This document summarizes research aimed at developing a more efficient system for screening potential drug inhibitors of P-glycoprotein (P-gp), an ATP-binding cassette transporter that pumps chemotherapeutics out of multidrug-resistant cancer cells. The researchers introduced the human MDR1 gene, which encodes P-gp, into a bacterial plasmid and transformed E. coli cells to express the protein. Sequence analysis confirmed the MDR1 gene was present in the plasmid as designed. Developing this bacterial screening system could allow high-throughput screening of potential P-gp inhibitors to identify drugs that could combat multidrug resistance in cancer.

1. Niharika Choudhury1, Collette Marchesseault2, Dr. John Wise2, and Dr. Pia Vogel2
1The Hockaday School, Dallas, TX; 2 Department of Biological Sciences, Southern Methodist University, Dallas, TX
High-Throughput Screening System for P-glycoprotein Inhibition
Introduction
While chemotherapeutics have seen some success in eliminating cancerous
cells, they have seen much failure as well. The overexpression of P-
glycoprotein (P-gp) has led to multidrug resistant (MDR) cancer cells that
keep chemotherapeutics from producing effective cytotoxicity in tumor
cells.
The ATP-binding cassette (ABC) transporter P-glycoprotein serves as a
pump that transports cytotoxins across its plasma membrane. In doing so,
P-gp protects the cells in the body from foreign substances and contributes
to the effectiveness of the blood-brain barrier or liver in pumping out toxins
and drugs; however, cancer cells that express P-gp in large amounts are
rendered multidrug resistant, as they do not respond effectively to
chemotherapeutics. Anti-cancer drugs that enter the cells are merely
pumped out by the protein, and higher doses of the chemotherapeutic are
required until the patient can no longer withstand treatment due to the
toxicity.
In response, there has been ongoing research to find drug inhibitors that
keep P-gp from pumping out chemotherapeutics while maintaining
reasonable toxicity levels in the body. Dr. John Wise and Dr. Pia Vogel’s lab
at the Center for Drug Discovery, Design, and Delivery at Southern
Methodist University has been working to find such drugs to inhibit the
activity of P-gp.
In order to screen compounds for inhibition, much time and money has
been spent on purifying the protein from yeast cells. In response, Collette
Marchessault in the Vogel-Wise lab has proposed a high-throughput
screening method to increase the number of compounds that could be
screened and, in turn, provide for a wider set of data to further the search
for effective inhibitors. I have worked with her this summer to introduce
the human protein P-gp, encoded with the MDR1 gene, into a more
versatile bacterial system in order to increase efficiency and reduce cost.
We have worked to get E. coli to take up the plasmid encoded with the
MDR1 gene so that it can express P-gp. Doing so would allow other
undergraduate and graduate students to screen for potential inhibitors of P-
gp in a more effective and efficient manner.
Methodology and Results
1. Ligation of pet24a-glpF and MDR1 (Fig. 1)
Acknowledgments
I would like to thank Dr. Wise and Dr. Vogel for this wonderful
opportunity and their continuous guidance throughout my time in the lab.
I also would like to thank Collette Marchesseault for taking me under her
wing and letting me contribute to her project. I extend my gratitude to the
rest of the Vogel-Wise lab for teaching me the various lab techniques and
being so welcoming. Finally, I thank Dr. Barbara Fishel from the
Hockaday School for facilitating this research opportunity.
Methodology and Results
2. Transformation of CM2 into BL21 and DH5α, two strains of E. coli
cells using the NEB High Efficiency Transformation Protocol.
3. Plated the cells, Incubated them overnight, and Counted colonies
4. Grew Overnights of the colonies in LB broth
5. Plasmid Preparation [Mini-Prep] on the overnights to isolate
plasmid DNA by means of centrifuging and re-suspending the formed
pellets along with a series of buffers and washes, as indicated by the
Zyppy Plasmid Prep protocol.
6. Polymerase Chain Reaction [PCR] of BL21 and DH5α cells to look
for the 5000bp insert to confirm that MDR1 is in the plasmid. Used
NEB Protocol for PCR Using Q5 High-Fidelity DNA Polymerase
along with T7 and T7term primers, which targeted the DNA region
that included glpF + MDR1 (Fig. 2).
7. DNA Purification: used a series of buffers and centrifuged PCR
products, as indicated by the Zymo DNA Clean and Concentrator Kit,
to isolate and purify DNA.
