Overlapping functions of Hdac1 and Hdac2 in cell cycle regulation and haematopoiesis
2010; Springer Nature; Volume: 29; Issue: 15 Linguagem: Inglês
10.1038/emboj.2010.136
ISSN1460-2075
AutoresRoel H. Wilting, Eva Yanover, Marinus R. Heideman, Heinz Jacobs, James W. Horner, Jaco van der Torre, Ronald A. DePinho, Jan‐Hermen Dannenberg,
Tópico(s)Cancer-related gene regulation
ResumoArticle22 June 2010free access Overlapping functions of Hdac1 and Hdac2 in cell cycle regulation and haematopoiesis Roel H Wilting Roel H Wilting Division of Molecular Genetics, Plesmanlaan 121, Amsterdam, The Netherlands Search for more papers by this author Eva Yanover Eva Yanover Division of Molecular Genetics, Plesmanlaan 121, Amsterdam, The Netherlands Search for more papers by this author Marinus R Heideman Marinus R Heideman Division of Immunology, Plesmanlaan 121, Amsterdam, The Netherlands Search for more papers by this author Heinz Jacobs Heinz Jacobs Division of Immunology, Plesmanlaan 121, Amsterdam, The Netherlands Search for more papers by this author James Horner James Horner Departments of Medical Oncology, Medicine and Genetics, Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA Search for more papers by this author Jaco van der Torre Jaco van der Torre Division of Molecular Genetics, Plesmanlaan 121, Amsterdam, The Netherlands Search for more papers by this author Ronald A DePinho Ronald A DePinho Departments of Medical Oncology, Medicine and Genetics, Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA Search for more papers by this author Jan-Hermen Dannenberg Corresponding Author Jan-Hermen Dannenberg Division of Molecular Genetics, Plesmanlaan 121, Amsterdam, The Netherlands Search for more papers by this author Roel H Wilting Roel H Wilting Division of Molecular Genetics, Plesmanlaan 121, Amsterdam, The Netherlands Search for more papers by this author Eva Yanover Eva Yanover Division of Molecular Genetics, Plesmanlaan 121, Amsterdam, The Netherlands Search for more papers by this author Marinus R Heideman Marinus R Heideman Division of Immunology, Plesmanlaan 121, Amsterdam, The Netherlands Search for more papers by this author Heinz Jacobs Heinz Jacobs Division of Immunology, Plesmanlaan 121, Amsterdam, The Netherlands Search for more papers by this author James Horner James Horner Departments of Medical Oncology, Medicine and Genetics, Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA Search for more papers by this author Jaco van der Torre Jaco van der Torre Division of Molecular Genetics, Plesmanlaan 121, Amsterdam, The Netherlands Search for more papers by this author Ronald A DePinho Ronald A DePinho Departments of Medical Oncology, Medicine and Genetics, Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA Search for more papers by this author Jan-Hermen Dannenberg Corresponding Author Jan-Hermen Dannenberg Division of Molecular Genetics, Plesmanlaan 121, Amsterdam, The Netherlands Search for more papers by this author Author Information Roel H Wilting1, Eva Yanover1,‡, Marinus R Heideman2,‡, Heinz Jacobs2, James Horner3, Jaco van der Torre1, Ronald A DePinho3 and Jan-Hermen Dannenberg 1 1Division of Molecular Genetics, Plesmanlaan 121, Amsterdam, The Netherlands 2Division of Immunology, Plesmanlaan 121, Amsterdam, The Netherlands 3Departments of Medical Oncology, Medicine and Genetics, Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA ‡These authors contributed equally to this work *Corresponding author. Division of Molecular Genetics, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel.: +31 20 512 1993; Fax: +31 20 512 2011; E-mail: [email protected] The EMBO Journal (2010)29:2586-2597https://doi.org/10.1038/emboj.2010.136 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Histone deacetylases (HDACs) counterbalance acetylation of lysine residues, a protein modification involved in numerous biological processes. Here, Hdac1 and Hdac2 conditional knock-out alleles were used to study the function of class I Hdac1 and Hdac2 in cell cycle progression and haematopoietic differentiation. Combined deletion of Hdac1 and Hdac2, or inactivation of their deacetylase activity in primary or oncogenic-transformed fibroblasts, results