Mouse Leukemia Cells Research Articles

LY294,002, a specific inhibitor of PI3K/Akt kinase pathway, antagonizes P-glycoprotein-mediated multidrug resistance

The transmembrane transport pump P-glycoprotein (P-gp) causes the efflux of chemotherapeutic agents from cells and is an important system that secures multidrug resistance (MDR) of neoplastic cells. In the present study drug sensitive L1210 and multidrug resistant L1210/VCR mouse leukemic cell lines were used as an experimental model. We found that LY 294,002, a specific inhibitor of PI3K/Akt kinase pathway, reduced the degree of vincristine resistance in L1210/VCR cells significantly and in a concentration-dependent manner. This was accompanied by decrease in IC 50 value to vincristine from 3.195 ± 0.447 to 1.898 ± 0.676 μmol/l for 2 μmol/l, to 0.947 ± 0.419 μmol/l for 4 μmol/l, and to 0.478 ± 0.202 μmol/l for 8 μmol/l LY294,002. The IC 50 value of sensitive cells for vincristine was about 0.010 μmol/l. FACS analysis of the proportion of cells in apoptosis or necrosis by annexin-V apoptosis kit showed the following: (i) vincristine-induced apoptosis in resistant cell to a much lower extent than in sensitive cells; (ii) LY294,002 alone did not induce apoptosis or necrosis in both sensitive and resistant cells; (iii) LY294,002 applied together with vincristine significantly increased the number of apoptotic cells. Transport activity of P-gp in resistant cells was monitored using calcein/AM as substrate and was depressed by LY294,002 in a concentration dependent manner. Significant differences in calcein retention were not observed when cells were preincubated with LY294,002 at different times from 0.5 to 24 h. Sensitive and resistant cells contain similar amounts of uncleaved (i.e., unactivated) caspase-3 but in latter cells the activation of caspase-3 by proteolytic cleavage was decreased. The reversal of vincristine resistance by LY294,002 was associated with marked activation of caspase-3. Western blot analysis revealed that the development of MDR phenotype in L1210/VCR cells was also associated with increased level of Bcl-2 protein. All the above findings point to the possible involvement of PI3K/Akt kinase pathway in modulation of P-gp mediated multidrug resistance in L1210/VCR mouse leukemic cell line. MDR reversal effect of LY294,002 is accompanied with this compound's influence on vincristine-induced apoptosis.

Molecular Imaging of EGFR Kinase Activity in Tumors with 124I-Labeled Small Molecular Tracer and Positron Emission Tomography

Positron emission tomography (PET) with epidermal growth factor receptor (EGFR) kinase-specific radiolabeled tracers could provide the means for noninvasive and repetitive imaging of heterogeneity of EGFR expression and signaling activity in tumors in individual patients before and during therapy with EGFR signaling inhibitors. We developed the synthesis and (124)I-radiolabeling of the (E)-But-2-enedioic acid [4-(3-[(124)I]iodoanilino)-quinazolin-6-yl]-amide-(3-morpholin-4-yl-propyl)-amide (morpholino-[(124)I]-IPQA), which selectively, irreversibly, and covalently binds the adenosine-triphosphate-binding site to the activated (phosphorylated) EGFR kinase, but not to the inactive EGFR kinase. The latter was demonstrated using in silico modeling with crystal structures of the wild type and different gain-of-function mutants of EGFR kinases. Also, this was demonstrated by selective radiolabeling of the EGFR kinase domain with morpholino-[(131)I]-IPQA in A431 human epidermoid carcinoma cells and Western blot autoradiography. In vitro radiotracer accumulation and washout studies demonstrated a rapid accumulation and progressive retention postwashout of morpholino-[(131)I]-IPQA in A431 epidermoid carcinoma and in U87 human glioma cells genetically modified to express the EGFRvIII mutant receptor, but not in the wild-type U87MG glioma cells under serum-starved conditions. Using morpholino-[(124)I]-IPQA, we obtained noninvasive PET images of EGFR activity in A431 subcutaneous tumor xenografts, but not in subcutaneous tumor xenografts grown from K562 human chronic myeloid leukemia cells in immunocompromised rats and mice. Based on these observations, we suggest that PET imaging with morpholino-[(124)I]-IPQA should allow for identification of tumors with high EGFR kinase signaling activity, including brain tumors expressing EGFRvIII mutants and nonsmall-cell lung cancer expressing gain-of-function EGFR kinase mutants. Because of significant hepatobiliary clearance and intestinal reuptake of the morpholino-[(124)I]-IPQA, additional [(124)I]-IPQA derivatives with improved water solubility may be required to optimize the pharmacokinetics of this class of molecular imaging agents.

