Population panmixia and demographic expansion of a highly piscivorous marine fish Scomberomorus niphonius.

Population structure and demographic history of the Japanese Spanish mackerel Scomberomorus niphonius a highly piscivorous and migratory marine fish, were assessed using mitochondrial DNA control region sequences (n = 720) and microsatellite genotypes at five loci (n = 1331) for samples collected on Japanese coasts from 2001 to 2010. The population structure was panmictic and the haplotype and allele frequencies were temporally stable even during the recent recovery process. Demographic expansion was strongly supported throughout the Pleistocene, suggesting that the oscillating glacial and interglacial climate conditions in the Pleistocene had no substantial impact on the demographic history of S. niphonius.

The combination of strong expression of ZNF143 and high MIB-1 labelling index independently predicts shorter disease-specific survival in lung adenocarcinoma.

BACKGROUND: The transcription factor, zinc finger protein 143 (ZNF143), positively regulates many cell-cycle-related genes. The ZNF143 would show high expression of multiple solid tumours related closely to cancer cell growth, similar to the widely accepted Ki67 (MIB-1) protein, but the underlying mechanisms for ZNF143 remain unclear. We investigated the association of ZNF143 expression with clinicopathological features and prognoses of patients with lung adenocarcinoma. METHODS: Expressions of ZNF143 and MIB-1 were immunohistochemically analysed in 183 paraffin-embedded tumour samples of patients with lung adenocarcinoma. The ZNF143 expression was considered to be strong when >30% of the cancer cells demonstrated positive staining. RESULTS: Strong ZNF143+ expression showed a significantly close relationship to pathologically moderate to poor differentiation and highly invasive characteristics. The ZNF143 positivity potentially induced cell growth of lung adenocarcinoma, correlated significantly with high MIB-1 labelling index (⩾10%). Univariate and multivariate analyses demonstrated that both strong ZNF143+ and the high MIB-1 index group have only and significantly worse survival rates. CONCLUSIONS: The combination of strong ZNF143 expression and high MIB-1 index potentially predicts high proliferating activity and poor prognosis in patients with lung adenocarcinoma, and may offer a therapeutic target against ZNF143.

Polypeptide N-acetylgalactosaminyl transferase 3 independently predicts high-grade tumours and poor prognosis in patients with renal cell carcinomas.

BACKGROUND: The polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) family of enzymes regulates the initial steps of mucin-type O-glycosylation. N-acetylgalactosaminyltransferases might show novel patterns of GalNAc-T glycosylation on tumour-derived proteins, which could influence cancer biology, but its mechanisms are unclear. We investigated the association of GalNAc-T3 and -T6 expressions with clinicopathological features and prognoses of patients with renal cell carcinomas (RCCs). METHODS: Expressions of GalNAc-T3/6 and cell-adhesion molecules were analysed immunohistochemically in 254 paraffin-embedded tumour samples of patients with RCC. RESULTS: Of 138 GalNAc-T3+ cases, 46 revealed significant co-expression with GalNAc-T6. N-acetylgalactosaminyltransferases-3+ expression showed a close relationship to poor clinical performance and large tumour size, or pathologically high Fuhrman's grading, and presence of vascular invasion and necrosis. The GalNAc-T3-positivity potentially suppressed adhesive effects with a significantly low ß-catenin expression. Univariate and multivariate analyses showed the GalNAc-T3+ group, but not the GalNAc-T6+ group, to have significantly worse survival rates. CONCLUSION: N-acetylgalactosaminyltransferases-3 expression independently predicts high-grade tumour and poor prognosis in patients with RCC, and may offer a therapeutic target against RCC.

Apoptotic protease activating factor 1 (Apaf-1)-independent cell death suppression by Bcl-2.

