Cell cycle regulators and bone: development and regeneration.

Cell cycle regulators act as inhibitors or activators to prevent cancerogenesis. It has also been established that they can play an active role in differentiation, apoptosis, senescence, and other cell processes. Emerging evidence has demonstrated a role for cell cycle regulators in bone healing/development cascade. We demonstrated that deletion of p21, a cell cycle regulator acting at the G1/S transition enhanced bone repair capacity after a burr-hole injury in the proximal tibia of mice. Similarly, another study has shown that inhibition of p27 can increase bone mineral density and bone formation. Here, we provide a concise review of cell cycle regulators that influence cells like osteoblasts, osteoclasts, and chondrocytes, during development and/or healing of bone. It is imperative to understand the regulatory processes that govern cell cycle during bone healing and development as this will pave the way to develop novel therapies to improve bone healing after injury in instances of aged or osteoporotic fractures.

Determination of albuterol and montelukast post-expiry drug strength by HPLC.

Background: Knowing the level of active ingredients in an expired drug is a matter of concern irrespective of its final disposition. This is also a matter of national security and defense as it has important implications on the nation's stockpile of prescription medications. Current literature has limited information about the strength of expired medications and any relevant trends. Objective: To utilize high performance liquid chromatography (HPLC) to determine the strength of selected drugs for asthma and chronic obstructive pulmonary disease (COPD) as a class of therapeutic agents commonly used in free clinics. Methods: Samples from expired lots of montelukast and albuterol pharmaceutical products were analyzed for their levels of their respective active ingredients. Two HPLC methods were developed, validated, and applied to achieve this goal. Quantitative analysis of each drug was performed using two different reversed phase C18 columns with a linear gradient of acetonitrile in 0.1% aqueous formic acid at a flow rate of 1 mL/min for both methods. Detection wavelength for montelukast and albuterol was 280 and 277 nm, respectively. Results: Expiry dates of analyzed batches ranged from 2003 to 2019. Despite the extended time range beyond expiry dates, levels of both drugs were relatively consistent and exceeded 90% of the listed strength in most analyzed lots. Conclusions: Our results introduce a new perspective towards reducing the financial burden resulting from disposal of expired medications with retained strength. They also offer supporting evidence to extend the use of out-of-date montelukast and albuterol preparations at home and in free clinics.

Astrocytes influence medulloblastoma phenotypes and CD133 surface expression.

The medulloblastoma (MB) microenvironment is diverse, and cell-cell interactions within this milieu is of prime importance. Astrocytes, a major component of the microenvironment, have been shown to impact primary tumor cell phenotypes and metastasis. Based on proximity of MB cells and astrocytes in the brain microenvironment, we investigated whether astrocytes may influence MB cell phenotypes directly. Astrocyte conditioned media (ACM) increased Daoy MB cell invasion, adhesion, and in vivo cellular protrusion formation. ACM conditioning of MB cells also increased CD133 surface expression, a key cancer stem cell marker of MB. Additional neural stem cell markers, Nestin and Oct-4A, were also increased by ACM conditioning, as well as neurosphere formation. By knocking down CD133 using short interfering RNA (siRNA), we showed that ACM upregulated CD133 expression in MB plays an important role in invasion, adhesion and neurosphere formation. Collectively, our data suggests that astrocytes influence MB cell phenotypes by regulating CD133 expression, a key protein with defined roles in MB tumorgenicity and survival.

Dual Specificity Phosphatase 5-Substrate Interaction: A Mechanistic Perspective.

The mammalian genome contains approximately 200 phosphatases that are responsible for catalytically removing phosphate groups from proteins. In this review, we discuss dual specificity phosphatase 5 (DUSP5). DUSP5 belongs to the dual specificity phosphatase (DUSP) family, so named after the family members' abilities to remove phosphate groups from serine/threonine and tyrosine residues. We provide a comparison of DUSP5's structure to other DUSPs and, using molecular modeling studies, provide an explanation for DUSP5's mechanistic interaction and specificity toward phospho-extracellular regulated kinase, its only known substrate. We also discuss new insights from molecular modeling studies that will influence our current thinking of mitogen-activated protein kinase signaling. Finally, we discuss the lessons learned from identifying small molecules that target DUSP5, which might benefit targeting efforts for other phosphatases. © 2017 American Physiological Society. Compr Physiol 7:1449-1461, 2017.

Dual Specificity Phosphatase 5 Is Essential for T Cell Survival.