Nitric Oxide Production by the Vacuolar-Type (H+)-ATPase Inhibitors Bafilomycin A1 and Concanamycin A and Its Possible Role in Apoptosis in RAW 264.7 Cells

In the mouse leukemic monocyte cell line RAW 264.7, the vacuolar-type (H(+))-ATPase (V-ATPase) inhibitors bafilomycin A1 and concanamycin A induced nitric oxide (NO) production through the expression of inducible nitric-oxide synthase mRNA and its protein and decreased cell growth and survival as determined by 3-(4,5-dimethyl(thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Bafilomycin A1 and concanamycin A activated nuclear factor (NF)-kappaB and activator protein-1 and decreased the level of IkappaB-alpha and increased that of phosphorylated c-Jun N-terminal kinase (JNK). NO production induced by these V-ATPase inhibitors was suppressed by the NF-kappaB inhibitor Bay 11-7082 [(E)3-[(4-methylphenyl)sulfonyl])-2-propenenitrile] and the JNK inhibitor SP600125 [anthra[1,9-cd]pyrazol-6(2H)-one] in parallel with the partial alleviation of the V-ATPase inhibitor-induced decrease in MTT response. The Na(+),K(+)-ATPase inhibitor dibucaine and the F-ATPase inhibitor oligomycin did not induce NO production at which concentrations the MTT response was decreased. The NO donor S-nitroso-N-acetyl-dl-penicillamine further lowered the V-ATPase inhibitor-induced decrease in the MTT response, and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, sodium salt (carboxy-PTIO) alleviated it partially. Mitochondrial depolarization, an index of apoptosis, was induced by bafilomycin A1 and concanamycin A. On treatment with the nitric-oxide synthase inhibitor N(G)-monomethyl-l-arginine acetate, the disruption of mitochondrial membrane potential induced by bafilomycin A1 and concanamycin A was alleviated partially in parallel with the decrease in NO production. Carboxy-PTIO also alleviated it partially. Our findings suggest that the V-ATPase inhibitors bafilomycin A1 and concanamycin A similarly induce NO production and the newly produced NO participates partially in the V-ATPase inhibitor-induced apoptosis in RAW 264.7 cells.

L1210 cells cultivated under the selection pressure of doxorubicin or vincristine express common mechanisms of multidrug resistance based on the overexpression of P-glycoprotein

Multidrug resistance of neoplastic tissue is often associated with the overexpression and increased drug transport activity of plasma membrane transporters like P-glycoprotein (P-gp), multidrug resistance associated proteins (MRPs) or breast cancer resistance protein, as well as with the elevation of the glutathione detoxification pathway. We have already described the overexpression of P-gp under the selection pressure of vincristine in L1210 mouse leukemia cells. In the present study, mechanisms of multidrug resistance induced in L1210 cells cultivated in the presence of doxorubicin were analyzed. The selection pressure of both vincristine (yielding a resistant subline of L1210 cells, R V) and doxorubicin (yielding a resistant subline of L1210 cells, R D) induced a dramatic depression of cell sensitivity to both drugs. Both R V and R D cells demonstrated a lack of ability to accumulate calcein/AM and fluo-3/AM as fluorescent substrates of P-gp and MRP. The retention of dyes could be reached in both cell sublines by the application of inhibitors of P-gp (like verapamil) but not by probenecid – an inhibitor of anion transporters, including MRPs. Massive protein bands, at a M r range of 130–180 kDa that interact with c219 antibody against P-gp, were detected in the crude membrane fraction isolated from both R V and R D (but not from L1210) cells by Western blot. The cytosolic activity of glutathione S-transferase was found to be similar in R V and R D cells and did not differ significantly from the activity ascertained in parental L1210 cells. Neither the R V nor R D cell sublines differed considerably, as measured by cell ultrastructure. In conclusion, based on P-gp overexpression, both doxorubicin and vincristine induce a common multidrug resistance phenotype in L1210 cells.