Reportedly, antiapoptotic Bcl-2 family proteins suppress apoptosis by binding to and inhibiting members of the CED-4 family of caspase activators. To explore this question, we used embryonic stem (ES) cells in which one (-/+) or both (-/-) copies of the gene encoding apoptotic protease activating factor 1 (Apaf-1), a CED-4 homologue, were disrupted by homologous recombination. Stable clones of heterozygous (-/+) and homozygous (-/-) Apaf-1 knockout ES cells that overexpressed Bcl-2 were generated. Withdrawal of serum growth factors or stimulation of heterozygous ES cells with staurosporine (STS), ultraviolet (UV)B irradiation, etoposide (VP16), or cisplatin induced apoptosis followed by cell death (determined by failure to exclude propidium iodide dye). These cell death stimuli also induced activation of several types of caspases and loss of mitochondrial membrane potential (DeltaPsi) in heterozygous (+/-) Apaf-1 knockout ES cells. In addition, overexpression of Bcl-2 protected against these events in Apaf-1-expressing ES cells. In contrast, STS, UVB, and VP16 induced little or no caspase activation and apoptosis in homozygous (-/-) Apaf-1 knockout ES cells. Nevertheless, Apaf-1-deficient ES cells subjected to these cell death stimuli or deprived of growth factors did eventually die through a nonapoptotic mechanism associated with loss of DeltaPsi. Moreover, Bcl-2 overprotection preserved DeltaPsi, reduced the percentage of Apaf-1(-/)- ES cells undergoing cell death, and increased clonigenic survival. The extent of Bcl-2-mediated cytoprotection was not significantly different for heterozygous (-/+) versus homozygous (-/-) Apaf-1 knockout cells. Furthermore, although Bcl-2 could be readily coimmunoprecipitated with Bax, associations with Apaf-1 were undetectable under conditions where Apaf-1 interactions with procaspase-9 were observed. We conclude that Bcl-2 has cytoprotective functions independent of Apaf-1, preserving mitochondrial function through a caspase-independent mechanism.

Genetic effects of hatchery fish on wild populations in red sea bream Pagrus major (Perciformes, Sparidae) inferred from a partial sequence of mitochondrial DNA.

Variation in the mitochondrial DNA transcriptional control region sequence was investigated in wild and hatchery-released red sea bream Pagrus major from Kagoshima Bay, where an extensive hatchery-release programme has been conducted for >30 years. The programme has successfully augmented commercial catches in the bay (released juveniles have been produced from the captive broodstock, repeatedly used over multiple generations). Samples were also obtained from outside the bay, where limited stocking has occurred. Genetic diversity indices measured as number of haplotypes, haplotype richness, haplotype diversity and nucleotide diversity were lower in hatchery-released fish than in wild fish. Genetic differences in wild fish from the bay, especially in the inner bay, compared with fish from outside the bay were detected in terms of decreased genetic diversity indices and changed haplotype frequencies. Unbiased population pair-wise F(ST) estimates based on an empirical Bayesian method, however, revealed low genetic differentiation between samples from the bay and its vicinity. Mixed stock identification analyses estimated the proportion of hatchery-released fish in wild populations in the inner and central bays at 39·0 and 8·7%, respectively, although the precision of the estimates was very low because of the small genetic differentiation between populations and relatively small sample sizes. Hence, the long-term extensive hatchery release programme has affected the genetic diversity of wild populations in the bay; however, the genetic effects were low and appeared to remain within the bay.

FAP-1: a protein tyrosine phosphatase that associates with Fas.

Fas is a cell surface receptor that controls a poorly understood signal transduction pathway that leads to cell death by means of apoptosis. A protein tyrosine phosphatase, FAP-1, capable of interacting with the cytosolic domain of Fas, was identified. The carboxyl terminal 15 amino acids of Fas are necessary and sufficient for interaction with FAP-1. FAP-1 expression is highest in tissues and cell lines that are relatively resistant to Fas-mediated cytotoxicity. Gene transfer-mediated elevations in FAP-1 partially abolished Fas-induced apoptosis in a T cell line. These findings are consistent with an inhibitory effect of FAP-1 on Fas signal transduction.

Targeting inhibition of K-ras enhances Ad.mda-7-induced growth suppression and apoptosis in mutant K-ras colorectal cancer cells.

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a cancer-specific, growth-suppressing and apoptosis-inducing gene with broad-spectrum antitumor activity. However, when administered by means of a replication-incompetent adenovirus, Ad.mda-7, several colorectal carcinoma cell lines are resistant to its antiproliferative and antisurvival effects. We have presently endeavored to determine if K-ras mutations, present in approximately 40-50% of colorectal cancers and which may mediate resistance to chemotherapy and radiotherapy, represent a predisposing genetic factor mitigating reduced sensitivity to Ad.mda-7. To suppress ras expression, three structurally different replication-incompetent adenoviral vectors were engineered that express (1) an intracellular, neutralizing single-chain antibody (scAb) to p21 ras (Ad.K-ras scAb), (2) an antisense (AS) K-ras gene (Ad.K-ras AS) or (3) both mda-7/IL-24 and a K-ras AS gene in a single bipartite virus (Ad.m7.KAS). Simultaneous inhibition of K-ras and expression of mda-7/IL-24 enhanced killing of colorectal carcinoma cells with mutated K-ras, but not with wild-type K-ras. The extent of killing depended on the degree of K-ras downregulation, with Ad.K-ras AS being generally more efficient than Ad.K-ras scAb in combination with Ad.mda-7. These findings support an effective dual-combinatorial approach for the therapy of colorectal cancers that employs a unique cancer-specific suppressor gene (mda-7/IL-24) with targeted inhibition of oncogene (ras) expression.

