scholarly journals Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures

2014 ◽  
Vol 112 (1) ◽  
pp. 141-155 ◽  
Author(s):  
Zhimei Du ◽  
David Treiber ◽  
John D. McCarter ◽  
Dina Fomina‐Yadlin ◽  
Ramsey A. Saleem ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Cultures ◽  
Small Molecule ◽  
Recombinant Proteins ◽  
Control Cell ◽  
Specific Productivity ◽  
Cho Cell ◽  
Cell Cycle Inhibitor

Manipulation of the sodium‐potassium ratio as a lever for controlling cell growth and improving cell specific productivity in perfusion CHO cell cultures

2018 ◽  
Vol 115 (4) ◽  
pp. 921-931 ◽  
Author(s):  
Samantha B. Wang ◽  
Alexandria Lee‐Goldman ◽  
Janani Ravikrishnan ◽  
Lili Zheng ◽  
Henry Lin
Keyword(s):  
Cell Growth ◽  
Cell Cultures ◽  
Specific Productivity ◽  
Cho Cell ◽  
Sodium Potassium

scholarly journals Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

2018 ◽  
Author(s):  
Evgeny Zatulovskiy ◽  
Daniel F. Berenson ◽  
Benjamin R. Topacio ◽  
Jan M. Skotheim
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Growth ◽  
Cell Size ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Inverse Correlation ◽  
Cell Types ◽  
Similar Amount ◽  
Cell Cycle Inhibitor

Cell size is fundamental to function in different cell types across the human body because it sets the scale of organelle structures, biosynthesis, and surface transport1,2. Tiny erythrocytes squeeze through capillaries to transport oxygen, while the million-fold larger oocyte divides without growth to form the ~100 cell pre-implantation embryo. Despite the vast size range across cell types, cells of a given type are typically uniform in size likely because cells are able to accurately couple cell growth to division3–6. While some genes whose disruption in mammalian cells affects cell size have been identified, the molecular mechanisms through which cell growth drives cell division have remained elusive7–12. Here, we show that cell growth acts to dilute the cell cycle inhibitor Rb to drive cell cycle progression from G1 to S phase in human cells. In contrast, other G1/S regulators remained at nearly constant concentration. Rb is a stable protein that is synthesized during S and G2 phases in an amount that is independent of cell size. Equal partitioning to daughter cells of chromatin bound Rb then ensures that all cells at birth inherit a similar amount of Rb protein. RB overexpression increased cell size in tissue culture and a mouse cancer model, while RB deletion decreased cell size and removed the inverse correlation between cell size at birth and the duration of G1 phase. Thus, Rb-dilution by cell growth in G1 provides a long-sought cell autonomous molecular mechanism for cell size homeostasis.

scholarly journals PP2ARts1 is a master regulator of pathways that control cell size

2014 ◽  
Vol 204 (3) ◽  
pp. 359-376 ◽  
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.

Halofuginone Exerts Antiproliferative and Antiangiogenic Actions on Acute Promyelocytic Leukemia Cells through Modulation of the TGFβ Pathway.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2850-2850
Author(s):  
Lorena L. Figueiredo-Pontes ◽  
Ana Silvia G. Lima ◽  
Barbara A. Santana-Lemos ◽  
Ana Paula A. Lange ◽  
Luciana C. Oliveira ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Acute Promyelocytic Leukemia ◽  
Cell Lines ◽  
Cell Cultures ◽  
Promyelocytic Leukemia ◽  
Cell Growth Inhibition ◽  
Tgfβ Signaling ◽  
Tgfβ Pathway

