Comparative Analysis of Multi-Drug Resistance Gene Expression Level - Lab Report Example

 Comparative Analysis of Multi-Drug Resistance Gene Expression Level Objective: 1 To understand the method of PCR. 2 To analyse the expression of MDR1 using agarose gel electrophoresis. 3 To relate the expression of MDR1 to the importance of pharmacogenomics. Introduction: MDR1 gene is located on the long arm of chromosome 7. The exact sequence of the gene cannot be stated with certainity, but it has been predicted to comprise of a core promoter region and 29 exons. MDR1, also known as ABCB1, encodes P-glycoprotein (Ueda et al., 1986).P-glycoprotein (Pgp) is a 170kD energy dependent integral membrane protein which acts as an efflux pump, alleviating the concentrations of multiple drugs and other xenobiotics in the system. It belongs to ATP binding cassette family of transport proteins with 2 ATP binding domains, requiring ATP binding to both domains for transport (Stein, 1997). It has a broad range of substrate specificity, binding to and transporting wide spectrum of cytotoxic as well as noncytotoxic compounds eg. digoxin (Greiner et al., 1999), etoposide (Lum et al, 1993), mitoxantrone (Solary et al., 1996), paclitaxel (Sparreboom et al., 1997), talinolol (Schwarz et al., 1999), tacrolimus (Floren et al., 1997). The pharmocological importance of the P-gp is based on three aspects og P-gp behaviour: (1). due to its broad substrate specificity and wide distribution in cells, it alters the behaviour of its substrates; (2) a number of clinically important compunds are known to alter the behaviour of P-gp eg. cortisol, erythromycin act as inhibitors while insulin, rifampin as inducers; (3) many clinically used compounds are known to be transported by P-gp. Thus a number of drug-drug interactions which remained unexplained till now are being found to be due to P-gp activity. Also in non cancerous cells efficacy of drugs limited by P-gp, can be improved by modifying the levels or activity of P-gp eg., P-gp is being examined as the reason for resistance of human immunodeficiency virus (HIV) to antiretroviral agents (Schrager, 1998).To study the genomics and expression of MDR1 several methods can be used, however detection at mRNA level is highly effctive, sensitive and reproducible. In the present experiment RT-PCR (reverse transcriptase polymerase chain reaction) is being used to assess the levels of expression of P-gp in breast and cervical carcinoma cell lines and to make a comparative study for the two. mRNA is isolated from HeLa (cervical carcinoma cells) and MCF-7 (breast carcinoma cells). A cDNA copy of this mRNA is now made using the enzyme reverse transcriptase and PCR is done with the cDNA. fig. 1 PCR cycle Polymerase chain reaction (PCR) allows specific DNA sequences to be copied or modified over a million fold in a simple enzyme reaction. Each PCR contains 4 basic components: 1 Template DNA containing the the target DNA sequence to be amplified, in this case MDR1. 2 Oligonucleotide primers- short single stranded DNA molecules that bind by comlimentary base pairing to opposite strands of template DNA, at either end of the DNA to be amplified. Primers are in vast excess to DNA to be amplified. They are oriented with their 3’ends facing each other so that the synthesis by DNA polymerase (which catalyzes growth in 5’3’ direction) extends across the segments of DNA between them. 3 DNA plymerase, the enzyme which copies target sequence and is thermostable. 4 dNTPs, the substrates for polymerase. PCR works in cycles every cycle comprising of 3 stages: 1 Denaturation- at a temperature >90.C the strands of DNA double helix are separated. 