Prashiela Manga, Ph.D.

Impact of population genetic substructure on association studies and risk assessment for melanoma
Lobach I; Belitskaya-Levy I; Goldberg JD; Ostrer H; Berman RS; Pavlick AC; Shapiro RL; Osman I; Manga P
2011 ;29(Suppl):?-? #8521, Journal of clinical oncology
Background: Genetic substructure due to varying allele frequencies between populations can confound association studies. Ancestry informative genetic marker (AIMs) data combined with statistical adjustment can reveal spurious associations and identify population specific risk markers. In melanoma, AIMs may also be risk markers, e.g. pigment genes contribute to melanoma susceptibility and segregate with ancestry. We have thus developed a strategy to adjust for population genetic substructure (PGS) using AIMs, while identifying potentially novel genes associated with melanoma. Methods: 326 melanoma patients and 400 controls of European ancestry from the New York area were studied. Tag SNPs spanning 14 candidate genes and 75 AIMs were genotyped and odds ratios (OR), unadjusted and adjusted for PGS, computed. Results: A PGS model based on all AIMs separated cases and controls, suggesting that some AIMs were associated with melanoma. An algorithm was developed to select AIMs least capable of separating cases and controls to infer PGS and validated using simulations. The resulting model, which was reproduced using 49 additional AIMs, separated Northern (NE) and Southern Europeans (SE) and was used to adjust ORs. Three classes of SNPs were identified 1. Associated before and after PGS correction in both groups (10 SNPs localized to MATP, TYR and ERCC5). 2. Not associated in unadjusted analysis, but significantly associated with melanoma in NEs (6 SNPs localized to XPC, ERCC4, OCA2, ASIP and TYR). 3. Associated with melanoma before but not after adjustment. To determine if AIMs that separate cases and controls can identify novel melanoma genes, we genotyped 16 SNPs localized to 4 genes that house candidate AIMs. Four SNPs at 2 different loci were associated with melanoma (e.g. AIM1: OR=0.35, p=0.01; AIM2: OR=1.77, p=0.03). Conclusions: Our approach demonstrated that ancestry is a significant confounding factor in identifying melanoma susceptibility genes. Melanoma risk markers vary significantly between groups and a DNA based risk assessment model will require adjustment for ancestry. We have also identified potentially novel susceptibility melanoma genes for futher study
— id: 132473, year: 2011, vol: 29, page: ?, stat: Journal Article,

Informed reasoning: repositioning of nitisinone to treat oculocutaneous albinism
Manga, Prashiela; Orlow, Seth J
2011 Oct 3;121(10):3828-3831, Journal of clinical investigation
Oculocutaneous albinism (OCA) is a group of genetic disorders characterized by hypopigmentation of the skin, hair, and eyes. Affected individuals experience reduced visual acuity and substantially increased skin cancer risk. There are four major types of OCA (OCA1-OCA4) that result from disruption in production of melanin from tyrosine. Current treatment options for individuals with OCA are limited to attempts to correct visual problems and counseling to promote use of sun protective measures. However, Onojafe et al., reporting in this issue of the JCI, provide hope for a new treatment approach for OCA, as they demonstrate that treating mice that model OCA-1b with nitisinone, which is FDA approved for treating hereditary tyrosinemia type 1, elevates plasma tyrosine levels, and increases eye and hair pigmentation
— id: 141072, year: 2011, vol: 121, page: 3828, stat: Journal Article,

Identifying novel therapeutic agents for vitiligo
Tan, A. U.; Orlow, S. J.; Manga, P.
2011 APR ;131(5):S121-S121, Journal of investigative dermatology
— id: 131840, year: 2011, vol: 131, page: S121, stat: Journal Article,

Role of oxidative stress and unfolded protein response in development of vitiligo
Toosi, S.; Orlow, S. J.; Manga, P.
2011 APR ;131(5):S120-S120, Journal of investigative dermatology
— id: 131839, year: 2011, vol: 131, page: S120, stat: Journal Article,

