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The Role of DCT/TYRP2 in Resistance of Melanoma Cells to Drugs and Radiation

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The Role of DCT/TYRP2 in Resistance of Melanoma Cells to Drugs and Radiation Chapter 32 / DOPAchrome Tautomerase-Mediated Drug and Radiation Resistance 577 32 The Role of DCT/TYRP2 in Resistance of Melanoma Cells to Drugs and Radiation Brian J. Pak and Yaacov Ben-David CONTENTS INTRODUCTION RETROVIRAL INSERTIONAL MUTAGENESIS AS A METHOD OF GENERATING RESISTANT CELL LINES IDENTIFICATION OF DOPACHROME TAUTOMERASE AS A MEDIATOR OF DRUG RESISTANCE MELANOGENESIS AND TYROSINASE-RELATED PROTEINS EXPRESSION OF DCT DCT AND RADIATION RESISTANCE DCT AND IMMUNOTHERAPY OF HUMAN MELANOMA POSSIBLE MECHANISMS OF DCT-MEDIATED RESISTANCE STRESS-DEPENDENT MECHANISMS OF RESISTANCE IN MELANOMAS CONCLUSIONS AND PERSPECTIVES REFERENCES Summary Intrinsic resistance to both chemotherapy and radiotherapy remains a major obstacle in the clinical treatment of malignant melanoma. Recent advances in cancer research have provided new insights into the molecular mechanisms governing their intrinsic resistance. We have recently demonstrated that DOPAchrome tautomerase (DCT), an enzyme that is well characterized for its function in melanin syn- thesis, is highly expressed in human melanoma cells that are resistant to both drug and radiation treat- ments. Conversely, melanoma cells expressing very low levels of DCT are highly susceptible to either type of treatment. Overexpression of DCT in melanoma cells by transfection could confer both radiore- sistance and chemoresistance. This review will summarize our findings, as well as discuss the possible mechanisms by which DCT overexpression contributes to intrinsic resistance of human malignant mela- noma. Key Words: Radioresistance; chemoresistance; malignant melanoma; DOPAchrome tauto- merase (DCT). From: From Melanocytes to Melanoma: The Progression to Malignancy Edited by: V. J. Hearing and S. P. L. Leong © Humana Press Inc., Totowa, NJ 577 578 From Melanocytes to Melanoma INTRODUCTION Conventional cancer therapies involve the selective targeting of actively dividing cells by either the systemic administration of antineoplastic agents or localized treatment with ionizing radiation. There are several different classes of chemotherapeutic drugs that exert their anticancer effects by distinct mechanisms. For example, drugs such as cis-diamminedichloroplatinum(II) (CDDP), cyclophosphamide, and dacarbazine gen- erate various forms of DNA damage (1). Other agents, such as paclitaxel and vinblastine bind tubulin, thereby inhibiting the formation of microtubules (2), whereas drugs such as methotrexate and 5-fluorouracil prevent nucleic acid synthesis (3). All of these agents act by blocking specific events during the mitotic cycle, thereby inducing cells to undergo apoptosis. Ionizing radiation, on the other hand, induces DNA strand breaks and dimer- ization, which, in turn, triggers the apoptotic cascade (4). Although radiation and chemotherapy remain first-line treatments for cancer, one of the major limitations of these approaches is that many malignancies either acquire resistance to these therapies during the course of treatment or are intrinsically resistant at the time of diagnosis. Several resistance pathways employed by malignant cells have been described in the literature. Drug efflux pumps consisting of groups of integral membrane proteins, such as P-glycoprotein (5), multidrug resistance protein (6), lung resistance protein (7), and breast cancer resistance protein, are expressed in a wide variety of different malignancies (8). Resistance to DNA damage by elevated expression of various DNA repair enzymes, including the nucleotide excision repair enzymes, ERCC1 and XPF, O6-alkylguanine-DNA alkyltransferase, and O6-methylguanine-DNA methyltransferase all have been widely described in resistant tumors (9,10). Resistance mechanisms employed by cancer cells are complex and multifactorial, and are likely heterogeneous within a tumor because of random mutations and differences in the tumor microenvironment. As such, a thorough understanding of these resistance pathways are of utmost importance in the effective clinical management of cancer using existing therapies, as well as in the development of novel therapeutic agents. The treatment of patients with melanoma can vary greatly depending on the stage at which melanoma is diagnosed. In its early stages, melanomas grow superficially on the epidermis and can be treated by surgical excision. However, once the primary tumor disseminates, melanomas become extremely difficult to treat. This is because most late- staged melanoma tumors display intrinsic resistance to chemotherapy and radiation therapy. Indeed, melanoma is virtually unresponsive to most chemotherapeutic drugs administered as a monotherapy (11,12). Combinations of such