Differential expression of splice variants in three phenotypically distinct neuroblastoma cell lines




Jillian K. Smith

Introduction

Neuroblastoma is a tumor stemming from neural crest cells that affects the nervous system (Riley 2004). Neuroblastoma tumors typically grow in young children under the age of four. In fact, 8-10 % of cancers found in children are neuroblastoma tumors (Riley 2004). The severity of the cancer ranges from highly malignant to spontaneous regression (Riley 2004). This range in the severity of the cancer may be due to the unique ability of the neuroblastoma cells to differentiate. Human neuroblastoma cells are phenotypically variable. N-type cells are small and rounded with cytoplasmic projections, and have weak substrate adherence (Biedler et al. 1997). Alternatively, S-type cells are larger, lack cell processes, and are highly adherent to substrates (Biedler et al. 1997). A third type, the I-type, is a phenotypic intermediate between the S and N-types. Aside from their morphological differences, phenotypically distinct cells can further be distinguished from each other by their biochemical characteristics. Many enzymes and immune response surface proteins expressed in the N-type are not expressed in the S- types (Biedler et al 1997). Additionally, S- type cells do not have neuronal activity (Biedler et al. 1997). The physical and biochemical properties in cell neuroblastoma cell types are important and research on differential cell characteristics may lead to a more complete cure in children who display more severe cases of the cancer.

The genes FAS, PDK1, and TP53I3 genes have known slice variants. The FAS gene codes for a protein in the TNF-receptor superfamily which regulates apoptosis and has been found to be a factor in various immune system diseases. There is evidence that the FAS gene plays a neuroprotective role. Reduced FAS expression was found to correlate with neurotoxic degeneration in mice (Landau et al. 2003). Alternatively, FAS, when interacting with its ligand FasL, may play a role in cell death in Alzheimers disease (Su et al. 2003). Alternative splicing is reported to occur in this gene (e.g. Liu et al. 1995; Cascino et al. 1996) and can result in up to seven separate isoforms. The gene may have up to 9 exons (Genbank). Differential expression of variants is thought to play an important role in cell surface expression. Liu et al. 1995 found that reduced expression of alternatively spliced mRNA results in a stronger expression at the cell surface in human peripheral blood mononuclear cells. Because of its role in regulating apoptosis, FAS may be important in the biology of neuroblastoma.

The PDK1 (pyruvate dehydrogenase kinase) gene activates protein kinases involved in homeostasis. PDK-1 protects cells from reactive oxygen speciesí production and can stop apoptosis induced by hypoxia (Kim 2006). PDK-1 has 11 exons (Genbank); however, alternative splicing can create different variations of the gene.

As with the FAS gene, the TP53I3 gene is also thought to be involved in apoptosis. Cell death is initiated in TP53I3 after induction by the tumor suppressor p53 (e.g. Polyak 1997; Nicholls et al. 2004). Activation of TP53I3 by p53 occurs when p53 interacts with a microsatellite region downstream of the promoter region (Nicholls et al. 2004). Polymorphisms in this microsatellite region are thought to be responsible for differential susceptibility to cancer (Nicholls et al. 2004). Alternative pre-mRNA splicing has been noted in TP53I3. There are five exons in the TP53I3 gene under normal conditions, however, when cells are exposed to UV light, a TP53I3 splice variant lacking exon 4 is expressed (Nicholls et al. 2004). The alternative splice variant has a short half life and rapidly gets degraded (Nicholls et al. 2004), which could leave cells more vulnerable to cancer.

To date, little is known on how these genes are expressed in neuroblastoma cells lines. Differential expression of these slice variants was tested in this study. It is predicted that splicing variants will form as they do in other cell lines, but that expression of these variants will vary among neuroblastoma phenotypes.



Figures


Figure 1- Differential gene expression of FAS gene isoforms in neuroblastoma N-type SH-SY5Y (N), S-type SH-EP1 (S), and I- type BE (2)-C (I) cell lines and a negative control (-).


Figure 2- Differential gene expression of PDK1 gene splice variants in neuroblastoma N-type SH-SY5Y (N), S-type SH-EP1 (S), and I- type BE (2)-C (I) cell lines and a negative control (-).


Figure 3- Differential gene expression of TP53I3 gene splice variants in neuroblastoma N-type SH-SY5Y (N), S-type SH-EP1 (S), and I- type BE (2)-C (I) cell lines and a negative control (-).


Figure 4- Splice variants amplified during RT-PCR. The 200 bp isoform of PDK-1 is lacking a piece of intron 3. The 265 bp isoform of TP53I3 lacks exon 4.


Alternatively spliced mRNAs are functionally different from one another, so it is important to recognize what variants are expressed and the consequences of that expression. Overall, alternative splicing was displayed in the neuroblastoma cells as well as differential expression of variants.

The malignant N-type SH-SY5Y cells appeared to have higher isoform expression ratios based on band intensity of the different variants.

The malignant I-type BE (2)-C is a phenotypic intermediate between the N and S cell types. Expression ratios of splicing variants in the I-type were low and variants were expressed more equally.

Lower expression ratios of slice variants S and I-type in the Tp53I3 gene were observed, which means that the more easily degraded 265 bp gene variant (Nicholls et al. 2004) is more equally expressed than the 462 bp variant.

Future research should include a more intensive look at the functional consequences of varying expression in neuroblastoma cell lines.

Full Paper

Acknowledgments

I would like to thank Joe Frezzo and Leleesha Samaraweera for their help throughout the course. I would also like to thank Leleesha Samaraweera for providing neuroblastoma cell types and Bo Liu for providing primer sets. I would also like to acknowledge Dr. Berish Rubin, Dr. John Wehr, and Dr. Sylvia Anderson for their assistance and guidance.


This document was last modified 05/09/2007.
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