Cdc7 and Dbf4 proteins form a complex in vivo
] to generate an active protein kinase in all organisms tested (reviewed by Sclafani [2
]). Human Cdc7-Dbf4 kinase activity is essential for cell proliferation by activating DNA replication [10,32,33
] (and data not shown). Although Cdc7 abundance is constant through the cell cycle, its kinase activity is strictly dependent on Dbf4, which significantly increases in abundance before S-phase entry.
The NCI-60 cell lines represent the most common forms of cancer in the United States [48
] and are widely used by the research community. Unfortunately, neither CDC7
nor either of the DBF4
-related genes were represented in the published microarray data for the NCI-60 cell lines [49–51
]. In this study, we showed that there was a wide range of Cdc7 protein expression among the NCI-60 cell lines and five additional leukemia cell lines. In contrast, we consistently observed low or undetectable Cdc7 protein in normal tissues and cell lines. Our results support and significantly extend a previous examination of CDC7
mRNA expression in cell lines and different primary tumors versus
normal tissues [29
]. Hess et al. [29
] found a significant number (15/48) of the tumor samples expressing CDC7
mRNA at higher levels than those seen in the normal tissue. We determined that ~50% of tumor cell lines we tested expressed high levels of Cdc7 and Dbf4 protein, including many leukemia cell lines. These data suggest that Cdc7-Dbf4 protein has a relatively low abundance in normal cells and is perhaps absent in nonproliferating tissues but has increased expression in some cancer cell lines derived from diverse tissues.
We also defined a significant correlation between Cdc7 protein expression level and the TP53
gene status. On the basis of published mutational data in tumor suppressor or oncogenes in the NCI-60 cell line set [43
], we established that Cdc7 overexpression is frequently associated with the presence of mutation(s) in the TP53
gene. We also found that CDC7
expression levels were highly correlated with TP53
status in primary breast tumors, suggesting either that p53 directly represses the CDC7
] or its loss indirectly activates CDC7
expression. However, we saw no difference in CDC7
expression in isogenic HCT-116 cell lines that differ only by deletion of TP53
. Also, induction of p53 in HCT-116 cells after exposure to bleomycin did not alter CDC7
mRNA expression. Because we also found some p53 mutant cell lines that had barely detectable Cdc7-Dbf4 protein expression, together these data indicate that p53 inactivation alone does not determine whether the protein kinase is highly expressed. It may be that p53 loss and an additional genetic alteration that frequently occurs during tumorigenesis give rise to increased Cdc7-Dbf4 expression.
Because Cdc7-Dbf4 is a two-subunit kinase, it was of interest to investigate whether Cdc7 and Dbf4 protein expressions were correlated in primary tumors and cancer cell lines. We analyzed the Dbf4 expression in 62 cancer cell lines with varying Cdc7 expression levels and found a tight correlation between the expression levels of both proteins. Dbf4 protein is frequently coexpressed with Cdc7 and low levels of Dbf4 correspond with low or undetectable levels of Cdc7 protein. Therefore, in the cancer cell lines we analyzed, the expression of the two partners is highly correlated. Expression of the two genes was also highly correlated in 678 tumors of diverse origin (P ≤ 2.2 x 10-16).
A comparative genomic hybridization analysis of 38 breast cancer cell lines showed that the 7q21–22 region, containing the DBF4
gene, is an amplification site (11% of the cases) [52
]. Moreover, the regional gene activation of this region was also found as a potential mechanism for acquired drug resistance, with or without changes in gene dosage and in breast and ovary cancer cell lines [53,54
]. We naturally wondered if gene amplification could explain the higher Dbf4 protein expression seen in a subset of tumor cell lines. Seven of 14 ovarian, breast, and prostate tumor cell lines had an increased copy number of the DBF4
gene (by FISH analysis) resulting from unbalanced chromosome translocations or increased chromosome 7 copy numbers. With the exception of MCF7, these cell lines also had increased Dbf4 protein levels. In contrast, FISH analysis revealed no evidence for increased CDC7
copy number, indicating that the increased Cdc7 protein expression was independent of 1p22 amplification in the cell lines we examined. Other mechanisms, such as epigenetic modification and chromatin remodeling, may contribute to the regional gene activation in this region. Recently, a group identified that melanoma cell lines with defects in the DNA damage G1
checkpoint displayed an enhanced expression of proliferation-associated genes such as CDC7
The results obtained by immunohistochemistry on primary tumors paralleled our immunoblot analyses on cell lines. We showed that Cdc7 protein could be detected directly in primary tumors but not in the normal tissues. Of 35 tumors from breast, colon, and lung, 25 exhibited high-level Cdc7 expression. Of particular interest was the finding that 15 of 20 breast carcinomas showed intense Cdc7 staining suggesting that increased Cdc7 expression may be a common feature of breast cancer. Because six metastatic breast carcinomas showed intense Cdc7 staining, increased Cdc7 expression occurs in more aggressive tumors as well. We further showed that most breast cancers surveyed increased CDC7 expression over normal controls (162/181 ≥ 1.5-fold and 123/181 ≥ 2-fold increase; ).
We found that DBF4
expression was increased in almost all 678 tumors we surveyed, especially tumors of the colon, lung, and ovary. Recently, it was shown that Dbf4 protein was not only associated 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 [32
]. On the basis of our data, it is possible that increased DBF4
copy number or p53 loss might contribute to Dbf4 protein overexpression. The Cdc6 and Mcm5 replication initiation proteins are also overexpressed in cervical and bladder carcinomas [26,28
] suggesting that increased expression of particular replication initiation proteins may be a common feature of certain tumors. Taken together, these data suggest that increased Cdc7 and Dbf4 expression may be selected during tumorigenesis in some tissue types.
It will be of interest to determine the functional consequences of altered Cdc7 and Dbf4 protein expression levels across multiple cell lines. Previous studies have shown that knockdown of Cdc7 in the HeLa cervical cancer cell line (but not normal cells) results in an apoptotic response [33
]. We saw the same effect but note that in two prostate cancer cell lines, loss of Cdc7 did not result in significant apoptosis. Because Cdc7-Dbf4 is required for entry into S-phase, higher levels of Cdc7-Dbf4 kinase might aid the proliferative capacity of tumor cells. However, increased Cdc7 expression was not correlated with the proliferative status of the cells (P
= .271). Therefore, increased Cdc7 expression is apparently not required for a high proliferation rate. In fact, transient overexpression of the hamster Cdc7 and/or Dbf4 caused a cell cycle arrest [56
], and in human cells, overexpression of both human Cdc7 and Dbf4 does not cause significant effects on cell cycle progression [57
]. Because Cdc7-Dbf4 has been implicated in the response to stalled forks or various forms of DNA damage [7,17–20
], increased Cdc7-Dbf4 kinase may instead aid recovery or repair of stalled replication forks to enhance survival of some tumor cells. Together, these observations suggest that increased Cdc7-Dbf4 expression may be a common and/or important step during tumorigenesis.