Biomedical subjects

Brian G Gabrielli

Publications and source records attributed to Brian G Gabrielli.

5 recordsLinked to original sources

PubMed2005

RNA interference against human papillomavirus oncogenes in cervical cancer cells results in increased sensitivity to cisplatin.

Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAi-based cancer therapies because the oncogenes that cause cervical cancer, E6 and E7, are expressed only in cancerous cells. We investigated whether targeting HPV E6 and E7 oncogenes yields cancer cells more sensitive to chemotherapy by cisplatin, the chemotherapeutic agent currently used for the treatment of advanced cervical cancer. We have designed siRNAs directed against the HPV E6 oncogene that simultaneously targets both E6 and E7, which results in an 80% reduction in E7 protein and reactivation of the p53 pathway. The loss of E6 and E7 resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. Interference was specific in that no effect on HPV-negative cells was observed. We demonstrate that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells. The IC50 for HeLa cells treated with cisplatin was 9.4 microM, but after the addition of a lentivirus-delivered shRNA against E6, the IC50 was reduced almost 4-fold to 2.4 microM. We also observed a decrease in E7 expression with a concurrent increase in p53 protein levels upon cotreatment with shRNA and cisplatin over that seen with individual treatment alone. Our results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, probably because of increased p53 levels.

Antineoplastic Agents↗

PubMed2005

Cdk1/Erk2- and Plk1-dependent phosphorylation of a centrosome protein, Cep55, is required for its recruitment to midbody and cytokinesis.

Centrosomes in mammalian cells have recently been implicated in cytokinesis; however, their role in this process is poorly defined. Here, we describe a human coiled-coil protein, Cep55 (centrosome protein 55 kDa), that localizes to the mother centriole during interphase. Despite its association with gamma-TuRC anchoring proteins CG-NAP and Kendrin, Cep55 is not required for microtubule nucleation. Upon mitotic entry, centrosome dissociation of Cep55 is triggered by Erk2/Cdk1-dependent phosphorylation at S425 and S428. Furthermore, Cep55 locates to the midbody and plays a role in cytokinesis, as its depletion by siRNA results in failure of this process. S425/428 phosphorylation is required for interaction with Plk1, enabling phosphorylation of Cep55 at S436. Cells expressing phosphorylation-deficient mutant forms of Cep55 undergo cytokinesis failure. These results highlight the centrosome as a site to organize phosphorylation of Cep55, enabling it to relocate to the midbody to function in mitotic exit and cytokinesis.

A Kinase Anchor Proteins↗

PubMed2004

Analyzing checkpoint controls in human skin.

A short-term whole-skin organ culture model has been established to enable the investigation of cell cycle perturbations in epidermal layer cells following exposure to ultraviolet radiation (UVR). This model affords the opportunity to manipulate the growth and nutrient conditions, and to perform detailed biochemical and immunohistochemical analysis of skin cells in their normal epidermal layer microenvironment. The use of this model is described in this chapter.

Bromodeoxyuridine↗

PubMed2004

Analysis of checkpoint responses to histone deacetylase inhibitors.

Cell cycle checkpoints respond to a wide range of stresses to prevent compromise to the integrity of the cell. The best studied checkpoints are those induced by genotoxic agents that cause DNA damage. Histone deacetylase inhibitors not only increase the acetylation state of chromatin histones, but they also perturb the cell cycle, causing both G1 and G2 phase arrests, the latter by initiating a checkpoint response. In this chapter we will describe the analysis of the histone deacetylase inhibitor-sensitive G2 checkpoint using synchronized cell populations.

Bromodeoxyuridine↗

PubMed2003

Mechanism of mitosis-specific activation of MEK1.

Activation of cyclin B-Cdc2 is an absolute requirement for entry into mitosis, but other protein kinase pathways that also have mitotic functions are activated during G(2)/M progression. The MAPK cascade has well established roles in entry and exit from mitosis in Xenopus, but relatively little is known about the regulation and function of this pathway in mammalian mitosis. Here we report a detailed analysis of the activity of all components of the Ras/Raf/MEK/ERK pathway in HeLa cells during normal G(2)/M. The focus of this pathway is the dramatic activation of an endomembrane-associated MEK1 without the corresponding activation of the MEK substrate ERK. This is because of the uncoupling of MEK1 activation from ERK activation. The mechanism of this uncoupling involves the cyclin B-Cdc2-dependent proteolytic cleavage of the N-terminal ERK-binding domain of MEK1 and the phosphorylation of Thr(286). These results demonstrate that cyclin B-Cdc2 activity regulates signaling through the MAPK pathway in mitosis.

CDC2 Protein Kinase↗