Publications by authors named "Tzvete Dentchev"

35 Publications

Fibrotic trochanters: A potential mechanism for stem cell depletion in scarring alopecias.

J Cutan Pathol 2021 Mar 14;48(3):458-460. Epub 2020 Oct 14.

Department of Dermatology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.

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http://dx.doi.org/10.1111/cup.13855DOI Listing
March 2021

Whole-Exome and Transcriptome Analysis of UV-Exposed Epidermis and Carcinoma In Situ Reveals Early Drivers of Carcinogenesis.

J Invest Dermatol 2021 Feb 8;141(2):295-307.e13. Epub 2020 Jul 8.

Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. Electronic address:

Squamous cell carcinoma in situ (SCCIS) is a prevalent precancerous lesion that can progress to cutaneous squamous cell carcinoma. Although SCCIS is common, its pathogenesis remains poorly understood. To better understand SCCIS development, we performed laser captured microdissection of human SCCIS and the adjacent epidermis to isolate genomic DNA and RNA for next-generation sequencing. Whole-exome sequencing identified UV-signature mutations in multiple genes, including NOTCH1-3 in the epidermis and SCCIS and oncogenic TP53 mutations in SCCIS. Gene families, including SLFN genes, contained UV/oxidative-signature disruptive epidermal mutations that manifested positive selection in SCCIS. The frequency and distribution of NOTCH and TP53 mutations indicate that NOTCH mutations may precede TP53 mutations. RNA sequencing identified 1,166 differentially expressed genes; the top five enriched gene ontology biological processes included (i) immune response, (ii) epidermal development, (iii) protein phosphorylation, (iv) regulation of catalytic activity, and (v) cytoskeletal regulation. The NEURL1 ubiquitin ligase, which targets Notch ligands for degradation, was upregulated in SCCIS. NEURL1 protein was found to be elevated in SCCIS suggesting that increased levels could represent a mechanism for downregulating Notch during UV-induced carcinogenesis. The data from DNA and RNA sequencing of epidermis and SCCIS provide insights regarding SCCIS formation.
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http://dx.doi.org/10.1016/j.jid.2020.05.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790860PMC
February 2021

Pharmacologic Activation of the G Protein-Coupled Estrogen Receptor Inhibits Pancreatic Ductal Adenocarcinoma.

Cell Mol Gastroenterol Hepatol 2020 4;10(4):868-880.e1. Epub 2020 May 4.

Perelman School of Medicine, Department of Dermatology, University of Pennsylvania, Philadelphia. Electronic address:

Background & Aims: Female sex is associated with lower incidence and improved clinical outcomes for most cancer types including pancreatic ductal adenocarcinoma (PDAC). The mechanistic basis for this sex difference is unknown. We hypothesized that estrogen signaling may be responsible, despite the fact that PDAC lacks classic nuclear estrogen receptors.

Methods: Here we used murine syngeneic tumor models and human xenografts to determine that signaling through the nonclassic estrogen receptor G protein-coupled estrogen receptor (GPER) on tumor cells inhibits PDAC.

Results: Activation of GPER with the specific, small molecule, synthetic agonist G-1 inhibited PDAC proliferation, depleted c-Myc and programmed death ligand 1 (PD-L1), and increased tumor cell immunogenicity. Systemically administered G-1 was well-tolerated in PDAC bearing mice, induced tumor regression, significantly prolonged survival, and markedly increased the efficacy of PD-1 targeted immune therapy. We detected GPER protein in a majority of spontaneous human PDAC tumors, independent of tumor stage.

Conclusions: These data, coupled with the wide tissue distribution of GPER and our previous work showing that G-1 inhibits melanoma, suggest that GPER agonists may be useful against a range of cancers that are not classically considered sex hormone responsive and that arise in tissues outside of the reproductive system.
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http://dx.doi.org/10.1016/j.jcmgh.2020.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578406PMC
May 2020

Voriconazole enhances UV-induced DNA damage by inhibiting catalase and promoting oxidative stress.

Exp Dermatol 2020 01 29;29(1):29-38. Epub 2019 Oct 29.

Department of Dermatology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.

Cutaneous squamous cell carcinoma (cSCC) is the second most common form of skin cancer and is associated with cumulative UV exposure. Studies have shown that prolonged voriconazole use promotes cSCC formation; however, the biological mechanisms responsible for the increased incidence remain unclear. Here, we show that voriconazole directly increases oxidative stress in human keratinocytes and promotes UV-induced DNA damage as determined by comet assay, 8-oxoguanine immunofluorescence and mass spectrometry. Voriconazole treatment of human keratinocytes potentiates UV-induced apoptosis and activation of the p38 MAP kinase and 53BP1 UV stress response pathways. The p38 MAP kinase activation promoted by voriconazole exposure can be mitigated by pretreating keratinocytes with N-acetylcysteine. Voriconazole increases oxidative stress in keratinocytes by directly inhibiting catalase leading to lower intracellular NADPH levels and the triazole moieties in voriconazole are critical for inhibiting catalase. Furthermore, voriconazole is shown to promote UV-induced dysplasia in an in vivo model. Together, these data demonstrate that voriconazole potentiates oxidative stress in UV-irradiated keratinocytes through catalase inhibition. Use of antioxidants may mitigate the pro-oncogenic effects of voriconazole.
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http://dx.doi.org/10.1111/exd.14038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401379PMC
January 2020

Topical kinase inhibitors induce regression of cutaneous squamous cell carcinoma.

