Mouse Leukemia Cells Research Articles

Blockage of SDF-1-CXCR4 Axis by AMD 3100 Can Be a Novel Therapy for Acute Lymphoblastic Leukemia by Targeting the Extramedullary Sites of Leukemic Cells.

Abstract 981Poster Board I-3 Background and Purpose:Acute lymphoblastic leukemia (ALL) is the most common type of childhood hematologic malignancy. Although the accumulated progresses in treatment regimen have raised the 5-year survival rate as high as 80% for whole pediatric patients, only poor prognosis, an overall survival rate of 30%, can be still now expected for the patients with relapsed diseases. Widespread extramedullary involvement such as liver, spleen, lymph nodes and central nervous system invasion is a well-known characteristic of ALL related to poor prognosis. Recently, bone marrow (BM) microenvironments supporting leukemic cells have been widely noticed as an important element which influences on treatment response and relapse of disease. Although the mechanism of extramedullary dissemination has been the most crucial issues in the study of leukemia, it still remains incompletely understood. In this study, we established a novel murine model of human ALL with NOD/SCID/γc null (NOG) mouse. Using this model, we examined the involvement of SDF-1-CXCR4 signaling axis in hepatomegary development in ALL. ResultPrimary bone marrow samples were collected from 13 children with ALL at the time of diagnosis with informed consent. The leukemic cells (1×106cells) were injected into the tail veins of non-irradiated 8- to 10-week old NOD/SCID/γc null (NOG) mice, a transgenic mouse with severe combined immunodeficiency and IL-2 receptor chain allelic mutation showing high potential to reconstitute the normal human hematopoietic system. Primary samples from 10 out of 13 patients were successfully engrafted into mice without any conditioning such as prior irradiation and DNA-damaging agents medication, and those engrafted leukemic cells were able to be serially transplanted into secondary, tertiary and quaternary recipients. Morphological and FACS analyses revealed as high as >80% blood chimerism and conserved blast phenotypes through serial transplantations. Moreover, extramedullary organs including liver, spleen and kidneys showed the leukemic invasion consistent with donor ALL disease. In contrast, no normal human hematopoiesis was observed in our xenotransplantation system without conditioning.CXCR4 is a known regulator of lymphocyte migration and has been suggested to be important for proliferation of normal leucocytes and leukemic cells. CXCR4 expression and function of leukemic cells in NOG mice were confirmed by flow cytometry and in vitro chemotaxis assays towards its known chemokine ligand SDF-1. Immunohistorical analysis of liver reveals that SDF-1 was detectable only in biliary duct endotherial cells. Finally, we demonstrated directly the effect of SDF-1-CXCR4 axis in our model by using the CXCR4 inhibitor AMD3100 in vivo and in vitro. Discussion:NOG mice model for engraftment of human leukemic cells provides significant insights into the biology of ALL and allows us to answer various questions concerning the molecular mechanism of extramedullaly invasion. This non-conditioning approach may prevent possible damage to the host microenvironment, thereby providing a more natural model for growth of human leukemic cells in mice. Our present study on the involvement of SDF-1-CXCR4 axis in ALL dissemination could rink to the novel therapies in future which target the extramedullary sites in order to perfectly overcome ALL. Disclosures:No relevant conflicts of interest to declare.

Synthesis and cytotoxic evaluation of β-alkyl or β-aryl-δ-methyl-α-methylene-δ-lactones. Comparison with the corresponding γ-lactones

We present a simple and general strategy for the synthesis of β,δ-disubstituted-α-methylene-δ-lactones starting from easily available tert-butyl 2-(diethoxyphosphoryl)alk-2-enoates. The elaborated synthetic protocol includes pyrrolidine-catalyzed Michael addition of acetone, diastereoselective reduction of the carbonyl group, lactonization and finally the Horner–Wadsworth–Emmons reaction with formaldehyde. All α-methylene-δ-lactones were evaluated in vitro against mouse leukemia cell line L-1210 and two human leukemia cell lines HL-60 and NALM-6. Comparison of cytotoxic activity with corresponding α-methylene-γ-lactones is also discussed.

