Carcinogenesis Advance Access originally published online on September 3, 2007
Carcinogenesis 2007 28(12):2501-2510; doi:10.1093/carcin/bgm197
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Identification and functional characterization of ASK/Dbf4, a novel cell survival gene in cutaneous melanoma with prognostic relevance
Department of Dermatology, Heinrich-Heine-University, Moorenstrasse 5, Duesseldorf D-40225, Germany
1 Institute of Pathology, Ruhr-University, Bürkle-de-la-Camp-Platz 1, Bochum D-44789, Germany
2 Institute of Functional Genomics for Microorganisms, Heinrich-Heine-University, Moorenstrasse 5, Duesseldorf D-40225, Germany
* To whom correspondence should be addressed. Tel: +49 211 811 8066; Fax: +49 211 811 8830; Email: ulrich.hengge{at}uni-duesseldorf.de
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Abstract |
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Malignant melanoma is one of the most aggressive and invasive metastatic tumors derived from melanocytes that have undergone malignant transformation by acquisition of genetic and epigenetic alterations. Oligonucleotide microarray-based screening of distinct stages in the tumor progression model of cutaneous melanoma identified ASK/Dbf4, as a novel determinant for melanoma development. Quantitative real-time polymerase chain reaction-based confirmation of ASK/Dbf4 on a series of benign nevi, dysplastic nevi, primary cutaneous melanomas and cutaneous melanoma metastases; and a number of other controls using normal human melanocytes as calibrator not only revealed a melanoma-specific over-expression but also revealed that higher ASK/Dbf4-expressing melanomas were associated with lower relapse-free survival. Additionally, we also confirmed the observed over-expression of ASK/Dbf4 in melanoma using western blot analysis and immunohistochemistry. As ASK/Dbf4 is known to be a cyclin-like regulatory subunit of mammalian Cdc7 from the studies in yeast, the present study investigated its role in melanoma cells. In keeping with its expected role, our data suggest that up-regulated ASK/Dbf4 is localized in the nucleus and binds to human Cdc7 to form Cdc7–ASK/Dbf4 complexes in several analyzed melanoma cell lines. Further, we demonstrate that small interfering RNA-mediated depletion of ASK/Dbf4 retarded melanoma cell survival and proliferation. In summary, we report the differential regulation of a novel gene, namely ASK/Dbf4, in melanoma and suggest that up-regulation of ASK/Dbf4 is a novel molecular determinant with prognostic relevance that confers a proliferative advantage in cutaneous melanoma.
Abbreviations: BrdU, 5-bromo 2'-deoxy-uridine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NHM, normal human melanocyte; siRNA, small interfering RNA
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Introduction |
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Cutaneous malignant melanoma is the most aggressive among skin cancers. Melanoma arises from neural crest-derived normal human melanocytes (NHMs) and has a strong propensity to metastasize (1). However, important genetic changes responsible for melanoma development and progression remain poorly characterized, and the heterogeneity of the clinical course is still unexplained (2).
Few reports have analyzed global gene expression patterns in cutaneous melanomas. By cDNA microarray analysis, Bittner et al. (3) identified a gene expression cluster that correlated with invasive behavior in vitro. Tschentscher et al. described a correlation between gene expression profiles and monosomy of chromosome 3 in uveal melanomas, whereas Hoek et al. investigated expression profile differences between NHMs and melanoma cells from advanced lesions (4,5). Recently, Haqq et al. (6) defined melanoma progression profiles using a cDNA microarray approach. However, the understanding of molecular mechanisms underlying melanoma development still remains incomplete. We therefore performed oligonucleotide microarray-based screening to further investigate and identify additional molecular predictors of melanoma development in patients. Toward this end, we compared the gene expression profiles of a series of benign congenital nevi, primary cutaneous melanomas and cutaneous melanoma metastases using the human AffymetrixTM GeneChip and identified, confirmed and functionally characterized the activator of S-phase kinase (ASK/Dbf4) as a novel determinant in cutaneous melanoma development.
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Materials and methods |
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Patients and tissue samples
Primary cutaneous melanoma, cutaneous melanoma metastases and congenital nevus samples were obtained from 88 patients who underwent surgical tumor removal at the Department of Dermatology, University of Duesseldorf, Germany. Patients signed an informed consent. Histopathological classification of each sample was performed according to the Breslow index and Clark level. All tumor specimens and nevi were microscopically dissected from the surrounding tissue, and immediately frozen and stored at –80°C.
