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1.
Biotechnol Bioeng ; 112(7): 1383-94, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25619381

ABSTRACT

Asparagine-linked glycosylation of the constant region of monoclonal antibodies (mAbs) plays an important role in their stability and efficacy and is a critical product quality attribute that needs to be consistent between various process changes and production lots. Exact product quality match is also of the utmost importance for the development of biosimilar protein therapeutics. This poses a process development challenge since mAb glycosylation profiles can fluctuate easily with changes in process parameters. Therefore, there is a need to identify methods to modulate glycosylation levels on therapeutic antibodies during a production run in order to maintain consistent product quality profiles between different drug lots. Here, we demonstrate the use of a small molecule ionophore, monensin, to increase high mannose levels on multiple therapeutic human immunoglobulins (IgGs) in both plate-based small scale production models as well as in production bioreactors. This method is simple to implement and readily applicable for multiple production cell lines. Moreover, high mannose levels can be increased without significant negative impact on titer or cell culture performance. As such, monensin gives us a manipulable product quality lever.


Subject(s)
Antibodies, Monoclonal/metabolism , CHO Cells/drug effects , CHO Cells/metabolism , Ionophores/metabolism , Mannose/metabolism , Monensin/metabolism , Animals , Antibodies, Monoclonal/genetics , Cricetulus , Glycosylation , Humans , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
2.
J Cell Physiol ; 228(8): 1784-92, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23389849

ABSTRACT

The serine/threonine kinase Akt/PKB promotes cancer cell growth and invasion through several downstream targets. Identification of novel substrates may provide new avenues for therapeutic intervention. Our study shows that Akt phosphorylates the cancer-related transcription factor Runx2 resulting in stimulated DNA binding of the purified recombinant protein in vitro. Pharmacological inhibition of the PI3K/Akt pathway in breast cancer cells reduces DNA-binding activity of Runx2 with concomitant reduction in the expression of metastasis-related Runx2 target genes. Akt phosphorylates Runx2 at three critical residues within the runt DNA-binding domain to enhance its in vivo genomic interactions with a target gene promoter, MMP13. Mutation of these three phosphorylation sites reduces Runx2 DNA-binding activity. However, Akt signaling does not appear to interefere with CBFß-Runx2 interactions. Consequently, expression of multiple metastasis-related genes is decreased and Runx2-mediated cell invasion is supressed. Thus, our work identifies Runx2 as a novel and important downstream mediator of the PI3K/Akt pathway that is linked to metastatic properties of breast cancer cells.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/secondary , Core Binding Factor Alpha 1 Subunit/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Binding Sites/genetics , Breast Neoplasms/genetics , Cell Line, Tumor , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor beta Subunit/metabolism , DNA, Neoplasm/metabolism , Female , Humans , Male , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Transgenic , Mutagenesis, Site-Directed , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Signal Transduction
4.
Ann N Y Acad Sci ; 1155: 4-14, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19250187

ABSTRACT

There is growing awareness that the fidelity of gene expression necessitates coordination of transcription factor metabolism and organization of genes and regulatory proteins within the three-dimensional context of nuclear architecture. The regulatory machinery that governs genetic and epigenetic control of gene expression is compartmentalized in nuclear microenvironments. Temporal and spatial parameters of regulatory complex organization and assembly are functionally linked to biological control and are compromised with the onset and progression of tumorigenesis. High throughput imaging of cells, tissues, and tumors, including live cell analysis, is expanding research's capabilities toward translating components of nuclear organization into novel strategies for cancer diagnosis and therapy.


Subject(s)
Cell Nucleus/genetics , Epigenesis, Genetic , Cell Proliferation , Cell Transformation, Neoplastic , Gene Expression , Humans , Mitosis , Transcription Factors/metabolism , Transcription, Genetic
5.
Biochem Cell Biol ; 87(1): 1-6, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19234518

ABSTRACT

Epigenetic control is required to maintain competency for the activation and suppression of genes during cell division. The association between regulatory proteins and target gene loci during mitosis is a parameter of the epigenetic control that sustains the transcriptional regulatory machinery that perpetuates gene-expression signatures in progeny cells. The mitotic retention of phenotypic regulatory factors with cell cycle, cell fate, and tissue-specific genes supports the coordinated control that governs the proliferation and differentiation of cell fate and lineage commitment.