Differential adaptation to chronic ER stress in wildtype and Oca2 melanocytes
Cheng T.; Manga P.; Bis S.; Knoll K.; Orlow S.S.
2010 ;23(5):710-710, Pigment cell & melanoma research
Accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR) to enable cells to recover from ER stress. If the UPR fails to restore normal ER function, apoptosis is induced instead. We have demonstrated that melanocytes have the capacity to adapt to chronic ER stress and escape from UPR-induced cell death. Mutations in the pinkeyed dilution/oculocutaneous albinism type 2 (Oca2)-gene result in altered tyrosinase processing and trafficking in melanocytes, accompanied by accumulation of misfolded tyrosinase in the ER. Despite this chronic overload of ER-retained tyrosinase, Oca2-mutant melanocytes do not show diminished viability suggesting that they have adapted to the ER stress. The aim of this study is to elucidate the process of adaptation to ER stress in melanocytes. Differential protein expression between wildtype (melan-a) and Oca2-melanocytes (melan-p) was analyzed using microarray assays and Western blotting. Adaptation to chronic ER stress was studied by comparing wildtype murine melanocytes dosed with the ER stressor thapsigargin to Oca2-mutants. We observed increased Ire1 expression in Oca2-melanocytes compared to wildtype, indicating that this UPR pathway is activated. Prolonged Ire1 signaling after UPR activation in stressed cells has been found to promote cell survival. In addition, expression of proteins involved in the pro-apoptotic Perk pathway appears to be decreased in Oca2- melanocytes. However, Oca2-mutants also demonstrated up-regulation of Chop, which typically promotes cell death by down-regulating expression of anti-apoptotic Bcl-2. Remarkably, the pro-apoptotic effects of Chop expression appear to be mitigated by up-regulation of Bcl-2 expression. Furthermore, expression of pro-apoptotic proteins such as Bid and caspase 1 were down-regulated in Oca2-mutant melanocytes as compared to wildtype cells. Acute ER stress (0-24 h) in wildtype melanocytes activated all three UPR pathways, but Ire1 signaling was attenuated in chronically stressed cells (12 days), while Perk signaling was maintained. Cell viability did not change during this period. Thus, wildtype melanocytes adapted to chronic ER stress despite sustained activation of pro-apoptotic pathways. These results indicate that chronically stressed wildtype melanocytes and Oca2-mutant melanocytes adapt to ER stress by differential mechanisms. Melanocytes may thus adapt to ER-stress by multiple mechanisms, some of which may account for the increased drug resistance observed in melanocytes and melanoma
— id: 113676, year: 2010, vol: 23, page: 710, stat: Journal Article,

Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation
Kadekaro, Ana Luisa; Leachman, Sancy; Kavanagh, Renny J; Swope, Viki; Cassidy, Pamela; Supp, Dorothy; Sartor, Maureen; Schwemberger, Sandy; Babcock, George; Wakamatsu, Kazumasa; Ito, Shosuke; Koshoffer, Amy; Boissy, Raymond E; Manga, Prashiela; Sturm, Richard A; Abdel-Malek, Zalfa A
2010 Oct;24(10):3850-3860, FASEB journal
The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to alpha-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to alpha-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively. These variants had different heterozygous effects on receptor function. Microarray data confirmed the observed differences in responses of melanocytes with functional vs. nonfunctional receptor to alpha-melanocortin and ultraviolet radiation, and identified DNA repair and antioxidant genes that are modulated by alpha-melanocortin. Our findings highlight the molecular mechanisms by which the melanocortin 1 receptor genotype controls genomic stability of and the mutagenic effect of ultraviolet radiation on human melanocytes
— id: 115707, year: 2010, vol: 24, page: 3850, stat: Journal Article,

