Evaluation of potential mechanisms underlying genotype-phenotype correlations in multiple endocrine neoplasia type 2.

Distinct dominant activating mutations in the RET proto-oncogene are responsible for the development of multiple endocrine neoplasia type 2 (MEN 2). Concise examination of the mutated codons led to the detection of a striking genotype-phenotype correlation between the mutated codon and the MEN 2 phenotype in terms of onset and aggressiveness of the disease, suggesting that manifestation and clinical progression is conditioned by the type of mutation. To gain insight into the molecular basis for this genotype-phenotype correlation, we analysed the impact of common and rare mutations identified in MEN 2A (C609Y, C634R), MEN 2B (A883F, M918T) and familial medullary thyroid carcinoma (Y791F) patients on several aspects of cell transformation, including proliferation, apoptosis, anchorage-independent growth and signaling. We found that tumor cells arising from distinct extracellular or intracellular MEN 2 mutations clearly differ in their proliferation properties owing to the activation of different molecular pathways, but importantly, also in resistance to apoptosis. Whereas MEN 2A mutants resulted in accelerated cell proliferation, MEN 2B-RET mutants significantly enhanced suppression of apoptosis, which may account, at least partially, for some of the clinical differences in MEN 2 patients.

Adenovirus-mediated TA-p73beta gene transfer increases chemosensitivity of human malignant melanomas.

Malignant melanoma is the most aggressive form of skin cancer and has proven to be highly resistant to conventional chemotherapy. Intriguingly, the p53 tumor suppressor, a main mediator of chemoresistance in other tumor types, is rarely mutated in melanoma. However, we have previously shown that anti-apoptotic isoforms of p73 (deltaTA-p73), another member of the p53 family, are overexpressed in metastatic melanomas. DeltaTA-p73 can oppose the pro-apoptotic functions of p53 and full length p73, and thus it could contribute to melanoma chemoresistance. In this study, we use an efficient adenoviral-based gene transfer approach to introduce a transcriptionally active form of p73 (TA-p73beta) in melanoma cells, with the objective of overcoming drug resistance. Interestingly, TA-p73beta significantly sensitized 5 out of 7 aggressive melanoma cell lines to the standard therapeutic agents adriamycin and cisplatin. More importantly, TA-p73beta displayed a synergistic effect in vivo allowing adriamycin or cisplatin to block melanoma cell growth in mouse xenograft models (p < 0.05). In summary, our data show that Ad-mediated TA-p73beta gene expression can markedly sensitize a subset of melanoma cell lines to adriamycin and cisplatin in vitro and in vivo, suggesting a new chemosensitization strategy for malignant melanomas.

Analysis of adenovirus gene transfer into adult neural stem cells.

Adult neural stem cells (aNSCs) represent an attractive source for the production of specific types of neurons in degenerative CNS diseases and for the development of new regenerative gene therapies. However, the use of adult NSCs for transplantation and gene replacement strategies requires efficient gene expression in the cells. Due to the low pathogenicity of adenovirus (Ad) for humans, its large delivery capacity, and long-term transgene expression, Ad vectors are widely used. Here, we tested the potential of the Ad vector system to transduce adult NSCs. Analysis of Ad receptor expression in primary aNSCs revealed a complete lack of the coxsackie-adenovirus receptor and no or low expression of alphanu- and beta5-integrins, respectively, on mRNA and protein level. Consistently, transduction at different multiplicities of infection using an Ad vector expressing the enhanced green fluorescent protein (GFP) showed that adult NSCs are particularly resistant to Ad infection even at highest MOI (1000) in contrast to differentiated types of neural cells.