Exp Dermatol 2019 05 13;28(5):609-613. Epub 2019 Mar 13.

Department of Dermatology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.

Actinic keratoses (AKs) and squamous cell carcinoma in situ (SCCIS) are precursor lesions for cutaneous squamous cell carcinoma (cSCC), the second most common form of cancer. Current topical therapies for AKs and SCCIS promote skin inflammation to eradicate lesions and do not directly target the biological mechanisms driving growth. We hypothesized that topical small molecule inhibitors targeting kinases promoting keratinocyte growth in AKs and SCCIS could induce regression of these lesions with less inflammation. To test this hypothesis, we determined the efficacy of topical dasatinib, 5-fluorouracil and BEZ-235 in inducing regression of cSCCs in the K14-Fyn Y528 transgenic mouse model. Topical dasatinib induced regression of cSCC with less inflammation, no ulceration and no mortality compared to 5-fluorouracil. Topical BEZ-235 induced cSCC regression similar to dasatinib without erythema or ulceration. These data indicate that topical small molecule kinase inhibitors targeting drivers of AK/SCCIS/cSCC growth represent a promising therapeutic approach to treat these common skin lesions.
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http://dx.doi.org/10.1111/exd.13902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520110PMC
May 2019

Expression of p15 in a spectrum of spitzoid melanocytic neoplasms.

J Cutan Pathol 2019 May 14;46(5):310-316. Epub 2019 Feb 14.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.

Background: Accurate classification of spitzoid melanocytic lesions is difficult due to overlapping clinical and histopathologic features between Spitz nevi, atypical Spitz tumors (ASTs), and spitzoid melanomas. Expression of p16 (CDKN2A) has been used as a marker of spitzoid lesions. However, its expression may be variable. p15 is a tumor suppressor encoded by CDKN2B, loss of which has been recently shown to promote transition from nevus to melanoma. We sought to determine whether p15 is a useful immunohistochemical marker to distinguish Spitz nevi from spitzoid melanomas and to compare p15 and p16 staining in this population.

Methods: Immunohistochemistry for p15 and p16 was performed on Spitz nevi (n = 19), ASTs (n = 41), and spitzoid melanomas (n = 17). Immunoexpression was categorized by a four-tiered system: 0 (negative), 1+ (weak), 2+ (moderate), 3+ (strong).

Results: 3+/strong p15 staining was observed in 68.4% of Spitz nevi, 34.2% of ASTs, and 17.7% of spitzoid melanomas. By contrast, we observed 3+ p16 staining in roughly equivalent percentages of Spitz nevi (57.9%), ASTs (56.1%), and spitzoid melanomas (58.8%).

Conclusion: These data illustrate that p15 may be more useful than p16 as a biomarker to help distinguish benign from malignant spitzoid lesions.
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http://dx.doi.org/10.1111/cup.13424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506222PMC
May 2019

Activation of G protein-coupled estrogen receptor signaling inhibits melanoma and improves response to immune checkpoint blockade.

Elife 2018 01 16;7. Epub 2018 Jan 16.

Perelman School of Medicine, Department of Dermatology, University of Pennsylvania, Philadelphia, United States.

Female sex and history of prior pregnancies are associated with favorable melanoma outcomes. Here, we show that much of the melanoma protective effect likely results from estrogen signaling through the G protein-coupled estrogen receptor (GPER) on melanocytes. Selective GPER activation in primary melanocytes and melanoma cells induced long-term changes that maintained a more differentiated cell state as defined by increased expression of well-established melanocyte differentiation antigens, increased pigment production, decreased proliferative capacity, and decreased expression of the oncodriver and stem cell marker c-Myc. GPER signaling also rendered melanoma cells more vulnerable to immunotherapy. Systemically delivered GPER agonist was well tolerated, and cooperated with immune checkpoint blockade in melanoma-bearing mice to dramatically extend survival, with up to half of mice clearing their tumor. Complete responses were associated with immune memory that protected against tumor rechallenge. GPER may be a useful, pharmacologically accessible target for melanoma.
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http://dx.doi.org/10.7554/eLife.31770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770157PMC
January 2018

p15 Expression Differentiates Nevus from Melanoma.

