To the Editor
Apoptosis is a biological process that plays a critical role in the development and homeostasis of multicellular organisms Citation[1]. The extrinsic apoptosis pathway initiates at the cell surface through the ligation of the death receptors Fas or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors by their ligands Citation[1]. While Fas directly binds with FADD, TRAIL receptors do not directly bond with FADD. Death-associated protein 3 (DAP3) serves as an adaptor protein, linking TRAIL receptor 1 and 2 to FADD Citation[2]. Also, a recent study reported that DAP3 localizes in the mitochondria and is involved in the process of mitochondrial fragmentation during apoptosis Citation[3]. DAP3 contains an ATP/GTP-binding motif (P-loop) that are also found in many apoptosis regulators, including CED-4, Apaf-1, ARTS, Nod1 and Nod2 Citation[1], Citation[2]. The DAP3 P-loop associates with TRAIL receptors and is essential for the DAP3-mediated caspase-8 activation Citation[2]. Also, the GTP-binding activity of DAP3 was essential for the fragmentation of mitochondria during apoptosis Citation[3].
Cells from human cancers have a reduced capability to undergo apoptosis in response to some physiological stimuli Citation[1]. Proapoptotic proteins are likely to be tumor suppressors and inactivation of apoptosis process by mutations of proapoptotic genes have been reported in many human cancers Citation[1], Citation[5]. Similarly, it could be hypothesized that DAP3, which play an important role in the apoptosis, could be mutated and be responsible to the apoptosis resistance of the cancer cells. To date, however, the data on the mutational status of DAP3 gene in human cancer is lacking. To explore the possibility, we analyzed 359 cases of human cancer tissues from various origins.
Methacarn-fixed tissues of 100 gastric carcinomas, 100 non-small cell lung cancers, 90 colorectal carcinomas and 69 hepatocellular carcinomas were randomly selected for this study. The gastric carcinoma samples consisted of 22 early and 78 advanced gastric carcinomas according to the depth of invasion. The non-small cell lung cancers consisted of 52 squamous cell carcinomas, 43 adenocarcinomas and five large cell carcinomas. The hepatocellular carcinomas consisted of Edmondson grade I (n = 8), grade II (n = 30) and grade III (n = 31) according to Edmondson and Steiner's criteria. The colorectal carcinomas originated from cecum (n = 2), ascending colon (n = 16), transverse colon (n = 5), descending colon (n = 2), sigmoid colon (n = 25) and rectum (n = 40). By microdissection, malignant cells and normal cells from the same patients were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle affixed to a micromanipulator Citation[5].
We analyzed the DNA sequences encoding the P-loop domain of DAP3 by a polymerase chain reaction (PCR) and a subsequent single strand conformation polymorphism (SSCP) analysis. Genomic DNA each from tumor cells and normal cells of the same patients were amplified by PCR with a primer pair covering the exon 5 of human DAP3 gene. The primer sequences were as follows; forward: 5′-AAAACAAAACAAAGCATAACTACTG-3′ and reverse: 5′-GGATAACTAAACCTAACAGACAGAAG-3′. Radioisotope ([32P]dCTP) was incorporated into the PCR products for detection by SSCP autoradiogram.
On the SSCP autoradiograms, all of the PCR products were clearly seen. However, the SSCP from the 359 carcinomas did not reveal any aberrantly migrating band compared to the wild-type bands from the normal tissues. To confirm the SSCP results, we repeated the experiments twice, including tissue microdissection, PCR, SSCP and direct DNA sequencing analysis to ensure the specificity of the results, and found that the data were consistent.
Because DAP3 acts as an important component of the apoptosis machinery both in intrinsic and extrinsic pathways, we expected to detect some DAP3 mutations in the cancer samples. However, we detected no DAP3 P-loop domain mutation in the samples, indicating that proapoptosis gene DAP3 P-loop mutation is rare in the cancers, and suggesting that mutational events in DAP3 P-loop may not contribute to the development of common human cancers, including gastric, lung, colorectal and hepatocellular carcinomas. Along with this study, we also analyzed the P-loop mutations of ARTS and Nod1 genes in cancer tissues from various origins, but found no mutation (unpublished data; Lee JW and Lee SH), suggesting that the P-loops in the proapoptotic proteins may not be mutational targets in human cancers.
This work was supported by a grant of the National Cancer Control R&D Program 2004, Ministry of Health & Welfare, Republic of Korea (0420040-1).
References
- Reed JC. Mechanisms of apoptosis. Am J Pathol 2000; 157: 1415–30
- Miyazaki T, Reed JC. A GTP-binding adapter protein couples TRAIL receptors to apoptosis-inducing proteins. Nat Immunol 2001; 2: 493–500
- Mukamel Z, Kimchi A. Death-associated protein 3 localizes to the mitochondria and is involved in the process of mitochondrial fragmentation during cell death. J Biol Chem 2004; 279: 36732–8
- Larisch S, Yi Y, Lotan R, Kerner H, Eimerl S, Tony Parks W, et al. A novel mitochondrial septin-like protein, ARTS, mediates apoptosis dependent on its P-loop motif. Nat Cell Biol 2000; 2: 915–21
- Kim HS, Lee JW, Soung YH, Park WS, Kim SY, Lee JH, et al. Inactivating mutations of caspase-8 gene in colorectal carcinomas. Gastroenterology 2003; 125: 708–15