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Experimental Therapeutics |
Laboratory of Molecular Pharmacology, Division of Basic Sciences [Y. Z., F. G. G., W. C. R., L. H. S., U. S., K. W. K., Y. P., J. N. W.], Microarray Facility, Advanced Technology Center, Division of Clinical Sciences [L. D. M.], and Office of the Director, Division of Clinical Sciences [E. T. L.], National Cancer Institute, NIH, Bethesda, Maryland 20892
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, using pin-spotted cDNA microarrays, we measured relative changes in expression of 1694 genes after brief CPT treatment of HCT116 cells. HCT116 is a MSH-2-deficient, nonapoptotic colon cancer-derived cell line that expresses functional p53 protein (13) . HCT116 cells arrest in G2-M phase after DNA damage, but unlike leukemia cell lines, they do not undergo rapid apoptosis (14) . This property was used to separate gene expression changes directly related to DNA damage from those associated with apoptosis. To obtain more homogeneous response to CPT treatment and distinguish changes in gene expression related to cell cycle from changes related to DNA damage, we carried out experiments using HCT116 cells synchronized in S-phase. The synchronization allowed us to discern cell cycle-dependent down-regulation and modest up-regulations that would have been buried in an unsynchronized (mixed) population of cells. By performing time- and dose-dependent experiments, we found that up-regulation of p53-related stress response genes was associated with high-dose CPT treatment or prolonged exposure to APH. In contrast, protracted expression of mitosis-related genes was correlated with cell cycle delay in G2. These results support the twin propositions that DNA damage can disrupt normal cell cycle-related gene expression and that this disruption may be involved in G2 cell cycle arrest. High and low concentrations of CPT produced qualitatively different changes in patterns of gene expression.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Cycle Synchronization and CPT Treatment
HCT116 cells, cultured to near confluence, were trypsinized and plated at 3.6 x 106 cells/150-mm2 dish (Becton Dickinson, Franklin Lakes, NJ). After a 24-h incubation, the cells were synchronized in medium containing 1 µM APH for 17 h or for the time indicated. CPT treatment was carried out as follows on synchronized cells after 17 h with APH. The APH-containing medium was removed, and the cells were washed twice with APH-free medium, and then cultured in drug-free medium for 2 h. Portions of the cells were then treated for 75 min with 0, 20, or 1000 nM CPT. In each case the medium contained 0.01% DMSO. The CPT/DMSO-containing medium was then removed, and the cells were washed twice with drug-free medium. Thereafter, cells were cultured in drug-free medium for the indicated time.
FACS Analysis of Cell Cycle Stage
Cells were trypsinized, and the resulting cell suspensions were centrifuged at 1000 rpm for 10 min. The cells were fixed by resuspending them in 0.5 ml of 70% ethanol for 30 min, centrifuging them at 1500 rpm for 10 min, and washing the pellets twice with ice-cold PBS to remove residual ethanol. The cell pellets were resuspended in 0.5 ml of PBS containing 50 µg/ml propidium iodide (Sigma Chemical Co.-Aldrich, St. Louis, MO) and 100 µg/ml RNase (Sigma-Aldrich), incubated at 37°C for 30 min, and then analyzed using a FACScan flow cytometer (Becton Dickinson).
cDNA Microarray Analysis of Gene Transcription
Total RNA was isolated from cells by the RNeasy Midi Kit (Qiagen, Valencia, CA) and concentrated to 5 µg/µl in diethyl pyrocarbonate-treated water (Research Genetics, Huntsville, AL). The RNA samples were aliquoted and stored at -70°C. cDNA microarray experiments were carried out using a protocol developed by the Laboratory of Cancer Genetics of the National Human Genome Research Institute,4
with minor modifications. Cy3 and Cy5 labeling was done with 54 and 72 µg of total RNA, respectively. Seventy-five µM Cy3-dUTP or Cy5-dUTP was used in each labeling reaction. Cy3- and Cy5-labeled cDNA samples, one experimental and the other a reference sample, were mixed in equal amounts. The mixture was then hybridized with a pin-spotted cDNA microarray. The microarrays were spotted robotically at the National Cancer Institute Microarray Facility, Advanced Technology Center. Each microarray contained 2076 cDNA spots corresponding to known cancer-related genes. Some of the genes were printed on the microarray more than once to provide replicates for reproducibility analysis.5
The hybridization data were acquired using the Avalanche Fluorescence Scanner (Molecular Dynamics, Sunnyvale, CA). Image analysis was carried out using a set of software tools developed by Y. Chen (15)
at the Laboratory of Cancer Genetics, National Human Genome Research Institute.6
To remove the systematic bias caused by the chemical difference between Cy3 and Cy5, each microarray study was performed twice using "reciprocal labeling," i.e., for the first microarray, the experimental sample was labeled with Cy3 and the reference sample was labeled with Cy5, whereas for the second microarray, the reference sample was labeled with Cy3 and the experimental sample was labeled with Cy5. Throughout the data analysis process, differential expression was represented by:
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Data Analysis
Data Preprocessing.
