Low level, long-term inorganic arsenite exposure causes generalized resistance to apoptosis in cultured human keratinocytes: Potential role in skin co-carcinogenesis Jingbo Pi 1 , Yuying He 2 , Carl Bortner 3 , Jianli Huang 2 , Jie Liu 1 , Tong Zhou 4 , Wei Qu 1 , Susan L. North 5 , Kazimierz S. Kasprzak 5 , Bhalchandra A. Diwan 6 , Colin F. Chignell 2 and Michael P. Waalkes 1 * 1 Laboratory of Comparative Carcinogenesis, National Cancer Institute at the National Institute of Environmental Health Sciences, NIH, Research Triangle Park, NC, USA 2 Laboratory of Pharmacology and Chemistry, NIEHS, NIH, Research Triangle Park, NC, USA 3 Laboratory of Signal Transduction, NIEHS, Research Triangle Park, NC, USA 4 Department of Pathology & Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 5 Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, MD, USA 6 Basic Research Program, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, MD, USA Inorganic arsenic is a human carcinogen that targets the skin. Car- cinogenesis is a multistep process in which acquired apoptotic resistance is a common event and prior work in non-skin cells shows acquired resistance to apoptosis occurs with chronic arsenite expo- sure. In the present study, when HaCaT cells, an immortalized, non-tumorigenic human keratinocyte cell line, were continuously exposed to low-level inorganic arsenite (as sodium arsenite; 100 nM) for 28 weeks, the cells acquired a generalized resistance to apoptosis. This included resistance to apoptosis induced by acute high concentrations of arsenite, ultraviolet A (UVA) irradiation, and several chemotherapeutic compounds (cisplatin, etoposide and doxorubicin). These arsenite-tolerant (As-TL) cells showed similar levels of UVA-induced reactive oxygen species (ROS) and oxidative DNA damage when compared to passage match control cells. Because cellular apoptosis is dependent on the balance between proapoptotic and survival pathways, the roles of protein kinase B (PKB), a key antiapoptotic molecule, in this acquired apoptotic resistance were investigated. Stimulation of apoptosis markedly decreased nuclear phosphorylated PKB (P-PKB) levels in control cells, but As-TL cells showed greatly increased stability of nuclear P-PKB. Pretreatment of the As-TL cells with LY294002 or Wort- mannin, which specifically inhibit PKB phosphorylation, completely blocked apoptotic resistance in As-TL cells, indicating acquired apoptotic resistance is associated with increased stability of nuclear P-PKB. Because arsenic and UV irradiation are co-carcinogenic in mouse skin, resistance to UV-induced apoptosis in As-TL cells may allow UV-damaged cells to escape normal cell population controls and initiate the carcinogenic cascade. The observation that As-TL cells show no lessening of UV-induced genotoxicity supports this possibility. ' 2005 Wiley-Liss, Inc. Key words: arsenite; apoptosis; ultraviolet; carcinogenesis; PKB Arsenic is a naturally occurring element that is present ubiqui- tously in the environment in both inorganic and organic forms. Human exposure to the generally more toxic inorganic arsenic compounds occur in occupational or environmental settings, as well as through medicinal arsenical use. 1 In recent years, arsenic contamination of drinking water has become a major public health concern worldwide, especially in Asia. 2,3 A primary concern for chronic arsenic exposure in human populations is its carcinogenic potential. Inorganic arsenic has been classified as a known human carcinogen based on strong epidemiological data. 1 Arsenic expo- sure in humans is associated with a marked increase in skin can- cer. 4,5 This is potentially due to the high affinity of arsenic for sulfhydryl groups, which leads to arsenic accumulation and reten- tion in keratin-rich skin tissue. 6 Characteristic arsenic-induced skin lesions are verrucous hyperkeratosis and pigmentation disor- ders. 3 Other proliferative skin lesions associated with human arsenic exposure include Bowen’s disease and squamous cell or basal cell carcinoma. 1,7,8 It is estimated that in the United States 1.3 million new cases of skin cancer are diagnosed annually. 9 Ultraviolet (UV) radiation from sunlight is the primary carcinogen in development of human skin cancer and can act as both an initiator and promoter. 9 Recent work in mouse skin models of carcinogenesis shows that arsenic acts as a co-carcinogen or co-promoter to induce tumors. 8,10 In particular, oral arsenic greatly enhances the incidence and progres- sion of skin malignancies induced by ultraviolet radiation. 10 Arsenic alone, however, does not induce skin tumor development in these model systems. 8,10 This points toward some sort of arsenic-induced enhancement of the carcinogenic potential of other agents, such as UV radiation, as a likely mechanism. Trans- placental inorganic arsenite exposure in mice acts alone to produce various internal cancers including liver and lung tumors, 11,12 but does not initiate skin cancers. 12 Events in the skin associated with arsenic carcinogenesis may be distinct from other target tissues and deserve additional investigation. Several lines of evidence indicate that tumorigenesis is a multi- step process involving progressive transformation of normal cells into malignant phenotypes along with the aberrant accumulation of such abnormal cells. 13,14 Agents that impact the carcinogenic process may act during malignant transformation or aberrant cell accumulation. In this regard, acquired resistance to apoptosis is a hallmark of most cancers. 13 Under normal circumstances apopto- sis eliminates DNA-damaged or mutated cells. 15 Acquired resist- ance to apoptosis may be a critical molecular event during carcinogenesis, and disruption of apoptosis has been shown to play a major role in tumor formation and malignant progression. 15 Dur- ing long-term low level arsenite exposure both rodent liver cells and human prostate epithelial cells acquire a remarkable general- ized resistance to apoptosis. 16,17 This includes resistance to potent chemotherapeutics as well as other agents that induce apoptosis as The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Serv- ices nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. *Correspondence to: Michael P. Waalkes, Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, NCI at NIEHS, PO Box 12233, Mail Drop F0-09, 111 Alexander Dr., Research Triangle Park, NC 27709. Fax: (919)541-3970. E-mail: waalkes@niehs.nih.gov Received 25 September 2004; Accepted after revision 17 December 2004 DOI 10.1002/ijc.20990 Published online 8 March 2005 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: As-TL, arsenic-tolerant cells; PDK-1, phosphoi- nositide-dependent kinase-1; PI3K, PI3 kinase; PKB, protein kin- ase B; P-PKB, phosphorylated PKB; PTEN, phosphatase and tensin homolog deleted on chromosome 10; ROS, reactive oxygen species; UV, ultraviolet; UVA, ultraviolet A. Int. J. Cancer: 116, 20–26 (2005) ' 2005 Wiley-Liss, Inc. Publication of the International Union Against Cancer