Chloroquine-Inducible Par-4 Secretion Is Essential for Tumor Cell Apoptosis and Inhibition of Metastasis

The induction of tumor suppressor proteins capable of cancer cell apoptosis represents an attractive option for the re-purposing of existing drugs. We report that the anti-malarial drug, chloroquine (CQ), is a robust inducer of Par-4 secretion from normal cells in mice and cancer patients in a clinical trial. CQ-inducible Par-4 secretion triggers paracrine apoptosis of cancer cells and also inhibits metastatic tumor growth. CQ induces Par-4 secretion via the classical secretory pathway that requires the activation of p53. Mechanistically, p53 directly induces Rab8b, a GTPase essential for vesicle transport of Par-4 to the plasma membrane prior to secretion. Our findings indicate that CQ induces p53- and Rab8b-dependent Par-4 secretion from normal cells for Par-4-dependent inhibition of metastatic tumor growth

Novel Role of Prostate Apoptosis Response-4 Tumor Suppressor in B-Cell Chronic Lymphocytic Leukemia

Prostate apoptosis response-4 (Par-4), a proapoptotic tumor suppressor protein, is downregulated in many cancers including renal cell carcinoma, glioblastoma, endometrial, and breast cancer. Par-4 induces apoptosis selectively in various types of cancer cells but not normal cells. We found that chronic lymphocytic leukemia (CLL) cells from human patients and from Eµ-Tcl1 mice constitutively express Par-4 in greater amounts than normal B-1 or B-2 cells. Interestingly, knockdown of Par-4 in human CLL-derived Mec-1 cells results in a robust increase in p21/WAF1 expression and decreased growth due to delayed G1-to-S cell-cycle transition. Lack of Par-4 also increased the expression of p21 and delayed CLL growth in Eμ-Tcl1 mice. Par-4 expression in CLL cells required constitutively active B-cell receptor (BCR) signaling, as inhibition of BCR signaling with US Food and Drug Administration (FDA)–approved drugs caused a decrease in Par-4 messenger RNA and protein, and an increase in apoptosis. In particular, activities of Lyn, a Src family kinase, spleen tyrosine kinase, and Bruton tyrosine kinase are required for Par-4 expression in CLL cells, suggesting a novel regulation of Par-4 through BCR signaling. Together, these results suggest that Par-4 may play a novel progrowth rather than proapoptotic role in CLL and could be targeted to enhance the therapeutic effects of BCR-signaling inhibitors

Oxidative Stress-Induced JNK/AP-1 Signaling is a Major Pathway Involved in Selective Apoptosis of Myelodysplastic Syndrome Cells by Withaferin-A

Myelodysplastic syndromes (MDS) are a diverse group of malignant clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, dysplastic cell morphology in one or more hematopoietic lineages, and a risk of progression to acute myeloid leukemia (AML). Approximately 50% of MDS patients respond to current FDA-approved drug therapies but a majority of responders relapse within 2-3 years. There is therefore a compelling need to identify potential new therapies for MDS treatment. We utilized the MDS-L cell line to investigate the anticancer potential and mechanisms of action of a plant-derived compound, Withaferin A (WFA), in MDS. WFA was potently cytotoxic to MDS-L cells but had no significant effect on the viability of normal human primary bone marrow cells. WFA also significantly reduced engraftment of MDS-L cells in a xenotransplantation model. Through transcriptome analysis, we identified reactive oxygen species (ROS)-activated JNK/AP-1 signaling as a major pathway mediating apoptosis of MDS-L cells by WFA. We conclude that the molecular mechanism mediating selective cytotoxicity of WFA on MDS-L cells is strongly associated with induction of ROS. Therefore, pharmacologic manipulation of redox biology could be exploited as a selective therapeutic target in MDS

MutS and UvrD proteins stimulate exonuclease action: insights into exonuclease-mediated strand repair

MutS and UvrD proteins individually stimulate Escherichia coli exonuclease VII activity on blunt-ended short duplex DNA substrates. Stimulation by both proteins is ATP-dependent but not mismatch-specific and is not accompanied by apparent strand separation. Under similar conditions, MutS and UvrD proteins in fact confer resistance to exonuclease VII action on ssDNA targets, thereby implying that a novel state of a double-stranded DNA intermediate, which we term a “destabilized duplex”, is involved in exonuclease-mediated strand degradation. We find that DNA strands in such a destabilized duplex can be displaced by the challenge of a molar excess of homologous single- and double-stranded DNA targets, in trans. Such an action of the UvrD protein is ATP-dependent. We discuss these results in relation to the (i) directional excision repair of E. coli MMR, (ii) downregulation of repeat deletions by exonucleases during replication slippage, and (iii) the fork reversal function of UvrD at stalled replication forks

Enhanced DNA dynamics due to cationic reagents, topological states of dsDNA and high mobility group box 1 as probed by PicoGreen

We report a study of dynamics with a dsDNA-specific dye called PicoGreen bound to plasmid DNA (3.4 kb), and show that at low dye/DNA phosphate ratios (1 : 100 and below), PicoGreen dynamics reflect the motional dynamics of dsDNA. We further evaluated the usefulness of this probe by measuring the time-resolved fluorescence dynamics of PicoGreen bound to dsDNA in the presence of cationic reagents that affect DNA dynamics [MgCl₂ and polyethyleneimine (PEI)] and also with plasmid DNA in different topological states. Among these conditions, MgCl₂, PEI and the supercoiled form of plasmid resulted in increases in the very short component (0.2–0.4 ns) of the rotational correlation time. Separately, HMGB1 protein enhanced DNA dynamics, as observed from the rotational correlation times of very short (0.2–0.4 ns) and short (2–4 ns) rotational correlation timescale components. By studying the effect of specific deletion mutants HMGB1-ΔA (deletion of 98 N-terminal amino acids) and HMGB1-ΔC (deletion of 30 C-terminal amino acids), we show that the acidic C-terminal tail is required for enhancement of DNA dynamics. We then discuss the possible mechanisms and implications of HMGB1-mediated flexibility of DNA in the context of formation of large nucleoprotein complexes

