John A. D’Orazio

PKA-Mediated Phosphorylation of ATR Promotes Recruitment of XPA to UV-Induced DNA Damage

The melanocortin 1 receptor (MC1R), which signals through cAMP, is a melanocytic transmembrane receptor involved in pigmentation, adaptive tanning, and melanoma resistance. We report MC1R-mediated or pharmacologically-induced cAMP signaling promotes nucleotide excision repair (NER) in a cAMP-dependent protein kinase A (PKA)-dependent manner. PKA directly phosphorylates ataxia telangiectasia and Rad3-related protein (ATR) at Ser435, which actively recruits the key NER protein xeroderma pigmentosum complementation group A (XPA) to sites of nuclear UV photodamage, accelerating clearance of UV-induced photolesions and reducing mutagenesis. Loss of Ser435 within ATR prevents PKA-mediated ATR phosphorylation, disrupts ATR-XPA binding, delays recruitment of XPA to UV-damaged DNA, and elevates UV-induced mutagenesis. This study mechanistically links cAMP-PKA signaling to NER and illustrates potential benefits of cAMP pharmacological rescue to reduce UV mutagenesis in MC1R-defective, melanoma-susceptible individuals.

Prolonged treatment of fair-skinned mice with topical forskolin causes persistent tanning and UV protection.

We previously reported that topical application of forskolin to the skin of fair‐skinned MC1R‐defective mice with epidermal melanocytes resulted in accumulation of eumelanin in the epidermis and was highly protective against UV‐mediated cutaneous injury. In this report, we describe the long‐term effects of chronic topical forskolin treatment in this animal model. Forskolin‐induced eumelanin production persisted through 3 months of daily applications, and forskolin‐induced eumelanin remained protective against UV damage as assessed by minimal erythematous dose (MED). No obvious toxic changes were noted in the skin or overall health of animals exposed to prolonged forskolin therapy. Body weights were maintained throughout the course of topical forskolin application. Topical application of forskolin was associated with an increase in the number of melanocytes in the epidermis and thickening of the epidermis due, at least in part, to an accumulation of nucleated keratinocytes. Together, these data suggest in this animal model, short‐term topical regular application of forskolin promotes eumelanin induction and that over time, topical forskolin treatment is associated with persistent melanization, epidermal cell accumulation, and skin thickening.

Melanocortin 1 Receptor: Structure, Function, and Regulation

The melanocortin 1 receptor (MC1R) is a melanocytic Gs protein coupled receptor that regulates skin pigmentation, UV responses, and melanoma risk. It is a highly polymorphic gene, and loss of function correlates with a fair, UV-sensitive, and melanoma-prone phenotype due to defective epidermal melanization and sub-optimal DNA repair. MC1R signaling, achieved through adenylyl cyclase activation and generation of the second messenger cAMP, is hormonally controlled by the positive agonist melanocortin, the negative agonist agouti signaling protein, and the neutral antagonist β-defensin 3. Activation of cAMP signaling up-regulates melanin production and deposition in the epidermis which functions to limit UV penetration into the skin and enhances nucleotide excision repair (NER), the genomic stability pathway responsible for clearing UV photolesions from DNA to avoid mutagenesis. Herein we review MC1R structure and function and summarize our laboratory’s findings on the molecular mechanisms by which MC1R signaling impacts NER.

Stem cell factor rescues tyrosinase expression and pigmentation in discreet anatomic locations in albino mice

The K14‐SCF transgenic murine model of variant pigmentation is based on epidermal expression of stem cell factor (SCF) on the C57BL/6J background. In this system, constitutive expression of SCF by epidermal keratinocytes results in retention of melanocytes in the interfollicular basal layer and pigmentation of the epidermis itself. Here, we extend this animal model by developing a compound mutant transgenic amelanotic animal defective at both the melanocortin 1 receptor (Mc1r) and tyrosinase (Tyr) loci. In the presence of K14‐Scf, tyrosinase‐mutant animals (previously thought incapable of synthesizing melanin) exhibited progressive robust epidermal pigmentation with age in the ears and tails. Furthermore, K14‐SCF Tyrc2j/c2j animals demonstrated tyrosinase expression and enzymatic activity, suggesting that the c2j Tyr defect can be rescued in part by SCF in the ears and tail. Lastly, UV sensitivity of K14‐Scf congenic animals depended mainly on the amount of eumelanin present in the skin. These findings suggest that c‐kit signaling can overcome the c2j Tyr mutation in the ears and tails of aging animals and that UV resistance depends on accumulation of epidermal eumelanin.

