Plasma membrane calcium ATPase (PMCA4): A housekeeper for RT-PCR relative quantification of polytopic membrane proteins
© Calcagno et al; licensee BioMed Central Ltd. 2006
Received: 02 June 2006
Accepted: 17 September 2006
Published: 17 September 2006
Although relative quantification of real-time RT-PCR data can provide valuable information, one limitation remains the selection of an appropriate reference gene. No one gene has emerged as a universal reference gene and much debate surrounds some of the more commonly used reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At this time, no gene encoding for a plasma membrane protein serves as a reference gene, and relative quantification of plasma membrane proteins is performed with genes encoding soluble proteins, which differ greatly in quantity and in targeting and trafficking from plasma membrane proteins. In this work, our aim was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues to be used for relative quantification of real-time RT-PCR data for ATP binding cassette (ABC) plasma membrane transporters.
In studies evaluating the expression levels of two commonly used reference genes coding for soluble proteins and two genes coding for membrane proteins, one plasma membrane protein, plasma membrane calcium-ATPase 4 (PMCA4), was comparable to the two reference genes already in use. In addition, PMCA4 expression shows little variation across eight drug-treated cell lines and was found to be superior to GAPDH and HPRT1, commonly used reference genes. Finally, we show PMCA4 used as a reference gene for normalizing ABC transporter expression in a drug-resistant lung carcinoma cell line.
We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors.
Relative quantification for real-time RT-PCR requires the use of a reference gene for normalization . An "ideal" reference gene should be expressed at similar levels in different cell types and under various treatment conditions. Many commonly used reference genes or "housekeeping" genes do not always possess these two necessary attributes. Several commonly used housekeeping genes were originally used as references for more qualitative assays such as Northern blots and conventional RT-PCR, and their use in quantitative RT-PCR was not originally re-evaluated. Therefore, the selection of the appropriate reference gene for performing relative quantification has been a topic of much discussion and evaluation [1, 3, 4]. This becomes more critical when examining the mRNA expression of membrane proteins, since many of the frequently used reference genes are soluble proteins, which differ significantly in total amount and in targeting and trafficking from membrane proteins.
Membrane proteins are a major component of the human genome, as they comprise nearly 30% of the total human genome . These proteins are also involved in approximately 85% of cell signalling pathways. The biogenesis of membrane proteins and cytosolic proteins varies greatly. At transcription, specific mRNA-binding proteins are added to nascent mRNA to ensure RNA integrity and to oversee RNA export from the nucleus, subcellular localization, translation and stability . Membrane localization of mRNA has been reported in membrane proteins, and this localization may facilitate cotranslational import of some membrane transporters as is seen in Atm1, a yeast ABC transporter of the inner mitochondrial membrane, and Ist2, a yeast plasma membrane putative ion channel . Investigators have also shown that mRNAs that encode for secreted or membrane proteins are preferentially found in membrane-bound polysomes . The translation of secretory, integral membrane proteins as well as that of luminal and membrane proteins of the endoplasmic reticulum (ER) take place on membrane-bound ribosomes of the ER , whereas the synthesis of cytosolic proteins occurs on the ribosomes within the cytoplasm. Polytopic membrane proteins have multiple membrane spanning domains, and they are believed to cotranslationally incorporate into the membrane of the ER with the assistance of the translocon .
An important superfamily of membrane proteins is the ATP-binding cassette (ABC) transporter family. Several ABC transporters are linked to disease conditions in humans including cystic fibrosis, Stargardt disease, Dubin-Johnson syndrome, Pseudoxanthoma elasticum and adrenoleukodystrophy . In addition, the overexpression of ABC transporters is the predominant cause of multidrug resistance in cancer , and understanding the triggers for overexpression of these proteins upon exposure to various drug regimens would help researchers find ways to circumvent multidrug resistance. Therefore, a reliable reference gene that does not change following drug treatment is vital for the relative quantification of ABC transporter gene expression. In this work, our goal was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues for normalization of real-time RT-PCR data for ABC transporters.
