Supplementary Table S1 from Histone Deacetylase Inhibitors (HDI) Cause DNA Damage in Leukemia Cells: A Mechanism for Leukemia-Specific HDI-Dependent Apoptosis?

Regulating the transition of cells such as T lymphocytes from quiescence (G(0)) into an activated, proliferating state involves initiation of cellular programs resulting in entry into the cell cycle (proliferation), the growth cycle (blastogenesis, cell size) and effector (functional) activation. We show the first proteomic analysis of protein interaction networks activated during entry into the first cell cycle from G(0). We also provide proof of principle that blastogenesis and proliferation programs are separable in primary human T cells. We employed a proteomic profiling method to identify large-scale changes in chromatin/nuclear matrix-bound and unbound proteins in human T lymphocytes during the transition from G(0) into the first cell cycle and mapped them to form functionally annotated, dynamic protein interaction networks. Inhibiting the induction of two proteins involved in two of the most significantly upregulated cellular processes, ribosome biogenesis (eIF6) and hnRNA splicing (SF3B2/SF3B4), showed, respectively, that human T cells can enter the cell cycle without growing in size, or increase in size without entering the cell cycle.

DNA replication is tightly regulated, but paradoxically there is reported to be an excess of MCM DNA replication proteins over the number of replication origins. Here, we show that MCM levels in primary human T cells are induced during the G 0 →G 1 transition and are not in excess in proliferating cells. The level of induction is critical as we show that a 50% reduction leads to increased centromere separation, premature chromatid separation (PCS) and gross chromosomal abnormalities typical of genomic instability syndromes. We investigated the mechanisms involved and show that a reduction in MCM levels causes dose-dependent DNA damage involving activation of ATR & ATM and Chk1 & Chk2. There is increased DNA mis-repair by non-homologous end joining (NHEJ) and both NHEJ and homologous recombination are necessary for Mcm7-depleted cells to progress to metaphase. Therefore, a simple reduction in MCM loading onto DNA, which occurs in cancers as a result of aberrant cell cycle control, is sufficient to cause PCS and gross genomic instability within one cell cycle.

We investigated functional epigenetic changes that occur in primary human T lymphocytes during entry into the cell cycle and mapped these at the single-nucleosome level by ChIP-chip on tiling arrays for chromosomes 1 and 6. We show that nucleosome loss and flanking active histone marks define active transcriptional start sites (TSSs). Moreover, these signatures are already set at many inducible genes in quiescent cells prior to cell stimulation. In contrast, there is a dearth of the inactive histone mark H3K9me3 at the TSS, and under-representation of H3K9me2 and H3K9me3 defines the body of active genes. At the DNA level, cytosine methylation (meC) is enriched for nucleosomes that remain at the TSS, whereas in general there is a dearth of meC at TSSs. Furthermore, a drop in meC also marks 3' transcription termination, and a peak of meC occurs at stop codons. This mimics the 3' nucleosomal distribution in yeast, which we show does not occur in human T cells.

We identified a G0→G1 commitment point in primary human T cells that controls entry into the cell cycle from quiescence. We demonstrated proof of principle that cellular pathways regulating cell cycle progression and effector functions that normally coincide during CD3/CD28 stimulation can be uncoupled experimentally. We have now used systems biology approaches to identify nuclear protein networks in primary human T cells that are regulated during the transition from quiescence into the cell cycle (G0→G1→S-phase). First we sequenced proteins that became bound to chromatin & nuclear matrix in G1 but were not bound in G0 and vice versa by mass spectrometry. Bioinformatic analysis identified 76 proteins specifically bound in G0 not G1 and 254 bound in G1 not G0. 179 of the 254 proteins bound in G1 not G0 (i.e. dynamic protein changes) were mapped to the 55,000 human protein interaction dataset. These are involved in numerous cellular functions, including epigenetics, transcription, RNA splicing & transport, and others. Cell cycle regulated chromatin/matrix binding of a subset was verified by western blotting (2/2 bound in G0 not G1 and 22/23 bound in G1 not G0). One of the proteins induced and bound in G1 was SAP145 (SF3B2). This is a component of the ubiquitous SF3b RNA splicing complex, involved in both major (U2-type) and minor (U12-type) spliceosomes. Since SAP145 is induced during G1 we investigated whether there was a role for SAP145 in regulating cell cycle progression. T cells depleted of SAP145 by siRNA enter G1 from G0 but progress poorly through S phase and die, probably by apoptosis. The same occurs if another component of the SF3B complex, SAP49 (SF3B4) is depleted with siRNA, indicating that the effect is due to depleting the complex rather than the individual SF3B protein. Proteins that are induced during G1 by CD3/CD28 stimulation e.g. cyclin D3, Cdc6 and cdc2 are produced normally when SAP145 is depleted, suggesting that their pre-mRNAs are spliced normally. In contrast, the expression of p107 and cyclin A2 are reduced markedly when SAP145 is depleted. Therefore, a systems biology approach to analysing cell cycle transitions identifies the splicing protein, SAP145 as rate-limiting for the G1 →S phase cell cycle transition but not for the transition from G0→G1.