Delineation of the unfolded protein response in melanocytes: Potential implications for vitiligo and UV response
Manga P.; Vega M.; Bis S.; Knoll K.; Orlow S.
2010 ;23(5):704-705, Pigment cell & melanoma research
Background: Accumulation of immature proteins in the Endoplasmic Reticulum (ER) causes organelle stress that is counteracted by the unfolded protein stress response (UPR). Three pathways compose the UPR and are initiated when Ire1, Perk and Atf6 respectively, are released from heterodimers formed with the ER chaperone BiP. The UPR signals down-regulation of global translation and increased ER-chaperone expression. Ire1 is phosphorylated, activating its nuclease activity, which leads to splicing of the X-box binding protein 1 (Xbp1). The spliced RNA encodes a transcription factor that regulates expression of a subset of genes that comprise one arm of the UPR. Apoptosis is initiated if homeostasis is not re-established following UPR activation. The Ire1 pathway has recently been shown to play arole in the development of vitiligo, while the UPR has been implicated in keratinocyte response to UVB and in drug resistance in melanoma. We therefore investigated the melanocyte response to ER stress induced by chemical ER disruptors and by oxidative stress (the mechanism by which we propose UV exposure perturbs the melanocyte ER). Method: Wild-type mouse melanocytes were treated with thapsigargin, which disrupts the calcium balance in the ER causing UPR induction, and cells harvested at 6, 12 and 24 h. Western blot and microarray analyses were performed and data evaluated to identify pathways activated by thapsigargin treatment. In addition, melanocytes were dosed with compounds that induce oxidative stress. RNA was harvested and evaluated for activation of the UPR by Xbp-1 splicing. Results: IRE1 expression was upregulated within 6 h of treatment with thapsigargin, which promoted splicing of XBP1 mRNA and activation of its transcription factor activity. PERK and its downstream target CHOP were phosphorylated and HA-tagged ATF6 was cleaved within 6-12 h of treatment. Up-regulation of BiP and ER chaperones such as Ero1 and down-regulation of tyrosinase were also observed. In addition, several p53-related pathways were modulated in response to thapsigargin. Induction of oxidative stress was found to induce Xbp1 splicing. Conclusions: The UPR may play an important role in melanocyte response to stress, including response to oxidative stress induced by UV exposure. Dysfunction of this response may contribute to initiation and progression of vitiligo and drug resistance in melanoma
— id: 113675, year: 2010, vol: 23, page: 704, stat: Journal Article,

The unfolded protein response in melanocytes: activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding
Manga, Prashiela; Bis, Sabina; Knoll, Kristen; Perez, Beremis; Orlow, Seth J
2010 Oct;23(5):627-634, Pigment cell & melanoma research
Summary Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways
— id: 112422, year: 2010, vol: 23, page: 627, stat: Journal Article,

From melanocyte to malignant metastatic melanoma. L
Manga, Prashiela; Hoek, Keith S; Davids, Lester M; Leachman, Sancy A
2010 ;2010:?-?, Dermatology research & practice
— id: 113806, year: 2010, vol: 2010, page: ?, stat: Journal Article,

Identification of tyrosinase polymorphisms for use in melanoma risk assessment
Pervolaraki E; Lobach I; Belitskaya-Levy I; Ostrer H; Goldberg JD; Polsky D; Shapiro RL; Berman RS; Osman I; Manga P
2010 ;28(15S):?-? #8570, Journal of clinical oncology
Background: Most skin cancer-related deaths are due to malignant melanoma. Risk assessment criteria for melanoma currently include skin phenotype, family and sun exposure history, factors that are subject to observer and recall bias. Genetic markers of susceptibility have been identified in association studies; however little progress has been made in developing them to improve screening and identification of individuals at risk of melanoma. Tyrosinase (TYR), a known susceptibility gene and a determinant of skin pigmentation, was thus investigated further to characterize its association with melanoma susceptibility and to identify markers which can be used in a risk assessment model. Methods: The cohort consisted of 326 individuals diagnosed with melanoma and 400 control subjects. TYR was interrogated using fifteen tag single nucleotide polymorphisms (SNPs) spanning the gene and statistical association tests performed. Additionally, ancestry informative markers were utilized to correct for population genetic sub-structure. Haplotype analysis was performed to determine if specific regions of the gene contributed more significantly to susceptibility. Coding regions of the gene are currently being sequenced and identified variants will be tested for impact on enzymatic function. Results: Of the 15 SNPs, 8 were associated with melanoma; 4 with decreased risk (Odds ratios 0.41-0.71) and 4 with increased risk (Odds ratios 1.43-1.96). SNPs localized to 2 regions of the gene (spanning exon 1 to intron 2 and intron 3 to 4) with markers of increased as well as decreased susceptibility present in both areas. With the exception of one coding region variant, SNPs were localized to introns. Conclusions: SNPs localized to TYR may serve as useful biomarkers for determining susceptibility to melanoma. We are currently sequencing the gene in our population in order to identify additional and potentially more potent markers of melanoma susceptibility. Coding region variants are being characterized for their effect on protein stability and enzyme activity such that functional active variants (most likely to affect susceptibility to melanoma) can be identified and assessed for their utility in melanoma risk assessment
— id: 111554, year: 2010, vol: 28, page: ?, stat: Journal Article,