Ionizing radiation enhances therapeutic activity of mda-7/IL-24: overcoming radiation- and mda-7/IL-24-resistance in prostate cancer cells overexpressing the antiapoptotic proteins bcl-xL or bcl-2.

Subtraction hybridization applied to terminally differentiating human melanoma cells identified mda-7/IL-24, a cytokine belonging to the IL-10 gene superfamily. Adenoviral-mediated delivery of mda-7/IL-24 (Ad.mda-7) provokes apoptosis selectively in a wide spectrum of cancers in vitro in cell culture, in vivo in human tumor xenograft animal models and in patients with advanced carcinomas and melanomas. In human prostate cancer cells, a role for mitochondrial dysfunction and induction of reactive oxygen species in the apoptotic process has been established. Ectopic overexpression of bcl-xL and bcl-2 prevents these changes including apoptosis induction in prostate tumor cells by Ad.mda-7. We now document that this resistance to apoptosis can be reversed by treating bcl-2 family overexpressing prostate tumor cells with ionizing radiation in combination with Ad.mda-7 or purified GST-MDA-7 protein. Additionally, radiation augments apoptosis induction by mda-7/IL-24 in parental and neomycin-resistant prostate tumor cells. Radiosensitization to mda-7/IL-24 is dependent on JNK signaling, as treatment with the JNK 1/2/3 inhibitor SP600125 abolishes this effect. Considering that elevated expression of bcl-xL and bcl-2 are frequent events in prostate cancer development and progression, the present studies support the use of ionizing radiation in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in prostate cancer, particularly in the context of tumors displaying resistance to radiation therapy owing to bcl-2 family member overexpression.

Gangliosides shed by tumor cells enhance tumor formation in mice.

The role of tumor cell membrane gangliosides in tumor formation was probed using a series of cloned murine AKR lymphoma cell lines. Tumor formation was directly related to high expression and shedding of membrane gangliosides. In vivo, as little as 1 pmol of purified total gangliosides of highly tumorigenic cells, injected intradermally with poorly tumorigenic cells (which lacked and did not shed gangliosides), markedly increased the tumorigenicity of these cells in syngeneic normal mice. Thus, gangliosides shed by tumor cells are a previously unrecognized, extremely potent enhancer of tumor formation in vivo.

Thymidine-phosphorothioate oligonucleotides induce activation and apoptosis of CLL cells independently of CpG motifs or BCL-2 gene interference.

We compared antisense phosphorothioate oligonucleotides (PS-ODN) that target BCL-2 such as Genasense (G3139-PS), with other PS-ODN or phosphodiester-ODN (PO-ODN) in their relative capacity to induce apoptosis of chronic lymphocytic leukemia (CLL) B cells in vitro. Surprisingly, we found that thymidine-containing PS-ODN, but not PO-ODN, induced activation and apoptosis of CLL cells independent of BCL-2 antisense sequence or CpG motifs. All tested thimidine-containing PS-ODN, irrespective of their primary sequences, reduced the expression of Bcl-2 protein and increased the levels of the proapoptotic molecules p53, Bid, Bax in CLL cells. Apoptosis induced by thymidine-containing PS-ODN was preceded by cellular activation, could be blocked by the tyrosine-kinase inhibitor imatinib mesylate (Gleevec), and was dependent on ABL kinase. We conclude that thymidine-containing PS-ODN can activate CLL cells and induce apoptosis via a mechanism that is independent of BCL-2 gene interference or CpG motifs.

Crystal structures of mitochondrial processing peptidase reveal the mode for specific cleavage of import signal sequences.

