Luis A. Martinez

Synthetic small inhibiting RNAs: Efficient tools to inactivate oncogenic mutations and restore p53 pathways

Single base pair mutations that alter the function of tumor suppressor genes and oncogenes occur frequently during oncogenesis. The guardian of the genome, p53, is inactivated by point mutation in more than 50% of human cancers. Synthetic small inhibiting RNAs (siRNAs) can suppress gene expression in mammalian cells, although their degree of selectivity might be compromised by an amplification mechanism. Here, we demonstrate that a single base difference in siRNAs discriminates between mutant and WT p53 in cells expressing both forms, resulting in the restoration of WT protein function. Therefore, siRNAs may be used to suppress expression of point-mutated genes and provide the basis for selective and personalized antitumor therapy.

Increased cell growth and tumorigenicity in human prostate LNCaP cells by overexpression to cyclin D1

Deregulated expression of cyclin D1 has been found in several types of human tumors. In order to investigate factors involved in human prostate cancer progression, we studied the effects of cyclin D1 overexpression on human prostate cancer cell proliferation and tumorigenicity by transfecting LNCaP cells with a retroviral vector containing human cyclin D1 cDNA. When compared to the parental and control-vector transfected LNCaP cells, these cyclin D1-transfected cells had more cells in S-phase and lower growth factor requirements. Furthermore, these cells grew more in androgen-free medium. We also detected higher levels of Rb phosphorylation and E2F-1 protein levels in LNCaP/cyclin D1 cells than that in the parental and vector control cells in medium with or without androgen. Cyclin D1 transfected clones formed tumors more rapidly than control and parental cells. These tumors were refractory to the androgen-ablation treatment by castration, whereas tumors from parental and vector-control LNCaP cells regressed within 4 weeks after castration. These results suggest that overexpression of cyclin D1 changes the growth properties, increases tumorigenicity and decreases the requirement for androgen stimulation in LNCaP cells both in vitro and in vivo.

Transgenic expression of cyclin-dependent kinase 4 results in epidermal hyperplasia, hypertrophy, and severe dermal fibrosis.

In a previous report we have described the effects of expression of D-type cyclins in epithelial tissues of transgenic mice. To study the involvement of the D-type cyclin partner cyclin-dependent kinase 4 (CDK4) in epithelial growth and differentiation, transgenic mice were generated carrying the CDK4 gene under the control of a keratin 5 promoter. As expected, transgenic mice showed expression of CDK4 in the epidermal basal-cell layer. Epidermal proliferation increased dramatically and basal cell hyperplasia and hypertrophy were observed. The hyperproliferative phenotype of these transgenic mice was independent of D-type cyclin expression because no overexpression of these proteins was detected. CDK4 and CDK2 kinase activities increased in transgenic animals and were associated with elevated binding of p27(Kip1) to CDK4. Expression of CDK4 in the epidermis results in an increased spinous layer compared with normal epidermis, and a mild hyperkeratosis in the cornified layer. In addition to epidermal changes, severe dermal fibrosis was observed and part of the subcutaneous adipose tissue was replaced by connective tissue. Also, abnormal expression of keratin 6 associated with the hyperproliferative phenotype was observed in transgenic epidermis. This model provides in vivo evidence for the role of CDK4 as a mediator of proliferation in epithelial cells independent of D-type cyclin expression.

Involvement of a pertussis-toxin sensitive G protein in the induction of gene expression by insulin☆

Binding of insulin to its receptor triggers multiple cellular responses, including changes in metabolism and in gene expression, resulting from the activation of multiple signalling pathways. Pertussis toxin has been shown to block an insulin-stimulated phospholipase C, resulting in an inhibition of the synthesis of phospholipid second messengers by insulin. In the present study, we investigated the significance of this pathway for the induction of growth-related genes by insulin treatment of H35 hepatoma cells. We found that pertussis toxin dramatically inhibits the induction of c-fos mRNA by insulin. Although c-jun and ornithine decarboxylase induction were also inhibited by pertussis toxin, they were much less sensitive than c-fos. These results indicate an important for lipid second messengers in mitogenic signalling by insulin and further demonstrate distinct roles for this pathway in the induction of c-fos and c-jun.