Cytotoxicity of bioactive polymeric fractions from grape cell culture on human hepatocellular carcinoma, murine leukemia and non-cancerous PK15 kidney cells

Previously, we isolated two fractions (TP-4 and TP-6) from grape cell culture that were potent catalytic inhibitors in a human DNA topoisomerase II assay for cancer chemoprevention. The objectives of this study were to further assess cytotoxicity of these fractions on cancerous and non-cancerous cells, and to subfractionate and characterize the composition of TP-6, a fraction that was selectively cytotoxic to carcinoma cell lines. Both TP-4 and TP-6 provided significant cytotoxicity to L1210 mouse leukemia cells. Only TP-6, a procyanidin-rich fraction, significantly reduced viability in HepG2 human liver cancer cells, yet unlike resveratrol, caused no cytotoxicity to non-cancerous PK15 pig kidney cells. After further subfractionation of TP-6 (maximal toxicity = 67.2%; ED 50 = 50.5 μM), the cytotoxicity of subfractions on HepG2 cells was TP-6-5 (maximal toxicity = 71.8%; ED 50 = 14.1 μM), TP-6-6 (maximal toxicity = 64.3%; ED 50 = 67.0 μM), and TP-6-4 (maximal toxicity = 27.6%; ED 50 = 118.0 μM) in descending order. LC–ESI/MS data suggested that cytotoxicity of these procyanidin mixtures to HepG2 cells was proportional to the degree of polymerization. Because TP-6 and its subfractions were selectively cytotoxic to cancerous cell lines tested, they warrant further investigation as potential natural anticancer agents.

Suppression of P-glycoprotein expression by antipsychotics trifluoperazine in adriamycin-resistant L1210 mouse leukemia cells

Multidrug resistance (MDR) to unrelated chemotherapeutic drugs can be mediated by overexpression of P-glycoprotein (P-gp), the mdr gene product. Trifluoperazine (TFP), a phenothiazine derivative antipsychotics, is known to reverse MDR of tumor cell lines by blocking P-gp efflux function. In the present study, we evaluated the effect of TFP on the expression of P-gp in multidrug-resistant L1210/Adr mouse leukemic cell lines, which are characterized by overexpession of P-gp. We found that TFP induced the downregulation of P-gp protein and mdr1b mRNA in a dose- and time-dependent manner in L1210/Adr cells. TFP reduction of mdr1b mRNA was paralleled by transcriptional suppression of the mdr1b promoter. Moreover, TFP restored the adriamycin-induced apoptosis in L1210/Adr cells. These results suggest that TFP may have utility as an adjuvant in the therapy of leukemia for the reversal of P-gp-dependent MDR as well as for the management of psychological symptoms in the cancer patients.

Bis(2-hydroxybenzylidene)acetone, a potent inducer of the phase 2 response, causes apoptosis in mouse leukemia cells through a p53-independent, caspase-mediated pathway

Bis(2-hydroxybenzylidene)acetone is a potent inducer of the phase 2 response through the Keap1-Nrf2-ARE pathway. This double Michael reaction acceptor reacts directly with Keap1, the sensor protein for inducers, leading to enhanced transcription of phase 2 genes and protection against oxidant and electrophile toxicities. In our efforts to identify potent chemoprotective agents, we found that in rapidly growing murine leukemia cells (L1210) low concentrations (in the submicromolar range) of bis(2-hydroxybenzylidene)acetone markedly increased the activities of NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) and glutathione reductase, and the levels of total glutathione, three markers of the phase 2 response. In contrast, at high concentrations (in the micromolar range) the same compound caused G2/M cell cycle arrest and apoptosis. Importantly, a mutant L1210 cell line (Y8), selected for resistance to deoxyadenosine and lacking expression of p53 protein, was considerably more sensitive to the apoptotic effects of bis(2-hydroxybenzylidene)acetone. When caspase activities were evaluated in cell-free extracts prepared from treated wild type or mutant L1210 cells, the activities of caspase-3, the terminal caspase in the cascade leading to apoptosis, and caspase-10 were found to be markedly elevated. The activities of other caspases measured, caspase-1, -6 and -8, were not appreciably affected. Thus, both induction of the phase 2 response and p53-independent, caspase-3-mediated apoptosis could act cooperatively in chemoprotection. The concentration-dependent differential effects on these two pathways should be carefully considered in mechanistic explanations and strategic designs.

