Dhan V. Kalvakolanu

Inhibition of Mcl-1 Promotes Senescence in Cancer Cells: Implications for Preventing Tumor Growth and Chemotherapy Resistance

ABSTRACT Although senescence in oncogenesis has been widely studied, little is known regarding the role of this process in chemotherapy resistance. Thus, from the standpoint of enhancing and improving cancer therapy, a better understanding of the molecular machinery involved in chemotherapy-related senescence is paramount. We show for the first time that Mcl-1, a Bcl-2 family member, plays an important role in preventing chemotherapy-induced senescence (CIS). Overexpression of Mcl-1 in p53+ cell lines inhibits CIS. Conversely, downregulation of Mcl-1 makes cells sensitive to CIS. Surprisingly, downregulation of Mcl-1 in p53− cells restored CIS to similar levels as p53+ cells. In all cases where senescence can be induced, we observed increased p21 expression. Moreover, we show that the domain of Mcl-1 responsible for its antisenescent effects is distinct from that known to confer its antiapoptotic qualities. In vivo we observe that downregulation of Mcl-1 can almost retard tumor growth regardless of p53 status, while overexpression of Mcl-1 in p53+ cells conferred resistance to CIS and promoted tumor outgrowth. In summary, our data reveal that Mcl-1 can inhibit CIS in both a p53-dependent and -independent manner in vitro and in vivo and that this Mcl-1-mediated inhibition can enhance tumor growth in vivo.

Cytokine-induced tumor suppressors: A GRIM story

Cytokines belonging to the IFN family are potent growth suppressors. In a number of clinical and preclinical studies, vitamin A and its derivatives like retinoic acid (RA) have been shown to exert synergistic growth-suppressive effects on several tumor cells. We have employed a genome-wide expression-knockout approach to identify the genes critical for IFN/RA-induced growth suppression. A number of novel genes associated with Retinoid-Interferon-induced Mortality (GRIM) were isolated. In this review, we will describe the molecular mechanisms of actions of one, GRIM-19, which participates in multiple pathways for exerting growth control and/or cell death. This protein is emerging as a new tumor suppressor. In addition, GRIM-19 appears to participate in innate immune responses as its activity is modulated by several viruses and bacteria. Thus, GRIMs seem to couple with multiple biological responses by acting at critical nodes.

Chromatin immunoprecipitation assay as a tool for analyzing transcription factor activity.

Differential gene expression is facilitated by transcriptional regulatory mechanisms and chromatin modifications through DNA-protein interactions. One of the widely used assays to study this is chromatin immunoprecipitation (ChIP) assay, which enables analysis of association of regulatory molecules to specific promoters and histone modifications in vivo. This is of immense value as ChIP assays can provide glimpse of the regulatory mechanisms involved in gene expression in vivo. This article outlines the general strategies and protocols to study ChIP assays in differential recruitment of transcriptional factors (TFs) and also global analysis of transcription factor recruitment is discussed. Further, the applications of ChIP assays for discovering novel genes that are dependent on specific transcription factors were addressed.

Interferons, Signal Transduction Pathways, and the Central Nervous System

The interferon (IFN) family of cytokines participates in the development of innate and acquired immune defenses against various pathogens and pathogenic stimuli. Discovered originally as a proteinaceous substance secreted from virus-infected cells that afforded immunity to neighboring cells from virus infection, these cytokines are now implicated in various human pathologies, including control of tumor development, cell differentiation, and autoimmunity. It is now believed that the IFN system (IFN genes and the genes induced by them, and the factors that regulate these processes) is a generalized alarm of cellular stress, including DNA damage. IFNs exert both beneficial and deleterious effects on the central nervous system (CNS). Our knowledge of the IFN-regulated processes in the CNS is far from being clear. In this article, we reviewed the current understanding of IFN signal transduction pathways and gene products that might have potential relevance to diseases of the CNS.

Bacteria and genetically modified bacteria as cancer therapeutics: Current advances and challenges.