Normal human primary melanocytes and melanoma cell lines
NHMs were obtained from PromoCell (Heidelberg, Germany) and cultured in phorbol-12-myristate-13-acetate-free medium. Human melanoma cell lines (A375, SK-MEL-28, BLM, MV3 and M13) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
RNA extraction and melanin removal
Total RNA from fresh tumors, NHMs and melanoma cell lines were obtained using TRIzol© (Invitrogen, Carlsbad, CA). Removal of melanin was performed using RNAeasy kits (Qiagen, Valencia, CA). RNA specimens were analyzed by microcapillary electrophoresis on LabChips using the Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Boeblingen, Germany).
Gene expression profiling on oligonucleotide arrays
The human genome Affymetrix GeneChip system HG-U133A microarray (Affymetrix, Santa Clara, CA) containing a total of 22 283 probe sets representing 18 400 transcripts was used. Two micrograms of total RNA was used for synthesizing cRNA using MessageAmpTM kit (#1750) from Ambion (Austin, TX). Synthesized and fragmented cRNAs were routinely analyzed by microcapillary electrophoresis as described above.
Microarray data analysis
Image acquisition, comparative analysis to the baseline array and scaling/normalization were performed using Affymetrix GeneChip® Operating Software. To normalize for sample loading and staining variation, the average of the fluorescent intensities of all probe sets on an individual array was scaled to a constant target signal intensity (target signal factor 100) and corrected for 3'/5' bias differences. Groups were defined based on the comparison performed; for example, group 1 = benign nevi, group 2 = primary cutaneous melanoma, group 3 = cutaneous melanoma metastases and group 4 = melanoma cell lines. Delta was chosen to limit the output gene list so that <0.11% predicted false positives would be included. Statistical testing, clustering and projection techniques were performed using the TIGR MeV 3.1 freeware (7). Supervised classification was based on biological annotations from gene ontology. Input parameters for multiclass significance analysis of microarrays are as follows: imputation engine is row average imputer, data are in log scale, number of permutations is 100, Random number generated seed is 1234567 and delta is 0.22797. Input parameters for two class-unpaired significance analysis of microarrays are as follows: imputation engine is row average imputer, data are in log scale, number of permutations is 100, Random number generated seed is 63158815 and delta is 0.36811.
Quantitative real-time polymerase chain reaction
cDNA synthesis of total RNA from fresh tumors was performed with TaqMan® Reverse Transcription Reagents #N8080234 (Applied Biosystems, Foster City, CA) using random hexamers and the primers and probes for ASK (Hs00272696_m1) and 18S (Hs99999901_s1) (Applied Biosystems). All samples were amplified simultaneously in triplicates.
Antibodies
The anti-human ASK/Dbf4 polyclonal antibody was raised against the amino acids 336–349 (C-FDFVEYEKDTPKKK-amide) in rabbits (BioGenes, Berlin, Germany). The pre-immune sera from the same animals were used as negative controls for western blot analysis and immunohistochemistry. The anti-Cdc7 mouse monoclonal antibody (DCS-341) was obtained from Abcam plc (Cambridge, UK).
Western blot analysis
Western blot analysis was performed according to the standard procedures using a polyclonal antibody against ASK/Dbf4 (dilution 1:500; BioGenes) and visualization by horseradish-labeled anti-rabbit antibodies (Cell Signaling, Danvers, MA) and the ECL chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, NJ). Equal loading was confirmed by using a β-actin antibody (Oncogene, San Diego, CA).
Immunohistochemistry
A biotin–streptavidin-amplified technique with an alkaline phosphatase (fast red chromogen) kit (Dako LSAB® 2 System, Glostrup, Denmark) was used to detect ASK/Dbf4 following the Dako standard protocol on acetone-fixed cryosections (5 µm) of normal skin, benign nevi, primary melanoma and melanoma metastasis specimens. Negative controls were tissue sections immunostained with rabbit IgG (pre-immune serum).
Immunofluorescence
A375 human melanoma cells were used upon acetone fixation for staining with primary antibodies (1:500 anti-ASK, 1:500 anti-Cdc7 in phosphate-buffered saline–0.05% Tween 20) as described earlier (8).
