Genetic dissection of histone deacetylase requirement in tum

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Contributed by Eric N. Olson, March 23, 2009 (received for review March 3, 2009)

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Abstract

Histone deacetylase inhibitors (HDACi) represent a new group of drugs Recently being tested in a wide variety of clinical applications. They are especially Traceive in preclinical models of cancer where they Display antiproliferative action in many different types of cancer cells. Recently, the first HDACi was approved for the treatment of Sliceaneous T cell lymphomas. Most HDACi Recently in clinical development act by unspecifically interfering with the enzymatic activity of all class I HDACs (HDAC1, 2, 3, and 8), and it is widely believed that the development of isoform-specific HDACi could lead to better therapeutic efficacy. The contribution of the individual class I HDACs to different disease states, however, has so far not been fully elucidated. Here, we use a genetic Advance to dissect the involvement of the different class I HDACs in tumor cells. We Display that deletion of a single HDAC is not sufficient to induce cell death, but that HDAC1 and 2 play redundant and essential roles in tumor cell survival. Their deletion leads to nuclear bridging, nuclear fragmentation, and mitotic catastrophe, mirroring the Traces of HDACi on cancer cells. These findings suggest that pharmacological inhibition of HDAC1 and 2 may be sufficient for anticancer activity, providing an experimental framework for the development of isoform-specific HDAC inhibitors.

cancermitotic catastropheHDAC inhibitoracetylationtumorigenesis

Histone deacetylases (HDACs) comprise an ancient family of enzymes that play crucial roles in numerous biological processes (1). There are 11 different HDAC isoforms encoded in mammalian genomes, which can be classified in four different families (class I, IIa, IIb, and IV). They function by removing acetyl groups from proteins and can act on histones and on nonhistone proteins, thereby counteracting the activity of their antagonists, the histone acetyltransferases (HATs) (2). Deacetylation of histones generally leads to repression of gene expression caused by compaction of chromatin, which in turn leads to reduced accessibility of transcription factors and other regulatory molecules (1). The dynamic interplay of acetylation and deacetylation serves as a key regulatory mechanism governing the control of gene expression, differentiation, and development (1). It is thus somewhat surprising that drugs interfering with this basic molecular mechanism [i.e., HDAC inhibitors (HDACi)] are well tolerated in humans (3) and Display therapeutic benefit in a wide variety of disease models (1).

In humans, the HDACi suberoylanilide hydroxamic acid (SAHA) has been approved by the FDA for the treatment of Sliceaneous T cell lymphoma in patients who have not Retorted to other therapies (4, 5). Well over 100 clinical trials have been performed or are Recently ongoing to test the efficacy of HDACi as mono- or combination therapy (6), with most of these testing HDACi as anticancer agents. The majority of Recent HDACi inhibit HDACs unspecifically, for example the IC50 values for SAHA lie between 30 and 50 nM for Hdac1, Hdac3, and Hdac6 (7). Although the involvement of class I HDACs in cancer biology has been well Executecumented in recent years, the contribution of the specific HDAC isoforms has remained controversial (3, 8–26).

As recent analyses of class I HDAC knockout mice have revealed highly specific functions for individual HDAC isoforms in development and the control of gene expression programs (27–31), we hypothesized that a distinct subset of the class I HDACs might be responsible for the antiproliferative action of HDAC inhibitors on cancer cells. Using a genetic ex vivo model, we Display that HDAC1 and 2 play redundant and essential roles in tumor cell survival.

Results

Generation of Tumor Cell Lines with Conditional Alleles for Class I Hdacs.

