Showing papers by "John A. D'Orazio published in 2017"

The melanocortin signaling cAMP axis accelerates repair and reduces mutagenesis of platinum-induced DNA damage

Abstract: Using primary melanocytes and HEK293 cells, we found that cAMP signaling accelerates repair of bi- and mono-functional platinum-induced DNA damage. Elevating cAMP signaling either by the agonistic MC1R ligand melanocyte stimulating hormone (MSH) or by pharmacologic cAMP induction by forskolin enhanced clearance of intrastrand cisplatin-adducts in melanocytes or MC1R-transfected HEK293 cells. MC1R antagonists human beta-defensin 3 and agouti signaling protein blocked MSH- but not forskolin-mediated enhancement of platinum-induced DNA damage. cAMP-enhanced repair of cisplatin-induced DNA damage was dependent on PKA-mediated phosphorylation of ATR on S435 which promoted ATR's interaction with the key NER factor xeroderma pigmentosum A (XPA) and facilitated recruitment of an XPA-ATR-pS435 complex to sites of cisplatin DNA damage. Moreover, we developed an oligonucleotide retrieval immunoprecipitation (ORiP) assay using a novel platinated-DNA substrate to establish kinetics of ATR-pS435 and XPA's associations with cisplatin-damaged DNA. Expression of a non-phosphorylatable ATR-S435A construct or deletion of A kinase-anchoring protein 12 (AKAP12) impeded platinum adduct clearance and prevented cAMP-mediated enhancement of ATR and XPA's associations with cisplatin-damaged DNA, indicating that ATR phosphorylation at S435 is necessary for cAMP-enhanced repair of platinum-induced damage and protection against cisplatin-induced mutagenesis. These data implicate cAMP signaling as a critical regulator of genomic stability against platinum-induced mutagenesis.

Paracrine regulation of melanocyte genomic stability: a focus on nucleotide excision repair

Abstract: UV radiation is a major environmental risk factor for the development of melanoma by causing DNA damage and mutations. Resistance to UV damage is largely determined by the capacity of melanocytes to respond to UV injury by repairing mutagenic photolesions. The nucleotide excision repair (NER) pathway is the major mechanism by which cells correct UV photodamage. This multistep process involves the basic steps of damage recognition, isolation, localized strand unwinding, assembly of a repair complex, excision of the damage-containing strand 3' and 5' to the photolesion, synthesis of a sequence-appropriate replacement strand, and finally ligation to restore continuity of genomic DNA. In melanocytes, the efficiency of NER is regulated by several hormonal pathways including the melanocortin and endothelin signaling pathways. Elucidating molecular mechanisms by which melanocyte DNA repair is regulated offers the possibility of developing novel melanoma-preventive strategies to reduce UV mutagenesis, especially in UV-sensitive melanoma-prone individuals.

Divergence of cAMP signalling pathways mediating augmented nucleotide excision repair and pigment induction in melanocytes.

Abstract: Loss-of-function melanocortin 1 receptor (MC1R) polymorphisms are common in UV-sensitive fair-skinned individuals and are associated with blunted cAMP second messenger signaling and higher lifetime risk of melanoma because of diminished ability of melanocytes to cope with UV damage. cAMP signaling positions melanocytes to resist UV injury by up-regulating synthesis of UV-blocking eumelanin pigment and by enhancing the repair of UV-induced DNA damage. cAMP enhances melanocyte nucleotide excision repair (NER), the genome maintenance pathway responsible for the removal of mutagenic UV photolesions, through cAMP-activated protein kinase (protein kinase A)-mediated phosphorylation of the ataxia telangiectasia mutated and Rad3 related (ATR) protein on the S435 residue. We investigated the interdependence of cAMP-mediated melanin upregulation and cAMP-enhanced DNA repair in primary human melanocytes and a melanoma cell line. We observed that the ATR-dependent molecular pathway linking cAMP signaling to the NER pathway is independent of MITF activation. Similarly, cAMP-mediated up-regulation of pigment synthesis is independent of ATR, suggesting that the key molecular events driving MC1R-mediated enhancement of genome maintenance (e.g. PKA-mediated phosphorylation of ATR) and MC1R-induced pigment induction (e.g. MITF activation) are distinct. This article is protected by copyright. All rights reserved.

