ProteasomeInhibitionUp-regulatesp53andApoptosis-Inducing FactorinChondrocytesCausingSevereGrowthRetardationinMice FarasatZaman, 1 Victoria Menendez-Benito, 2 Emma Eriksson, 1 Andrei S. Chagin, 1 MasaharuTakigawa, 4 BengtFadeel, 3 NicoP.Dantuma, 2 Dionisios Chrysis, 1 andLarsSa ¨vendahl 1 1 Department of Woman and Child Health, Pediatric Endocrinology Unit, Astrid Lindgren Children’s Hospital, 2 Department of Cell and Molecular Biology, The Medical Nobel Institute, and 3 Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Stockholm, Sweden; and 4 Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan Abstract Proteasome inhibitors (PI), a novel class of anticancer drugs, arerelativelywelltoleratedandhaverecentlybeenintroduced into the clinic for the treatment of multiple myeloma. The tumor selectivity and low toxicity of PIs are surprising, given the crucial role of the ubiquitin/proteasome system in a multitude of cellular processes. Here, we show that systemic administration of PIs specifically impairs the ubiquitin/ proteasome system in growth plate chondrocytes. Impor- tantly,youngmicedisplayedseveregrowthretardationduring treatment as well as 45 days after the cessation of treatment with clinically relevant amounts of MG262 (0.2 Mmol/kg body weight/injection) or bortezomib (1.0 mg/kg body weight/ injection). Dysfunction of the ubiquitin/proteasome system was accompanied by the induction of apoptosis of stem-like and proliferative chondrocytes in the growth plate. These results were recapitulated in cultured fetal rat metatarsal bones and chondrocytic cell lines (rat, human). Apoptosis was associated with up-regulation of the proapoptotic molecules, p53 and apoptosis-inducing factor (AIF), both in vitro and in vivo .InadditiontotheobservationthatAIFisexpressedin thegrowthplate,wealsoprovideevidencethatAIFservesasa direct target protein for ubiquitin, thus explaining its prominent up-regulation upon proteasome inhibition. Sup- pression of p53 or AIF expression with small interfering RNAs partly rescued chondrocytes from proteasome inhibition– induced apoptosis (35% and 41%, respectively). Our observa- tions show that proteasome inhibition may selectively target essential cell populations in the growth plate causing signif- icant growth failure. These findings could have important implications for the use of proteasome inhibitors in the treat- ment of childhood cancer. [Cancer Res 2007;67(20):10078–86] Introduction Longitudinal bone growth takes place in the growth plate, a thin layer of cartilage localized at each end of the long bones. The growth plate is mainly composed of chondrocytes and could be morphologically and functionally divided into three different zones: resting, proliferative, and hypertrophic. Resting zone chondrocytes, also known as stem-like chondrocytes (1, 2), are stimulated to proliferate and undergo hypertrophy, a process which involves several stages of maturation (3). Disturbed chondrocyte activity, either due to disruption in normal cell division or excessive cell death, may lead to defective longitudinal bone growth. Survivors of childhood cancer often have severe long-term complications secondary to previously administered life-saving treatment, em- phasizing the importance of minimizing these undesired effects. The proteasome is a large proteolytic complex that resides in the nucleus and cytosol of all eukaryotic cells. The selectivity of the system is safeguarded by the small protein modifier ubiquitin, which is covalently conjugated to proteins destined for proteaso- mal degradation. The proteasome preferentially binds and degrades ubiquitinated proteins (4). The critical involvement of the ubiquitin/proteasome system in the regulation of a number of cellular processes, as well as protein quality control, suggests that compounds or conditions that interfere with this process would be inherently toxic to eukaryotic cells. In vitro experiments confirm this notion as cells undergo apoptosis when cultured in the presence of proteasome inhibitors, making these agents attractive candidates for cancer therapy (5). Presently, there are five major classes of specific proteasome inhibitors: peptide aldehydes, peptide vinyl sulfones, peptide boronates, peptide epoxyketones, and h-lactones. Only recently, bortezomib (also known as PS341 or Velcade), a boronate proteasome inhibitor, was approved as a third-line agent in the treatment of multiple myeloma because of its profound antitumor effect (6), which was also documented in a clinical phase 1 trial in children (7). The most prevalent side effects of proteasome inhibitors, thrombocytopenia and peripheral neuropathy, are only observed in a minority of treated patients (8). However, there is no previous information available on any specific side effects of proteasome inhibitors in young, rapidly growing individuals. The molecular mechanism for proteasome inhibitor–induced cell death of cancer cells remains enigmatic and it is therefore difficult to anticipate which somatic cells may be at risk during treatment with proteasome inhibitors. To address this, the effects of two highly selective boronate proteasome inhibitors, MG262 (9) and bortezomib (7, 8), were analyzed in young mice. A transgenic reporter mouse model for the ubiquitin/proteasome system was used to monitor the in vivo proteasome inhibition. Our studies revealed functional impairment of ubiquitin-dependent proteasomal degradation in growth plate chondrocytes. Using these transgenic mice, as well as cell lines and metatarsal organ cultures as model systems, evidence for the role of caspase- dependent and -independent induction of apoptosis in chondro- cytes upon proteasome inhibition has been observed. Importantly, severe growth retardation was seen in mice treated with the Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org). V. Menendez-Benito and E. Eriksson contributed equally to this work. Current address for V. Menendez-Benito: Division of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, the Netherlands. Requests for reprints: Farasat Zaman, Pediatric Endocrinology Unit, Q2:08, Karolinska University Hospital, 171 76 Stockholm, Sweden. Phone: 46-8517-72382; Fax: 46-8517-75128; E-mail: Farasat.Zaman@ki.se. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-3982 Cancer Res 2007; 67: (20). 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