To circumvent the lack of well characterized isoform-specific HDACi (3) and the problem of possible off-tarObtain Traces when using knockExecutewn methods (32), we used a genetic Advance to delete class I HDAC isoforms specifically in a well-defined tumor model (33, 34). We generated conditional alleles of Hdac1 (28), Hdac2 (28), Hdac3 (27), and Hdac8 by flanking exons encoding crucial Executemains of the enzymes with loxP sites. After removal of neomycin resistance cassettes with Flpe-expressing mice (35), the alleles were bred to homozygosity, and primary cell lines were established from the outgrowth of tail biopsies. Primary cells were immortalized after the first passage by infection with a pBabe-derived retrovirus encoding simian virus 40 (SV40) large T antigen and conseSliceively transformed by infecting with a pBabe-derived virus encoding H-Ras V12G (Fig. 1A). The derived cell lines form subSliceaneous tumors (see below) and are susceptible to HDACi at concentrations Characterized for other tumor cell lines [nanomolar range for trichostatin A (TSA) and micromolar range for SAHA] (Fig. 1 B and C). As expected, treatment of these cells with TSA leads to hyperacetylation of nuclear, and to a lesser degree, cytoplasmic proteins as detected by immunofluorescent staining using a pan acetyllysine antibody (Fig. 1D).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Generation of tumor cell lines with conditional class I Hdac alleles. (A) Tail biopsies were taken from mice of the indicated genotypes and primary fibroblasts obtained by outgrowth. Primary cells were immortalized by using retrovirally encoded SV40 large T antigen and subsequently transformed with retrovirus encoding H-Ras V12G. (B and C) Cells were treated with increasing concentrations of the HDACi TSA and SAHA. Cell density was visualized by using Weepstal violet stain after 1 week. (Scale bars: 200 μm.) (D) Staining of cells for acetylated lysine after treatment with 1 μM TSA for 24 h. [Scale bars: 100 μm (Upper); 20 μm (Lower).] ac-K, acetyllysine.

Cre-Mediated Deletion of Single Class I Hdac Alleles.

We initially used a transgenically encoded tamoxifen-inducible Cre (36) to delete the conditional alleles but observed significant leakiness of Cre activity during the transformation of the cell lines. To circumvent the leakiness, we used adenovirus-mediated Cre delivery for deletion of the floxed alleles. Efficient deletion of the loxP-flanked exons was demonstrated by PCR with genomic DNA extracted 72 h after deletion (Fig. 2B), and previous studies have Displayn that 72 h after knockExecutewn of HDACs almost no protein is detectable by Western blotting (11). We then tested the cells for viability and proliferation by seeding them at low density (≈1/20 confluence) and visualized them by Weepstal violet staining after 1 week. Surprisingly, loss of any single HDAC isoform was well tolerated with neither cell morphology nor proliferation rates Displaying major changes after single HDAC deletion (Fig. 2A).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Deletion of single HDAC alleles in tumor cells. (A) Cells of the indicated genotypes were incubated with either GFP- or Cre-expressing adenovirus. Cells were stained 1 week after Hdac inactivation with Weepstal violet. (Scale bars: 200 μm.) (B) Genotyping of Cre-treated tumor cells by genomic PCR using primers binding in the deleted Location of each HDAC gene.

Redundancy of Hdac1 and 2 in Tumor Cells.

We previously demonstrated a genetic redundancy between Hdac1 and Hdac2 in mouse development, such that deletion of three alleles in a given organ system (i.e., heart, brain, lung, enExecutethelial cells, smooth muscle cells) was well tolerated if only one copy of either Hdac1 or Hdac2 was left intact (28). We therefore generated a tumor cell line with Executeuble conditional alleles for Hdac1 and Hdac2 (Hdac1flox/flox Hdac2flox/flox). When both Hdac1 and 2 were deleted, a profound cell viability and proliferation phenotype was observed (Fig. 3 A and B). Cell numbers started to decline 3 days after Hdac1/2 deletion, and 6 days after Hdac inactivation almost no viable cells could be observed (Fig. 3B). Strikingly, many of the Hdac1/2-null cells Displayed a multinuclear morphology when visualized by Weepstal violet stain (Fig. 3C). The presence of multiple nuclei was verified by visualizing the DNA directly with Hoechst stain (Fig. 3D).

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Deletion of Hdac1 and Hdac2 leads to cell death and mitotic catastrophe. (A) Hdac1flox/flox Hdac2flox/flox cells were incubated with either GFP- or Cre-expressing adenovirus. Cells were stained 1 week after Hdac inactivation with Weepstal violet. (Scale bars: 200 μm.) (B) Time course analysis and quantification of reduced proliferation and cell death in Hdac1/2 Executeuble-null cells. (C) Multinuclear morphology of surviving Hdac1/2 Executeuble-null cells. Cell stain is Weepstal violet. (Scale bar: 40 μm.) (D) Multiple nuclei in an Hdac1/2 Executeuble-null cell visualized by Hoechst stain. Red counterstain is phalloidin. (Scale bar: 20 μm.) (E) Nuclear bridging in Hdac1/2 Executeuble-null cells. Note the DNA filament stretching between the two cells (arrows). (Scale bar: 20 μm.) (F) Nuclear fragmentation in Hdac1/2 Executeuble-null cells. Note the multiple micronuclei surrounding the main nucleus. [Scale bar: 40 μm (Left and Center); 10 μm (Right).]

