In this paper, we have shown that Siva-1, a pro-apoptotic protein that lacks a true BH3 domain, behaves like a small BH3 only protein and plays a significant and essential but partial role in genotoxic (p53-induced) apoptosis. Using the colorectal cancer cell line HCT116, we have clearly shown the dependence of Siva-1 intracellular expression on functional p53. The expressed Siva-1 targets mitochondria and induces Bax oligomerization and caspase-9 activation and the twenty amino acid putative helical region in Siva-1 (SAH) is the principal mediator.
Our data demonstrating the dependence of endogenous Siva-1 expression on induced p53 activity is in accordance with previous reports published by Pommmier and coworker (2003) using gene profiling. They showed that the topoisomerase inhibitor topotecan induces Siva-1 expression (transcripts) in a p53 dependent manner.[12
] A previous study of differential gene expression analysis using cell cycle and apoptosis DNA microarray in cisplatin treated Hep3B cells also reveals significant induction of Siva expression.[11
] In addition, our data is also in agreement with a report previously published by Slack and coworkers that p53 as well as E2F1 directly activate the transcription of Siva-1.[4
] It is therefore not surprising that p53, a powerful tumor suppressor, induces apoptosis in susceptible cells by upregulating transcription of the pro-apoptotic gene Siva-1. As shown in , Siva-1 knockdown alone appears to promote cell survival; however, we have observed this only in HCT116 but not in Jurkat cells and, therefore, disregarded the observation. The maximum effect of Siva-1, however, was seen in cells treated with various apoptotic stimuli.[7
Although it lacks significant homology to the Bcl-2 family per se
, we have shown here that the ability of Siva-1 to bind anti-apoptotic proteins like Bcl-xL
may consequently result in direct activation of Bax and cytochrome C release. The unique putative twenty amino acid SAH region in Siva-1 was shown by us to be essential for its apoptotic function. Based on our earlier observation,[10
] it appears to behave like the BH3 only peptide of Bid, in inducing apoptosis.[16
] The time dependent increase in expressed Siva-1 in heavy membrane fraction, upon cisplatin treatment, is highly likely to be due its localization to mitochondria, Here we have shown that treatment of mitochondrial fraction with the above peptide results in cytochrome C leakage; in our previous studies, we demonstrated that it can specifically interact with Bcl2 or Bcl-xl but not Bax.[13
] Thus, induction of Siva-1 by p53 (genotoxic stress) is likely to result in its translocation to mitochondria and neutralize the pro-survival effects of Bcl2 anti-apoptotic members. This explains the enhanced Bax oligomerization seen in cells forced to overexpress Siva-1 .
A large body of work supports the idea that the antiapoptotic members such as Bcl2 and Bcl-xL counteract the apoptotic effects of Bax and Bak and thus act as sentinels of cell survival. Under conditions where the DNA is damaged and irreparable (genotoxic stress), the relatively small proapoptotic molecules such as Bid, Bad and Puma interfere with the above interaction, resulting in the release of Bax and Bak and their oligomerization. Oligomerization of Bax and Bak, in turn, results in loss in mitochondrial integrity and leakage of cytochrome c. The above events are typically induced by small BH3 only apoptotic molecules that are induced by p53. Siva-1, although induced by p53, is not a member of the Bcl2 family and yet induces oligomerization of Bax.
P53 is known to induce intrinsic apoptosis in a transcriptional independent as well as transcriptional dependent manner. The most well characterized proapoptotic proteins that are transcriptionally regulated by p53 are Bax and the BH3 only protein Puma.[17
] In normal cell, the levels of p53 are very low; however, they are rapidly elevated upon induction of DNA damage. This results in accumulation of p53 in cytosol. The apoptotic activity of p53 is neutralized by Bcl2 and Bcl-xl. Under the above conditions, Puma levels are also known to increase. It has a high affinity for Bcl2 and Bcl-xL and, thus, displaces p53 from Bcl2 and Bcl-xl, resulting in intrinsic apoptosis.[18
] There appears to be a strong dependence of Puma for p53, since it fails to induce significant apoptosis in p53 null cells.[2
] In this sense, Siva-1 appears to be different from that of Puma. Its ability to promote intrinsic cell death does not require p53, since expression of Siva-1 in p53 null cells also enhances cisplatin-induced cell death .[13
] Since elimination of DNA damaged cells to prevent tumorigenesis is a very important function, it appears to be regulated by redundant and overlapping pathways. For instance, p53 regulated Noxa appears to have a definitive role in ROS-induced cell death, whereas Puma is an essential mediator of DNA damage-induced cell death. The role of Puma appears to be somewhat limited by functional p53, although its transcription can also be regulated by E2F1.
Recently, an essential role for Siva-1 was also shown in campotheticin-induced apoptosis in cerebellar neuronal granules, that was also p53 dependent.[23
] The fact that expression of Siva-1 can potentiate cisplatin-induced apoptosis even in the absence of a functional p53 means that Siva-1 is likely to play a role in genotoxic stress-induced apoptosis, even if p53 is mutated and non-functional, as is the case with almost 50% of cancers.
Although we have shown that Siva-1 plays an essential and significant role in cisplatin-induced intrinsic cell death, despite the knockdown of Siva-1 expression, the cells still undergo considerable apoptosis. This could be explained by the fact that p53 induces transcription of several apoptotic genes, other than Siva-1, and these could be responsible for the residual cell death.[17