ORIGINAL PAPERS Histonedeacetylaseinhibitorsspecificallykillnonproliferatingtumourcells Andrew Burgess 1 , Astrid Ruefli 2 , Heather Beamish 1 , Robyn Warrener 1 , Nicholas Saunders 1 , Ricky Johnstone 2 and Brian Gabrielli* ,1 1 Cancer Biology Program, Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland 4102, Australia; 2 Peter MacCallum Cancer Institute, Melbourne Victoria, Australia Conventional chemotherapeutic drugs target proliferating cells,relyingonoftensmalldifferencesindrugsensitivityof tumour cells compared to normal tissue to deliver a therapeutic benefit. Consequently, they have significant limiting toxicities and greatly reduced efficacy against nonproliferating compared to rapidly proliferating tumour cells. This lack of selectivity and inability to kill nonproliferating cells that exist in tumours with a low mitoticindexaremajorfailingsofthesedrugs.Arelatively new class of anticancer drugs, the histone deacetylase inhibitors (HDI), are selectively cytotoxic, killing tumour and immortalized cells but normal tissue appears resistant. Treatment of tumour cells with these drugs causes both G1phase cell cycle arrest correlated with increase p21 expression, and cell death, but even the G1 arrested cells died although the onset of death was delayed. We have extended these observations using cells that were stably arrested by either serum starvation or expression of the cyclin-dependent kinase inhibitor p16 ink4a . We report that histone deacetylase inhibitors have similar cytotoxicity towards both proliferating and arrested tumour and immortalized cells, although the onset of apoptosis is delayedby24hinthearrestedcells.Bothproliferatingand arrested normal cells are unaffected by HDI treatment. Thus, the histone deacetylase inhibitors are a class of anticancer drugs that have the desirable features of being tumour-selective cytotoxic drugs that are equally effective in killing proliferating and nonproliferating tumour cells and immortalized cells. These drugs have enormous potentialforthetreatmentofnotonlyrapidlyproliferating tumours, but tumours with a low mitotic index. Oncogene (2004) 23, 6693–6701.doi:10.1038/sj.onc.1207893 Published online 5 July 2004 Keywords: histone deacetylase inhibitors; quiescent; apoptosis; chemotherapy; cell cycle Introduction Many of the currently used anticancer drugs are effective in killing proliferating cancer cells, but are less effective against nonproliferating cells that exist in the core of a solid tumour (Drewinko et al., 1981). This presents a problem with residual disease, the quiescent cancer cells surviving the course of chemotherapy then presenting subsequently as relapse of the disease. The identification of new drugs or treatment modalities that circumvent this limitation would be an invaluable addition to the current arsenal available for the treatment of cancer. Histonedeacetylaseinhibitors(HDIs)areanewclass of drugs with anticancer potential. A number of these are now undergoing stage I/II clinical trial (Gabrielli et al., 2002; Marks et al., 2001). These drugs have both cytostatic and cytotoxic activities, depending on the dose of drug used. At low doses, these drugs have cytostaticactivitycharacterizedbyaG1phasecellcycle arrestthatisassociatedwiththeincreasedexpressionof the cyclin-dependent kinase (cdk) inhibitor p21 WAF1/CIP1 . Thecytostaticactivityofthesedrugsisnotrestrictedto tumour cells, since normal cells also arrest in G1phase (Qiu et al., 2000). At higher doses, these drugs are selectivelycytotoxic,killingawiderangeoftumourand transformed cell lines but not normal cell lines (Qiu et al., 2000; Burgess et al., 2001). We have shown that the selective cytotoxicity is a consequence of the functional status of an HDI-sensitive G2phase cell cycle checkpoint. An intact checkpoint protects drug- resistant cells, whereas drug-sensitive cells have a checkpoint defect (Qiu et al., 2000). Activation of this checkpointisonlyobservedwhendrug-resistantcellsare treatedwithhighdosesofdrugthatwouldbecytotoxic for drug-sensitive cells (Burgess et al., 2001; Richon et al.,2000).Treatmentofthemajorityofdrug-sensitive cell lines with high doses of HDI kills a proportion of cells (20–50%) within 24h, but also causes a G1phase arrestinthesurvivingcells.Thesearrestedcellsstilldie, although the onset of cell death is slower than in cells thatdonotarrestinG1(Burgess et al.,2001;Qiu et al., 2000), suggesting that HDIs may be able to kill nonproliferating as well as proliferating cells. Other groupshavereportedthattheleukaemiccelllineCCRF- CEM engineered to inducibly express the cdk inhibitor p16 ink4a is refractory to the effects of HDI treatment when arrested in G1phase by p16 ink4a induction (Bern- hard et al.,1999;Peart et al.,2003).Thisconflictingdata promptedustoexaminemorecloselythecytotoxicityof high-dose HDI treatment with nonproliferating cells to Received 18 February 2004; revised 13 April 2004; accepted 11 May 2004; published online 5 July 2004 *Correspondence: B Gabrielli; Email: bgabrielli@cicr.uq.edu.au Oncogene (2004) 23, 6693–6701 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc