Expression levels of chaperone proteins Hsp70 and Hsp90 in reverse transformed neuroblastoma cell lines.




Jim Boylan

Introduction

Multidrug resistance is a reoccurring obstacle faced in the chemotherapeutic treatments of cancer. Multidrug resistance (MDR) is characterized by high levels of resistance to a number of chemically unrelated drugs. MDR is established in cell lines by expressing high levels of plasma membrane efflux pumps such as P-glycoprotein and MRP-1, both members of the ABC superfamily of transporters. These transporters decrease cellular levels of a particular drug, and through a molecular mechanism not yet identified, also create cellular resistance for other drugs. Interestingly, in a number of cases associated with MDR, cell lines have shown signs of reverse transformation. Reverse transformation is a phenomenon that occurs in transformed cell lines, in which the malignant cells revert back to a normal phenotype and become significantly less tumorigenic.
Hsp70 and Hsp90, Heat Shock Protein of about 70 and 90 kilodaltons, respectively, are chaperone proteins that have both constitutive and inducible forms, and have a number of vital cellular functions. For example, Hsp70 proteins have an essential role in cells to aid in proper protein folding and degradation, membrane translocation, and regulatory processes. Similarly, Hsp90 aids in the proper folding of transcription factors and protein kinases and preventing protein aggregation. Originally, it was conceived that heat shock proteins were only involved in response to thermal stress. However, it has been found that expression levels of these proteins are elevated under all kinds of stressful conditions: oxidant injury, fever, toxic metals, inflammation, trauma to tissues and cancer.
This study looks at expression levels of Hsp70 and Hsp90? mRNA in reverse transformed, multidrug-resistant neuroblastoma cell lines using RT-PCR. These reverse transformed neuroblastoma lines are under stress from high-level exposure to chemotherapeutic drugs. In a previous study Spengler et al, (2006) an increase of Hsp70 protein was seen in reverse transformed MDR neuroblastoma cell lines using Western Blotting. This prompted an examination of the mRNA levels of Hsp70 as well as other heat shock proteins to determine if the elevated amounts of protein were a result of increase levels of mRNA expression or protein stabilization.

Figures


Figure 1-RT-PCR product results from primers located in exon 7 (nucleotides 1063-1081 of Accession number NM_002154.3) and exon 9 (nucleotides 1403-1384 of Accession number NM_002154.3) using 20ng of RNA from cell lines BE(2)-C, VCR20, and ACT0.2, respectively. Hsp70 samples were subjected to 33 cycles of amplification. The PCR for the GAPDH primers was performed for 22 cycles (nucleotides 100-119 and 327-308 of accession number NM_002046). Samples were then run on a 1% agarose gel with ethidium bromide and visualized using UV light.


Figure 2-Sequence analysis of RT-PCR product for primers targeting Hsp70. 68 nucleotides of the RT-PCR product was compared to the NCBI database. 66 of the 68 nulceotides matched exactly. Values of N on the Hsp70 gel were the result of unclear sequence. Alignment was done using MacVector.


Figure 3-RT-PCR product from primers specific for Hsp90 and GAPDH. Primers for Hsp90 were located in exon 1 (nucleotides 130-150 of accession number NM_001017963.1) and the reverse in exon 2 (nucleotides 509-490 of accession number NM_001017963.1). Primers for GAPDH were the same as in Figure 1. Samples from the cell lines BE(2)-C, VCR20, and ACT0.2 were amplified for 35 cycles for Hsp90, and for 22 cycles for GAPDH.


Figure 4-Sequence analysis of RT-PCR product for primers targeting Hsp90. 76 nucleotides of the RT-PCR product was compared to the NCBI database. 73 of the 76 nucleotides matched exactly. Values of N on the Hsp90 gel were the result of unclear sequence reaction result. Alignment was done using Mac Vector.


A slight increase in mRNA levels for Hsp70 was seen in reverse transformed MDR lines.

Increased protein expression of Hsp70 in the reverse transformed MDR lines does not account for the total difference previously seen using immunoblotting.

The difference in Hsp70 present in the reverse transformed multidrug resistant lines is not due to increased amounts of mRNA.

Hsp90 mRNA levels did not consistently change between reverse transformed MDR and parental I-type cells.

Full Paper

Acknowledgments

I would like to thank Dr. Silvia Anderson and Dr. Rubin for the use of their lab and guidance in carrying out this project. To Jinsong Qiu and Lisa Sarran, you have my gratitude for your constant patience and answers to all the questions throughout this project. Thanks to Dr. Robert Ross and Barbara Spengler for their help in establishing this project, data analysis, cell lines, and RNA. Congratulations to the rest of the class for undertaking such interesting and diverse projects.


This document was last modified 05/17/2006.
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