Functional Profiling: From Microarrays via Cell-Based Assays to Novel Tumor Relevant Modulators of the Cell Cycle Free

Proliferation of mammalian cells requires that DNA replication is strictly regulated and done with high fidelity. The commitment of cells to enter the S phase is the primary decision point at which they control their division. Alterations in expression and activity of proteins that are involved in this process can cause unrestrained growth and are prerequisite for the development and progression of cancer (1). In this line, tumor-associated changes in mRNA transcription are frequently examined in tumor microarray studies. Whereas such analyses typically identify hundreds or thousands of genes (2–4), often, the function of the encoded proteins is unknown and their role in the disease process is not well understood (5). Long lists of differentially expressed genes reflect the complex interplay of biological processes that are involved in cancer (e.g., inflammation, angiogenesis, metabolism, as well as cell cycle control, and apoptosis; refs. 6–9). The distinction between causative changes in gene expression from secondary effects remains a key challenge.

We adopted the idea of functional profiling (10), developed a cancer relevant automated high-throughput assay, and applied it to genes that were associated with cancer by whole genome microarray analysis (11, 12). This identified novel proteins that modified the rate of DNA replication in mammalian cells, this being the first direct indication of a causative relation to oncogenic processes. We refined the functional profiles for 16 candidate genes and proteins with assays that probed other cell cycle– and cancer-related processes, extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, and anchorage-independent growth. Functional profiling distinguished causative from secondary effects and is therefore able to bridge the gap between whole genome tumor microarray studies and single candidate analysis.

Selection of novel full-length cDNAs. Tumors of the brain, breast, kidney, and gastrointestinal stroma were screened for cancer-associated changes in mRNA transcription using a 31,500-element whole transcriptome cDNA array (11, 12). Genes with differential expression between normal and tumor samples or between tumor types were selected by a two-sample t test. From these, we selected 103 open reading frames (ORF) based on the availability of full-length cDNAs for the cell-based bromodeoxyuridine (BrdUrd) incorporation assay. The cDNAs had been isolated from libraries of human fetal brain (library DKFZp564), human fetal kidney (DKFZp566), human amygdala (DKFZp761), human uterus (DKFZp586), and human testis (DKFZp434; ref. 13). ORFs were cloned and sequence validated in Gateway (14) vectors, then shuttled into NH₂-terminal cyan fluorescent protein (CFP) and COOH-terminal yellow fluorescent protein (YFP) to produce mammalian expression constructs under control of the cytomegalovirus promoter (15). Subcellular localization of proteins was determined as described (15). Initial annotation of protein sequences was done with automated tools (16).

Automated cell-based assay to assess DNA replication. Expression plasmids were isolated in a 96-well format using a MultiProbe IIex-Robot (Perkin-Elmer, Wellesley, MA) and Montage Plasmid Miniprep96 Kits (Millipore, Bedford, MA). DNA masterplates were generated with a final concentration of 27.5 ng/μL and a total volume of 80 μL per well. Cells (10,000; NIH 3T3 cells: ATCC CRL-1658) were transfected (Effectene, Qiagen, Hilden, Germany) using 100 ng plasmid DNA per well in an array of 12 chamber slides (MWG Biotech, Ebersberg, Germany) to mimic a 96-well plate. Each CFP and YFP expression construct was included in duplicate. Control cDNAs (CFP-cyclin A, cyclin A-YFP as positive controls for activating, CFP subunit A of phosphatase 2, subunit A of phosphatase 2-YFP as positive controls for repressing effects on DNA replication; CFP and YFP served as negative controls) were placed at fixed positions within the plate. After 24 hours, cells were incubated in presence of 1 mmol/L BrdUrd for 6 hours before fixation with 4% paraformaldehyde. Transfection, BrdUrd immunostaining with a mouse anti-BrdUrd primary antibody (Roche Diagnostics, Penzberg, Germany), incubation with a Cy5-labeled goat anti-mouse secondary antibody (Molecular Probes, Karlsruhe, Germany), and 4′,6-diamidino-2-phenyindole (DAPI) staining were done using a MultiProbe robot.