in a senescence-like G1 cell cycle arrest, accompanied by up-regulation of the cyclin-dependent kinase inhibitor p21Cip. Notably, concomitant genetic inactivation of p53 or p21Cip indicates that Hdac1 and Hdac2 regulate p53–p21Cip-independent pathways critical for maintaining cell cycle progression. In vivo, we show that Hdac1 and Hdac2 are not essential for liver homeostasis. In contrast, total levels of Hdac1 and Hdac2 in the haematopoietic system are critical for erythrocyte-megakaryocyte differentiation. Dual inactivation of Hdac1 and Hdac2 results in apoptosis of megakaryocytes and thrombocytopenia. Together, these data indicate that Hdac1 and Hdac2 have overlapping functions in cell cycle regulation and haematopoiesis. In addition, this work provides insights into mechanism-based toxicities observed in patients treated with HDAC inhibitors. Introduction Post-translational modifications (PTMs) such as phosphorylation, methylation, ubiquitination, and acetylation are crucial regulatory modules at the heart of biological processes in the cell and are tightly regulated by a multitude of enzymes that catalyse the addition or removal of PTMs (Campos and Reinberg, 2009). Lysine acetylation of histones and non-histone proteins is controlled by histone acetyl transferases and histone deacetylases (HDACs). HDACs can be classified on the basis of their homology to yeast counterparts (Yang and Seto, 2008). Class I HDACs (HDAC1, -2, -3 and -8) are highly homologous to Saccharomyces cerevisiae Rpd3. Class IIa HDACs (HDAC4, -5, -7 and -9) and Class IIb HDACs (HDAC6 and -10) consist of S. cerevisiae Hda1 homologues. HDAC11 is the sole member of the Class IV HDACs, based on homology to both class I and class II HDACs (Gregoretti et al, 2004). Although the high sequence similarity between class I HDACs might anticipate a significant overlap in function, genetic studies in mice have revealed redundant as well as specific functions of these enzymes (Haberland et al, 2009c). Deletion of Hdac1 results in embryonic lethality as early as E9.5 of development (Lagger et al, 2002). In contrast, Hdac2 deficiency results in viable mice with reduced body weight (Trivedi et al, 2007; Zimmermann et al, 2007; Guan et al, 2009). Others have reported that Hdac2 deficiency is not compatible with life because of cardiac myopathy (Montgomery et al, 2007). The basis for these different phenotypes is not clear, but may relate to genetic background of the mice. Hdac2 also has a specific function in repression of genes involved in synaptogenesis, as evidenced by enhanced synapse formation, learning and memory in Hdac2-deficient mice (Guan et al, 2009). Hdac3 deletion results in early embryonic lethality and this enzyme has a critical function in cell cycle regulation and cardiac metabolism (Bhaskara et al, 2008; Knutson et al, 2008; Montgomery et al, 2008). Finally, Hdac8 has an important function in the differentiation of neural crest cells (Haberland et al, 2009b). A prime function of HDACs relates to their classical function as transcriptional co-repressors through deacetylation of lysine residues in histone tails. This results in a closed chromatin structure and diminished accessibility for the basal transcription machinery. Class I HDACs are present in a variety of repressor complexes such as SIN3A, NuRD, REST and N-CoR/SMRT, which acquire their regional activities in part by interacting with sequence-specific transcription factors (Yang and Seto, 2008). The intimate link between class I HDACs and proteins involved in tumourigenesis, such as Mad/Mxi, pRB, p53 and PML–RAR fusion proteins, has established important functions for HDACs in tumourigenic processes. Correspondingly, pharmacological inhibition of HDACs, using chemical HDAC inhibitors (HDACi), results in cell cycle arrest and apoptosis of tumour cells (Minucci and Pelicci, 2006). Moreover, the use of relative selective HDACi-targeting class I HDACs has produced anti-tumourigenic effects, and genetic inactivation of class I Hdac1 and Hdac2 in transformed murine cells results in cessation of tumourigenic potential (Rasheed et al, 2008; Haberland et al, 2009a). Despite the clinical efficacy of HDACi, treatment of