Murine Lymphocytic Leukemia Cell Research Articles

Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL

AbstractDysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5′ untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation–related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

In vitro evaluation of immunobiological activity of simple mannolipids

Immunomodulation, cytotoxicity and anti-cancer activity of selected amphiphilic non-ionic (thio)alkyl α-D-mannosides (with aglycone of C6-C12) were investigated in vitro in human cervix epitheloid carcinoma cell line HeLa, murine melanoma cancer cells B16, murine lymphocytic leukemia cell line L1210, murine fibroblast cell line NIH 3 T3 and murine macrophage cell line RAW 264.7. Toxicological studies revealed structure-dependent immunobiological effectivity based on a tight interaction with relevant cells. The results demonstrated diverse immunomodulation of macrophage cell-line RAW264.7 proliferation and production of Th1 and Th2 cytokines, and induction of pro-inflammatory interleukins IL-1α, TNFα, IL-6, IL-12 and IL-17 and anti-inflammatory IL-10 following (thio)alkyl α-D-mannosides 24 and 48 h exposure. Direct application of alkyl mannosides MOC10 and MOC12 and their thio analogues MSC10 and MSC12 in reconstructed human EpiDerm™ and MOC12 and MSC12 in EpiOcular™ model assays for dermal and ocular irritation together with quantification of human proinflammatory cytokines IL-1α, TNFα, IL-6 and IL-8 culture media release was used to ascertain toxicological safety.

Lymphoma Immunochemotherapy: Targeted Delivery of Doxorubicin via a Dual Functional Nanocarrier.

Chemotherapy drug (paclitaxel, PTX) incorporated in a dual functional polymeric nanocarrier, PEG-Fmoc-NLG, has shown promise as an immunochemotherapy in a murine breast cancer model, 4T1.2. The formulation is composed of an amphiphilic polymer with a built-in immunotherapy drug NLG919 that exhibits the immunostimulatory ability through the inhibition of indoleamine 2,3-dioxygenase 1 (IDO-1) in cancer cells. This work evaluates whether the PEG-derivatized NLG polymer can also be used for delivery of doxorubicin (Dox) in treatment of leukemia. The Dox-loaded micelles were self-assembled from PEG-Fmoc-NLG conjugate, which have a spherical shape with a uniform size of ∼120 nm. In cultured murine lymphocytic leukemia cells (A20), Dox-loaded PEG-Fmoc-NLG micelles showed a cytotoxicity that was comparable to that of free Dox. For in vivo studies, significantly improved antitumor activity was observed for the Dox/PEG-Fmoc-NLG group compared to Doxil or the free Dox group in an A20 lymphoma mouse model. Flow cytometric analysis showed that treatment with Dox/PEG-Fmoc-NLG micelles led to significant increases in the numbers of both total CD4+/CD8+ T cells and the functional CD4+/CD8+ T cells with concomitant decreases in the numbers of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Treg). Dox/PEG-Fmoc-NLG may represent a promising immunochemotherapy for lymphoma, which warrants more studies in the future.

