Expression of Caspase-9 in Neuroblastoma Cell Lines




Bo Liu

Introduction

Human neuroblastoma, one of the most common solid tumors of infancy, is a cancer in the neural crest, and is commonly categorized by their location, age at diagnosis, spread or metastasis, and degree of cellular maturation and heterogeneity (1). Histopathological examination of tumors has revealed the presence in neuroblastomas of a variety of cellular phenotypes, especially neuroblasts and nonneuronal (Schwann cell, glia, melanoblast) precursor cells (2). Scientists have shown that cell diversity also characterizes cultured human neuroblastoma cell lines and have defined three prominent cell types. Most common are sympathoadrenal neuroblasts (N-type), which grow as poorly attached aggregates of small, rounded cells with neuritic processes. A second cell type (S-type) resembles neural crest-derived, nonneuronal precursor cells. These large, flattened cells attach strongly to the substrate, and show contact inhibition of cell growth. A third cell type (I-type), an intermediate stem cell from which the first two may arise, is morphologically intermediate between N-type and S-type (1, 2). Recently, these three cell types have been demonstrated to occur in human neuroblastoma tumors as well. The three cell types display different tumorigenic potentials. S-type nonneuronal cells are nonmalignant, whereas N-type cells and I-type cells are malignant. I-type cells have the highest malignant potential among these three cell types (2, 3). Amplification of the oncogene MYCN in neuroblastoma strongly correlates with unfavorable outcome and is used as a marker for poor prognosis (4). However, it has also been reported that malignant potential of the neuroblastoma is defined by the malignant stem cell phenotypes, not by the amplification of MYCN or the level of expression (3, 5).

Apoptosis is a process of programmed cell death that depends on a number of genes highly conserved. Multiple pathways tightly control the coordinated action of these genes and ultimately lead to cell death. Proper regulation of apoptosis is essential for normal homeostasis and tissue development (6). Aberrations in apoptosis may contribute to the pathogenesis of cancer, and therefore are likely to be important in the neuroblastoma progression. The apoptotic program involves activation of a group of cysteine proteases termed caspases, which are present in cells as inactive or low active proenzymes (7). Caspase-9 is an initiator caspase that plays an important role in many forms of drug-induced cell death triggered by release of cytochrome c from the mitochondria into the cytosol. In the cytosol, procaspase-9 is activated through the formation of a multi-protein complex, termed an apoptosome, by the binding of cytochrome c to the activating factor Apaf-1 (6, 8). Once the apoptosome is formed and caspase-9 is activated, downstream effector caspases, such as caspase-3 and caspase-7, can then be activated to promote the cell death program (6, 7). It has been reported caspase-9 and its cofactor Apaf-1 play an important role in controlling tumor development (9). Recent studies also found the differential expression of caspase-9 gene in neuroblastoma tumors with unfavorable versus favorable biology (10). Considering these, caspase-9 seems a potential therapeutic target in neuroblastoma.

In this study, we examined expression of caspas-9 in three neuroblastoma cell lines (Table 1), on both mRNA levels and protein levels, to determine whether the expression of caspase-9 gene is correlated with distinct cell phenotypes or amplification status of MYCN gene.

Figures


Figure 1-Expression of caspase-9 mRNA in different neuroblastoma cell lines. RT-PCR, using primers located in exon 2 and exon 3, was performed on RNA isolated from three different cell lines. The resulting amplified products were fractionated on a 1% agarose gel. RT-PCR amplification of GAPDH mRNA performed on all three cell lines was used to monitor the amount of RNA present in the samples.


Figure 2-BLAST alignment of RT-PCR product with caspase-9 mRNA (NM_001229) from NCBI.


Figure 3-Western blot analysis of procaspase-9 in different neuroblastoma cell lines using a monoclonal antibody against procaspase-9. An antibody against GAPDH was used to detect GAPDH as an internal control.