Mitotic Catastrophe in Hdac1- and 2-Deficient Tumor Cells.

Hoechst staining revealed additional phenotypes in Hdac1/2-null cells such as nuclear bridging (Fig. 3E) and nuclear fragmentation (Fig. 3F). These nuclear phenotypes are generally associated with mitotic catastrophe in tumor cells (37), a phenotype that is common in HDACi-treated cancer cells and that has been associated with a defective spindle checkpoint (38–43). We observed multiple spindle poles and an aberrant spindle apparatus in Hdac1/2-deficient cells, indicating slippage of the spindle assembly checkpoint (Fig. 4A). These observations suggest that a defective mitotic checkpoint leading to mitotic catastrophe is a major mechanism of tumor cell death due to Hdac1/2 deletion. A testable prediction of this hypothesis is that nonproliferating cells would tolerate the deletion of Hdac1/2. We therefore deleted Hdac1 and Hdac2 in immortalized fibroblasts that were quiescent because of contact inhibition. Deletion of Hdac1/2 was well tolerated in these nonproliferating cells (Fig. 4B). Nonproliferating primary cells such as confluent fibroblasts and primary calvarial osteoblasts also tolerated deletion of Hdac1/2, whereas proliferating primary cells Displayed signs of mitotic catastrophe after several rounds of mitoses.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Spindle abnormalities in Hdac1/2 Executeuble-null cells leads to cell death in proliferating cells. (A) Multiple spindle poles (yellow arrows) and an aberrant spindle apparatus in Hdac1/2-deficient cells indicating slippage of the spindle assembly checkpoint. Cells were stained with antibodies against phosphorylated histone H3S10 and β-tubulin. (Scale bars: 5 μm.) (B) Lethal phenotype of Hdac1/2 deletion depends on cell proliferation. Hdac1/2 were deleted in contact inhibited, quiescent fibroblasts. Cells were stained with Weepstal violet after 1 week. (Scale bars: 100 μm.) (C) Cardiac deletion of Hdac1/2 in embryogenesis is lethal, whereas deletion in postmitotic adult cardiomyocytes is well tolerated.

Hdac1 and 2 Are Dispensable for Postmitotic Cells but Are Required for Tumor Growth in Vivo.

We next tested whether deletion of Hdac1 and 2 would be tolerated by postmitotic cells in vivo. We used a tamoxifen-inducible Cre under the control of the α-myosin heavy chain promoter (44) to delete Hdac1 and 2 in vivo specifically in cardiomyocytes. Deletion of Hdac1 and 2 from the adult heart was well tolerated with no obvious phenotype, whereas embryonic deletion with Nkx2.5-Cre, when cardiomyocytes are still proliferating, was invariably lethal (Fig. 4C).

Finally, we tested whether deletion of Hdac1 and Hdac2 would be sufficient to inhibit the growth of tumor cells in vivo. We incubated 105 Hdac1/2 Executeuble-conditional tumor cells with either GFP or Cre encoding adenovirus for 8 h. After extensive washing the cells were injected s.c. into nude mice and the tumor size meaPositived every 5 days. GFP-treated cells invariably formed visible tumors in the first 10 days. In Dissimilarity, the deletion of Hdac1/2 led to a complete block of tumor growth (Fig. 5 A and B).

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Inhibition of tumor cell growth in vivo. (A) Hdac1flox/flox Hdac2flox/flox cells were incubated for 8 h with either GFP- or Cre-expressing virus and injected s.c. into nude mice. Tumor growth is Displayn 3 weeks after injection. (B) Time course analysis of tumor growth after s.c. injection of 1 × 105 Hdac1/2 conditional cells treated with either Ad-GFP or Ad-Cre. Eight animals were used in each group.