Preparation of protein lysates and immunoprecipitation
Protein lysates were extracted from melanoma cell lines BLM, A375, MV3 and M13 cells using KALB lysis buffer [150 mM NaCl, 50 mM Tris (pH 7.5), 1% (vol/vol) Triton X-100 and 1 mM ethylenediaminetetraacetic acid, 1 mM Na3VO4 and 1 mM NaF] containing protease inhibitors (complete cocktail tablets; Roche, Basel, Switzerland) (8). Immunoprecipitation studies were performed with 400 µg of proteins and 1–2 µg of anti-human Cdc7 antibody (Abcam plc, Cambridge, UK) for 12 h at 4°C according to the published protocols. Cell extracts incubated with protein A/G sepharose in the absence of anti-Cdc7 antibody were used as negative control. HeLa cell extract was used as positive control for ASK/DBf4 and control IgG was used to ascertain that the observed band was from ASK/Dbf4 contained in the anti-Cdc7-precipitated immune complex.
Small interfering RNAs and transfection
Small interfering RNA (siRNA) specific to ASK/DBf4, negative control (scrambled) and silencer siRNA transfection kit (#1630) from Ambion were used on synchronized 0.5 x 105 MV3 cells. Transfection of 100 nM siRNA was conducted as per the manufacturer's recommendation. siRNA treatments were repeated every 24 h.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
After siRNA transfection, cells were incubated with 10 µl of 5 mg/ml stock of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St Louis, USA) for 2 h at 37°C. Upon solubilization, the number of surviving cells was measured at 540 nm.
5-Bromo 2'-deoxy-uridine incorporation assay
After siRNA transfection, 5-bromo 2'-deoxy-uridine (BrdU) assays were performed using the cell proliferation enzyme-linked immunosorbent assay BrdU (Colorimetric) (#11 647 229 001) according to the manufacturer's protocol (Roche Diagnostics, Mannheim, Germany).
Assessment of apoptosis by annexin-V–phycoerythrin staining
The annexin-V–phycoerythrin Apoptosis Detection Kit (PharMingen, San Diego, CA) was used according to the manufacturer's recommendations. Analysis was performed using a FACSCalibur and the Cell Quest software system (Becton Dickinson, San Diego, CA).
Kaplan–Meier analysis
The time to the occurrence of metastasis was plotted in relation to the strength of marker expression to estimate the percentage of relapse-free survival using the software MedCalc Version 9.2.01. The median expression of the individual genes as well as that of the four-gene summation in primary melanoma was used to categorize patients as having a high or a low expression. Differences in relapse-free survival were analyzed using the log-rank test. A P value of <0.5 was considered to indicate statistical significance.
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Screening for novel differentially expressed candidates in cutaneous melanoma development
As summarized in supplementary data 1 (available at Carcinogenesis Online), 37 well-characterized specimens of four different classes (11 congenital nevi, 10 primary cutaneous melanoma, 11 cutaneous melanoma metastases and 5 melanoma cell lines) were studied. Gene identification from Affymetrix HG-U133A array analysis was based on comparison with NHMs as calibrator. Multiclass significance analysis of microarrays was performed to identify genes that significantly differ among the four classes (9) (Table I). Unsupervised hierarchical clustering yielded a general separation of three patterns (patterns A, B and C) based on their expression profiles (Figure 1A and Table I). Patterns A and B represented genes whose expression distinguished benign nevi from malignant melanomas, whereas pattern C not only distinguished primary tumors from nevi but also primary tumors from cutaneous metastases (Figure 1A).