Chronic lymphocytic leukemia of Eμ-TCL1 transgenic mice undergoes rapid cell turnover that can be offset by extrinsic CD257 to accelerate disease progression

AbstractResults of heavy-water labeling studies have challenged the notion that chronic lymphocytic leukemia (CLL) represents an accumulation of noncycling B cells. We examined leukemia cell turnover in Eμ-TCL1 transgenic (TCL1-Tg) mice, which develop a CLL-like disease at 8 to 12 months of age. We found that leukemia cells in these mice not only had higher proportions of proliferating cells but also apoptotic cells than did nonleukemic lymphocytes. We crossed TCL1-Tg with BAFF-Tg mice, which express high levels of CD257. TCL1×BAFF-Tg mice developed CLL-like disease at a significantly younger age and had more rapid disease progression and shorter survival than TCL1-Tg mice. Leukemia cells of TCL1×BAFF-Tg mice had similar proportions of proliferating cells, but fewer proportions of dying cells, than did the CLL cells of TCL1-Tg mice. Moreover, leukemia cells from either TCL1×BAFF-Tg or TCL1-Tg mice produced more aggressive disease when transferred into BAFF-Tg mice than into wild-type (WT) mice. Neutralization of CD257 resulted in rapid reduction in circulating leukemia cells. These results indicate that the leukemia cells of TCL1-Tg mice undergo high levels of spontaneous apoptosis that is offset by relatively high rates of leukemia cell proliferation, which might allow for acquisition of mutations that contribute to disease evolution.

Antitumor antibiotic fostriecin covalently binds to cysteine-269 residue of protein phosphatase 2A catalytic subunit in mammalian cells

Fostriecin is a phosphate monoester with excellent antitumor activity against mouse leukemia, and it is a potent inhibitor of protein phosphatase (PP) 2A. This compound has been predicted to covalently bind to the Cys269 residue of the PP2A catalytic subunit (PP2Ac) at the α,β-unsaturated lactone via a conjugate addition reaction. However, this binding has not yet been experimentally proven. To confirm such binding, we synthesized biotin-labeled fostriecin (bio-Fos), which has an inhibitory activity against the proliferation of mouse leukemia cells. We showed that fostriecin directly binds to PP2Ac in HeLa S3 cells by pull-down assays using bio-Fos. Moreover, we directly demonstrated that fostriecin covalently binds to the Cys269 residue of PP2Ac by matrix assisted laser desorption/ionization time-of-flight mass spectrometry analysis. From these results, the inhibitory mechanism of fostriecin on PP2A activity is discussed.

TSC-22 contributes to hematopoietic precursor cell proliferation and repopulation and is epigenetically silenced in large granular lymphocyte leukemia

Aberrant methylation of tumor suppressor genes can lead to their silencing in many cancers. TSC-22 is a gene silenced in several solid tumors, but its function and the mechanism(s) responsible for its silencing are largely unknown. Here we demonstrate that the TSC-22 promoter is methylated in primary mouse T or natural killer (NK) large granular lymphocyte (LGL) leukemia and this is associated with down-regulation or silencing of TSC-22 expression. The TSC-22 deregulation was reversed in vivo by a 5-aza-2'-deoxycytidine therapy of T or NK LGL leukemia, which significantly increased survival of the mice bearing this disease. Ectopic expression of TSC-22 in mouse leukemia or lymphoma cell lines resulted in delayed in vivo tumor formation. Targeted disruption of TSC-22 in wild-type mice enhanced proliferation and in vivo repopulation efficiency of hematopoietic precursor cells (HPCs). Collectively, our data suggest that TSC-22 normally contributes to the regulation of HPC function and is a putative tumor suppressor gene that is hypermethylated and silenced in T or NK LGL leukemia.