2 Annealing- cooling the reaction to 40-60.C allows primer to bind to single stranded template DNA. 3 Extension- reaction when heated to 72.C when target DNA sequence is copied. Each molecule of target DNA synthesized acts as a template for the synthesis of new target molecules in next cycle. This leads to rapid increase in amount of target DNA with successive cycles. Typically 30 cycles of PCR results in a 228-fold amplification of a discrete target or gene, which can be visualized on an agarose gel. Agarose gel electrophoresis separates linear DNA on the basis of size by migration of DNA through a matrix under the influence of an electric field. Agarose is a polysaccharide which forms a solid gel when dissolved in aqueous solutions and depending on the range of length of DNA to be analyzed agarose concentrations of 0.5 to 2% (w/v) are used. Wells are loaded with sample DNA fragments in specific order to enable comparative study. When an electric field is applied to an agarose gel in presence of a buffer solution which conducts electricity, DNA fragments move through the gel in specific lanes towards the positive electrode, the DNA being negatively charged; at a rate which is dependent on size and shape. Small linear fragments move quickly than larger ones which are retarded by entanglement with the network of agarose fibres forming the gel. The added dye also moves along with DNA samples and is used to track the movement of DNA. DNA is stained by ethidium bromide, which intercalates with DNA, and fluoresces as an orange band in presence of UV light, thus making DNA visible. Tris/acetate/EDTA (TAE) is the buffer used for gel electrophresis, since it has a good resolution for DNA. fig. 2 Agarose gel electrophoresis A DNA ladder is used as standard for reference, to enable deducing sample DNA length by comparison. The commercially available 100bp ladder is used in the present experiment. fig. 3 100bp DNA ladder The sample PCR product that is a portion of MDR1 gene is given for this experiment. To analyze and compare the expression level of MDR1 from HeLa cells and MCF-7 cells, agarose gel electrophresis is carried out. The gel is prepared of concentrations suitable for the DNA sample fragment length. The PCR products are then loaded onto sample wells alonwith DNA ladder in a specific sequence to ensure accurate gene comparisons. The gel is run at an optimal voltage and for time suitable for sample DNA separation. Finally the gel is visualized with UV illumination. MDR1 is responsible for resistance of cancer cells to lipophilic drugs such as vincristin, adriamycin etc. This can further lead to development of resistance to other lipophilic drugs as well and therefore will render chemotherapy ineffective (Berger et al., 1994). Thus, it is important to evaluate expression of MDR1 in different tissues. Previous studies have shown the level of P-gp to be low in breast carcinoma cell lines and negligible in cervical carcinoma cell lines (Zhang et al., 1999), however the same has been found to be increased after exposure to chemotherapeutic agents (Hochhauser and Harris, 1991). And therefore, this study is essential for effective treatment of cancers. Table 1 mdr-1 gene expression in 151 specimens (%) Site of tumors n H M L/N H&M Esophagus 46 10.9 26.1 63.0 37.0 Gastric cardia 35 17.1 11.9 71.0 29.0 Stomach 16 25.0 12.5 72.5 27.5 Colorectum 16 18.8 12.5 68.7 31.3 Lung 9 22.0 33.0 45.0 55.0 Breast 15 6.6 6.6 86.8 13.2 Thyroid 10 20.0 20.0 60.0 40.0 Uterine cervix 4 0.0 0.0 100.0 0.0 Total 151 15.2 18.6 66.2 33.8 H: high expression, M: middle expression, L(N): lower/no expression (Zhang et al, 1999) Method: 1 Added 1µL of DNA loading dye to each of five PCR samples, labelled HeLa P-gp and MCF-7 P-gp, HeLa GAPDH and MCF-7 GAPDH, and negative control. 2 Weighed out agarose to make an agrose solution of 1% final concentration in 100ml of TAE in an Erlenmeyer flask. 3 The agarose/TAE solution was microwaved for 20 seconds at a time and the process was repeated until agarose is completely dissolved. 4 The glassware was allowed to cool slightly. Handling the glass carefully, enough ethidium bromide was added to make a final concentration of 0.5µg/ml. 5 The gel was poured in gel tray with gel combs in place and allowed to solidify. 6 The gel was placed in the electrophoresis tank and TAE buffer was added to cover the gel for a depth of 1mm. 7 Separate wells were loaded with 11µL of sample, one in each well, followed by a 100bp DNA ladder using a pipetteman. 