HighlightsFacultative anaerobic bacteria grow in hypoxic areas of tumors and suppress growth.Bacteria can present tumor‐associated peptides to the immune system.Bacterially‐delivered mammalian expression plasmids can suppress tumor growth.Bacterial species that can stay in human blood offer more promise to target tumors. ABSTRACT Bacteria act as pro‐ or anti‐ tumorigenic agents. Whole bacteria or cytotoxic or immunogenic peptides carried by them exert potent anti‐tumor effects in the experimental models of cancer. The use of attenuated microorganism(s) e.g., BCG to treat human urinary bladder cancer was found to be superior compared to standard chemotherapy. Although the phase‐I clinical trials with Salmonella enterica serovar Typhimurium, has shown limited benefits in human subjects, a recent pre‐clinical trial in pet dogs with tumors reported some subjects benefited from this treatment strain. In addition to the attenuated host strains derived by conventional mutagenesis, recombinant DNA technology has been applied to a few microorganisms that have been evaluated in the context of tumor colonization and eradication using mouse models. There is an enormous surge in publications describing bacterial anti‐cancer therapies in the past 15 years. Vectors for delivering shRNAs that target oncogenic products, express tumor suppressor genes and immunogenic proteins have been developed. These approaches have showed promising anti‐tumor activity in mouse models against various tumors. These can be potential therapeutics for humans in the future. In this review, some conceptual and practical issues on how to improve these agents for human applications are discussed.

Microglia and necroptosis: The culprits of neuronal cell death in multiple sclerosis

Multiple Sclerosis is an inflammatory demyelinating and degenerative disease of the central nervous system in which activated microglia contribute to oligodendroglial, neuronal, and axonal damage. A recent study (summarized here) provided evidence for a role of necroptosis in MS brain tissue based on reduced caspase-8 and increased expression of cFLIP in microglial cells. In addition, activation of RIPK1, RIPK3, and MLKL (molecules characteristic of necroptosis) was demonstrated in cortical lesions from MS brain specimens. Defective caspase-8 activity in microglia adds a new dimension to microglial role in MS and provides a potential therapeutic target in the progressive forms of the disease.

Cytokine signaling in cancer: Novel players and pathways

Cancer, like many others, is a disease due to dysfunction of cytokine-regulated networks. In this background, the 1st Aegean conference meeting focused on the Cytokine Signaling in Cancer was organized and held at Chania, Crete, Greece in May of 2015. A number of novel aspects of the cytokine signaling and their relevance to oncogenic processes were presented at that meeting. This special issue of Cytokine captures snap shots of this meeting in the form of reviews. In addition to these reviews, several other original research articles on the roles of cytokines and chemokines in various cancers are also present in this issue.

GRIM-19: A Double-edged Sword that Regulates Anti-Tumor and Innate Immune Responses.

Gene associated with retinoid-interferon-β-induced mortality (GRIM)—19, was originally identified as a critical regulatory protein necessary for Interferon-β-Retinoic acid-induced cell death. Overexpression of GRIM-19 activates cell death and its suppression or inactivation promotes cell growth. GRIM-19 targets multiple proteins/pathways for exerting growth control and cell death. However, GRIM-19 is also required for normal cellular processes. In addition, viruses ‘hijack’ GRIM-19 for their survival. Intracellular bacterial infections and bacterial products have been reported to induce the expression of GRIM-19. In this review, we will discuss the current status of GRIM-19 in growth control and innate immune response.

Immortalization of Murine Bone Marrow-Derived Macrophages

Macrophages are specialized phagocytes that display a variety of important functions for the host immune system. They are particularly important for the recognition of exogenous and endogenous danger signals, forming the defensive front line as part of innate immune response. As such, murine macrophages are commonly used for in vitro cell-based assays examining the mechanisms of innate immune activation, which can require the ongoing breeding and housing of a large number of genetically modified mouse strains. Here, we describe a robust protocol for the generation of immortalized bone marrow-derived macrophages (iBMDMs) from primary murine bone marrow cells. We further provide general protocols for harvesting, freezing, and thawing of bone marrow cells, maintaining iBMDMs in culture and generation of monoclonal iBMDM populations by single-cell cloning.