Initiation of the unfolded protein response in melanocytes and melanoma
Bis, SG; Knoll, KE; Lolis, MS; Orlow, SJ; Manga, P
2009 APR ;129(5):S139-S139, Journal of investigative dermatology
— id: 97878, year: 2009, vol: 129, page: S139, stat: Journal Article,

Association of MDM2 SNP309, age of onset, and gender in cutaneous melanoma
Firoz, Elnaz F; Warycha, Melanie; Zakrzewski, Jan; Pollens, Danuta; Wang, Guimin; Shapiro, Richard; Berman, Russell; Pavlick, Anna; Manga, Prashiela; Ostrer, Harry; Celebi, Julide Tok; Kamino, Hideko; Darvishian, Farbod; Rolnitzky, Linda; Goldberg, Judith D; Osman, Iman; Polsky, David
2009 Apr 1;15(7):2573-2580, Clinical cancer research
PURPOSE: In certain cancers, MDM2 SNP309 has been associated with early tumor onset in women. In melanoma, incidence rates are higher in women than in men among individuals less than 40 years of age, but among those older than 50 years of age, melanoma is more frequent in men than in women. To investigate this difference, we examined the association among MDM2 SNP309, age at diagnosis, and gender among melanoma patients. EXPERIMENTAL DESIGN: Prospectively enrolled melanoma patients (N = 227) were evaluated for MDM2 SNP309 and the related polymorphism, p53 Arg72Pro. DNA was isolated from patient blood samples, and genotypes were analyzed by PCR-restriction fragment length polymorphism. Associations among MDM2 SNP309, p53 Arg72Pro, age at diagnosis, and clinicopathologic features of melanoma were analyzed. RESULTS: The median age at diagnosis was 13 years earlier among women with a SNP309 GG genotype (46 years) compared with women with TG+TT genotypes (59 years; P = 0.19). Analyses using age dichotomized at each decade indicated that women with a GG genotype had significantly higher risks of being diagnosed with melanoma at ages <50 years compared with women >or=50 years, but not when the comparison was made between women <60 and >or=60 years. At ages <50 years, women with a GG genotype had a 3.89 times greater chance of being diagnosed compared with women with TG+TT genotypes (P = 0.01). Similar observations were not seen among men. CONCLUSIONS: Our data suggest that MDM2 may play an important role in the development of melanoma in women. The MDM2 SNP309 genotype may help identify women at risk of developing melanoma at a young age
— id: 104875, year: 2009, vol: 15, page: 2573, stat: Journal Article,

Developing genetic markers for melanoma risk assessment
Manga P.; Goldberg J.D.; Belitskaya-Levy I.; Lobach I.; Polsky D.; Pavlick A.; Shapiro R.; Berman R.; Osman I.; Ostrer H.
2009 ;27(15 Suppl 1):9046-9046, Journal of clinical oncology
Background: Risk assessment for melanoma is currently based on phenotype, family and exposure history. This approach is subject to recall bias and excludes at-risk groups such as those with darker skin pigmentation. Poorly stratified risk pools also result in unnecessary dermatologist visits and biopsies for those at lower risk. Use of genetic markers may improve risk assessment; however few susceptibility markers have been developed to date. There have been a number of reports of association between melanoma and genetic markers though few have been replicated or validated. In addition, these studies frequently utilized specific coding region variants as markers and failed to test the entire gene. We have therefore assembled a case-control cohort in which to search for potential biomarkers for melanoma risk by interrogating genes using recently developed tools for genetic analysis. A pilot study was performed to test the utility of our cohort. Methods: A cohort of 326 individuals diagnosed with melanoma and treated at the New York University Langone Medical Center and 400 controls obtained from the New York Cancer project was assembled. Candidate genes were selected based on involvement in determining melanoma predisposition factors (skin pigmentation and DNA repair capability) and previous studies showing association. Three genes, ERCC1, ERCC4 (DNA repair) and MATP (skin pigmentation) were selected. Tag Single Nucleotide Polymorphisms (tSNPs) were selected using Haploview (Hapmap.org) and DNA genotyped (Sequenom Inc, San Diego, CA). Odds ratios and confidence intervals were computed for each SNP. Results: An association was found between SNP rs11615 at the ERCC1 locus and melanoma (Odds ratio = 1.718, 95% Confidence interval: 1.259 - 2.343 for TT vs TC/CC). Conclusions: A tSNP approach is thus useful in identifying associations in our melanoma case-control cohort. Sequence variation at the ERCC1 locus contributes to melanoma risk and the gene will now be screened for clinically useful susceptibility biomarkers. Additional DNA repair and pigmentation genes will also be interrogated using this approach. Genes found to be associated with melanoma will be screened by high- density SNP analysis to identify the most appropriate biomarker/s for use in risk assessment
— id: 111805, year: 2009, vol: 27, page: 9046, stat: Journal Article,

