The effect of chemically induced differentiation on caspase 8 and 10 mRNA level in neuroblastoma cells




Yingyu Mao

Introduction

Caspase 8 and caspase 10 are aspartate-specific cysteine proteases involved in programmed cell death initiation. Caspase 8 can be activated by Fas/TNFR/DR5 (TRAIL receptor) activation in the extrinsic apoptotic pathway or participate in the intrinsic pathway of apoptosis, where it truncates Bid to give rise to tBid, and in turn increase the release of cytochrome c and Smac/DIABLO. Both pathways lead to caspase 3 activation. The deficiency of caspase 8 expression is observed in a majority (Jiang, 2008; Eggert, 2001) of neuroblastoma (a pediatric solid tumor, arises from neural crest) cell lines and primary tumor tissues. Their resistance to FasL, TNF-α (Kim, 2004), TRAIL (Eggert, 2001) or p53 mediated apoptosis (Ding, 2000) and other chemo- (Hannum, 1997)/immuno-/radiation therapy is correlated to this deficiency.

Caspase 10 can also be activated by Fas/TNFR/DR5 in the same way as caspase 8. Although CASP10 collocates with CASP8 at 2q33, they are said to have different downstream cleavage substrates (Wang, J., et al, 2001). Vincenz, et al (1997) showed that caspase 10 can cleave poly (ADP-ribose) polymerase (PARP) directly, bypassing the traditional caspase 8 – caspase 3 – PARP pathway. Caspase 10 is also shown to be deficient in a majority of neuroblastomas (Eggert, 2001; Harada, 2002) and may be a crucial factor in influencing their apoptotic potential.

RA (or ATRA, all-trans retinoic acid) was recently been shown to have an inductive effect on caspase 8 expression in primary tumors and neuroblastoma cell lines and increase the apoptotic responses of these cells to TNF-α and other cytotoxic agents (Jiang, 2008). To see whether the differentiated I-type cell line BE-(2)-C10 by RA and 5-bromo-2’-deoxyuridine (BUdR) exhibit an enhancement of caspase 8 and 10 expression level as compared to their I-type progenitor, the amount of these two transcript were semiquantified in the untreated cells and cells that are treated with the two differentiation agents, which induce N-type and S-type cell differentiation respectively.

Figures


Figure 1-Primers for caspase 8 and caspase 10 and their position on the genes.


Figure 2-Gel pictures of RT-PCR products amplified by caspase 8 primers in three repeated experiments. The table shows the corresponding band intensity relative to that of GAPDH and the average increases (fold) of caspase 8 mRNA amount in RA- and BUdR- treated cells as compared to untreated cells.


Figure 3-Gel pictures of RT-PCR products amplified by caspase 10 primers in three repeated experiments. The table shows the corresponding band intensity relative to that of GAPDH and the average increases (fold) of caspase 10 mRNA amount in RA- and BUdR- treated cells as compared to untreated cells.


Figure 4-Alignment of caspase 8 and 10 PCR products’ sequences with the corresponding mRNA sequences in NCBI database.


The mRNA amount of caspase 8 in RA-/BUdR- treated cells rose to more than two fold as compared to untreated cells, while the increase in caspase 10 is not obvious. This is probably because the basal mRNA amount of caspase 10 in untreated cells is higher than that of caspase 8. To determine whether there is a rescuing effect of caspase 10 on caspase 8 deficiency, further study needs to be done in other neuroblastoma cell lines and tumor samples.

The lack of correlation of caspase 8 and 10 mRNA level is consistent with previous findings (Harada, 2002), suggesting that even though the two genes are genetically related, their transcription might be regulated through distinct pathways or their transcripts might have different stability.

After all, the differentiation induction of RA and BUdR did enhance BE-(2)-C cells’ caspase 8 mRNA level. It implies the potentialities of both RA and BUdR being used to sensitize neuroblastoma cells to apoptotic signals. Further study of protein amount, especially the functional (cleaved) form of caspase 8 and 10, will help with drawing a final conclusion about their influences on neuroblastoma cell lifetime.

Please refer to the attached full paper for more details.

Full Paper

Acknowledgments

I would like to thank Dr. Rubin for his guidance and advices. Thank Bo Liu and Leleesha Samaraweera for their help in the lab work of this project. I also appreciate Dr. Ross’s suggestions on the project and Barbara Spengler’s kindly provision of neuroblastoma cell lines.


This document was last modified 05/13/2008.
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