We hypothesized that plasma membrane proteins could function as reference genes and that a plasma membrane reference gene would be most appropriate for relative quantification of membrane proteins consisting of 4–12 membrane spanning helices otherwise known as polytopic membrane proteins. As mentioned previously, membrane protein biogenesis, in particular for polytopic membrane proteins, involves several steps that are specific to membrane proteins; therefore, a reference gene encoding for a protein which undergoes similar process of biogenesis would be best to use as a reference measurement for gene expression. First of all, to evaluate this hypothesis, two commonly used soluble protein reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyl transferase 1 (HPRT1), and two commonly expressed plasma membrane proteins, plasma membrane calcium-ATPase 4 (PMCA4) and the alpha subunit of the Na+ /K+-ATPase (ATP1A1), were evaluated using real-time RT-PCR with a variety of human tissue RNA samples. PMCA4 and ATP1A1 were chosen because both are ubiquitously expressed polytopic plasma membrane proteins. We determined that the consistency of PMCA4's expression profile was comparable to that of the commonly used reference genes across a panel of tissues. We then compared these four genes in eight drug-treated cell lines, and PMCA4 was found to be superior to GAPDH and HPRT1. Finally, we used PMCA4 to normalize ABC transporter expression from real-time RT-PCR for drug-resistant lung carcinoma cells.
Results and Discussion
PMCA4 shows comparable expression to GAPDH and HPRT1 in various tissues
Comparison of Housekeeping Gene Expression in Various Cell Linesa
Human Salivary Gland cells
19.62 ± 0.38
16.96 ± 0.11
21.33 ± 0.21
20.61 ± 0.14
HCT-116 (colorectal carcinoma)
20.4 ± 0.67
17.33 ± 0.4
21.71 ± 0.31
21.7 ± 0.36
KB-3-1 (Cervical squamous cell carcinoma)
20.57 ± 0.36
17.73 ± 0.07
20.49 ± 0.57
22.13 ± 0.1
19.77 ± 0.27
17.49 ± 0.15
21.9 ± 0.2
19.77 ± 0.17
20.62 ± 0.34
17.01 ± 0.43
21.48 ± 0.28
21.61 ± 0.13
MCF-7 (breast adenocarcinoma)
18.49 ± 0.28
17.1 ± 0.33
20.69 ± 0.13
21.39 ± 0.24
18.66 ± 0.8
16.31 ± 0.5
21.22 ± 0.25
20.58 ± 0.3
COR-L23P (lung carcinoma)
18.14 ± 0.34
18.12 ± 0.24
20.54 ± 0.21
21.03 ± 0.17
PMCA4 expression is unchanged under a variety of treatment conditions
PMCA4, on the other hand, was found to be superior to all other reference genes tested in this study. An F-test was used to individually compare the variance of the PMCA4 samples to the variance of the samples for the other genes. The variances for ATP1A1 and GAPDH, p = 0.027 and 0.00001, respectively, were statistically different than the variance for PMCA4. No statistical difference was found between PMCA4 and HPRT1, p = 0.146. From these results, we first identified a reference gene which was foremost, a membrane protein and which would also be consistent for all cell lines examined, regardless of drug selection conditions. PMCA4 was chosen as the reference gene for our subsequent work because of the convincing data from these studies. To our knowledge, this is the first report of the use of a gene encoding a membrane protein for normalization of real time RT-PCR data for polytopic membrane transport proteins.
PMCA4 is a good reference gene for polytopic plasma membrane proteins
Evaluation of ABCC1 Fold Change Using Different Housekeeping Genesa
Gene Used for Relative Quantification
Fold Change of ABCC1 in COR-L23R cells Compared to parental COR-L23P cells
90.7 ± 7.1
407.8 ± 98.9
152.3 ± 58.9
No other ABC transporter in our panel (consisting of the 12 ABC transporters linked to multidrug resistance) was overexpressed, and only the transporter which was previously reported remained the ABC transporter conferring resistance as seen by Western blotting (Fig. 4C and 4D). ABCC1 was not detectable in the parental COR-L23P cells, as seen in this Western Blot. Calcein-AM efflux assays using MK-571, the typical ABCC1 inhibitor, further verified that COR-L23R cells overexpress functional ABCC1 (Fig. 4E). These results are consistent with previous reports on the drug resistance profiles of these cells .