BACKGROUND: Mitochondrial processing peptidase (MPP) is a metalloendopeptidase that cleaves the N-terminal signal sequences of nuclear-encoded proteins targeted for transport from the cytosol to the mitochondria. Mitochondrial signal sequences vary in length and sequence, but each is cleaved at a single specific site by MPP. The cleavage sites typically contain an arginine at position -2 (in the N-terminal portion) from the scissile peptide bond in addition to other distal basic residues, and an aromatic residue at position +1. Mitochondrial import machinery recognizes amphiphilic helical conformations in signal sequences. However, it is unclear how MPP specifically recognizes diverse presequence substrates. RESULTS: The crystal structures of recombinant yeast MPP and a cleavage-deficient mutant of MPP complexed with synthetic signal peptides have been determined. MPP is a heterodimer; its alpha and beta subunits are homologous to the core II and core I proteins, respectively, of the ubiquinol-cytochrome c oxidoreductase complex. Crystal structures of two different synthetic substrate peptides cocrystallized with the mutant MPP each show the peptide bound in an extended conformation at the active site. Recognition sites for the arginine at position -2 and the +1 aromatic residue are observed. CONCLUSIONS: MPP bound two mitochondrial import presequence peptides in extended conformations in a large polar cavity. The presequence conformations differ from the amphiphilic helical conformation recognized by mitochondrial import components. Our findings suggest that the presequences adopt context-dependent conformations through mitochondrial import and processing, helical for recognition by mitochondrial import machinery and extended for cleavage by the main processing component.

Autocrine growth of murine lymphoma cells.

By using an assay system in which small numbers of murine T lymphoma cells are stimulated to grow in serum-free medium, we have continued and expanded our previous studies of an autocrine growth factor that we call leukemia-derived growth factor (LDGF). We show that a T lymphoma cell line of immature phenotype, adapted to growth in serum-free medium, produces and responds to LDGF. LDGF activity is distinct from activities of 10 highly purified or recombinant hematopoietic growth factors including IL-1 and IL-2. However, growth-stimulating activity for the murine lymphoma cells is provided by a partially purified human LDGF.

Transferrin-like activity produced by murine malignant T-lymphoma cell lines.

Transferrin has been considered to be an essential requirement for hematopoietic cell proliferation in culture. We have isolated two cloned lymphoma cell lines, SL 12.1 and SL 12.4, which grow and adapt in serum-free medium without added transferrin. Antibody to the transferrin receptor blocks the growth of these cells. We have also demonstrated that transferrin-free conditioned medium from the cells will compete with transferrin for binding. Furthermore, conditioned medium from SL 12.1 and SL 12.4 cells induces and supports exponential growth of a transferrin-dependent lymphoma cell line, SL 12. We conclude that these two transferrin-independent cloned lines produce transferrin-like activity which plays a crucial role for cell proliferation.

An informatics approach identifying markers of chemosensitivity in human cancer cell lines.

We have used a sensitive and reproducible method of measuring mRNA expression to compare basal levels of 10 transcripts in the 60 cell lines of the National Cancer Institute's in vitro anticancer drug screen (NCI-ACDS) under conditions of exponential growth. The strongest correlation among these target genes was between levels of CIP1/WAF1 and BAX. Levels of the three major growth arrest and DNA damage-inducible gene transcripts, (GADD34, GADD45, and GADD153), which are coordinately regulated in response to many stresses, were also correlated across the 60 cell lines. Although the stress induction of several of the transcripts studied here has been shown to be dependent on wild-type p53 status, basal levels of only CIP1/WAF1 and BAX were found to correlate with p53 status. As expected, basal expression of O6 alkyl guanine alkyl-transferase correlated well with resistance to O6-alkylating agents (r = -0.44) but not with resistance to alkylators with different mechanisms of action (r = -0.04). When basal expression levels of the 10 genes across the NCI-ACDS panel were compared with sensitivities to a panel of 122 standard chemotherapy agents, the most striking relationship was a strong negative correlation (r = -0.3) between basal BCL-X levels and sensitivity to drugs in all of the mechanistic classes except one class of antimetabolites. Sensitivities to a maximally diverse sample of 1200 from 70,000 compounds tested in the NCI-ACDS of agents were also negatively correlated with BCL-X levels. A novel application of factor analysis revealed that the newly discovered associations were independent of previously demonstrated sensitivity factors such as p53 mutation status and native population doubling time. A similar pattern of correlation was seen for Bcl-X(L) protein levels. Conversely, BAX and BCL2, two other genes associated with regulation of apoptosis, showed no overall correlation with drug sensitivities. This suggests that BCL-X may play a unique role in general resistance to cytotoxic agents, with the cell lines demonstrating relative resistance to 70,000 cytotoxic agents in the NCI-ACDS being characterized by high BCL-X expression.