Evaluation of the melanocortin-1-receptor gene in melanoma predisposition, progression and recurrence
Sidash S; Ostrer H; Goldberg JD; Belitskaya-Levy I; Lobach I; Polsky D; Shapiro RL; Berman RS; Osman I; Manga P
2009 ;27:15S-15S, Journal of clinical oncology
— id: 102306, year: 2009, vol: 27, page: 15S, stat: Journal Article,

Developing a multidisciplinary prospective melanoma biospecimen repository to advance translational research
Wich, Lindsay G; Hamilton, Heather K; Shapiro, Richard L; Pavlick, Anna; Berman, Russell S; Polsky, David; Goldberg, Judith D; Hernando, Eva; Manga, Prashiela; Krogsgaard, Michelle; Kamino, Hideko; Darvishian, Farbod; Lee, Peng; Orlow, Seth J; Ostrer, Harry; Bhardwaj, Nina; Osman, Iman
2009 ;1(1):35-43, American Journal of Translational Research
Several challenges face the development and operation of a biospecimen bank linked to clinical information, a critical component of any effective translational research program. Melanoma adds particular complexity and difficulty to such an endeavor considering the unique characteristics of this malignancy. We describe here a review of biospecimen bank and our experience in establishing a multi-disciplinary, prospective, integrated clinicopathological-biospecimen database in melanoma. The Interdisciplinary Melanoma Cooperative Group (IMCG), a prospective clinicopathological and biospecimen database, was established at the New York University (NYU) Langone Medical Center. With patients' informed consent, biospecimens from within and outside NYU, clinicopathological data, and follow-up information are collected using developed protocols. Information pertaining to biospecimens is recorded in 35 fields, and clinicopathological information is recorded in 371 fields within 5 modules in a virtual network system. Investigators conducting research utilizing the IMCG biospecimen resource are blind to clinicopathological information, and molecular data generated using biospecimens are linked independently with clinicopathological data by biostatistics investigators. This translational research enterprise acts as a valuable resource to efficiently translate laboratory discoveries to the clinic
— id: 105566, year: 2009, vol: 1, page: 35, stat: Journal Article,

Inhibition of mitochondrial protein translation sensitizes melanoma cells to arsenic trioxide cytotoxicity via a reactive oxygen species dependent mechanism
Bowling, Benjamin D; Doudican, Nicole; Manga, Prashiela; Orlow, Seth J
2008 Dec;63(1):37-43, Cancer chemotherapy & pharmacology
PURPOSE: Current standard chemotherapeutic regimens for malignant melanoma are unsatisfactory. Although in vitro studies of arsenic trioxide (ATO) have demonstrated promise against melanoma, recent phase II clinical trials have failed to show any significant clinical benefit when used as a single agent. To enhance the efficacy of ATO in the treatment of melanoma, we sought to identify compounds that potentiate the cytotoxic effects of ATO in melanoma cells. Through a screen of 2,000 marketed drugs and naturally occurring compounds, a variety of antibiotic inhibitors of mitochondrial protein translation were identified. METHODS: The mechanism of action for the most effective agent identified, thiostrepton, was examined in a panel of melanoma cells. Effects of combinatorial ATO and thiostrepton treatment on cytotoxicity, apoptosis, mitochondrial protein content, and reactive oxygen species (ROS) were assessed. RESULTS: Thiostrepton (1 microM) sensitized three out of five melanoma cell lines to ATO-mediated growth inhibition. Treatment with thiostrepton resulted in reduced levels of the mitochondrial-encoded protein cytochrome oxidase I (COX1). Exposure to thiostrepton in combination with ATO resulted in increased levels of cleaved poly (ADP-ribose) polymerase and cellular ROS. The growth inhibitory and pro-apototic effects of addition of the ATO/thiostrepton combination were reversed by the free radical scavenger N-acetyl-L-cysteine. CONCLUSIONS: Our data suggest that thiostrepton enhances the cytotoxic effects of ATO through a ROS-dependent mechanism. Co-administration of oxidative stress-inducing drugs such as thiostrepton in order to enhance the efficacy of ATO in the treatment of melanoma warrants further investigation
— id: 91428, year: 2008, vol: 63, page: 37, stat: Journal Article,