drugs, however, have been shown to improve tumor response, but rarely lead to complete remission or cures. Currently, the mechanisms underlying intrinsic resistance in melanomas remain poorly understood. Mutations in the tumor suppressor protein, p53, have been shown to enhance drug resistance in many cancer models. However, because most melanomas express wild-type p53, it is widely believed that this resistance phenotype is independent of a p53 mutation (13–19). It has been reported that more than 90% of melanomas express the antiapoptotic protein Bcl-2 (20–24) raising the possibility that Bcl-2 expression may contribute to their intrinsic resistance. Studies by Jansen and colleagues (25) have shown that the downregulation of Bcl-2 expression in xenograft models using antisense oligo- nucleotide therapy targeting Bcl-2, improved sensitivity of human melanoma cells to both dacarbazine and CDDP. Such results have also been obtained by treating melanoma Chapter 32 / DOPAchrome Tautomerase-Mediated Drug and Radiation Resistance 579 Fig. 1. Restriction map of murine stem cell virus retrovirus. cells with small interfering RNA (26,27). Other antiapoptotic factors, such as Bcl-xL, Mcl-1, and Survivin have also been reported to be highly expressed in melanoma cells and, therefore, may also contribute to the intrinsic resistance to therapies (28–31). RETROVIRAL INSERTIONAL MUTAGENESIS AS A METHOD OF GENERATING RESISTANT CELL LINES In 1994, we described the use of retroviral insertional mutagenesis as a means of rapidly generating resistant variants of drug-sensitive melanoma cell lines (32). This method involves the infection of comparatively drug-sensitive melanoma cells with replication-defective amphotrophic retroviruses to generate mutants of the parental cell line. The integration of proviruses into the host cell genome generates random mutations that can lead to the activation or inactivation of cellular genes depending on the site of integration. For example, if the provirus integrates within a gene, the expression of the gene may be disrupted. Conversely, if the provirus integrates between genes, the transactivating property of the viral long terminal repeats may activate the transcription of adjacent genes. Therefore, mutant cells that harbor a dominantly selectable phenotype can be isolated and characterized. This approach has been used by our group, as well as others, and has yielded several important genes associated with cancer initiation, pro- gression, and resistance, including Fli-1, Spi-1, p53, NF-E2/p45, and CRL-1 (33–38). In an attempt to identify genes that may be associated with drug and radiation resis- tance in melanoma cells, retroviral insertional mutagenesis was used to create mutants of an early-staged, drug- and radiation-sensitive human melanoma cell line. The retrovirus used was a replication-defective, neo-containing murine stem cell virus with an amphotrophic host range (39). Murine stem cell virus is devoid of env sequences, and includes an extended packaging region (P+) for high viral titer and a modified 5'-untrans- lated region with a primer-binding site for tRNAGln instead of the usual tRNAPro. Long terminal repeats and pgk promoters are used for the expression of viral and neo RNA transcripts, respectively (Fig. 1). Melanoma mutants generated by retroviral insertional mutagenesis were subjected to treatments of lethal doses of CDDP. The rationale was that among the wild-type popu- lation of cells, some cells would have acquired a mutation that would confer CDDP resistance. These cells can then be isolated, expanded in culture, and characterized to determine which genes may be associated with CDDP resistance. By this approach, several independently derived CDDP-resistant clones were established. Quite interest- ingly, more than half of these clones showed the integration of a single provirus at an 580 From Melanocytes to Melanoma identical site, which was designated “CDDP resistance locus 1” (CRL-1). CRL-1 was mapped to human chromosome 3p21 (B. J. Pak et al., unpublished data). An analysis of approx 20 kb of genomic DNA flanking CRL-1 failed to identify exonic sequences. These results were not unexpected because proviruses often alter the expression of target genes located as much as 80 kb from the site of integration (40). Attempts to determine the gene(s) that are directly regulated by proviral integration have, thus far, been unsuc- cessful. IDENTIFICATION OF DOPACHROME TAUTOMERASE AS A MEDIATOR OF DRUG RESISTANCE To screen for genes that are differentially expressed in the CDDP-resistant mutants, subtractive hybridization was employed. Messenger RNA from wild-type cells and from one of the virally derived mutant cells were compared and yielded a single transcript, DCT, also termed tyrosinase-related protein (TYRP)-2, which was upregulated in the CDDP-resistant clone (41). Subsequent
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