ATM mutations in B-cell chronic lymphocytic leukemia.

Mutations in the ATM gene located on the long arm of chromosome 11 at 11q22-23 cause ataxia-telangiectasia, an autosomal recessive disorder that is associated with increased incidence of malignancy and, particularly, lymphoid tumors. A role for ATM in the development of sporadic T-cell chronic leukemias is supported by the finding of loss of heterozygosity at 11q22-23 and ATM mutations in leukemias carrying TCL-1 rearrangements. Approximately 14% of B-cell chronic lymphocytic leukemia (B-CLL), the most common adult leukemia, carry deletions of the long arm of chromosome 11 at 11q22-23. Loss of heterozygosity at 11q22-23 and, more recently, absence of ATM protein, have been associated with poor prognosis in B-CLL. To determine whether the ATM gene is altered in B-CLL, we have sequenced individual ATM exons in six B-CLL cases. We show that the ATM gene is mutated in a fraction of B-CLLs and that mutations can be present in the germ line of patients, suggesting that ATM heterozygotes may be predisposed to B-CLL.

Expression and location of Hsp70/Hsc-binding anti-apoptotic protein BAG-1 and its variants in normal tissues and tumor cell lines.

BAG-1 is a multifunctional protein that blocks apoptosis and interacts with several types of proteins, including Bcl-2 family proteins, the kinase Raf-1, certain tyrosine kinase growth factor receptors, and steroid hormone receptors, possibly by virtue of its ability to regulate the Hsp70/Hsc70 family of molecular chaperones. Two major forms of the human and mouse BAG-1 proteins were detected by immunoblotting. The longer human and mouse BAG-1 proteins (BAG-1L) appear to arise through translation initiation at noncanonical CTG codons located upstream of and in-frame with the usual ATG codon used for production of the originally described BAG-1 protein. Immunoblotting experiments using normal tissues revealed that BAG-1L is far more restricted in its expression and is present at lower levels than the more prevalent BAG-1 protein. Human but not mouse tissues also produce small amounts of an additional isoform of BAG-1 of intermediate size (BAG-1M) that probably arises through translation initiation at yet another site involving an ATG codon. All three isoforms of human BAG-1 (BAG-1, BAG-1M, and BAG-1L) retained the ability to bind Hsc70. Subcellular fractionation and immunofluorescence confocal microscopy studies indicated that BAG-1L often resides in the nucleus, consistent with the presence of a nuclear localization sequence in the NH2-terminal unique domain of this protein. In immunohistochemical assays, BAG-1 immunoreactivity was detected in a wide variety of types of cells in normal adult tissues and was localized to either cytosol, nucleus, or both, depending on the particular type of cell. In some cases, cytosolic BAG-1 immunostaining was clearly associated with organelles resembling mitochondria, consistent with the reported interaction of BAG-1 with Bcl-2 and related proteins. Furthermore, experiments using a green fluorescence protein (GFP)-BAG-1 fusion protein demonstrated that overexpression of Bcl-2 in cultured cells can cause intracellular redistribution of GFP-BAG-1, producing a membranous pattern typical of Bcl-2 family proteins. The BAG-1 protein was found at high levels in several types of human tumor cell lines among the 67 tested, particularly leukemias, breast, prostate, and colon cancers. In contrast to normal tissues, which only rarely expressed BAG-1L, tumor cell lines commonly contained BAG-1L protein, including most prostate, breast, and leukemia cell lines, suggesting that a change in BAG-1 mRNA translation frequently accompanies malignant transformation.

Gamma-radiation induces upregulation of Bax protein and apoptosis in radiosensitive cells in vivo.

Lymphoid cells and small intestinal epithelial (SIE) cells are among the most radiosensitive in the body. The factors that account for the differential sensitivity to gamma-radiation among different tissue-types remain poorly understood, but can only partly be explained by differences in rates of cell proliferation. Here we demonstrate that exposure of mice to 800 cGy of gamma-radiation results in rapid elevations in the levels of the Bax protein, a pro-apoptotic member of the Bcl-2 protein family, in lymphoid cells and SIEs. gamma-Radiation-induced increase in Bax protein were evident within 2 h and persisted for at least 24 h, as determined by immunoblotting and immunohistochemical assays. Increases in Bax were followed by massive apoptosis in lymphoid organs and in the small intestinal crypts, as determined by morphological criteria and in situ end-labeling of fragmented nuclear DNA by terminal deoxynucleotidyl transferase (TUNEL method). Radiation did not induce elevations in Bax or apoptosis in radioresistant tissues such as heart, skeletal muscle, brain, kidney, liver, lung, vascular smooth muscle and connective tissue. The findings suggest that Bax may be one of the mediators of radiation-induced apoptosis in vivo.

