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Quantitation of c-myc Amplification by a Competitive PCR Assay System

Seth P. Harlow and Carleton C. Stewart

Departments of Surgical Oncology and Flow Cytometry, Roswell Park Cancer Institute, Buffalo, New York 14263

Gene amplification is a common Gene amplification, particularly am- being possible. A number of variables, event in the progression of human plification of the growth-promoting however, must be accounted for to de- cancers. The detection and quantita- proto-oncogenes, is a common event in termine the true gene amplification level tion of certain amplified oncogenes the progression of many human can- in a population of cells. These include has been shown to have prognostic cers. (~ Detection and quantitation of cell number, cell cycle phase (cells in G 2 importance in certain human malig- certain specific amplified may or mitosis will have twice the gene cop- nancies. A method is described that have the potential for predicting patient ies as cells in G O or G1), and chromo- utilizes the principles of competitive outcome or response to therapy for a some ploidy (genes on aneuploid chro- PCR for quantitation of the c-mu number of different human tumor mosomes generally are not considered gene copy number in relation to the types. (2'3) Recently, a number of meth- amplified). To account for all of these copy number of a reference gene (tis- ods have been described to accomplish variables, quantitation of an internal sue plasminogen activator It-PAl this goal by a variety of techniques (4-6~ control or reference gene has been used gene) located on the same chromo- differing from the standard Southern routinely in Southern techniques. The some (8) as the c-mu gene. This ratio blot technique. (7~ A significant advance reference gene ideally should be located gives the true level of amplification in the quantitation of specific nucleic on the same chromosome as the gene of of the c-myc gene, accounting for acid sequences was made with the intro- interest and should be located at a dis- variables such as cell number, cell cy- duction of quantitative PCR methods. tance from that gene such that it would cle phase, and chromosome 8 ploidy. The technique of competitive PCR, (s~ not likely be coamplified with it. In ad- The determination of gene amplifica- which uses an external DNA standard, dition, the reference gene should not be tion depends on the precise measure- has been well documented as a precise a gene that is known to be amplified or ment of the ratio of target and refer- and accurate method for quantitating deleted on its own as part of the known ence genes. An important feature of specific gene sequences. (9'1~ tumor progression. Gene amplification this assay is that the competitive ref- Competitive PCR methods rely on the can then be calculated simply as follows erence standards used for target addition of a known amount of external (number of gene copies/number of refer- gene c-mu and reference gene t-PA DNA standard to several PCR reaction ence gene copies) in a given sample. have been linked to form a hybrid. tubes containing equivalent amounts of In this paper we describe a novel This simple modification guarantees sample DNA. The external standard is a modification of the competitive PCR that both reference gene and target double-stranded DNA molecule identical methodology for quantitating gene am- gene assay tubes get identical to the genomic sequence of interest with plification of the c-myc proto-oncogene. amounts of the competitive template either a small deletion, insertion, or new This method utilizes a competitive refer- for each gene, thereby eliminating a restriction site, such that it can be easily ence standard (CRS) molecule, which is a significant source of error. This discriminated from the genomic se- competitive external DNA standard for method has the same desirable at- quence by size on gel electrophoresis. the c-myc gene (chromosome 8q24) (11~ tributes of standard PCR in that very Thus, the external standard has the same linked to a competitive external DNA small sample sizes are required and primer binding sites and has a similar standard for the tissue plasminogen that results can easily be obtained in amplification efficiency as the genomic activator (t-PA) gene (chromosome <24 hr. In addition, this technique sequence. Quantitation of the sample 8p12). 02~ This CRS molecule acts as an does not require the use of radioac- gene dosage can then be made by a titra- external standard to control for the rate tivity or expensive DNA detection tion of external standard and extrapolat- and efficiency of the PCR reaction and as kits, and thus, may give it wider ap- ing where the genomic product molarity an internal standard to control for cell plicability for the study of human is equal to that of the external standard. number, cell cycle phase, and ploidy ab- ca n ce rs. Competitive PCR methods can there- normalities. This method shares the ad- fore give precise information on the vantages of standard PCR techniques in number of gene sequences in a given that it is relatively quick and can be used sample with sensitivities <1 attomole on samples of very small size (as few as