Result
To study the differential expression of caspase-9 mRNA in these three different neuroblastoma cell lines with different cell phenotypes and MYCN amplification status, RNA isolated from three cell lines were amplified by RT-PCR using primers located in exon 2 and 3 of caspase-9 gene. RT-PCR amplification of GAPDH was used to monitor the amount of RNA in samples. PCR products of caspase-9 mRNA and GAPDH mRNA were then analyzed on 1% agarose gel (Figaure 1). Caspase-9 mRNA was detected in all three neuroblastoma cell lines. Significant difference in caspase-9 mRNA expression was found among these cell lines, while no clear difference was found in GAPDH mRNA expression. With same MYCN amplification status, I-type BE(2)-C cells have lower expression of caspase-9 mRNA than N-type BE(2)-M17 cells. Between the two I-type cell lines, MYCN amplified BE(2)-C cells express caspase-9 RNA at a lower extent as compare to MYCN nonampified SK-N-ER cells. This result indicates that the expression of caspase-9 mRNA may be regulated by both cell phenotypes and MYCN amplification.
PCR product was purified and sequenced by Sangerís dideoxy method. A BLAST search using the sequencing result of 179 bases was performed against NCBI database. One hundred percent homology between 179 bases of PCR-product and caspase-9 mRNA (NM_001229) confirmed the PCR product was amplified from the expected segment of caspase-9 mRNA (Figure 2).
Western blot analysis was performed on cellular extracts prepared from three neuroblastoma cell lines, using a monoclonal antibody against both procaspase-9 and processed caspase-9 (Figure 3). Expression of procaspase-9 (47kD) was found in all three cell lines. While no significant difference in the expression of GAPDH (35kD) was found among three cell lines, procaspase-9 was found to be expressed to a lower extent in BE(2)-C cells as compared to BE(2)-M17 cells and SK-N-ER cells. The consistent results in expression analysis on both mRNA levels and protein levels indicate the expression of caspase-9 may be regulated by both cell phenotype and MYCN amplification status in neuroblastoma cells. Since only three neuroblastoma cell lines were studied in this project, another possibility is that expression of caspase-9 is specifically lower in the BE(2)-C cells, without correlation with cell phenotypes or MYCN amplification. A further study on a larger number of neuroblastoma cell lines with different phenotypes and different MYCN amplification is needed to confirm the correlation between expression of caspase-9 and cell phenotypes or MYCN amplification.

Discussion
It has been proposed that apoptosis plays an important role in neuroblastoma progression (10). In a tumor, a balance between cell proliferation and apoptotic cell death is a crucial determinant of its net growth rate (11). Accordingly, repressed apoptosis may lead to tumor progression, and enhanced apoptosis may lead to tumor regression. Expression of caspase-9 was found in all three cell lines. Although the N-type cells and I-type cells are both malignant, expression of caspase-9 in these neuroblastoma cells is not silenced. This may be responsible for the possible spontaneous regression in neuroblastoma. This study appears to show differential expression of caspase-9 in different neuroblastoma cells lines. Both the cell phenotypes and MYCN amplification status may have the regulatory effect on expression of caspase-9 in neuroblastoma cells. Lower expression of caspase-9 in I-type BE(2)-C cells comparing to N-type BE(2)-M17 cells may be one possible reason for the higher malignant potential of I-type cells than N-type cells. Since advanced staged neuroblastoma tumors have a higher frequency of I-type cells (3), our result is consistent with an earlier study in which scientists found lower expression levels of caspase-9 in advanced tumors (10).
We also found that the MYCN amplified I-type BE(2)-C cells have lower expression of caspase-9 than I-type cells without MYCN amplification (SK-N-ER). Amplification of MYCN is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis (12). MYCN amplification results in high level of N-myc protein, a transcription factor, which perturbs the finely tuned interplay of N-myc and Max and eventually induces abnormal expression of target genes (13). Caspase-9 gene may be among the target genes, and downregulated by N-myc protein. Another possible reason is the frequently loss of heterozygosity of chromosome region 1p36, where caspase-9 gene is located, in MYCN amplified neuroblastomas (14). Loss of one allele leads to the reduction in caspase-9 expression.
Although a further study on a larger number of neuroblastoma cell lines is required to confirm the correlation of expression of caspase-9 with the cell phenotypes and MYCN amplification status, the present study indicates that caspase-9, the initiator caspase in the mitochondrial apoptosis pathway, may play an important role in regulation of apoptosis in neuroblastoma cells. Experiments have found that caspase-9 was a necessary response to the cytotoxic drugs, such as doxorubicin and cisplatin, within the drug-treated neuroblastoma cells (15). Recently, the specific activation of the mitochondrial apoptotic pathway using cyclooxygenase-2 inhibitors effectively induces apoptosis of neuroblastoma cells both in vitro and in vivo (16). The low expression of caspase-9 may be responsible for drug resistance in advanced neuroblastoma. An apparent imbalance between mitochondrial pro-apoptotic and anti-apoptotic mediators found in advanced stages of neuroblastoma tumors suggests that the mitochondrial apoptotic pathway might have a decisive effect on the development and aggressive behavior of advanced neuroblastoma (10). Future studies on other members in mitochondrial apoptotic pathway, including Apaf-1 and Bcl-2 family proteins, are required to fully explore the role of mitochondrial apoptotic pathway in neuroblastoma. Research on mitochondrial apoptotic pathway in neuroblastoma cells should have significance with regard to the design of novel therapies for neuroblastoma.

Full Paper

Acknowledgments

I would like to thank Jinsong Qiu and Brian Fox for their continuous help through this project. I am grateful to Barbara Spengler and Dr. Robert Ross for the RNA samples, cells and their helpful advices. Sincere thanks to Dr. Berish Rubin for his guidance and providing the opportunity to do the present project.

References
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