A role for the pink-eyed dilution protein in tyrosinase folding
Manga, P; Knoll, K; Fenton, J; Orlow, SJ
2008 APR ;21(2):261-262, Pigment cell & melanoma research
— id: 78651, year: 2008, vol: 21, page: 261, stat: Journal Article,

Does melanin have an SPF and can it be measured?
Epstein, H; Manga, P; Koshoffer, A; Story, D; Simion, T; Boissy, R
2007 SEP-OCT ;58(5):596-597, Journal of cosmetic science
— id: 87151, year: 2007, vol: 58, page: 596, stat: Journal Article,

A Role for Tyrosinase-Related Protein 1 in 4-tert-Butylphenol-Induced Toxicity in Melanocytes: Implications for Vitiligo
Manga, Prashiela; Sheyn, David; Yang, Fan; Sarangarajan, Rangaprasad; Boissy, Raymond E
2006 Nov;169(5):1652-1662, American journal of pathology
Vitiligo presents with depigmented cutaneous lesions following localized melanocyte death. Multiple factors contribute to cell death, including genetically determined susceptibility to trauma, and environmental factors, such as exposure to 4-tert-butylphenol (4-TBP). We demonstrate that 4-TBP induces oxidative stress that is more readily overcome by melanocytes from normally pigmented individuals than from two individuals with vitiligo. The antioxidant catalase selectively and significantly reduced death of melanocytes derived from two individuals with vitiligo, indicating a role for oxidative stress in vitiligo pathogenesis. In normal melanocytes, oxidative stress results in reduced expression of microphthalmia-associated transcription factor (MITF). Melanocyte-stimulating hormone-induced expression of MITF protein caused increased sensitivity to 4-TBP, whereas sensitivity of melanomas correlated with MITF expression. MITF stimulates melanin synthesis by up-regulating expression of melanogenic enzymes such as tyrosinase-related protein-1 (Tyrp1). Although melanin content per se did not affect sensitivity to 4-TBP, expression of Tyrp1 significantly increased sensitivity. Melanocytes and melanomas that express functional Tyrp1 were significantly more sensitive to 4-TBP than Tyrp1-null cells. Thus, normal melanocytes respond to 4-TBP by reducing expression of MITF and Tyrp1. We hypothesize that melanocytes in vitiligo demonstrate reduced ability to withstand oxidative stress due, partly, to a disruption in MITF regulation of Tyrp1
— id: 69049, year: 2006, vol: 169, page: 1652, stat: Journal Article,

On the etiology of contact/occupational vitiligo
Boissy, Raymond E; Manga, Prashiela
2004 Jun;17(3):208-214, Pigment cell research
Vitiligo is an acquired depigmentary disorder of the skin that results from the selective destruction of melanocytes, generally during the second decade of life and affecting approximately 1% of the population worldwide. Loss of cutaneous pigment appears to render the skin susceptible to premature aging and cancer. In addition this disease can be socially devastating for afflicted individuals. The etiology of vitiligo is poorly understood. The present dogma suggests that genetic factors render the melanocyte fragile thus predisposing individuals to developing vitiligo. When subjected to instigating factors, these susceptible, fragile melanocytes undergo apoptosis. Autoimmune factors then perpetuate the removal of the melanocyte component from the skin. In the majority of cases the instigating factors are not known (idiopathic vitiligo), however a small sub-set of individuals develop contact/occupational vitiligo following exposure to particular chemicals. Many of these chemicals have been implicated in both contact/occupational vitiligo and chemical leukoderma. Both conditions present with well-defined, depigmented skin lesions that develop following exposure. Only in the case of vitiligo does the depigmentation spread beyond the areas of contact, probably via an immune-mediated mechanism. The largest class of chemicals known to trigger contact/occupational vitiligo is the phenolic/catecholic derivatives. Many have been demonstrated to be preferentially cytotoxic to melanocytes, with high-dose exposure resulting in the initiation of apoptosis. Phenolic/catecholic derivatives are structurally similar to the melanin precursor tyrosine, and therefore tyrosinase was originally implicated as a mediator of cytotoxicity. However, our data suggests that tyrosinase-related protein-1, rather than tyrosinase, facilitates toxicity, possibly by catalytic conversion of the compounds, which results in the generation of radical oxygen species. The ensuing oxidative stress then triggers activation of cellular free radical scavenging pathways to prevent cell death. Genetic inability of melanocytes to tolerate and/or respond to the oxidative stress may underlie the etiology of contact/occupational vitiligo
— id: 68898, year: 2004, vol: 17, page: 208, stat: Journal Article,