Normal DNA replication must be accurate and occur only once per cell cycle. Sites of DNA replication are specified by binding the origin recognition complex, that includes minichromosome maintenance (MCM) proteins. Paradoxically, in higher eukaryotes MCM proteins are present in >20 fold excess of that required for DNA replication. They are also downregulated by elevated expression of proteins such as cyclin E that occurs in cancers, including AML and breast cancer. We investigated why human cells need “excess” MCM proteins and whether the reduction of MCM protein levels might contribute to a malignant phenotype. We determined the consequences of reducing the levels of MCM proteins in primary human T cells in which cell cycle controls and DNA damage responses are normal. Mass spectrometry sequencing of chromatin/nuclear matrix-bound proteins and western blotting identified that Mcm7 is not present in quiescent, normal primary human T cells. Mcm7 is induced in mid G1after the G0→G1 commitment point, the point beyond which T cells are committed to entering the cell cycle. Reduction of Mcm7 with siRNA to <5% of normal during G0→G1→S-phase reduces chromatin-binding of each of the MCM proteins that form the DNA helicase. However, these cells still enter S-phase and replicate DNA. Reducing MCM levels by titrating siRNA causes dose-dependent DNA-damage responses involving activation of ATR & ATM and Chk1 & Chk2. However, cells depleted of Mcm7 do not undergo apoptosis, rather reducing MCM levels even by 50% causes gross non-clonal chromosomal abnormalities normally found in genomic instability syndromes. M-FISH identified chromosome translocations, as well as loss and gain of individual chromosomes, which can occur individually or together in the same cell. Reducing MCM levels also causes misrepair by non-homologous end joining (NHEJ), and both NHEJ and homologous recombination (HR) are necessary for chromosomal abnormalities to occur. Therefore, “excess” MCM proteins that are present in a normal, proliferating cell are necessary for maintaining genome stability and reduction of MCM loading onto DNA that occurs in cancers is sufficient to cause genomic instability.

p73 is a member of the p53-family of transcription factors (p73, p53 and p63). A truncated form of p73 (ΔNp73) also exists that lacks the transcriptional activation domain and inhibits both p53 & p73. However, ΔNp73 transcripts have not been reported in normal tissues or peripheral blood. We now show for the first time that ΔNp73 mRNA and protein are not present in quiescent (G0), primary human T cells but are induced post the G0→G1 cell cycle commitment point following stimulation with anti-CD3/CD28 or PMA/ionomycin. The ΔNp73 transcript could be produced either by activation of a promoter in intron 3 or from the 5′ P1 promoter by alternative splicing. Bisulfite sequencing shows that the intron 3 promoter is hypermethylated in T cells in G0 and throughout the cell cycle while the 5′ P1 promoter remains largely unmethylated. 5′-RACE results with quiescent and stimulated T cells verify that ΔNp73 transcripts originate from the P1 promoter. Additionally, RT-PCR analyses show that the transcript originating from the P1 promoter is present in G0 and mature ΔNp73 mRNA is produced by cell cycle-dependent splicing during the G0→G1 transition. We investigated the function of ΔNp73 during G0→G1 by inhibiting its induction with two different siRNAs. Microarray analyses show that expression of mRNA encoding a number of cell cycle regulators like E2F1, TCFL2 and CDT1 during G0→G1 are dependent on ΔNp73 and ΔNp73 represses the expression of the pro-apoptotic PUMA, a known p73 target. We conclude that ΔNp73 is produced in normal, human T cells during cell cycle entry and regulates normal gene expression. Moreover, the intron 3 promoter is hypermethylated, the ΔNp73 transcript originates at the P1 promoter and ΔNp73 mRNA is produced by cell cycle-dependent splicing.

