Patients with a tumor of
Patients with a tumor of 4 cm or greater were more likely to have lymph node or tumor metastasis (Fisher’s test p = 0.0001 and 0.0003, respectively). In this smaller patient group a significant correlation between higher DAPK-1 mRNA K-252c in tumor and normal tissue in tumor-free patients compared to that in patients who later had metastatic disease was detected (p = 0.04 and 0.02, respectively). When considering lymph node status in this subgroup, DAPK-1 mRNA expression in tumor tissue but not in normal tissue achieved statistical significance (p = 0.009 and 0.08, respectively, table 3).
Discussion We recently described new pro-apoptotic p53 target genes that are frequently methylated and to high levels in clear cell RCC. Methylation of a gene promoter region may lead to transcriptional silencing following inactivation of the gene. In RCC a correlation between methylation and subsequent silencing was described for RASSF1A in vitro. To our knowledge we report for the first time the relation between methylation and mRNA expression levels of the APAF-1 and DAPK-1 genes in vivo using highly sensitive and quantitative detection methods. In nonadvanced and in advanced RCC matched pair analysis showed a significant correlation between increased APAF-1 methylation and decreased mRNA expression levels, suggesting the regulation of APAF-1 mRNA expression in RCC by promoter methylation. In contrast, the methylation of APAF-1 or DAPK-1 in normal tissue as a mechanism of down-regulation of mRNA expression seems to be unlikely because methylation was rarely observed and to low levels. In the group of patients with positive lymph nodes APAF-1 as well as DAPK-1 expression was significantly lower compared to that in the group with negative lymph nodes. Some investigators consider a tumor size of greater than 4 to 5 cm as a prognostic factor. In the subgroup of these patients such a correlation could not be detected for APAF-1 expression but DAPK-1 expression remained significantly lower in tumor tissue from patients with positive lymph nodes or recurrent disease. What was also intriguing was the observation that in corresponding normal tissue taken distal of the tumor significantly lower levels of APAF-1 or DAPK-1 mRNA expression were detected compared to those in tumor tissue. Lowest expression levels of these tumor suppressor genes were detected in normal kidney tissue from patients who already had lymph node metastasis or who later had metastatic disease. This may lead to the hypothesis that at early tumor stage higher expression of APAF-1 and DAPK-1 helps prevent further tumor development. After expression levels decrease below a specific threshold the cell is unable to induce apoptosis and the metastatic process might become activated. However, this remains highly speculative. We admit that interpreting a small data set for prognostic purposes is of limited value and it should be reserved for larger studies. Moreover, measurement of the corresponding protein levels has not been performed to our knowledge. In leukemic blasts Fu et al noted that APAF-1 protein levels did not correlate with the mRNA expression of APAF-1 and they concluded that APAF-1 protein levels might also be controlled at the post-transcription level. Wethkamp et al observed unaltered protein expression levels of DAPK-1 even in the presence of DAPK-1 promoter methylation, also suggesting control at a posttranslational level. APAF-1 is a central component of the intrinsic pathway that catalyzes the auto-activation of pro-caspase-9 to caspase-9, leading to the activation of downstream caspase-3,6 and 7. In cells in which APAF-1 function is deactivated the execution of the intrinsic apoptotic pathway is defective. Promoter hypermethylation may not always lead to gene inactivation. Fu et al found relatively unaltered protein levels of APAF-1 in leukemic cell lines with APAF-1 methylation despite treatment with demethylating 5-Aza-CdR. In contrast, Furukawa et al reported low or undetectable mRNA expression of APAF-1 in the majority of the leukemic cell lines investigated. To our knowledge our study shows for the first time that APAF-1 is transcriptionally down-regulated in RCC in vivo. The fact that the lowest levels of APAF-1 mRNA were detected in tumors in cases with positive lymph nodes suggests a role of APAF-1 in the control of the intrinsic apoptotic pathway, not only in vitro, but also in vivo. Consequently the application of demethylating agents may reactivate APAF-1 function and, thus, restore pro-apoptotic function in APAF-1 methylated tumor cells.