patients with HDACi results in undesirable haematological side effects, such as anaemia and thrombocytopenia (Prince et al, 2009). Currently, it is unclear whether these side effects are due to the targeting of (multiple) HDACs or because of off-target effects on non-HDAC proteins. These issues prompted us to explore more precisely the function of class I Hdac1 and Hdac2 in cell proliferation and haematopoietic development. Results Normal cell cycle regulation and increased levels of Hdac1 in Hdac2-deficient mouse embryonic fibroblasts To examine the function of Hdac2 in cell cycle regulation in primary cells, we generated mouse embryonic fibroblasts (MEFs) deficient for Hdac2. Relative to wild-type controls, both Hdac2+/− and Hdac2−/− MEFs do not show any alterations in proliferation under normal culture conditions as well as under growth-restricting conditions, such as low serum, oncogene-induced senescence (OIS) and irradiation (data not shown). These results prompted us to investigate possible compensatory mechanisms of other class I Hdac members, Hdac1, -3 and -8. Western blot analysis of Hdac2-deficient MEF protein lysates revealed that Hdac1 protein levels are increased as compared with Hdac2-proficient MEFs. In contrast, protein levels of Hdac3 and Hdac8 remained unchanged (Figure 1A). Interestingly, ablation of Hdac1 results in an increase of Hdac2 protein levels (Figure 3A; Supplementary Figure 3A). These results indicate reciprocal compensatory mechanisms between Hdac1 and Hdac2 and suggest functional redundancy between these two class I Hdacs. Figure 1.Hdac1 and Hdac2 collectively control cell cycle progression. (A) Western blot analysis of Hdac2-deficient MEF protein lysates for indicated proteins. Tubulin served as a loading control. (B) Representative photographs of MEF cell cultures with indicated genotypes grown without 4-OHT or with 200 nM 4-OHT. Representative details of MEF cultures with indicated genotypes are shown in the third row. Note the presence of large, flat cells in 4-OHT-treated RCM2+;Hdac1L/L;Hdac2−/− cultures. Bottom panels show representative pictures of senescence-associated β-galactosidase-stained MEF cultures with indicated genotypes. (C) Growth curve analysis of Hdac1KO (closed squares), Hdac2KO (closed triangles) or DKO MEFs (open circles). All experiments were performed in triplicate. (D) Percentage of SA-bgalactosidase positive cells in MEF cultures with indicated genotypes. (E) Cell cycle analysis of wild-type and DKO MEFs for G1, S and G2/M cell cycle phases by BrdU-PI FACS. Values represent the average of three independent experiments. Download figure Download PowerPoint Hdac1 and Hdac2 collectively regulate cell cycle progression To directly explore possible functional redundancy between Hdac2 and its most homologous family member, Hdac1, we generated a cell culture system in which Hdac1 and Hdac2 can be deleted individually or simultaneously. Therefore, we used an Hdac1 conditional knock-out (cKO) allele in which exon 2 is flanked by loxP recombination sites (Supplementary Figure 1A). Cre-recombinase-mediated deletion of Hdac1 exon 2 produces an Hdac1-null allele (Supplementary Figure 1A) and results in embryonic lethality of Hdac1−/− mice consistent with earlier studies (Lagger et al, 2002; Montgomery et al, 2007). Intercrosses using Hdac1 cKO mice, Hdac2 cKO mice (Guan et al, 2009) and Rosa26CreERT2 (RCM2) mice (Hameyer et al, 2007) allowed us to generate a series of isogenic MEFs in which, on addition of tamoxifen (4-OHT), Hdac1 and Hdac2 could be deleted (Supplementary Figure 1B). Ablation of Hdac2 (resulting in Hdac2KO MEFs) or Hdac1 (resulting in Hdac1KO MEFs) did not result in an overt phenotype in MEFs under normal growth conditions (Figure 1B and C). In contrast, somatic deletion of Hdac1 in germ-line Hdac2−/− cells (referred to as DKO MEFs) results in a dramatic growth arrest and induction of a large and flat, senescence-like, cell morphology (Figure 1B and C). Accordingly, up to 80% of all DKO cells stained positive for senescence-associated β-galactosidase (SA-β-gal) activity, whereas Hdac1 or Hdac2 single null cells showed wild-type-staining patterns (Figure 1B and D). To dissect the