Subject(s)
Cell Lineage/genetics , Epigenesis, Genetic , Transcription Factors/metabolism , Animals , Cell Nucleus/genetics , Cell Proliferation , Humans , Mitosis
6.
J Cell Physiol ; 218(3): 473-9, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19006109

ABSTRACT

Runx proteins are tissue-specific transcriptional scaffolds that organize and assemble regulatory complexes at strategic sites of target gene promoters and at intranuclear foci to govern activation or repression. During interphase, fidelity of intranuclear targeting supports the biological activity of Runx1 and Runx2 proteins. Both factors regulate genes involved in cell cycle control and cell growth (e.g., rRNA genes), as well as lineage commitment. Here, we have examined the subcellular regulatory properties of the third Runx member, the tumor suppressor protein Runx3, during interphase and mitosis. Using in situ cellular and biochemical approaches we delineated a subnuclear targeting signal that directs Runx3 to discrete transcriptional foci that are nuclear matrix associated. Chromatin immunoprecipitation results show that Runx3 occupies rRNA promoters during interphase. We also find that Runx3 remains associated with chromosomes during mitosis and localizes with nucleolar organizing regions (NORs), reflecting an interaction with epigenetic potential. Taken together, our study establishes that common mechanisms control the subnuclear distribution and activities of Runx1, Runx2, and Runx3 proteins to support RNA polymerase I and II mediated gene expression during interphase and mitosis.


Subject(s)
Chromosomes/metabolism , Core Binding Factor Alpha 3 Subunit/metabolism , Epigenesis, Genetic , Mitosis , Tumor Suppressor Proteins/metabolism , Animals , HeLa Cells , Humans , Interphase , Nuclear Localization Signals , Nuclear Matrix/metabolism , Nucleolus Organizer Region/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Transport , RNA, Ribosomal/genetics , Rats , Transcription, Genetic
7.
J Cell Sci ; 121(Pt 23): 3981-90, 2008 Dec 01.
Article in English | MEDLINE | ID: mdl-19001502

ABSTRACT

RUNX1/AML1 is required for definitive hematopoiesis and is frequently targeted by chromosomal translocations in acute myeloid leukemia (AML). The t(8;21)-related AML1-ETO fusion protein blocks differentiation of myeloid progenitors. Here, we show by immunofluorescence microscopy that during interphase, endogenous AML1-ETO localizes to nuclear microenvironments distinct from those containing native RUNX1/AML1 protein. At mitosis, we clearly detect binding of AML1-ETO to nucleolar-organizing regions in AML-derived Kasumi-1 cells and binding of RUNX1/AML1 to the same regions in Jurkat cells. Both RUNX1/AML1 and AML1-ETO occupy ribosomal DNA repeats during interphase, as well as interact with the endogenous RNA Pol I transcription factor UBF1. Promoter cytosine methylation analysis indicates that RUNX1/AML1 binds to rDNA repeats that are more highly CpG methylated than those bound by AML1-ETO. Downregulation by RNA interference reveals that RUNX1/AML1 negatively regulates rDNA transcription, whereas AML1-ETO is a positive regulator in Kasumi-1 cells. Taken together, our findings identify a novel role for the leukemia-related AML1-ETO protein in epigenetic control of cell growth through upregulation of ribosomal gene transcription mediated by RNA Pol I, consistent with the hyper-proliferative phenotype of myeloid cells in AML patients.


Subject(s)
Chromosomes, Human, Pair 21/genetics , Chromosomes, Human, Pair 8/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Genes, rRNA/genetics , Leukemia, Myeloid, Acute/genetics , Mitosis/genetics , Nucleolus Organizer Region/metabolism , Oncogene Proteins, Fusion/genetics , Cell Line, Tumor , Cell Nucleolus/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , Fluorescent Antibody Technique , Humans , Oncogene Proteins, Fusion/metabolism , RUNX1 Translocation Partner 1 Protein , Translocation, Genetic/genetics
8.
J Cell Biochem ; 104(6): 2016-26, 2008 Aug 15.
Article in English | MEDLINE | ID: mdl-18615590

ABSTRACT

The regulatory machinery that governs genetic and epigenetic control of gene expression is compartmentalized in nuclear microenvironments. Temporal and spatial parameters of regulatory complex organization and assembly are functionally linked to biological control and are compromised with the onset and progression of tumorigenesis providing a novel platform for cancer diagnosis and treatment.