ERK5 Knockdown Generates Mouse Leukemia Cells with Low MHC Class I Levels That Activate NK Cells and Block Tumorigenesis

Tumor cell-based vaccines are currently used in clinical trails, but they are in general poorly immunogenic because they are composed of cell extracts or apoptotic cells. Live tumor cells should be much better Ags provided that they are properly processed by the host immune system. We show herein that stable expression of a small hairpin RNA for ERK5 (shERK5) decreases ERK5 levels in human and mouse leukemic cells and leads to their elimination by NK cells in vivo. The shERK5 cells show down-regulation of MHC class I expression at the plasma membrane. Accordingly, ectopic activation of the ERK5 pathway induces MHC class I gene expression. Coinjection of shERK5-expressing cells into the peritoneum diminishes survival of engrafted wild-type tumor cells. Moreover, s.c. injection of shERK5-expressing cells strongly diminishes tumor development by wild-type cells. Our results show that shERK5 expression in leukemia cells effectively attenuates their tumor activity and allows their use as a tumor cell-based vaccine.

ANTIGENOTOXIC, FIBRINOLYTIC AND IMMUNOMODULATING ACTIVITY OF TRADITIONALLY FERMENTED SOY PRODUCT, CHUNGKUKJANG

Fibrinolytic, antigenotoxic and immunomodulating properties of chungkukjang were evaluated. The purified fibrinolytic enzyme was characterized as an alkaline serine protease with molecular weight of 22 kDa determined by fibrin zymography and stable over pH range 6.0-10.0 below 50C. The comet assay showed that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage level (190.8 AU) in 3T3 cells was considerably decreased to 121.2 AU with chungkukjang extracts (CB) showing 49.7% of protection. Incubation of the 3T3 cells with CB significantly reduced the cytotoxicity of MNNG. [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay revealed that the addition of CB enhanced the proliferation of RAW 264.7 (mouse leukemic monocyte macrophage cell line) macrophages up to about 150% of the control levels. RAW 264.7 macrophages incubated with CB induced a strong increase in nitric oxide (NO), tumor necrosis factor-a (TNF-a) and interleukin-1α (IL-1α) with a dose-dependent manner. After 24 h incubation with 1 mg/mL of CB, NO, TNF-a and IL-1α showed the highest production of 511, 2.46 and 0.258 ng/mL, respectively.

The dark and the bright side of Stat3: proto-oncogene and tumor-suppressor

Stat transcription factors have been implicated in tumorigenesis in mice and men. Stat3 and Stat5 are considered powerful proto-oncogenes, whereas Stat1 has been demonstrated to suppress tumor formation. We demonstrate here for the first time that a constitutive active version of Stat3alpha (Stat3alphaC) may also suppress transformation. Mouse embryonic fibroblasts (MEFs) deficient for p53 can be transformed with either c-myc or with rasV12 alone. Interestingly, transformation by c-myc is efficiently suppressed by co-expression of Stat3alphaC, but Stat3alphaC does not interfere with transformation by the rasV12-oncogene. In contrast, transplantation of bone marrow cells expressing Stat3alphaC induces the formation of a highly aggressive T cell leukemia in mice. The leukemic cells invaded multiple organs including lung, heart, salivary glands, liver and kidney. Interestingly, transplanted mice developed a similar leukemia when the bone marrow cells were transduced with Stat3beta, which is also constitutively active when expressed at significant levels. Our experiments demonstrate that Stat3 has both - tumor suppressing and tumor promoting properties.

DNA cleavage and antitumour activity of platinum(II) and copper(II) compounds derived from 4-methyl-2-N-(2-pyridylmethyl)aminophenol: spectroscopic, electrochemical and biological investigation

