The three Ad-INSM1p-HSV-tk-treated mice that did not have visible tumors were monitored for an additional 4 weeks without any tumor growth. infection and ganciclovir (GCV) sensitivity studies indicated that theINSM1promoter could mediate specific expression of the HSV-tkgene Pramiracetam and selective Pramiracetam killing of INSM1-positive PNETs.In vivointratumoral adenoviral delivery demonstrated that theINSM1promoter could direct HSV-tkgene expression in a nude mouse tumor model and effectively repressed tumor growth in response to GCV treatment. Taken together, our data show that theINSM1promoter is specific and effective for targeted cancer gene therapy in PNETs. == Introduction == Cancer gene therapyhas gained much interest as an alternative or combination treatment modality. The emergence of improved viral delivery vectors and the significant progress made in understanding the process of oncogenesis have allowed the field of cancer gene therapy to expand (Seth,2005). The herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene system is amongst the Rabbit Polyclonal to TDG oldest and most widely tested concepts for cancer gene therapy (Mooltenet al.,1990). Although initial studies of the HSV-tk/GCV system demonstrated excellent potential, the major roadblock to its usefulness is the significant liver toxicity (Chenet al.,1995; O’Malleyet al.,1995; Brandet al.,1997; van der Ebet al.,1998). Ultimately, the goal of any cancer therapy is to efficiently eliminate the tumor cells with minimal deleterious side effects to the normal surrounding tissue. One strategy to ensure tumor-selective expression is through transcriptional targeting with a tissue-specific promoter. The current challenge is to identify potential regulatory elements that are highly active in tumor cells and have low to ideally no activity in normal cells, especially the liver. Primitive neuroectodermal tumors (PNETs) are a heterogeneous group of tumors that generally occur in pediatric patients and include medulloblastoma, retinoblastoma, and neuroblastoma. Medulloblastoma is the most common pediatric central nervous system malignancy. Medulloblastoma is an aggressive, embryonal tumor derived from cerebellum. With combined surgery, irradiation, and chemotherapy, the 5-year recurrence-free survival rate has reached 6065% (Packeret al.,1999; Oyharcabal-Bourdenet al.,2005). However, nearly all patients exhibit debilitating side effects from Pramiracetam radiation including cognitive impairment, psychiatric disorders, endocrine dysfunction, and skeletal Pramiracetam growth retardation. Neuroblastoma is a solid tumor derived from cells of neural crest origin. Despite intensive therapy for high-risk neuroblastoma, the 5-year survival is 33% (Berthold and Hero,2000). Therefore, alternative treatment options for these forms of cancer are needed. Gene therapy approaches focused toward high-risk neuroblastoma and central nervous system tumors such as medulloblastoma have used common neuronal-specific promoters from the genes encoding midkine, tyrosine hydroxylase, neural crest homeobox (NCX), and Mash1 (Naritaet al.,2001; Adachiet al.,2002; Steffenset al.,2004; Arvidssonet al.,2005). Despite promising results from these gene therapy strategies, one significant barrier is the continuous expression of these gene products, with the exception ofMash1, in normal adult neuronal cells. This aspect limits their usefulness for cancer gene therapy especially if delivered systemically. Therefore, new genes with tumor-selective expression need to be identified and tested. INSM1 is a novel transcriptional repressor that was originally identified in an insulinoma subtractive hybridization screen (Gotoet al.,1992). INSM1 expression is both temporally and spatially restricted to the embryonic peripheral and central nervous system in cells of neuroendocrine origin including the pituitary, pancreas, thymus, thyroid, retina, olfactory bulb, brain, and spinal cord and is silenced during normal postnatal development (Zhuet al.,2002; Xieet al.,2002; Mellitzeret al.,2006). Strikingly,INSM1is reactivated in tumors of neuroendocrine origin including insulinomas, pituitary tumors, pheochromocytomas, medullary thyroid carcinomas, small-cell lung carcinomas, medulloblastomas, neuroblastomas, and retinoblastomas (Gotoet al.,1992; Breslinet al.,2003; DeSmaeleet al.,2008). These features make it an excellent candidate for use in promoter-regulated cancer gene therapy. One study usingINSM1promoter-targeted HSV-tk/GCV gene therapy for small-cell lung cancer demonstrated effective killing of INSM1-expressing lung cancer cellsin vitro(Pedersenet al.,2006). In the present study, we have defined a 1.7-kilobase (kb) region of theINSM1promoter that faithfully recapitulates endogenousINSM1gene expression in PNETs. We tested both adult and childhood tumors for response Pramiracetam to the suicide gene therapy, including neuroblastoma (IMR-32), medulloblastoma (D283 Med), retinoblastoma (Y79), and glioblastoma (U-87 MG). Both Northern blot and luciferase reporter gene assays indicated thatINSM1mRNA and promoter activities were specifically restricted in normal fetal brain and PNETs. Adenoviral INSM1p-HSV-tkconstructs showed that theINSM1promoter could mediate selective expression of the HSV-tkgene and in the presence of GCV induced killing of INSM1-positive tumor cell lines.