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Preclinical studies of anticancer drugs are typically performed using cancer cell lines maintained in two-dimensional (2D) cultures, ignoring the influences of the extracellular matrix (ECM) and three-dimensional (3D) microenvironment. In this study, we evaluated the microenvironmental control of human breast cancer cells responses to doxorubicin (DOXO) using the 3D laminin-rich ECM (3D lrECM) cell culture model. Under 3D culture conditions, MCF-7 cells displayed drastic morphological alterations, a decrease in proliferation and elevated sensitivity to DOXO. Interestingly, the chemotherapy-mediated activation of autophagy was compromised in the 3D matrix, suggesting an association between the increased cytotoxicity of DOXO and hindered autophagy induction. Indeed, while chloroquine or ATG5 knockdown potentiated DOXO-induced cell death under the 2D culture conditions, the autophagy inducer rapamycin improved the resistance of 3D-cultured cells to this drug. Moreover, in the monolayer-cultured cells, DOXO treatment led to increases in p53 and DRAM-1 expression, which is a p53-dependent activator of autophagy that functions in response to DNA damage. Conversely, p53 and DRAM-1 expression was impaired in 3D-cultured cells. The knockdown of p53 by shRNA blocked DRAM-1 activation, impaired autophagy induction and sensitized only those cells maintained under 2D conditions to DOXO. In addition, 2D-cultured MDA-MB-231 cells (a p53-mutated breast cancer cell line) not only showed increased sensitivity to DOXO compared with MCF-7 cells but also failed to induce DRAM-1 expression or autophagy. Similar to p53 silencing, DRAM-1 knockdown potentiated DOXO cytotoxicity only in 2D-cultured cells. These results suggest that the 3D tissue microenvironment controls tumor cell sensitivity to DOXO treatment by preventing p53-DRAM-autophagy axis activation. [ABSTRACT FROM AUTHOR]

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumours (malignant gliomas). The mechanism of TMZ-induced glioma cell death is unknown. Here, we show that malignant glioma cells undergo apoptosis following treatment with the methylating agents N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and TMZ. Cell death determined by colony formation and apoptosis following methylation is greatly stimulated by p53. Transfection experiments with O6-methylguanine-DNA methyltransferase (MGMT) and depletion of MGMT by O6-benzylguanine showed that, in gliomas, the apoptotic signal originates from O6-methylguanine (O6MeG) and that repair of O6MeG by MGMT prevents apoptosis. We further demonstrate that O6MeG-triggered apoptosis requires Fas/CD95/Apo-1 receptor activation in p53 non-mutated glioma cells, whereas in p53 mutated gliomas the same DNA lesion triggers the mitochondrial apoptotic pathway. This occurs less effectively via Bcl-2 degradation and caspase-9, -2, -7 and -3 activation. O6MeG-triggered apoptosis in gliomas is a late response (occurring >120 h after treatment) that requires extensive cell proliferation. Stimulation of cell cycle progression by the Pasteurella multocida toxin promoted apoptosis whereas serum starvation attenuated it. O6MeG-induced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by γH2AX formation. Glioma cells mutated in DNA-PKcs, which is involved in non-homologous end-joining, were more sensitive to TMZ-induced apoptosis, supporting the involvement of DSBs as a downstream apoptosis triggering lesion. Overall, the data demonstrate that cell death induced by TMZ in gliomas is due to apoptosis and that determinants of sensitivity of gliomas to TMZ are MGMT, p53, proliferation rate and DSB repair.Oncogene (2007) 26, 186–197. doi:10.1038/sj.onc.1209785; published online 3 July 2006 [ABSTRACT FROM AUTHOR]

The hepatitis B virus surface antigen (HBsAg) gene, under control of the inducible mouse metallothionein I gene promoter, was inserted in an expression vector based on the Epstein-Barr virus (EBV). This vector was introduced into human cells by DNA transfection and clones were selected for their resistance to hygromycin B. The recombinant EBV vector replicates efficiently as an episome in human cells and approximately six copies per cell were found in one clone of hygromycin-B-resistant cells. These cells produce high levels of HBsAg in the presence of metals. The protein is mainly found in the cell medium, suggesting that the HBsAg is secreted from the cells. [ABSTRACT FROM AUTHOR]