Synthesis and Biological Evaluation of Purealin and Analogues as Cytoplasmic Dynein Heavy Chain Inhibitors

Cytoplasmic dynein plays important roles in membrane transport, mitosis, and other cellular processes. A few small-molecule inhibitors of cytoplasmic dynein have been identified. We report here the first synthesis of purealin, a natural product isolated from the sea sponge Psammaplysilla purea, which is known to inhibit axonemal dynein. Also described are the first syntheses, by modular amide coupling reactions, of the natural product purealidin A (a component of purealin) and a small library of analogues. The library was examined for inhibition of cytoplasmic dynein heavy chain and cell growth. The compounds showed effective antiproliferative activity against a mouse leukemia cell line but selective activities against human carcinoma cell lines. Purealin and some of the analogues inhibited the microtubule-stimulated ATPase activity of recombinant cytoplasmic dynein heavy chain motor domain. The inhibitory effect of purealin was concentration dependent and uncompetitive, supporting the hypothesis that it does not compete with the binding of ATP.

Diverse biomodulatory effects of glucomannan from Candida utilis

Using four experimental model systems, it was demonstrated that glucomannan (GM) isolated from the cell wall of the industrial yeast Candida utilis revealed a broad range of protective activities. This effect depended on the nature and mode of action of the counteracting genotoxic compound as well as on the experimental model system used. In the Saccharomyces bioprotectivity assay, GM increased resistance towards ofloxacin-induced toxicity in the wild type and recombination repair-deficient yeast strains significantly enhancing survival of the cells. In the chromosomal aberration assay, GM exerted anticlastogenic effect against maleic hydrazide induced clastogenicity in Vicia faba L. In the DNA-topology assay, GM protected plasmid DNA from the breaks induced by Fe 2+ ions, but enhanced damage induced by bleomycin and hydrogen peroxide. In the cell-revitalization assay, it enhanced cytotoxic/cytostatic effect of teniposide applied to mouse leukemia cells. Thus, depending on the experimental model, GM acted as antimutagen, anticlastogen, DNA breaks inhibitor or inducer, and as cytotoxic/cytostatic effect enhancer. Several possible mechanisms of bioprotective action underlying the observed activities are suggested including iron chelation and free radical scavenging. The results imply that GM is a polysaccharide with marked biological activities and suggest its potential biomedical application, especially in combination with other bioactive compounds.

Structures, biogenetic relationships, and cytotoxicity of pimarane-derived diterpenes from Petalostigma pubescens

Extraction of Petalostigma pubescens heartwood followed by chromatographic purifications and crystallizations afforded five tricyclic diterpenes: 5,9-syn-rosanes petalostigmones A and B ( 1 and 2), the erythroxylane petalostigmone C ( 3), the norditerpene lactone pubescenone ( 4), and the known ent-cleistanthane diterpene (−)-sonderianol ( 5). The structures and relative stereochemistry were elucidated by means of spectroscopic methods, chemical correlations, and, in the cases of 1 and 4, by X-ray crystallographic analyses. The new isolates 1– 4 are assumed to belong to the same absolute configurational family (9αCH 3) of ent-pimarane-derived diterpenes as the known co-occurring (−)- 5 (10αCH 3). Biogenetic schemes originating from a common ent-copalyl diphosphate intermediate are presented to rationalize the structures of these natural products. A novel ring contraction–ring expansion mechanism is suggested to account for the 7-membered B ring of pubescenone. Compounds 1– 5 were evaluated for their cytotoxicity; sonderianol ( 5) showed the highest activity against mouse leukemia cell lines L1210, P388 and mouse liver cancer cells HEPA1c1c7.

Targeting BCR-ABL Kinase Activity-Independent Signaling Pathways and Leukemia Stem Cells Is Essential for Curative Therapy of Philadelphia Chromosome Positive (Ph+) Leukemia.

