Journal of Undergraduate Research
Volume 5, Issue 3 - December 2003

Head Involution Defective (hid) Iap Antagonist - A Possible Transcriptional Gene Target for Tumor Suppressor Gene p53

Serggio Lanata

ABSTRACT

Our investigation focused on elucidating the molecular mechanisms of irradiation-induced apoptotic cell-death mediated by P53, with an emphasis on the Iap pathway. P53 is an important transcription factor that activates the cell death machinery when a cell is damaged. P53 is often mutated in cancer cells. This mutation allows cancer cells to escape Iaps inhibition and thus proliferate abnormally. Iaps act by suppressing the enzymatic activity of caspases - protease enzymes responsible for apoptotic cell death. Iaps can be released by action of specific Iap antagonists, thus allowing apoptosis to occur. We established a system to over-express p53 gene in alternate body segments of drosophila embryos. We then performed RNA in situ hybridization for four Iap antagonists, or cell-death genes, to determine if they are transcriptional targets of p53. This system allowed us to test how P53 leads to the activation of caspases and cell death. We found that over-expression of p53 is sufficient to drive the expression of hid Iap antagonist gene but not the others. Elucidating the molecular mechanism of P53 mediated irradiation induced cell death will help us develop alternative cancer treatment options.

INTRODUCTION

The importance of apoptosis in maintaining healthy tissue homeostasis

Our life, since its beginning, is a homeostatic balance between cellular life and death. During the fifth week of development, for example, a fetus’s fingers are joined by a thin sheath of skin. By the sixth week, cells located in this sheath die, separating the fingers and shaping the human hand as we know it. Throughout embryogenesis, our tissues and organs differentiate similarly. This highly coordinated cell elimination process is called programmed cell death (PCD), or apoptosis; without apoptosis, an embryo could not develop into an adult.

In adulthood, the role of apoptosis in maintaining physiological balance is equally important. Approximately 50 to 70 billion cells die throughout the day of an average adult as a result of apoptosis [4]. Such a massive cell-death process, aimed at eliminating obsolete or damaged cells, makes space available for the billions of new cells produced daily in self-renewing tissues such as the skin, gut, and bone marrow.

Similar apoptotic processes have been described in many other animal species; thus indicating that apoptosis is an evolutionarily-conserved process critical in maintaining adequate tissue homeostasis. If tissue homeostasis is disturbed, disease usually follows. In fact, disturbances of this homeostatic balance are the basis of many chronic diseases, cancer being the most prominent.

Apoptosis and cancer

Molecularly, a cell becomes cancerous when it fails to respond to natural environmental controls of cell growth and death. Cells that are potentially detrimental to an organism’s homeostasis, such as those with genetic alterations induced by exposure to UV sunrays, are eliminated by apoptosis - a safe-guarding. [As a result of apoptosis, for example, our skin peels off after severe sunburn]. Defects in these cell-death regulatory pathways can extend the lifespan of a noxious cell. When this happens, genetically altered cells proliferate uncontrollably and grow abnormally – they become neoplastic. In such an apoptosis-absent environment, the chances for cells to develop carcinogenic mutations increase dramatically. Therefore, cancer can be seen as a failure of genetically compromised cells to undergo apoptosis.

Irradiation-induced apoptosis

Our investigation focuses on irradiation-induced apoptosis; specifically, x-ray radiation. X-ray irradiation causes direct DNA damage. If the DNA cannot be repaired, the cell undergoes apoptosis. Irradiation-induced apoptosis is be mediated by three signal transduction pathways: the mitochondria pathway, the death receptor pathway, and the inhibitor of apoptosis protein (Iap) pathway [1]; our investigation focuses on the Iap pathway.

Ultimately, all three pathways share the same core cell-death machinery: Cell-death genes (specific to each pathway) activate a family of intracellular proteases called caspases (for cysteine aspartic acid-specific protease). Caspases fragment cellular material and cause the formation of lysosome-like vacuoles which store the fragmented cellular material. [This process maintains the cell membrane intact, thus internalizing secretions and cellular debris that would create an inflammatory response if spilled outside the cell]. The immune system clears the fragmented cells through phagocytosis.

Irradiation, Iaps, and p53

Our investigation focuses on the induction of apoptosis mediated by tumor suppressor gene p53 as a result of x-ray radiation, emphasizing p53’s interaction with the Iaps pathway. Iaps inhibit the enzymatic activity of caspases through direct interaction; they may also participate in caspase degradation [1]. In this respect, Iaps are very efficient at inhibiting apoptosis. Inhibition can be successfully released by Iap antagonists (rpr, hid, grim, corps). Iap antagonists, in turn, are possibly upregulated by p53. P53 is a tumor-suppressor protein (proapoptotic protein) present in every cell; its role in X-ray induced apoptosis is crucial.