nature of the cellular proliferation arrest in DKO MEFs, BrdU-PI fluorescence-activated cell sorting (FACS) analysis was used to determine the cell cycle distribution in wild-type and DKO MEFs. As compared with wild-type controls, DKO MEFs displayed a two-fold reduction in S-phase cells and a 10% increase in the G1-phase cells (Figure 1E). Thus, Hdac1 and Hdac2 cooperate to control G1 to S transition and their absence provokes a senescence-like, G1 cell cycle arrest. Hdac1 or Hdac2 catalytic activity is required to maintain cell cycle progression HDACs remove acetyl groups from lysine residues through a mechanism that involves deacetylase activity, which is dependent on other proteins present in HDAC multi-protein complexes (Sengupta and Seto, 2004). Inactivation of Hdac1 and Hdac2 in our experiments resulted in a complete removal of these proteins, raising the question whether the observed growth arrest in DKO MEFs is due to dissociation of HDAC protein complexes or because of the absence of HDAC activity. To address this question, we generated Hdac1 and Hdac2 catalytic inactive mutants by mutating conserved residues found to be critical for deacetylase activity in HDAC8 (Supplementary Figure 2A–D; Vannini et al, 2007). Retroviral expression in MEFs resulted in near-physiological protein levels and proper cellular localization of Hdac1D99A, Hdac1Y303F, Hdac2D100A or Hdac2Y304F mutants, similar to wild-type Hdac1 or Hdac2 (Figure 2A and B). Subsequently, expression of catalytic inactive mutants as well as wild-type Hdac1 and Hdac2 was tested for its ability to rescue a growth arrest in DKO MEFs. Although MEFs deficient for Hdac1 and Hdac2 were unable to proliferate, identical MEFs expressing exogenous wild-type Hdac1 or wild-type Hdac2 were fully rescued with respect to their proliferation capacity. In contrast, DKO MEFs expressing Hdac1D99A, Hdac1Y303F, Hdac2D100A or Hdac2Y304F catalytic inactive mutants were unable to proliferate (Figure 2C). These results further corroborate functional redundancy between Hdac1 and Hdac2, as expression of either Hdac1 or Hdac2 is sufficient to rescue DKO MEFs. Furthermore, our results establish that the deacetylase activity of Hdac1 or Hdac2 is essential for cellular proliferation. Figure 2.Hdac1 or Hdac2 deacetylase activity is required for cell cycle progression. (A) Western blot analysis of DKO MEFs expressing wild-type Hdac1, Hdac1D99A, Hdac1Y303F (left panel), wild-type Hdac2, Hdac2D100A or Hdac2Y304F (right panel). Lysates prepared from wild-type (control) and DKO MEFs (vector) were used as a positive and negative control, respectively. Cdk4 served as a loading control. (B) Subcellular localization of wild-type and mutant Hdac1 and Hdac2 ectopically expressed in DKO MEFs by immunofluorescence staining using antibodies for Hdac1 (left panels) or Hdac2 (right panels). Note the presence of a Hdac1-proficient nucleus in vector-treated DKO MEFs because of a non-recombined Hdac1 cKO allele. (C) Growth curve analysis of DKO MEFs expressing either wild-type or mutant Hdac1 or Hdac2. Download figure Download PowerPoint Hdac1 and Hdac2 cooperatively regulate p21Cip expression Cellular senescence is a potent proliferation arrest induced upon oncogene expression, DNA-damage or suboptimal cell culture conditions of normal, non-transformed cells. The cell cycle inhibitors p16Ink4a and p19Arf are up-regulated upon senescence-inducing conditions and activate the Retinoblastoma protein (pRb) and p53-tumour suppressors, respectively (Campisi, 2005). To see whether Hdac1 and Hdac2 prevent cellular senescence by repressing the expression of senescene-associated cell cycle inhibitors, we analysed p16Ink4a and p19Arf protein levels in wild-type, Hdac1KO, Hdac2KO and DKO MEFs. Western blot analysis revealed no up-regulation of p16Ink4a or p19Arf in the absence of Hdac1 and Hdac2 (Figure 3B; Supplementary Figure 3A). In addition, p53 protein levels were not stabilized in DKO MEFs, suggesting that Hdac1 and Hdac2 do not control p53 protein levels under normal culture conditions (Figure 3A). It also suggests that deficiency for Hdac1 and Hdac2 does not result in a p53-activating DNA-damage response. p21Cip, a cell cycle inhibitor