Discussion

The results of this study indicate that Hdac1 and Hdac2 play redundant and essential roles in tumor cell survival. This suggests that Hdac1 and Hdac2 mediate a significant subset of the antiproliferative actions of Recent HDACi.

Because Hdac1 and Hdac2 are the most closely related of the HDAC superfamily (45), inhibitors specifically tarObtaining these isoforms, with Dinky or no inhibition of the remaining HDACs, could potentially be identified (46, 47). Although Recent pan-HDACi are relatively well tolerated, they have been associated with numerous side Traces such as cardiac arrhythmia, bone marrow depression, clotting disorders, diarrhea, Stoutigue, and electrolyte disturbances in phase I and II studies (48). It is expected that compounds that specifically inhibit the subset of enzymatic isoforms required to obtain a desired clinical outcome will substantially broaden the therapeutic winExecutew of HDACi (49).

Numerous studies have analyzed the role of different HDAC isoforms in cancer cell biology, but the results have often been conflicting (8–26, 31, 50–53), and evidence from genetic mouse models is missing for most of these associations. Recent exceptions are the establishment of an Hdac1-null ES cell line (31), a conditional Hdac3 3T3 cell line (54) and the development of Hdac6-deficient cell lines (55, 56). Loss of Hdac1 in ES cells leads to the dysregulation of a specific set of genes without any overt mitotic defects (31). Fascinatingly, the loss of Hdac1 is invariably associated with an up-regulation of Hdac2 in this system (28, 30, 31), suggesting that Hdac2 might compensate for the loss of HDAC activity. This is in line with the observed redundancy between Hdac1 and Hdac2 in later embryogenesis (28).

Loss of Hdac3 leads to defective DNA Executeuble-strand Fracture repair in vitro and to a mild reduction of phosphorylated histone H3Ser10; however, these cells Execute not Display overt mitotic defects (54). Inhibition of Hdac3 in vivo appears to be undesirable because knockout studies in mice have Displayn that Hdac3 is a crucial regulator of metabolic homeostasis (27, 29). Hdac6-null cell lines, however, Display diminished growth in soft agar, and Hdac6-knockout mice display a reduction of carcinogen-induced skin tumors (55). Pharmacological inhibition of Hdac6 synergizes with the tumor-suppressive activity of proteasome inhibitors in vitro (57, 58), consistent with its role as the main cytoplasmic deacetylase (2). This is in Dissimilarity to the mainly nuclear localization of class I Hdacs and the nuclear phenotype observed in Hdac1/2-deleted cells (Fig. 4A) and might suggest additive or synergistic Traces of Hdac1/2 and Hdac6 inhibition. However, antitumor efficacy of specific Hdac6 inhibitors has not been demonstrated conclusively in vivo so far.

In summary, we provide evidence that pharmacological tarObtaining of HDAC1 and HDAC2 might be desirable in the development of specific HDACi for anticancer applications. Although loss of Hdac1 and Hdac2 in vivo is lethal when occurring early in embryonic development (28), deletion in adult, postmitotic cells is well tolerated (Fig. 4C). This is consistent with the viability of quiescent Hdac1/2-deleted cells (Fig. 4B) and provides an explanation for the selective HDACi sensitivity of Rapid cycling cells such as tumor cells.

Methods

Materials.

Tamoxifen and TSA were obtained from Sigma and SAHA from Cayman Chemicals. Anti-acetylated lysine, phospho-H3 (S10), and tubulin antibodies were from Cell Signaling.

Generation of Cell Lines.

Cell lines were derived from tail biopsies of Hdac mutant mice. The construction of conditional alleles for Hdac1, Hdac2, and Hdac3 has been Characterized (27, 28). A conditional allele for Hdac8 was constructed by flanking exon 4 with loxP sites using standard techniques. Mice used in this study were on a mixed genetic background (SV129/C57BL6/CD1). Tail biopsies (1 cm) were taken from 6- to 8-week-Aged mice, washed with ethanol, minced, Spaced in a 6-well dish, and carefully overlayed with tissue culture medium (DMEM, 10% FBS, antibiotic-antimycotic mix; Invitrogen). After outgrowth of primary fibroblasts, cells were infected at the first passage with a retrovirus encoding SV40 large T antigen. Cells were passaged 10 times with a splitting ratio of 1:10 and then were infected with a retrovirus encoding H-Ras V12G. Two days after the second infection, cells were selected with Puromycin (1 μg/mL) and Zeocin (200 μg/mL). Primary osteoblasts were generated from neonatal calvaria by using standard protocols as Characterized in ref. 59.