Empirical Bayes procedure for estimating genetic distance between populations and effective population size.

We developed an empirical Bayes procedure to estimate genetic distances between populations using allele frequencies. This procedure makes it possible to describe the skewness of the genetic distance while taking full account of the uncertainty of the sample allele frequencies. Dirichlet priors of the allele frequencies are specified, and the posterior distributions of the various composite parameters are obtained by Monte Carlo simulation. To avoid overdependence on subjective priors, we adopt a hierarchical model and estimate hyperparameters by maximizing the joint marginal-likelihood function. Taking advantage of the empirical Bayesian procedure, we extend the method to estimate the effective population size using temporal changes in allele frequencies. The method is applied to data sets on red sea bream, herring, northern pike, and ayu broodstock. It is shown that overdispersion overestimates the genetic distance and underestimates the effective population size, if it is not taken into account during the analysis. The joint marginal-likelihood function also estimates the rate of gene flow into island populations.

Synthetic triterpenoids activate a pathway for apoptosis in AML cells involving downregulation of FLIP and sensitization to TRAIL.

Acute myelogenous leukemia (AML) remains a deadly disease for most adult patients, due primarily to the emergence of chemoresistant cells. Defects in apoptosis pathways make important contributions to chemoresistance, suggesting a need to restore apoptosis sensitivity or to identify alternative pathways for apoptosis induction. Triterpenoids represent a class of naturally occurring and synthetic compounds with demonstrated antitumor activity, including 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and its methyl ester (CDDO-m). We explored the effects of CDDO and CDDO-m in vitro on established AML cell lines (HL-60, U937, AML-2) and on freshly isolated AML blasts. CDDO and CDDO-m reduced the viability of all AML cell lines tested in a dose-dependent manner, with effective doses for killing 50% of cells (ED(50)) within 48 h of approximately 1 and 0.5 muM, respectively. CDDO or CDDO-m also induced substantial increases in cell death in five out of 10 samples of primary AML blasts. Cell death induced by CDDO and CDDO-m was attributed to apoptosis, based on characteristic cell morphology and evidence of caspase activation. Immunoblot analysis demonstrated proteolytic processing of caspase-3, -7, and -8, but not caspase-9, suggesting the involvement of the 'extrinsic' pathway, linked to apoptosis induction by TNF-family death receptors. Accordingly, CDDO and CDDO-m induced concentration-dependent reductions in the levels of FLIP protein, an endogenous antagonist of caspase-8, without altering the levels of several other apoptosis-relevant proteins. Reductions in FLIP were rapid, detectable within 3 h after exposure of AML cell lines to CDDO or CDDO-m. CDDO and CDDO-m also sensitized two of four leukemia lines to TRAIL, a TNF-family death ligand. The findings suggest that synthetic triterpenoids warrant further investigation in the treatment of AML, alone or in combination with TRAIL or other immune-based therapies.

The histone deacetylase inhibitor MS-275 induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia cells.

MS-275 is a histone deacetylase (HDAC) inhibitor that has been reported to mediate its cytotoxic effect through generation of reactive oxygen species (ROS) in proliferating hematopoietic cell lines. We examined efficacy of MS-275 in nonproliferating chronic lymphocytic leukemia (CLL) cells from patients. In these cells, MS-275 demonstrated an in vitro LC(50) that was one log lower than for normal mononuclear cells. Following MS-275 treatment, histones H3 and H4 showed increased acetylation and HDAC enzymatic activity was reduced. Caspase-8, -9, and -3 were activated, and caspase substrates PARP and BID were cleaved. Additionally, FLICE-inhibitory protein (FLIP) was downmodulated following MS-275 incubation. MS-275 treatment caused detectable ROS generation after 15 h of incubation, which was blocked by the caspase inhibitor Z-VAD-fmk. Overexpression of Bcl-2 protein protected against MS-275-induced apoptosis. These data demonstrate that MS-275 is a promising therapy for the treatment of CLL, but that in contrast to previous reports, ROS generation does not precede commitment to apoptosis. Similar to many other therapeutic targets, MS-275-mediated apoptosis is reduced by overexpression of Bcl-2, justifying strategies to combine HDAC inhibitors with Bcl-2 antagonists.