typical speckled localization pattern into fewer and larger clusters of splicing compartments (Huang et al., 1994). Microtubules were selectively destructed by inhibitors of phosphatases (Merrick et al., 1997) and tubulin disruption was recently shown as a mechanism by which inhibitors of HDACs reduce the secretion of interleukin-1 (Carta et al., 2006).
An overall impact of the drug as would have been expected in the case of cytotoxicity would have certainly led to changes in the morphology of the investigated cellular structures. However immunofluorescence with antibodies against nucleoli, speckles and microtubules did not reveal variations in the appearance of the stained compartments after chaetocin treatment on the light-microscopic level.
Despite lacking morphological changes and positive tests for S-phase in drug treated cells, (hetero)chromatin condensation as observed after Chaetocin treatment in HFbs is not inconsistent with cytotoxicity.
Cells commiting apoptosis also display chromatin aggregated into dense compact masses (Fadeel and Orrenius, 2005; Lawen, 2003). However it was shown that de novo chromatin
condensation normally seen during mitosis does not occur when cells undergo apoptosis. Instead, the noticed condensed chromatin results from aggregation of the typical constitutive heterochromatin (Hendzel et al., 1998).
Mitotic cell death (MCD) also known as “mitotic catastrophy” is another form of cell death to eliminate damaged cells and is mainly described in morphological terms (Blank et al., 2006). To date, evidence of premature mitosis in damaged cells relies primarily on the appearance of uneven chromatin condensation (UCC), which is the formation of hypercondensed chromatin aggregates (Roninson et al., 2001). Since MCD was identified as a prominent response of cells to different anticancer drugs (Lock and Stribinskiene, 1996; Tounekti et al., 1993) (Torres and Horwitz, 1998), this process might be an explanation for the observed effects after Chaetocin treatment.
Surprisingly, an overall decrease in H3K9me3 staining intensity, at least by visual inspection, was not observed after drug application, suggesting that SUV39H1 activity and therefore H3K9me3 levels are not altered in Chaetocin-treated cells. This is at least partly in consistency with in vivo inhibition of SU(VAR)3-9 in Drosophila SL-2 cells which show mainly
a reduction of H3K9me2 rather than H3K9me3 in mass spectrometry (MS) (Greiner et al.,
2005). However in another experiment the authors could also observe a substantial drop in H3K9me3.
In HFbs Chaetocin treatment led to a rearrangement of chromatin that could be highlighted by changes in the patterns after H3K9me3 staining and DAPI-counterstaining. The relocated
chromatin is characterized by very large clusters, of what is mostly but not exclusively (peri)- centromeric heterochromatin. However, keeping in mind the amount of chromatin that seems
to be rearranged, it is very likely that heterochromatic as well as euchromatic regions are affected by drug treatment.
Taking into account the fact that Chaetocin treatment induces changes in chromatin formation in general it is reasonable to suppose that the appearance of H3K9me3 clusters is a consequence of this overall chromatin alteration rather than a direct effect of SUV39H1 inhibition. However information about SUV39H1 enzyme activity remains to be obtained by enzyme activity assays.
Western blot analysis of SUV39H, H3K9me3 and H3 (as a control) could provide a much better resolution compared to simple visual inspection and is essential for any final conclusions on this topic.
The mechanisms leading to the rearrangement of heterochromatin that are described here remain unclear. Taken all observations together it seems rather unlikely that the observed chromatin rearrangements in HFbs were simply based on cytotoxicity.
Another important question to be answered is why a similar impact of Chaetocin was only observed in HFbs but not in the cancer cell-lines DLD-1 and MCF-7.
An explanation might be the development of several mechanisms for drug resistance in cancer cells. Each cancer cell has specific genetic and epigenetic alterations (Ballestar and Esteller, 2005). Hence tumor cells express different arrays of drug-resistance genes conferring simultaneous resistance to many different drugs, a phenomenon called multidrug resistence (Gottesman et al., 1994). Much data is provided about mechanisms that alter accumulation of drugs within cells (Ambudkar et al., 1999; Borst et al., 2000). There are two ways for drug resistance of cancer cells that should be also taken into account for the discussed observations. A first mechanism can increase drug efflux from cancer cells and a second mechanism leads to reduced uptake of drugs. The latter argument does not fit the finding that all cells from all cell-lines died at 0,1µM Chaetocin.
Most tumor cells show ABC-transporter (ATP-binding cassette)-mediated multidrug resistance (Higgins, 1992). This type of resistance was already discovered in 1976 (Juliano and Ling, 1976). The major mechanism of multidrug resistance in cultured cancer cells was the expression of an energy-dependent drug efflux pump, known alternatively as P- glycoprotein (Pgp) (Ueda et al., 1987). It is the product of the MDR1 gene in humans and highly expressed in cancer cells (Chen et al., 1986). Although big efforts have been made recently for overcoming this feature of tumor cells (Borowski et al., 2005) it is still a major cause of failure in anti-tumor therapy. The resistence is due to the expression of ABC transporter glycoproteins which participate actively in effluxing the drug out of the cell thus preventing the accumulation of substances. These general findings in tumor cells would