Doublecortin, Caspase-1, and Interleukin-18 mRNA Expression in Neuroblastoma Cell lines and the Detection of an Alternatively Spliced Transcript




Katherine Reid

Introduction

Neuroblastoma (NB) is a cancer of the sympathetic nervous system that arises from neural crest cells (Walton et. al 2004). NB accounts for approximately 9% of pediatric cancers with about 700 new cases reported each year in the US (Theiele, 1999). NB cell lines express a great deal of heterogeneity and frequently contain three phenotypically and biochemically distinct cell types: neuroblastic (N), substrate-adherent (S) and intermediate (I) (Walton et. al. 2004). I-type cells display the greatest malignancy, followed by N-type, while S-type cells are considered nonmalignant due to their lack of ability of anchorage-independent growth in soft agar (Walton et. al. 2004). It is suggested that the I-type cell is a neural crest stem cell from which the other two cell types may arise based on its ability to express specific enzymatic markers of both N-type and S-type cells, namely neuronal and melanocytic enzyme markers (Walton et. al. 2004). Although the cause of neuroblastoma remains unclear, many attempts toward effective treatments are currently being pursued. Agents that induce apoptosis, which destroy aberrant cells and thereby inhibit tumor progression, and induce cellular differentiation, are current theories behind treatment. One line of cancer treatment involves the administration of retinoids. While the details behind the mechanism of retinoids remain elusive, they have shown to modulate cellular differentiation and apoptosis (Sidell et. al. 1983).

Doublecortin is a 40kDa microtubule-associated protein that is expressed in migrating and differentiating neurons in both the central and peripheral nervous systems (LoTurco, 2004).DCX is also a reliable and commonly used molecular marker for neurogenesis (Couillard-Depres, S. et al. 2005) DCX contains microtubule-binding domains that direct migration of microtubules and increase their stability. However, the direct mechanism of how DCX aids in neuronal migration is poorly understood (Polloneal et. al 2006). DCX has also been recently proposed to be a tumor marker for minimal residual disease in neuroblastoma. A previous study has also recently reported that retinoic acid treatment decreased DCX expression in the SK-N-SH cell line(Oltra et. al. 2005; Messi et. al. 2008). This offers valuable insight into another cellular mechanism in which retinoic acid may help combat neuroblastoma.

Caspases are a family of cytokine proteases that play an integral role in the apoptotic and proinflammatory processes in the cell. Caspase-1 (CASP-1) cleaves and, thereby, activates proinflammatory cytokines interleukin-1-beta and interleukin-18(IL-18). After proteolytic processing of caspase-1, the proinflammatory cytokine interleukin-18 induces the production of IFN-gamma. IFN-gamma has been shown to have anti-tumor effects in cells and has also been suggested as a treatment approach in neuroblastoma (Feng et. al. 2004).

Here we have investigated DCX, CASP-1 and IL-18 expression in three neuroblastoma cell lines. We have also investigated their expression in untreated, RA treated, and BUdR (5-bromo-2’deoxyuridine) treated I-type cells (which are differentiating agents that induce I-type cells into N-type and S-type , respectively).

Figures


Figure 1-Detection of an alternatively spliced transcript. DCX was expressed in only N-type and I-type of the KCN83n and BE(2)-C respectively. DCX expression was upregulated after RA treatment and down regulated after BUdR treatment in the I-type BE(2)-C cell line.


Figure 2- CASP-1 expression in only BE(2)-C S-type cells. CASP-1 expression was only detected after treatment with BUdR.


Figure 3-IL-18 expression in only the S-type cell of BE(2)-C cell line. IL-18 expression was only detected after BUdR treatment.


We observed that DCX is expressed in only N-type and I-type cells. DCX expression was increased when I-type cells were induced to the neuronal phenotype. DCX expression decreased when I-type were induced toward the S-cell phenotype. These findings suggest that DCX is exclusively expressed in N-type and I-type cells and may be linked to cellular invasiveness in neuroblastoma.

We have also shown that both CASP-1 and IL-18 were only expressed in S-type cells. CASP-1 and IL-18 were also only expressed when I-type cells were induced over to the S-type phenotype by BUdR treatment. These findings demonstrate that CASP-1 and IL-18 are restricted to the non-malignant S-type cell in the cell lines studied.

Altogether, these findings merit further investigation on why CASP-1 and IL-18 are only expressed in the non-invasive S-type cells and on the correlation of DCX expression with the invasiveness and progression of this cancer.

Full Paper

Acknowledgments

I would like to thank Dr. Robert Ross for generously supplying the cells. I would like to thank Bo Liu for his unyielding patience and guidance throughout this project. Also I would like to give a special thanks to Leleesha Samaraweera for her advice and support.I would like to thank the support and encouragement that my classmates have given me over the course of this project. And finally, I would like to offer a sincere thank you to Dr. Berish Rubin for his guidance and making this project possible.


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