Modulation of Mcl-1 sensitizes glioblastoma to TRAIL-induced apoptosis.

Glioblastoma (GBM) is the most aggressive form of primary brain tumour, with dismal patient outcome. Treatment failure is associated with intrinsic or acquired apoptosis resistance and the presence of a highly tumourigenic subpopulation of cancer cells called GBM stem cells. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising novel therapy for some treatment-resistant tumours but unfortunately GBM can be completely resistant to TRAIL monotherapy. In this study, we identified Mcl-1, an anti-apoptotic Bcl-2 family member, as a critical player involved in determining the sensitivity of GBM to TRAIL-induced apoptosis. Effective targeting of Mcl-1 in TRAIL resistant GBM cells, either by gene silencing technology or by treatment with R-roscovitine, a cyclin-dependent kinase inhibitor that targets Mcl-1, was demonstrated to augment sensitivity to TRAIL, both within GBM cells grown as monolayers and in a 3D tumour model. Finally, we highlight that two separate pathways are activated during the apoptotic death of GBM cells treated with a combination of TRAIL and R-roscovitine, one which leads to caspase-8 and caspase-3 activation and a second pathway, involving a Mcl-1:Noxa axis. In conclusion, our study demonstrates that R-roscovitine in combination with TRAIL presents a promising novel strategy to trigger cell death pathways in glioblastoma.

The Bcl-2 repertoire of mesothelioma spheroids underlies acquired apoptotic multicellular resistance.

Three-dimensional (3D) cultures are a valuable platform to study acquired multicellular apoptotic resistance of cancer. We used spheroids of cell lines and actual tumor to study resistance to the proteasome inhibitor bortezomib in mesothelioma, a highly chemoresistant tumor. Spheroids from mesothelioma cell lines acquired resistance to bortezomib by failing to upregulate Noxa, a pro-apoptotic sensitizer BH3-only protein that acts by displacing Bim, a pro-apoptotic Bax/Bak-activator protein. Surprisingly, despite their resistance, spheroids also upregulated Bim and thereby acquired sensitivity to ABT-737, an inhibitor of anti-apoptotic Bcl-2 molecules. Analysis using BH3 profiling confirmed that spheroids acquired a dependence on anti-apoptotic Bcl-2 proteins and were 'primed for death'. We then studied spheroids grown from actual mesothelioma. ABT-737 was active in spheroids grown from those tumors (5/7, ∼70%) with elevated levels of Bim. Using immunocytochemistry of tissue microarrays of 48 mesotheliomas, we found that most (33, 69%) expressed elevated Bim. In conclusion, mesothelioma cells in 3D alter the expression of Bcl-2 molecules, thereby acquiring both apoptotic resistance and sensitivity to Bcl-2 blockade. Mesothelioma tumors ex vivo also show sensitivity to Bcl-2 blockade that may depend on Bim, which is frequently elevated in mesothelioma. Therefore, mesothelioma, a highly resistant tumor, may have an intrinsic sensitivity to Bcl-2 blockade that can be exploited therapeutically.

Pancreas-graft immunogenicity and pretreatment with anti-class II monoclonal antibodies.

Pretreatment of organ allografts to reduce graft immunogenicity is an attractive and potentially clinically applicable concept. We have studied the effect of perfusing rat pancreases with anti-class II monoclonal antibody (MoAb), to remove class II- positive accessory cells from the intact organ, on prolongation of allograft survival after transplantation. The capacity of pancreatic islets obtained from these perfused organs to stimulate proliferation of allogeneic T-lymphocytes was studied in a mixed islet-lymphocyte culture (MILC). There was a significant prolongation in pancreas-allograft survival when intact pancreases were transplanted after a 3-h normothermic perfusion with MoAb reactive with class II antigens (16.2 +/- 3.6 days, n = 19) compared with control animals (11.0 +/- 1.4 days, n = 24). In vitro treatment of islets with MoAb and complement (CI) inhibited their stimulatory capacity in the MILC, as measured by [3H]thymidine uptake. Similarly, the stimulatory capacity of islets removed from perfused pancreases was also abrogated when MoAb was included in the perfusate. Although reduction in graft immunogenicity, by increasing allograft survival, was achieved by a 3-h pretreatment regimen, it was not sufficient to inhibit rejection altogether in our transplant model.

Specificity in formation of triple-stranded nucleic acid helical complexes: studies with agarose-linked polyribonucleotide affinity columns.

The binding of a variety of deoxyribo and ribo homo- and copolynucleotide complementary duplexes to agarose-linked homopolynucleotide affinity columns has been studied. The results provide information concerning the specificity of recognition of complementary base pairs of nucleic acids through a mechanism that involves triple-helix formation under physiological conditions of ionic strength, pH, and temperature. The method employed made it possible, for the first time, to survey the full range of base triplets conceivable from the canonical nucleic acid bases and, in addition, hypoxanthine and thereby to differentiate between those triplets which can and cannot form. Certain previously observed features of the stereochemistry of double-helical targets for third-strand binding are confirmed, and some unrecognized features are elaborated. These include a general requirement for clusters of purine residues in one strand, protonation of third-strand C residues, the ability of natural third-strand residues to distinguish between A.T/U and G.C base pairs, and a capacity of third-strand (unnatural) I residues to recognize all base pairs within such clusters. Thus, the basis for a third-strand binding code is demonstrated.