Pink-eyed dilution protein controls the processing of tyrosinase
Chen, Kun; Manga, Prashiela; Orlow, Seth J
2002 Jun;13(6):1953-1964, Molecular biology of the cell
The processing of tyrosinase, which catalyzes the limiting reaction in melanin synthesis, was investigated in melan-p1 melanocytes, which are null at the p locus. Endoglycosidase H digestion showed that a significant fraction of tyrosinase was retained in the endoplasmic reticulum. This retention could be rescued either by transfection of melan-p1 cells with an epitope-tagged wild-type p transcript or by treatment with either bafilomycin A1 or ammonium chloride. We found that the endoplasmic reticulum contains a significant amount of p protein, thus supporting a role for p within this compartment. Using immunofluoresence, we showed that most mature full-length tyrosinase in melan-p1 cells was located in the perinuclear area near the Golgi, in contrast to its punctate melanosomal pattern in wild-type melanocytes. Expression of p in melan-p1 cells restored tyrosinase to melanosomes. Triton X-114 phase separation revealed that an increased amount of tyrosinase was proteolyzed in melan-p1 cells compared with wild-type melanocytes. The proteolyzed tyrosinase was no longer membrane bound, but remained enzymatically active and a large proportion was secreted into the culture medium of melan-p1 cells. We conclude that p regulates posttranslational processing of tyrosinase, and hypopigmentation in melan-p1 cells is the result of altered tyrosinase processing and trafficking
— id: 34784, year: 2002, vol: 13, page: 1953, stat: Journal Article,

Pink-eyed Dilution Protein Modulates Arsenic Sensitivity and Intracellular Glutathione Metabolism
Staleva, Liliana; Manga, Prashiela; Orlow, Seth J
2002 Dec;13(12):4206-4220, Molecular biology of the cell
Mutations in the mouse p (pink-eyed dilution) and human P genes lead to melanosomal defects and ocular developmental abnormalities. Despite the critical role played by the p gene product in controlling tyrosinase processing and melanosome biogenesis, its precise biological function is still not defined. We have expressed p heterologously in the yeast Saccharomyces cerevisiae to study its function in greater detail. Immunofluorescence studies revealed that p reaches the yeast vacuolar membrane via the prevacuolar compartment. Yeast cells expressing p exhibited increased sensitivity to a number of toxic compounds, including arsenicals. Similarly, cultured murine melanocytes expressing a functional p gene were also found to be more sensitive to arsenical compounds compared with p-null cell lines. Intracellular glutathione, known to play a role in detoxification of arsenicals, was diminished by 50% in p-expressing yeast. By using the glutathione-conjugating dye monochlorobimane, in combination with acivicin, an inhibitor of vacuolar gamma-glutamyl cysteine transpeptidase, involved in the breakdown of glutathione, we found that p facilitates the vacuolar accumulation of glutathione. Our data demonstrate that the pink-eyed dilution protein increases cellular sensitivity to arsenicals and other metalloids and can modulate intracellular glutathione metabolism
— id: 34781, year: 2002, vol: 13, page: 4206, stat: Journal Article,

Abnormal tyrosinase processing and fate in melanocytes lacking the pink-eyed dilution gene
Chen, K; Manga, P; Orlow, S
2001 AUG ;117(2):510-510, Journal of investigative dermatology
— id: 54904, year: 2001, vol: 117, page: 510, stat: Journal Article,