8 The lid was placed on the gel case. 9 After ensuring that the lead end was connected correctly, the voltage was set to 80V/cm and the gel was run till the DNA tracking dye reached about 1-2cms from the end. 10 Power supply was turned off. 11 The gel was brought up to the demonstrator and the DNA was viewed using UV light source. Expeiment Protocol: HeLa cell MCF-7 cell Lyse cells Isolate m RNA Prepare RNA for Reverse Transcription Reverse Transcription cDNA PCR Sample DNA Agarose Gel Electrophresis Result Result: P-gp expression was the same as control GAPDH expression. 100bp DNA ladder shows a prominent mark at 500bp (fig.3). The result shows expression of sample genes in all four DNA samples at a spot close to 500 bp reference point (fig.4) fig. 4 result Discussion: The result that the expression levels of P-gp in breast and cervical carcinoma cells is same as GAPDH, has important consequences in the pharmacogenomics of MDR1, considering the fact that GAPDH is a housekeeping gene. A housekeeping gene is responsible for maintenance of cell functions and therefore, is not only normally produced in all cells, but is also produced in high quantities. This experiment proves the fact that MDR1 is upregulated in breast and cervical carcinoma cells. In tumor cells P-gp leads to reduced concentrations of drugs , thereby reducing the accessibilty of chemotherapeutic agents to their site of action and hence lowering their efficacy. This corroboarates with previous studies which have shown that expression of P-gp is elevated in tumour cells transformed from cells that normally produce P-gp (colon cancer), as well as in tumour of cells which otherwise do not produce P-gp eg. breast cancer making tumours resistant to the wide range of chemotherapeutic agents which are P-gp substrates (table 1). Thus the gene name MDR1 or multi drug resistance gene. Thus in clinical field further knowledge of P-gp expression in normal and tumourous cells, in addition to in depth understanding of its substrate pharmacokinetics, would help better understanding of the efficacy of individual and coadministered drugs and thus help in clinical monitoring. Because of high spectrum of P-gp substrate and its pharmocological importance it is imperative to understand the genomics of MDR1. References: 1 Berger W., Elbling L. & Minai-Pour M., (1994). Intrinsic MDR 1 gene and P-glycoprotein expression in human melanoma cell lines. Int J Cancer, 59, 717. 2 Floren L. C., Bekersky I., Benet L. Z. et al., (1997). Tacrolimus bioavailabilty doubles with coadministration of ketoconazole. Clin Pharmacol Thar, 62, 41-9. 3 Greiner B., Eichelbaum M., Fritz P. et al., (1999). The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin. J Clin Invest, 104, 147-53. 4 Hochhauser D. & Harris A. L., (1991). Drug resistance. Br Med Bull, 47, 178-96. 5 Lum B. L., Gosland M. P., Kaubisch S. & Sikic B. I., (1993). Molecular targets in oncology: implications of the multidrug resistance gene. Pharmacotherapy, 13, 88-109. 6 Schrager L. K. & D’Souza M. P., (1998). Cellular and anatomical reservoirs of HIV-1 patients receiving potential antiretroviral combination therapy. JAMA, 280, 67-71. 7 Schwarz U. I., Gramatte T., Krappweis J. et al., (1999). Unexpected effect of verapamil on oral bioavalabilty of the beta- blocker talinolol in humans. Clin Pharmacol Ther, 65, 283-90. 8 Solary E., Witz B., Calliot D. et al., (1996). Combination of quinine as a potential reversing agent with mitoxantrone and cytarabine for the treatment of acute leukemias: a randomized multicenter study. Blood, 88, 1198-205. 9 Sparreboom A., van Asperen J., Mayer U. et al., (1997). Limited oral bioavailabilty and active epithelial excretion of pacitael (Taxol) caused by P-glycoprotein in the intestine. Proc Nat Acad Sci USA, 94, 2031-5. 10 Stein, W. D., (1997). Kinetics of multidrug transporter (P-glycoprotein) and its reversal. Physiol Rev, 77, 545-90. 11 Ueda K., Cornwell M. M., Gottesman M. M., et al., (1986). The mdr 1 gene, responsible for multidrug resistance, codes for P-glycoprotein. Biochem Biophy Res Commun, 141, 956-72. 12 Zhang L., Chen K., Xu G., Xing H. & Shi X. T., (1999). Congenital expression of mdr-1 gene in tissues of carcinoma and its relation with pathomorphology and prognosis. World journal of gastroenterology, 5(1), 53-6. Read More