The reaction of the redox-active ligand, Hpyramol (4-methyl-2-N-(2-pyridylmethyl)aminophenol) with K(2)PtCl(4) yields monofunctional square-planar [Pt(pyrimol)Cl], PtL-Cl, which was structurally characterised by single-crystal X-ray diffraction and NMR spectroscopy. This compound unexpectedly cleaves supercoiled double-stranded DNA stoichiometrically and oxidatively, in a non-specific manner without any external reductant added, under physiological conditions. Spectro-electrochemical investigations of PtL-Cl were carried out in comparison with the analogue CuL-Cl as a reference compound. The results support a phenolate oxidation, generating a phenoxyl radical responsible for the ligand-based DNA cleavage property of the title compounds. Time-dependent in vitro cytotoxicity assays were performed with both PtL-Cl and CuL-Cl in various cancer cell lines. The compound CuL-Cl overcomes cisplatin-resistance in ovarian carcinoma and mouse leukaemia cell lines, with additional activity in some other cells. The platinum analogue, PtL-Cl also inhibits cell-proliferation selectively. Additionally, cellular-uptake studies performed for both compounds in ovarian carcinoma cell lines showed that significant amounts of Pt and Cu were accumulated in the A2780 and A2780R cancer cells. The conformational and structural changes induced by PtL-Cl and CuL-Cl on calf thymus DNA and phiX174 supercoiled phage DNA at ambient conditions were followed by electrophoretic mobility assay and circular dichroism spectroscopy. The compounds induce extensive DNA degradation and unwinding, along with formation of a monoadduct at the DNA minor groove. Thus, hybrid effects of metal-centre variation, multiple DNA-binding modes and ligand-based redox activity towards cancer cell-growth inhibition have been demonstrated. Finally, reactions of PtL-Cl with DNA model bases (9-Ethylguanine and 5'-GMP) followed by NMR and MS showed slow binding at Guanine-N7 and for the double stranded self complimentary oligonucleotide d(GTCGAC)(2) in the minor groove.

Multidrug resistant P-glycoprotein positive L1210/VCR cells are also cross-resistant to cisplatin via a mechanism distinct from P-glycoprotein-mediated drug efflux activity

P-glycoprotein (P-gp, a drug transporter found in the plasma membrane)-mediated multidrug resistance of leukemia cells represents a real obstacle in the effective chemotherapeutic treatment of leukemia. While cisplatin (CisPt) is known to be a substance that is untransportable by P-gp, P-gp positive cells were often found to be resistant to CisPt. The aim of the current paper is to study this phenomenon using P-gp positive mouse leukemia cells L1210/VCR in which the overexpression of P-gp was induced by its ability to adapt to growth on vincristine (VCR). L1210/VCR cells are also resistant to CisPt. However, resistance to this substance could not be reversed by addition of the known P-gp inhibitor verapamil. CisPt induced more pronounced entry into apoptosis, as measured using the annexin V/propidium iodide kit, in sensitive L1210 cells than in resistant L1210/VCR cells. In addition, CisPt induced an increase in the proportion of L1210 cells that were in the g2 phase of the cell cycle when compared to L1210/VCR cells, as measured by staining with propidium iodide. Similarly, a higher release of cytochrome c from the mitochondria to the cytosol was induced by CisPt treatment in L1210 than in L1210/VCR cells. While similar levels of Bax and Bcl-2 proteins were observed in sensitive and resistant cells, CisPt induced a more pronounced decrease of the Bcl-2 levels in L1210 cells than in L1210/VCR cells. Consistent with this observation, CisPt induced a larger decrease of the Bcl-2 content in the Bcl-2:Bax heterooligomer in L1210 cells than in L1210/VCR cells. Moreover, CisPt induced a similar apoptotic DNA fragmentation pattern in both resistant and sensitive cells. All of the above observations indicated that L1210/VCR cells are also resistant to CisPt and that this resistance is related to the differences in the regulatory mechanisms responsible for CisPt-induced apoptosis in L1210/VCR cells without any contribution from the drug efflux activity of P-gp.

Polycomb Group Ring Finger 5 (PCGF5) Is a Notch Transcriptional Target and Regulates Cell Size and Cell Cycle in Hematopoietic Progenitors.

