Lymphocyte antigen 6K signaling to aurora kinase promotes advancement of the cell cycle and the growth of cancer cells, which is inhibited by LY6K-NSC243928 interaction.

Lymphocyte antigen 6K (LY6K) is a small GPI-linked protein that is normally expressed in testes. Increased expression of LY6K is significantly associated with poor survival outcomes in many solid cancers, including cancers of the breast, ovary, gastrointestinal tract, head and neck, brain, bladder, and lung. LY6K is required for ERK-AKT and TGF-ß pathways in cancer cells and is required for in vivo tumor growth. In this report, we describe a novel role for LY6K in mitosis and cytokinesis through aurora B kinase and its substrate histone H3 signaling axis. Further, we describe the structural basis of the molecular interaction of small molecule NSC243928 with LY6K protein and the disruption of LY6K-aurora B signaling in cell cycle progression due to LY6K-NSC243928 interaction. Overall, disruption of LY6K function via NSC243928 led to failed cytokinesis, multinucleated cells, DNA damage, senescence, and apoptosis of cancer cells. LY6K is not required for vital organ function, thus inhibition of LY6K signaling is an ideal therapeutic approach for hard-to-treat cancers that lack targeted therapy such as triple-negative breast cancer.

Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue.

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted. Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study. The principle and protocol of this methodology have been described in the literature. Additionally, alternate methodologies to isolate primary microglia are well described. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures. It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Evaluation of the in vitro and in vivo antitumor activity of histone deacetylase inhibitors for the therapy of retinoblastoma.

PURPOSE: To evaluate the potential utility of histone deacetylase inhibitors (HDACi) for treatment of retinoblastoma (RB). EXPERIMENTAL DESIGN: Growth-inhibitory effects of HDACi [trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), or MS-275] were assessed in human and transgenic murine RB cells. Effects of TSA and MS-275 were also assessed in combination with standard therapeutic agents for RB. Proapoptotic effects of MS-275 and TSA were evaluated by caspase-3/7 activity, Annexin V translocation, and/or Bim expression analyses. Effects of MS-275 on cell cycle distribution and reactive oxygen species levels were determined by flow cytometry. Retinal tissue morphology was evaluated in mice after local administration of MS-275. Analysis of retinal acetyl-histone levels was used to assess MS-275 delivery after systemic administration. Therapeutic effects of MS-275 were determined in transgenic mouse and rat ocular xenograft models of RB after i.p. injection of 20 mg/kg every other day for 21 or 13 days, respectively. RESULTS: TSA, SAHA, and MS-275 dose dependently reduced RB cell survival. TSA and MS-275 showed additive growth-inhibitory effects in combination with carboplatin, etoposide, or vincristine. TSA and MS-275 increased caspase-3/7 activity. MS-275 increased Annexin V membrane translocation and induced G1 arrest. Cytotoxicity of MS-275 was dependent on increased reactive oxygen species levels and was reversed by antioxidant pretreatment. Intraocular administration of 1 microL of 10 micromol/L MS-275 did not alter ocular tissue morphology. Increased acetyl-histone levels confirmed MS-275 delivery to retinal tissue after systemic administration. MS-275 significantly reduced tumor burden in both mouse and rat models of RB. CONCLUSIONS: HDACi should be considered for clinical trials in children with RB.

Endogenous 2-oxoacids differentially regulate expression of oxygen sensors.

Adaptations to change in oxygen availability are crucial for survival of multi-cellular organisms and are also implicated in several disease states. Such adaptations rely upon gene expression regulated by the heterodimeric transcription factors HIFs (hypoxia-inducible factors). Enzymes that link changes in oxygen tensions with the stability and transcriptional activity of HIFs are considered as oxygen sensors. These enzymes are oxygen-, iron- and 2-oxoglutarate-dependent dioxygenases that hydroxylate key proline and asparagine residues in HIFalpha subunits. The constitutive inhibitory action of these enzymes on HIFs is relieved by hypoxia and by agents that displace iron or 2-oxoglutarate. Two of the enzymes, HPH (HIF prolyl hydroxylase)-1 and HPH-2, are known to be inducible by hypoxia in a HIF-dependent manner. This suggests the existence of a novel feedback loop for adjusting hypoxia-regulated gene expression. We have recently shown that HIF-1alpha stability, HIF-1 nuclear translocation and HIF-mediated gene expression in human glioma cell lines can be stimulated by pyruvate independently of hypoxia. In the present study we show that the endogenous 2-oxoacid oxaloacetate can also activate HIF-mediated gene expression. Pyruvate and oxaloacetate treatment of cells also up-regulates HPH-1 and HPH-2, but not HPH-3 or the HIF asparaginyl hydroxylase FIH-1 (factor inhibiting HIF). Regulation of HIF-1 and the expression of HPH homologue genes can thus be influenced by specific glycolytic and tricarboxylic acid cycle metabolites. These findings may underlie important interactions between oxygen homoeostasis, glycolysis, the tricarboxylic acid cycle and gluconeogenesis.