Cdc7-Dbf4 kinase overexpression in multiple cancers and tumor cell lines is correlated with p53 inactivation

Abstract

Cdc7 is a conserved serine/threonine kinase essential for the initiation of DNA replication, likely by activating the MCM DNA helicase at the G(1-) to S-phase transition. Cdc7 kinase activity requires association with its regulatory subunit Dbf4/activator of S-phase kinase. Cdc7-Dbf4 is also downstream of the conserved Ataxia telangectasia and RAD3-related kinase that responds to stalled replication forks or DNA damage. In this study, we found that Cdc7 protein was very low or undetectable in normal tissues and cell lines but had increased expression in approximately 50% of the 62 human tumor cell lines we examined. Most cell lines with increased Cdc7 protein levels also had increased Dbf4 abundance, and some tumor cell lines had extra copies of the DBF4 gene. A high expression of Cdc7 protein was also detected in primary breast, colon, and lung tumors but not in the matched normal tissues. We also found a high correlation between p53 loss and increased CDC7 and DBF4 expression in primary breast cancers (P = 3.6 x 10(-9) and 1.8 x 10(-10), respectively) and in the cancer cell lines we studied. Therefore, increased Cdc7-Dbf4 abundance may be a common occurrence in human malignancies.

Figure 1

Abundance of Cdc7 and Dbf4 proteins in normal and tumor cell lines. Twenty micrograms of protein sample from whole-cell extracts was separated on 10% SDS-PAGE gels for normal tissues of different origins (A), and immortalized IMR-90 and WI-38 fibroblast cell lines (B). Panels (C–K) represent tumor cell lines of the central nervous system, colon, non-small cell lung, leukemia, melanoma, kidney, ovary, prostate, and breast. After transfer, membranes were probed with a Cdc7- or Dbf4-specific antibody. Equal loading was confirmed by probing with a β-actin-specific antibody. Asterisk (*) indicates the cell lines that were shown to be derived from the same tumor [41,43].

Figure 2

Increased DBF4 copy number in cancer cell lines. Fluorescence in situ hybridization experiments were done with a centromeric probe specific to the chromosome 7 (CEP7; SpectrumGreen-labeled) versus the DBF4-specific probe (SpectrumOrange-labeled) on interphase nuclei and on metaphase spreads. (A) Control FISH experiment was done on phytohemagglutinin-stimulated lymphocytes derived from a karyotypically normal male donor. Pictures are also shown for two ovary cancer cell lines OVCAR-8 and OVCAR-4 (B and C), respectively.

Figure 3

Immunostaining of Cdc7 in primary breast and colon tumors. (A) Cdc7 protein staining of normal breast tissue (left) and the corresponding breast carcinoma (right) shown at low and high (below) magnifications. (B) Normal colon tissue (left) and tumor (right) stained for Cdc7 protein at low and high (below) magnifications. (C) Fluorescence in situ hybridization experiments were done as described in Figure 2. A representative picture of nuclei from a breast primary tumor showing increased DBF4 gene copy number is shown.

Figure 4

Overexpression of CDC7 and DBF4 in human cancers. (A) The gene expression level of CDC7 and DBF4 in tumor samples compared with corresponding normal tissue (see the Materials and Methods section). Plotted is the fold increase or fold decrease in gene expression in each tumor sample (n = 678). Red bars highlight the tumors that have increased gene expression. (B) CDC7 and DBF4 expression data described in (A) as a scatter plot with correlation coefficient, significance value (boxed), and best-fit line generated using linear regression.

Figure 5

siRNA knockdown of CDC7 in tumor cell lines causes growth arrest and apoptosis. (A) Cdc7 immunoblots at 48 and 72 hours after transfection for mock and Cdc7 siRNA-transfected HeLa, DU-145, and PC-3 cells. Actin loading control is shown below. (B) Graphs showing cell number for control-transfected (◆) and Cdc7 knockdown cells (▪) over time. Data represent the mean of at least two experiments ± SD. (C) The percentage of TUNEL-positive cells are shown for each cell line for control transfected (light gray bars) and Cdc7 knockdown cells (dark gray bars) +/- SD. (D) Immunoblots of PARP full length (FL) and cleavage product (CL) are shown for the transfection time course.