Therapeutic efforts for Philadelphia chromosome positive (Ph+) leukemia have focused on targeting mainly BCR-ABL kinase activity with kinase inhibitors, since it has generally been believed that shutting down BCR-ABL kinase activity will completely inhibit its functions, leading to inactivation of downstream signaling pathways. Inhibition of BCR-ABL kinase activity by imatinib mesylate (Gleevec) is highly effective in treating human Ph+ chronic myeloid leukemia (CML) in chronic phase, but not Ph+ B-cell acute lymphoblastic leukemia (B-ALL) and CML blast crisis. The reasons for this are not well understood, but the fact that imatinib is a strong inhibitor of BCR-ABL kinase activity suggests that BCR-ABL kinase activity-independent pathways also play a critical role in the development of both forms of Ph+ leukemia. We have previously shown that the SRC family kinases LYN, HCK, and FGR are activated by BCR-ABL in pre-B leukemic cells and are required for the development of B-ALL (Hu et al, Nat Genet 36:453, 2004). Others have shown that cells from imatinib-resistant patients imatinib expressed an activated form of LYN (Donato et al, Blood 101:690, 2003), and that a BCR-ABL mutant with no kinase activity was still able to activate HCK (Warmuth et al, J Biol Chem 272:33260, 1997). Based on these observations, we hypothesized that inhibition of BCR-ABL kinase by imatinib might not inactivate SRC kinases activated by BCR-ABL in pre-B leukemic cells, which may explain the relatively poor activity of imatinib against Ph+ B-ALL and lymphoid blast crisis CML. We find that SRC kinases activated by BCR-ABL remain fully active in imatinib-treated mouse leukemic cells and this BCR-ABL kinase activity-independent pathway is essential for leukemic cell survival and proliferation. Blockade of this pathway also prevents CML transition to lymphoid blast crisis. In mice with B-ALL, inhibition solely of BCR-ABL kinase activity by imatinib is not curative, but inhibition of both SRC and BCR-ABL kinase activities by the novel, oral, multi-targeted kinase inhibitor dasatinib (BMS-354825), while not killing leukemic stem cells, affords complete remission, maintained as long as treatment is continued. In these mice, we identified the B-ALL leukemic stem cells as B220+CD43+ pro-B cells. CML mice treated with dasatinib lived significantly longer than those treated with imatinib, which correlated with significantly lower numbers of BCR-ABL-expressing leukemic cells in bone marrow, peripheral blood, and spleens of dasatinib-treated CML mice versus placebo- or imatinib-treated mice. However, neither dasatinib nor imatinib were curative in these mice, which was attributed to an inability of both drugs to completely kill Lin-c-kit+CD34-Hoe- CML stem cells. Our studies indicate that complete eradication of leukemic cells in B-ALL and CML mice requires not only targeting BCR-ABL kinase activity-dependent and SRC-dependent pathways, but also killing BCR-ABL-expressing stem cells insensitive to both imatinib and dasatinib. However, the rapid and striking hematologic response of B-ALL mice to dasatinib suggests that the pro-B progenitors with acquired self-renewal capacity are the major source of highly proliferating B-lymphoid leukemic cells in B-ALL mice, and that complete inhibition of growth of this leukemic population with dasatinib could achieve long-term survival in B-ALL.

Expression of P-glycoprotein in L1210 cells is linked with rise in sensitivity to Ca 2+

L1210/VCR cell line (R) was obtained by adaptation of the L1210 mouse leukaemia cells (S) to vincristine and showed P-glycoprotein (P-gp) mediated multidrug resistance (MDR). R cells were observed to be more sensitive to high external calcium as parental S. More pronounced calcium uptake was observed for R cells. Moreover, differences in intracellular calcium cell localization between S and R cells were found ultrastructurally following a calcium precipitating cytochemical method. In S cells, calcium precipitates were found to be localized predominantly along the cell surface coat and within mitochondria delineating the cristae. In R cells, precipitates were also found inside nuclei, at the border of heterochromatin clumps, and scattered within the cytoplasm. High extracellular calcium did not influence the P-gp mediated extrusion of calcein/AM as P-gp substrate. These results indicate that calcium enters and consequently damages the MDR cells to a higher extent than parental cells.

