Ann-Marie Broome

S100 protein subcellular localization during epidermal differentiation and psoriasis.

S100 proteins are calcium-activated signaling proteins that interact with target proteins to modulate biological processes. Our present studies compare the level of expression, and cellular localization of S100A7, S100A8, S100A9, S100A10, and S100A11 in normal and psoriatic epidermis. S100A7 and S100A11 are present in the basal and spinous layers in normal epidermis. These proteins appear in the nucleus and cytoplasm in basal cells but are associated with the plasma membrane in spinous cells. S100A10 is present in basal and spinous cells, in the cytoplasm, and is associated with the plasma membrane. S100A8 and S100A9 are absent or are expressed at minimal levels in normal epidermis. In involved psoriatic tissue, S100A10 and S100A11 levels remain unchanged, whereas, S100A7, S100A8, and S100A9 are markedly overexpressed. The pattern of expression and subcellular localization of S100A7 is similar in normal and psoriatic tissue. S100A8 and S100A9 are strongly expressed in the basal and spinous layers in psoriasis-involved tissue. In addition, we demonstrate that S100A7, S100A10, and S100A11 are incorporated into detergent and reducing agent-resistant multimers, suggesting that they are in vivo trans-glutaminase substrates. S100A8 and S100A9 did not form these larger complexes. These results indicate that S100 proteins localize to the plasma membrane in differentiated keratinocytes, suggesting a role in regulating calcium-dependent, membrane-associated events. These studies also indicate, as reported previously, that S100A7, S100A8, and S100A9 expression is markedly altered in psoriasis, suggesting a role for these proteins in disease pathogenesis.

Deep Penetration of a PDT Drug into Tumors by Noncovalent Drug-Gold Nanoparticle Conjugates

Efficient drug delivery to tumors is of ever-increasing importance. Single-visit diagnosis and treatment sessions are the goal of future theranostics. In this work, a noncovalent PDT cancer drug-gold nanoparticle (Au NP) conjugate system performed a rapid drug release and deep penetration of the drug into tumors within hours. The drug delivery mechanism of the PDT drug through Au NPs into tumors by passive accumulation was investigated via fluorescence imaging, elemental analysis, and histological staining. The pharmacokinetics of the conjugates over a 7-day test period showed rapid drug excretion, as monitored via the fluorescence of the drug in urine. Moreover, the biodistribution of Au NPs in this study period indicated clearance of the NPs from the mice. This study suggests that noncovalent delivery via Au NPs provides an attractive approach for cancer drugs to penetrate deep into the center of tumors.

A Regulatory Role for p38δ MAPK in Keratinocyte Differentiation EVIDENCE FOR p38δ-ERK1/2 COMPLEX FORMATION

p38 MAPK isoforms are important in the regulation of a variety of cellular processes. Among the four described p38 isoforms, p38α, β, and δ are expressed in keratinocytes (Dashti, S. R., Efimova, T., and Eckert, R. L. (2001) J. Biol. Chem. 276, 8059–8063). However, very little is known about how individual p38 isoforms regulate keratinocyte function. In the present study, we use okadaic acid (OA) as a tool to study the role of p38 MAPKs as regulators of keratinocyte differentiation. We demonstrate that OA activates p38δ but not other p38 isoforms. p38δ activation is increased as early as 0.5 h after OA addition, and activity is maximal at 8 and 24 h. ERK1 and ERK2 activity are reduced on an identical time course. We show that p38δ forms a complex with ERK1/2, and overexpression of p38δ inhibits ERK1/2 activity without reducing ERK1/2 level. Thus, p38δ may directly suppress ERK1/2 activity. Additional studies show that p38δ is expressed in the epidermis, suggesting a role for p38δ in regulating differentiation. To evaluate its function, we show that increased p38δ activity is associated with increased levels of AP1 and CAATT enhancer binding protein factors, increased binding of these factors to the involucrin (hINV) promoter, and increased expression. Moreover, these responses are maintained in the presence of SB203580, an agent that inhibits p38α and β, further suggesting a central role for the p38δ isoform. Dominant-negative p38 also inhibits these responses. These unique observations suggest that p38δ is the major p38 isoform driving suprabasal hINV gene expression and that p38δ directly regulates ERK1/2 activity via formation of a p38δ-ERK1/2 complex.

Protein Kinase Cδ Regulates Keratinocyte Death and Survival by Regulating Activity and Subcellular Localization of a p38δ-Extracellular Signal-Regulated Kinase 1/2 Complex

ABSTRACT Protein kinase Cδ (PKCδ) is an important regulator of apoptosis in epidermal keratinocytes. However, little information is available regarding the downstream kinases that mediate PKCδ-dependent keratinocyte death. This study implicates p38δ mitogen-activated protein kinase (MAPK) as a downstream carrier of the PKCδ-dependent death signal. We show that coexpression of PKCδ with p38δ produces profound apoptosis-like morphological changes. These morphological changes are associated with increased sub-G1 cell population, cytochrome c release, loss of mitochondrial membrane potential, caspase activation, and PARP cleavage. This death response is specific for the combination of PKCδ and p38δ and is not produced by replacing PKCδ with PKCα or p38δ with p38α. A constitutively active form of MEK6, an upstream activator of p38δ, can also produce cell death when coupled with p38δ. In addition, concurrent p38δ activation and extracellular signal-regulated kinase 1/2 (ERK1/2) inactivation are required for apoptosis. Regarding this inverse regulation, we describe a p38δ-ERK1/2 complex that may coordinate these changes in activity. We further show that this p38δ-ERK1/2 complex relocates into the nucleus in response to PKCδ expression. This regulation appears to be physiological, since H2O2, a known inducer of keratinocyte apoptosis, promotes identical PKCδ and p38δ-ERK1/2 activity changes, leading to similar morphological changes.

