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A synthetic peptide that may block HIV reproduction is described. The peptide, called Tat10-biotin, resembles the HIV Tat protein. This protein binds to viral RNA and begins the viral reproductive process. Tat10-biotin can also bind to viral RNA and block viral reproduction. The peptide inhibited HIV reproduction in cell culture and also suppressed the production of the viral enzyme reverse transcriptase, which is required for viral reproduction.

Infection by the human immunodeficiency virus (HIV) causes a wide variety of disease ranging from positive blood tests for HIV infection without accompanying symptoms, to the extensive deterioration of the immune system. Characteristics of the HIV life cycle may influence the disease process of HIV infection. HIV specifically attacks and destroys CD4 cells, a type of immune cell. CD4 cells are involved in the production of immune proteins called antibodies, another type of immune cell. Antibodies are produced in the presence of a foreign substance such as a virus, and can specifically bind and inactivate the virus. Hence, HIV destruction of CD4 cells decrease the production of antibodies and reduce the ability of the patient to fight viral infection. The viral envelope, or outer structure of the virus, consists of antigens, components that can activate an immune response. Because the viral envelope of HIV is highly variable, the ability of the patient's immune response to control HIV infection is limited. The persistence of HIV results from the ability of the virus to integrate its genetic material into the cells of the infected individual. This viral genetic material can then be activated by cell signals that control normal immune cell activation. In this way, HIV replication is be linked with the normal cell functions of the patient. These characteristics contribute to the chronic and progressive nature of HIV infection. (Consumer Summary produced by Reliance Medical Information, Inc.)

BCCIP was originally identified as a BRCA2 and CDKN1A/p21 interaction protein. Although a partial loss of BCCIPfunction is sufficient to trigger genomic instability and tumorigenesis, complete deletion of BCCIPis lethal to cells. Using Rosa26-CreERT2 mouse models, we found that induced Bccipdeletion in adult mice caused an acute intestinal epithelial denudation that cannot be relieved by co-deletion of Trp53. The critical role of Bccipin intestine epithelial renewal was verified with a Villin-CreERT2 mouse model. The epithelium degeneration was associated with a rapid loss of the proliferative capability of the crypt progenitor cells in vivo, lack of crypt base columnar stem cell markers, and a failure of in vitrocrypt organoid growth. RNA-Seq analysis of freshly isolated intestinal crypt cells showed that Bccipdeletion caused an overwhelming down-regulation of genes involved in mitotic cell division but an up-regulation of genes involved in apoptosis and stress response to microbiomes. Our data not only indicate that intestinal epithelium is the most sensitive tissue to whole-body deletion of Bccipbut also point to Bccipas a novel and critical factor for the proliferation of the intestinal progenitors. These findings have significant implications for understanding why a hypomorphic loss of BCCIP functions is more relevant to tumorigenesis.

Osteoblasts and adipocytes are derived from a common precursor, mesenchymal stem cells (MSCs). Alterations in the normal fate of differentiating MSCs are involved in the development of obesity and osteoporosis. Here, we report that viable motheaten(mev) mice, which are deficient in the SH2-domain-containing phosphatase-1 (SHP1), develop osteoporosis spontaneously. Consistently, MSCs from mev/mevmice exhibit significantly reduced osteogenic potential and greatly increased adipogenic potential. When MSCs were transplanted into nude mice, SHP1-deficient MSCs resulted in diminished bone formation compared with wild-type MSCs. SHP1 was found to bind to GSK3β and suppress its kinase activity by dephosphorylating pY216, thus resulting in β-catenin stabilization. Mice, in which SHP1 was deleted in MSCs using SHP1fl/flDermo1-cre, displayed significantly decreased bone mass and increased adipose tissue. Taken together, these results suggest a possible role for SHP1 in controlling tissue homeostasis through modulation of MSC differentiation via Wnt signaling regulation.

An imbalance between normal adipogenesis and osteogenesis by mesenchymal stem cells (MSCs) has been shown to be related to various human metabolic diseases, such as obesity and osteoporosis; however, the underlying mechanisms remain elusive. We found that the interaction between osteopontin (OPN), an arginine‐glycine‐aspartate‐containing glycoprotein, and integrin αv/β1 plays a critical role in the lineage determination of MSCs. Although OPN is a well‐established marker during osteogenesis, its role in MSC differentiation is still unknown. Our study reveals that blockade of OPN function promoted robust adipogenic differentiation, while inhibiting osteogenic differentiation. Re‐expression of OPN restored a normal balance between adipogenesis and osteogenesis in OPN−/−MSCs. Retarded bone formation by OPN−/−MSCs was also verified by in vivoimplantation with hydroxyapatite‐tricalcium phosphate, a bone‐forming matrix. The role of extracellular OPN in MSC differentiation was further demonstrated by supplementation and neutralization of OPN. Blocking well‐known OPN receptors integrin αv/β1 but not CD44 also affected MSC differentiation. Further studies revealed that OPN inhibits the C/EBPs signaling pathway through integrin αv/β1. Consistent with these in vitroresults, OPN−/−mice had a higher fat to total body weight ratio than did wild‐type mice. Therefore, our study demonstrates a novel role for OPN‐integrin αv/β1 in regulating MSC differentiation. StemCells2014;32:327–337

