Differential expression of cancer-related genes by single and permanent exposure to bone morphogenetic protein 2

Abstract

Purpose: Bone morphogenetic proteins (BMPs) are multifunctional regulators of various cell functions. The BMP-signalling network plays a pivotal role during embryogenesis and tumorigenesis. BMPs, e.g. BMP-2 exert their biological function in a time and concentration-dependent manner but also modulated by the context of the cellular microenvironment. In this study, we investigated the effect of a steady high level of BMP-2 versus a single application of BMP-2 on the breast cancer cell line MCF-7.

Methods: The effect of the incubation regimes was analysed by DNA microarray expression profiling. Data were verified by real-time PCR. The protein expression of apoptosis-related genes was studied by western blot analysis.

Results: We found a clear difference in the altered gene expression between the constant high level and the single application of BMP-2. After grouping the genes of interest into the biological processes of Gene Ontology, the group of apoptosis-related genes like BAX, BAG5 or PKR, was predominantly affected under the single-application regime of BMP-2. Among these protein kinase R was the most prominently regulated. Further studies on the protein level showed activation of PKR after 4 h with a subsequent enhanced phosphorylation of the PKR substrate eIF2alpha for several hours.

Conclusions: The duration of treatment and the concentration of BMP-2 affect the global expression pattern of MCF-7 cells. Among the regulated cancer-related genes, the cohort of the apoptosis-related genes showed the pronounced alterations. Our data point to a novel role of BMP-2 in the regulation of the PKR pathway in tumorigenesis.

Fig. 1

Distribution of genes showing altered expression after single or chronic application of BMP-2. Genes were annotated to biological processes according to Gene Ontology (The Gene Ontology Consortium 2000). All processes sum up at least 7% of the affected genes of one of the two subsets. Bars represent the number of genes (in %) of subset “overexpression” (black bars), subset “incubation” (grey bars) and the portion of the entire process related to the GO term “Biological process” (white bars). *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 2

Validation of microarray data. Genes showing a significantly altered expression after single application of BMP-2 (MCF-7/BMP-2inc.4h vs. MCF-7/sf) or under chronic application of BMP-2 (MCF-7/BMP-2 vs. MCF-7/3.1) in the microarray analysis were further studied by real-time PCR. All but one analysed representative gene showed a similar expression pattern in the microarray setting as well as in real-time PCR. Black bars down-regulation of the gene and grey bars up-regulation of the gene

Fig. 3

Validation of BMP-2-dependent alterations of the expression of apoptosis-related genes obtained by microarray analysis. Real-time PCR confirmed the observed alterations in all but one case. Black bars real-time PCR after 4 h treatment of MCF-7 cells with BMP-2 (log 2 ratio MCF-7/BMP-2inc.4h vs. MCF-7/sf); grey bars microarray analysis after 4 h treatment of MCF-7 cells with BMP-2 (log 2 ratio MCF-7/BMP-2inc.4h vs. MCF-7/sf)

Fig. 4

BMP-2 altered the phosphorylation status of PKR and its substrate eIF2alpha. MCF-7 cells were treated with 100 ng/ml BMP-2. At the time-points indicated, cells were harvested and analysed by western blot. BMP-2 induced a rapid activation of PKR after 4 h. The phosphorylation of eIF2alpha was increased after 16 h and remained elevated for at least 8 h