Melanoma cell lines are susceptible to histone deacetylase inhibitor TSA provoked cell cycle arrest and apoptosis

Karita Peltonen, Taija M. Kiviharju, Pӓivi M. Jӓrvinen, Runar Ra and Marikki Laiho

Haartman Institute and Molecular Cancer Biology Program,

Biomedicum Helsinki, University of Helsinki, Helsinki, Finland

Summary:

Melanomas are highly aggressive skin cancers that are resistant to most available anti-cancer therapies. However, histone deacetylase (HDAC) inhibitors, including trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA), and depsipeptide FR901228, have demonstrated the ability to induce differentiation, growth arrest, and apoptosis in tumor cells. Due to their ability to affect transformed cell lines with little or no toxicity to normal cells, TSA, SAHA, and depsipeptide FR901228 have shown potential as therapeutic agents in in vivo models and in clinical trials. These HDAC inhibitors function by blocking chromatin deacetylation, thereby relaxing the chromatin and enabling gene transcription. HDAC enzymes also have non-histone substrates that help regulate both the stability and activity of several transcription factors, including the tumor suppressor p53. And while melanoma cells often retain the wild-type p53 protein, the p53 mediated apoptosis pathway is nonetheless typically suppressed.

Dr. Peltonen and colleagues at the Haartman Institute and Molecular Cancer Biology Program in Helsinki, Finland, have investigated the action of the HDAC inhibitor TSA and its mechanism of action in relation to p53 on a number of melanoma cell lines, including those with wild-type p53 (A375, G361, Malme 3M, and WM 239) and mutant p53 (RPMI 7951, SK-Mel 2, and SK-Mel 28). Previous papers have detailed both p53-dependent and p53-independent apoptotic responses to HDAC inhibitors, and Dr. Peltonen and colleagues investigated the response of melanoma cells to TSA. Their group has determined in prior studies that UV damage causes wild-type p53 to accumulate with some retained transactivitation capacity. They therefore hypothesized that the function of the p53 pathway is typically restrained in melanoma, but that wild-type p53 may be activated by appropriate stimuli. It is possible that one mechanism of p53 pathway inactivation is improper methylation and acetylation of the promoter areas of the p53 proapoptotic target genes. As p53 is regulated by histone acetyltransferases (HATs) and HDACs, the Peltonen group carried out experiments to determine whether TSA (a HDAC inhibitor) stabilizes and activates wild-type p53 in melanoma cell lines.

The Peltonen group used several techniques to determine whether apoptosis was induced by p53-dependent or p53-independent mechanisms. They obtained and cultured cell lines, ran immunoblots using SDS-PAGE, gathered RNA analysis using Northern blots, and determined cell growth and apoptosis using growth curve and flow cytometric analysis. The group first examined changes in both p53 levels and levels of its transcriptional targets HDM2 and p21 in response to TSA. They found that TSA decreased the levels of p53 in melanomas with wild-type p53. The decrease in p53 occurred quite late (after twelve hours) and was modest in melanoma cells with mutant p53. The decrease in p53 was also found to not be a result of an increase in HDM2, as levels of HDM2 were either unaltered or slightly decreased. It was also found that p21 cyclin dependent kinase inhibitor expression, which is a marker of p53 activity in cellular stress, was induced by TSA. However, because p21 induction by HDAC inhibitors is directly mediated by binding sites in the p21 promoter, it was determined that p21 induction is at least partially independent of p53. Further studies ruled out the possibility that the decrease in p53 was a result of p53 proteasomal degredation by other E-3 ligases.

The observation that p53 was stabilized while its protein levels were reduced suggested that TSA exerts transcriptional control over p53 mRNA (Figure 2). TSA stabilized wild-type p53, and p53 accumulation in the cells in turn led to downregulation of P53 mRNA and therefore an overall decrease in p53 protein. They also found that growth arrest occurred in all seven cell lines, and apoptosis occurred in six of the seven – however, these effects were independent of the p53 status of the cell. Regardless, HDAC inhibitors are still promising as anti-cancer agents, and more work must be done to determine their effectiveness.

Significance:

The results of these studies indicate that the HDAC inhibitor TSA could be used as a potential anti-cancer therapy, as it induces cell growth arrest and apoptosis in melanomas. Further clinical investigation is needed to determine any harmful effects of TSA on normal-functioning cells, and more research into the mechanism of apoptosis and cell growth arrest must be conducted.

Critique:

Dr. Peltonen and colleagues have crafted a very detailed paper with sound and logical experiments. They have numerous controls to account for a host of possible confounding variables, including proteasome-induced p53 degradation and p21/HDM2 expression as a result of TSA. However, the paper concludes with the belief that TSA and other HDAC inhibitors may prove to be viable anti-cancer treatments solely because of the apoptotic properties of the HDAC inhibitors. In fact, the paper states that much more work must be done to determine the exact mechanisms of apoptosis and cell growth arrest, and also notes the large amount of variability in the mechanism of p53 control between different cell lines. Perhaps the authors should have refrained from making such bold claims with the knowledge that more research must be conducted.