PDCD2 is an evolutionarily conserved eukaryotic protein with unknown function. The DrosophilaPDCD2 ortholog Zfrp8 has an essential function in fly hematopoiesis. Zfrp8mutants exhibit marked lymph gland hyperplasia that results from increased proliferation of partially differentiated hemocytes, suggesting Zfrp8 may participate in cell growth. Based on the above observations we have focused on the role of PDCD2 in human cancer cell proliferation and hypothesized that aberrant PDCD2 expression may be characteristic of human malignancies. We report that PDCD2 is highly expressed in human acute leukemia cells as well as in normal hematopoietic progenitors. PDCD2 knockdown in cancer cells impairs their proliferation, but not viability relative to parental cells, supporting the notion that PDCD2 overexpression facilitates cancer cell growth. Prospective analysis of PDCD2 in acute leukemia patients indicates PDCD2 RNA expression correlates with disease status and is a significant predictor of clinical relapse. PDCD2’s role in cell proliferation and its high expression in human malignancies make it an attractive, novel potential molecular target for new anti-cancer therapies.

Mesenchymal stromal cells (MSCs) tend to infiltrate into tumors and form a major component of the tumor microenvironment. These tumor-resident MSCs are known to affect tumor growth, but the mechanisms are largely unknown. We found that MSCs isolated from spontaneous lymphomas in mouse (L-MSCs) strikingly enhanced tumor growth in comparison to bone marrow MSCs (BM-MSCs). L-MSCs contributed to greater recruitment of CD11b+Ly6C+monocytes, F4/80+macrophages, and CD11b+Ly6G+neutrophils to the tumor. Depletion of monocytes/macrophages, but not neutrophils, completely abolished tumor promotion of L-MSCs. Furthermore, L-MSCs expressed high levels of CCR2 ligands, and monocyte/macrophage accumulation and L-MSC-mediated tumor promotion were largely abolished in CCR2−/−mice. Intriguingly, TNFα-pretreated BM-MSCs mimicked L-MSCs in their chemokine production profile and ability to promote tumorigenesis of lymphoma, melanoma, and breast carcinoma. Therefore, our findings demonstrate that, in an inflammatory environment, tumor-resident MSCs promote tumor growth by recruiting monocytes/macrophages.

Both the canonical and noncanonical nuclear factor κB (NF-κB) pathways have been linked to tumorigenesis. However, it remains unknown whether and how the 2 signaling pathways cooperate during tumorigenesis. We report that inhibition of the noncanonical NF-κB pathway significantly delays tumorigenesis mediated by the viral oncoprotein Tax. One function of noncanonical NF-κB activation was to repress expression of the WWOX tumor suppressor gene. Notably, WWOX specifically inhibited Tax-induced activation of the canonical, but not the noncanonical NF-κB pathway. Mechanistic studies indicated that WWOX blocked Tax-induced inhibitors of κB kinaseα (IKKα) recruitment to RelA and subsequent RelA phosphorylation at S536. In contrast, WWOX Y33R, a mutant unable to block the IKKα recruitment and RelA phosphorylation, lost the ability to inhibit Tax-mediated tumorigenesis. These data provide one important mechanism by which Tax coordinates the 2 NF-κB pathways for tumorigenesis. These data also suggest a novel role of WWOX in NF-κB regulation and viral tumorigenesis.

Both the canonical and noncanonical nuclear factor κB (NF-κB) pathways have been linked to tumorigenesis. However, it remains unknown whether and how the 2 signaling pathways cooperate during tumorigenesis. We report that inhibition of the noncanonical NF-κB pathway significantly delays tumorigenesis mediated by the viral oncoprotein Tax. One function of noncanonical NF-κB activation was to repress expression of the WWOXtumor suppressor gene. Notably, WWOX specifically inhibited Tax-induced activation of the canonical, but not the noncanonical NF-κB pathway. Mechanistic studies indicated that WWOX blocked Tax-induced inhibitors of κB kinaseα (IKKα) recruitment to RelA and subsequent RelA phosphorylation at S536. In contrast, WWOX Y33R, a mutant unable to block the IKKα recruitment and RelA phosphorylation, lost the ability to inhibit Tax-mediated tumorigenesis. These data provide one important mechanism by which Tax coordinates the 2 NF-κB pathways for tumorigenesis. These data also suggest a novel role of WWOX in NF-κB regulation and viral tumorigenesis.