In vitro 웰니스 화합물 (Ochnaflavone)에 의한 암세포 성장 저해

많은 식물들은 부작용이 적고, 가격이 저렴하며, 다양한 웰니스 융복합 화합물들을 함유하고 있기 때문에 다양한 제재에 이용되고 있다. 이 연구에서 뉴질랜드 식물인 Quintinia acutifolia (Q. acutifolia)로부터 쥐 백혈병 세포(P388 murine lymphocytic leukemia cells)의 성장을 저해하는 활성을 MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide] assay에 의해 평가하였다. P388 murine lymphocytic leukemia 세포의 성장을 저해하는 2,3,2,3-tetrahydroochanaflavone (1)과 2,3-dihydroochana-flavone (3)을 1D/2D-NMR와 다른 분광학적 분석법에 의해 분리하였고, 구조를 규명하였다. 이 두 화합물은 두 개의 플라보노이드 기본구조를 갖는 바이플라보노이드 (biflavonoid)로써 2,3,2,3-Tetrahydroochnaflavone (1)과 2,3-dihydroochana-flavone (3) 화합물은 P388 murine lymphocytic leukemia세포에 대해 50%의 성장저해를 나타내는 농도가 각각 $8.2{\mu}g/mL$ 와 $3.1{\mu}g/mL$ 로 나타났다. 특히 2,3-dihydroochana-flavone (3) 화합물은 2,3,2,3-tetrahydroochanaflavone (1)의 B 링(ring)에 쌍으로 결합되지 않은 플라본 구조 (unconjugated flavonone system)를 갖는 것으로 나타났다. 그럼으로 두 화합물은 향후 항암 치료제 개발에 이용될 수 있으며, 더 많은 연구가 요구된다. 【Medicinal plants containing wellness-fusion-complex compound are increasingly being pursued as suitable alternative sources of various biological properties. In this study, inhibitory effect of Quintinia acutifolia, which is a New Zealand plant, on P388 murine lymphocytic leukemia cells using MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide] assay. Based on $^1H-NMR$ , $^{13}C-NMR$ spectral data and other spectral analysis, 2,3,2,3-tetrahydroochanaflavone (1) and 2,3-dihydroochana-flavone (3) inhibited the leukemia cells were purified from the plants. 2,3,2,3-tetrahydroochanaflavone (1) and 2,3-dihydroochana-flavone (3) are biflavonoids possessing two basic flavonoids and actively inhibited growth of P388 murine lymphocytic leukemia cells with a 50% inhibitory concentration ( $IC_{50}$ ) of $8.2{\mu}g/mL$ and $3.1{\mu}g/mL$ , respectively. Specially, 2,3-dihydroochana-flavone (3) possessed unconjugated flavonone system, which isn't consist of a pair with B ring of 2,3,2,3-tetrahydroochanaflavone (1). Therefore, the two compounds could be considered as a candidate for development of anticancer drugs and need to much studies in the future.】

Mouse bone marrow-derived mesenchymal stem cells inhibit leukemia/lymphoma cell proliferation in vitro and in a mouse model of allogeneic bone marrow transplant.

The allogeneic hematopoietic stem cell (HSC) transplantation of mesenchymal stem cells (MSCs) contributes to the reconstitution of hematopoiesis by ameliorating acute graft-versus-host disease (aGVHD). However, the role of MSCs in graft-versus-leukemia remains to be determined. In the present study, we co-cultured C57BL/6 mouse bone marrow (BM)-derived MSCs with A20 murine B lymphoma, FBL3 murine erythroleukemia and P388 murine acute lymphocytic leukemia cells. Cell proliferation, apoptosis, cell cycle progression and the amount of cytokine secretion were then measured using a Cell Counting kit-8, Annexin V/propidium iodide staining, flow cytometry and ELISA, respectively. We also established a model of allogeneic bone marrow transplantation (BMT) using BALB/c mice. Following the administration of A20 cells and MSCs, we recorded the symptoms and the survival of the mice for 4 weeks, assessed the T cell subsets present in peripheral blood, and, after the mice were sacrifice, we determined the infiltration of MSCs into the organs by histological staining. Our results revealed that the MSCs inhibited the proliferation of the mouse lymphoma and leukemia cells in vitro, leading to cell cycle arrest and reducing the secretion of interleukin (IL)-10. In our model of allogeneic BMT, the intravenous injection of MSCs into the mice injected wth A20 cells decreased the incidence of lymphoma, improved survival, increased the fraction of CD3+CD8+ T cells, decreased the fraction of CD3+CD4+ T cells and CD4+CD25+ T cells in peripheral blood, and ameliorated the manifestation of aGVHD. The results from the present study indicate that MSCs may be safe and effective when used in allogeneic BMT for the treatment of hemotological malignancies.

Enhancement of immunogenicity of murine lymphocytic leukemia cells by transfection with BCG heat shock protein 70 gene

The effects of BCG heat shock protein 70 (BCG HSP70) gene transfection on tumorigenicity and immunogenicity of murine lymphocytic leukemia cell line (L1210) were studied. After HSP70 gene transfection, the tumor cells became strongly immunogenic and lost their tumorigenicity in syngeneic mice. It mainly exhibited that tumor growth was slow or without the formation of tumor, mean survival time of mice was significantly prolonged and a marked stimulating effect on L1210 specific Th1 cells detected by IFN-γ ELISPOT assay. Tumor-bearing mice treated with the L1210-HSP70 cells showed thorough coagulation necrosis and abundant CD8+ T lymphocyte infiltration. Meanwhile, as the tumor vaccine, the HSP70-transfected tumor cells could induce a protective immune response in vivo. It showed that the tumor growth was significantly inhibited, tumor diameter was markedly reduced and the survival time of tumor-bearing mice was further prolonged. Immunization with it also resulted in regression of the established L1210 tumor and prolonged survival time of mice. These results suggest that gene transfection of BCG HSP70 could effectively improve the immunogenicity of tumor cells and it may be used as a suitable candidate gene-modified cell vaccine for cancer immunotherapy.