Am J Pathol 2016 12 14;186(12):3094-3099. Epub 2016 Nov 14.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address:

Most melanomas are driven by BRAF(V600E)-activating mutations, while nevi harboring the same mutations have growth arrest. Although decreased p16 expression has been associated with melanoma formation, in recent work, p15 represented a primary effector of oncogene-induced senescence in nevomelanocytes that was diminished in melanomas. This study determined whether decreased p15 levels represent a general biomarker for the transition from nevus to melanoma. We performed p15 and p16 IHC analyses on a random series of nevi and melanomas. Staining was evaluated and graded for percentage and intensity to determine the H score. For real-time quantitative RT-PCR analysis of p15, RNA was extracted from FFPE sections from 14 nevus and melanoma samples via macrodissection. A two-sided t-test was used to evaluate between-group differences in mean H scores and qΔCt values. p15 Expression was significantly increased in melanocytic nevi compared with melanomas (mean H scores, 254.8 versus 132.3; P < 0.001). On p15 staining, the H score differential was greater than that with p16 staining [122.5 (P < 0.001) and 64.8 (P = 0.055), respectively]. Real-time quantitative RT-PCR analysis revealed a lower mean qΔCt value in melanomas, consistent with lower p15 expression (P = 0.018). Together, these data support the hypothesis that decreased p15 expression is a robust biomarker for distinguishing nevus from melanoma.
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http://dx.doi.org/10.1016/j.ajpath.2016.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225287PMC
December 2016

CDKN2B Loss Promotes Progression from Benign Melanocytic Nevus to Melanoma.

Cancer Discov 2015 Oct 16;5(10):1072-85. Epub 2015 Jul 16.

Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

Unlabelled: Deletion of the entire CDKN2B-CDKN2A gene cluster is among the most common genetic events in cancer. The tumor-promoting effects are generally attributed to loss of CDKN2A-encoded p16 and p14ARF tumor suppressors. The degree to which the associated CDKN2B-encoded p15 loss contributes to human tumorigenesis is unclear. Here, we show that CDKN2B is highly upregulated in benign melanocytic nevi, contributes to maintaining nevus melanocytes in a growth-arrested premalignant state, and is commonly lost in melanoma. Using primary melanocytes isolated directly from freshly excised human nevi naturally expressing the common BRAF(V600E)-activating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAF(V600E), we show that BRAF activation results in reversible, TGFβ-dependent, p15 induction that halts proliferation. Furthermore, we engineer human skin grafts containing nevus-derived melanocytes to establish a new, architecturally faithful, in vivo melanoma model, and demonstrate that p15 loss promotes the transition from benign nevus to melanoma.

Significance: Although BRAF(V600E) mutations cause melanocytes to initially proliferate into benign moles, mechanisms responsible for their eventual growth arrest are unknown. Using melanocytes from human moles, we show that BRAF activation leads to a CDKN2B induction that is critical for restraining BRAF oncogenic effects, and when lost, contributes to melanoma.
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http://dx.doi.org/10.1158/2159-8290.CD-15-0196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4592422PMC
October 2015

Filaggrin-2 barrier protein inversely varies with skin inflammation.

Exp Dermatol 2015 Sep 24;24(9):720-2. Epub 2015 Jul 24.

Department of Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

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http://dx.doi.org/10.1111/exd.12749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547533PMC
September 2015

Rational development and characterization of humanized anti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.

Sci Transl Med 2015 Feb;7(275):275ra22

Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 19104, USA. Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.

Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).
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http://dx.doi.org/10.1126/scitranslmed.aaa4963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467166PMC
February 2015

Ionizing radiation selectively reduces skin regulatory T cells and alters immune function.

PLoS One 2014 24;9(6):e100800. Epub 2014 Jun 24.

Division of Infectious Diseases, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

The skin serves multiple functions that are critical for life. The protection from pathogens is achieved by a complicated interaction between aggressive effectors and controlling functions that limit damage. Inhomogeneous radiation with limited penetration is used in certain types of therapeutics and is experienced with exposure to solar particle events outside the protection of the Earth's magnetic field. This study explores the effect of ionizing radiation on skin immune function. We demonstrate that radiation, both homogeneous and inhomogeneous, induces inflammation with resultant specific loss of regulatory T cells from the skin. This results in a hyper-responsive state with increased delayed type hypersensitivity in vivo and CD4+ T cell proliferation in vitro. The effects of inhomogeneous radiation to the skin of astronauts or as part of a therapeutic approach could result in an unexpected enhancement in skin immune function. The effects of this need to be considered in the design of radiation therapy protocols and in the development of countermeasures for extended space travel.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0100800PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4069168PMC
November 2015

Comprehensive analysis of gene expression in human retina and supporting tissues.

Hum Mol Genet 2014 Aug 14;23(15):4001-14. Epub 2014 Mar 14.

Department of Ophthalmology

Understanding the influence of gene expression on the molecular mechanisms underpinning human phenotypic diversity is fundamental to being able to predict health outcomes and treat disease. We have carried out whole transcriptome expression analysis on a series of eight normal human postmortem eyes by RNA sequencing. Here we present data showing that ∼80% of the transcriptome is expressed in the posterior layers of the eye and that there is significant differential expression not only between the layers of the posterior part of the eye but also between locations of a tissue layer. These differences in expression also extend to alternative splicing and splicing factors. Differentially expressed genes are enriched for genes associated with psychiatric, immune and cardiovascular disorders. Enrichment categories for gene ontology included ion transport, synaptic transmission and visual and sensory perception. Lastly, allele-specific expression was found to be significant for CFH, C3 and CFB, which are known risk genes for age-related macular degeneration. These expression differences should be useful in determining the underlying biology of associations with common diseases of the human retina, retinal pigment epithelium and choroid and in guiding the analysis of the genomic regions involved in the control of normal gene expression.
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http://dx.doi.org/10.1093/hmg/ddu114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297232PMC
August 2014

CD164 and FCRL3 are highly expressed on CD4+CD26- T cells in Sézary syndrome patients.