Before any of the analyses, we removed the data from empty spots and reagent-control spots [poly(dA), Cot I, Cy3, and Cy5]. On each array, 2076 of the original 2208 spots remained, and 242 of those spots were in duplicate. We also removed data from spots identified as visibly flawed (<20 in all for the 36 arrays). We then normalized the ratio data by Gaussian-kernel fitting7
and normalized the resulting values for each cell type so that the sum of the log-transformed values was the same for each cell sample. We next filtered the data to obtain genes whose expression differed by more than 1.7-fold in the reciprocal-averaged ratio (GMRi). This threshold criterion was, to a degree, arbitrary. It was based, however, on quality control experiments in which we found that >99% of spots had reciprocal-averaged ratios between 1.7 and 1/1.7 when two identical samples were analyzed (Fig. 3)
. In addition, to further remove possible outliers (such as, e.g., the three points in the right panel of Fig. 3
), we selected only spots that met the 1.35-fold change criterion for both of the duplicate arrays. We further required that the data point meet these criteria for more than 1 design point in the set of 18. This procedure selected a total of 33 different genes, including 3 that were represented twice in the microarray.
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RESULTS |
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. To obtain a relatively homogeneous cell population before CPT treatment, we synchronized HCT116 cells using APH. APH, an inhibitor of DNA polymerase 
, blocks the cell cycle in S-phase. HCT116 cells have a doubling time of 16 h, and incubation of HCT116 cells with 1 µM APH for 17 h arrested most cells in S-phase. Cell cycling resumed immediately after removal of APH. After 17 h of exposure to APH and 2 h in APH-free medium, 
80% of the cells were still in S-phase. The cells were then treated with or without CPT for 75 min and finally incubated in CPT-free medium for the times indicated in Fig. 1
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, the cells not treated with CPT resumed cell cycle progression after removal of APH, and the time taken to complete the first cycle was close to the normal doubling time of HCT116 cells. The cells treated with 20 nM CPT slowed down slightly in S-phase and were significantly delayed in G2-M. The apparent G2-M phase of cells treated with 20 nM CPT was approximately twice as long as that of the control cells (0.01% DMSO). The cells treated with 1000 nM CPT showed significant S-phase delay (8–10 h), and G2-M arrest was not reversed even after a 60-h incubation in drug-free medium (data not shown).
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, and the reference sample was total RNA isolated from cells harvested at the end of the 17-h APH synchronization (2 h before CPT treatment). This series generated 36 microarray data sets, i.e., 18 reciprocally labeled pairs of arrays. Included were duplicate sets for cells collected 5 h after treatment with 1000 nM CPT.
The second series of experiments examined the effects of APH treatment on gene expression. In these experiments, total RNA isolated from APH-treated cells was compared with total RNA isolated from nontreated cells. The APH treatment was carried out for 6, 12, 17, and 24 h. This series of experiments produced 10 microarray data sets, i.e., 5 reciprocally labeled pairs of arrays. Included were duplicate data sets for cells harvested after 17 h of treatment with APH. In addition, to evaluate random variation in the experimental procedure, we carried out 17-h APH synchronization three different times using the same conditions, and the three independent samples were compared with each other in pairwise fashion. After calibrating the signals from both the Cy3 and Cy5 channels and averaging the ratios of two reciprocally labeled duplicates, we found that >99% of the genes in the three control data sets showed ratio differences <1.7-fold (Fig. 3)
. Data reproducibility was also assured by the high correlation between duplicate gene pairs (represented by the same clone or different clones). As illustrated in Fig. 4
, the duplicate clones clustered together.
