Ras protein farnesyltransferase: A strategic target for anticancer therapeutic development.

Ras proteins are guanine nucleotide-binding proteins that play pivotal roles in the control of normal and transformed cell growth and are among the most intensively studied proteins of the past decade. After stimulation by various growth factors and cytokines, Ras activates several downstream effectors, including the Raf-1/mitogen-activated protein kinase pathway and the Rac/Rho pathway. In approximately 30% of human cancers, including a substantial proportion of pancreatic and colon adenocarcinomas, mutated ras genes produce mutated proteins that remain locked in an active state, thereby relaying uncontrolled proliferative signals. Ras undergoes several posttranslational modifications that facilitate its attachment to the inner surface of the plasma membrane. The first-and most critical-modification is the addition of a farnesyl isoprenoid moiety in a reaction catalyzed by the enzyme protein farnesyltransferase (FTase). It follows that inhibiting FTase would prevent Ras from maturing into its biologically active form, and FTase is of considerable interest as a potential therapeutic target. Different classes of FTase inhibitors have been identified that block farnesylation of Ras, reverse Ras-mediated cell transformation in human cell lines, and inhibit the growth of human tumor cells in nude mice. In transgenic mice with established tumors, FTase inhibitors cause regression in some tumors, which appears to be mediated through both apoptosis and cell cycle regulation. FTase inhibitors have been well tolerated in animal studies and do not produce the generalized cytotoxic effects in normal tissues that are a major limitation of most conventional anticancer agents. There are ongoing clinical evaluations of FTase inhibitors to determine the feasibility of administering them on dose schedules like those that portend optimal therapeutic indices in preclinical studies. Because of the unique biologic aspects of FTase, designing disease-directed phase II and III evaluations of their effectiveness presents formidable challenges.

Radiation therapy and ferromagnetic hyperthermia in the treatment of murine transgenic retinoblastoma.

BACKGROUND: Combined modality therapy for childhood retinoblastoma holds the potential of decreasing treatment-related morbidity while maintaining excellent tumor control rates. OBJECTIVE: To evaluate the efficacy of external beam radiation therapy (EBRT), ferromagnetic hyperthermia (FMH), and the combination of both modalities in the control of ocular tumors in a transgenic murine model of retinoblastoma. METHODS: One hundred sixty-six mouse eyes from 4-week-old animals transgenically positive for simian virus 40 large T antigen were treated with a total dose of 10, 15, 20, 30, 40, 45, or 50 Gy of EBRT in 5-Gy fractions twice daily, with 48 degrees C or 54 degrees C FMH for 20 minutes, or with combined EBRT at 10 or 30 Gy and 48 degrees C or 54 degrees C FMH for 20 minutes. Serial histologic sections, obtained 8 weeks after treatment, were examined for the presence of tumor. RESULTS: The tumor control dose for 50% of eyes (TCD50) treated with EBRT occurred at 27.6 Gy. Ferromagnetic hyperthermia at 48 degrees C cured 30% (6/20) of eyes, while 54 degrees C FMH resulted in a 100% (20/20) cure rate. Combined treatment with 48 degrees C FMH and EBRT exhibited a TCD50 at 3.3 Gy. The thermal enhancement ratio was 8.4. Ferromagnetic hyperthermia at 54 degrees C exhibited tumor cure in all animals, but 25% of eyes were lost owing to secondary treatment complications. CONCLUSIONS: This represents the first documentation of tumor control via EBRT, ocular FMH, and a combination of these treatment modalities in this murine transgenic retinoblastoma model. The extent of treatment synergy in this model suggests that combined treatment application may allow a reduction in total ocular and periocular radiation dose while maintaining excellent local tumor control.

Immortalized hypothalamic gonadotropin-releasing hormone neurons.

The neuroendocrine hypothalamus has been intensively studied using whole animals and tissue slices. However, it has been difficult to approach questions at the molecular and cellular level. By targeting expression of the oncogene product, simian virus 40 T antigen, in transgenic mice using the regulatory domain of the rat gonadotropin-releasing hormone (GnRH) gene, we have produced specific hypothalamic tumours. These tumours have been cultured to produce clonal cell lines (GT-1 cells) that express T antigen, GnRH and many other neuronal markers, but do not express other hypothalamic hormones. These immortal cell lines have a distinctive neuronal phenotype, process the GnRH peptide accurately and secrete GnRH in a pulsatile pattern. Thus, by targeting oncogenesis to a defined population of neurons using the regulatory region of a gene that is expressed late in differentiation of that cell lineage, we have succeeded in immortalizing hypothalamic GnRH neurons. The GT-1 cell lines are an excellent model for future molecular, cell biological, physiological and biochemical investigations into the mechanisms involved in regulation of GnRH and the characteristics of an isolated central nervous system neuron. Their derivation demonstrates the utility of targeting tumorigenesis to specific differentiated neurons of the central nervous system in transgenic mice.

Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis.

By genetically targeting tumorigenesis to specific hypothalamic neurons in transgenic mice using the promoter region of the gonadotropin-releasing hormone (GnRH) gene to express the SV40 T-antigen oncogene, we have produced neuronal tumors and developed clonal, differentiated, neurosecretory cell lines. These cells extend neurites, express the endogenous mouse GnRH mRNA, release GnRH in response to depolarization, have regulatable fast Na+ channels found in neurons, and express neuronal, but not glial, cell markers. These immortalized cells will provide an invaluable model system for study of hypothalamic neurosecretory neurons that regulate reproduction. Significantly, their derivation demonstrates the feasibility of immortalizing differentiated neurons by targeting tumorigenesis in transgenic mice to specific neurons of the CNS.