8. Ran PCR Products on a 0.6% Agarose Gel (Fig. 3)
Conclusions
Based on the sequence data, we can conclude that the E. coli did, indeed,
take up our plasmid with the MDR1 gene. In its entirety, the sequence
highlighted some point mutations in the MDR1, but none of them
impacted which amino acid was encoded and were thus deemed
irrelevant. Furthermore, there were 6 cysteine to alanine mutations, all of
which were accounted for since they were engineered to be there. Two
other mutations were merely natural variants in the gene, and the final
one was a glutamic acid to cysteine mutation, engineered to keep the
protein inactive during this stage of experimentation. The sequence data
thus proved that the MDR1 sequence was complete and correct in the
newly engineered bacterial plasmid, CM2.
Discussion
Since we now know that we have the MDR1 gene in the plasmid, the next
step would include activating the gene so that it expresses the protein P-
gp in the bacterial system. Activating the gene would require a
mutagenesis experiment to mutate the cysteine back to glutamic acid in
order to produce active protein. With P-gp in the bacterial system, high-
throughput screening would be possible and prove to be an efficient and
effective means of finding inhibitors for P-gp.
Furthermore, experimentation has begun to induce the E. coli cells
to produce the glpF-MDR1 fusion protein. In order to do so, the bacteria
has to be grown in the presence of IPTG, a molecule that binds to the
repressor that keeps the glpf-MDR1 sequence from being transcribed.
Once the repressor is removed, the bacteria should be able to produce the
fusion protein. P-gp can then be isolated from the bacteria and used for
further experimentation.
This bacterial system is much more versatile and effective model
system than yeast. Once P-gp is expressed and isolated, research on
potential inhibitors can be conducted in an efficient manner.
Figure 1. Plasmid CM1 shows plasmid pet24a with the addition of the glpF gene
between the two enzyme cut sites. Plasmid CM2 shows the result of the ligation of
pet24a-glpf (CM1) and MDR1.
Figure 2. PCRs use thermal cycling for DNA melting and enzymatic replication of DNA.
The Polymerase enzymatically assembles new DNA strands from nucleotides by using
DNA strands as templates and DNA primers for DNA synthesis. The DNA primers are
complementary to a DNA region that is then targeted for amplification
For further information
Please contact Dr. John Wise ( jwise@smu.edu) at the Southern Methodist University or
visit the website for the Center for Drug Discovery, Design, and Delivery for more
information or to learn more about the ongoing research at SMU.
http://www.smu.edu/Dedman/Academics/InstitutesCenters/CD4
Figure 3. For our gel, we filled the first lane with the 1kb DNA ladder, the next three
with purified DNA from BL21, and the final three with purified DNA from DH5α. Gel
electrophoresis separates DNA fragments by length in order to estimate the DNA size;
all samples were at the 5000bp mark, indicating that MDR1 was in the plasmid.
Furthermore DH5α-10 yielded the brightest band (and therefore had the highest
concentration of DNA), so it was used for the subsequent PCRs.
Methodology and Results
9. Subsequent PCRs on cleaned DH5α-10 resulted in PCR products that
could be used for DNA sequencing. Since the MDR1 gene is almost 4000
nucleotides long, we had to run a series of PCRs in which we used
different forward and reverse primers to target different sections of the
plasmid until we had all of the DNA fragments. For these PCRs, we used
the NEB PCR Protocol for Taq DNA Polymerase with Standard Taq
Buffer.
10. Cleaned the PCR Products with the Zymo DNA Clean and Concentrator
Kit and Ran the products on a 0.6% Agarose Gel. We had estimated the
sizes of the DNA segments earlier based on the location of the primers,
but we ran the gel to confirm the sizes and determine the concentration of
DNA for each segment of the plasmid (Fig. 4).
11. Sequenced the DNA through LoneStar Labs to see if the entire MDR1
gene was in the plasmid (Figs. 5, 6)
Figure 4. Two of the results of
the gel electrophoresis on the
cleaned PCR products of c10 are
shown to the left. “1471” and
“1624” refer to the locations of
the targeted DNA.
Figure 5. Chromatogram data showing the DNA sequence of a portion of the
MDR1 gene. Each color correlates to a different nucleotide, labelled at the top of
the graph: Adenine, Guanine, Cytosine or Thymine.
Figure 6. The sequence data (portion shown above) matched up with the genetic
sequence of MDR1 and thus allowed us to conclude that MDR1 was, indeed, in
our pet24a-glpF-MDR1 plasmid.