J Invest Dermatol 2014 Jan 21;134(1):229-236. Epub 2013 Jun 21.

Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Sézary syndrome (SS) cells express cell surface molecules also found on normal activated CD4 T cells. In an effort to find a more specific surface marker for malignant SS cells, a microarray analysis of gene expression was performed. Results showed significantly increased levels of mRNA for CD164, a sialomucin found on human CD34+ hematopoietic stem cells, and FCRL3, a molecule present on a subset of human natural T regulatory cells. Both markers were increased in CD4 T cells from SS patients compared with healthy donors (HD). Flow cytometry studies confirmed the increased expression of CD164 and FCRL3 primarily on CD4+CD26- T cells of SS patients. Importantly, a statistically significant correlation was found between an elevated percentage of CD4+CD164+ T cells and an elevated percentage of CD4+CD26- T cells in all tested SS patients but not in patients with mycosis fungoides and atopic dermatitis or HD. FCRL3 expression was significantly increased only in patients with high tumor burden. CD4+CD164+ cells displayed cerebriform morphology and their loss correlated with clinical improvement in treated patients. Our results suggest that CD164 can serve as a marker for diagnosis and for monitoring progression of cutaneous T-cell lymphoma (CTCL)/SS and that FCRL3 expression correlates with a high circulating tumor burden.
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http://dx.doi.org/10.1038/jid.2013.279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3869886PMC
January 2014

IL-31 is produced by the malignant T-cell population in cutaneous T-Cell lymphoma and correlates with CTCL pruritus.

J Invest Dermatol 2013 Dec 22;133(12):2783-2785. Epub 2013 May 22.

Department of Dermatology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA. Electronic address:

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http://dx.doi.org/10.1038/jid.2013.227DOI Listing
December 2013

Viral-associated trichodysplasia: characterization of a novel polyomavirus infection with therapeutic insights.

Arch Dermatol 2012 Feb;148(2):219-23

Department of Dermatology, University of Pennsylvania, 3600 Spruce St, Second Floor, Maloney Bldg, Philadelphia, PA 19104, USA.

Background: Viral-associated trichodysplasia of immunosuppression is a rare cutaneous eruption that is characterized by follicularly based shiny papules and alopecia with characteristic histopathologic findings of abnormally anagen follicules with excessive inner root sheath differentiation. Prior reports have described the histopathologic characteristics on vertical sections; however, to our knowledge, immunohistochemical analysis of polyomavirus proteins has not been previously performed.

Observations: We discuss the thorough diagnostic evaluation and therapy of an unusual case of viral-associated trichodysplasia due to a newly described human polyomavirus that occurred in a patient with posttreatment chronic lymphocytic leukemia and an abnormal white blood cell count. Unique to our study is the immunohistochemical staining for the polyomavirus middle T antigen, which demonstrated positive staining of cellular inclusions within keratinocytes that compose the inner root sheath. Further evaluation with scanning electron microscopy and polymerase chain reaction analysis of viral DNA confirmed the presence of the virus. Treatment with topical cidofovir resulted in dramatic clinical improvement and hair regrowth.

Conclusions: Several tools, including immunohistochemical staining for the polyomavirus middle T antigen, can be used to identify the pathogenic virus associated with viral-associated trichodysplasia. This case highlights the utility of multiple diagnostic modalities and a robust response to a topical therapeutic agent, cidofovir.
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http://dx.doi.org/10.1001/archdermatol.2011.1413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346264PMC
February 2012

Srcasm inhibits Fyn-induced cutaneous carcinogenesis with modulation of Notch1 and p53.

Cancer Res 2009 Dec;69(24):9439-47

Department of Dermatology, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104, USA.

Src family tyrosine kinases (SFK) regulate cell proliferation, and increased SFK activity is common in human carcinomas, including cutaneous squamous cell carcinomas (SCC) and its precursors. The elevated SFK activity in cutaneous SCCs was modeled using K14-Fyn Y528F transgenic mice, which spontaneously form punctate keratotic lesions, scaly plaques, and large tumors resembling actinic keratoses, SCC in situ, and SCCs, respectively. Lesional tissue showed increased levels of activated SFKs, PDK1, STAT3, and ERK1/2, whereas Notch1/NICD protein and transcript levels were decreased. p53 levels also were decreased in SCC in situ and SCCs. Increasing Srcasm levels using a K14-Fyn Y528F/K14-Srcasm double transgenic model markedly inhibited cutaneous neoplasia. In contrast, increased expression of a nonphosphorylatable Srcasm mutant maintained the neoplastic phenotype. Increasing Srcasm levels decreased levels of Fyn, activated SFKs, ERK1/2, PDK1, and phospho-STAT3, and increased Notch1/NICD and p53 levels. Analysis of human specimens revealed that levels of Fyn and activated SFKs were elevated in SCCs compared with adjacent nonlesional epidermis. In addition, Notch1 and Srcasm protein and transcript levels were decreased in human SCCs compared with nonlesional epidermis. Therefore, the SCCs produced by the Fyn Y528F mice resemble their human counterparts at the molecular level. K14-Fyn Y528F mice represent a robust model of cutaneous carcinogenesis that manifests precancerous lesions and SCCs resembling human disease. The Fyn/Srcasm signaling nexus modulates activity of STAT3, PDK1, ERK1/2, Notch1, and p53. Further study of Fyn and Srcasm should provide insights into the mechanisms regulating keratinocyte proliferation and skin carcinogenesis.
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http://dx.doi.org/10.1158/0008-5472.CAN-09-2976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794931PMC
December 2009

