Suicide cancer gene therapy using pore-forming toxin, streptolysin O.

We cloned the streptolysin O gene from the Streptococcus pyogenes genome and tested the possibility of using it as an anticancer reagent. Transient transfection of the streptolysin O gene efficiently killed 293T cells after 12 hours of transfection as determined by lactate dehydrogenase release and propidium iodide uptake. No caspase activity was observed and necrosis was prominent during streptolysin O-induced cell death. Biochemical analysis of streptolysin O protein revealed that the deletion of only 5 amino acids from the COOH-terminal region of streptolysin O, which is essential for cholesterol binding activity, abolished its cell-killing activity, whereas the NH2-terminal region was more resilient, i.e., up to 115 amino acids could be deleted without changing its cell-killing activity. We generated a streptolysin O-expressing adenovirus and injected it into human cervical cancer cell-derived tumors grown in a nude mouse model. Twenty-one days postinjection, the average size of tumors in the streptolysin O adenovirus-injected group was 29.3% of that of the control PBS-treated group. Our results show that the genes of pore-forming toxins, like streptolysin O protein, have the potential to establish a novel class of suicide gene therapeutic reagents.

Conversion of Bfl-1, an anti-apoptotic Bcl-2 family protein, to a potent pro-apoptotic protein by fusion with green fluorescent protein (GFP).

Human Bfl-1 is an anti-apoptotic Bcl-2 family member. Here, we found that Bfl-1 was converted into a potent death-promoting protein by green fluorescent protein (GFP) fusion with its N-terminus. The transient expression of GFP-Bfl-1 induced cytochrome c release and triggered apoptosis in 293T cells, which depended on the mitochondrial localization of GFP-Bfl-1. Apoptosis induced by GFP-Bfl-1 was significantly blocked by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone, but was not blocked by either Bcl-xL or Bfl-1. Our findings provide a useful model for understanding the structural basis of Bcl-2 family proteins that act in an opposite way despite sharing structural similarity between anti-apoptotic and pro-apoptotic proteins.

C-terminal region of Bfl-1 induces cell death that accompanies caspase activation when fused with GFP.

Previously, we reported that anti-apoptotic Bfl-1 is converted to a pro-apoptotic protein following fusion at its N-terminus with green fluorescent protein (GFP) (GFP-Bfl-1). In this study, we performed a Bfl-1 deletion study in order to elucidate the underlying mechanism of GFP-Bfl-1-induced cell death. We found that the Bcl-2 homology (BH) domains in Bfl-1 are dispensable with respect to cell death and that GFP fusion with the 29 amino acids of the C-terminal region of Bfl-1 (GFP-BC) is sufficient to induce cell death. Moreover, when BC was fused with other tagging partners like GST or MBP, little cell death was observed, implying that the GFP region is as important as the BC region for GFP-BC-induced cell death. Further deletion analysis defined a region of GFP as a determinant of GFP-BC-induced cell death. Confocal microscopic analysis showed that GFP-chimeras containing the BC region of Bfl-1 are located mainly in mitochondria. The GFP-BC-induced cell death accompanied cellular caspase activation, and treatment with the pan-caspase inhibitor, Boc-D-FMK, partially inhibited GFP-BC-induced cell death. However, the over-expression of anti-apoptotic molecules, such as Bcl-x(L) and CrmA, did not block GFP-BC-induced cell death. In summary, GFP-BC induces cell death with caspase activation through mitochondria dependent process.