Apoptosis of K562 leukemia cells by Abnobaviscum F®, a European mistletoe extract

Evidence suggests that mistletoe extract has the potential to be used as an anticancer agent. AbnobaviscumF® is a European mistletoe extract from the host tree Fraxinus. We investigated the effect of AbnobaviscumF on the growth and survival of different leukemia cell lines. AbnobaviscumF treatment strongly reduced survival and induced apoptosis of K562 (human myeloid leukemia), RPMI-8226 (human plasmacytoma) and L1210 (murine lymphocytic leukemia) cells in culture. Using K562 cells to further investigate the mechanism of action of AbnobaviscumF, we showed that AbnobaviscumF-induced cell death was associated with the activation of caspase-9, JNK-1/2 and p38 MAPK, as well as with the downregulation of Mcl-1, and inhibition of ERK-1/2 and PKB phosphorylation. Moreover, AbnobaviscumF treatment led to both a reduction of cellular glutathione (GSH) and the induction of ER stress (GRP78 and CHOP induction and eIF-2α phosphorylation). By contrast, AbnobaviscumF did not impact the expression of the DR4 and DR5 death receptors. The AbnobaviscumF-induced apoptosis of K562 cells was blocked by pretreatment with either GSH, z-VAD-fmk or SP600125. Our results, therefore, show that AbnobaviscumF induces apoptosis of K562 cells through the activation of the intrinsic caspase pathway, the phosphorylation of JNK-1, the reduction of cellular GSH, and the induction of ER stress.

Phenylpropanoid triesters from Daucus glaber

Three new phenylpropanoid triesters, glaberins A–C ( 1– 3), were isolated from the leaves and stems of Daucus glaber (Forssk.) Thell. (Apiaceae). Their gross structures were determined on the basis of 2D NMR experiments, and the absolute structure of 1 by X-ray crystallography and chemical methods. Glaberins A ( 1) and B ( 2) were evaluated for cytotoxicity against P-388 murine lymphocytic leukemia cells.

Prevention of MDR development in leukemia cells by micelle-forming polymeric surfactant

Doxorubicin (Dox) incorporated in nanosized polymeric micelles, SP1049C, has shown promise as monotherapy in patients with advanced esophageal carcinoma. The formulation contains amphiphilic block copolymers, Pluronics, that exhibit the unique ability to chemosensitize multidrug resistant (MDR) tumors by inhibiting P-glycoprotein (Pgp) drug efflux system and enhancing pro-apoptotic signaling in cancer cells. This work evaluates whether a representative block copolymer, Pluronic P85 (P85) can also prevent development of Dox-induced MDR in leukemia cells. For in vitro studies murine lymphocytic leukemia cells (P388) were exposed to increasing concentrations of Dox with/without P85. For in vivo studies, BDF1 mice bearing P388 ascite were treated with Dox or Dox/P85. The selected P388 cell sublines and ascitic tumor-derived cells were characterized for Pgp expression and functional activity (RT-PCR, Western Blot, rhodamine 123 accumulation) as well as Dox resistance (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). The global gene expression was determined by oligonucleotide gene microarrays. We demonstrated that P85 prevented development of MDR1 phenotype in leukemia cells in vitro and in vivo as determined by Pgp expression and functional assays of the selected cells. Cells selected with Dox in the presence of P85 in vitro and in vivo exhibited some increases in IC 50 values compared to parental cells, but these values were much less than IC 50 in respective cells selected with the drug alone. In addition to mdr1, P85 abolished alterations of genes implicated in apoptosis, drug metabolism, stress response, molecular transport and tumorigenesis. In conclusion, Pluronic formulation can prevent development of MDR in leukemia cells in vitro and in vivo.