protein that is transcriptionally regulated by Hdac1 in embryonic stem (ES) cells (Lagger et al, 2002), was modestly up-regulated in Hdac1KO MEFs and Hdac2KO MEFs as compared with control MEFs (Figure 3B). In contrast, DKO MEFs showed strong induction of p21Cip. Expression of a closely related cell cycle inhibitory protein p27Kip did not correlate with the cell cycle arrest in DKO MEFs, as it was up-regulated in Hdac2KO as well as DKO MEFs (Figure 3B). Collectively, these data point to p21Cip as a potential point of action in Hdac1/2-mediated regulation of the cell cycle. Figure 3.Hdac1 and Hdac2 regulate cell cycle progression independent of p53 or p21Cip. (A) Western blot analysis of wild-type (WT), Hdac1KO and DKO MEF protein lysates for Hdac1, Hdac2 and p53. γ-Irradiated wild-type cells expressing either control or p53 shRNA were used as a positive and negative control, respectively. Tubulin served as a loading control. (B) Western blot analysis of protein lysates for Hdac1, Hdac2, p21Cip, p27Kip and p16Ink4a of MEFs with indicated genotypes infected with retroviruses expressing control shRNA (C), p21Cip shRNA (p21) or p53 shRNA (p53). Tubulin served as a loading control. (C) Representative pictures of wild-type, Hdac1KO, Hdac2KO or DKO MEFs infected with retroviruses expressing control shRNA, p21Cip shRNA or p53 shRNA. (D) Growth curve analysis of wild-type, Hdac1KO, Hdac2KO and DKO MEFs, expressing either control shRNA or shRNA directed against p21Cip and p53. Download figure Download PowerPoint Hdac1 and Hdac2 regulate cell cycle progression independent of p53–p21Cip To examine whether the increased expression of p21Cip is responsible for the cell cycle arrest induced by Hdac1 and Hdac2 ablation, we infected control and DKO MEFs with empty retroviruses (vector) or retroviruses expressing a short-hairpin RNA (shRNA) against p21Cip (referred to as p21KD) (Figure 3B). Despite efficient knock-down of p21Cip, cells lacking both Hdac1 and Hdac2 still entered a cell cycle arrest identical to p21Cip-proficient cells lacking Hdac1 and Hdac2 (Figure 3C and D). As p53 is a major regulator of the G1/S transition in cellular senescence and functions as a transcriptional activator of p21Cip (el-Deiry et al, 1993), we tested whether shRNA-mediated knock-down of p53 (p53KD) is required for both p21Cip induction and cell cycle arrest in DKO MEFs. Although p53 knock-down blocked p21Cip induction (Figure 3B), DKO MEFs still underwent a cell cycle arrest, indicating that p53 is required for the induction of p21Cip expression in the absence of Hdac1 and Hdac2 (Figure 3B) and independently confirm our results obtained with p21Cip knock-down (Figure 3C and D). These data indicate that the p53–p21Cip axis is dispensable for the cell cycle arrest as a result of Hdac1 and Hdac2 deficiency. Although we obtained hardly detectable levels of p21Cip using p21Cip or p53 shRNA-mediated knock-down, it is conceivable that residual levels of p21Cip are accountable for the observed cell cycle arrest in the absence of Hdac1 and Hdac2. To address this issue, we generated Hdac2+/−p21−/− mice, which were intercrossed to obtain Hdac2−/−p21−/− MEFs (referred to as Hdac2KO;p21−/− MEFs). To down-regulate Hdac1 levels in this cell system, we generated retroviruses expressing Hdac1 shRNA, resulting in efficient knock-down of Hdac1 (Supplementary Figure 3A). Similar to the results obtained in DKO MEFs, knock-down of Hdac1 (Hdac1KD) in Hdac2KO MEFs, resulted in a senescence-like G1 cell cycle arrest accompanied by SA-β-gal activity and increased p21Cip protein levels (Supplementary Figure 3B–D). In contrast, expression levels of p19Arf, p16Ink4a and p27Kip did not correlate with the observed phenotype (Supplementary Figure 3A). Subsequently, we infected Hdac2WT;p21+/−, Hdac2KO; p21+/+, Hdac2KO;p21+/− and Hdac2KO;p21−/− MEFs with retroviruses expressing Hdac1 shRNA or control shRNA. Hdac1KD resulted in an increased expression of p21Cip only in Hdac2KO;p21+/+ and to a lesser extent in Hdac2KO; p21+/− MEFs (Figure 4A). Regardless of p21Cip status, proliferation ceased dramatically in Hdac1KD;Hdac2KO MEFs (Figure 4B and C). During the growth curve analysis, we noted that Hdac2KO;p21−/− MEF