harakiri, a novel regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting proteins Bcl-2 and Bcl-XL

Abstract

Programmed cell death is essential in organ development and tissue homeostasis and its deregulation is associated with the development of several diseases in mice and humans. The precise mechanisms that control cell death have not been elucidated fully, but it is well established that this form of cellular demise is regulated by a genetic program which is activated in the dying cell. Here we report the identification, cloning and characterization of harakiri, a novel gene that regulates apoptosis. The product of harakiri, Hrk, physically interacts with the death‐repressor proteins Bcl‐2 and Bcl‐XL, but not with death‐promoting homologs, Bax or Bak. Hrk lacks conserved BH1 and BH2 regions and significant homology to Bcl‐2 family members or any other protein, except for a stretch of eight amino acids that exhibits high homology with BH3 regions. Expression of Hrk induces cell death which is inhibited by Bcl‐2 and Bcl‐XL. Deletion of 16 amino acids including the conserved BH3 region abolished the ability of Hrk to interact with Bcl‐2 and Bcl‐XL in mammalian cells. Moreover, the killing activity of this mutant form of Hrk (Hrk ΔBH3) was eliminated or dramatically reduced, suggesting that Hrk activates cell death at least in part by interacting with and inhibiting the protection afforded by Bcl‐2 and Bcl‐XL. Because Hrk lacks conserved BH1 and BH2 domains that define Bcl‐2 family members, we propose that Hrk and Bik/Nbk, another BH3‐containing protein that activates apoptosis, represent a novel class of proteins that regulate apoptosis by interacting selectively with survival‐promoting Bcl‐2 and Bcl‐XL.

Evaluation of a new dual-specificity promoter for selective induction of apoptosis in breast cancer cells

“…It is a member of the BH3 -only subfamily of pro -apoptotic Bcl -2 proteins that act, at least in part, by binding to and inactivating the antiapoptotic proteins Bcl -2 and Bcl -x L . 32 We have observed quick and massive cell death in permissive cells transfected with the pBEHhrk plasmid or infected with AdEHhrk when the expression of the gene was activated by estrogens and /or hypoxia. The construction of a conventional E1 -deleted adenovirus was useful to demonstrate that the system retains its properties in the context of this kind of vectors.…”

“…The plasmid pBEHhrk contains the harakiri gene under the control of the ERE /HRE promoter. It was obtained by blunt -end ligation of the HindIII ± XbaI fragment of pCDNA -hrk ( containing the harakiri gene ) 32 into the BglII ±EcoNI sites of pBERE/ HRE.…”

“…As a positive control for induction of apoptosis, we used a plasmid with constitutive expression of harakiri ( pcDNA3 -hrk ). 32 …”

“…Hrk, the product of the harakiri gene, is a potent inductor of apoptosis that was originally characterized using HEK 293T cells. 32 We performed transient transfections of pBEHhrk in MCF7 cells, together with a lower amount of a -galactosidase reporter plasmid ( pCMV ) to identify the transfected cells after X -Gal staining. The cells had been deprived of estrogens before and during the transfection.…”

“…28 Several pro -apoptotic genes have been used in gene therapy approaches to fight cancer. 29 ± 31 In this work, we describe the construction and characterization of a replication-deficient adenovirus vector that expresses harakiri, a potent pro -apoptotic gene, 32 under the control of the new ER and hypoxia-specific promoter. We show evidence that this promoter can be used to direct the expression of a lethal gene and achieve controlled killing of breast cancer cells.…”