the-fastest-western-town-contemporary-twist-on-classic-western-blot

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

University of California, San Francisco

Poly(ADP-ribose) metabolism plays a major role in DNA repair, transcription, replication, and recombination. Poly(ADP-ribose) polymerases are localized primarily to the nucleus, whereas significant levels of poly(ADP-ribose) glycohydrolase (PARG) are believed to be located in the cytoplasm. Only one PARG gene has been identified, but prior studies have reported multiple products of this gene. Here we studied PARG activity and PARG gene expression in several CNS cell types that span the cell growth spectrum: rapidly dividing C6 glioma tumor cells, dividing astrocytes, non-dividing astrocytes (due to contact inhibition), and post-mitotic neurons. Activity assays showed no overall differences between these cell types, but the nuclear to cytoplasmic ratio of PARG activity was highest in C6 glioma cells and lowest in neurons. Western blotting revealed full-length PARG as well as lower molecular weight PARG species in all four cell types.

Expression and activity of poly(ADP-ribose) glycohydrolase in cultured astrocytes, neurons, and C6 glioma cells.

Maternal inheritance of a pathogenic point mutation within complex I of the mitochondrial genome causes Leber's hereditary optic neuropathy (LHON), resulting in the neurodegeneration and demyelination of the optic nerve. The integrated stress response (ISR), a signaling pathway that responds to various stresses by activating a common set of genes, has been linked to both mitochondrial defects and demyelinating diseases. Therefore, we wanted to determine whether mitochondrial dysfunction induced by complex I inhibition with rotenone can activate the ISR, specifically by the ER kinase PERK, in oligodendroglial cells. Our complex I-deficient oligodendroglial model reproduced similar biochemical defects as in LHON by decreasing ATP synthesis and ATP levels. The same doses of rotenone that reduced ATP production also induced dose-dependent increases in PERK and eIF2alpha phosphorylation as well as activated the ISR stress genes, ATF4 and CHOP. In addition, complex I inhibition at these same concentrations induced a PERK-dependent activation of the cell death kinase, JNK, and inhibited oligodendroglial proliferation. Taken together, our results demonstrate that activation of the ISR may be one example of mitochondrial retrograde signaling in response to complex I deficiency and we suggest that this response mechanism may be relevant to the pathophysiology of LHON.

Inhibition of mitochondrial function induces an integrated stress response in oligodendroglia.

KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDA) but remains an intractable pharmacologic target. Consequently, defining RAS effector pathway(s) required for PDA initiation and maintenance is critical to improve treatment of this disease. Here, we show that expression of BRAF(V600E), but not PIK3CA(H1047R), in the mouse pancreas leads to pancreatic intraepithelial neoplasia (PanIN) lesions. Moreover, concomitant expression of BRAF(V600E) and TP53(R270H) result in lethal PDA. We tested pharmacologic inhibitors of RAS effectors against multiple human PDA cell lines. Mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) inhibition was highly effective both in vivo and in vitro and was synergistic with AKT inhibition in most cell lines tested. We show that RAF→MEK→ERK signaling is central to the initiation and maintenance of PDA and to rational combination strategies in this disease. These results emphasize the value of leveraging multiple complementary experimental systems to prioritize pathways for effective intervention strategies in PDA.

A central role for RAF→MEK→ERK signaling in the genesis of pancreatic ductal adenocarcinoma.

Mutationally activated BRAFV600E cooperates with PTEN silencing in the conversion of normal melanocytes to metastatic melanoma cells, but the mechanism underlying this cooperation is poorly understood. Here the consequences of pharmacological blockade of BRAFV600E or PI3'-kinase signaling were explored using pathway-targeted inhibitors and a panel of human BRAF mutated melanoma-derived cell lines. Blockade of BRAFV600E→MEK1/2→ERK1/2 or class I PI3'-kinases inhibited melanoma proliferation, whereas inhibition of AKT had only modest effects, even in cells with mutated or amplified AKT. Although single-agent inhibition of either BRAFV600E or PI3'-kinase signaling elicited anti-proliferative effects, combinatorial inhibition was more potent. Analysis of signaling downstream of BRAFV600E or PI3'-kinase revealed that these pathways cooperated to regulate protein synthesis through AKT-independent, mTORC1-dependent effects on p70S6K, ribosomal protein S6 and 4E-BP1 phosphorylation. Moreover, inhibition of mTORC1/2 inhibited cell proliferation as profoundly as single-agent inhibition of either BRAFV600E or PI3'-kinase signaling. These data reveal a mechanism by which BRAFV600E and PI3'-kinase signaling cooperate to regulate melanoma proliferation through AKT-independent effects on protein translation. Furthermore, this study provides a potential foundation for pathway-targeted combination therapy designed to enhance the therapeutic benefit to melanoma patients with combined alterations in BRAF and PI3'-kinase signaling. Implications: PI3'-kinases, but not AKT, represent potential targets for melanoma therapy.

Article	Year
Expression and activity of poly(ADP-ribose) glycohydrolase in cultured astrocytes, neurons, and C6 glioma cells. Brain research. Molecular brain research\| PubMed ID: 14559156	2003
Inhibition of mitochondrial function induces an integrated stress response in oligodendroglia. Neurobiology of disease\| PubMed ID: 19233273	2009
A central role for RAF→MEK→ERK signaling in the genesis of pancreatic ductal adenocarcinoma. Cancer discovery\| PubMed ID: 22628411	2012
BRAFV600E Cooperates with PI3'-Kinase Signaling, Independent of AKT, to Regulate Melanoma Cell Proliferation. Molecular cancer research : MCR\| PubMed ID: 24425783	2014

Jillian M. Silva

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