In this work, we identified a consistent housekeeping gene, PMCA4, for plasma membrane proteins which was comparable to other established reference genes when examined in a diverse panel of tissues and cell lines. Our work investigated the effects of drug treatment and tissue type on the selection of a housekeeping gene; additional work evaluating other conditions is currently under way. PMCA4 was also found to be superior to these reference genes, since it showed less variation regardless of drug treatment for the KB-3-1 and MCF-7 cells. We also thoroughly characterized the ABC transporter gene expression profile for a multi-step doxorubicin-selected cell line using PMCA4 as the reference gene. Overexpression of ABCC1 was verified in the COR-L23R cells at both the mRNA and protein levels. Although we have found that PMCA4 works quite well for the described conditions, as for all known housekeeping genes, there are conditions where alternative housekeeping genes may work best. For PMCA4, other investigators reported that increased Pi levels in cattle resulted in increased PMCA4 levels; neuronal development was also linked to increased PMCA4 levels [24, 25]. PMCA4 appears to be a suitable housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters, ATPases and receptors.
Chemicals and other reagents
Acetoxy-methyl ester of calcein (calcein-AM) was purchased from Molecular Probes (Eugene, OR). Doxorubicin was purchased from Calbiochem (San Diego, CA). RT-PCR reagents were purchased from Roche Applied Sciences (Indianapolis, IN). RNA from various tissue types examined in Fig. 2 was purchased from Clontech (Mountain View, CA).
The human large-cell lung tumor line COR-L23P and its doxorubicin-selected MRP1-overexpressing multidrug-resistant variant COR-L23R were a generous gift from Margery A. Barrand (Department of Pharmacology, University of Cambridge, Cambridge, UK) . Both COR-L23P and COR-L23R cells were cultured in RPMI 1640 medium with 10% (v/v) fetal calf serum supplemented with 100 units of penicillin/streptomycin/ml (Invitrogen) at 37°C in 5% CO2 humidified air. The COR-L23R cells were also maintained in the presence of 0.2 μg/ml doxorubicin.
The cervical epidermal carcinoma cell line KB-3-1 and its drug resistant subline, KB-A1, were generous gifts of Michael M. Gottesman (Laboratory of Cell Biology, National Cancer Institute, NIH, Bethesda, MD) , and the MCF-7 breast cancer cell line and the subline MCF-7/ADR were a generous gift of Kapil Mehta (MD Anderson Cancer Center, Houston, TX) . These cell lines were cultured in DMEM with 10% (v/v) fetal calf serum supplemented with 2 mM glutamine and 100 units of penicillin/streptomycin/ml (Invitrogen) at 37°C in 5% CO2 humidified air. The drug resistant subline KB-A1 was kept under constant selection in 1 μg/ml doxorubicin while 0.5 μg/ml doxorubicin was added to the MCF-7/ADR cells every other passage.
RNA was isolated from cells grown in 6-well plates to characterize reference gene expression in all cell lines and to characterize ABC transporter expression in the parental COR-L23P and drug-resistant COR-L23R cell lines. In these studies, 6-well plates were seeded at 200,000 cells per well for each cell type, and RNA was isolated following 72 hr of growth. Drug-resistant cell lines were plated with the appropriate concentration of drug during the 72 hr period. The medium and any detached cells were removed from the wells. RNA isolation was performed on the cells that remained attached using the Qiagen RNeasy kit (Valencia, CA) as per the manufacturer's protocol with a 15 minute on-column DNAse incubation step. RNA samples were isolated in triplicate. The pure RNA was quantified using the Nanodrop ND-1000 Spectrophotometer (Wilmington, DE). The integrity of the RNA was verified using the Agilent 2100 Bioanalyzer (Palo Alto, CA) with the Eukaryote Total RNA Nano assay. The RNA samples were stored at -80°C until needed.
List of Primers used for RT-PCR
Position of primer
Forward oligo sequence
Reverse oligo sequence
Two hundred nanograms of total RNA with 250 nM primers were used to evaluate the primers for the studies comparing the four reference genes. The LightCycler RNA Master SYBR Green Kit and LightCycler 480 instrument (Roche Biochemicals, Indianapolis, IN) were utilized in these studies to determine which of the four reference genes would be the most consistent across tissue types and treatment conditions. The average crossing point values for 4 housekeeping genes across different tissue samples with standard deviation were determined, and the RT-PCR conditions were as described in the manufacturer's protocol for the RNA Master SYBR Green kit. Negative controls consisting of no-template (water) reaction mixtures were run with all reactions.