Introduction to a Special Issue on Cytokines in Neuroinflammation and Immunity

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating and degenerative disease of the central nervous system (CNS). It is pathologically characterized by infiltration of activated inflammatory cells across the blood–brain barrier into the brain and spinal cord tissue resulting in demyelination and loss of cells, including oligodendrocytes, neurons, and axons. The inciting antigen has not been characterized and is likely to be heterogeneous. Genetic and environmental risk factors have been identified and their interaction is believed to underlie the immune dysregulation that leads to MS. MS is clinically heterogeneous, but the most common phenotype is relapsing remitting (RRMS), which ultimately transitions into secondary progressive (SPMS) with accumulation of disability. Fewer patients begin with a progressive form of the disease from the outset without relapses or remissions, referred to as the primary progress form (PPMS). No consistent immune differences among the different forms of the disease have been identified. While currently available immunomodulatory drugs are relatively effective in RRMS; unfortunately, they do not impact the course of PPMS. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS induced either by active immunization of the animal with myelin proteins and complete Freund’s adjuvant, or by passive transfer of encephlitogenic T-cells. Although not a prefect model of MS, EAE is commonly used to study MS immunopathogenesis and to screen for potential therapies. In this special issue of Journal of Interferon & Cytokine Research ( JICR), leading experts in neuroimmunology address current hot topics in MS, including pathogenesis and disease triggers, relevant signaling pathways, biomarkers, and potential novel therapeutic targets. Ben Segal highlights MS as a dynamic disease with stage-specific immune alterations. He reviews potential biomarkers for different phases of the disease and how these biomarkers could be utilized as outcome measures in clinical trials. V. Wee Yong and colleagues discuss the interactions between microglia and T cells in MS. They present evidence for reciprocal stimulation between the two cell types that could result in injurious or neurotrophic outcomes. They propose that this interaction could be targeted therapeutically. Kiel and Kasper review gut commensalism and its relationship to CNS demyelination. They discuss the role of the gut microbiome in disease severity as well as in disease-free state and the contribution of T-helper, T-regulatory, and B cells to immune homeostasis within the gut. Two contributions to this issue highlight the importance of specific signaling pathways in MS. Benveniste and colleagues discuss the involvement of the JAK/STAT signaling pathway in MS and its animal model EAE and how this pathway mediates the biological effects of the cytokines believed to contribute to MS pathogenesis. Racke and colleagues propose that the transcription factor T-bet is critical for T-cell encephalogenecity and could be targeted therapeutically in MS. Interferon-beta (IFN-B) was the first drug to be approved for the treatment of MS more than two decades ago. Nallar and Kalvakolanu reviewed the known mechanisms of IFN action and the recently emerged ones in their article. Some of these signaling pathways are involved in neurotoxicity and others in neuroprotection. A number of experimental animal models that may have potential relevance to the CNS and IFN signaling have been identified in that review. However, a direct relevance of the proposed players needs to be established in the context of MS with more detailed studies. Thus, there is a great need to fill the gap between IFN action and its proand anti-CNS effects. Tony Reder discusses how IFN-B works in MS and comments on some unexpected mechanistic effects for IFNB in the context of risk factors and other therapeutic interventions in MS. Graber and Dhib-Jalbut review potential biomarkers to monitor clinical efficacy of IFN-B therapy in MS. Finally, Makar and colleagues discuss the role of neurotrophic factors—specifically, brain-derived neurotrophic factor—in EAE and how this molecule could be utilized therapeutically using genetically modified stem cell delivery into the CNS. We hope that this special issue of JICR will advance the reader’s appreciation of the cytokines and signaling pathways involved in MS and how knowledge of these intricate pathways can potentially lead to better therapies and animal models of the disease. We realize that this issue is not an