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RT-PCR Primers for the Study of Apoptosis


Investigating programmed cell death

Wei-ping Yang Danny Hoang Douglas McKenzie Peter Pingerelli
Stratagene

The apoptotic response is a complex biological process that requires the regulation of a variety of genes that interact with and respond to intracellular and extracellular stimuli. Stratagene is introducing RT-PCR primer sets that are specific for genes involved in inducing and regulating the apoptotic response. These primers are specific for genes encoding proteins from the Fas and Fas ligand, Bcl-2 and ICE protein families. The new RT-PCR primer sets will find application for diverse disciplines, including developmental biology, immunology, and tumor biology.

Apoptosis, the gene-directed process of programmed cell death, plays a central role during development and homeostasis in most organisms. The activation of apoptosis is associated with many diseases, such as cancer, acquired immunodeficiency syndrome (AIDS), and neurodegenerative disorders. The basic machinery to carry out apoptosis appears to be present in essentially all mammalian cells at all times, but the activation of apoptosis is regulated by many different signals that originate from both the intracellular and the extracellular milieu. Components in these signaling pathways are varied, and many agents have been characterized either as inducers or inhibitors of the apoptotic response. During the last few years, the cell death pathway has been extensively studied, and three protein families have been identified according to their functions in apoptosis. These proteins are Fas and Fas ligands, Bcl-2 (and analogs), and the ICE families.

The activation of death receptors, such as the Fas ligand, may induce the apoptosis pathway. The Fas antigen, which is located on the cell surface of most mammalian cells, was discovered in 1989.1,2 By virtue of its structure, the Fas membrane protein was identified as a member of the tumor necrosis factor (TNF) and nerve growth factor (NGF) receptor family. When the Fas ligand binds to Fas, the death pathway of the target cell is initiated.3 Cases of both up-regulation and down-regulation of the Fas system have been identified; such regulation characterizes Fas-mediated diseases. In certain animal disease models, such as the mouse spontaneous mutations lpr (lymphoproliferation), the expression of Fas is decreased. A similar observation is also seen in human Systemic Lupus Erythematosus (SLE). In various cell lines, the expression of the Fas protein is up-regulated by interferon-g (IFN-g).

Fas-mediated apoptosis requires the activation of a class of cysteine proteases, which include the ICE family.4 In humans, the ICE family includes CPP-32 and LAP-3 proteases. Triggering of Fas by either the Fas ligand or the anti-Fas antibody rapidly stimulates the proteolytic activity of ICE proteins. Overexpression of the ICE proteins strongly potentiates Fas-mediated cell death; inhibitors of ICE proteins inhibit the apoptotic process. Thus, determining the expression of ICE family proteins is an important marker for monitoring the cell death process.

During normal cell growth stages, the family of Bcl-2 proteins is present as the antagonists of cell death. When the death signal is presented to the cell, the expression of Bcl proteins is down-regulated, which helps the death signal to trigger the activation of the ICE protease family. Consequently, the cell undergoes a characteristic apoptosis process that includes endonuclease activation, cell surface alteration, and cytoskeletal reorganization. Eventually, the cell undergoes phagocytosis. In apoptosis, the dying cell is eliminated although the inflammatory response is not induced.

Primer Design

Stratagene has developed reverse transcriptase-mediated polymerase chain reaction (RT-PCR) primer sets for genes whose products are important in the induction or regulation of the apoptotic response. These primers will find application in diverse disciplines, including developmental biology, immunology, and tumor biology. By using these primers, researchers will be able to analyze the transcription state of genes in uninduced cells and cells induced to undergo apoptosis.

The RT-PCR primer sets for studying the apoptotic response were designed to meet several criteria: (1) The primers sets, based on known genomic sequences, amplify a region that spans at least one intron. (2) To distinguish the amplification products from genomic sequences, which are longer than the cDNA products, the primer sets amplify PCR products that are 400 bp to 650 bp in length. (3) The primers were synthesized as 18- to 27-mer oligonucleotides and maintain close to 50% GC content whenever possible. (4) Each set of primers amplifies only a specific target

Testing Primers with cDNA Templates

Table 1
Target Genes and Expected Size of the PCR Product

Target Gene

Size of PCR Product

Mouse ICE (a,b,g) Splice Variants

a: 610 bp, b: 520 bp, g: 380 bp

Mouse ICE (all)

470 bp

Human ICE-b

600 bp

CPP32

540 bp

ICE-Lap-3

600 bp

Human Fas Antigen

653 bp

Rat Fas Antigen

661 bp

Mouse Fas Antigen

590 bp

Mouse Fas Ligand

551 bp

A variety of cell lines were used to generate cDNA libraries to test the specificity of the RT-PCR primers for studying apoptosis. The target genes and the expected size of the PCR product are listed in Table 1. Each 50-l PCR amplification contained 10- to 15-l aliquots of cDNA libraries, 5 l of 10X Taq polymerase buffer, 2 l of 100 M primer sets, 4 l of 2.5 mM dNTP mixture, 0.5 l of Taq2000 DNA polymerase (5 U/l), and 28.5 l of distilled, deionized H2O. Mineral oil (100 l) was added on each tube.