Synthesis and Evaluation of Novel N-Substituted-6-methoxynaphthalene-2-Carboxamides as Potential Chemosensitizing Agents for Cancer

A novel class of molecules with structure N-[3-(heteroaryl)propyl]-6-methoxynaphthalene-2-carboxamides 8-13 were synthesized by condensing 6-methoxy-2-naphthoyl chloride 1 with 3-(heteroaryl)propyl amines 2-7. Compounds 8-12 were evaluated in vitro, in P388 murine lymphocytic leukemia cell line (P388) using SRB assay for cytotoxicity and in adriamycin resistant P388 murine lymphocytic leukemia cell line (P388/ADR) using MTT assay for resistant reversal activity. Compounds 8-12 were non-toxic at lower dose of 20 microg/ml, and effectively reversed adriamycin resistance. However, at higher doses (40, 80 microg/ml) they showed significant cytotxicity and hence reversal potency was not determined at these concentrations.

The synthesis, chemical and biological properties of dichlorido(azpy)gold(III) chloride (azpy = 2-(phenylazo)pyridine) and the gold-induced conversion of the azpy ligand to the chloride of the novel tricyclic pyrido[2,1-c][1,2,4]benzotriazin-11-ium cation

A new Au(III) coordination compound with the ligand 2-(phenylazo)pyridine has been synthesized and fully characterized by means of elemental analysis, IR, UV–visible, conductivity measurements, NMR, electrospray ionization (ESI-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES). The chemical stability of the cation in this compound, [Au(azpy)Cl2]+ (abbreviated: Au-azpy), was analyzed by means of several physicochemical methods. While stable in the solid state, stability studies performed with the gold compound in solution showed an unexpected and unprecedented reactivity. A cationic organic derivative of 2-(phenylazo)pyridine, (abbreviated: pyrium), was produced from the solution and has been isolated as its chloride salt and characterized by crystal structure determination, elemental analysis, NMR, ESI-MS and conductivity studies in solution. This cyclization reaction is reported for the first time in the case of gold coordination compounds.The Au adduct and the pyrium cation were investigated as potential cytotoxic and anticancer agents, and both show moderate to high cytotoxic properties in cisplatin-sensitive and cisplatin-resistant ovarian carcinoma cell lines, A2780; and cisplatin-sensitive and cisplatin-resistant murine lymphocytic leukemia cell lines, L1210. Significant anticancer activity against the cisplatin resistant cell lines was found for the pyrium salt, ruling out the occurrence of cross resistance phenomena.

Vascular targeting of doxorubicin using cationic liposomes

Tumor vessel has been recognized as an important target for anticancer therapy. Cationic liposomes have been shown to selectively target tumor endothelial cells, thus can potentially be used as a carrier for chemotherapy agents. In this study, cationic liposomes containing 20 mol% cationic lipid dimethyl dioctadecyl ammonium bromide (DDAB) and loaded with doxorubicin (DOX) were prepared and characterized. The cationic liposomal DOX showed 10.8 and 9.1 times greater cytotoxicity than control PEGylated liposomal DOX in KB oral carcinoma and L1210 murine lymphocytic leukemia cells, and 7.7- and 6.8-fold greater cytotoxicity compared to control neutral non-PEGylated liposomal DOX, repectively, in these two cell lines. Although cationic liposomal DOX had higher tumor accumulation at 30 min after intravenous administration compared to control liposomes ( p < 0.05), DOX uptake of these liposomes at 24 h post-injection was similar to that of PEGylated liposomal DOX ( p > 0.05) and approximately twice the levels of the free drug and non-PEGylated liposomes. In a murine tumor model generated using L1210 cells, increased survival rate was obtained with cationic liposomal DOX treatment compared to free DOX ( p < 0.01), neutral liposome control ( p < 0.01), as well as PEGylated liposomes ( p < 0.05). In conclusion, the cationic liposomal DOX formulation produced superior in vitro cytotoxicity and in vivo antitumor activity, and warrants further investigation.

Design, synthesis and evaluation of naphthalene-2-carboxamides as reversal agents in MDR cancer

A novel class of molecules with structure N-[3-(4-substituted-1-piperazinyl) propyl]-6-methoxy naphthalene-2-carboxamides were designed by generating a pharmacophore for potent MDR reversal activity, using Elacridar (GF 120918) as a query molecule and using MOE software. They were synthesized by condensing 6-methoxynaphthalene-2-carboxylic acid with N-[3-(4-substituted-1-piperazinyl) propyl] amines in the presence of DCC in DMF. They were evaluated in P388 murine lymphocytic leukemia cell line (P388) in vitro using SRB assay for cytotoxicity and in adriamycin-resistant P388 murine lymphocytic leukemia cell line (P388/ADR) using MTT assay for resistant reversal activity. Test compounds were non-toxic at the doses studied (upto 80 μg/ml). They effectively reversed adriamycin resistance at the doses studied (40 and 80 μg/ml). The percentage enhancement in adriamycin activity was in the range 33.58 −90.67 (at 40 μg/ml) and 8.80–46.04 (at 80 μg/ml) and the corresponding reversal potency values were in the range 1.33–1.90 and 1.08–1.46, respectively. Test compounds 2, 3, and 5 exhibited better activity as compared to the standard resistant reversal agent (Verapamil), at same concentration.

120 Synthesis and cytoxicity studies of platinum nucleobase adducts

A new Au(III) coordination compound with the ligand 2-(phenylazo)pyridine has been synthesized and fully characterized by means of elemental analysis, IR, UV–visible, conductivity measurements, NMR, electrospray ionization (ESI-MS) and inductively coupled plasma optical emission spectrometry (ICP-OES). The chemical stability of the cation in this compound, [Au(azpy)Cl2]+ (abbreviated: Au-azpy), was analyzed by means of several physicochemical methods. While stable in the solid state, stability studies performed with the gold compound in solution showed an unexpected and unprecedented reactivity. A cationic organic derivative of 2-(phenylazo)pyridine, (abbreviated: pyrium), was produced from the solution and has been isolated as its chloride salt and characterized by crystal structure determination, elemental analysis, NMR, ESI-MS and conductivity studies in solution. This cyclization reaction is reported for the first time in the case of gold coordination compounds.The Au adduct and the pyrium cation were investigated as potential cytotoxic and anticancer agents, and both show moderate to high cytotoxic properties in cisplatin-sensitive and cisplatin-resistant ovarian carcinoma cell lines, A2780; and cisplatin-sensitive and cisplatin-resistant murine lymphocytic leukemia cell lines, L1210. Significant anticancer activity against the cisplatin resistant cell lines was found for the pyrium salt, ruling out the occurrence of cross resistance phenomena.

Synthesis and cytotoxic activity of 2-dialkylaminoethylamino substituted xanthenone and thioxanthenone derivatives

The synthesis and biological evaluation of some new pyranoxanthenones and pyranothioxanthenones, substituted with flexible amino side-chains, and their evaluation as potential antitumor agents is described. The cytotoxic activity of the compounds and their eventual selective effect on a phase of the cell cycle were evaluated in vitro, using the murine lymphocytic L1210 leukemia cell line. The new aminoderivatives exhibited highly potent cytotoxicity against the leukemia L1210 cell line when compared to acronycine. All the compounds induced a partial accumulation of cells in the G2+M phase of the cell cycle.

Antineoplastic agents 440. Asymmetric synthesis and evaluation of the combretastatin A-1 SAR probes (1S,2S)- and (1R,2R)-1, 2-dihydroxy- 1-(2',3'-dihydroxy-4'-methoxyphenyl)-2-(3' ',4' ',5' '-trimethoxyphenyl)-ethane.

The synthetic (E)-isomer (3b) of natural combretastatin A-1 (1a) isolated from the African bushwillow Combretum caffrum was the focus of chiral hydroxylation (Sharpless) reactions as part of a structure-activity relationship study. The resulting (R,R)- and (S,S, )-diols (6 and 7) and synthetic intermediates were evaluated against a series of cancer cell lines, microorganisms, and tubulin. Chiral diols 6 and 7 showed increased activity against the P-388 murine lymphocytic leukemia cell line with ED(50) values of 3.9 and 2.9 microg/mL, respectively, when compared to the precursor (E)-stilbene 3b. In contrast, (E)-stilbene 3b exhibited more potent antibiotic activity than the chiral diols (6 and 7). Both diols, (R,R)-6 and (S, S)-7, displayed less cancer cell growth inhibition and less antibiotic activity than did natural combretastatin A-1 (1a) (P-388 ED(50) 0.25 microg/mL).