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1000 cells). These benefits make this downstream primer and the comple- plifying from the small CRS templates. methodology attractive for analysis of ment to the t-PA upstream primer. The The primers used in this study are as fol- clinical samples where available tissue is resulting product was a combined c-myc! lows: c-myc (~1) primers--sense (5' end) often limited. t-PA CRS that could be used for the de- position 6721-6740, 5'-AAGGTCAGAG- termination of c-myc gene amplification TCTGGATCAC-3'; antisense (3' end) po- (Fig. 2). The templates were linked to re- sition 7059-7078, 5'-TAACTACCTTGG- MATERIALS AND METHODS duce the inaccuracies produced by hav- GGGCCTTT-3', PCR product gener- CRS ing to quantitate and dilute two inde- ated-- 358 bp. t-PA (~2) primers--sense (5' By using site-specific mutagenesis tech- pendent templates. Molar amounts of end) position 30,805-30,824, 5'-GC- niques, as described previously, (13) com- the CRS were determined by quantita- CACCTGCGGCCTGAGACA-3'; antisense petitive double-stranded DNA templates tion of OD at 260 nm after purification (3' end) position 30,959-30,978, 5'-AG- to the c-myc and t-PA genes were made. of the CRS on low-melting-point agarose AGAGAATCCAGCAGGAGC-3'; PCR prod- Each DNA template had a novel SmaI re- gels. uct generated = 174 bp. striction endonuclease recognition site (5'-CCCGGG-3') incorporated with mu- PCR Primers Cell Lines tagenesis primers (Fig. 1). Both tem- plates were found to digest completely Primers used for the competitive PCR re- Cell lines used in this study were ob- into two fragments by the SmaI restric- actions were selected to lie in a nested tained from ATCC (American Type Cul- tion enzyme before linking. The two fashion in relation to the ends of the ture Collection, Rockville, MD) and competitive templates were then linked CRS sequence. We have found that using grown according to the instructions sup- by PCR, using an oligonucleotide primer nested primers improves PCR amplifica- plied. The cell lines studied were HL60 that was a combination of the c-myc tion sensitivity and specificity when am- (human promyelocytic leukemia), SKCO1 (human colon adenocarcinoma), and Colo 320DM (human colon carcinoma). Cells were harvested without enzy- matic digestion or EDTA to avoid inter- A ference with the PCR. Cells were centri- fuged and resuspended in sterile Genomic Sequence 5' CCCAGG z' phosphate-buffered saline. They were Position (6940-6945) counted by hemocytometer and ali- quoted so that study solutions would Competitive Template contain the equivalent of -1000 cells/t~l Same Position 5' CCCGGG 3' of lysis solution (with the exception of the COLO320 DM cell line, where -500 cells/l~l of lysis solution were used). c-myc Competitive Template with 222 BP's 136 BP's sma-1 site ~ Cell Lysis Solution (lengths are those Prior to competitive PCR reactions, cells obtained with nested were lysed in the following solution, as primers) has been described previously: (14) 10 mM Tris-HC1 (pH 8.3), 2.5 mM MgC12, 50 mM KC1, 0.1 mg/ml gelatin, 0.45% NP-40 B (LKBProdukterAB, Bromma, Sweden), 0.45% Tween 20 (Sigma Chemical, St. Genomic Sequence Louis, MO), and 200 l~g/ml of proteinase Position (30922-30927) 5' CCCGGA , 3' K. Samples were incubated at 60~ for 60 min, then at 94~ for 10 min to inacti- vate proteinase K. Competitive Template CCCGGG Same Position Competitive PCR Reactions

