- Original Articles
- Open Access
Identification of mRNAs Differentially Expressed in Quiescence or in Late G1 Phase of the Cell Cycle in Human Breast Cancer Cells by Using the Differential Display Method
© Molecular Medicine 1996
- Published: 1 July 1996
The decision for a cell to enter the DNA synthesis (S) phase of the cell cycle or to arrest in quiescence is likely to be determined by genes expressed in the late G1 phase, at the restriction point. Loss of restriction point control is associated with malignant cellular transformation and cancer. For this reason, identifying genes that are differentially expressed in late G1 phase versus quiescence is important for understanding the molecular basis of normal and malignant growth.
Materials and Methods
The differential display (DD) method detects mRNA species that are different between sets of mammalian cells, allowing their recovery and cloning of the corresponding cDNAs. Using this technique, we compared mRNAs from synchronized human breast cancer cells (21PT) in quiescence and in late G1.
Six mRNAs differentially expressed in late G1 or in quiescence were identified. One mRNA expressed 10 hr after serum induction showed 99% homology to a peptide transporter involved in antigen presentation of the class I major histocompatibility complex (TAP-1) mRNA. Another mRNA expressed specifically in quiescence and down-regulated 2 hr following serum induction showed 98% homology to human NADP+-dependent cytoplasmic malic enzyme (EC22.214.171.124) mRNA, which is an important enzyme in fatty acid synthesis and lipogenesis. Three others showed high homology to different mRNAs in the GeneBank, corresponding to genes having unknown functions. Finally, one mRNA revealed no significant homology to known genes in the GeneBank.
We conclude that DD is an efficient and powerful method for the identification of growth-related genes which may have a role in cancer development.
Under the influence of external factors, cells make decisions in late G1 phase of the cell cycle between alternative programs, including going into quiescence (G0) and DNA replication. These decisions are likely to be determined by gene products that are expressed in late G1 phase around the restriction point (1–3). Passage through the restriction point results in loss of requirements for growth factor stimulation and less sensitivity to protein synthesis inhibitors (i.e., cyclohexamide), suggesting that all the protein synthesis required for initiation and progression of these programs are completed in late G1 (1–3). Thus, the late G1 regulatory mechanism most likely controls both the onset of DNA replication and the transcription of mRNAs involved in this process. Loss of restriction point control is found to be associated with malignant transformation and cancer (1,3).
Identification of genes induced or down-regulated in late G1 compared with quiescence (G0) should include genes involved in the cellular programs controlled around the restriction point. In order to identify and clone such genes, we first synchronized human breast cancer cells in G0 and in late G1 phase of the cell cycle and then compared the expression of mRNAs by using differential display (DD). This method, originally developed in our laboratory, has now been used successfully for cloning numerous genes (4–8). DD is directed toward the identification of differentially expressed genes, detecting individual mRNA species that are changed in different sets of mammalian cells, and permitting recovery and cloning of their cDNAs (8).
In this study, we synchronized human breast cancer cells (21PT) in late G1 by either using a plant amino acid mimosine, which is a reversible G1 blocker (9, 10), or collecting the cells at 8, 10, and 12 hr after serum induction. Quiescence in G0 was maintained by serum starvation. Comparing mRNA expressions in quiescence and in late G1 by using DD, we cloned several differentially expressed mRNAs. Here we report that five corresponding cDNAs showed strong sequence homology to mRNAs from the GeneBank, including TAP-1 (a peptide transporter involved in antigen presentation of class I major histocompability complex [MHC]) (11) and human NADP+-dependent cytoplasmic malic enzyme (an important enzyme in fatty acid synthesis and lipogenesis) (12, 13). One cDNA clone had no significant homology to known genes. Possible involvement of identified genes in the growth process and the significance of DD as a method of choice in this study are discussed.
The 21PT cell line used in this study, obtained from R. Sager (Harvard Medical School, Boston, MA, U.S.A.) was derived from a human primary breast cancer specimen as described (14). Cells (7.5 × 105) were plated in 150-mm tissue culture plates and grown in α-minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1 µg/ml insulin, 12.5 ng/ml epidermal growth factor, 2.8 µM hydrocortisone, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 10 mM HEPES buffer, incubated at 37°C in a humidified incubator containing 6.5% C02.