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. The gene definitions were based on human Unigene build #95.
Clustering the genes brings out patterns in the data by grouping together genes with similar expression profiles. Of the 33 genes that exhibited changes according to the criteria established, 27 clustered tightly within three groups (Fig. 4A)
. Group I genes were up-regulated in the control and low-dose CPT-treated cells, but the up-regulation took place at a later time in cells treated with low-dose CPT. The difference in timing of up-regulation closely matched the difference in the timing of mitosis. Those genes were not up-regulated after high-dose CPT treatment. Group II genes were down-regulated during the period of time when low-dose CPT-treated cells were recovering from G2 delay. In the case of high-dose CPT treatment, some of these genes showed an abortive down-regulation shortly after drug removal. Group III genes were up-regulated in the cells treated with high-dose CPT during S-phase delay and/or G2 arrest, but these genes did not change in the cells treated with low-dose CPT. Time-dependent expression changes for the 36 spots are shown in a line graph in Fig. 4B
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The changes in expression identified by cDNA microarray were largely consistent with the results obtained using Northern blot analysis (Table 1)
. In summary, Northern blot analyses were run for five of the genes, each over eight experimental conditions. IMP dehydrogenase 1, which showed no significant difference in ratio from sample to sample in the microarray analysis, was used as a normalization reference. The overall Pearson correlation coefficients (r) relating microarray and Northern expression levels for the eight conditions were as follows: for p21, r = 0.88; for 14-3-3
, r = 0.92; for cyclin B1, r = 0.79; for aurora/IPL1, r = 0.91; for p16-INK4a, r = 0.78. The overall average of the correlation coefficients for the five genes was, therefore, r = 0.86
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according to the cluster order generated from the CPT experimental data. The results are shown in the five columns at the right side of the CIM in Fig. 4
. The group I genes were down-regulated in most of the APH-treated cells, whereas some of the group III genes were up-regulated in the cells treated with APH for 17 and 24 h. Group II genes did not show any consistent changes. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To study dose- and time-dependent effects of CPT treatment on cell cycle progress and gene expression, we carried out the experiments using APH-synchronized cells treated with 1000, 20, or 0 nM CPT (all in 0.01% DMSO). The time dependence of treatment effects was revealed by comparing each sample with an internal second-color control harvested after synchronization with APH but without any other treatment. This direct comparison of all CPT- and sham-treated samples with a single internal control permitted us to map out the time course of expression levels after treatment.
Because the cytotoxicity of CPT is cell-cycle specific and because CPT treatment directly affects cell cycle progression, we reasoned that we could optimize our chance of seeing down-regulations and modest up-regulations of genes if we synchronized the cells and then treated them at the appropriate point in the cell cycle. Among the possible methods (none of them perfect) for synchronizing cells, we selected the commonly used technique based on incubation with APH, an inhibitor of DNA polymerases. However, APH itself can induce gene expression changes. It can activate p53 and up-regulate p21/WAF1 expression (20)
. Mitosis may also be perturbed immediately after treatment with high-dose APH (21)
. To minimize (although not completely eliminate) such confounding influences, we used a minimum concentration of APH for synchronization. In this study, we did not observe perturbation of mitosis after removal of APH (Fig. 2)
, and a colony formation assay did not indicate significant toxicity (data not shown). To monitor gene expression changes caused by APH treatment, we compared gene expression in cells incubated with and without APH. Stress-related responses, including moderate elevation of p21/WAF1 transcript, were observed after prolonged APH treatment (Fig. 4A)
, but they had little effect on cell cycle progression after removal of APH. Because the internal control for the APH experiments was an unsynchronized cell sample and that for the CPT-treatment experiments was an APH-treated sample, the appearance of up-regulated gene expression after CPT treatment indicated an additional up-regulation above the already increased expression caused by APH. For example, p53-responsive genes were up-regulated after APH treatment. Subsequent treatment with high-dose CPT induced additional up-regulation, whereas treatment with low-dose CPT or no CPT largely maintained the already elevated transcription levels.