cultures expressing Hdac1 shRNA regained proliferation capacity after 6–8 days on plating (Figure 4C). Western blot analysis of these MEF cultures, along with Hdac2WT;p21+/−MEF cultures expressing Hdac1 shRNA, revealed loss of Hdac1 knock-down specifically in Hdac2KO;p21−/− MEF cultures (Figure 4D). These results indicate a strong selection against Hdac1KD in Hdac2KO;p21−/− MEFs, supporting our results that loss of Hdac1 and Hdac2, even in the absence of p21Cip, is not compatible with proliferation. Collectively, these data strongly indicate and independently confirm that p21Cip is dispensable for establishing the cell cycle arrest in the absence of Hdac1 and Hdac2. Figure 4.Genetic ablation of p21Cip, p16Ink4a or p19Arf does not allow proliferation of DKO MEFs. (A) Western blot analysis of protein lysates of indicated MEFs expressing either control shRNA (C) or Hdac1 shRNA (KD) for Hdac1, Hdac2 and p21Cip. Cdk4 served as a loading control. (B) Representative pictures of MEFs with indicated genotypes infected with retroviruses expressing either control shRNA or Hdac1 shRNA. (C) Growth curve analysis of Hdac2KO;p21+/+(squares), Hdac2WT;p21−/− (triangles), Hdac2KO;p21+/− (diamonds) and Hdac2KO;p21−/− (circles), MEFs expressing either control shRNA (left panel) or Hdac1 shRNA (right panel). (D) Western blot analysis of protein lysates for Hdac1 and Hdac2 of two independent Hdac2WT;p21−/− and Hdac2KO;p21−/− MEF clones infected with either control (#1, #3, #5, #7) or Hdac1 shRNA (#2, #4, #6, #8), isolated at day 8 of the growth curve analysis as shown in (C). The clone numbers in (C) correspond with the clones and genotypes as shown in (D). Cdk4 served as a loading control. (E) Left panel: western blot analysis of Hdac2WT;Cdkn2a−/− and Hdac2KO;Cdkn2a−/− MEFs expressing either control (control) or Hdac1 shRNA (Hdac1KD) for Hdac1, Hdac2, p16Ink4a and p19Arf. Cdk4 was used as a loading control. As a control for p16Ink4a and p19Arf expression, we used late passage wild-type MEFs. Right panel: growth curve analysis of Hdac2WT;Cdkn2a−/− (squares) and Hdac2KO;Cdkn2a−/− (circles) MEFs expressing either control (filled tags) or Hdac1 shRNA (open tags). Download figure Download PowerPoint p16Ink4a- and p19Arf-independent cell cycle arrest in Hdac1;Hdac2-deficient MEFs It is conceivable that other cell cycle inhibitor proteins besides p21Cip are involved in the senesence-like cell cycle arrest in the absence of Hdac1 and Hdac2. p16Ink4a and p19Arf, encoded by the Cdkn2a allele, are two major cell cycle inhibitors involved in cellular senescence and activate the pRb- and p53-tumour suppressor pathways by inhibition of cyclinD/cdk4 and Mdm2, respectively. To test whether deletion of these cell cycle inhibitors allows a bypass of the observed cell cycle arrest, we generated Hdac2WT;Cdkn2a−/− and Hdac2KO;Cdkn2a−/− MEFs and subsequently down-regulated Hdac1 by expression of Hdac1 shRNA. Despite the absence of p16Ink4a and p19Arf, simultaneous inactivation of Hdac1 and Hdac2 still resulted in a cell cycle arrest, indicating that these cell cycle inhibitors are not required for inducing a cell cycle arrest in Hdac1- and Hdac2-deficient MEFs (Figure 4E). Oncogenic-transformed cells harbour a senescence-like programme suppressed by Hdac1 or Hdac2 OIS is viewed as a mechanism to protect cells from oncogenic transformation (Mooi and Peeper, 2006). Expression of oncogenic RasV12 induces the expression of the cell cycle inhibitors p16Ink4a and p19Arf, thereby activating the pRb- and p53-tumour suppressor proteins. Inactivation of p53 allows bypass of RasV12-induced senescence and as a consequence oncogenic transformation (Campisi, 2005). We wished to address whether the senescence-like arrest in the absence of Hdac1 and Hdac2 is still functional in oncogenic-transformed cells that have bypassed OIS. To this end, we oncogenically transformed control and RCM2+;Hdac1L/L;Hdac2−/− MEFs with retroviruses expressing p53 shRNA as well as oncogenic Ras (RasV12) (Figure 5A). Upon addition of 4-OHT to these cells, we obtained wild-type, Hdac1-deficient (Hdac1KO) and Hdac1/Hdac2-deficient (DKO)-transformed fibroblasts. Cells deficient for either Hdac1 or Hdac2 did not show an