t-PA Competitive All competitive PCR reactions were per- Template with 120 BP's 54 BP's formed in 100 t~1 total volume and cy- sma-1 site ~ cled in a Perkin-Elmer Cetus (Norwalk, (lengths are those CT) DNA Thermal Cycler with an overlay obtained with of mineral oil. The reaction solution nested primers) constituents were as follows: 10 mM Tris- FIGURE 1 The sites of directed point mutations to create the competitive templates for the c-myc HC1 (pH 8.3), 1.5 mM MgC12, 50 mM KC1, gene (A) and the t-PA gene (B). The size of each portion of the template fragment after digestion 0.1 mg/ml of gelatin, 150 i~molefliter of with SmaI restriction endonuclease is also shown. dATP, 150 i~mole/liter of dCTP, 150

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sense~myc t-PA wan, Republic of China) at a resolution 5'~ 3' of 300 dots per inch and 256 gray-scale levels. The digitized images were stored 3' 5' on an Apple Macintosh IIci computer (Apple Computer, Inc., Cupertino, CA). antisensemyc antisense t-PA Images were inverted from positive to negative using Adobe photoshop (Adobe Systems, Inc., Mountain View, CA) be- c-myc 447 BP's t-PA 190 BP's fore densitometric analysis. Individual band densities were determined with the (amplified segment 358 BP's) (amplifiedsegment 174 BP's) Scan Analysis densitometry program FIGURE 2 Illustration of the CRS molecule used to determine c-myc gene amplification in this (Biosoft, Inc., Ferguson, MO) using a study. The two competitive templates for c-myc and t-PA have been linked end to end. (Left) The matched background subtraction. This c-myc competitive template; (right) the t-PA competitive template. The arrows identify the sites of program allows densitometric measure- primer binding for sense and antisense primers. The antisense c-myc and sense t-PA primers lie ment of defined areas outlined by a side by side. All primers are nested within the ends of the CRS molecule. The location of the mouse. Even bands that may be sepa- incorporated point mutation encoding the SmaI restriction site is illustrated by the notched areas rated just barely on a gel (as is the case of the DNA molecule. with some of the t-PA product bands in this study), can be quantitated accu- rately. Densitometry measurements i~mole/liter of dGTP, 150 i~mole/liter of Analysis of Competitive were made for each of the bands [two dTTP, 40 pmoles of sense primer, 40 PCR Reactions SmaI-cut bands (CRS) and one uncut pmoles of antisense primer, 1.0 i~l of cell band]. Sma/Digestion lysate (1000 cells/l~l), CRS (see below), and 2.5 units of AmpliTaq DNA poly- After completion of the competitive PCR Calculation of Gene Copy Number merase (Perkin-Elmer Cetus, Norwalk, reactions, aliquots of 30 i~1 of each reac- CT). tion were digested with 20 units of SmaI Plots were made comparing the ratio of For each set of competitive PCR reac- restriction endonuclease (New England SmaI-cut/uncut densitometry values on tions performed, master mixes were Biolabs, Beverly, MA) at 25~ overnight. the y-axis versus the amount of CRS on made (CRS added separately) so that This was clone by the simple addition of the x-axis. Gene copy number per tube constituents would be equal in all reac- SmaI enzyme directly to the PCR solu- (1000 cells) could then be determined by tion tubes. For additional specificity of tion (we have found that SmaI has full extrapolating the amount of competitive the PCR reactions, the dNTP solution activity in this buffer). template that would give a cut/uncut ra- was not added to tubes until the tubes tio of 1.0. This value would be equal to reached 94~ in the thermal cycler, a the molar amount of genomic DNA se- process called hot start. (ls~ Thermal cy- Gel Electrophoresis quences per tube at the start of the PCR cler settings were 94~ for 3 min (initial- reaction. Relative gene amplification is SmaI-digested samples underwent elec- izing step), 94~ for 1 min, 60~ for 1 determined by dividing the copy num- trophoresis on 2% agarose gels in Tris- min, (stepwise for 30 cycles), 72~ for 2 ber for the c-myc gene by that for the acetate/EDTA (TAE) buffer. min, 72~ for 5 min (final extension), t-PA gene. and a 4~ soak. The amount of cell lysate and com- Gel Staining RESULTS petitive template added to reaction tubes for this study can be seen in Table 1. Gels were incubated in TAE buffer with Figures 3, 4, and 5 illustrate the negative ethidium bromide (50 i~g/l) for 10 min image of the ethidium bromide-stained with gentle rocking, and washed with gels and the plots of CRS (cut)/genomic deionized water for 10 min. (uncut) band densities versus the TABLE 1 Cell Lysate and amount of CRS for the specimens ana- Competitive Template lyzed. Because there were two bands Photography formed by the SmaI digestion of each Sample Number of Amount of CRS CRS fragment, the sum of these two tube lysed cells (attomole) Gels were visualized on a UV transillu- band densities was used for its densito- minator and photographed with Po- A 1000 0 metry value in each plot. Lanes A and I laroid (Cambridge, MA) type 667 instant B 1000 0.003 from each gel represent control lanes for black-and-white film through a UV filter. C 1000 0.006 each reaction. There was a small amount D 1000 0.012 of undigested product in lane I of some E 1000 0.024 gels, which was never greater than 5% of Densitometry F 1000 0.048 the band densities; this most likely rep- G 1000 0.096 Photographs were scanned with a Mi- resented incompletely digested CRS se- H 1000 0.192 I 0 0.048 crotek ScanMaker 600GS scanner (Mi- quences and did not influence results crotek International, Inc., Hsinchu, Tai- significantly. In addition, not shown are

