Abstracts
2001 Digestive Disease Week

# 335 Downstream Apoptosis Effectors APAF-1 and CLARP are Markedly Reduced in Colon Cancer
Sergio Huerta, David Heber, Julio Licinio, Ma-Li Wong, Jonas Hannestad, Edward H. Livingston, Los Angeles, CA

Background/Objectives: Loss of apoptosis is associated with the development of malignant cancers and apoptosis induction a possible approach for cancer treatment. Each major apoptosis mediator has been shown to contribute in some way to the pathogenesis of colon cancer, however, the relative importance of individual mediators remains unknown. Only simultaneous analysis of them in a model of colon cancer progression can resolve which has the greatest potential impact on apoptosis. Utilizing a cell line system representing a primary tumor and subsequent metastatic lesion we simultaneously analyzed 9,600 cDNA's for apoptosis-related gene expression.

Methods: Cell lines SW480 (primary colon cancer) and SW620 (metastatic lesion from the same patient) were obtained from the ATCC and grown under standard conditions. mRNA was extracted as previously described (Roesler et al. Am. J. Surg 174:251-257, 1997.), converted to cDNA then hybridized to a GeneChip as per standard protocol for the device.

Results: See Table. Expression was also reduced in SW620 relative to SW480 for: TNF-related apoptosis inducing ligand, TNFR-related death receptor-6, apoptosis-related protein TFAR15, CSE1, and apoptosis inhibitor surviving gene s.

Conclusion: Bcl-2 was the most highly expressed apoptosis regulatory molecule in SW480 and its expression markedly reduced in the metastatic SW620 cell line. The apoptosis facilitator BAX had slightly increased expression in SW620. However, the downstream effector molecules APAF-1 and Caspase-like apoptosis regulatory protein (CLARP) were markedly reduced in both cell lines. BCL-2 and BAX act reciprocally at the initiation stage for apoptosis. The BCL-APAF-cytochrome c-caspase complex activates down stream caspases that enzymatically break down cell component during apoptosis. Loss of the downstream-effector molecules predominated in both cell lines. Downstream APAF and Caspase systems should be targeted for increased expression if enhanced apoptosis is desired as a cancer therapy.

Expression of Apoptotic mediators_(% Relative Expression Compared to GAPDH)

Cell Line BCL-2 14-3-3 BAX p53 Cytoch. C APAF-1 CLARP

SW480 87.2 13.7 2.8 3.4 12.9 0.3 2.5

SW620 12.2 2.7 3.9 0.4 10.9 0.0 0.9