The highly conserved Notch gene is activated by mutation in more than half of human T cell acute lymphoblastic leukemia (T-ALL) cases. The Notch protein is a transmembrane receptor which, upon binding of its ligand, is cleaved in a series of proteolytic steps releasing the intracellular portion (ICN) to translocate to the nucleus where it acts as a transcriptional activator for target genes such as HES1, Deltex, preTalpha, and c-Myc. To better understand the mechanism by which Notch causes leukemogenesis, microarray gene expression profiling experiments where conducted on five human Notch signaling-dependent T-ALL cell lines which where either mock-treated or treated with a gamma-secretase inhibitor (GSI) to prevent the release of Notch from the membrane. The Polycomb Group gene PCGF5 was identified as one of the genes most strongly downregulated upon Notch inhibition. This regulation was subsequently confirmed by quantitative RT-PCR in both human and mouse leukemia cell lines. Our interest in this gene was encouraged by its homology to the well-known oncogene Bmi-1, which acts to modify chromatin and silence expression of several genes including the cyclin-dependent kinase inhibitors p16 and p19ARF within the CDKN2a locus. Interestingly, we found that inhibition of Notch signaling by GSI treatment in both human and mouse leukemia cells resulted in an increase of both p16 and p19ARF at the mRNA and protein levels. This suggested that Notch may be responsible for maintaining expression of a transcriptional repressor that suppresses p16 and p19ARF. We hypothesize that PCGF5 may be acting in a manner analogous to Bmi-1 in this cellular context and thus mediating p16/p19ARF repression. Studies to test this hypothesis are currently in progress.To further investigate the role of PCGF5 in hematopoiesis, mouse bone marrow progenitors were transduced with retrovirus to express PCGF5 constitutively and transplanted into lethally irradiated recipients. Our results show long term reconstitution by PCGF5-expressing cells with as yet no evidence of PCGF5-induced hematopoeitic malignancy in a small cohort up to 6 months post-transplant. However, we did observe cells expressing high levels of PCGF5 to be skewed toward myeloid lineages, while mid-level expressing cells develop efficiently into lymphocytes. We detected no defects in B cell maturation; however, PCGF5-expressing T cell numbers were significantly lower than controls in the peripheral blood and spleen of recipient animals. Consistent with this observation, fetal thymic organ culture of PCGF5-transduced fetal liver hematopoietic progenitors showed accumulation in the early double negative thymocyte stages. Additionally, we found PCGF5-expressing B and T lymphocytes to be larger than control cells, and preliminary data suggests these cells may be arrested in G2/M phase of the cell cycle. Biochemical studies are also in progress to assess participation of PCGF5 in the Polycomb Repressive Complex PRC-1 and its effect on chromatin structure. In sum, these preliminary data suggest enforced PCGF5 expression, though not oncogenic, alters normal lymphoid/myeloid fate selection by hematopoietic progenitors and may affect lymphoid cell size by altering cell cycle progression.

Cbfb-MYH11 Can Block Hematopoiesis and Alter Gene Expression through Mechanisms Independent of Cbfb and Runx1 Repression