As mentioned earlier, apoptosis is activated in response to irreversible DNA damage from x-ray radiation. Specifically, X-ray radiation induces an enzymatic reaction which inactivates the binding site of Mdm2 on p53 (Mdm2 is a negative regulator of p53 expression, thus inactivating p53 expression). Without its regulator protein, p53 is over-expressed and effectively induces the transcription of cell death regulatory genes which, in their protein forms, function upstream of one or more caspases. Caspases initiate the apoptotic process, as described above.

In short, p53 recognizes cell damage, and when this damage cannot be fixed, p53 initiates the cell-death machinery. This way apoptosis prevents the accumulation of harmful mutations that could give rise to cancer. Considering that 50% of cancerous cells have a mutated (non-functional) p53 gene, understanding p53’s interaction with the Iap antagonists and other cell-death genes is of crucial importance.

Drosophila Melanogaster as a model to study mammalian cancer development

More than 60% of genes implicated in human disease have Drosophila Melanogaster orthologues [3]. This makes the fruit fly an adequate model to indirectly study human disease. In fact, at least three of the major mammalian cell-death regulatory pathways are conserved in Drosophila.

Furthermore, Drosophila contains a p53 orthologue - Dmp53 - which shares many important functional and structural components with p53. Most notoriously, both proteins bind at similar DNA sites and act as transcriptional activators to mediate apoptosis.

METHODS

Gal4/UAS system

We used the Gal4/UAS system to generate a fly model that would enable us to test the phenotypic effect of p53 expression and identify transcriptional targets of p53 responsible for mediating x-ray induced cell-death. The Gal4/UAS system was originally derived from the yeast Saccharomyces cerevisiae and has been successfully adapted into Drosophila. It is one of the best tools to indirectly observe the function of genes involved in human apoptosis and cancer.

Gal4 protein products directly bind to the Upstream Activating Sequence (UAS) element and drive the expression of genes located downstream to the UAS element. Direct binding of Gal4 to UAS is essential for the transcriptional activation of the Gal4-regulated genes. In this sense, Gal4 is referred to as the driver of the responder genes located downstream from the UAS element (Fig 1).

Each Gal4 driver has a unique transcriptional pattern that enables the experimenter to monitor and control the expression of the responder gene. Fly strains with single Gal4 drivers and other strains carrying desired responder genes downstream to a UAS element are available.

Fly strains and genetic crosses

A fly strain with a [UAS p53]/[UAS p53] responder line was mated to a fly strain carrying a prdGal4/TM3 driver (Fig. 2). PrdGal5 drives the expression of p53 in a characteristic striped pattern in the fly embryos. Thus, absence of prdGal4 in the progeny containing the responder line, UASp53, maintains the embryos transcriptionally silent. To activate the transcription of p53, progeny flies must contain both the responder line and the prdGal4 driver.

Accordingly, half of the F1 progeny had a [UAS p53]/[prdGal4] genotype and therefore expressed the characteristic striped phenotypic pattern of the prdGal4 driver; the other half of the F1 progeny has a [UAS p53]/TM3 genotype, and therefore remains transcriptionally silent.

Embryo fixation

Embryos were collected after a 0 – 12 hour incubation period, at room temperature; embryo-collecting plates contained grapefruit concentrate with brewers yeast. Control and experimental groups were selected randomly. The Experimental group was exposed to X-ray radiation (40 Gy) and then set at room temperature (90 minutes) before fixation; experimental and control groups were fixed simultaneously.

RNA probe labeling, In situ hybridization

In situ hybridization using Dig-labeled complementary RNA (cRNA) was used to identify embryos of the F1 generation that were transcriptionally active. cRNA probes were used to identify corresponding sequences of mRNA in embryonic Drosophila tissue. Hydrogen-bonding between cRNA probes and mRNA occurred within specific regions (those determined by the transcriptional activity of the prdGal4 driver).

cRNA probes for p53 and four Iap antagonists - hid, grim, rpr, and corps - were synthesized using a bacterial vector and T3 RNA polymerase. We used Digoxigenin (Dig) labeled nucleotides (non-radioactive). Dig-labeled probes provide high sensitivity, resolution, and stability. After transcription (two hours at 37°C) the mRNA products (Dig-labeled cRNA probes) of p53, hid, rpr, sickle, and corps were run through a gel, isolated, and precipitated in LiCl and ethanol.