Iron chelation protects the retinal pigment epithelial cell line ARPE-19 against cell death triggered by diverse stimuli.

Invest Ophthalmol Vis Sci 2009 Mar 31;50(3):1440-7. Epub 2009 Jan 31.

F. M. Kirby Center for Molecular Ophthalmology and the.

Purpose: Cell death can be induced by exogenous reactive oxygen species (ROS). Endogenous ROS can also play a role in cell death triggered by agents that are not themselves ROS. One of the most potent ROS-generating systems is the iron-catalyzed Fenton reaction. Herein, the authors tested whether iron plays an important role in cell death induced by diverse stimuli in retinal pigment epithelial (RPE) cells.

Methods: The ability of the iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) to chelate intracellular labile iron was tested in the human cell line ARPE-19. The ability of SIH to protect against RPE cell death induced by hydrogen peroxide, staurosporine, anti-Fas, and exposure to A2E plus blue light was determined. ROS production by staurosporine was assessed in the presence and absence of SIH. The protective activity of SIH was compared with that of other iron chelators and an antioxidant.

Results: Acute exposure to SIH was nontoxic and at least partially protective against cell death induced by all tested agents. On a molar basis, SIH was more protective against hydrogen peroxide than other iron chelators and an antioxidant. SIH decreased levels of staurosporine-induced ROS.

Conclusions: Iron chelation with SIH can decrease levels of ROS and protect RPE cells against cell death induced by diverse stimuli. These results suggest a central role for iron in cell death pathways, potentially involving the generation of oxidative stress. SIH or related iron chelators may prove useful for protection against diseases involving RPE death, such as AMD.
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http://dx.doi.org/10.1167/iovs.08-2545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665187PMC
March 2009

Iron prochelator BSIH protects retinal pigment epithelial cells against cell death induced by hydrogen peroxide.

J Inorg Biochem 2008 Dec 24;102(12):2130-5. Epub 2008 Aug 24.

Department of Chemistry, Duke University, Durham, NC 27708-0346, USA.

Dysregulation of localized iron homeostasis is implicated in several degenerative diseases, including Parkinson's, Alzheimer's, and age-related macular degeneration, wherein iron-mediated oxidative stress is hypothesized to contribute to cell death. Inhibiting toxic iron without altering normal metal-dependent processes presents significant challenges for standard small molecule chelating agents. We previously introduced BSIH (isonicotinic acid [2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzylidene]-hydrazide) prochelators that are converted by hydrogen peroxide into SIH (salicylaldehyde isonicotinoyl hydrazone) chelating agents that inhibit iron-catalyzed hydroxyl radical generation. Here, we show that BSIH protects a cultured cell model for retinal pigment epithelium against cell death induced by hydrogen peroxide. BSIH is more stable than SIH in cell culture medium and is more protective during long-term experiments. Repetitive exposure of cells to BSIH is nontoxic, whereas SIH and desferrioxamine induce cell death after repeated exposure. Combined, our results indicate that cell protection by BSIH involves iron sequestration that occurs only when the cells are stressed by hydrogen peroxide. These findings suggest that prochelators discriminate toxic iron from healthy iron and are promising candidates for neuro- and retinal protection.
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http://dx.doi.org/10.1016/j.jinorgbio.2008.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2662444PMC
December 2008

MiRNA expression in the eye.

Mamm Genome 2008 Aug 22;19(7-8):510-6. Epub 2008 Jul 22.

Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA 19104-6055, USA.

MiRNAs are a newly discovered class of small noncoding RNAs that regulate gene expression by translational repression and mRNA degradation. It has become evident that miRNAs are involved in many important biological processes, including tissue differentiation and development. The role of miRNAs in the eye is beginning to be explored following their recent detection by miRNA expression analyses. Many of the target genes for these ocular miRNAs remain undefined. This review summarizes the current information about ocular miRNA expression. Future research should focus on the function of ocular miRNAs in eye development.
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http://dx.doi.org/10.1007/s00335-008-9127-8DOI Listing
August 2008

Ceruloplasmin/hephaestin knockout mice model morphologic and molecular features of AMD.