Image acquisition and analysis. A fully automated high-content screening microscope ( 17) was used to measure on average 19 frames from each well. The image frames consisted of three color channels (CFP/YFP, Cy5, and DAPI) each at 1,280 × 1,024 pixels at 12-bit resolution. Images were analyzed by an automated segmentation and quantification algorithm that we implemented in Labview (National Instruments, http://ni.com).

Within-well analysis. The data from one well were modeled as

where b_i, t_i, and d_i are the BrdUrd, transfection, and DAPI intensities, respectively, for cell i; m is a smooth regression function, f(i) is the frame in which cell i was found; 𝛉_f is the background for the fth frame; κ is a spectral crosstalk coefficient; and ε_i is a noise term. We introduced the correction terms 𝛉_f and κd_i to address empirically observed systematic effects: 𝛉_f accounts for the different baseline levels of the fluorescence intensities in different frames and κ for spectral overlap from the DAPI signal into the CFP detection channel. A separate coefficient κ was fitted for each 96-well plate to account for apparent long-time drift of the equipment. Values for κ were between 0 and 0.06. Spectral overlap between DAPI and YFP was negligible. The smooth local regression function m, its derivative m′, and confidence bands for m′ were fitted using the R package locfit (18). A constant bandwidth α = 1 was used throughout. To determine the intensity t* that corresponds to very weakly expressing cells we calculated the midpoint of the shorth, which is the shortest interval that contains half of the t_i. As a measure of the effect, we used the z score, which is the ratio of the estimated slope and its SE.

Between-well analysis. To obtain a ranking of the proteins, we compared the set of z scores from the replicate wells for each ORF to the overall distribution of z scores via the two-sided Wilcoxon test. We considered an effect significant when P < 0.05. Multiple testing adjustment for control of the false discovery rate was done following Benjamini and Hochberg (19).

We provide software for statistical procedures and visualizations through the package prada, which is part of the bioinformatics platform Bioconductor (http://www.bioconductor.org).

Assay for quantification of anchorage-independent growth. Flat-bottomed 96-well plates were coated with poly(2-hydroxylethyl methacrylate) [poly(HEMA), 120 mg/mL of 95% ethanol] as described (20). Kidney clear cell carcinoma cells Caki-1 (ATCC HTB-46) and Caki-2 (ATCC HTB-47) cells were cultured in RPMI 1640 with 1% penicillin/streptomycin, 1 % l-glutamine, 1% nonessential amino acids, and 10% fetal bovine serum (Life Technologies, Gaithersburg, MD) and transfected with Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. Proteins found to activate DNA replication were transfected into Caki-2 cells to measure their potential to stimulate anchorage-independent growth of this cell line. Repressors of DNA replication were analyzed in Caki-1 cells for their potential to reduce anchorage-independent growth. Twenty-four hours after transfection, 3,500 Caki-1 or Caki-2 cells were spread in each well of the poly(HEMA)-coated plates in a total volume of 200 μL RPMI 1640. Medium was exchanged after 4 days of culture. Measurement of cell growth was done as described by Fukazawa et al. (20).

Assay automation. We designed a functional profiling strategy that took genes that had been associated with cancer by microarray studies and screened the encoded proteins in tumor relevant assays (Fig. 1). ORFs of previously uncharacterized genes were subcloned from publicly available full-length cDNA resources (13, 21) into expression vectors (15) and transfected into NIH 3T3 cells to overexpress the encoded proteins. All ORFs were fused to NH₂-terminal CFP or COOH-terminal YFP variants of the green fluorescent protein (GFP), and their expression was monitored by automated microscopy. The protein localization at the subcellular level was visually inspected for all constructs (15, 22). This information was used in the subsequent assays, only data obtained with the relevant expression constructs were considered (Fig. 1).

A DNA replication assay was fully automated with help of liquid handling robots and customized statistics software. Transient transfections were carried out in 96-well plates; analysis of protein overexpression and induced effects was 24 hours later. We applied automated high-content screening microscopy (17, 23) to achieve a single-cell resolution (Fig. 2) and traced individual cells within their microenvironment of the well. We restricted our analysis to cells having small levels of protein expression to minimize the perturbation of the cellular system. Whereas we cannot completely rule out nonphysiologic effects that might be induced by even minor increases in protein content, our strategy is superior to other approaches, like plate reader assays and RNA interference, where a correlation of protein levels with effects is not possible at a single-cell resolution. Additionally, we could simultaneously identify activators and repressors of DNA replication (i.e., potential oncogenes and tumor suppressor genes; Figs. 2 and 3). Cyclin A and phosphatase 2A were used in the assay set up and in every screening plate as positive controls for activation and repression, respectively.