Transcriptional Repression of Bim by a Novel YY1-RelA Complex Is Essential for the Survival and Growth of Multiple Myeloma

<div><p>Abstract</p><p>Multiple Myeloma (MM) is an incurable plasma cell cancer that is caused by several chromosomal translocations and gene deletions. Although deregulation of several signaling pathways including the Nuclear Factor-Kappa B (NF-κB) pathway has been reported in MM, the molecular requirement and the crosstalk between NF-κB and its target genes in MM cell survival has been largely unclear. Here, we report that Yin Yang1 (YY1), a target gene for NF-κB, is hyperexpressed in most MM tumor cells obtained from human patients, exhibits constitutive nuclear localization, and is essential for survival of MM cells. Mechanistically, we report a novel YY1-RelA complex formation, which is essential to transcriptionally repress a proapoptotic gene Bim. In line with this, depletion of YY1 or RelA resulted in elevated levels of Bim and apoptosis. Moreover, both YY1 and RelA are recruited to the Bim promoter and are required to repress the Bim promoter. Importantly, depletion of YY1 or RelA almost completely impaired the colony forming ability of MM progenitor cells suggesting that both RelA and YY1 are essential for the survival and growth of MM progenitor cells. Moreover, depletion of either YY1 or RelA completely inhibited MM tumor growth in xenograft models for human myeloma. Thus, a novel RelA-YY1 transcriptional repression complex is an attractive drug target in MM.</p></div

Hyper expression and activation of YY1 in MM tumors.

<p>(<b>A</b>) Lysates from Primary MM tumor cells and normal healthy human B-cells were analyzed for YY1 levels as indicated by immunoblotting for the indicated proteins. LDH levels were analyzed as loading controls. (<b>B</b>) Cytoplasmic (Cyto) and nuclear (Nuc) extracts from the indicated MMCLs were analyzed by immunoblotting for the indicated proteins. LDH and HDAC1 levels were analyzed for the purity of cytoplasmic and nuclear extracts respectively. Note the constitutive nuclear localization of YY1.</p

YY1 and RelA repress Bim promoter.

<p><b>(A) Luciferase Reporter Assay.</b> A 2.5 kb Bim promoter was cloned into pGL2-basic vector (pGL2-Bim-Basic). HEK-293T cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066121#pone.0066121-Vallabhapurapu2" target="_blank">[43]</a> were cotransfected with the pGL2-Bim-Basic vector together with the empty vectors or vectors expressing YY1 and RelA as indicated. 24 hours later, lysates were analyzed for the luciferase activity and the relative light units were plotted as indicated. Note that combined presence of YY1 and RelA was required to actively repress Bim promoter. (<b>B</b>) <b>Schematic Representation of a 145-bp Bim promoter</b>. A 145-bp region of the Bim promoter spanning −300 to −156 was shown. YY1 and NF-κB recognition sites were identified using Mat Inspector software and were shown in bold and italic. Double stranded DNA corresponding to the YY1 and NF-κB recognition sites with the core recognition site in bold and italics were also shown. Within this region YY1 and NF-κB recognition sites were separated by a 85-bp region (<b>C</b>) <b>Recruitment of RelA and YY1 to the Bim promoter</b>. Chromatin from KMM1 cells was immunoprecipitated using α-RelA or α-YY1 antibodies. Normal IgG was used as a control. Recruitment of YY1 and NF-κB to the Bim promoter was analyzed by PCR-amplification of the immunoprecipitated DNA using specific primers that amplified a 145-bp region (−300 to −156) close to the Bim gene transcriptional start site. As a Control, another region spanning −1374 to −1128 of the Bim promoter was also amplified using specific primers where RelA and YY1 were not recruited. Amplified PCR products were analyzed by running on agarose gels as indicated.</p

YY1 is essential for the survival and growth of MM tumors.

<p>(<b>A</b>) KMM1 cells were infected with lentiviruses expressing control-ShRNA or Sh-RNA targeting YY1. 48 hours post infection lysates were analyzed by immunoblotting for the efficiency YY1 silencing as indicated. (<b>B</b>) KMM1 cells were infected with lentiviruses expressing control-ShRNA or ShRNA targetting YY1. 5 days later viability was measured by flow cytometry upon staining with Annexin-V and 7AAD. Numbers in the quandrants represent % of cells that are positive or negative for Annexin-V and/or 7AAD. A representative figure from 3 independent experiments was shown. (<b>C</b>) KMM1 cells were infected with lentiviruses expressing control-ShRNA or YY1-ShRNA. Two days later cell viability was measured to be equal between the control-Sh and YY1-Sh cells. 3×10⁶ cells were subcutaneously injected in nude mice as indicated (Solid arrows indicate injection of control cells where as dotted arrows indicate injection of YY1-depleted cells). Mice were euthanized when the tumor size has reached to a size about 1 cm. (<b>D</b>) KMM1 cells were infected with lentiviruses expressing control-ShRNA or YY1-ShRNA. 24 hours after infection cells were washed and 2000 cells for both control-ShRNA and YY1-ShRNA were seeded in methylcellulose cultures. Colonies were counted 10 days later and plotted as indicated. (<b>E</b>) Colony pictures were taken by a Axiovert S100TV microscope and were shown as indicated. Note that depletion of YY1 had completely impaired colony formation by MM progenitor cells.</p