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The molecular changes associated with transition of benign nevi to primary cutaneous melanoma were distinct and discernible. The genes up-regulated in cutaneous melanoma development (pattern A) represented a variety of functions including genes associated with signal transduction pathways, e.g. mitogen-activated protein kinase 1 (MAPK1), signal transducer and activator of transcription 3 (STAT3), ELK1 (member of EIS oncogene family), cell-cycle regulation; e.g. activator of S-phase kinase (ASK/Dbf4), cyclin A1 (CCNA1), cyclin B1 (CCNB1), cyclin E2 (CCNE2), cell motility and extracellular matrix and e.g. vimentin (VIM), chondroitin sulfate proteoglycan 4 (CSPG4) and other functions such as translocated promoter region/tumor potentiating region (Tpr), dihydrofolate reductase (DHFR), etc. Analysis of the collated data showed that genes reported over-expressed in earlier non-array-based studies like melanoma antigen family A12 (MAGE A12), melanoma antigen family A5 (MAGE A5), melanoma adhesion molecule (MCAM/MUC18) and MET proto-oncogene/hepatocyte growth factor receptor (c-MET) were also highly expressed in most of our primary melanoma and metastasis patients, but were significantly lower in most nevi (Figure 1A and Table I). Likewise, some of the up-regulated genes such as CXCL1 (melanoma growth-stimulating activity), minichromosome maintenance-deficient 4 (MCM4), cell division cycle 2 (Cdc2), reticulocalbin 2 (RCN2), dihydrofolate reductase (DHFR) and cathepsin B (CTSB) were found to distinguish melanoma from benign nevi in our study (Table II) and in another recent cDNA array-based study (6). In contrast, osteopontin (OPN) (10) and keratin expression (11) previously reported in melanoma were not significantly up-regulated in our study.
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Genes down-regulated during cutaneous melanoma development (pattern B) included spondin 1 (SPON1), interleukin-18 (IL-18), etc., whereas pattern C represented genes that distinguished nevi from primary cutaneous melanoma as well as primary cutaneous melanoma from cutaneous melanoma metastasis, including nuclear matrix protein (NXP2) and lamin A/C (LMNA), for example (Figure 1A and Table I).
The ASK/Dbf4 gene was further selected for confirmation and characterization based on having the highest significance (see significance score in Table I).
Quantitative real-time reverse transcription–polymerase chain reaction confirmation of ASK/Dbf4
We used quantitative real-time polymerase chain reaction using TaqMan® Gene Expression Assay (Applied Biosystems) to confirm the expression profile of ASK/Dbf4 in a larger sample group. This sample group included an additional set of benign nevi (n = 9), dysplastic nevi (n = 5), primary cutaneous melanoma (n = 18), cutaneous melanoma metastasis (n = 15), melanoma cell lines (n = 5), non-melanoma type skin cancers (n = 3) and primary keratinocytes with NHMs as calibrator (Table II). Some identical RNA samples from the earlier microarray study (e.g. P61, P84, P85, P171 and P172) were included in this sample set as internal controls.
ASK/Dbf4 showed a median over-expression of 17.9 and 13.2-fold relative to NHM in primary cutaneous melanoma and cutaneous melanoma metastasis, respectively, whereas the majority of benign and dysplastic nevi expressed similar or lower levels than in NHM (Table II). To ensure that the observed expression of ASK/Dbf4 was indeed from melanoma cells, we needed to confirm that the basal transcript levels in normal human keratinocytes and peripheral blood mononuclear cells were not significantly higher than in NHMs (Table II). ASK/Dbf4 was associated not only with a higher relative change in the transition from nevi to cutaneous melanoma but was also up-regulated in a high number of primary melanoma and melanoma metastasis patients (66.7 and 86.7%, respectively). The expression of ASK/Dbf4 in non-melanoma skin cancer samples was negligible in comparison with the over-expression observed in melanoma demonstrating that ASK/Dbf4 over-expression was specific (Table II).
Expression of ASK/Dbf4 and relapse-free survival
To analyze the predictive value of the identified ASK/Dbf4 for the occurrence of first metastasis, we extracted the patients' disease history from their medical records following excision of primary melanoma. The levels of expression of ASK/Dbf4 was used to partition primary melanoma patients described in Table II on the basis of the strength of ASK/Dbf4 expression; the time to occurrence of metastatic disease was computed against the time of relapse-free survival in Kaplan–Meier plots (Figure 1B). The median follow-up time for the relapse-free survival analysis in our study was 130.4 weeks (minimum 45.7 and maximum 186.8 weeks). High ASK/Dbf4 expression indicated early occurrence of metastasis (Figure 1B). The percentage of relapse-free survival for high ASK/Dbf4-expressing group at 186 weeks was 51.4 as opposed to 82% in the low ASK/Dbf4-expressing patients (Figure 1B). On a different note, we were not able to show a correlation of Breslow index with ASK/Dbf4 RNA expression in this patient cohort.