On the basis of cluster analysis (Fig. 4A)
, the 33 changing genes fell into three predominant groups based on their patterns of gene expression:
Group I Genes.
Group I genes tended to be maximally up-regulated during the time that cells were in transit through G2-M. Their increased expression correlated well with the period of time during which most of the APH-treated control cells or the low-dose CPT-treated cells were beginning to exit from mitosis. Among six genes in this group, cyclin B1, aurora/STK15, Nek2, and hRad6 are directly involved in mitotic regulation. Through formation of a complex with CDK1 (p34cdc2), cyclin B1 promotes entry into mitosis, and its expression is tightly regulated in the cell cycle. Its expression is low in G1 but up-regulated throughout the S and G2 phases (22)
. Cyclin B1 associates with centrosomes at about the time of transition of the centrosome’s microtubule organizing center from an interphase to a mitotic pattern (Ref. 23
and references cited therein). STK15, a member of the aurora/IPL1 kinase family, is a short-lived protein associated with the mitotic spindle and centrosome. It is required for cell cycle progression and genome stability (24
, 25)
. The STK15 gene is amplified and overexpressed in multiple human tumor cell types and can induce excessive centrosome duplication. Nek2 is another protein kinase whose expression is up-regulated during the S and G2 phases. Nek2 is a core component of the centrosome. It is probably required for separation of centrosomes and for the G2-to-M transition (26, 27, 28)
. hRad6 codes a ubiquitin-conjugating enzyme critical for degradation of proteins that maintain the G2 or M states of the cell (29)
. When analyzing a CIM of the entire 1694-gene data set (results not shown), we identified a tight cluster branch that contained all of the group I genes as well as other genes showing expression patterns similar to those of group I but not meeting the criteria used to select the set of 33 genes. That branch is shown in Fig. 5
. Most of the additional genes are mitosis related. Among them, ckshs2 and CENP-F kinetochore protein have been shown to have regulated expression patterns similar to that of cyclin B1 (30)
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The implication of TRAF5 and p16-INK4a in group I is not clear at this point. TRAF5 is an adapter protein that links signaling from tumor necrosis factor receptor to nuclear factor-
B and to the apoptosis-regulating kinase ASK1 (37
, 38)
. Via both mechanisms, TRAF5 tends to protect cells from apoptosis. p16-INK4a tends to block entry into S-phase by binding to cdk4/6, thereby preventing the activation of cdk4/6 by cyclin D. In all of our experiments, p16-INK4a exhibited expression patterns very similar to those of cyclin B1. This behavior is consistent with reports of p16 accumulation during G2 delay of UV-treated HeLa cells (Ref. 39
and references cited therein).
Group II Genes.
These genes were down-regulated during the period of G2 delay in the cells treated with low-dose CPT. This down-regulation seemed not to be associated with G2 per se because it was not seen in control cells as they passed through G2, nor in the early period of G2 delay in the cells treated with low-dose CPT. It was only during the extension of G2 that the down-regulation occurred. This finding suggests that the down-regulation of group II genes is intimately related to a checkpoint that reversibly delays the transition from G2 to mitosis. The absence of down-regulation in the high-dose CPT experiment suggests that most of the cells that were in S-phase during CPT treatment never reached a normal G2 state. Some genes in this experiment showed an immediate transient down-regulation, perhaps reflecting the subpopulation of cells that were already at or near G2 at the time of CPT treatment (see Fig. 2
). The group II genes are heterogeneous in function, and they have not been associated previously with DNA damage response or normal cell cycle regulation. Some of them are associated with calcium-dependent pathways, cellular metabolism, and signal transduction pathways.
Group III Genes.
