppression networks and the cellular damage response, which may ultimately promote genomic instability and tumor formation. Our findings provide insights into the role of ASPP2 in lymphomagenesis and reveal possible new targets for cancer therapy. Disclosures: No relevant conflicts of interest to declare.

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Combination Therapy with the Histone Deacetylase Inhibitor Vorinostat Plus the Novel Aurora Kinase A Inhibitor MK-5108 Leads to Enhanced Lymphoma Cell Death Due to Acetylation of p53 and Repression of c-Myc, hTERT, and miRNA Levels.

Blood ◽

10.1182/blood.v114.22.1690.1690 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1690-1690

Author(s):

Leo Kretzner ◽

Anna Scuto ◽

Kowolik Claudia ◽

Richard Jove ◽

Stephen J Forman ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Cell Growth ◽

Research Funding ◽

Aurora Kinase ◽

Aurora Kinase A ◽

Non Hodgkin Lymphoma ◽

Deacetylase Inhibitor ◽

Kinase A

Abstract Abstract 1690 Poster Board I-716 Background Patients with relapsed or refractory Hodgkin (HL) and Non Hodgkin Lymphoma (NHL) have few options after salvage therapy and transplant, and new agents are thus needed. MK-5108 is a novel aurora kinase inhibitor (AKI) with specificity against aurora kinase A, that produces G2/M phase cell cycle arrest. We show that addition of vorinostat, a histone and protein deacetylase inhibitor, to AKI treatment results in reactivation of proapoptotic genes and enhanced lymphoma cell death. A panel of HL and NHL cell lines was studied with either drug or the combination, using cell growth, apoptosis, and flow cytometry assays, followed by molecular studies. Results MK-5108 alone at 0.1 – 3 mM results in significant growth inhibition and apoptosis in multiple cell lines representing Hodgkin, Burkitt, and Non-Hodgkin lymphoma types, interestingly,DHL-4 and DHL-6 cells were more sensitive to this agent than to the pan-AKI MK-0457. Vorinostat alone at a dose range of 0.5 – 3 mM reduces cell growth by 50% or more in all lines tested. The combination of 1.5 mM vorinostat and 100 nM MK-5108 results in over 85% apoptosis of multiple lymphoma lines tested at 72 hours. Cell cycle analyses by FACS of MK-5108 treated cells show an increased percentage of cells in G2/M with few cells in sub-G1, whereas in combination with vorinostat the G2/M peak decreases and there is a significant increase in the apoptotic sub-G1 population. Real-time PCR analysis and immunoblotting of L540 cells treated with either single agent or in combination revealed that vorinostat treatment leads to alteration in pro-apoptosis, growth arrest, and DNA damage response genes. Myc mRNA and protein levels are reduced by vorinostat, and repression of microRNAs (miRNAs) in the Myc-regulated polycistronic cluster of miRNAs of chromosome 13, such as miR-17.5p, -17.3p, and 18, occurs with vorinostat and TSA. Prosurvival genes such as bcl-XL and hTERT are downregulated five-fold by vorinostat treatment, while the proapoptotic BAK gene is upregulated 1.5 – 2-fold. Vorinostat treatment leads to enhanced acetylation of p53, with a corresponding increase in the p53 target genes p21 and Noxa. To analyze the role of Myc inhibition in the sensitization by vorinostat of lymphoma cells to MK-5108, siRNA-mediated knock-down of Myc expression in L540 cells was performed. The siRNA-Myc transfected L540 cells showed enhanced sensitivity to MK-5108 as compared to control siRNA-null cells, as well as decreased hTERT levels, confirming the role of Myc inhibition by vorinostat as an integral part of the sensitization of lymphoma cells to MK-5108. Conclusions The HDACi vorinostat leads to both transcriptional and post-transcriptional changes that create a pro-apoptotic milieu, sensitizing the cell to centrosome-acting agents such as the aurora kinase A inhibitor MK-5108. These preclinical data support clinical trials of MK-5108 plus vorinostat in patients with relapsed or refractory lymphomas. [We acknowledge Merck Inc for providing Vorinostat, MK-0457, MK-5108, and research support.] Disclosures Kretzner: Merck: Research Funding. Yen:Merck: Research Funding. Kirschbaum:Merck: Research Funding, Speakers Bureau.

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