RESUMO
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.
Assuntos
Anticorpos Monoclonais/metabolismo , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Ionóforos/metabolismo , Manose/metabolismo , Monensin/metabolismo , Animais , Anticorpos Monoclonais/genética , Cricetulus , Glicosilação , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/secundário , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Sítios de Ligação/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/metabolismo , DNA de Neoplasias/metabolismo , Feminino , Humanos , Masculino , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de SinaisRESUMO
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.
Assuntos
Núcleo Celular/genética , Núcleo Celular/metabolismo , Transformação Celular Neoplásica/metabolismo , Senescência Celular/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Animais , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/deficiência , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Marcação de Genes , Instabilidade Genômica/fisiologia , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Osteoblastos/patologiaRESUMO
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.
Assuntos
Cromossomos/metabolismo , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Epigênese Genética , Mitose , Proteínas Supressoras de Tumor/metabolismo , Animais , Células HeLa , Humanos , Interfase , Sinais de Localização Nuclear , Matriz Nuclear/metabolismo , Região Organizadora do Nucléolo/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , RNA Ribossômico/genética , Ratos , Transcrição GênicaRESUMO
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.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Apoptose/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fatores de Ribosilação do ADP/genética , Oxirredutases do Álcool , Animais , Nucléolo Celular/metabolismo , Células Cultivadas , Proteínas Correpressoras , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/efeitos da radiação , Humanos , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/efeitos da radiação , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos da radiação , Transporte Proteico , RNA Interferente Pequeno , Proteína Supressora de Tumor p53/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Raios UltravioletaRESUMO
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.
Assuntos
Núcleo Celular/genética , Epigênese Genética , Neoplasias/genética , Neoplasias/patologia , Animais , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Linhagem da Célula/genética , Epigênese Genética , Fatores de Transcrição/metabolismo , Animais , Núcleo Celular/genética , Proliferação de Células , Humanos , MitoseRESUMO
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.
Assuntos
Núcleo Celular/genética , Epigênese Genética , Proliferação de Células , Transformação Celular Neoplásica , Expressão Gênica , Humanos , Mitose , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
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.