DNA replication was quantified by monitoring the rate of BrdUrd incorporation with help of a Cy5-labeled antibody. In addition, cells were stained with DAPI to facilitate autofocus of the microscope and to aid the image segmentation algorithm with the cell finding. Thus, three fluorescence intensities (GFP/YFP, Cy5, and DAPI) were recorded for each cell. The effect of the candidate proteins' expression on DNA replication was determined by local regression of the Cy5 signal (BrdUrd) on the GFP/YFP signal (expression). For each well, this resulted in an estimated regression slope and a z score that is the estimate standardized by its SE. Data from replicate wells per protein were summarized by the Wilcoxon statistic and P value for the comparison of their z scores to the overall distribution of z scores. This resulted in a ranking of the proteins for effect strength and a list of 16 proteins with Ps < 0.05.

Assay performance. The results of all microscopic frames (19 on average) from within one well were combined, taking into the account disturbance factors as spectral overlaps and frame-specific background values. One such frame is shown in Fig. 2A. The intracellular amount of recombinant protein varied over a broad range. The majority of cells were either untransfected (cells left of the midpoint of the shorth in Fig. 2B) or expressed the protein at low levels (right of the midpoint of the shorth). Few cells strongly expressed the recombinant protein with signal intensities of >100 in the YFP channel. A scatterplot of expression level versus BrdUrd incorporation is shown in Fig. 2C. Even the population of nonexpressing cells showed a considerable intrinsic variation in DNA replication as reflected by varying BrdUrd-Cy5 signals. A systematic trend was observed, however, for the population of cyclin A–expressing cells towards the right end of the plot: the higher the expression level, the more cells had incorporated BrdUrd during the experiment. This trend is visualized by the local regression curve in Fig. 1C. This curve was calculated based on the data of nonexpressing and weakly overexpressing cells. Strongly overexpressing cells (e.g., signal intensity >100 in Fig. 2B-C) were downweighed as in these the level of recombinant protein could induce nonphysiologic effects (e.g., apoptosis). A z score was calculated as a summary of the data for the cells within one well.

To obtain a visual overview of the protein effects from a 96-well experiment, we produced plate plots (Fig. 2D). Z scores for positive (activators) and negative (repressors) slopes are indicated in color. The red color for cyclin A is indicative of this protein to be an activator of DNA replication. Interactive versions of the plate plots are provided in the web supplement (http://www.dkfz.de/mga/home/fctassay/sphase). Scatterplots similar to and including the one shown in Fig. 2C can be viewed by mouse clicking on a well. To assess statistical significance, we compared the results from multiple wells for the same protein through histograms of the z scores for both tag orientations (Fig. 3).

Candidate selection. We selected 103 genes (Fig. 1; Table S1) for a cellular screen based on the following criteria: (i) their transcripts were contained in a set of 396 differentially expressed genes from genome-wide tumor microarray studies (11, 12); (ii) the encoded proteins had no public functional annotation, other than from high-throughput projects such as microarray analysis; (iii) we had determined the subcellular localization of the proteins (15, 22). This provided an initial piece of information that connected the proteins to their respective cellular environment and thus limited their potential for protein interactions. In addition, use of full-length proteins ensured that all motifs determining, for example, protein localization, activity, and regulation were present.

DNA replication assay and functional profiling. Analyzing the 103 proteins in the BrdUrd incorporation assay, cells were detected in 2,462 of 2,880 wells. Fluorescence intensities were recorded for CFP/YFP, Cy5 (BrdUrd), and DAPI signals individually, resulting in 3 × 46,221 images. A total of 2,251,739 cells were analyzed, giving over 6.75 million data points. Sixteen of the 103 proteins showed a significant effect on the passage through the S phase (individual P < 0.05; false discovery rate <0.16). Seven of these were activators and nine were repressors of BrdUrd incorporation and thus of DNA replication (Fig. 3; Table 1). The 16 proteins were then examined for a possible effect on ERK1/2 activation, to measure effects on G₁-S transition (24). There we found one activating and one inhibiting protein (Fig. S1; Table 1). Repressors of DNA replication were further subjected to a poly(HEMA) growth assay to test their potential to modulate anchorage-independent cell proliferation. Three proteins were positive in this assay (Fig. 4; Table 1). We annotated all 16 genes and proteins using tumor microarray results from Oncomine (ref. 25; Table S1). Taqman RNA quantification was done in two kidney cancer cell lines (Caki-1 and Caki-2) to refine analysis of the poly(HEMA) assay and was included in the annotation as well as protein and domain classifications from Source (26) and Interpro (ref. 27; Table S1). Together, this integrated data set provided us with candidates for detailed and ongoing studies. In the following, four such genes will be described to show the synergy that is achieved with the functional profiling strategy.