Western blot and immunohistochemistry analysis of ASK/Dbf4 expression in cutaneous melanoma development
ASK/Dbf4 expression was investigated in a series of normal skin, NHM and primary cutaneous melanoma by western blot analysis of frozen specimens using an ASK/Dbf4-specific polyclonal antibody (Figure 2A–D). In normal skin, ASK/Dbf4 immunostaining was very weak, whereas it was strong in several melanoma cell lines and most melanoma specimens analyzed (Figure 2C and D). Immunohistochemistry analysis of a series of normal skin, benign nevi, primary melanoma, melanoma metastasis specimens and melanoma cell lines revealed that in normal skin and benign nevi, ASK/Dbf4 immunostaining was weak, whereas it was strong in primary melanoma, melanoma metastasis and melanoma cell lines (Figure 2E and F; supplementary data 2 is available at Carcinogenesis Online). In addition, the specificity of the antibody for the protein during western blot analysis and immunohistochemistry analysis was demonstrated by using pre-immune serum as control (Figure 2E and F).
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Association of ASK/Dbf4 and human Cdc7 in human melanoma
Cdc7 is a serine/threonine kinase that is required for the initiation of DNA replication and is conserved from yeasts to human (12,13). Although the level of Cdc7 is constant throughout the cell cycle, Cdc7 kinase activity peaks at late G1 through S phase (14,15). This cyclic control, which is observed in yeasts and humans reflects changes in the abundance of its regulatory subunit ASK/Dbf4 (16–18).
To further characterize the expression of ASK, we examined its subcellular distribution in human melanoma cells. Immunofluorescence with an ASK-specific antibody in A375 melanoma cells detected the endogenous ASK protein predominantly in the nuclei as bright speckles (Figure 3). Similarly, huCdc7 was detected in both cytoplasm and nuclei under the same conditions (Figure 3). Co-immunofluorescence with ASK and anti-Cdc7 antibodies in A375 cells suggested that huCdc7 and ASK proteins are co-localized in the nuclei of melanoma cells (Figure 3), although it is not known whether they are present at the same subnuclear localization. We confirmed the expression of Cdc7 in the melanoma cell lines BLM, A375, MV3 and M-13 (Figure 4A) and also performed immunoprecipitation studies with anti-Cdc7 antibodies, revealing the presence of ASK/Dbf4 protein in human Cdc7 immunocomplexes in three (A375, MV3 and M-13) of the four investigated melanoma cell lines (Figure 4B). These results clearly demonstrate that human Cdc7 and ASK/Dbf4 form a functional complex in cutaneous melanoma.
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ASK/Dbf4 confers a proliferative advantage in cutaneous melanoma
To elucidate the role of ASK/Dbf4 in human melanoma, we used a siRNA-mediated strategy to knock down endogenous ASK/Dbf4 in MV3 melanoma cells. At 48 h, ASK/Dbf4 protein levels decreased partially by 55.2% in siRNA–ASK as compared with siRNA-scrambled treated cells, whereas at 72 h there was a nearly complete loss (decrease of 83.7%) of ASK/Dbf4 protein in siRNA–ASK-treated cells (supplementary data 3 is available at Carcinogenesis Online). To elucidate a cause-and-effect association between loss of endogenous ASK/Dbf4 and melanoma cell growth, we investigated the kinetics of (i) cell survival, as measured by conversion of MTT to formazan (MTT assay) and (ii) cell proliferation per cells in S phase, as measured by BrdU incorporation at various times after siRNA treatment. At 72 h, a significant reduction in both cell survival and cell proliferation (Figure 5A–D) without any significant change in apoptosis (measured by annexin-V staining) in comparison with the respective siRNA-scrambled control was detected (Figure 5E). Therefore, endogenous ASK/Dbf4 confers a proliferative advantage to melanoma cells. Collectively, these results strongly indicate that ASK/Dbf4 is essential for cell proliferation and DNA synthesis in melanoma cells.
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Discussion |
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Melanoma is a complex multigenic disease, susceptibility to which is determined by several parallel and stepwise progressive pathways affecting growth control, differentiation, cell adhesion and survival. Inspite of advances in melanoma research, the understanding of molecular mechanisms underlying melanoma development still remains incomplete. In this study, we report ASK/Dbf4, a novel gene in cutaneous melanoma development, and focused on the functional characterization of its role in melanoma.
