Differential Expression of S-Phase Kinase-Associated Protein 2 (Skp2) mRNA in Phenotypically Distinct Neuroblastoma Cell Lines




Travis J. Bernardo

Introduction

Neuroblastoma is a cancer which develops from the neural crest during early childhood development. It is one of the most common cancers in children, accounting for approximately 10% of all pediatric cancers. Diagnosis typically involves categorizing a neuroblastic tumor according to its location, the patient’s age at diagnosis, the presence and degree of metastasis, and the extent of cellular maturation and homogeneity. Neuroblastoma cells can be separated into three unique phenotypes: neuroblastic/neuroendocrine precursor N-type cells, I-type stem cells, and Schwannian/substrate-adherent S-type cells. These cell types display distinct morphological features and growth patterns in tissue culture.

Among the various features distinguishing these phenotypes, it has been shown that malignancy, as assessed by both tumor formation in nude mice and anchorage-independent growth in soft agar, is significantly more pronounced in I-type cells than in both N-type and S-type cells. Tumorigenicity studies involving both nude mice and soft agar have revealed that S-type cells are non-tumorigenic, forming few to no tumors in soft agar and no tumors in nude mice. N-type cells possess intermediate malignancy, forming tumors in nude mice with a frequency of 33-100% and having colony-forming efficiencies of 4-32%. I-type cells have the greatest malignancy potential, forming tumors in 100% of mice and exhibiting plating efficiencies 3-fold higher than that of N-type cells.

Previous studies have shown that Notch1, a signaling protein involved in cellular differentiation during embryonic development, is capable of inducing embryonic stem cells to pursue a neural lineage. Preliminary studies have further suggested that Notch1 expression may be another distinguishing factor between the three neuroblastoma phenotypes. I-type cells have been shown to express greater Notch1 mRNA and protein levels compared to N-type and S-type cells. Notch1 has been linked in previous studies to the cell cycle machinery by its ability to regulate entry into S-phase. This is accomplished through induction by Notch1 of transcription of the S-phase kinase-associated protein 2 (Skp2) mRNA. Skp2 serves as an F-box protein subunit in the SCF ubiquitin-ligase complex. The F-box subunit binds particular substrates to the complex, allowing it to target specific proteins for proteasome-mediated degradation. Skp2 binds the cyclin-dependent kinase inhibitor p27Kip1, targeting it for degradation. This CKI has been shown to play an important role in timing of the G1-S phase transition by inhibiting cellular progression into the S-phase. Thus, an up-regulation of Skp2 (resulting from an increased presence of Notch1) brings about p27Kip1 degradation, which in turn leads to entry into S-phase and hence uncontrolled cell replication.

The purpose of this current investigation is to determine whether the up-regulation of Notch1 in I-type cells corresponds to higher levels of Skp2 mRNA in I-types than in N-type or S-type cells. Confirmation of this link may aid in the search for the cause of tumorigenicity in neuroblastoma. Additionally, differential expression between the two reported transcriptional isoforms of Skp2 will also be examined. These results lay the groundwork for future studies to determine whether there are functional and regulatory differences between the two isoforms.

Figures


Figure 1-
A) RT-PCR amplification of the 5' region of Skp2 on 10 ng total RNA extract from each of three neuroblastoma cell lines: I-type line CB-JMN (lane 1), S-type SH-EP-1 (lane 2), N-type SH-SY5Y (lane 3). Comparison to a 100 bp ladder confirmed the predicted product sizes. Samples were subjected to both 29 and 28 cycles of amplification. Amplification of GAPD mRNA was used as a loading control. Samples were subjected to 22 cycles of amplification. B) RT-PCR amplification of a central region of Skp2 for 10 ng total RNA extract from each of three neuroblastoma cell lines: I-type line CB-JMN (lane 1), S-type SH-EP-1 (lane 2), N-type SH-SY5Y (lane 3). Samples were subjected to both 28 and 27 cycles of amplification. Amplification of GAPD mRNA was used as a loading control. Samples were subjected to 22 cycles of amplification.


Figure 2-
A) RT-PCR amplification of isoform 1 of Skp2 on 10 ng total RNA extract from each of three neuroblastoma cell lines: I-type line CB-JMN (lane 1), S-type SH-EP-1 (lane 2), N-type SH-SY5Y (lane 3). Primers amplified exon 10, which has an isoform-specific sequence. Comparison to a 100 bp ladder confirmed the predicted product sizes. Samples were subjected to both 32 and 30 cycles of amplification. Amplification of GAPD mRNA was used as a loading control. Samples were subjected to 22 cycles of amplification.B) RT-PCR amplification of isoform 2 of Skp2 on 10 ng total RNA extract from each of three neuroblastoma cell lines: I-type line CB-JMN (lane 1), S-type SH-EP-1 (lane 2), N-type SH-SY5Y (lane 3). Primers amplified exon 10, which has an isoform-specific sequence. Samples were subjected to both 32 and 30 cycles of amplification. Amplification of GAPD mRNA was used as a loading control. Samples were subjected to 22 cycles of amplification.


Figure 3-
Partial sequence alignment of purified RT-PCR product with reported Skp2 mRNA sequence (NCBI). Alignment for the 5’ region primer product (top) shows 100% homology to a 107 bp region of Skp2. Alignment for the primer product amplifying a central Skp2 region (bottom) shows 98% homology to a 116 bp segment.


Figure 4-
Compiled data (N=2) for RT-PCR amplification of Skp2 mRNA versus GAPD in CB-JMN, SH-EP-1, and SH-SY5Y. Four regions of Skp2 mRNA were amplified: the 5’ region (1), a central region (2), the 3’ region unique to the first isoform (3), and the 3’ region unique to the second isoform (4). Data were normalized to EP-1, which was set at 100% expression.


Conclusions


• Skp2 appears to be expressed as a function of Notch1 levels as well as cell phenotype in neuroblastoma. Expression does not seem to depend on one particular isoform over another. However, a greater sample size must be used before any conclusions can be reached.

• Skp2-like transcripts have been previously reported in the GenBank database at NCBI. These three transcripts (alpha, beta, and gamma) are homologous to Skp2 but lack a 5’ UTR and have unique 3’ ends. The results of this investigation suggest that neuroblastoma does not express these unusual transcripts, since 5’ amplification (with a primer situated in the 5’ UTR) and central region amplification result in the same expression levels between the cell lines. A uniform 1 ½ to 2-fold increase was seen in central region amplification versus 5’ amplification, but the reason for this is unclear.

• N-myc amplification, a common feature in neuroblastoma cells, was not accounted for in this study, since all cell lines were non-amplified. Future studies should include amplified lines to determine whether N-myc plays a role in the Skp2 signaling pathway.

• Given the difficulty in finding suitable primers targeting specific Skp2 transcripts (two different isoforms and three Skp2-like transcripts), RT-PCR has limited usefulness in examining Skp2. Future studies should include Northern blotting, as well as investigation of protein levels via Western blotting. Other studies may include altering the putative upstream pathway of Skp2 and looking for any expression differences that result.

Full Paper

Acknowledgments

I would like to thank Jinsong Qiu and Lisa Sarran for their invaluable help throughout the course of this project. I would also like to acknowledge Dr. Robert Ross, Barbara Spengler, Brooke Grandinetti, and Jeanette Walton for their expertise and assistance in developing both my research as well as my abilities as a researcher. Finally, I would like to thank Dr. Berish Rubin for his guidance and for allowing me the opportunity to carry out this project.


This document was last modified 05/16/2006.
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