The effect of steroid on myeloid leukemic cells: The potential of short-course high-dose methylprednisolone treatment in inducing differentiation, apoptosis and in stimulating myelopoiesis

Several in vitro studies have shown that dexamethasone (Dex) and prednisolone can induce differentiation of some mouse and human myeloid leukemic cells to macrophages and granulocytes. Based on in vitro experiments, we have shown that short-course (3–7 days) high-dose methylprednisolone (HDMP) (20–30 mg/kg/day) treatment can induce differentiation of myeloid leukemic cells in vivo in children with different subtypes of acute myeloblastic leukemia (AML) (AML-M1, -M2, -M3, -M4, -M7). We have also shown that induction of apoptosis of myeloid leukemic cells with or without differentiation is possible by short-course HDMP treatment. In addition, short-course HDMP treatment has been shown to be effective in accelerating leukocyte recovery, possibly stimulating normal CD34-positive hematopoietic progenitor cells. Addition of HDMP to mild cytotoxic chemotherapy (low-dose cytosine arabinoside (LD-Ara-c), weekly mitoxantrone and Ara-c or 6-thioguanine) increased the remission rate (87–89%) and improved the outcome of AML children. We believe that the results of our 17-year clinical experience will provide important benefits to AML patients.

Transplantation of human acute myeloid leukemia (AML) cells in immunodeficient mice reveals altered cell surface phenotypes and expression of human endothelial markers

To better characterize acute myeloid leukemia (AML) development in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice, we transplanted samples from patients with AML or KG-1 and EOL-1 cell lines. We found 9/12 primary AML samples and both cell lines to engraft within 2–8 weeks, with 5–80% human cells in bone marrow. Compared with freshly isolated AML cells, percentages of human CD33 +, CD38 +, CD31 + CD13 + or CD15 + subpopulations increased after transplantation, whereas percentages of CD34 + cells decreased. Engrafted mice frequently showed expression of human endothelial cell markers. Thus, transplantation of human AML into NOD/SCID mice reveals expression of hematopoietic and endothelial differentiation markers.

Resistance to 9-3-D-Arabinofuranosyl-2-Fluoroadenine due to Reduced Incorporation into DNA from Competition by Excess Deoxyadenosine Triphosphate: Implications for Different Sensitivities to Nucleoside Analogues

The cytotoxic action of the deoxyadenosine analogue 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) depends on the incorporation into DNA after being phosphorylated to F-ara-A triphosphate (F-ara-ATP) by deoxycytidine kinase (dCK). The mechanisms of resistance to F-ara-A were investigated in a newly established variant of L1210 mouse leukemia cells (L1210/F). L1210/F was more than 41-fold more resistant to F-ara-A than the parental cell line and had a 55% lower dCK activity. Interestingly, L1210/F showed a modest level of cross-resistance to deoxycytidine analogues phosphorylated by dCK, for instance, 1-beta-D-arabinofuranosylcytosine (ara-C). The comparative study of F-ara-A and ara-C demonstrated that the difference in the accumulation of their respective triphosphates was minor. In contrast, the incorporation of F-ara-A into DNA was strikingly suppressed compared with that of ara-C. In general, the high natural triphosphate levels interfere with corresponding analogue incorporation into DNA. The deoxyadenosine triphosphate (dATP) and deoxycytidine triphosphate pool sizes in L1210/F cells were increased by 4.9-fold and 1.9-fold, respectively, compared with the parental cells. Treatment with hydroxyurea increased the ratio of F-ara-ATP to dATP 2.1-fold and enhanced the action of F-ara-A in L1210/F. This is the first cell line to show that the profoundly defective incorporation of F-ara-A into DNA during competition with excess dATP confers a high degree of resistance to F-ara-A.

Experimental Study on Apoptosis in Leukemia Cells Induced by Econazole

To investigate apoptosis of mouse leukemia cell (WEHI-3) induced by econazole and its mechanism, apoptosis induced by econazole was examined by flow cytometry, while free calcium ([Ca(2+)]i) was determined by Fura-2 fluorescein load technique. The protein was isolated from endoplasmic reticulum of WEHI-3 cells, and then the expression of caspase-12 and caspase-7 was evaluated by Western blot. The results showed that WEHI-3 exhibited typical change of apoptosis when it was treated by econazole, [Ca(2+)]i was significantly higher in comparison with the control. The expression of caspase-12 and caspase-7 enhanced as the econazole concentration increased. In conclusion, econazole can induce WEHI-3 cell apoptosis and the caspase-12 plays a key role in this process.