Infection with the human T-cell leukemia virus-1 (HTLV-1) results in a variety of diseases including adult T-cell leukemia/lymphoma (ATL). Although the pathogenesis of these disorders is poorly understood, it involves complex interactions with the host immune system. Activation of infected T cells may play an important role in disease pathogenesis through induction of the oncogenic HTLV-1 Tax transactivator protein. To test this hypothesis, we employed transgenic mice in which Tax is regulated by the HTLV-1 LTR. T-cell receptor stimulation of LTR-Tax CD4+ T cells induced Tax expression, hyper-proliferation, and immortalization in culture. The transition to cellular immortalization was accompanied by markedly increased expression of the antiapoptotic gene, mcl-1, previously implicated as important in T-cell survival. Immortalized cells exhibited a CD4+CD25+CD3− phenotype commonly observed in ATL. Engraftment of activated LTR-Tax CD4+ T cells into NOD/Shi-scid/IL-2Rγ null mice resulted in a leukemia-like phenotype with expansion and tissue infiltration of Tax+, CD4+ lymphocytes. We suggest that immune activation of infected CD4+ T cells plays an important role in the induction of Tax expression, T-cell proliferation, and pathogenesis of ATL in HTLV-1–infected individuals.

Treatment of cultured PANC-1, MIA PaCa-2, and BxPC-3 human pancreatic adenocarcinoma cells with 0.1 to 1.6 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 96 h inhibited the proliferation of these cells in a dose-dependent manner, and PANC-1 and MIA PaCa-2 cells were more sensitive to TPA than BxPC-3 cells. Inhibition of proliferation by TPA in PANC-1 cells was associated with an increase in the level of p21, but this was not observed in MIA PaCa-2 or BxPC-3 cells. The TPA-induced increase of p21 in PANC-1 cells was blocked by bisindolylmaleimide or rottlerin (inhibitors of protein kinase C). Studies in NCr-immunodeficient mice with well established PANC-1 tumor xenografts indicated that daily i.p. injections of TPA strongly inhibited tumor growth, increased the percentage of caspase-3-positive cells, and decreased the ratio of mitotic cells to caspase-3-positive cells in the tumors. Studies with BxPC-3 tumors in NCr mice receiving daily i.p. injections of vehicle, TPA, all-trans retinoic acid (ATRA), or a TPA/ATRA combination showed that TPA had an inhibitory effect on tumor growth, but treatment of the animals with the TPA/ATRA combination had a greater inhibitory effect on tumor growth than TPA alone. Treatment with the TPA/ATRA combination resulted in a substantially decreased ratio of the percentage of mitotic cells to the percentage of caspase-3-positive cells in the tumors compared with tumors from the vehicle-treated control animals. The inhibitory effects of TPA on tumor growth occurred at clinically achievable blood levels.

Proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin-1β, and lipopolysaccharides (LPS), suppress the gene expression of cytochrome P-450 1A1 (cyp1a1). The mechanism of the suppression is not well understood. In present study, we show that activation of nuclear factor-κB (NF-κB) is a critical event leading to the suppression ofcyp1a1gene expression, thus providing an underlying mechanism for the TNF-α- and LPS-induced cyp1a1suppression. We demonstrated that: (i) inducible RelA expression down-regulated aryl hydrocarbon receptor (AhR) activated reporter gene; (ii) the suppressive effects of LPS and TNF-α on the AhR-activated reporter gene could be blocked by pyrrolidine dithiocarbamate, which is known to inhibit NF-κB action; and (iii) TNF-α and LPS-imposed repression could be reversed by the NF-κB super repressor (SRIκBα), thus demonstrating the specific involvement of NF-κB. Furthermore, nuclear receptor coactivators p300/CBP and steroid receptor coactivator-1 act individually as well as cooperatively to reverse the suppressive effects by NF-κB on the AhR-activated reporter gene, suggesting that these transcriptional coactivators serve as the common integrators for the two pathways, thereby mediating the cross-interactions between AhR and NF-κB. Finally, using the chromatin immunoprecipitation assay, we demonstrated that AhR ligand induces histone H4 acetylation at the cyp1a1promoter region containing the TATA box, whereas TNF-α inhibits this acetylation, suggesting that AhR/NF-κB interaction converges at level of transcription involving chromatin remodeling.