The RT-PCR reaction for ABC transporters was performed on 300 ng total RNA with 250 nM specific primers under the following conditions in the LightCycler II: reverse transcription (20 min at 61°C), one cycle of denaturation at 95°C for 30 seconds, and PCR reaction of 45 cycles with denaturation (15s at 95°C), annealing (12s at 58°C), and elongation (30s at 72°C with a single fluorescence measurement). The PCR reaction was followed by a melting curve program (65–95°C with a heating rate of 0.1°C per second and a continuous fluorescence measurement) and then a cooling program at 40°C. Negative controls consisting of no-template (water) reaction mixtures were run with all reactions. PCR products were also run on agarose gels to confirm the formation of a single product at the desired size. Crossing points for each transcript were determined using the 2nd derivative maximum analysis with the arithmetic baseline adjustment. Crossing point values for each transporter were normalized to the respective crossing point values for reference gene plasma membrane calcium ATPase 4. Data are presented as a fold change in gene expression for the drug resistant cell lines compared to the parental cells using the delta delta Ct method.
For the Western blotting assays, cells were harvested and the cell lysates were prepared from drug-resistant and parental cells using a lysis buffer containing 10 mM Tris, pH 8.0, 1% Triton X 100, 2 mM DTT, 1% aprotinin, 1 mM AEBSF, and DNase. Following brief sonication and the addition of SDS, equivalent numbers of cells were loaded and separated by 7% NuPAGE gels. The electrophoressed proteins from the gel were transferred to a nitrocellulose membrane and probed with the appropriate primary antibodies specific for the ABC protein of interest. Immunoblotting was performed using the C219 (1:2000) antibody for ABCB1, MRPr1 (1:1000) (Alexis) for ABCC1, Anti-BCRP (1:1000) for ABCG2, and the anti-mouse IgG-horseradish peroxidase (HRP) conjugated (1:10000) secondary antibody. HRP-dependent luminescence was developed using Western lighting chemiluminescence reagent plus (PerkinElmer, Wellesley, MA). The blot was exposed to Hyperfilm within a Hypercassette for various times. The intensity of bands on the blot was quantitated using a scanner and ImageQuant software (Piscataway, NJ).
Assay of transport of fluorescent substrates by flow cytometry
A FACSort flow cytometer equipped with Cell Quest software (Becton-Dickinson, Franklin Lakes, NJ) was used for FACS analysis. Briefly, 300,000–500,000 cells were harvested after trypsinization by centrifugation at 500 × g and then resuspended in IMDM supplemented with 5% Fetal Bovine Serum. 0.25 μM calcein-AM was added to the cells in 4 ml of IMDM in the presence or absence of a specific inhibitor for the ABC transporter evaluated. For example, 20 μM MK571 was used to specifically inhibit ABCC1. The cells were incubated in a water bath at 37°C in the dark for 10 minutes for a calcein-AM efflux assay prior to being pelleted by centrifugation at 500 × g. The cell pellet was then resuspended in 300 μl of PBS containing 0.1 % BSA and then analyzed immediately using the flow cytometer as described previously .
Comparisons between the expression profiles for PMCA4 and each of the other genes was performed using a one-tailed F-test two sample for variances, with a confidence level of 0.05.
ATP binding cassette
calcein acetoxy-methyl ester
multidrug resistance protein
(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-(dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid
polyacrylamide gel electrophoresis
Hypoxanthine phosphoribosyl transferase 1
Plasma Membrane Calcium- ATPase 4
We thank Dr. Michael M. Gottesman for his encouragement and helpful discussions. We also thank Dr. Margery Barrand (University of Cambridge, UK) for the gift of COR-L23 cell lines, Dr. Susan Bates (NCI, NIH, Bethesda, MD) for providing the MCF-7 cell lines and Dr. Kapil Mehta (MD Anderson Cancer Center, Houston, TX) for the MCF-7 and MCF-7/ADR cell lines. We thank Dr. Zuben Sauna for his helpful suggestions and George Leiman for his editorial assistance. This research was supported by the Intramural Research Program of the National Cancer Institute, NIH, Center For Cancer Research. AMC was supported by the NIGMS Pharmacology Research Associate(PRAT) Program.
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