Instead, the vast majority is located in the cytoplasm,17, 32 although the underlying mechanism that regulates cytoplasmic and nuclear localization of Twist1 is not clear. It has been reported that the NLS in Twist1 can lead to the interaction between Twist1 and the nuclear membrane pore channel and the NLS can also induce Twist1 to enter the

nucleus and act as a transcription factor.33 In the present study we provide data to show that the up-regulation of Twist1 reaches its peak level 24 hours after hypoxia, whereas the expression of Twist1 decreased after 24 hours, as many of these APO866 ic50 cells die due to continued hypoxia. When hypoxia was relieved after 24 hours, the high expression level signaling pathway of Twist1 can be sustained for more than 24 hours. Interestingly, the antiapoptotic protein Bcl-2 also exhibited an expression peak and trend similar to Twist1 within the same period (24 hours). This result indicated that Bcl-2 and Twist1 possibly acted during the stress phase in the same cell and followed similar kinetics. Bcl-2 and its family members

have been found to mediate the apoptosis process. They have also been found to participate in protein modification and to form a complex with other proteins for participating in complicated processes of cell metabolism.2, 3, 34 In tumor tissues, Bcl-2 expression in the nucleus correlates with poor prognosis. Our data provide evidence that Bcl-2 may form a complex with Twist1 and synergistically to promote the transcription of downstream target genes which can lead a cascade changes in proliferation, adhesion, migration, infestation, clone formation, and tubal formation of tumor cells. The formation of Bcl-2 with Twist1 as a protein complex to stimulate the transcription was unexpected. Bcl-2

has long been considered as a mitochondrial membrane protein. However, reports on the effects of Bcl-2 and its other family members in more complex biological processes are limited. The present study revealed that specific amino acids within Bcl-2 and Linifanib (ABT-869) Twist1 are involved in the binding of two proteins and form a novel functional complex and jointly enter the nucleus, which leads to changes of multiple downstream target genes. Such a heterodimer is more potent in stimulating the transcription of multiple downstream target genes than Twist1 alone. Although the detailed mechanisms for interaction between Twist1 and Bcl-2 are not clear at this time, we speculate that the following mechanisms may be involved. Bcl-2 may be initially associated with the nuclear membrane pore structure, and assists Twist1 in entering the nucleus. In the nucleus, Bcl-2 and Twist1 forms a protein complex and functions in synergy on the promoters of different target genes to regulate their transcription.