Hormonal Regulation of the Repair of UV Photoproducts in Melanocytes by the Melanocortin Signaling Axis.

Abstract: Melanoma is the deadliest form of skin cancer because of its propensity to spread beyond the primary site of disease and because it resists many forms of treatment. Incidence of melanoma has been increasing for decades. Though ultraviolet radiation (UV) has been identified as the most important environmental causative factor for melanoma development, UV-protective strategies have had limited efficacy in melanoma prevention. UV mutational burden correlates with melanoma development and tumor progression, underscoring the importance of UV in melanomagenesis. However, besides amount of UV exposure, melanocyte UV mutational load is influenced by the robustness of nucleotide excision repair, the genome maintenance pathway charged with removing UV photoproducts before they cause permanent mutations in the genome. In this review, we highlight the importance of the melanocortin hormonal signaling axis on regulating efficiency of nucleotide excision repair in melanocytes. By understanding the molecular mechanisms by which nucleotide excision repair can be increased, it may be possible to prevent many cases of melanoma by reducing UV mutational burden over time. This article is protected by copyright. All rights reserved.

Retraction Note: The melanocortin signaling cAMP axis accelerates repair and reduces mutagenesis of platinum-induced DNA damage

Abstract: Using primary melanocytes and HEK293 cells, we found that cAMP signaling accelerates repair of bi- and mono-functional platinum-induced DNA damage. Elevating cAMP signaling either by the agonistic MC1R ligand melanocyte stimulating hormone (MSH) or by pharmacologic cAMP induction by forskolin enhanced clearance of intrastrand cisplatin-adducts in melanocytes or MC1R-transfected HEK293 cells. MC1R antagonists human beta-defensin 3 and agouti signaling protein blocked MSH- but not forskolin-mediated enhancement of platinum-induced DNA damage. cAMP-enhanced repair of cisplatin-induced DNA damage was dependent on PKA-mediated phosphorylation of ATR on S435 which promoted ATR’s interaction with the key NER factor xeroderma pigmentosum A (XPA) and facilitated recruitment of an XPA-ATR-pS435 complex to sites of cisplatin DNA damage. Moreover, we developed an oligonucleotide retrieval immunoprecipitation (ORiP) assay using a novel platinated-DNA substrate to establish kinetics of ATR-pS435 and XPA’s associations with cisplatin-damaged DNA. Expression of a non-phosphorylatable ATR-S435A construct or deletion of A kinase-anchoring protein 12 (AKAP12) impeded platinum adduct clearance and prevented cAMP-mediated enhancement of ATR and XPA’s associations with cisplatin-damaged DNA, indicating that ATR phosphorylation at S435 is necessary for cAMP-enhanced repair of platinum-induced damage and protection against cisplatin-induced mutagenesis. These data implicate cAMP signaling as a critical regulator of genomic stability against platinum-induced mutagenesis.

Getting a tan without getting SIK.

Abstract: It has been more than a decade since the Fisher lab reported that epidermal eumelanin could be robustly up-regulated by topical application of forskolin, an adenylyl cyclase activator that bypassed a defective melanocortin 1 receptor (Mc1r) to promote melanin synthesis. Eumelanin induction protected fair-skinned mice from UV damage using an animal model that recapitulates many of the features of pigmented human epidermis. Through constitutive production of stem cell factor (Kit ligand) in the basal epidermis, the K14-Scf transgenic mouse retains interfollicular melanocytes in the skin and therefore accumulates melanin pigments in the epidermis. This article is protected by copyright. All rights reserved.