Cancer-relevant cell cycle modulators. The protein encoded by cDNA DKFZp434P2235, an activator of passage through the S phase, localizes to the plasma membrane. The corresponding gene has been independently reported as PRC17 oncogene (28). Pei et al. found it amplified in 15% of prostate cancers and highly overexpressed in about half of metastatic prostate tumors. We found the transcription of the PRC17 (DKFZp434P2235) gene up-regulated in kidney tumors (Table 1). The gene is also expressed in the kidney carcinoma cell line Caki-1, which shows anchorage-independent growth but not in Caki-2 which does not (Figs. S2 and S3). The encoded protein interacts with Rab5 and activates its GTPase activity (28). Pei et al. discuss that expression of PRC17 could affect the amount of inactive Rab5, which should inhibit receptor endocytosis and result in prolonged growth factor signaling. This hypothesis is consistent with our finding that the overexpressed DKFZp434P2235 protein promotes DNA synthesis in the S phase (Fig. 3). Whereas the PRC17 oncogene takes one candidate from our list of truly novel genes, the discovery of this protein in the DNA replication assay shows the power of our integrated approach to identify proteins with cancer relevance.

The gene for DKFZp566A0646 was found down-regulated in kidney tumors (Table 1) and to have a lower expression in Caki-1 compared with Caki-2 cells (Fig. S2). The overexpressed protein inhibited DNA synthesis in the S phase (Fig. 3) and blocked anchorage-independent growth of Caki-1 cells (Fig. 4). These findings suggest a repressing effect on tumorigenicity in vitro. The protein encoded by cDNA DKFZp566A0646 contains a domain common to the Rab subfamily of Ras small GTPases (IPR003579). The function of its orthologous protein p34 in rat is not established; however, in a yeast two-hybrid screen, it was shown to be a binding partner for the γ and α subunits of the AP1 and AP2 complexes, respectively (29). These adaptor complexes participate in intracellular clathrin-coated vesicle trafficking. We found that Rab5 as well as its effector early endosome antigen-1 (EEA-1) are down-regulated in kidney tumors (11). Together with the up-regulation of the Rab5 GTPase-activating enzyme PRC17, this would imply reduced receptor internalization through endocytosis. In addition, Rab GDP-dissociation inhibitor α (GDIα) was found up-regulated and Rab11b down-regulated in kidney tumors, supporting the hypothesis that endocytosis is inhibited in kidney cancer. Changing expression levels of Rab genes have also been described for other cancer types (30–32).

The protein encoded by cDNA DKFZp564D152 has 36% identity over 186 residues with the SET protein of Drosophila melanogaster (SOURCE; ref. 26), which belongs to the nucleosome assembly protein (NAP) family. The NAP family is a group of histone chaperone-like proteins that are known to be regulators of transcriptional control (33). Nucleosome assembly and disassembly are essential processes in the S phase of the cell cycle. In proliferating cells, the synthesis of histones during the S phase is coupled to DNA synthesis. This ensures the packing of chromatin. The protein NAP-1 is thought to be a cell cycle regulator and interacts with Kap114p (34). That protein is a member of the karyopherin/importin family, which executes the nuclear import of histone 2A and histone 2B. The activating effect of the DKFZp564D152 protein on DNA replication (Fig. 3), in conjunction with its nuclear and cytoplasmic localization support the assumption that it might be involved in cytoplasmic-nuclear transfer of histones.

