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In JoVE (1)
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Articles by Chandreyee Mukherjee in JoVE
JoVE Immunology and Infection
Emballagen HIV-eller FIV-baserede Lentivector ekspressionskonstruktioner og transduktion af VSV-G-pseudotypet virale partikler
Lentivirale ekspressionsvektorer er de mest effektive køretøjer til stabilt at udtrykke forskellige effektormolekyler eller reporterkonstruktioner i delende og ikke-delende mammale celler og hele organismer. Her giver vi en protokol om, hvordan man pakker lentivector ekspressionskonstrukter i pseudoviral partikler og transducere målceller ved hjælp af de pseudoviral partikler.
Other articles by Chandreyee Mukherjee on PubMed
DNA Damage Response and MCL-1 Destruction Initiate Apoptosis in Adenovirus-infected Cells
Expression of adenovirus E1A deregulates cell proliferation to facilitate viral DNA replication, prompting the initiation of apoptosis signaled primarily through proapoptotic BAK in productively infected cells. We demonstrate here that in uninfected cells, BAK is complexed with the anti-apoptotic BCL-2 family member Myeloid Cell Leukemia 1 (MCL-1). E1A expression during infection resulted in the specific down-regulation of MCL-1 through destabilization of the protein and loss of the mRNA. Upon loss of the MCL-1-BAK complex, BAK complexed with either BAX in proapoptotic E1B mutant adenovirus-infected cells, or with the adenovirus BCL-2 homolog E1B 19K in cells infected with the wild-type virus in which apoptosis is inhibited. Loss of MCL-1 was required to initiate the apoptotic pathway in infected cells as restoration of MCL-1 expression rescued infected cells from E1A-induced apoptosis. Analogous to E1A expression, DNA damage down-regulates MCL-1, and adenovirus infection resulted in the accumulation of phosphorylated H2AX and ataxia-telangiectasia mutant protein (ATM), hallmarks of DNA double-strand breaks. Thus, MCL-1 may function by maintaining BAK in an inactive state, and the loss of MCL-1 upon activation of the DNA damage response, perhaps through replication stress induced in virus infected cells, may be required to initiate the apoptotic response.
Caspase-dependent Processing Activates the Proapoptotic Activity of Deleted in Breast Cancer-1 During Tumor Necrosis Factor-alpha-mediated Death Signaling
Deleted in breast cancer-1 (DBC-1) was initially cloned from a homozygously deleted region in breast and other cancers on human chromosome 8p21, although no function is known for the protein product it encodes. We identified the generation of amino-terminally truncated versions of DBC-1 during tumor necrosis factor (TNF)-alpha-mediated apoptosis. Full-length 150 kDa DBC-1 underwent caspase-dependent processing during TNF-alpha-mediated death signaling, to produce p120 DBC-1 and p66 DBC-1 carboxy-terminal fragments. Endogenous DBC-1 localized to the nucleus in healthy cells, but localized to the cytoplasm during TNF-alpha-mediated apoptosis, consistent with the loss of the amino-terminus containing the nuclear localization signal. Overexpression of an amino-terminal truncated DBC-1, resembling p120 DBC-1, caused mitochondrial clustering, mitochondrial matrix condensation, and sensitized cells to TNF-alpha-mediated apoptosis. The carboxy-terminal coiled-coil domain of DBC-1 was responsible for the cytoplasmic and mitochondrial localization, and for the death-promoting activity of DBC-1. Thus, caspase-dependent processing of DBC-1 may act as a feed-forward mechanism to promote apoptosis and possibly also tumor suppression. DBC-1, like its homolog cell cycle and apoptosis regulatory protein-1 (CARP-1), may function in the regulation of apoptosis.
Autophagy Promotes Tumor Cell Survival and Restricts Necrosis, Inflammation, and Tumorigenesis
Defective apoptosis renders immortalized epithelial cells highly tumorigenic, but how this is impacted by other common tumor mutations is not known. In apoptosis-defective cells, inhibition of autophagy by AKT activation or by allelic disruption of beclin1 confers sensitivity to metabolic stress by inhibiting an autophagy-dependent survival pathway. While autophagy acts to buffer metabolic stress, the combined impairment of apoptosis and autophagy promotes necrotic cell death in vitro and in vivo. Thus, inhibiting autophagy under conditions of nutrient limitation can restore cell death to apoptosis-refractory tumors, but this necrosis is associated with inflammation and accelerated tumor growth. Thus, autophagy may function in tumor suppression by mitigating metabolic stress and, in concert with apoptosis, by preventing death by necrosis.
Role of the Polarity Determinant Crumbs in Suppressing Mammalian Epithelial Tumor Progression
Most tumors are epithelial-derived, and although disruption of polarity and aberrant cellular junction formation is a poor prognosticator in human cancer, the role of polarity determinants in oncogenesis is poorly understood. Using in vivo selection, we identified a mammalian orthologue of the Drosophila polarity regulator crumbs as a gene whose loss of expression promotes tumor progression. Immortal baby mouse kidney epithelial cells selected in vivo to acquire tumorigenicity displayed dramatic repression of crumbs3 (crb3) expression associated with disruption of tight junction formation, apicobasal polarity, and contact-inhibited growth. Restoration of crb3 expression restored junctions, polarity, and contact inhibition while suppressing migration and metastasis. These findings suggest a role for mammalian polarity determinants in suppressing tumorigenesis that may be analogous to the well-studied polarity tumor suppressor mechanisms in Drosophila.
Defective Ubiquitin-mediated Degradation of Antiapoptotic Bfl-1 Predisposes to Lymphoma
The antiapoptotic Bcl-2 family member Bfl-1 is up-regulated in many human tumors in which nuclear factor-kappaB (NF-kappaB) is implicated and contributes significantly to tumor cell survival and chemoresistance. We previously found that NF-kappaB induces transcription of bfl-1 and that the Bfl-1 protein is also regulated by ubiquitin-mediated proteasomal degradation. However, the role that dysregulation of Bfl-1 turnover plays in cancer is not known. Here we show that ubiquitination-resistant mutants of Bfl-1 display increased stability and greatly accelerated tumor formation in a mouse model of leukemia/lymphoma. We also show that tyrosine kinase Lck is up-regulated and activated in these tumors and leads to activation of the IkappaB kinase, Akt, and extracellular signal-regulated protein kinase signaling pathways, which are key mediators in cancer. Coexpression of Bfl-1 and constitutively active Lck promoted tumor formation, whereas Lck knockdown in tumor-derived cells suppressed leukemia/lymphomagenesis. These data demonstrate that ubiquitination is a critical tumor suppression mechanism regulating Bfl-1 function and suggest that mutations in bfl-1 or in the signaling pathways that control its ubiquitination may predispose one to cancer. Furthermore, because bfl-1 is up-regulated in many human hematopoietic tumors, this finding suggests that strategies to promote Bfl-1 ubiquitination may improve therapy.