The mitogen-activated protein kinase (MAPK) pathway regulates many key cellular processes such as differentiation, apoptosis, and survival. The final proteins in this pathway, ERK1/2, are regulated by dual specificity phosphatase 5 (DUSP5). DUSP5 is a nuclear, inducible phosphatase with high affinity and fidelity for ERK1/2. By regulating the final step in the MAPK signaling cascade, DUSP5 exerts strong regulatory control over a central cellular pathway. Like other DUSPs, DUSP5 plays an important role in immune function. In this study, we have utilized new knockout mouse reagents to explore its function further. We demonstrate that global loss of DUSP5 does not result in any gross phenotypic changes. However, loss of DUSP5 affects memory/effector CD8+ T cell populations in response to acute viral infection. Specifically, Dusp5-/- mice have decreased proportions of short-lived effector cells (SLECs) and increased proportions of memory precursor effector cells (MPECs) in response to infection. Further, we show that this phenotype is T cell intrinsic; a bone marrow chimera model restricting loss of DUSP5 to the CD8+ T cell compartment displays a similar phenotype. Dusp5-/- T cells also display increased proliferation, increased apoptosis, and altered metabolic profiles, suggesting that DUSP5 is a pro-survival protein in T cells.

Identification of inhibitors that target dual-specificity phosphatase 5 provide new insights into the binding requirements for the two phosphate pockets.

BACKGROUND: Dual-specificity phosphatase-5 (DUSP5) plays a central role in vascular development and disease. We present a p-nitrophenol phosphate (pNPP) based enzymatic assay to screen for inhibitors of the phosphatase domain of DUSP5. METHODS: pNPP is a mimic of the phosphorylated tyrosine on the ERK2 substrate (pERK2) and binds the DUSP5 phosphatase domain with a Km of 7.6 ± 0.4 mM. Docking followed by inhibitor verification using the pNPP assay identified a series of polysulfonated aromatic inhibitors that occupy the DUSP5 active site in the region that is likely occupied by the dual-phosphorylated ERK2 substrate tripeptide (pThr-Glu-pTyr). Secondary assays were performed with full length DUSP5 with ERK2 as substrate. RESULTS: The most potent inhibitor has a naphthalene trisulfonate (NTS) core. A search for similar compounds in a drug database identified suramin, a dimerized form of NTS. While suramin appears to be a potent and competitive inhibitor (25 ± 5 µM), binding to the DUSP5 phosphatase domain more tightly than the monomeric ligands of which it is comprised, it also aggregates. Further ligand-based screening, based on a pharmacophore derived from the 7 Å separation of sulfonates on inhibitors and on sulfates present in the DUSP5 crystal structure, identified a disulfonated and phenolic naphthalene inhibitor (CSD (3) _2320) with IC50 of 33 µM that is similar to NTS and does not aggregate. CONCLUSIONS: The new DUSP5 inhibitors we identify in this study typically have sulfonates 7 Å apart, likely positioning them where the two phosphates of the substrate peptide (pThr-Glu-pTyr) bind, with one inhibitor also positioning a phenolic hydroxyl where the water nucleophile may reside. Polysulfonated aromatic compounds do not commonly appear in drugs and have a tendency to aggregate. One FDA-approved polysulfonated drug, suramin, inhibits DUSP5 and also aggregates. Docking and modeling studies presented herein identify polysulfonated aromatic inhibitors that do not aggregate, and provide insights to guide future design of mimics of the dual-phosphate loops of the ERK substrates for DUSPs.

Protein expression, characterization and activity comparisons of wild type and mutant DUSP5 proteins.

BACKGROUND: The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function. RESULTS: In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P. CONCLUSION: Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.

The cancer stem cell paradigm: a new understanding of tumor development and treatment.

IMPORTANCE OF THE FIELD: Cancer is the second leading cause of death in the United States, and therefore remains a central focus of modern medical research. Accumulating evidence supports a 'cancer stem cell' (CSC) model - where cancer growth and/or recurrence is driven by a small subset of tumor cells that exhibit properties similar to stem cells. This model may provide a conceptual framework for developing more effective cancer therapies that target cells propelling cancer growth. AREAS COVERED IN THIS REVIEW: We review evidence supporting the CSC model and associated implications for understanding cancer biology and developing novel therapeutic strategies. Current controversies and unanswered questions of the CSC model are also discussed. WHAT THE READER WILL GAIN: This review aims to describe how the CSC model is key to developing novel treatments and discusses associated shortcomings and unanswered questions. TAKE HOME MESSAGE: A fresh look at cancer biology and treatment is needed for many incurable cancers to improve clinical prognosis for patients. The CSC model posits a hierarchy in cancer where only a subset of cells drive malignancy, and if features of this model are correct, has implications for development of novel and hopefully more successful approaches to cancer therapy.