We have studied adenovirus-mediated cytotoxicity after infection of malignant cells obtained from patients with chronic lymphocytic leukemia (CLL). Our studies indicate that adenoviruses can infect primary CLL cells and that infection of CLL cells with a replication-competent strain of human adenovirus 5 (Ad5dl309) results in cytotoxicity. Adenovirus-mediated cytotoxicity was also seen after infection of CLL cells with a variety of viruses attenuated by mutations in the adenovirus early region 1 (E1) or early region 2 (E2). Even viruses attenuated by deletion of the entire E1 region resulted in cytotoxicity after infection of the CLL cells obtained from some patients. Although there was variability in the degree of cytotoxicity induced by different viruses in different patients cells, a virus with a mutation in the E1B 19K gene resulted in the greatest degree of cytotoxicity in most of the CLL samples tested. These studies demonstrate that infection of CLL cells by attenuated adenoviruses with specific mutations in the E1 or E2 region results in cell death. Attenuated adenoviruses should be developed further as therapeutic agents for patients with CLL.

The Ah receptor (AhR) mediates many of the toxic responses induced by polyhalogenated and polycyclic hydrocarbons (PAHs) which are ubiquitous environmental contaminants causing toxic responses in human and wildlife. NF-κB is a pleiotropic transcription factor controlling many physiological functions adversely affected by PAHs, including immune suppression, thymus involution, hyperkeratosis, and carcinogenesis. Here, we show physical interaction and mutual functional repression between AhR and NF-κB. This mutual repression may provide an underlying mechanism for many hitherto poorly understood PAH-induced toxic responses, and may also provide a mechanistic explanation for alteration of xenobiotic metabolism by cytokines and compounds that regulate NF-κB.

Experimental gene transfer and viral infections can result in the accumulation of unintegrated DNA in target cells. The effects of such accumulation on target cell metabolism have not been directly studied. The experiments reported in this paper show that transfection of cloned retroviral long-terminal-repeat (LTR) DNA, or of a variety of eukaryotic promoters, into proliferating HeLa cells results in rapid, sequence-specific, and dose-dependent cell death. Plasmids containing the Rous sarcoma virus LTR or the human immunodeficiency virus LTR cloned in pUC-related plasmids are 5 to 10 times more toxic than pUC19. The demonstrated sensitivity of eukaryotic cells to exogenously introduced DNA has important implications for the interpretation of gene transfer experiments and may be relevant to the pathogenic mechanisms in the course of retroviral infections such as AIDS.

Human immunodeficiency virus type 1 (HIV-1) Tat-mediatedtrans-activation requires the structural integrity of TAR RNA and the cooperative interaction of human host cell proteins. The TAR domain, minimally required fortatresponse, includes the Tat binding pyrimidine bulge, the TAR RNA upper stem, and the loop sequences. However, little is known about the significance of the 5′-stem structure of TAR in the regulation of viral growth. We designed viral mutations, specifically in the TAR RNA lower stem structure, and studied their effects on the kinetics of viral growth in T-lymphocyte cell lines and in activated human peripheral blood mononuclear cells. Mutations that destabilized the lower TAR stem structure inhibited viral growth to various degrees in different[formula]T-cells. These results suggest that the structural integrity of the lower stem structure of TAR plays an important role in viral growth, presumably by binding to specific host cell proteins that stabilize Tat–TAR interactions.

Cytokines such as interleukin-1β (Il-1) are produced in the brain during development and during inflammatory processes that result from lesions or disease. One function of Il-1 in the brain appears to be the stimulation of astrocytes to proliferate and produce a variety of cytokines and trophic factors, including nerve growth factor. The mechanisms by which Il-1 exerts its actions on astrocytes remain poorly defined. We present evidence that this cytokine elicits activation of the NFκB transcription factor and that this transcription factor mediates effects of Il-1 on nerve growth factor mRNA expression. Elucidation of the processes by which cytokines activate astrocytes and influence trophic factor expression may provide insight into mechanisms governing inflammatory processes within the central nervous system.