The PCR amplifications were cycled using the RoboCycler temperature cycler and the following parameters: 1 cycle of 94C for 5 minutes; 40 cycles of 94C for 1 minute, 60C for 2 minutes and 72C for 2 minutes; 1 cycle of 72C for 10 minutes. The reactions were stored at 6C. From each PCR amplification, 20 l was analyzed by electrophoresis on a 1.2% agarose gel in TAE buffer with 1 mg/ml of ethidium bromide for staining. The results were visualized using Stratagenes Eagle Eye II still video system.

Mouse ICE Family

Figure 1

A mouse macrophage cDNA library was used as template for the PCR amplifications with two sets of primers (Figure 1). One set, mouse ICE (a,b,g), amplifies the ICE gene and distinguishes the splice variants of ICE. These variants generate PCR products of different lengths: 610-bp PCR product for ICE-a, 520-bp PCR product for ICE-b, and 380-bp PCR product for ICE-g. In contrast, the ICE (all) primer set does not distinguish among the splice variants and generates a 470-bp product for all ICE variants.

Human ICE Family and Fas Antigen

Figure 2

To test human ICE-b , CPP32, ICE-Lap-3, and Fas antigen RT-PCR primers, five human cDNA libraries were used as templates (Figure 2 and Figure 3). Human genomic DNA was used as the genomic DNA control, and mouse and rat cDNA were tested as negative controls. Each amplification yielded a specific band at the expected length: For ICE- b, a 600-bp band was observed; for CPP32, a 540-bp fragment was observed and for ICE-Lap-3, a 600-bp band was observed. The genomic DNA and mouse and rat cDNA did not generate similar PCR products. The relatively weak bands seen for CPP32 may reflect the abundance of the CPP32 cDNA present in the cDNA library. Increasing the concentration of template in the reaction by 50% did not result in a significant increase of PCR products.

Figure 3

The ICE-Lap-3 and Fas antigen primer sets did not amplify the expected PCR product from the human muscle cDNA library. Repeated PCR attempts generated the same results. This may be explained by the lack of specific cDNA target in the library. A control primer, human b-actin, was used to test the cDNA library. An expected band was amplified for all human cDNA libraries tested (data not shown).

Mouse Fas Antigen and Fas Ligand

Several different mouse cDNA library templates were used to amplify Fas antigen and Fas ligand gene products (Figure 4). Both mouse Fas antigen and mouse Fas ligand RT-PCR primer sets amplified PCR products of the expected sizes. The Fas ligand gene has a wide distribution range in different tissues; however, the Fas antigen gene is differentially expressed in certain tissues. No amplification occurred in the control rat and human cDNA libraries.

Figure 4

Rat Fas Antigen

The rat Fas antigen gene was amplified in rat brain, lung, and heart cDNA libraries (Figure 5). The rat heart cDNA library generated a PCR product larger in size than expected (lane 4). This may be a spliced version of Fas antigen found in testes or an artifact. Again, the amplification of the rat Fas antigen gene is specific. No band appeared on the control lanes with mouse and human cDNA libraries.

Figure 5

Conclusions

Apoptosis is one of the most actively studied areas in the biological sciences. Stratagenes RT-PCR primer sets for studying apoptosis are specific for genes involved in inducing and regulating the apoptotic response. These primer sets target human, rat, and mouse genes that encode proteins from the Fas and Fas ligand, Bcl-2, and ICE families. The specificity of these primers has been tested and confirmed in a variety of RT-PCR reactions. The availability of these primer sets will enhance research efforts in disciplines such as developmental biology, immunology, and tumor biology.

Acknowledgments

We wish to kindly thank Mark Dycaico for providing human genomic DNA

REFERENCES

  1. Tranth, B.C., et al. (1989) Science 245: 301.

  2. Yonehara, S., Ishii, A., and Yonehara, M. (1989) J. Exp. Med. 169: 17471.

  3. Nagata, S., et al. (1994) Adv. Immunol. 57: 129.

  4. Douglas, P., et al. (1992) Science 256. 97.


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