Berberine inhibits arylamine N-acetyltransferase activity and gene expression in mouse leukemia L 1210 cells

N-acetyltransferases (NATs) are recognized to play a key role in the primary step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylators, which are being thought to involve cancer risk related to environmental exposure. Berberine has been shown to induce apoptosis and affect NAT activity in human leukemia cells. The purpose of this study is to examine whether or not berberine could affect arylamine NAT activity and gene expression (NAT mRNA) and the levels of NAT protein in mouse leukemia cells (L 1210). N-acetylated and non-N-acetylated AF were determined and quantited by using high performance liquid chromatography. NAT mRNA was determined and quantited by using RT-PCR. The levels of NAT protein were examined by western blotting and determined by using flow cytometry. Berberine displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by berberine for up to 24 h. The NAT1 mRNA and NAT proteins in mouse leukemia cells were also inhibited by berberine. This report is the first demonstration, which showed berberine affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and levels of NAT protein.

Bcr-Abl-mediated suppression of normal hematopoiesis in leukemia

A variety of experimental evidence including findings in various mouse models indicates that the BCR-ABL oncogene is the cause of chronic myeloid leukemia (CML). Since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we determined whether this is an active process mediated by the leukemia cells. The lipocalin 24p3 was reported to be secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells. Here, we show that BCR-ABL+ mouse hematopoietic cells induced persistent expression and secretion of 24p3. Importantly, BCR-ABL+ hematopoietic cells were resistant to the apoptotic effects of 24p3. The expression of the Bcr-Abl oncoprotein and its tyrosine kinase were required for induction of 24p3 expression. Co-culture studies showed that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Antisense 24p3/siRNA expression reduced the level of 24p3 protein in both BCR-ABL+ cells and in conditioned medium (CM) obtained from these cells. CM from BCR-ABL+ cells expressing antisense 24p3/siRNA had reduced apoptotic activity for target cells; 24p3 antibody also reduced the apoptotic activity of the CM. Leukemic mice induced by BCR-ABL+ cells expressing either antisense 24p3 or 24p3 siRNA had increased levels of normal hematopoiesis and reduced invasion of leukemia cells in marrow and spleen tissues. These findings indicate that suppression of normal hematopoiesis in BCR-ABL-induced leukemia is an active process involving secretion of the cell death-inducing factor 24p3 by mouse leukemia cells, raising the possibility that similar factors are involved in BCR-ABL+ CML.

Functional analysis of the effect of forced activation of STAT3 on M1 mouse leukemia cells

M1 mouse myeloid leukemia cells exhibit growth arrest and differentiation to monocytes/macrophages in response to leukemia inhibitory factor (LIF) stimulation. Although recent studies have demonstrated that STAT3 plays a central role in this process, it is unknown whether STAT3 activation alone is sufficient. To address this issue, we have established M1/STAT3ER cells, where STAT3 is selectively activated by 4-hydroxytamoxifen (4HT). 4HT stimulation did not have any effect on growth and morphology of M1/ STAT3ER cells, and did not induce the down-regulation of mRNA of c-myc and c-myb, which is necessary for M1 cell differentiation. On the other hand, mRNA of jun-B, IRF1 and p19 was increased by 4HT. DNA precipitation assay indicated that both stimulation of LIF and 4HT similarly activated STAT3ER. Introduction of a constitutive active MAP kinase kinase (MEK1) into M1/STAT3ER cells did not induce differentiation either. Together, our present data suggest that signaling other than the activation of STAT3 and MEK1 may be necessary for M1 cell-growth arrest and differentiation, while a set of early genes of LIF are induced by only STAT3 activation.

Luotonin A: A Lead toward Anti-Cancer Agent Development

Luotonin A, a novel pyrroloquinazolinoquinoline alkaloid isolated from the aerial parts of Peganum nigellastrum Bunge, showed a cytotoxic activity against mouse leukemia cells (P-388) and an inhibitory activity against topoisomerase I and II. This review covers the isolation, structural determination, synthesis and biological activity of luotonin A and its related derivatives. A water-soluble topoisomerase I inhibitor, 14-azacamptothecin analogous to luotonin A and camptothecin, was also discussed.