For G0 synchronization by serum starvation exponentially growing cultures were washed twice with PBS and placed in α-MEM medium containing 0.4% FBS for 85 hr. For late G1 synchronization, two different methods were used. After 85 hr of serum starvation, cells were either released into α-MEM medium containing 10% FBS with 400 µM mimosine for 24 hr or at Time 0 cells were stimulated by addition of α-MEM medium containing 10% FBS and samples were taken for RNA extraction at 8, 10 and 12 hr. For the analysis of cell cycle-dependent expression of genes, cells synchronized at G0 were stimulated by α-MEM containing 10% FBS, and samples were taken for RNA extraction at indicated times.
Total Cellular RNA Extraction and Northern Blot Analysis
Total cellular RNA extraction was performed with RNAzol-B RNA Extraction Solution (Biotech Lab. Inc., Houston, TX, U.S.A.) according to manufacturer’s instructions. Northern blot analyses were performed as described with minor variations (15). Briefly, 20 µg of total RNA from each sample was electrophoresed in 1.1% agarose gel containing 7% formaldehyde, transferred to 0.45-µm nylon membranes (MSI, Westboro, MA, U.S.A.) with 10× SSC, and cross-linked by ultraviolet (UV) irradiation. Prehybridization and hybridization reactions were performed in a microhybridization oven (Belco Glass Inc., Vineland, NJ, U.S.A.) at 42°C using a hybridization buffer containing 50% formamide, 6× SSPE, 5× Denhardt’s reagent, 0.1% SDS, and 0.1 mg/ml sheared salmon sperm DNA. Reamplified cDNA probes were purified by 1.5% agarose gel electrophoresis using the QIAEX kit from QIAGEN (Chatsworth, CA, U.S.A.) and labeled with [32P]-dCTP by the random prime labeling kit from Boehringer Mannheim Biochemicals, (Indianapolis, IN) essentially as instructed except 1 µl of 10 µM corresponding oligo dT primer was also included during the labeling. The membranes were washed with 2× SSC, 0.1% SDS for 30 min at room temperature, followed by a wash for 10 min at 55°C in 0.2× SSC, 0.1% SDS. The membranes were then exposed to Kodak X-OMAT AR X-ray films with intensifying screens at −70°C.
Cell Cycle Analysis
Fluorescence-activated cell sorter (FACS) analysis was performed as described (16). Briefly, cells were trypsinized, washed with phosphate-buffered saline (PBS), fixed with 70% ethanol for 30 min at 4°C, treated with 10 µg/ml RNase for 30 min at 37°C, stained with 70 µM propidium iodide in 38 mM sodium citrate at room temperature for 30 min, and analyzed with a Becton Dickinson cell sorter at the Dana-Farber Cancer Institute FACS analysis facility. CELL-FIT (Becton-Dickinson, Mountain View, CA, U.S.A.) software was used for graphics and statistics.
Differential display (DD) was performed as previously described with minor modifications (8,17). Briefly, 50 µg of total cellular RNA extracted from each sample was treated with DNase I (MessageClean kit; GeneHunter, Brookline, MA, U.S.A.) for removal of chromosomal DNA contamination. Two tenths of a microgram per sample of total RNA from the cells at different time points were reverse transcribed with oligo dT primers and then amplified by polymerase chain reaction (PCR) with arbitrary primers and corresponding oligo dT primers in the presence of 1 µCi/reaction α-[33P]dATP (2000 Ci/mmole; New England Nuclear, Boston, MA, U.S.A.). The following PCR conditions were used: 94°C for 30 sec, 40°C for 2 min, 72°C for 30 sec for 40 cycles, then 72°C for 5 min. After PCR amplification, the products were resolved by denaturing 6% Polyacrylamide gel electrophoresis. After drying the gel and performing autoradiography for screening the bands, differentially expressed cDNAs were cut from the gel and reamplified by PCR with the previous corresponding primer sets. Reamplified cDNAs confirmed by Northern blot analysis were cloned into pCRIII TA cloning vector (Invitrogene, San Diego, CA, U.S.A.), sequenced (Sequenase kit; United States Biochemical Co., Cleveland, OH, U.S.A.) and reprobed for Northern blot analysis after isolation from the plasmid. Differentially expressed cDNA sequences were compared with sequences in GeneBank and EMBL databases via the BLAST network server.
