There is growing evidence for the existence of a pathway to gene inactivation in cancer which does not involve the loss or alteration of the primary sequence of DNA, but rather a change in the activation status of the gene through epigenetic means . Alterations in DNA methylation have been the type of epigenetic change most closely studied and it has been found that hypermethylation is associated with the silencing of key genes in many cancers [15, 16]. Inactivation of the H19 gene by methylation is implicated in a number of childhood cancers, including Wilms' tumour and embryonal rhabdomyosarcoma . Wilms' tumors (WT) often occur in association with Beckwith-Weidemann Syndrome (BWS) and it is the subset of BWS patients with methylation of the maternal allele of H19 which usually develop WT [17, 18]. In some cases of BWS and of Wilms' tumors the methylation and inactivation of the H19 gene is seen in preneoplastic kidney tissue, indicating that it is an early event in tumorigenesis [10–12]. The H19 gene is part of a cluster of imprinted genes on human chromosome 11p15 which includes the IGF2 gene and several others . In normal tissues, methylation on the paternal allele of H19 inactivates the promoter of the gene. In addition, there is a region upstream which acts as an insulator, preventing Igf2 from interacting with the enhancers downstream of H19 on the maternal allele. On the paternal allele, the insulator, like the H19 promoter, is also methylated and inactivated, allowing IGF2 to interact with the enhancers. The maternal LOH commonly seen in tumors such as WT and RD eliminates the active maternal H19 gene and usually duplicates the active paternal IGF2 gene. Methylation of the normally unmethylated maternal H19 allele in these tumors has a similar effect, as both copies of H19 are now silent and IGF active biallelically. Deletion of the H19 locus or of the insulator upstream  by homologous recombination in mice causes the silent maternal allele of Igf2 to become active. In contrast, removal of methylation from the maternal H19 gene by inactivation of the major methyltransferase activity in the cell causes activation of the silent paternal H19 allele and a concomitant decrease in Igf2 expression . Treatment of RD cells by AzaC has also been shown previously to have this effect .
Recent work has indicated that DNA methylation and histone deacetylation may act along the same pathway of inactivation. Three proteins which bind to methylated DNA, MeCP2, MBD1 and MBD2 have been shown repress transcription through a mechanism which involves histone deacetylases . Additionally, both the major DNA methyltransferase enzyme DNMT1  and the CTCF protein  which binds the upstream insulator region between H19 and IGF2 may cause histone deacetylation either directly or by interacting with HDACs. The inactive alleles of H19 and Igf2 have also recently been shown to have lower levels of histone acetylation than their active counterparts , as would be expected if methylation and deacetylation were coupled. If methylation must be followed by histone deacetylation to inactivate a gene, then it is possible that reacetylation of histones associated with a silenced gene may be sufficient to relieve repression and allow transcription. Alternatively, it may be necessary to use a combined treatment which both demethylates the gene and acetylates its associated histones to reactivate a silenced gene.
We found in the work described here that the HDAC inhibitors TSA and sodium butyrate did cause clear increases in H4 acetylation both globally and at the H19 locus in RD cells treated for 6 hrs with these drugs. These increases, while insufficient to reactivate the silent H19 gene, caused up-regulation of the already active TPA gene, which has previously been shown to be responsive to changes in histone acetylation levels . This is similar to the findings of Cameron et al, who showed that TSA alone could up-regulate the basally transcribed CDKN2B and CDKN1A (p21) genes, but not reactivate the silent MLH1 and TIMP3 genes in the colorectal carcinoma cell line RKO . Our results also agree with previous work in mice , where it was found that TSA did not reactivate the silenced H19 allele in the vast majority of cells in the mouse embryo. TSA failed to reactivate H19 in RD cells at any concentration from one low enough to show no effect on histone acetylation (25 nM) to a level high enough to have significant cytotoxic side-effects (1 mM) and for any length of time tested from 6 hr to 12 days. In contrast to these results, treatment of RD cells with as little as 500 nM AzaC for a period as short as 15 hrs was sufficient to reactivate H19 in some RD cells, indicating that methylation is a far more important determinant of H19 inactivation in RD cells. These results agree with earlier findings from treatment of RD cells  or cells from uniparental embryos  with AzaC and also with studies on mice deficient in Dnmt1 activity [8, 24]. H19 seems particularly sensitive to changes in methylation levels compared to other imprinted genes in the same cluster as shown by our previous work  and that of other laboratories [24, 25].
Cameron et al had also found that treatment of cells with a combination of HDAC and methylation inhibitors could reactivate silent genes which were unresponsive to the HDAC inhibitors alone . While Pedone and co-workers found that H19 could be reactivated in this way in cultured mouse fibroblasts when RT-PCR assays were used for detection , our own results from RD cells presented here and those of El Kharroubi et al from cultured mouse fibroblasts  show that treatment of cells with both types of inhibitor together in fact decreases the levels of H19 mRNA detected relative to those seen with AzaC treatment alone. We found this to be the case at all timepoints and concentrations of inhibitors tested. The evidence therefore suggests that under most circumstances the silent paternal H19 allele cannot be reactivated by treatment with HDAC inhibitors on their own or in combination with demethylating agents.
We also found that the silent Igf2 allele could not be significantly reactivated on treatment with HDAC inhibitors in mouse primary cells. Again, this was true of all drug concentrations and times used, while we could show clear effects on histone acetylation and on the control gene, Tpa in this system. The use of cells which were derived from embryos carrying an Igf2 deletion allowed for sensitive assaying for reactivation against a background of zero expression, similar to the situation for H19 in RD cells. The results agree with those from RD cells, where no increase in transcription from the active IGF2 allele is seen on treatment with acetylase inhibitors and are in agreement with some earlier reports, which have either found no effect [21, 27] or some inhibitory effect  of HDAC inhibitors on Igf2 expression. In order to be certain that a significant reactivation of the silenced Igf2 or H19 is occurring, we chose to examine expression using Northern blots against a zero expressing background rather than RT-PCR assays, which are prone to artifact nonlinearity. It can be noted that levels of reactivation detected by RT-PCR for p57Kip2 or Peg3 in some experiments using acetylase inhibitors are only a small fraction of the wild-type levels . We have previously successfully used Northern blots to detect two-fold differences in expression of H19 and Igf2 in Dnmt1 mutant mice  and in AzaC-treated RD cells  and here we could detect very low levels of H19 expression which are a result of 15 hr treatment with AzaC (Fig 1B). We also confined our studies to a very recently-derived cell line with no H19 expression or to primary mouse cells which have never been passaged and have a Igf2 null background, to increase sensitivity. Using these methods, we found no evidence that hyperacetylation caused significant reactivation of Igf2 or H19 in either RD cells or primary mouse cells.