Neuroblastoma is an embryonal cancer of the postganglionic sympathetic nervous system, derived from neural crest cells. Neuroblastoma is commonly present at birth, with 96% of cases occurring before ten years of age.
Figure 1-RT-PCR analysis of the MDR1 gene in the BE(1), BE(2)-C, and BE(2)-C/VCR(10) cell lines. (a). 60ng of RNA isolated from each of the cell lines was amplified for 33 cycles with primers recognizing nucleotides 3781-3761and 3952-3971 of the MDR1 transcript, accession number NM_000927 (NCBI). (b). 20 ng of RNA isolated from each of the cell lines was amplified for 26 cycles with actin primers recognizing nucleotides 553-573 and 1161-1141 of the β-actin transcript, accession number BC001301 (NCBI).
Figure 2-Alignment of MDR1 mRNA (NCBI) with the BE(2)-C RT-PCR product.
Figure 3-Western blot analysis of MDR1 in the BE(1), BE(2)-C, and BE(2)-C/VCR(10) cell lines. 30µg of protein lysate from BE(1), BE(2)-C, and BE(2)-C/VCR(10) were fractionated by SDS-PAGE and subjected to Western blot analysis using mouse monoclonal antibody to MDR1 (A) and GAPDH (B). To determine the molecular weights of the detected proteins, prestained standards were run in parallel.
The phenomenon of clinical drug resistance has been implicated in the failure of chemotherapeutic agents to eradicate tumorigenic growth in numerous human cancers. MDR1 P-glycoprotein is an ABC-transporter that pumps drugs targeting cancer cells, along with many other cellular macromolecules (such as lipids), out of the cytosol. Accumulation of drugs in the cell is thus prevented and consequently, therapeutic levels of chemotherapeutic agents are difficult to reach. For three neuroblastoma cell lines, BE(1), BE(2)-C, and BE(2)-C/VCR(10), MDR1 gene and protein expression were measured. While RT-PCR showed similar levels of MDR1 transcript in the three cell lines, Western blot analysis revealed elevated levels of MDR1 protein in cells treated with vincristine and cells that have acquired high resistance to this agent. This discrepancy remains unaccounted for and thus, future studies need to be conducted to further characterize the correspondence between MDR1 gene and protein expression in human neuroblastoma.
Sincere thanks to Brian Fox and Jinsong Qui for their patience, guidance, and encouragement throughout this project. Thanks are due to Dr. Robert Ross for providing the cell lines and to Barbara Spengler for her insightful conversations and personal interest in this work. Much thanks to Dr. Berish Rubin for giving me the foundation to pursue this project and to both Drs. Rubin and Kandpal for so generously opening their labs to the students. Thanks to Dr. Sylvia Anderson for ordering the primers and to Fordham University Department of Biology for funding this research.
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