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FIGURE 3 Negative images of ethidium bromide-stained gels for the competitive PCR reactions to determine c-myc amplification in the SKCO1 cell line. (Left) The c-myc gel; (right) the t-PA gel. Lanes A-I correspond to tubes A-I described in Table 1. The uppermost band for each gel represents the uncut, or genomic sequence, band. The lower two bands for each represent the two uncut, or CRS sequence, bands. The ratio of cut/uncut densitometry values vs. the amount of CRS is plotted below each gel. Only the linear portion of each plot is shown (RZ: c-myc plot, 0.9712; t-PA plot, 0.9428). Calculated gene dosage: c-myc gene, 0.015 attomoles; t-PA gene, 0.007 attomoles. Gene amplification for the c-myc gene was 0.015/0.007, or twofold. the results of negative controls (no CRS and the regression coefficients of each SKCO1 line. Sufficient quantities of or cell lysate) run with each group for are given. From these plots we calculated product were formed from as few as 500 which no bands were seen. the molar amount of each gene. The lev- lysed cells to be readily visualized and As has been described in prior com- els of c-myc gene amplification for the quantitated using ethidium bromide petitive PCR studies, (s) we found the lin- cell lines studied were 2-fold for SKCO1, staining. ear range when comparing CRS/genomic 6-fold for HL60, and 15-fold for the densitometry values versus the amount COLO320 DM line. These levels of am- of CRS to be located near the point plification for the HL60 and Colo320 DISCUSSION where the CRS/genomic ratio equaled DM cell lines correlate well with those 1.0. Values that fell at a distance from described in the literature for these lines An easy assay procedure for the quanti- that point were generally out of the lin- when analyzed by Southern tech- tation of gene copy number and gene ear range. The plots shown in each figure niques; (16'17) we were unable to find sim- amplification of the c-myc proto-onco- represent this linear portion for each, ilar data on amplification levels in the gene has been described. This procedure

FIGURE 4 Gels and gene dosage plots for the HL60 cell line. (Left) The c-myc gel, (right) the t-PA gel. Lanes A-I correspond to tubes A-I described in Table 1 (R2: c-myc plot, 0.9821; t-PA plot, 0.8832). Calculated gene dosage: c-myc gene, 0.075 attomoles; t-Pa gene, 0.0126 attomoles. Gene amplification for the c-myc gene was 0.075/0.0126, or sixfold.