Invest Ophthalmol Vis Sci 2008 Jun 7;49(6):2728-36. Epub 2008 Mar 7.

FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Purpose: Iron is an essential element in human metabolism but also is a potent generator of oxidative damage with levels that increase with age. Several studies suggest that iron accumulation may be a factor in age-related macular degeneration (AMD). In prior studies, both iron overload and features of AMD were identified in mice deficient in the ferroxidase ceruloplasmin (Cp) and its homologue hephaestin (Heph) (double knockout, DKO). In this study, the location and timing of iron accumulation, the rate and reproducibility of retinal degeneration, and the roles of oxidative stress and complement activation were determined.

Methods: Morphologic analysis and histochemical iron detection by Perls' staining was performed on retina sections from DKO and control mice. Immunofluorescence and immunohistochemistry were performed with antibodies detecting activated complement factor C3, transferrin receptor, L-ferritin, and macrophages. Tissue iron levels were measured by atomic absorption spectrophotometry. Isoprostane F2alpha-VI, a specific marker of oxidative stress, was quantified in the tissue by gas chromatography/mass spectrometry.

Results: DKOs exhibited highly reproducible age-dependent iron overload, which plateaued at 6 months of age, with subsequent progressive retinal degeneration continuing to at least 12 months. The degeneration shared some features of AMD, including RPE hypertrophy and hyperplasia, photoreceptor degeneration, subretinal neovascularization, RPE lipofuscin accumulation, oxidative stress, and complement activation.

Conclusions: DKOs have age-dependent iron accumulation followed by retinal degeneration modeling some of the morphologic and molecular features of AMD. Therefore, these mice are a good platform on which to test therapeutic agents for AMD, such as antioxidants, iron chelators, and antiangiogenic agents.
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http://dx.doi.org/10.1167/iovs.07-1472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2569891PMC
June 2008

Isoprostane F2alpha-VI, a new marker of oxidative stress, increases following light damage to the mouse retina.

Mol Vis 2007 Feb 7;13:190-5. Epub 2007 Feb 7.

F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA.

Purpose: A number of studies have suggested that retinal light damage involves oxidative stress. These include demonstration of protection by antioxidants, immunohistochemical detection of oxidative stress markers, and upregulation of antioxidant enzymes. Recently a new specific marker of lipid peroxidation (LPO), isoprostane F(2alpha)-VI, has been developed. This prostaglandin isomer is produced by nonenzymatic oxidation of membrane-linked arachidonic acid. Because it provides an unusually stable and specific measure of LPO, we sought to determine whether its levels would increase following retinal light damage.

Methods: Balb/c mice were exposed to bright fluorescent light for 7 h. Twenty-eight h after light exposure, photoreceptor death was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis. Isoprostane F(2alpha)-VI was quantified in retinal extracts by gas chromatography/mass spectrometry. Retinal isoprostane was localized by immunofluorescence.

Results: TUNEL analysis demonstrated photoreceptor cell death after light exposure. Compared with controls, retina extracts from mice exposed to fluorescent light had a significant increase in isoprostane F(2alpha)-VI levels following light damage. Immunohistochemistry confirmed an increase in retinal isoprostane.

Conclusions: Elevated levels of isoprostane F(2alpha)-VI, a stable, highly specific marker of lipid peroxidation, confirm earlier reports of light-mediated retinal lipid peroxidation, potentially an important mechanism of retinal degeneration. Further, since levels of isoprostane F(2alpha)-VI are readily quantified, its measurement provides a new means to specifically monitor retinal oxidative damage caused by prooxidants such as light.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2610371PMC
February 2007

Retinal localization and copper-dependent relocalization of the Wilson and Menkes disease proteins.

Invest Ophthalmol Vis Sci 2006 Jul;47(7):3129-34

F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

Purpose: Menkes and Wilson diseases are associated with retinal degeneration. The Menkes and Wilson genes are homologous copper transporters, but differences in their expression pattern lead to different disease manifestations. To determine whether the Wilson and Menkes genes may act locally in the retina, this study was undertaken to assess retinal Wilson and Menkes expression and localization.

Methods: RT/PCR was used to test for the presence of Wilson and Menkes mRNAs in mouse and human retinas and retinal pigment epithelial cell lines. The Menkes and Wilson proteins were immunolocalized in human and mouse retinas and in the ARPE-19 cell line.

Results: The Menkes mRNA and protein were present in the RPE and neurosensory retina whereas the Wilson mRNA and protein were limited to the RPE. In the RPE, both proteins localized to the Golgi. Increased copper concentration led to relocalization of the Wilson protein to a diffuse cytoplasmic distribution.

Conclusions: Both the Menkes and Wilson proteins are present in the RPE. Since the RPE is a blood-brain barrier, these proteins most likely regulate not only their own copper levels but also copper levels of the overlying photoreceptors. Because the Wilson protein delivers copper to the ferroxidase ceruloplasmin in the liver, it is likely that the Wilson and/or Menkes proteins provide copper to ceruloplasmin made in the RPE. Retinopathy in Wilson and Menkes diseases may result not only from abnormal systemic copper levels but also from loss of retinal Wilson or Menkes protein.
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http://dx.doi.org/10.1167/iovs.05-1601DOI Listing
July 2006

The iron carrier transferrin is upregulated in retinas from patients with age-related macular degeneration.

