UPDATE 09/18/2013

Which suicide gene?

Why suicide gene?

In our project, we try to build a circuit that can prevent the iPSC-differentiated tissues from tumor formation. Instead of using some medicines to kill them, like the traditional gene therapies did, what we want to achieve here is to make the tumor cells kill themselves, in other word, automatically commit suicide. So we decided to use an exogenous suicide gene.

The ideal suicide genes that fit our design here will be genes that can successfully induce apoptosis in cells and do not introduce any harmful effect to normal tissues. However, many apoptosis pathways are reported to be blocked in tumor cells. From recent reports and papers, we found out that there have been many genes reported to have important roles in mammalian cell apoptosis. After a long time comparison and consideration, our final candidates of suicide gene are listed below:

- hBax & hBax mutant

- delta TK

- caspase3

- rip1

- rip3

- apoptin

The introduction of each genes about its suicide mechanisms and the reasons why we choose them are as follows:

Which suicide gene?

hBax & hBax mutant

hBax is a member of the Bcl-2 related protein family from human. This Family contains pro-apoptotic and anti-apoptotic proteins, and the balance among them determines the cell survival. hBax is the pro-apoptotic protein. During apoptosis, hBax will be inserted into the mitochondrial outer membrane and form permeable channels, release pro-apoptotic signals, finally lead to apoptosis[1]. The pathway of apoptosis is showed in Figure 1.

Figure1. apoptosis pathway induced by hBax

hBax S184a[3] is a mutant of hBax that can constantly insert into mitochondrial outer membrane, thus we guess that it may have a stronger apoptosis-induced effect than normal hBax.

It have been reported that over expression of this gene can successfully induce apoptosis in both Hela cell line and HEK-293 cell line[2]. Due to its generality, we finally determined to use it as one of our suicide genes candidates.

However, our experiments results finally showed to us that, when expressed in Hep G2 cell line, which is a typical hepatoma cell line, they can not induce observable apoptosis. However, although it can not successfully kill Hep G2 cell line, we figured out that the pathway is conserved in yeast[4] ,so we also tried to transfect the gene and its mutant in yeast and finally proved that the killing effect is observable and even dose-dependent (BBa_K1061006). So this extra result may be useful for other team in the future who want to apply safety device in yeast. We finally submitted the hBax as a biobrick and its mutant form as an improvement of the pre-existing part of part registry.

(show results)

Caspase 3

Caspase 3 is the last downstream executer of apoptosis in most mammalian cells. Almost every apoptosis process needs the execution of caspase 3. As a cysteine protease, it can directly cleavage proteins inside cells and take part in DNA fragmentation[4]. Caspase 3 contains two subunits, p17 and p12, which are translated in the same ORF. When cleavaged by caspase 9, another kind of protease involving in apoptosis, they will form a dimer that will act as an active form[5].

Actually we split its gene into two parts, p17 and p12, and we used leuzine zipper to direct the dimerization of the two subunits[5].

However, in our project we finally decided to drop trying this gene, mainly for 3 reasons:

1. The apoptotic effect needs two subunit to be expressed simultaneously, which would increase the complication of our circuit.
2. To overcome the anti-apoptotic protein XIAP[6], which is high-expressed in Hep G2 cell line[7], we may need an extremely high expression of caspase 3, which would also be a problem for our experiment.
3. We have mistakenly clone the wrong ORF of two subunits from the plasmid, so it leave us no time to do the experiment of caspase 3 before regional jamboree=.=.

Besides, we may try to test another version of active caspase 3---the reconstitute caspase 3 [8]in following days.

RIP 1

RIP1 is the abbreviation of Receptor Interacting Protein kinase 1 in mammalian cells. It is an important regulator of cell survival and death, taking part in several program cell death pathways[9]. It has been reported that over expression of RIP 1 can induce both apoptosis and necrosis[10] in certain cell lines. So it is a potential suitable suicide gene that we can use in our device.

We tested rip1 in several cell lines, including HTC-75, Bosc, and Hep G2 cell lines. What we have observed is a mix of both apoptosis and necrosis( result ). Although it can induce necrosis in cancer cell lines, which may potentially cause inflammation in tissues, we still consider it as our choice of suicide gene. The reasons are as follows:

1. We know that in iPSC differentiation period, only a small fraction of cells will become cancerous at a certain time, so the necrosis of these cells would not likely to lead to severe inflammation.
2. Many apoptosis pathways have been reported to be blocked in tumor cells, but there is some evidence revealing that when the apoptosis pathways are blocked the necrosis pathway will be activated[11]. Besides, some recent papers showed that RIP1 can trigger cell necrosis only with extra exogenous medicine. In other circumstances RIP1 could lead to apoptosis.
3. We have cloned another receptor interacting protein kinase, RIP 3, which has been proved to interact with RIP 1 and lead to necrosis[12], so we think that even the over expression of RIP 1 can not successfully kill the cells, it may also co-express with RIP 3.

Surely, we will keep trying to find genes that only lead to apoptosis but not necrosis in the future, the better solution, in our mind, may be a combination of multiple apoptotic genes.

RIP 3

　　 RIP3, like RIP 1, is also a member of Receptor Interacting Protein family. It works via the interaction with RIP 1, and thus induces the necrosis pathway which RIP 1 mediated.[1]

However, it have also been reported that over expression of RIP 3 in certain cell lines can induce apoptosis[15].In our experiment, we found out that RIP 3 can lead to cell death in several cell lines, including HTC-75 cell lines and HEK-293 cell lines.

Because RIP 3 mainly induces necrosis, we select it as our candidate of suicide genes for the same reasons as we select RIP 1. And we also suggest that when using RIP 3 to cure cancer, tight control expression system may be necessary.

Apoptin

Apoptin, a protein first isolated from Chicken anemia virus, has been regarded as a potential drug for cancer treatment[16]. It has been reported in over 70 cell lines that apoptin can selectively kill cancer cells but not normal cells[17]. The result of in vivo test in mice is very exciting: the intraperitoneal injection of vector carrying the apoptin seems not confer any observable side effect on mice[18].

The mechanism of how apoptin works is not fully understood. It probably works via the non-p53 apoptosis pathway[17],hence is not easy to be blocked. The localization of apoptin in cancer cells is in nuclear, while in normal cells it locates in cytoplasm[17] . This was proved by our experiment results.

A special character of apoptin is that it works like a sensor, probably by recognizing certain early signals of cancer formation[17]. These signals may be general, which explained the reason why the apoptin can kill such a broad spectrum of cancer cells. So the construct of EF-1alpha-apoptin may provide a general circuit for safety issues of gene therapy and renew the concept of sensor. Making use of the by-stander effect, TAT-apoptin[20] or SP-TAT-apoptin[21] may be powerful and provide a "safe environment" for the cells under genetic manipulation.

However, we still have to say that although Apoptin has been proved to be safe for many normal cell types, it has not been proved in all normal cell types. So we should consider and test thoroughly about the immune reaction effect, making sure that it does not happen or can be controlled.