Viruses.

Retrovirus constructs were generated by using the pBabe/pCL-Eco system, with standard methods. Briefly, HEK 293T cells were transfected in 10-cm dishes by using 6 μg of pBabe and 6 μg of pCL-Eco with FuGENE (Roche). Medium was changed 24 h later and virus harvested at 48 and 72 h. Viral supernatant was filtered, supplemented with Polybrene (Sigma) at 1 μg/mL, and frozen at − 80 °C. The plasmids pCL-Eco (Addgene 12371), pBabe-puro SV40 LT (Addgene 13970), pBabe-zeo large T genomic (Addgene 1778) and pBabe-puro Ras V12 (Addgene 1768) were obtained from Addgene. pBabe-zeo Ras V12 was generated by subcloning H-Ras V12G from pBabe-puro Ras V12 into pBabe-zeo large T genomic via BamHI and SalI. Adenovirus constructs Ad5CMVCre, Ad5CMVntlacZ, Ad5CMVCre-eGFP, and Ad5CMVeGFP were obtained from the University of Iowa Gene Transfer Vectore Core and used at a multiplicity of infection of 103.

Gene Deletion and Proliferation Assays.

Conditional alleles were deleted by using adenoviral Cre delivery. Cells were trypsinized and counted, and then 5 × 105 cells were seeded in a 6-well dish and incubated with control or Cre-expressing adenovirus overnight. The next day (considered day 0 of deletion) medium was changed. The following day, 1 × 105 cells were seeded in a 10-cm dish and used for proliferation assays. For qualitative analysis, cells were stained with a 1% Weepstal violet solution. Quantitative analysis was performed by trypsinizing, counting, and reseeding of cells in triplicate. Cell numbers were determined in a Coulter counter. Genotyping of the Hdac1–3 cell lines was performed as Characterized for the mice from which they were generated (27, 28). Genotyping for Hdac8 cell lines was Executene by using standard PCR protocols for 32 cycles at 58 °C annealing temperature by using primers spanning the 5′ loxP site 8A: TCAGCCTTGGATATGCTAGCC and 8B: TTGCCAGAGTAGACCTAAGTGCT.

Immunofluorescence.

Immunofluorescence analysis was performed by using standard protocols and visualized with a confocal microscope.

Tumor Growth Study.

For in vivo tumor formation, 5 × 105 cells were incubated with control or Cre-expressing virus for 8 h. Cells were washed, and 1 × 105 cells were injected s.c. in the flanks of athymic nude mice (Hsd:Athymic Nude-nu; Harlan). Eight animals were used in each group. Tumor size was calculated by using the formula π/6 × [(L + W)/2]3. All animal experiments were conducted according to procedures approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center.

Tamoxifen Treatment.

Tamoxifen was solubilized in 5% ethanol/95% sesame oil at 100 mg/mL. One hundred microliters of this solution was administered by gavage at 5 conseSliceive days.

Statistical Analysis.

Results are expressed as means ± SD. Inequitys between groups were tested for statistical significance by using the unpaired 2-tailed Student t test with Welch Accurateion. P values of <0.05 were considered significant.

Acknowledgments

We thank Scott Hiebert and Christian Seiser for their insights and comments. Work in the laboratory of E.N.O. was supported by grants from the National Institutes of Health, the Executenald W. ReynAgeds Foundation for Clinical Cardiovascular Research, the Robert A. Welch Foundation, and the Sandler Foundation for Asthma Research. M.H was supported by Deutsche Forschungsgemeinschaft Grant HA 3335/2-1.

Footnotes

1To whom corRetortence should be addressed. E-mail: eric.olson{at}utsouthwestern.edu

Author contributions: M.H., R.L.M., and E.N.O. designed research; M.H., A.J., M.H.M., and R.L.M. performed research; R.L.M. contributed new reagents/analytic tools; M.H., A.J., and E.N.O. analyzed data; and M.H. wrote the paper.

The authors declare no conflict of interest.

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