impairment of proliferation. Similar to our observations in primary MEFs, Hdac1 deficiency resulted in increased Hdac2 levels, suggesting a compensatory function for Hdac2 in the absence of Hdac1 (Figure 5A). Indeed, ablation of Hdac1 and Hdac2 in transformed cells resulted in a senescence-like growth arrest in short- and long-term proliferation assays (Figure 5B and C). Despite the fact that these cells have bypassed the p53-dependent RasV12-induced senescence checkpoint, we still observed SA-β-gal activity in the majority (up to 80%) of DKO cells (Figure 5D). Similar to our observations in primary MEFs, p53 knock-down in transformed fibroblasts prevented p21Cip up-regulation (Figure 5A), but not a cell cycle arrest in the absence of Hdac1 and Hdac2, suggesting that also in transformed cells Hdac1 and Hdac2 function independent of p53 and p21Cip in maintaining cellular proliferation. Together, these data show an essential and redundant function of Hdac1 and Hdac2 in suppressing a p53–p21Cip-independent pathway that is able to evoke a senescence response, even in cells that have escaped OIS. Figure 5.Hdac1 and Hdac2 collectively suppress a senescence-inducing pathway in transformed cells. (A) Western blot analysis of 4-OHT-treated MEFs with indicated genotypes, expressing RasV12 and p53 shRNA for Hdac1, Hdac2, p21Cip and RasV12. Tubulin served as loading control. DKO MEFs served as a control for p21Cip expression. (B) Representative pictures of oncogenic-transformed (RasV12;p53KD) MEF cultures with indicated genotypes in the absence (top panels) or presence (lower panels) of 4-OHT. (C) Growth curve analysis of wild-type (triangles), Hdac1KO (diamonds) and DKO (open circles) oncogenic-transformed MEFs. (D) Quantification and representative pictures of SA-β-galactosidase positive cells in cultures of wild-type and DKO oncogenic-transformed cells. Shown are average values of six different microscopic fields. Download figure Download PowerPoint Ablation of Hdac1 and Hdac2 results in anaemia and thrombocytopenia Treatment of cancer patients using HDACi is complicated by the adverse clinical impact on the haematopoietic system (Prince et al, 2009). The growth arrest conferred by simultaneous deletion of Hdac1 and Hdac2 in primary fibroblasts prompted us to explore whether the HDACi-related toxicities can be explained by selective targeting of (multiple) HDACs or to off-target effects. Deletion of Hdac1 and/or Hdac2 in the haematological compartment of the mouse will enable us to address these questions. To this end, we generated mice harbouring the interferon-inducible MxCre transgene (Kuhn et al, 1995) and cKO alleles for Hdac1 and Hdac2. Administration of polyinosine-polycytidylic acid (pI;pC) induces an interferon response, thereby activating MxCre expression predominantly in the haematopoietic system and liver. pI;pC-induced MxCre expression resulted in successful deletion of Hdac1 and Hdac2 in bone marrow (Supplementary Figure 4) and liver (Supplementary Figure 5). Ablation of Hdac1, or simultaneous deletion of Hdac1 and Hdac2 in the liver, did not result in histological abnormalities, indicating that Hdac1 and Hdac2 are not critical in the maintenance of hepatocytes (Supplementary Figure 5). In contrast, although pI;pC-treated MxCre+ or MxCre+; Hdac1L/L (referred to as MxCre+;Hdac1KO) mice appeared normal, similar treated MxCre+;Hdac1L/L;Hdac2L/L (referred to as MxCre+;DKO) mice became rapidly moribund at approximately 9 days after pI;pC injections displaying anaemic features and internal bleedings (Figure 6A). Indeed, peripheral blood analysis showed a five-fold reduction in red blood cells and thrombocyte numbers were 16-fold reduced in MxCre+;DKO mice as compared with MxCre+ mice. Although thrombocyte numbers in MxCre+;Hdac1KO mice were decreased, this reduction is not significant (P>0.05) (Figure 6B). Histological examination of bone-marrow sections as well as total bone-marrow cell counts revealed a reduction in total cell numbers in MxCre+;DKO mice compared with MxCre+ mice (Figure 6B and C). Strikingly, as compared with MxCre+ and MxCre+;Hdac1KO mice, MxCre+;DKO bone marrow contained
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