Invest Ophthalmol Vis Sci 2006 May;47(5):2135-40

Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

Purpose: Iron can cause oxidative stress, and elevated iron levels have been associated with several neurodegenerative diseases including age-related macular degeneration (AMD). Transferrin, an iron transport protein, is expressed at high levels in the retina. The purpose of this study was to assess transferrin involvement in AMD by determining the expression profile of transferrin in retinas with AMD compared with retinas without evidence of disease.

Methods: Postmortem retinas were obtained from AMD and non-AMD eyes. Expression of transferrin was assessed in a microarray dataset from 33 retinas of unaffected donors and 12 retinas of patients with AMD (six with neovascular AMD and six with non-neovascular AMD). Quantitative real-time RT-PCR (QPCR) was used to confirm the microarray results. Transferrin protein expression was assessed by semiquantitative Western blot analysis and immunohistochemistry.

Results: In comparison to unaffected retinas, mean transferrin mRNA levels, as measured by microarray analysis were elevated 3.5- and 2.1-fold in non-neovascular and neovascular AMD retinas, respectively. Semiquantitative Western blot analysis demonstrated a 2.1-fold increase in transferrin protein in AMD eyes. Immunohistochemistry showed more intense and widespread transferrin label in AMD maculas, particularly in large drusen, Müller cells, and photoreceptors.

Conclusions: These data demonstrate that transferrin expression is increased in the retinas of patients with AMD relative to those of healthy control patients of comparable age. Along with previous studies that have demonstrated elevated iron levels in AMD retinas, early onset drusen formation in a patient with retinal iron overload resulting from aceruloplasminemia, and retinal degeneration with some features of macular degeneration in the iron-overloaded retinas of ceruloplasmin/hephestin knockout mice, the present study suggests that altered iron homeostasis is associated with AMD.
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http://dx.doi.org/10.1167/iovs.05-1135DOI Listing
May 2006

Disruption of ceruloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration.

Proc Natl Acad Sci U S A 2004 Sep 13;101(38):13850-5. Epub 2004 Sep 13.

The F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

Mechanisms of brain and retinal iron homeostasis have become subjects of increased interest after the discovery of elevated iron levels in brains of patients with Alzheimer's disease and retinas of patients with age-related macular degeneration. To determine whether the ferroxidase ceruloplasmin (Cp) and its homolog hephaestin (Heph) are important for retinal iron homeostasis, we studied retinas from mice deficient in Cp and/or Heph. In normal mice, Cp and Heph localize to Müller glia and retinal pigment epithelium, a blood-brain barrier. Mice deficient in both Cp and Heph, but not each individually, had a striking, age-dependent increase in retinal pigment epithelium and retinal iron. The iron storage protein ferritin was also increased in Cp-/-Heph-/Y retinas. After retinal iron levels had increased, Cp-/-Heph-/Y mice had age-dependent retinal pigment epithelium hypertrophy, hyperplasia and death, photoreceptor degeneration, and subretinal neovascularization, providing a model of some features of the human retinal diseases aceruloplasminemia and age-related macular degeneration. This pathology indicates that Cp and Heph are critical for CNS iron homeostasis and that loss of Cp and Heph in the mouse leads to age-dependent retinal neurodegeneration, providing a model that can be used to test the therapeutic efficacy of iron chelators and antiangiogenic agents.
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http://dx.doi.org/10.1073/pnas.0405146101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC518844PMC
September 2004

Immunolocalization and regulation of iron handling proteins ferritin and ferroportin in the retina.

Mol Vis 2004 Aug 26;10:598-607. Epub 2004 Aug 26.

F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, Philadelphia, PA, USA.

Purpose: CNS iron accumulation is associated with several neurodegenerative diseases, including age-related macular degeneration. Intracellular overload of free iron is prevented, in part, by the iron export protein, ferroportin, and the iron storage protein, ferritin. The purpose of this study was to assess retinal localization and regulation of ferroportin and ferritin.

Methods: Normal murine retinas were analyzed by immunohistochemistry to localize ferroportin, cytosolic ferritin, and mitochondrial ferritin, with double-labeling using cell-specific markers to identify cell types. Retinas deficient in the ferroxidases, ceruloplasmin and hephaestin, accumulate iron in their retinas and RPE, while retinas deficient in iron regulatory proteins (IRPs) lack the ability to regulate several proteins involved in iron metabolism; retinas from these knockout mice along with their age matched wild type littermates were also examined to study regulation of ferritin and ferroportin. To enable visualization of label in the retinal pigment epithelial cells, sections from pigmented mice were bleached with H2O2 prior to IHC, a novel use of this technique for study of the RPE.

Results: In normal retinas, cytosolic ferritins were found predominantly in rod bipolar cells and photoreceptors. Ferroportin was found in RPE and Müller cells. Iron accumulation in mice deficient in ceruloplasmin and hephaestin was associated with upregulation of ferritin and ferroportin. Mice deficient in IRPs showed upregulation of ferritin and ferroportin, likely because of their inability to repress translation.