Inv(16)(p13;q22), the chromosomal inversion commonly found in patients with AML M4Eo, results in the fusion gene CBFB-MYH11. It has been proposed that this fusion gene acts by dominantly repressing the activity of the CBFβ/RUNX1 heterdimeric transcription factor. It has been shown that the fusion protein contains high-affinity RUNX binding sites that are thought to preferentially bind RUNX1 and block its normal gene regulatory functions. Data from mouse models support this hypothesis as embryos heterozygous for the knock-in allele of Cbfb-MYH11 (Cbfb+/MYH11) have the same phenotype as mice homozygous null for either Cbfb (Cbfb−/−) or Runx1 (Runx1−/−) embryos: failure of definitive hematopoiesis, severe central nervous system hemorrhaging, and embryonic lethality. However, we also observed defects in primitive hematopoiesis in the Cbfb+/MYH11 embryos, which were not described in initial reports of Cbfb−/− or Runx1−/− embryos, raising the possibility that Cbfb-MYH11 has activities not related to Cbfb inhibition. To address this hypothesis, we further characterized primitive blood cells in Cbfb+/MYH11, Cbfb−/−, and Runx1−/− embryos. We found that Cbfb-MYH11 caused defects in differentiation, proliferation, and apoptosis that were undetectable or less severe in Cbfb−/− and Runx1−/− embryos, indicating that Cbfb-MYH11 has activities independent of Cbfb/Runx repression during primitive hematopoiesis. Gene expression profiling showed a large number of changes in primitive blood cells of Cbfb+/MYH11, but not Cbfb−/−, embryos, with 95% of them being up-regulation. These gene expression changes are potentially important for leukemogenesis as most of the genes (76%) upregulated in the blood of Cbfb+/MYH11 embryos also showed expression in leukemic cells of mice expressing Cbfb-MYH11. In addition, analysis of published gene expression profiling studies of patients with Inv16+ AML indicates that many of these genes are also expressed in human Cbfb-MYH11 expressing AML cells. Of the over 400 genes that show deregulated expression uniquely in Cbfb+/MYH11 embryos, and expression in mouse leukemic cells, we further examined the expression of 3 genes: Csf2rb, Il1rl1, and Gata2. These genes were chosen because they were among the genes with highest expression changes in the embryos and they have multiple functional roles during hematopoiesis. They are also all linked, either directly or indirectly, with eosinophil development. Abnormal eosinophils are a hallmark of Inv16+ AML, and are not associated with AMLs involving other translocations that disrupt CBFB/RUNX function, such as t(8:21), therefore genes involved in eosinophil development are likely downstream targets of CBFB-MYH11. In agreement with our gene expression studies, we found that the Csf2rb, Il1rl1, and Gata2 proteins are expressed at high levels in leukemic cells from mice expressing Cbfb-MYH11. Interestingly, we found that in mice with a conditional allele of Cbfb-MYH11, a transient population of myeloid progenitor-like cells expressing both Csf2rb and Il1rl1 was seen in the peripheral blood soon after induction of Cbfb-MYH11 expression. This implies that upregulation of Csf2rb and Il1rl1 are early events after Cbfb-MYH11 expression and that they may be direct targets of the fusion protein. In addition, the promoters of both of the genes, as well as GATA2, contain multiple RUNX binding sites, raising the possibility that Cbfb-MYH11 is affecting their transcription through aberrant transactivation by RUNX1. Collectively this study indicates that, in addition to its ability to repress CBFB/RUNX, Cbfb-MYH11 has other activities that are likely important for leukemogenesis. This finding has important implications for future efforts to develop new treatments for Inv16+ AML.

Activity of elaeochytrin A from Ferula elaeochytris on leukemia cell lines

Phytochemical investigation of the roots of Ferula elaeochytris made it possible to isolate two sesquiterpene esters, 6-anthraniloyljaeschkeanadiol (elaeochytrin A) and 4β-hydroxy-6α-( p-hydroxybenzoyloxy)dauc-9-ene (elaeochytrin B), as well as eight known compounds: 6-angeloyljaeschkeanadiol, teferidin, ferutinin, 6-( p-hydroxybenzoyl)epoxyjaeschkeanadiol, 6-( p-hydroxybenzoyl)lancerotriol, 5-caffeoylquinic acid, 1,5-dicaffeoylquinic acid and sandrosaponin IX. The cytotoxic activities of all compounds were investigated on K562R (imatinib-resistant) human chronic myeloid leukaemia and DA1-3b/M2 BCR-ABL (dasatinib-resistant) mouse leukemia cell line. Elaeochytrin A was the most active compound on both cell lines (IC 50 = 12.4 and 7.8 μM, respectively). It was also tested on non-resistant human promyelocytic leukemia cells (HL60, IC 50 = 13.1 μM) and was not toxic to normal peripheral blood mononuclear cells up to 100 μM.

Potentiation of cytostatic effect of sulfur- or chlorine-containing purines and related ribonucleosides by amphotericin-B on cultured mouse leukemia cells.

Synergistic effects of sulfur-containing purines and related ribonucleosides (6-thioinosine, 6-thioguanine, 6-thiocyanatopurine, 6-methylthioinosine, 6-thiocyanatoguanine, 6-thiocyanatoguanosine, 6-phenacylthioinosine, 6-nitrobenzlythioinosine, 6-(p-chlorobenzyl)thioinosine, 6-(p-nitrobenzyl)thioguanosine, 6-benzylthioinosine, 6-ethylthioinosine, 6-benzylthioguanine, 6-benzylthiopurine, 6-methylthiopurine, and 6-thiocyanatoinosine) and chlorine-containing purine and its ribonucleoside, (6-chloropuine and 6-chloropurine riboside), in combination with the polyene antibiotic, Amphotericin-B, on cell survival and synthesis of DNA were examined in mouse leukemia L5178Y cells. 6-Methylthioinosine, 6-thiocyanatopurine, 6-thiocyanatoinsoine, 6-methylthiopurine, and 6-thiocyanatoguanine (or -guanosine) among sulfur-containing compounds were strongly potentiated by Amphotericin-B, and 6-chloropurine riboside, which is electronically analogous to methylthioinosine, was also enhanced by the polyene. Thiocyanoto or methylthio group at position 6 of the purine ring seems to be important for the polyene-mediated potentiation. 6-Methylthioinosine alone had much greater effect on DNA synthesis of HeLa cells than on L5178Y cells, and Amphotericin-B failed to potentiate the action of 6-methylthioinosine in HeLa cells.