Group III genes are characterized by increased expression after high-dose, but not low-dose, CPT treatment. These genes are inducible by DNA damage and are associated with cell cycle arrest and apoptosis. Five of the eight genes in the group are known to block cell cycle progression and to be activated by p53 in response to DNA damage: (a) the cyclin-dependent kinase inhibitor p21 (35
, 40)
; (b) the chaperone 14-3-3 (14
, 41)
; (c) the type IIC protein phosphatase Wip1 (42
, 43)
; (d) the apoptosis-inducer CD95/Fas (44)
; and (e) the damage-specific DNA-binding protein subunit DDB2 gene (45)
. HCT116 is p53 wild type and exhibits p53 function (13)
. It is possible that the up-regulation of p53-responsive genes in HCT116 after high-dose CPT treatment was attributable to activation of p53. Although we do not have direct data on p53 protein in these experiments, this pattern of up-regulation of five p53-responsive genes in these p53 wild-type cells strongly suggests that p53 protein activity was up-regulated after high-dose CPT treatment. These observations are consistent with present understanding of the transcriptional role of p53 in cellular responses to DNA damage.
In contrast to the observation with high-dose CPT, group III genes showed no up-regulation after low-dose CPT treatment, suggesting that p53-dependent cell death- and cell cycle-related genes were not significantly up-regulated under conditions of reversible cell cycle arrest. Two of the p53-responsive genes (p21 and 14-3-3) were in fact down-regulated, perhaps reflecting recovery from the up-regulation induced by exposure to APH. This behavior suggests a remarkable distinction between the reversible G2 delay seen in these experiments after low-dose CPT treatment and the more permanent G2 arrest that followed extensive DNA damage after high-dose CPT.
In addition to p53-responsive genes, group III also included the genes for TGF-ß superfamily protein and IFN-induced 17-kDa protein. The TGF-ß superfamily protein is involved in signal transduction events that regulate cell growth and differentiation (46) . It has been reported to be up-regulated in cells treated with the DNA topoisomerase II inhibitor VP-16 (47) . The highly correlated expression pattern seen here indicates a possible relationship of TGF-ß superfamily protein to DNA damage and p53 activity, although such associations have not previously been reported.
In conclusion, we find it striking that 27 of the 33 genes selected objectively from the overall set of 1694 represented on the microarrays fell coherently into three tightly clustered patterns of behavior. These patterns appear to define signatures for the initial molecular consequences of CPT treatment. Even more unexpected, changes in gene expression during the first cell cycle after treatment revealed patterns after a relatively nontoxic dose of CPT that were qualitatively different from those after a highly toxic dose, although there was no gross cell death during the course of the experiments in either case. We were not surprised to see that the high dose precipitated broad changes not seen at the low dose, but we were surprised to find that the reverse was also true, that the low dose led to changes not seen at the high dose. On the basis of these observations, we propose that there is a fundamental difference between the gene expression changes associated with reversible G2 delay that follows mild DNA damage and permanent G2 arrest that follows more extensive DNA damage. The fate of cells after DNA damage is probably determined by coordination and balancing of multiple parallel processes at both the transcription and protein levels. Directed experimental investigations will be required to test the hypotheses generated by these microarray experiments. In particular, it will be instructive to follow up the suggestion of these experiments that the differences involve patterns of regulation in the pathways of p53 and other stress-response genes, as well as in the mitotic machinery, especially with regard to centromere formation and function.
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FOOTNOTES |
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1 Present address: Cellular Genomics Inc., 36 East Industrial Drive, Branford, CT 06405. 
2 To whom requests for reprints should be addressed, at Laboratory of Molecular Pharmacology, DBS, NCI, NIH, Building 37/5D-02, 37 Convent Drive MSC 4255, Bethesda, MD 20892-4255. Phone: (301) 496-5971; Fax: (301) 402-0752; E-mail: weinstein{at}dtpax2.ncifcrf.gov
3 The abbreviations used are: CPT, camptothecin; APH, aphidicolin; FACS, fluorescence in situ sorting; CIM, clustered image map; TRAF5, tumor necrosis factor receptor-associated factor 5; TGF, tumor growth factor.
4 http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/protocol.html.
5 The gene list can be found at http://nciarray.nci.nih.gov/gi_acc_ug_title.shtml.
6 http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/.
7 L. H. Smith et al., New methods for analysis of two-color microarray data, manuscript in preparation.
8 A versatile program for producing CIMs can be found at http://discover.nci.nih.gov.
Received 6/ 1/01. Accepted 1/14/02.
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, treatment with 0.01% DMSO; 
, treatment with 20 nM CPT in 0.01% DMSO; 
, treatment with 1000 nM CPT in 0.01% DMSO.