Conclusions: Normal retinas contain ferritin and ferroportin, whose levels are regulated by iron-responsive, iron regulatory proteins. Ferroportin colocalizes with ceruloplasmin and hephaestin to RPE and Müller cells, supporting a potential cooperation between these ferroxidases and the iron exporter. Cytosolic ferritin accumulates in rod bipolar synaptic terminals, suggesting that ferritin may be involved in axonal iron transport. Mitochondrial ferritin increases with iron accumulation, suggesting a role in iron storage.
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August 2004

Increased metallothionein in light damaged mouse retinas.

Exp Eye Res 2004 Aug;79(2):287-93

F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

Oxidative stress plays a role in human age-related macular degeneration and in the light damage model of retinal degeneration. Metallothionein (MT), an antioxidant, has been reported to protect retinal pigment epithelial cells against apoptosis and oxidative stress. The purpose of this study was to evaluate changes in MT expression level and retinal localization following light damage. To accomplish this, Balb/c mice were exposed to cool white fluorescent light (10,000 lx) for 7 hr. In three independent experiments, at several intervals after the light injury, retinal MTs were studied at the protein level by immunohistochemistry (IHC) and Western analysis, and at the mRNA level by quantitative PCR with isoform-specific primers. Western analysis and IHC indicated an increase in metallothionein protein following light damage. MT localized to the retinal pigment epithelium and several layers of neural retina. Quantitative PCR identified the expression of MT I-III isoforms, not the MT IV isoform in the mouse retina, and, following light damage, showed increased expression of retinal MT-I and MT-II mRNAs by 8- and 22-fold, respectively. Increased expression of the antioxidant MT in the light damaged mouse retina suggests that upregulation of MT is an important acute retinal response to photo-oxidative stress.
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http://dx.doi.org/10.1016/j.exer.2004.05.004DOI Listing
August 2004

Light damage induced changes in mouse retinal gene expression.

Exp Eye Res 2004 Aug;79(2):239-47

F.M. Kirby Center for Molecular Ophthalmology Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

Oxidative stress plays a role in the light damage model of retinal degeneration as well as in age-related macular degeneration. The purpose of this study is to identify retinal genes induced by acute photo-oxidative stress, which may function as mediators of apoptosis or as survival factors. To accomplish this, Balb/c mice were exposed to bright cool white fluorescent light for 7 hr. Retinas were then isolated for total RNA preparation followed by Affymetrix DNA microarray analysis to compare gene expression in light damaged mice to unexposed controls. Three independent light damage experiments were carried out and statistical filters were applied to detect genes with expression changes averaging at least two-fold. Quantitative PCR was carried out to confirm altered gene expression. Seventy genes were upregulated at least two-fold immediately following light damage. QPCR confirmed upregulation of all 10 genes tested. The upregulated genes fall into several categories including antioxidants: ceruloplasmin, metallothionein, and heme oxygenase; antiapoptotic gene: bag3, chloride channels: clic1 and clic4; transcription factors: c-fos, fra1, junB, stat1, krox-24 and c/ebp; secreted signaling molecules: chitinase 3-like protein 1 and osteopontin; inflammation related genes: MCP-1 and ICAM1 and others. Upregulation of five interferon-gamma responsive genes suggests elevated interferon levels after light damage. Upregulation of three components of the AP-1 transcription factor is consistent with previous evidence implicating AP-1 in light damage pathogenesis. Four copper or iron binding proteins were upregulated, suggesting that photo-oxidative stress may affect metal homeostasis. The genes found upregulated by light damage may affect the survival of photoreceptors subjected to photo-oxidative stress.
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http://dx.doi.org/10.1016/j.exer.2004.05.002DOI Listing
August 2004

Attenuated nef DNA vaccine construct induces cellular immune response: role in HIV-1 multiprotein vaccine.

Immunol Lett 2003 Oct;89(2-3):207-14

Department of Infectious Diseases and Microbiology, University of Pittsburgh/GSPH, 130 DeSoto Street, Pittsburgh, PA 15261, USA.

HIV-1 positive patients generate Nef-specific CTL response, indicating that Nef is a potent immunogen. However, Nef is also known to down regulate the expression of CD4 and MHC-I molecules, thereby protecting virally infected target cells. We compared the immunogenicity of non-functional nef vaccine constructs to wild type functional nef as potential immunogen. Mice were immunized with different nef constructs and assessed for their ability to induce cellular immune responses. Evaluation of T cell immune responses in mice showed that non-functional nef vaccine constructs are capable of inducing a significant T cell immune response measured by IFN-gamma ELISPOT. Further epitope mapping studies indicate that one of our attenuated constructs, Nef R-38, has multiple CTL epitopes spanning throughout the gene. Our results indicate that functionally attenuated Nef antigen might be a better candidate for future multiprotein HIV-1 vaccine.
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http://dx.doi.org/10.1016/s0165-2478(03)00141-xDOI Listing
October 2003