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FIGURE 5 Gels and gene dosage plots for the COLO320 DM cell line. (Left) The c-myc gel; (right) the t-PA gel. Lanes A-I correspond to tubes A-I described in Table 1 (RZ: c-myc plot, 0.9478; t-PA plot, 0.919). Calculated gene dosage: c-myc gene, 0.134 attomoles; t-Pa gene, 0.009 attomoles. Gene amplification of the c-myc gene was 0.134/0.009, or 15-fold.

does not require large numbers of sam- each standard. The technique can be ap- that these techniques may be used to ple cells, organic extractions of sample plied to the quantitation of any gene se- study tumor cell subpopulations, which DNA, or radioactive probes, and it can be quence of interest without having to run can be isolated by means of fluores- accomplished from start to finish in <24 standardizing curves to assess PCR effi- cence-activated cell sorters. Gene ampli- hr. The technique of competitive PCR ciency. Also, it may be useful for detect- fication levels of samples enriched for has theoretical advantages over other ing gene deletions as well as gene ampli- tumor cells could be quantitated without PCR-based quantitative assays, such as fications. the diluting effect of nonmalignant stro- differential PCR, (4~ in which two differ- The method of competitive PCR is mal and inflammatory cells that are ent gene sequences are amplified in a not completely without problems. Some present in varying amounts in all solid single PCR reaction (a study and a refer- investigators have described heterodu- tumors. Also, specific tumor cell subpop- ence sequence). Such techniques assume plex formation between the point mu- ulations could be studied for differences that two completely different PCR reac- tated and nonmutated DNA strands as a in gene copy number to assess whether tions will proceed at identical rates and possible confounding factor with this such populations influence clinical out- reach plateaus at identical times, an as- method. (8~ In general, however, this has come. These tools will provide the abil- sumption that may not always be true. not been found to be a significant factor ity for in-depth studies of the molecular Another advantage of the competitive if starting sample sizes are small and PCR of tumor development and pro- PCR method is that numerous data cycles are kept below 40. If it becomes a gression and intratumoral variability, points are collected, increasing confi- problem, specific measures to account which have been lacking in the past. dence that the results are accurate. This for it have been developed. (18) Incom- method as described uses both an exter- plete digestion of PCR products by the nal (the competitive template) and an restriction endonuclease is another po- ACKNOWLEDGMENTS internal control (the t-PA quantitation), tential problem. This is best addressed This research was supported by National in contrast with single-sequence com- (as we have done) by including a control Institutes of Health T32 training grant petitive PCR, in which only an external tube that has no cell lysate but only CRS. CA-09581 and by U.S. Public Health Ser- control is used. With this method, accu- If a significant proportion of this PCR vices RO1 grant 60201A. rate estimations of c-myc amplification product is not cleaved (>5%), then re- from samples of unknown cell number suits of that assay should be questioned. are more reliable; however it introduces Finally, it is hoped that use of this or REFERENCES similar techniques will make molecular the possibility for significant error if the 1. Schwab, M. 1990. Oncogene amplifica- concentrations of the two standards are analysis of human cancers easier and tion in neoplastic development and pro- not identical. To eliminate this error, the more reliable in the future. The sensitiv- gression of human cancers. Crit. Rev. On- two standards have been linked to- ity of the PCR method allows for molec- cogen. 2: 35-51. gether, which guarantees that all reac- ular studies of small samples, making it 2. Seeger, R.C., G.M. Brodeur, H. Sather, A. tion tubes receive identical amounts of of great clinical benefit. It is also hoped Dalton, S.E. Siegel, K.Y. Wong, and D.

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168 PCR Methods and Applications Downloaded from genome.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press

Quantitation of c-myc gene amplification by a competitive PCR assay system.

S P Harlow and C C Stewart

Genome Res. 1993 3: 163-168

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