Immune dysfunction is a hallmark of B-cell chronic lymphocytic leukemia (B-CLL) which occurs through loss of normal cell function as the malignant clone expands, as a result of therapy or because of immunoregulatory properties of the tumor itself. It has previously been shown that B-CLL cells are poor stimulators of the allogeneic mixed lymphocyte reaction (MLR) and we first determined whether this is due to lack of stimulatory activity or active immunosuppression by examining the effect of B-CLL contact and tumor supernatant (TSN) on a 3rd party MLR. Incorporation of B-CLL cells in a 5 day MLR inhibited 3H proliferation by responders in 2/10 cases, whereas TSN inhibited in all 10 cases. Studies in which normal T cells were stimulated by CD3/28 beads for 72 hours in the absence or presence of TSN showed a reduction in cell cycle entry measured by PI and FITC staining with 26+/−4.6% and 13.7+/−4.3% of cells in S +G2M in the absence and presence of TSN respectively (p<0.0001). Studies performed using CFSE labelled normal T cells showed that TSN reduced the number of T cells undergoing one or more cell divisions from a mean of 81.8+/−1.65% to 58.2+/−4.4% (p=0.0072). It is known that T cells in B-CLL have an acquired defect in CD40L expression, which has been ascribed to downregulation by CD40 present on tumor cells. Our experiments confirm that this defect is reversible since purification of B-CLL T cells restores activation induced CD40L upregulation to normal. We further demonstrate that B-CLL TSN from all 17 patients tested inhibits CD40L upregulation by normal T cells in response to PMA and ionomycin or CD3/28 beads (to a mean of 51%+/−5.6%, p<0.0001, of those activated in the absence of TSN) and a parallel inhibition of IL-2 secretion (correlation with CD40L inhibition: p=0.006, r2 = 0.54). In addition to the effects of TSN on T proliferation and activation, B-CLL TSN also induced Th2 polarisation of normal T cells. When activated using CD3/28 beads in control medium, normal T cells show an increase in IL-2, γ-interferon and TNF-α secretion consistent with the expected Th1 response. When incubated in TSN however, 10 and 1000 fold increases in IL-4 and IL6 release were observed respectively consistent with a shift to a Th2 response. B-CLL cells are known to secrete a number of cytokines and in order to determine which might be responsible for the observed effects a number were assayed either by enzyme-linked or cytokine bead array assay. The effects of TSN were not due to TGF-β , IL-10 or soluble CD40 and depletion of soluble CD25 using bead conjugated anti-CD25 had no effect on the immunosuppressive activity. High levels of IL-6 were detected in TSN from all cases studied (n=5). When normal T cell were activated in TSN, a 100 fold further increase in IL-6 level was observed suggesting that this cytokine may be responsible for at least some of the observed effects of TSN. Antibody neutralization of the IL-6 in TSN demonstrated an increase in both Th1 cytokine production and CD40L expression. Furthermore, addition of recombinant IL-6 to T cells activated in media inhibited CD40L upregulation. In summary, B-CLL cells secrete factor(s) which inhibit T cell activation and proliferation and promote Th2 polarisation. These factors might contribute to the disease phenotype by impairing T cell responses to infection, predisposing to autoimmunity and promoting the growth of the malignant clone through the action of IL-6.

To investigate the potential functional cooperation between p27(Kip1) and p130 in vivo, we generated mice deficient for both p27(KiP1) and p130. In p27(Kip1-/-);p130(-/-) mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27(Kip1-/-) counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27(Kip1-/-);p130(-/-) animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27(Kip1) in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27(Kip1-/-) splenocytes. The finding that the p27(Kip1-/-);p130(-/-) splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27(Kip1) to regulate hematopoietic cell proliferation in vivo.

Abstract Histone deacetylase inhibitors (HDI) increase gene expression through induction of histone acetylation. However, it remains unclear whether increases in specific gene expression events determine the apoptotic response following HDI administration. Herein, we show that a variety of HDI trigger in hematopoietic cells not only widespread histone acetylation and DNA damage responses but also actual DNA damage, which is significantly increased in leukemic cells compared with normal cells. Thus, increase in H2AX and ataxia telangiectasia mutated (ATM) phosphorylation, early markers of DNA damage, occurs rapidly following HDI administration. Activation of the DNA damage and repair response following HDI treatment is further emphasized by localizing DNA repair proteins to regions of DNA damage. These events are followed by subsequent apoptosis of neoplastic cells but not normal cells. Our data indicate that induction of apoptosis by HDI may result predominantly through accumulation of excessive DNA damage in leukemia cells, leading to activation of apoptosis. (Mol Cancer Res 2006;4(8):563–73)