Recently, we demonstrated that Pseudomonas aeruginosa Exotoxin T (ExoT) employs two distinct mechanisms to induce potent apoptotic cytotoxicity in a variety of cancer cell lines. We further demonstrated that it can significantly reduce tumour growth in an animal model for melanoma. During these studies, we observed that melanoma cells that were transfected with ExoT failed to undergo mitosis, regardless of whether they eventually succumbed to ExoT-induced apoptosis or survived in ExoT's presence. In this report, we sought to investigate ExoT's antiproliferative activity in melanoma. We delivered ExoT into B16 melanoma cells by bacteria (to show necessity) and by transfection (to show sufficiency). Our data indicate that ExoT exerts a potent antiproliferative function in melanoma cells. We show that ExoT causes cell cycle arrest in G1 interphase in melanoma cells by dampening the G1/S checkpoint proteins. Our data demonstrate that both domains of ExoT; (the ADP-ribosyltransferase (ADPRT) domain and the GTPase activating protein (GAP) domain); contribute to ExoT-induced G1 cell cycle arrest in melanoma. Finally, we show that the ADPRT-induced G1 cell cycle arrest in melanoma cells likely involves the Crk adaptor protein. Our data reveal a novel virulence function for ExoT and further highlight the therapeutic potential of ExoT against cancer.

中文翻译：

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铜绿假单胞菌 ExoT 诱导黑色素瘤细胞 G1 细胞周期停滞

最近，我们证明铜绿假单胞菌外毒素 T (ExoT) 采用两种不同的机制在多种癌细胞系中诱导有效的凋亡细胞毒性。我们进一步证明它可以显着减少黑色素瘤动物模型中的肿瘤生长。在这些研究中，我们观察到转染 ExoT 的黑色素瘤细胞无法进行有丝分裂，无论它们最终是死于 ExoT 诱导的细胞凋亡还是在 ExoT 存在的情况下存活。在本报告中，我们试图研究 ExoT 在黑色素瘤中的抗增殖活性。我们通过细菌（以显示必要性）和转染（以显示充分性）将 ExoT 递送至 B16 黑色素瘤细胞中。我们的数据表明 ExoT 在黑色素瘤细胞中发挥有效的抗增殖功能。我们发现 ExoT 通过抑制 G1/S 检查点蛋白导致黑色素瘤细胞的细胞周期停滞在 G1 间期。我们的数据表明 ExoT 的两个域；（ADP-核糖基转移酶（ADPRT）结构域和GTP酶激活蛋白（GAP）结构域）；有助于 ExoT 诱导的黑色素瘤 G1 细胞周期停滞。最后，我们发现 ADPRT 诱导的黑色素瘤细胞 G1 细胞周期停滞可能涉及 Crk 接头蛋白。我们的数据揭示了 ExoT 的新毒力功能，并进一步强调了 ExoT 抗癌的治疗潜力。