The protein encoded by DKFZp434C2415 was found to repress DNA replication in the S phase (Fig. 3). However, it had a significant activating effect on ERK1/2 activation in serum starved as well as in stimulated cells (Table 1; Fig. S1). The protein was recently described as UBA5, which is an E1-like enzyme in the ubiquitin degradation pathway (35). Ubiquitin activating enzymes regulate the rate of ubiquitin conjugation and ubiquitin mediated protein degradation, which are essential control mechanisms in the transition from G₁ to S phase. Several studies show that cell cycle regulating proteins are directly targeted for ubiquitin-mediated destruction through ERK1/2 (36, 37). Thus, DKFZp434C2415 could regulate ERK1/2 activity through ubiquitin mediated degradation of, for example, phosphatases, the activated ERK1/2 would then mark cell cycle–regulating proteins for degradation.

The DKFZp566F123 protein was identified as a BTB/POZ zinc finger protein (POK protein). The BTB/POZ domain (IPR000210) mediates protein/protein interaction, whereas the C2H2 zinc finger (IPR007087) is a DNA-binding domain. DKFZp566F123 shares closest homology to the transcriptional repressor BCL-6. The latter protein is involved in hematopoiesis, oncogenesis, and immune response (38) and multiple roles in cell survival and differentiation are described (39). Zhang et al. hypothesize that the protein encoded by DKFZp566F123 might also be involved in these biological processes and we found that overexpression of this protein has indeed a repressing effect on passage through the S phase (Fig. 3). Thus, we postulate that DKFZp566F123 is a transcriptional repressor that regulates the expression of proteins that are essential for control of cell cycle progression, and its down-regulation would lead to enhanced cell growth. Also for other POK proteins (e.g., the tumor suppressor HIC1; ref. 40), it has been shown that their inactivation is associated with cell transformation (39).

We have developed a fully automated cell-based assay that is designed to identify modulators of DNA replication. It has a single-cell resolution with exceptionally high sensitivity and specificity. The assay was applied to narrow down a list of cancer associated genes from microarray studies, identifying novel candidate modulators of the cell cycle. Here we selectively discerned proteins based on their property to affect a specific activity of cells (DNA replication); however, this transfection screen is adaptable for analyzing diverse cellular pathways and processes.

The endogenous expression of genes in tumor and normal cells in many cases correlated with the outcome of the DNA replication assay. For example, the gene encoding cDNA DKFZp434I0515 was found down-regulated in kidney tumor profiling, whereas it is expressed in normal kidney. Gene expression was not detectable in Caki-1 and Caki-2 cells. The encoded protein had repressing effects on DNA replication. Such correlation should however not be regarded obligatory (41), because cellular effects of endogenous and overexpressed proteins may be tissue and cell type specific that are tuned by cellular regulatory networks.

Depending on the outcome of the initial DNA replication screen additional assays were carried out with protein subsets to further characterize the candidates. Whereas it is clear that the described high-throughput assays do not uncover all molecular and cellular functions of the respective proteins, they effectively bridge the gap between gene lists from tumor microarray studies and the capacity of single gene and protein analysis that is needed to comprehensively characterize a pathway with traditional biochemistry and cell biology methodologies.

This concept holds the potential for clustering genes based on their functional profiles (42) and epistatic analyses, to quantitatively elucidate complex genetic networks. Quantitative cell-based analysis offers an advantage by permitting the detection of gene functions that are associated with subtle, possibly buffered effects. These are basis for the complex interactions of multiple processes that are involved in the onset and progression of most diseases (43). Combined with the mapping of protein interaction networks (44) and other large-scale cancer relevant assays (45–49), this will lay the ground for the comprehensive exploitation of genomic resources and information and ultimately lead to an accelerated functional understanding of the cellular systems that control health and disease.

Grant support: National Genome Research Network grants 01GR0101 and 01GR0420 (German Cancer Research Center) and Bundesministerium für Bildung und Forschung grants 01KW9987 and 01KW0012 (German Cancer Research Center) and 01KW0013 (European Molecular Biology Laboratory) within the German Genome Project.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Claudio Schneider (LNCIB, Trieste, Italy) for kindly providing NIH 3T3 cells; Heike Wilhelm, Angelika Duda, Kerstin Hettler, and Saskia Stegmüller for excellent technical assistance; and Jan Mollenhauer and Patricia McCabe for discussions, suggestions, and critical reading of the article.