Abstract The effects of TGFβ signaling in tumorigenesis is both cell type and context-dependent. Although this cytokine may behave as tumor suppressor in early stages of malignant transformation, tumor progression is often accompanied by altered TGFβ responsiveness and increased angiogenesis. Acute Promyelocytic Leukemia (APL) is a distinct subtype of Acute Myelogenous Leukemia characterized by rearrangements involving the PML and RARα genes on chromosomes 15 and 17, respectively. The expression of the PML/RARα oncoprotein leads to PML delocalization and functional impairment. Among its physiological roles, PML is a regulator of the TGFβ pathway, and the expression of PML-RARα has been associated with TGFβ resistance to differentiation and cell growth inhibition. Moreover, TGFβ is known to regulate Vascular Endothelial Growth Factor (VEGF) production and response. APL patients present increased bone marrow microvessel density, and the APL cell line NB4 was shown to secrete high levels of VEGF. Our aim was to test on APL the effect of Halofuginone (HF), an alkaloid that has been shown to inhibit TGFβ in other cell types. Cell cultures of NB4 and NB4-R2 cell lines, this latter resistant to ATRA, were treated with increasing doses of HF (6.25, 12.5, 25, 50, 100 ng/ml) and 10−6M of ATRA during 72 hours. Cell proliferation and apoptosis were accessed by flow cytometry using a simultaneous staining with bromodeoxyuridine and 7AAD. In NB4, there was significant cell growth inhibition with HF doses superior to 25 ng/ml (P <0.001). In addition, a 1.5 fold increase in apoptosis was seen with 100 ng/ml (P <0.001). In NB4-R2, cell growth inhibition was observed with 50 and 100 ng/ml and apoptosis with 100 ng/ml of HF (P < 0.001). HF was able to block the cell cycle progression at G1/S transition and, simultaneously, reduce Bcl2 protein expression in both cell lines. Concomitantly, mRNA expression of TGFβ target genes involved in cell cycle regulation was evaluated by real time PCR. Results showed the upregulation of p15, SMAD3, TGFβ and TGFβRI, and downregulation of c-MYC by treatment with high doses of HF (75 and 100 ng/ml). VEFG and TGFβ production was measured by ELISA in supernatants after 72 hours of culture. Significant reduction of VEGF levels was detected in samples treated with HF at doses higher than 25 ng/ml or with ATRA (P=0.018) and a decrease of TGFβ secretion was observed with 50 and 100 ng/ml of HF (P=0.026). Nuclear extracts from cell cultures treated as above were obtained, and western blot analysis showed that higher doses of HF (50 to 100 ng/ml) reduced TGFβ and Smad 4 expression. Our results indicate that HF was able to inhibit TGFβ at protein level and consequently to reduce VEGF production and thus may revert APL aberrant angiogenesis. As TGFβ transcription is at least in part auto-regulated, HF treatment was associated with an increase of TGFβ transcripts. These effects were independent of ATRA sensitivity, since both cell lines presented the same behavior. Although the disruption of TGFβ signaling itself is not sufficient to initiate malignant transformation, it may be a critical second step that contributes to leukemia progression. In this context, HF may have therapeutic potential in APL.

Productivity and Quality of Recombinant Proteins Produced by Stable CHO Cell Clones can be Predicted by Transient Expression in HEK Cells

2012 ◽  
Vol 54 (2) ◽  
pp. 497-503 ◽  
Author(s):  
Carolin Diepenbruck ◽  
Matthias Klinger ◽  
Thomas Urbig ◽  
Patrick Baeuerle ◽  
Rüdiger Neef
Keyword(s):  
Transient Expression ◽  
Recombinant Proteins ◽  
Cho Cell ◽  
Hek Cells ◽  
Cell Clones

scholarly journals Growth-dependent signals drive an increase in early G1 cyclin concentration to link cell cycle entry with cell growth

2020 ◽  
Author(s):  
Robert A. Sommer ◽  
Jerry T. DeWitt ◽  
Raymond Tan ◽  
Douglas R. Kellogg
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Budding Yeast ◽  
Control Cell ◽  
Transcriptional Repressor ◽  
Cell Cycle Entry ◽  
G1 Cyclin

AbstractEntry into the cell cycle occurs only when sufficient growth has occurred. In budding yeast, the cyclin Cln3 initiates cell cycle entry by inactivating a transcriptional repressor called Whi5. Growth-dependent changes in the concentrations of Cln3 or Whi5 have been proposed to link cell cycle entry to cell growth. However, there are conflicting reports regarding the behavior and roles of Cln3 and Whi5. Here, we found no evidence that changes in the concentration of Whi5 play a major role in controlling cell cycle entry. Rather, the data suggest that cell growth triggers cell cycle entry by driving an increase in the concentration of Cln3. We further found that accumulation of Cln3 is dependent upon homologs of mammalian SGK kinases that play roles in control of cell growth and size. Together, the data are consistent with models in which Cln3 serves as the crucial link between the cell cycle and signals that control cell growth and size.

