A gene encoding a putrescine oxidase (PuO Rh , EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (K M =8.2 μM, k cat = 26 s −1 ). PuO Rh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuO Rh is a reasonably thermostable enzyme with t 1/2 at 50°C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuO Rh , which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuO Rh .

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine and a selective inducer of apoptosis in a range of tumour cells, but not in normal, untransformed cells. A large number of chemotherapeutics as well as biological agents are being tested for their potential to sensitise resistant tumour cells to TRAIL as a means to broaden the range of tumours treatable with TRAIL. However, because of the incomplete understanding of the mechanism(s) underlying TRAIL resistance in non-malignant cells, it is unpredictable whether the effect of these sensitisers will be restricted to tumour cells or they would also sensitise non-transformed cells causing unwanted toxicity. In this study, we carried out a systematic analysis of the mechanisms driving TRAIL resistance in non-transformed cells. We found that cellular FLICE-like inhibitory protein, anti-apoptotic B-cell lymphoma 2 proteins, and X-linked inhibitor of apoptosis protein were independently able to provide resistance to TRAIL. Deficiency of only one of these proteins was not sufficient to elicit TRAIL sensitivity, demonstrating that in non-transformed cells multiple pathways control TRAIL resistance and they act in a redundant manner. This is contrary to the resistance mechanisms found in tumour cell types, many of them tend to rely on a single mechanism of resistance. Supporting this notion we found that 76% of TRAIL-resistant cell lines (13 out of 17) expressed only one of the above-identified anti-apoptotic proteins at a high level (≥1.2-fold higher than the mean expression across all cell lines). Furthermore, inhibition or knockdown of the single overexpressed protein in these tumour cells was sufficient to trigger TRAIL sensitivity. Therefore, the redundancy in resistance pathways in non-transformed cells may offer a safe therapeutic window for TRAIL-based combination therapies where selective sensitisation of the tumour to TRAIL can be achieved by targeting the single non-redundant resistance pathway.

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Acute myeloid leukemia (AML) is an aggressive stem cell malignancy that is difficult to treat. There are limitations to the current treatment regimes especially after disease relapse, and therefore new therapeutic agents are urgently required which can overcome drug resistance whilst avoiding unnecessary toxicity. Among newer targeted agents, both tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and proteasome inhibitors show particular promise. In this report we show that a combination of the proteasome inhibitor bortezomib and TRAIL is effective against AML cell lines, in particular, AML cell lines displaying myelomonocytic/monocytic phenotype (M4/M5 AML based on FAB classification), which account for 20-30% of AML cases. We show that the underlying mechanism of sensitization is at least in part due to bortezomib mediated downregulation of c-FLIP and XIAP, which is likely to be regulated by NF-κB. Blockage of NF-κB activation with BMS-345541 equally sensitized myelomonocytic AML cell lines and primary AML blasts to TRAIL.