Subject(s)
Cell Nucleus/genetics , Epigenesis, Genetic , Neoplasms/genetics , Neoplasms/pathology , Animals , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Transcription Factors/metabolism
9.
Proc Natl Acad Sci U S A ; 104(50): 19861-6, 2007 Dec 11.
Article in English | MEDLINE | ID: mdl-18077419

ABSTRACT

The osteogenic Runt-related (Runx2) transcription factor negatively regulates proliferation and ribosomal gene expression in normal diploid osteoblasts, but is up-regulated in metastatic breast and prostate cancer cells. Thus, Runx2 may function as a tumor suppressor or an oncogene depending on the cellular context. Here we show that Runx2-deficient primary osteoblasts fail to undergo senescence as indicated by the absence of beta-gal activity and p16(INK4a) tumor suppressor expression. Primary Runx2-null osteoblasts have a growth advantage and exhibit loss of p21(WAF1/CIP1) and p19(ARF) expression. Reintroduction of WT Runx2, but not a subnuclear targeting-defective mutant, induces both p21(WAF/CIP1) and p19(ARF) mRNA and protein resulting in cell-cycle inhibition. Accumulation of spontaneous phospho-H2A.X foci, loss of telomere integrity and the Mre11/Rad50/Nbs1 DNA repair complex, and a delayed DNA repair response all indicate that Runx2 deficiency leads to genomic instability. We propose that Runx2 functions as a tumor suppressor in primary diploid osteoblasts and that subnuclear targeting contributes to Runx2-mediated tumor suppression.


Subject(s)
Cell Nucleus/genetics , Cell Nucleus/metabolism , Cell Transformation, Neoplastic/metabolism , Cellular Senescence/genetics , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Animals , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/deficiency , Core Binding Factor Alpha 1 Subunit/physiology , Cyclin-Dependent Kinase Inhibitor p21/physiology , Gene Targeting , Genomic Instability/physiology , Mice , Mice, Knockout , Osteoblasts/metabolism , Osteoblasts/pathology
10.
Mol Cell Biol ; 26(6): 2360-72, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16508011

ABSTRACT

ARF encodes a potent tumor suppressor that antagonizes MDM2, a negative regulator of p53. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF or p53 singly null mice, results in a broadened tumor spectrum and decreased tumor latency. To investigate the mechanism of p53-independent tumor suppression by ARF, potential interacting proteins were identified by yeast two-hybrid screen. The antiapoptotic transcriptional corepressor C-terminal binding protein 2 (CtBP2) was identified, and ARF interactions with both CtBP1 and CtBP2 were confirmed in vitro and in vivo. Interaction with ARF resulted in proteasome-dependent CtBP degradation. Both ARF-induced CtBP degradation and CtBP small interfering RNA led to p53-independent apoptosis in colon cancer cells. ARF induction of apoptosis was dependent on its ability to interact with CtBP, and reversal of ARF-induced CtBP depletion by CtBP overexpression abrogated ARF-induced apoptosis. CtBP proteins represent putative targets for p53-independent tumor suppression by ARF.


Subject(s)
ADP-Ribosylation Factors/metabolism , Apoptosis/physiology , DNA-Binding Proteins/metabolism , Phosphoproteins/metabolism , Tumor Suppressor Protein p53/metabolism , ADP-Ribosylation Factors/genetics , Alcohol Oxidoreductases , Animals , Cell Nucleolus/metabolism , Cells, Cultured , Co-Repressor Proteins , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/radiation effects , Humans , Mice , Phosphoproteins/genetics , Phosphoproteins/radiation effects , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/radiation effects , Protein Transport , RNA, Small Interfering , Tumor Suppressor Protein p53/genetics , Two-Hybrid System Techniques , Ubiquitin/metabolism , Ultraviolet Rays
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