ABSTRACTExpression of human T-cell leukemia virus type 1 (HTLV-1) is regulated both by the HTLV-1 Tax transactivator and by cellular transcriptional factors binding to the viral long terminal repeat (LTR), suggesting that cellular signals may play a role in regulating viral expression. Treatment of cells chronically infected with HTLV-1, which express low levels of HTLV-1 RNAs and Tax protein, with phorbol esters (i.e., phorbol12-myristate 13-?acetate [PMA]), phytohemagglutinin (PHA), sodium butyrate, or combinations of cytokines resulted in induction of HTLV-?1 gene expression. PMA or PHA treatment following cotransfection of HTLV-1 Tax expression plasmids resulted in synergistic activation of HTLV-1 LTR-directed gene expression, apparently involving tyrosine ki- nase-?mediated pathways. These results suggest that cellular activation stimuli may cooperate with HTLV-1 Tax to enhance expression of integrated HTLV-1 genomes and thus may play a role in the pathogenesis of HTLV-1 disease.

JΔK cells were isolated as a chronically infected survivor cell line, following infection of Jurkat CD4+ T cells with dI-NF, a mutated strain of human immunodeficiency virus type 1 (HIV-1) containing a deletion of the long terminal repeat (LTR) NF-κB sites. JΔK cells exhibited very low levels of constitutive HIV production. HIV-1 expression was activated from JΔK cells by treatment with phorbol myristate acetate (PMA), sodium butyrate (NAB), or hexamethylene bisacetamide (HMBA), but not tumor necrosis factor α (TNF-α), confirming the role of NF-κB in mediating TNF-α induction of HIV transcription. The strong induction of HIV expression by NaB or HMBA in JΔK cells clearly demonstrates the existence of NF-κB-independent mechanisms of HIV activation in chronically infected cells. JΔK cells may provide a useful model for characterizing NF-κB-independent transcriptional activation of the HIV LTR.

The long terminal repeats (LTRs) of primate lentiviruses contain conserved binding sites for the NF-κB and Sp1 cellular transcription factors. In order to study the role that these sites play in simian immunodeficiency virus (SIV) replication, we have introduced mutations that disrupt either the NF-κB or Sp1 binding sites in the LTR of an infectious molecular clone of SIVmac239. An additional mutation also disrupted the SF3 transcription factor binding site that overlaps the NF-κB site. Viruses containing point mutations or deletions of the NF-κB, SF3, or Sp1 binding sites retained the ability to replicate efficiently in the CEMx174 and MT4 cell lines, as well as in PHA-stimulated primary rhesus macaque peripheral blood mononuclear cells (PBMCs). Efficient replication of SIVs mutated in either NF-κB or Sp1 binding sites suggests that the SIV LTR promoter contains multiple functionally redundant elements capable of supporting sufficient transcription to allow productive viral replication.

Mammalian cells contain multiple copies of endogenous type C retroviral DNA sequences1–4. Among these sequences are complete, potentially infectious proviruses5–7, pro viral DNA that is expressed only in the form of viral antigens8–10, retroviral segments that may contribute portions of envelope (env) genes during the generation of recombinant polytropic viruses11,12, and many subgenomic viral DNA segments12that may not be expressed at all. We have previously reported the identification and molecular cloning of type C retroviral sequences from human DNA13and have shown that the partial nucleotide and deduced amino acid sequences of one of the clones obtained (λ51) are homologous to Moloney MuLV (MoMuLV) in the gag and pol regions14. The λ51 clone as well as several others isolated from a human DNA library contained approximately 4.3 kilobases (kb) of retroviral sequences, were deleted in the env region14, and were flanked by tandem repeats unlike the long terminal repeats (LTRs) typically found in proviral DNAs (P.E.S., in preparation). We describe here the characterization of a full-length human retroviral clone (λ4-1) containing LTR elements as well as a putative env region. DNA–RNA hybridization experiments reveal that human cells contain species of poly(A)+RNA that anneal to segments of the full-length retroviral DNA clone.

Steward: UMDNJ: Patents & Royalties. Schaar:UMDNJ: Patents & Royalties. Sabaawy:UMDNJ: Patents & Royalties.

To understand the potential role of placental tissue in the pathogenesis of neonatal AIDS, the distribution of human immunodeficiency virus type 1 (HIV-l) receptor and the infectability of placental tissue by HIV-l were studied. Both the mRNA and the protein for the HIV receptor (CD4) were present in fetal-derived placenta. By immunofluorescent microscopy, a number of different cell types appeared to be CD4+; positive cells were observed in the lining and stroma of the chorionic villi. Some of these CD4+ cells dual-labeled with the trophoblastic marker placental lactogen. In addition, CD4+ cells were observed within the lining of placental blood vessels. Organ cultures of first-trimester and term placentas were infectable by HIV as monitored by reverse transcriptase activity of culture supernatants and by immunofluorescent labeling of HIV antigens. One potential route of congenital HIV transmission may be direct placental infection by HIV as early as the first trimester, with subsequent transplacental spread of the virus.