Parathyroid hormone-related peptide (PTHrP) was initially discovered as the agent responsible for humoral hypercalcemia in malignancy. PTHrP is the product of a gene that, in humans, exhibits a pluri-exonic organization with three transcriptional start sites leading to generation of multiple mRNA variants by alternative promoter usage and splicing mechanisms. cDNA-deduced encoded polypeptides of either 139, 141 or 173 amino acids display N-terminal sequence homology with parathyroid hormone (PTH), allowing binding to the same G protein-linked receptor and accounting for the PTH-like activity mediated by the N-terminal domain of PTHrP. Recent evidence suggests that PTHrP may be a polyprotein that undergoes proteolytic processing into smaller bioactive forms, which may function as local autocrine/paracrine modulators of cellular phenotypic expression (1)
. We have previously reported that discrete domains of PTHrP ([1–34], [67–86] and, to a minor extent, [107–139]) are biologically-active on breast cancer cells (2)
. In addition, the recently-characterized PTHrP [38–94] domain, which is likely to be the midregion PTHrP domain present in the extracellular fluid (4)
, has been found to impair the in vitro growth and invasive behavior of MDA-MB231 breast carcinoma cells (5)
. These data prompted a more detailed analysis of the interaction between MDA-MB231 cells and PTHrP [38-94], to examine whether differences in cellular gene expression occur after PTHrP treatment. Here we report the successful utilization of M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant (Cat.# M3682) and Taq DNA Polymerase, to generate mRNA differential display electrophoretograms, which are powerful tools for identifying genes that are selectively expressed under particular experimental conditions.
MDA-MB231 breast cancer cells were routinely cultured in RPMI 1640 medium (Gibco), supplemented with 10% fetal calf serum (Gibco) and antibiotics. Before mRNA extraction, cells were cultured in serum-free RPMI medium, with or without 1nM PTHrP [38–94] (kindly donated by Prof. A.F. Stewart, University of Pittsburgh Medical School, Pittsburgh, PA, USA). After 24 hours, fresh medium of the same composition was added and the cells were incubated for a further 24 hours (2)
Messenger RNA Extraction and Reverse Transcription
Isolation of polyA+ mRNA from MDA-MB231 cell monolayers was performed using a commerically available mRNA isolation kit (Roche). cDNA synthesis was performed according to the protocol provided with M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant using 200 units of enzyme, 50ng of Random Hexamers (Cat.# C1181) as primers, and 1µg of mRNA.
Before DD-PCR experiments, the cDNA preparations obtained from the reverse transcriptase reaction were checked by amplification in the presence of primers specific for human beta-actin as reported by Luparello et al. (6)
. PCR was performed using 2.5 units of Taq DNA Polymerase and 100pmol of each primer in a 50µl reaction mixture; the thermal cycling profile was as follows:
94°C for 2 minutes, then:
94°C for 30 seconds
55°C for 30 seconds
72°C for 45 seconds
For 45 cycles, then:
72°C for 5 minutes
For differential expression analysis, DD-PCR was performed using the arbitrary 10-mer amplimers designed by Sokolov and Prockop (Table 1)(7)
, in pairs.
Table 1. Primers Used for DD-PCR Amplification (7).
PCR amplification was performed using 25pmol of each primer, 1µl of cDNA template, 200µM dNTP mix, 1.5mM MgCl2 and 5 units Taq DNA Polymerase in a total reaction volume of 50µl. The thermal cycling profile was as follows:
94.5°C for 3 minutes, then:
94.5°C for 1 minute
34°C for 1 minute
72°C for 1 minute
For 45 cycles, then:
72°C for 10 minutes
After PCR amplification, 15µl of the products were separated by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining. In addition, 8µl of the products were separated on a non-denaturing 6% polyacrylamide gel in a sequencing apparatus at a constant voltage of 55W as reported by Doss (8)
. After electrophoresis, the banding pattern was visualized using SILVER SEQUENCE™ DNA staining reagents according to the SILVER SEQUENCE™ DNA Sequencing System Technical Manual #TM023. For reamplification of selected bands, the silver-stained gel was exhaustively washed with double-distilled water. The bands of interest were then carefully scratched from the gel with a sterile needle and used as template for further PCR amplification.
Results and Conclusions
To search for genes selectively switched on or off in MDA-MB231 breast cancer cells after treatment with PTHrP [38–94], we amplified cDNA from mRNA isolated from PTHrP-treated and untreated cells by reverse transcription using degenerate hexanucleotides. Prior to DD-PCR analysis, the integrity of the cDNA preparations was checked by amplification in the presence of primers specific for human β-actin (Figure 1).
Internal regions of the isolated cDNAs were then subjected to PCR amplification using combinations of two 10mer primers (Table 1) with arbitrary but defined sequences (7)
. As shown in Figure 2, three of the combinations of arbitrary primers (BS57/BS58, BS70/BS73 and BS76/BS78) generated electropherograms that showed differential gene expression.
Preliminary analysis by agarose gel electrophoresis (Figure 2) showed a general decrease in the transcriptional activity of MDA-MB231 cells in response to PTHrP[38–94] treatment. This is evident by the lower number of bands obtained after PCR amplification of cDNA from PTHrP[38–94]-treated (lanes 1, 3 and 5) versus control preparations (lanes 2, 4 and 6). When the same amplification reactions were run on a polyacrylamide gel and silver-stained, the resolution of the band pattern improved enormously (Figure 3).
Three bands, one from untreated and two from treated cells, were chosen for further analysis and submitted to re-amplification with the same primers. The selected bands are denoted by arrows (Figure 3). Each of the cDNA fragments present in these bands gave a robust yield of amplified product (Figure 4).
When a single band is obtained (Figure 4, lane 3), the amplification product can be directly sequenced after purification from the gel. Alternatively, when more than one band appears in the electrophoretogram (e.g., Figure 4, lane 2), it is necessary to cut and elute the discrete bands from the gel matrix, and to submit the eluted material to further cycle(s) of ampification, electrophoresis and purification until preparations suitable for sequencing are obtained.
In conclusion, we can recommend the use of Promega's M-MLV Reverse Transcriptase, RNase H Minus Point Mutant for the preparation of cDNA for use in DD-PCR, and Taq DNA Polymerase for the efficient production of cDNA fragments suitable for sequencing after elution from DD-PCR gels.