We used a protease enzyme to increase the membrane permeability of the embryos during the in situ hybridization; and we used formaldehyde to maintain the embryo’s structural integrity.

RESULTS

We observed expression of p53 tumor suppressor gene in alternate body segments - the prdGal4/UASp53 system worked successfully. After performing the Dig-labeled p53 cRNA in situ hybridization, both the experimental group and the control group showed transcription of p53 gene in the recognizable alternate segment pattern of the prdGal4 driver (Fig. 3).

P53 is sufficient in directing the expression of hid but not other cell death genes.We performed Dig-labeled in situ hybridization for grim, rpr, hid, and corps in both experimental and control group embryos. Grim, rpr, and corps showed evidence of transcriptional activity, but not in the prdGal4 pattern. Dig-labeled in situ hybridization of hid in both experimental and control groups showed evidence of transcriptional activity in the striped pattern of prdGal4 (Fig. 4).

P53 alone is not sufficient in increasing the sensitivity to x-ray induced cell death.

Figure 4: hid in situ hybridization. A) Transversal view; B) sagittal view.

DISCUSSION

The controlled expression system worked for p53. A stripped, alternate pattern of transcription, characteristic of the prdGal4 driver, was observed in prdGal4/UASp53 embryos.

The controlled-expression system is sufficient in driving the expression of hid but not rpr, corps, and grim; however, all four are induced after x-ray treatment. This result indicates that, together with p53, other factors are involved in mediating x-ray induced cell death.

Over-expression of p53 is sufficient to drive the expression of hid Iap antagonist gene, suggesting that hid is a direct transcriptional target of p53. According to our results, p53 alone is not sufficient to activate the transcription of the other Iap antagonist genes tested: rpr, grim, and corps.

Our results are supported by the fact that Dig-labeled cRNA in situ hybridization using hid and p53 probes in the prdGal4/UASp53 controlled system embryos showed the striped transcriptional pattern characteristic of prdGal4 driver. Because the Gal4/UAS system was constructed to express the p53 responder gene only, the fact that hid was transcribed in these embryos in the same pattern as p53 suggests that p53 is sufficient to drive the transcription of hid. Interestingly, hid expression is observed in both the control and the experimental embryos; indicating that P53 can activate the expression of hid in the absence of x-ray irradiation.

This is not the case of the other Iap antagonists, or cell-death genes. Grim, rpr, and corps do not show the transcriptional pattern observed for p53, as indicated by Dig-labeled cRNA in situ hybridization in both the control and experimental embryos of the prdGal4/UASp53 system. This fits with the observation that over-expression of p53 is not sufficient to increase the sensitivity to x-ray induced cell death. In fact, in situ hybridization showed that transcriptional activity for rpr, hid, and grim resembles that of wild type embryos. This result indicates that p53 alone is not sufficient in driving the expression of these pro-apoptotic genes. Perhaps the molecular mechanism behind the expression of these Iap antagonists involves different pathways that not necessarily include p53; or P53 must function with other molecules to drive the expression of these genes.

We have our own orthologues of Drosophila Iap antagonist genes - the smac and HtrA3/omi genes. In this respect, it will be interesting to see whether smac orthologue is also a transcriptional target of p53. As mentioned earlier, p53 is mutated in over 50% of cancerous cells; thus, identifying those Iap antagonist cell-death genes that are transcriptional targets of p53 may help us bypass p53’s role and directly induce the transcription of these cell-death genes in order to overcome the cell’s resistance to apoptosis. This way we could successfully kill cancerous cells.

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REFERENCES

1. Zhou L, Yuan R, Lanata S. 2003. Molecular mechanisms of irradiation-induced apoptosis. Frontiers in Bioscience 8:9-19
   
   
2. Duffy B. 2002. GAL4 System in Drosophila: A Fly Geneticist’s Swiss Army Knife. Genesis 34:1-15.
   
   
3. Bernards A, Hariharan I. 2001. Of flies and men – studying human disease in Drosophila. Current Opinion in Genetics and Development 11:274 – 278.
   
   
4. Reed J. 1999. Dysregulation of Apoptosis in Cancer. Journal of Clinical Oncology 17:2941-2953.
   
   
5. Wing J, Zhou L, Schwartz L, Nambu J. 1998. Distinct cell killing properties of the Drosophila reaper, head involution defective, and grim genes. Cell Death and Differentiation 5:930-939.

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