Inorganic arsenic inhibits the nucleotide excision repair pathway and reduces the expression of XPC

Chronic exposure to arsenic, most often through contaminated drinking water, has been linked to several types of cancer in humans, including skin and lung cancer. However, the mechanisms underlying its role in causing cancer are not well understood. There is evidence that exposure to arsenic can enhance the carcinogenicity of UV light in inducing skin cancers and may enhance the carcinogenicity of tobacco smoke in inducing lung cancers. The nucleotide excision repair (NER) pathway removes different types of DNA damage including those produced by UV light and components of tobacco smoke. The aim of the present study was to investigate the effect of sodium arsenite on the NER pathway in human lung fibroblasts (IMR-90 cells) and primary mouse keratinocytes. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6-4 photoproducts (6-4 PP) and cyclobutane pyrimidine dimers (CPDs). We find a concentration-dependent inhibition of the removal of 6-4 PPs and CPDs in both cell types treated with arsenite. Treatment of both cell types with arsenite resulted in a significant reduction in the abundance of XPC, a protein that is critical for DNA damage recognition in NER. The abundance of RNA expressed from several key NER genes was also significantly reduced by treatment of IMR-90 cells with arsenite. Finally, treatment of IMR-90 cells with MG-132 abrogated the reduction in XPC protein, suggesting an involvement of the proteasome in the reduction of XPC protein produced by treatment of cells with arsenic. The inhibition of NER by arsenic may reflect one mechanism underlying the role of arsenic exposure in enhancing cigarette smoke-induced lung carcinogenesis and UV light-induced skin cancer, and it may provide some insights into the emergence of arsenic trioxide as a chemotherapeutic agent.

Melanoma — Epidemiology, Genetics and Risk Factors

The U.S. National Cancer Institute’s Surveillance Epidemiology and End Results (SEER) Cancer Statistics Review estimates over 70,000 people will be diagnosed and 9,000 will die from melanoma in the United States in 2012. Though melanoma can affect persons of essen‐ tially any age, it is mainly a disease of adulthood, with median ages of diagnosis and death between 61 and 68 years, respectively (Weinstock, 2012). Nonetheless, melanoma incidence is increasing across age groups, over the past several decades in the United States (Fig. 1) (Ekwueme et al., 2011). In 1935, the average American individual had a 1 in 1,500 lifetime risk of developing melanoma. In 2002, the approximate risk of developing melanoma increased to 1 in 68 individuals (Rigel, 2002). Globally, Australia and New Zealand have the highest incidence rate of melanoma, an abundance of fair-skinned residents living in a UV-rich geography widely believed to be a major factor (Lens and Dawes, 2004). The current melanoma risk for Australian and New Zealander populations may be as high as 1 in 50 (Rigel, 2010). Considering melanoma is being diagnosed more often in young adults, could be prevented by UV-avoiding behaviors, and can be associated with extensive morbidity and mortality, it is truly an emerging public health concern. Part of the apparent increase in melanoma incidence may be due to better surveillance and earlier detection (Erdmann et al., 2012) however, even with heightened melanoma awareness and screening, there seems to have been a real increase in melanoma incidence over the past several decades.

Purification and growth of melanocortin 1 receptor (Mc1r)-defective primary murine melanocytes is dependent on stem cell factor (SFC) from keratinocyte-conditioned media

The melanocortin 1 receptor (MC1R) is a transmembrane Gs-coupled surface protein found on melanocytes that binds melanocyte-stimulating hormone and mediates activation of adenylyl cyclase and generation of the second messenger cyclic AMP (cAMP). MC1R regulates growth and differentiation of melanocytes and protects against carcinogenesis. Persons with loss-of-function polymorphisms of MC1R tend to be UV-sensitive (fair-skinned and with a poor tanning response) and are at high risk for melanoma. Mechanistic studies of the role of MC1R in melanocytic UV responses, however, have been hindered in part because Mc1r-defective primary murine melanocytes have been difficult to culture in vitro. Until now, effective growth of murine melanocytes has depended on cAMP stimulation with adenylyl cyclase-activating or phosphodiesterase-inhibiting agents. However, rescuing cAMP in the setting of defective MC1R signaling would be expected to confound experiments directly testing MC1R function on melanocytic UV responses. In this paper, we report a novel method of culturing primary murine melanocytes in the absence of pharmacologic cAMP stimulation by incorporating conditioned supernatants containing stem cell factor derived from primary keratinocytes. Importantly, this method seems to permit similar pigment expression by cultured melanocytes as that found in the skin of their parental murine strains. This novel approach will allow mechanistic investigation into MC1R’s role in the protection against UV-mediated carcinogenesis and determination of the role of melanin pigment subtypes on UV-mediated melanocyte responses.