A Novel Tumor Suppressor Protein Promotes Keratinocyte Terminal Differentiation via Activation of Type I Transglutaminase

Tazarotene-induced protein 3 (TIG3) is a recently discovered regulatory protein that is expressed in the suprabasal epidermis. In the present study, we show that TIG3 regulates keratinocyte viability and proliferation. TIG3-dependent reduction in keratinocyte viability is accompanied by a substantial increase in the number of sub-G1 cells, nuclear shrinkage, and increased formation of cornified envelope-like structures. TIG3 localizes to the membrane fraction, and TIG3-dependent differentiation is associated with increased type I transglutaminase activity. Microscopic localization and isopeptide cross-linking studies suggest that TIG3 and type I transglutaminase co-localize in membranes. Markers of apoptosis, including caspases and poly(ADP-ribose) polymerase, are not activated by TIG3, and caspase inhibitors do not stop the TIG3-dependent reduction in cell viability. Truncation of the carboxyl-terminal membrane-anchoring domain results in a complete loss of TIG3 activity. The morphology of the TIG3-positive cells and the effects on cornified envelope formation suggest that TIG3 is an activator of terminal keratinocyte differentiation. Our studies suggest that TIG3 facilitates the terminal stages in keratinocyte differentiation via activation of type I transglutaminase.

Addressing Brain Tumors with Targeted Gold Nanoparticles: A New Gold Standard for Hydrophobic Drug Delivery?

EGF-modified Au NP-Pc 4 conjugates showed 10-fold improved selectivity to the brain tumor compared to untargeted conjugates. The hydrophobic photodynamic therapy drug Pc 4 can be delivered efficiently into glioma brain tumors by EGF peptide-targeted Au NPs. Compared to the untargeted conjugates, EGF-Au NP-Pc 4 conjugates showed 10-fold improved selectivity to the brain tumor. This delivery system holds promise for future delivery of a wider range of hydrophobic therapeutic drugs for the treatment of hard-to-reach cancers.

Transferrin receptor-targeted theranostic gold nanoparticles for photosensitizer delivery in brain tumors

Therapeutic drug delivery across the blood-brain barrier (BBB) is not only inefficient, but also nonspecific to brain stroma. These are major limitations in the effective treatment of brain cancer. Transferrin peptide (Tfpep) targeted gold nanoparticles (Tfpep-Au NPs) loaded with the photodynamic pro-drug, Pc 4, have been designed and compared with untargeted Au NPs for delivery of the photosensitizer to brain cancer cell lines. In vitro studies of human glioma cancer lines (LN229 and U87) overexpressing the transferrin receptor (TfR) show a significant increase in cellular uptake for targeted conjugates as compared to untargeted particles. Pc 4 delivered from Tfpep-Au NPs clusters within vesicles after targeting with the Tfpep. Pc 4 continues to accumulate over a 4 hour period. Our work suggests that TfR-targeted Au NPs may have important therapeutic implications for delivering brain tumor therapies and/or providing a platform for noninvasive imaging.

Biodistribution and clearance of a filamentous plant virus in healthy and tumor-bearing mice

AIM Nanoparticles based on plant viruses are emerging biomaterials for medical applications such as drug delivery and imaging. Their regular structures can undergo genetic and chemical modifications to carry large payloads of cargos, as well as targeting ligands. Of several such platforms under development, only few have been characterized in vivo. We recently introduced the filamentous plant virus, potato virus X (PVX), as a new platform. PVX presents with a unique nanoarchitecture and is difficult to synthesize chemically. METHODS Here, we present a detailed analysis of PVX biodistribution and clearance in healthy mice and mouse tumor xenograft models using a combination of ex vivo whole-organ imaging, quantitative fluorescence assays and immunofluorescence microscopy. RESULTS & CONCLUSION While up to 30% of the PVX signal was from the colon, mammary and brain tumor tissues, remaining particles were cleared by the reticuloendothelial system organs (the spleen and liver), followed by slower processing and clearance through the kidneys and bile.

Mutations in DCHS1 cause mitral valve prolapse.

Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1+/− mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1+/− mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

An Optical Probe for Noninvasive Molecular Imaging of Orthotopic Brain Tumors Overexpressing Epidermal Growth Factor Receptor

We have developed a near-infrared (NIR) probe that targets cells overexpressing the EGF receptor (EGFR) for imaging glioblastoma brain tumors in live subjects. A peptide specific for the EGFR was modified with various lengths of monodiscrete polyethylene glycol (PEG) units and a NIR Cy5.5 fluorescence dye. The lead compound, compound 2, with one unit of PEG displayed good binding (8.9 μmol/L) and cellular uptake in glioblastoma cells overexpressing EGFR in vitro. The in vivo studies have shown that the probe was able to selectively label glioblastoma-derived orthotopic brain tumors. In vivo image analyses of peptide binding to the tumors using fluorescence-mediated molecular tomography revealed that the compound could distinguish between tumors expressing different levels of EGFR. The data presented here represent the first demonstration of differential quantitation of tumors expressing EGFR in live animals by a targeted NIR fluorescence probe using a molecular imaging device. Mol Cancer Ther; 11(10); 2202–11. ©2012 AACR.