Synchronization of 21PT Cells in Quiescence (G0) and Late G1
Differentially Expressed mRNAs Identified by Differential Display
Characteristics of cDNA clones identified by differential display from human primary breast cancer cells (21PT)
SM-20 rat serum-inducible mRNA
Human malic enzyme
Human partial cDNA
Human ORF cDNA
Cell Cycle-Dependent Expression of Differentially Expressed Genes
To analyze the cell cycle-dependent expression of identified genes and examine whether the differentially expressed mRNAs identified from the first set are the consequence of specifically expressed mRNAs at late G1 or the consequence of mimosine treatment itself, we made time course membranes from total RNA taken at indicated time points after serum stimulation of G0 cells. Northern blot analysis with time course membranes revealed that five of six genes were expressed in cell cycle-dependent manner. Band #1 and Clone 15d (human ORF mRNA) were detected at 10 hr and disappeared in the following 2 hr (Figs. 2D and 3H). Band #1 was also detected at 24 hr after serum induction (Fig. 2D). Expression of 11c and 12b (TAP-1) was undetectable in quiescent cells, up-regulated in mid and late G1, and continuously expressed in the rest of the cell cycle (Fig. 3 D and F). Clone 40b (cytoplasmic malic enzyme) was expressed in quiescent cells, and its expression was downregulated after serum stimulation throughout the rest of the cell cycle (Fig. 2K). Although 34a was up-regulated following mimosine treatment (Fig. 2 E–G), we could not detect this up-regulation in cells induced by serum with mimosine-free medium. Thus, we concluded that the up-regulation of 34a in late G1 was not a consequence of serum stimulation but an effect of mimosine itself.
The biochemical and molecular mechanisms that regulate cell proliferation are a major field of interest in our laboratory. Our early hypothesis is that cell proliferation is importantly controlled 2 hr before the sudden onset of DNA synthesis in the cell cycle (1,2). The decisions of cells to enter the DNA synthesis (S) phase or to arrest in G0 are likely to be determined by genes expressed in late G1 phase around the restriction point. We proposed that mRNAs differentially expressed in late G1 compared with G0 might be involved in the growth process. In order to identify genes differentially expressed in quiescence versus late G1, we synchronized human breast cancer cells (21PT) in quiescence and in late G1 by two different approaches and compared the mRNA expressions by the powerful DD method. Several technical advantages of DD over alternative methods such as subtractive hybridization made it the method of choice in our study. First, DD is less laborious and much faster. A very large number of mRNAs can be screened and several differentially expressed ones can be identified and cloned in a week. Second, very little RNA is required (0.2 µg/sample). Third, both under-and overexpressed genes can be simultaneously detected on the same DD gel. Fourth, mRNAs can be visualized at each step so that working blindly for about 2 months, as in subtractive hybridization, is eliminated. On the other hand, there are a few technical difficulties with DD, such as false positivity and some DD bands’ containing more than one sequence. Here, we report the identification, partial cloning, and cell cycle-dependent expression of six genes by using this strategy.
Three of six differentially expressed cDNAs showed strong homologies to known genes, namely SM-20 (Clone 34a), NADP+-dependent cytoplasmic malic enzyme (EC 126.96.36.199) (Clone 40b), and TAP-1 (Clone 12b). Clone 1a showed no significant homology to any known sequences in the GeneBank. Clones 11c and 15d showed very high homology to cDNA sequences with unknown functions in the GeneBank.