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

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.

Venous Thromboembolic Disease in Children and Adolescents

The VTE is mainly a disease of the older adult, though its incidence has increased significantly in the pediatric population over the past several years. This trend is likely due to enhanced awareness and recognition of VTE, as well as increased prevalence of thromboembolic associated risk factors, such as increases in the proportion of children with predisposing medical conditions. The evaluation and management of a child with VTE is similar to that of adults, however pediatric patients have their own distinct aspects of care, stemming from particularities of the hemostatic system, age-related risk factors and differences in response to anticoagulant and antithrombotic therapy. This review addresses the risk factors and the evaluation and management of children with VTE.

Autoimmune Hemolytic Anemia in a 2-Year-Old Child With Pneumococcal Pneumonia

A 2-year-old boy presented to his primary care provider with a 2-week history of daily fevers (to 39°C), malaise, increasing pallor, decreased appetite, and a 3-lb weight loss. Review of systems was otherwise negative (no history of cough, vomiting, diarrhea, or rash). The child was fully immunized and had no relevant past medical problems. His first cousin had been treated for autoimmune hemolytic anemia (AIHA) 3 years prior. An officebased complete blood count found leukocytosis (39 000 cells/mL) and profound anemia (hemoglobin 6.3 g/dL); platelets were read as “clumped” and therefore were uninterpretable. A presumptive diagnosis of acute leukemia was entertained, and the child was transferred to our institution for further management. The child was well developed and in no distress. Height and weight plotted at the 95th and 25th percentiles, respectively. Temperature was 37.2°C, heart rate was 176 beats per minute, and regular and blood pressure, initially obtained at 148/99 mm Hg in the first minutes of hospitalization, later normalized. Respirations were regular and unlabored at 38 per minute, and resting oxygen saturation was 93% as measured by pulse oximetry. HEENT examination was unremarkable and there was no evidence of exanthem, purpurae, or abnormal lymphadenopathy. There was tachycardia without murmur. Auscultation of the chest revealed the presence of breath sounds on both sides and no rales or rhonchi. Only when auscultory findings were directly compared from one side to the other were decreased breath sounds on the left side appreciated. The abdomen was soft and there was no hepatosplenomegaly. Neurologic examination, including gait, was normal for age. A complete blood count confirmed a leukocytosis (40 100 cells/mL), but the differential was inconsistent with acute leukemia; there were only 1% blasts. Rather, neutrophils and bands together accounted for 80% of total white cells. In addition, there were 14% lymphocytes, 3% plasma cells, and 2% nucleated red blood cells. Hemoglobin was 5.1 g/dL, hematocrit 14.8%, and platelet count 657 000/mL. The mean corpuscular volume was 96 fL, and the reticulocyte count was 6.4%. The peripheral blood smear revealed thrombocytosis, a leftshifted leukocytosis including scattered early myeloid forms and blasts, moderate erythrocyte polychromasia, anisocytosis, and slight macrocytosis (Figure 1). Serum electrolytes and uric acid were essentially normal, but lactate dehydrogenase was elevated at 423 U/L. Flow cytometric analysis of the peripheral blood, performed to investigate the possibility of a clonal process such as leukemia, revealed a polyclonal, reactive leukocytosis of normal blood cells. We interpreted the elevated reticulocyte count to be consistent with a destructive red cell process and investigated various causes of hemolysis. A direct Coomb’s test was strongly positive for both IgG and deposition of C3 complement. We next considered the child’s tachypnea, hypoxia, and differential breath sounds. The chest radiograph was markedly abnormal, showing near-complete opacification of the left hemithorax (Figure 2A). To differentiate a primary pneumonic process from a pleural accumulation, a chest computed tomography scan was performed (Figure 2B). The computed tomography scan found evidence of a large left pleural effusion with almost complete collapse of the underlying lung and a slight rightward mediastinal shift. A blood culture rapidly grew gram positive cocci in pairs and chains, later identified as Streptococcus pneumoniae. The child was thus diagnosed with AIHA associated with pneumococcal pneumonia, empyema, and bacteremia. He was transfused with red blood cells and treated with a combination of ceftriaxone and vancomycin. The next day, video-assisted thoracoscopic surgery (VATS) with decortication was performed to remove infectious material surrounding the left lung. A moderate amount of thick fluid within the left chest