Mislocalization of melanosomal proteins in melanocytes from mice with oculocutaneous albinism type 2
Manga P; Boissy RE; Pifko-Hirst S; Zhou BK; Orlow SJ
2001 Jun;72(6):695-710, Experimental eye research
More than 10% of admissions worldwide to institutions for the visually impaired are due to some form of albinism. The most common form, oculocutaneous albinism type 2, results from mutations at the p locus. The function of the p gene is yet to be determined. It has been shown that melanocytes from p -null mice exhibit an abnormal melanosomal ultrastructure in addition to alterations in activity and localization of tyrosinase, a critical melanogenic enzyme. In light of these observations, we examined tyrosinase trafficking in p -null vs wildtype mouse melanocytes in order to explore p function. Electron microscopy of wildtype melan-a and p -null melan-p1 cells demonstrated accumulation of tyrosinase in 50 nm vesicles throughout the cell in the absence of p, an observation corroborated by an increase in tyrosinase activity in vesicle-enriched fractions from melan-p1 compared to melan-a cells. Misrouting in the absence of p was not limited to tyrosinase; a second melanosomal protein, tyrosinase-related protein 1, also trafficked incorrectly. In melan-p1, mislocalization led to secretion of tyrosinase into the medium. Adding tyrosine to the medium was found to partially correct tyrosinase trafficking and to reduce secretion; the cysteine protease inhibitor E64 also reduced secretion. We propose that p is required by melanocytes for transport of melanosomal proteins. In its absence, tyrosinase accumulates in vesicles and, in cultured melanocytes, is proteolysed and secreted.
— id: 21192, year: 2001, vol: 72, page: 695, stat: Journal Article,

Inverse correlation between pink-eyed dilution protein expression and induction of melanogenesis by bafilomycin A1
Manga P; Orlow SJ
2001 Oct;14(5):362-367, Pigment cell research
The pink-eyed dilution protein (p) plays a pivotal role in the synthesis of eumelanin. In its absence, critical melanosomal proteins fail to traffic to the melanosome. Pink-eyed dilution gene (P) mutations are the most common cause of tyrosinase-positive oculocutaneous albinism worldwide. Thus, reports that bafilomycin A1 was able to induce synthesis of melanin in tyrosinase-positive melanomas led us to test the drug on p-null murine melanocytes. We found that in melanocytes lacking p, bafilomycin A1 was able to induce melanin synthesis. These cells, once transfected with an expression vector encoding an epitope-tagged p transcript, failed to respond to the drug. The increase in melanin synthesis is accompanied by a reduction in tyrosinase protein cleavage and secretion with subsequent accumulation within the melanocyte. Bafilomycin A1 has also been reported to induce pigmentation of normal Caucasian melanocytes. Based on these data we hypothesize that p may serve as a key control point at which ethnic skin color variation is determined
— id: 34788, year: 2001, vol: 14, page: 362, stat: Journal Article,

Mutational analysis of the modulation of tyrosinase by tyrosinase-related proteins 1 and 2 in vitro
Manga P; Sato K; Ye L; Beermann F; Lamoreux ML; Orlow SJ
2000 Oct;13(5):364-374, Pigment cell research
The albino (tyrosinase, Tyrc), brown (tyrosinase-related protein 1, Tyrp1b) and slaty (tyrosinase-related protein 2, tyrp2slt) loci are all involved in the regulation of melanogenesis. Phenotypes of inbred mice mutant at two or more of these loci are not always explicable by simple summation of the established or suspected catalytic functions of the gene products. These phenotypes suggest that relationships among the proteins extend beyond the obvious fact that they catalyze different steps in the same melanogenic pathway, and that they may also interact intimately in such a way that a mutation in one impacts the function of the other(s). Previous studies have attributed catalytic activities to each member of this trio; however, it has been difficult to study the proteins individually, either in vivo or in tissues or cells. Therefore, we undertook to transfect the genes, in revealing combinations, into COS-7 cells (which have no melanogenic apparatus of their own) to clarify the interacting functions of their encoded proteins. Specifically, we attempted to evaluate the effects of Tyrp1 and Tyrp2 proteins on tyrosinase protein. We report evidence that Tyrp1 stabilizes tyrosinase, confirming previous observations, and, in addition, demonstrate that Tyrp1 decreases tyrosinase activity. By contrast, Tyrp2 increases tyrosinase activity by stabilizing the protein. We conclude that both Tyrp1 and Tyrp2, in addition to other catalytic functions they may possess, act together to modulate tyrosinase activity
— id: 34794, year: 2000, vol: 13, page: 364, stat: Journal Article,

The pink-eyed dilution gene and the molecular pathogenesis of tyrosinase-positive albinism (OCA2)
Manga P; Orlow SJ
1999 Nov;26(11):738-747, Journal of dermatology
— id: 11863, year: 1999, vol: 26, page: 738, stat: Journal Article,