SM-20 was originally identified and cloned from rat vascular smooth muscle cells after the induction of platelet-derived growth factor (PDGF) (18). Clone 34a showed very similar features to SM-20, including 80% sequence identity, which suggested that Clone 34a might be the as yet unknown human homolog of rat SM-20 gene. Although Clone 34a was identified to be up-regulated in mimosine-treated cells, failure to show the same expression pattern on a mimosine-free time course membrane suggested that up-regulation in late G1 is an effect of mimosine itself. SM-20 was shown to be expressed at 30 min after serum induction and disappeared in 2 hr, which represents an immediate early gene (18). The function of this gene is unknown and remains to be determined.
Clone 40b is expressed in quiescent cells and down-regulated in 2 hr after serum induction (Fig. 2 I–K). Sequence analysis of Clone 40b revealed 98% homology to NADP+-dependent cytoplasmic malic enzyme (ME) cDNA, which is involved in fatty acid synthesis in lipogenesis and catalysis the oxidative decarboxylation of malate into pyruvate (12,13). By this reaction, NADPH is generated from NADP+, which is required for fatty acid synthesis. ME mRNA and protein expression is elevated by triiodothyronine (T3), insulin, and dietary factors such as high carbohydrate, glucose intake, and low fat intake (19). Peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimer, which is important in adipocyte differentiation, transcriptionally activates ME (20). Induction of ME by insulin and T3 is strongly suppressed by EGF (19). The suppressive effect of EGF on ME expression might explain the early inhibition of ME expression after serum induction. Taken together, we showed that ME mRNA expression is significant in quiescent human breast cancer cells and is down-regulated in 2 hr following serum induction. Although there is no evidence about the direct effect of ME on growth regulation, we suggested that some of the upstream regulators of ME such as EGF and PPAR/RXR are involved in the positive and negative regulation of cell growth, respectively.
Clone 12b was not detectable in quiescent cells and was found to be up-regulated in late G1 (Fig. 3F). Computer analysis of clone 12b from the GeneBank showed 99% homology to TAP-1 mRNA, which belongs to the ABC (ATP binding cassette) superfamily of transporters (11). ABC superfamily includes the human multidrug-resistance protein and a series of transporters from bacteria and eucaryotic cells capable of transporting a range of substances, including peptides (11). In one model, TAP-1 protein was proposed to form a heterodimer complex with another ABC superfamily member protein TAP-2 for peptide transporter activity associated with antigen processing necessary for normal assembly of major histocompatibility complex (MHC) class I molecules on the cell membrane (21). Mutation of TAP genes or inhibition of their function by some viral proteins significantly decreases the expression of MHC class I molecules on the cell surface (22,23). Here, we showed that TAP-1 mRNA expression is cell cycle dependent and differentially expressed in proliferating human breast cancer cells. We suggest that immunogenecity of somatic cells might be controlled in part by growth regulation.
#1 and Clone 1a both detected at least three transcripts, of which only one was differentially expressed (Fig. 2 B–D). The differentially expressed transcript was about 8 kb and was expressed at 10 and 24 hr after serum induction (Fig. 2D). Sequence analysis of Clone 1a revealed no significant homology with known sequences. Expression timing coincidentally overlaps with late G1 and late S phase of the cell cycle, suggesting a cyclin E or A type expression pattern. Clones 11c and 15d showed very strong homology to human cDNAs with unknown function in the GeneBank. Clone 11c was detected in mid G1 and in the rest of cell cycle (Fig. 3D). Clone 15d detected a transcript expressed only at 10 hr after serum induction (Fig. 3H). Coincident expression of #1 and Clone 15d at 10 hr after serum induction supports the significance of late G1 following serum stimulation.
In conclusion, we identified six cDNAs differentially expressed genes in late G1 or in quiescence by using the DD method and showed the cell cycle-dependent expression patterns of these genes. We conclude that DD is an efficient and powerful method for the identifying of known and unknown growth-related genes as well as many other genes with different functions.
We thank Dr. R. Sager (Harvard Medical School, Boston) for providing 21PT cells. This work was supported by the National Institutes of Health grant, 5-R01 GM24571-15.
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