The influence of 1,25-dihydroxyvitamin D3 and 1,24-dihydroxyvitamin D3 on alphavbeta3 integrin expression in cancer cell lines.

Integrins are cell-surface receptors engaged in important cancer invasion processes, such as adhesion, migration, proliferation and differentiation. The aim of this study was to evaluate the effect of 1,25-dihydroxyvitamin D3 (calcitriol) and its metabolite 1,24-dihydroxyvitamin D3 (PRI-2191) on alphavbeta3 integrin expression in various cancer cell lines. The expression levels of the beta3 and alphav integrins were reduced only in the WEHI-3 and LLC cell lines by the two compounds. Calcitriol or PRI-2191 treatment caused differentiation of WEHI-3 mouse leukemia cells, but apoptosis of LLC cells. WEHI-3 and LLC cells exposed to calcitriol or PRI-2191 lost their migratory and adhesive potentials. The inhibition of migratory potential was higher in the LLC cells than in the WEHI-3 cells and appeared to correlate with the increased down-regulation of alphavbeta3 integrin by calcitriol or PRI-2191. The observed in vivo effects (antitumor and antimetastatic) in mice bearing subcutaneously transplanted LLC cancer are possibly associated with inhibited migratory potential as a consequence of the lowered integrin expression caused by calcitriol or PRI-2191.

Activity of Indenoisoquinolines against African Trypanosomes

African trypanosomiasis (sleeping sickness), caused by protozoan Trypanosoma brucei species, is a debilitating disease that is lethal if untreated. Available drugs are antiquated, toxic, and compromised by emerging resistance. The indenoisoquinolines are a class of noncamptothecin topoisomerase IB poisons that are under development as anticancer agents. We tested a variety of indenoisoquinolines for their ability to kill T. brucei. Indenoisoquinolines proved trypanocidal at submicromolar concentrations in vitro. Structure-activity analysis yielded motifs that enhanced potency, including alkylamino substitutions on N-6, methoxy groups on C-2 and C-3, and a methylenedioxy bridge between C-8 and C-9. Detailed analysis of eight water-soluble indenoisoquinolines demonstrated that in trypanosomes the compounds inhibited DNA synthesis and acted as topoisomerase poisons. Testing these compounds on L1210 mouse leukemia cells revealed that all eight were more effective against trypanosomes than against mammalian cells. In preliminary in vivo experiments one compound delayed parasitemia and extended survival in mice subjected to a lethal trypanosome challenge. The indenoisoquinolines provide a promising lead for the development of drugs against sleeping sickness.

Structure-property relationship for description of apoptosis (programmed cell death) induction by phenol derivatives

Structure-property relationship based on fragment descriptors of molecular structure was used to assess the ability of phenol derivatives to induce apoptosis in L1210 leukemia cells in mice. Simple models that provide quite accurate estimates for the apoptic activity of various phenol compounds were suggested. Such models allow one to clearly interpret the contributions of structural elements of phenol derivatives and to predict how their apoptic activity will vary with structural modification.

