Ropero and Esteller reply:

We are delighted to learn that Ree et al. have confirmed our results showing the presence of inactivating mutations in HDAC2 that lead to loss of HDAC2 protein in Co115 colorectal cancer cells1. We are also glad that these same authors have validated our finding of the presence of a truncating mutation in HDAC2 that results in loss of HDAC2 protein in the original RKO colorectal cancer cells used in our study1. The only minor difference we can see between our study and that of Ree et al. is that, using another batch of RKO cells, they observed only a reduction of HDAC2 protein, whereas we observed minimal expression. The minimal expression of the HDAC2 protein in RKO mutant cells has also been confirmed by others (J.G. Herman, Johns Hopkins Medical Institutions, personal communication). We analyzed 700 SNPs and found complete identity between the original RKO cells and a recent batch obtained from the American Type Culture Collection (data not shown). Ree et al. did not provide their cells for further tests.

Another interesting issue is the response to hydroxamic HDAC inhibitors, such as trichostatin A (TSA), according to the HDAC2 mutational status. We observed a reduced biochemical and cellular response to these drugs in cells that harbor the mutation in HDAC2 (ref. 1). A similar resistance to HDAC inhibitor–induced apoptosis has been found in another set of independent RKO HDAC2 mutant cells (J.G. Herman, personal communication). Ree et al. try to suggest that histone acetylation upon TSA treatment might be independent of HDAC2 mutational status. The problem is that the histone acetylation profiles induced by TSA administration provided by Ree et al. contradict the previously published data from these same authors based on the same colorectal cancer cell lines, drug and conditions2. This inconsistency precludes drawing any further valid conclusion from their data.

Overall, the body of data from these groups and others3 and our recent finding that HDAC2 impairment leads to aberrant gene expression4 support the presence of HDAC2-inactivating mutations in a subset of unstable microsatellite human tumors that renders these cells more resistant to the usual antiproliferative and proapoptotic effects of hydroxamic-based histone deacetylase inhibitors.