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Bcl-xL is expressed in neuroblastoma cells and modulates chemotherapy-induced apoptosis
M G Dole1, R Jasty, M J Cooper
1Department of Pediatrics, University of Michigan, Ann Arbor 48109-0684, USA.
Abstract:
bcl-x is a new member of the bcl-2 gene family and is highly expressed in neural tissues. The present study was designed to determine the expression of the bcl-x gene products in neuroblastoma (NB) and their role in the modulation of chemotherapy-induced apoptosis. Twenty-seven NB cell lines were screened by quantitative immunoprecipitation for Bcl-xL, Bcl-xS, and Bcl-2 expression. None of the cell lines expressed Bcl-xS. Twenty-four of 27 (88%) of the NB cell lines expressed Bcl-xL and 21 of 27 (78%) were positive for Bcl-2. The level of Bcl-xL and Bcl-2 expression was variable among the lines analyzed. Bcl-2 expression was restricted to cells of chromaffin lineage, whereas Bcl-xL was seen in both chromaffin and nonchromaffin lines. To determine whether Bcl-xL could mediate chemotherapy resistance, a NB cell line expressing negligible levels of Bcl-xL was transfected with a bcl-xL expression vector, and unique clones were generated expressing variable levels of Bcl-xL. Cells were treated either with cisplatinum (CP), 4-hydroperoxy-cyclophosphamide (4-HC), or etoposide (VP-16) to induce apoptosis, and cell viability and DNA degradation were determined. Following treatment with CP or 4-HC, Bcl-xL-expressing cells showed significantly increased viability as compared to vector-transfected controls (P < 0.005). Flow cytometric analysis of propidium iodide-stained nuclei following CP or 4-HC treatment revealed significantly increased DNA degradation in controls as compared to Bcl-xL-expressing lines (P < 0.004). DNA analysis by pulsed-field gel electrophoresis revealed high molecular weight (approximately 40 kb) DNA degradation in controls, whereas the DNA in cells expressing Bcl-xL was largely intact. In contrast to CP and 4-HC, results with VP-16 revealed a short-term delay in the onset of apoptosis in Bcl-xL-expressing cells with no long-term survival advantage. The results of these studies indicate Bcl-xL is expressed in NB cells and functions in a manner analogous to Bcl-2 by inhibiting chemotherapy-induced apoptosis.
Insights
Bcl-xL protein is expressed in most neuroblastoma cells and inhibits chemotherapy-induced apoptosis, similar to Bcl-2. This suggests Bcl-xL plays a role in neuroblastoma chemoresistance.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- The Bcl-2 gene family regulates apoptosis.
- Bcl-x is a newly identified member of this family, highly expressed in neural tissues.
- Neuroblastoma (NB) is a pediatric cancer with variable treatment responses.
Purpose of the Study:
- To investigate the expression of Bcl-x gene products (Bcl-xL and Bcl-xS) in neuroblastoma.
- To determine the role of Bcl-xL in modulating chemotherapy-induced apoptosis in neuroblastoma cells.
Main Methods:
- Screening of 27 neuroblastoma cell lines for Bcl-xL, Bcl-xS, and Bcl-2 expression using quantitative immunoprecipitation.
- Transfection of a neuroblastoma cell line with a Bcl-xL expression vector to generate clones with varying Bcl-xL levels.
- Treatment of transfected cells with cisplatinum (CP), 4-hydroperoxy-cyclophosphamide (4-HC), or etoposide (VP-16) to induce apoptosis.
- Assessment of cell viability, DNA degradation (flow cytometry, pulsed-field gel electrophoresis).
Main Results:
- Bcl-xS was not detected in any neuroblastoma cell lines.
- Bcl-xL was expressed in 88% and Bcl-2 in 78% of the cell lines.
- Bcl-xL expression significantly increased cell viability and reduced DNA degradation following treatment with CP or 4-HC.
- Bcl-xL conferred a short-term delay in apoptosis with VP-16 but no long-term survival advantage.
- Bcl-2 expression was limited to chromaffin lineage cells, while Bcl-xL was found in both chromaffin and nonchromaffin lines.
Conclusions:
- Bcl-xL is expressed in neuroblastoma cells and inhibits chemotherapy-induced apoptosis, similar to Bcl-2.
- Bcl-xL contributes to chemoresistance in neuroblastoma.
- These findings highlight Bcl-xL as a potential therapeutic target in neuroblastoma treatment.