Targeting 11q23 positive acute leukemia cells with high molecular weight-melanoma associated antigen-specific monoclonal antibodies

Acute leukemia with 11q23 aberrations is associated with a poor outcome with therapy. The lack of efficacy of conventional therapy has stimulated interest in developing novel strategies. Recent studies have shown that 11q23-positive acute leukemia cells express the high molecular weight-melanoma associated antigen (HMW-MAA). This tumor antigen represents a useful target to control growth of human melanoma tumors in patients and in severe combined immunodeficient (SCID) mice, utilizing antibody-based immunotherapy. This effect appears to be mediated by inhibition of the HMW-MAA function such as triggering of the focal adhesion kinase/proline-rich tyrosine kinase 2 (Pyk2) pathways. Therefore, in this study we tested whether HMW-MAA-specific monoclonal antibodies (mAb) could inhibit growth of 11q23-positive leukemia cells in SCID mice. HMW-MAA-specific mAb were tested for their ability to inhibit the in vitro proliferation of an 11q23-positive acute myeloid leukemia (AML) cell line and blasts from four patients with 11q23 aberrations and their in vivo growth in subcutaneous and disseminated xenograft models. The HMW-MAA-specific mAb did not affect in vitro proliferation although they down-regulated phosphorylated (P) Pyk2 expression. Furthermore, the mAb enhanced the in vitro anti-proliferative effect of cytarabine. In vivo the mAb inhibited the growth of leukemic cells in a dose-dependent fashion. However, the difference did not reach statistical significance. No effect was detected on P-Pyk2 expression. Furthermore, HMW-MAA-specific mAb in combination with cytarabine did not improve tumor inhibition. Lastly, the combination of two mAb which recognize distinct HMW-MAA determinants had no detectable effect on survival in a disseminated xenograft model. HMW-MAA-specific mAb down-regulated P-Pyk2 expression and enhanced the anti-proliferative effect of cytarabine in vitro, but had no detectable effect on survival or growth of leukemia cells in vivo. Whether the HMW-MAA-specific mAb can be used as carriers of toxins or chemotherapeutic agents against 11q23-acute leukemia remains to be determined.

Thalidomide inhibits epidermal growth factor-induced cell growth in mouse and human monocytic leukemia cells via Ras inactivation

The effect of thalidomide on epidermal growth factor (EGF)-induced cell growth was examined. Thalidomide inhibited EGF-induced cell growth in mouse and human monocytic leukemia cells, RAW 264.7, U937 and THP-1. Thalidomide inhibited EGF-induced phosphorylation of extracellular signal regulated kinase (ERK) 1/2, but not p38 and stress-activated protein kinase (SAPK)/JNK. The phosphorylation of MEK1/2 and Raf at Ser 338 as the upstream molecules of ERK 1/2 was also prevented by thalidomide. Further, it inhibited EGF-induced Ras activation through preventing the transition to GTP-bound active Ras. Thalidomide inhibited the Ras activation induced by lipopolysaccharide (LPS) and vascular endothelial growth factor (VEGF) as well as EGF. There was no significant difference in the expression and function of EGF receptor between thalidomide-treated and non-treated cells. Therefore, thalidomide was suggested to inhibit EGF-induced cell growth via inactivation of Ras.

Capture of Phosphopeptides Using α-Zirconium Phosphate Nanoplatelets

Alpha-zirconium phosphate nanoplatelets (alpha-ZrPN) were studied as a binding agent for phosphopeptides. Nanoplatelets of alpha-zirconium phosphate were incubated overnight with zirconium oxychloride, followed by centrifugation, and washed twice with water followed by an aqueous solution of 80% acetonitrile to form the binding agent. Alpha-ZrPN were able specifically to capture phosphoserine-containing peptides from a tryptic digest of a complex peptide mixture in which its abundance was only 0.05%. Alpha-ZrPN also bound peptides containing phosphothreonine and phosphotyrosine. The limit of detection for phosphopeptides is approximately 2 fmol, based on using matrix-assisted laser desorption/ionization mass spectrometry. Alpha-ZrPN were applied for the analysis of tryptic digests of mouse liver and leukemia cell phosphoproteomes and succeeded in identifying 158 phosphopeptides (209 phosphorylation sites) from 101 phosphoproteins in mouse liver lysate and 78 phosphopeptides (104 phosphorylation sites) from 59 phosphoproteins in leukemia cell extract. For these two tryptic digests, the alpha-ZrPN approach is able to capture more phosphopeptides than that obtained from TiO2 particles or from Fe(3+)-IMAC beads, but each method is able to bind some phosphopeptides that the others do not.