ASK/Dbf4 has been reported to be an essential molecule in eukaryotic chromosomal replication as inferred from functional studies in lower organisms and general mammalian models (14,15). DNA replication during the G1–S transition requires the activation of two S-phase-promoting kinases: cyclin-dependent kinases, Cdks, and the ASK/Dbf4-dependent kinase, Cdc7. The activation of Cdc7 kinase is in turn specifically achieved by its own regulatory subunit, the ASK/Dbf4 protein (12). However, such functional roles of cellular molecules implied from generalized models might not always represent its functional role within the perspective of tumor development and progression. For instance, the oncogenic activity of cyclin E does not exclusively rely on its ability as a positive regulator of G1 progression rather, cyclin E harbors other functions, independent of Cdk2 activation and p27Kip1 binding that contribute significantly to its oncogenic activity (19). Recent data obtained in cyclin E1 and cyclin E2 knockout mice (20) indicate that cyclin E is dispensable for proliferation of normal cells and that low-molecular weight cyclin E forms in cutaneous melanoma and functions to generate angiogenic tumors with prominent perineural invasion (21). The biological significance of these findings together with the absence of the previous reports of up-regulation of ASK/Dbf4 in melanoma prompted us to investigate whether the pro-proliferative role of ASK/Dbf4 is preserved within the context of cutaneous melanoma development. First, we confirmed the melanoma-specific over-expression of ASK/Dbf4 transcript and demonstrated that higher ASK/Dbf4-expressing melanomas were associated with lower relapse-free survival. Additionally, we also confirmed the over-expression of ASK/Dbf4 in melanoma using western blot analysis and immunohistochemistry. Through immunofluorescence-based experiments, we demonstrated that ASK/Dbf4 and Cdc7 were co-localized in the nuclei of melanoma cells. Further, the presence of ASK/Dbf4 protein in human Cdc7 immunocomplexes in melanoma cells was elucidated through immunoprecipitation studies, suggesting that the observed over-expression of ASK/Dbf4 in cutaneous melanoma was indeed for associating with human Cdc7. Subsequently, the cause–effect relationship of ASK/Dbf4 in cutaneous melanoma was demonstrated using the siRNA-mediated specific depletion of ASK in melanoma cell line MV3. The depletion of ASK/Dbf4 protein in MV3 cells at 72 h after siRNA treatment was found to be associated with a significant retardation in cell survival and proliferation without any significant difference in apoptosis. These results demonstrated that the essential role of ASK/Dbf4 in cell proliferation and DNA synthesis was conserved in cutaneous melanoma cells. Interestingly, it was also observed that a near to complete loss of ASK/Dbf4 protein at 72 h after siRNA–ASK treatment was associated with only 40.7 and 65% decrease in cell survival and proliferation, respectively, as compared with scrambled siRNA. This inability to produce a complete loss of cell survival and proliferation may be explained by the redundancy of pro-proliferative pathways in melanoma (8). Alternatively, it is also possible that in the absence of ASK/Dbf4, its homolog Drf1/ASKL1 may have a role in driving cell proliferation (22). Interestingly, both benign nevi and dysplastic nevi showed lower levels of ASK/Dbf4 expression. This observation coupled with the fact that loss of ASK/Dbf4 expression in malignant melanoma cells resulted in a proliferative disadvantage, indicated that ASK/Dbf4 over-expression may not be an initial event toward acquiring a malignant potential, but rather more importantly, in coping with the high-proliferative pressure of malignant melanoma cells.
In addition to its role in activating Cdc7 in mammalian cells, Tenca et al. (23) recently demonstrated that genotoxic agents do not down-regulate Cdc7·ASK/Dbf4 complexes, suggesting that they are correctly positioned to regulate proteins required for genome replication. Therefore, a combination of specific Cdc7 or ASK/Dbf4 inhibitors along with drugs that impair the elongation reaction of DNA synthesis may hold potential to treat melanoma.
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Supplementary material |
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Supplementary data can be found at http://carcin.oxfordjournals.org
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Funding |
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Deutsche Forschungsgemeinschaft (DFG 1383/9-1) and the Joachim Kuhlmann-Stiftung, Essen, Germany.
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Acknowledgments |
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We thank the Affymetrix-Core-Facility, Institute of Oncological Chemistry, Heinrich-Heine-University, for performing the chip hybridizations.
Conflict of Interest Statement: None declared.
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References |
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