The Influence of pH on Cell Growth and Specific Productivity of Two CHO Cell Lines Producing Human Anti Rh D IgG

2001 ◽  
pp. 197-199 ◽  
Author(s):  
Maria J. De Jesus ◽  
M. Bourgeois ◽  
G. Baumgartner ◽  
P. Tromba ◽  
Martin Jordan ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Specific Productivity ◽  
Cho Cell ◽  
Influence Of Ph

Synergistic Killing of Leukemic Cells by Small Molecule Inhibitors of the Pim Protein Kinases and Inactivators of Additional Signal Transduction Pathways.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 409-409
Author(s):  
Yingwei Lin ◽  
Zanna M Beharry ◽  
Elizabeth G Hill ◽  
Jin H. Song ◽  
Wenxue Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Signal Transduction ◽  
Cell Growth ◽  
Small Molecule ◽  
Single Agent ◽  
Signal Transduction Pathways ◽  
Pim Kinases ◽  
Cycle Arrest ◽  
Mtorc1 Pathway ◽  
P70 S6k

Abstract Abstract 409 The serine/threonine Pim kinases are up regulated in specific hematologic neoplasms, and play an important role in key signal transduction pathways, including those regulated by c-Myc, N-Myc, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. Pim protein kinases were first identified as a proviral integration site in c-Myc overexpressor mice and function to greatly enhance lymphoma development. Here we demonstrate that SMI-4a, a novel benzylidene-thiazolidine-2, 4-dione small molecule inhibitor of the Pim kinases supplied by Vortex Biotechnology (Mt. Pleasant, SC), kills a wide range of both myeloid and lymphoid cell lines with precursor T-cell lymphoblastic leukemia/lymphoma (pre T-LBL/T-ALL) being the most sensitive. Incubation of pre T-LBL cells with SMI-4a induced G1 phase cell cycle arrest secondary to a dose dependent induction of p27Kip1, apoptosis through the mitochondrial pathway, inhibition of mTORC1 pathway based on decreases in phosphorylation of p70 S6K and 4E-BP1, two substrates of this enzyme, and down regulation of c-myc. We demonstrate that treatment with 60 mg/kg twice daily by oral gavage of SMI-4a inhibits subcutaneous growth of pre T-LBL tumors by an average of 47.9% (p< .05) in immuno-deficient animals without notable toxicity to weight, blood counts, cell morphology, or blood chemistries. To enhance the killing effect of SMI-4a we have examined a number of potential combination therapies. First, because we find in animals and cell culture that single agent SMI-4a treatment up regulates the ERK pathway and in the spleen and thymus of Pim1/2/3 knock out mice there is increased phosphorylation of ERK1/2, we combined SMI-4a and a MEK1/2 inhibitor, U0126 or PD184352. Our results demonstrate that this combination is highly synergistic in killing pre T-LBL cells in culture. Secondly, because SMI-4a shares a number of important properties with γ-secretase inhibitors (GSI), Notch1 pathway inhibitor, including inhibition of pre T-LBL cell growth, cell cycle arrest, induction of p27Kip1, mTORC1 inhibition, and c-Myc down regulation, we tested the possibility that these agents could be synergistic. We find that single agent treatment with SMI-4a at 5 μM or treatment with the GSI Z-IL-CHO at 10 μM kills less than 20% of pre T-LBL cells, whereas in combination these drugs kill 78% of these cells, suggesting a high degree of synergy. Finally, because SMI-4a inhibits the mTORC1 pathway decreasing the phosphorylation of two mTOR substrates, p70 S6K and 4E-BP1, and because Pim plays an essential role in the FLT3/ITD signaling pathway, we examined the activity of SMI-4a with or without rapamycin in myeloid leukemic MV4-11 carrying both MLL-AF4 and FLT3-ITD and the RS4-11 cell line containing only MLL-AF4. We find that these two agents are highly synergistic in culture. SMI-4a alone inhibited growth 18% and rapamycin 40% but when combined 76% of the cell growth was blocked. SMI-4a had no effect on RS 4-11 cells. Our results demonstrate that unique combinations of a potent Pim inhibitor, SMI-4a, and small molecule blockade of either the mTORC1, ERK or Notch pathways has promise as a novel combination strategies for the treatment of human leukemia. Disclosures: No relevant conflicts of interest to declare.

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