The Murine PKR Tumor Suppressor Gene Is Rearranged in a Lymphocytic Leukemia

The double-stranded RNA-dependent kinase, PKR, is encoded by an interferon inducible gene and is largely responsible for the anti-viral effects of this cytokine. Recent studies have shown that PKR may also play a role in the regulation of normal cellular growth. Although numerous examples of viral strategies for inactivation of PKR exist, there is no evidence of PKR inactivation in tumors. We demonstrate here that the Tik gene, which encodes a dual-specificity kinase, is the murine homolog of PKR, the dsRNA-dependent kinase, and has undergone a rearrangement of one allele in a murine lymphocytic leukemia cell. We have cloned a cDNA that corresponds to a mutated transcript from the rearranged mPKR gene and show that while the mutated polypeptide retains its ability to dimerize and bind dsRNA, it is catalytically inactive. Although this mutated mPKR lacks apparent dominant-negative function, the net effect of reduced PKR activity in these cells may be significant.

Characterization of a novel Na+-dependent, guanosine-specific, nitrobenzylthioinosine-sensitive transporter in acute promyelocytic leukemia cells.

NB4 cells are the only bona fide in vitro model of human acute promyelocytic leukemia. We have examined cytidine and guanosine transport in this cell line and characterized a novel guanosine-specific transporter. Cytidine transport occurred predominately by equilibrative nitrobenzylthioinosine (NBMPR)-sensitive (es) transport. In the presence of Na+, guanosine at various concentrations accumulated at least 6-fold above equilibrium. The initial rate of guanosine transport in Na+ buffer decreased by 75% with the addition of 1 microM NBMPR and the IC50 for NBMPR inhibition was 0.7 +/- 0.1 nM. Replacement of Na+ with choline also resulted in a 75% decrease in total guanosine transport. The potent inhibition of guanosine transport by NBMPR and the loss of transport in choline suggested that a Na+-dependent NBMPR-sensitive transporter was responsible for the majority of guanosine uptake. This concentrative, sensitive transporter is Na+ dependent with a stoichiometric coupling ratio of 1:1. This novel transporter, referred to as csg, is guanosine-specific with total guanosine transport inhibited by only 50% in the presence of 1 mM competing nucleosides. HL-60, acute myelocytic leukemia cells, do not exhibit csg activity while L1210, murine acute lymphocytic leukemia cells, exhibit csg transport. The presence of the csg transporter suggests an important role for guanosine in particular forms of leukemia and may provide a new target for cytotoxic therapy.

A novel beta-galactoside-binding lectin in cultured murine lymphocytic leukemia cells.

Using affinity chromatography on lactosyl-Sepharose, a beta-galactoside-binding protein of 38 kDa was detected in mouse L1210 lymphocytic leukemia cells. Immunoblotting analysis revealed that it is distinct from any known larger molecular weight galectin. The partial amino acid sequences of the 38 kDa protein indicated that it is a novel member of the galectin family. This 38 kDa lectin is expressed in lymphocytic cell lines but not macrophage-like cell lines.

Photoaffinity Labeling of a Cell Surface Polyamine Binding Protein

Intracellular polyamine pools are partially maintained by an active transport apparatus that is specific for and regulated by polyamines. Although mammalian transport activity has been characterized by kinetic studies, the actual protein itself has yet to be identified, purified, or cloned. As one approach to this problem, we attempted photoaffinity labeling of plasma membrane proteins using two specifically designed and synthesized polyamine conjugates as photoprobes. The first is a spermidine conjugate bearing the photoreactive moiety 4-azidosalicylic acid at the N4 position via an alkyl linkage, and the second is a norspermine conjugate with 4-azidosalicylic acid at the N4 position via an acyl linkage. Labeling of murine L1210 lymphocytic leukemia cells was carried out at 4 degrees C to promote selective alkylation of cell surface proteins. Separation of plasma membrane proteins from cells cross-linked with the N4-spermidine conjugate by SDS-polyacrylamide gel electrophoresis revealed two heavily labeled proteins at approximately 118 and approximately 50 kDa (designated p118 and p50, respectively). Band p118 was more well defined and much more intensely labeled. Analogous proteins were also observed in human U937 lymphoma cells. Specificity of labeling was strongly suggested by competition with polyamines and analogs during labeling and further indicated by the nearly identical labeling of the same protein by the N1-norspermine photoprobe but not by the unconjugated photoreagent. Neuraminidase pretreatment of L1210 cells increased mobility of the p118, suggesting that it was glycosylated and, thus, of plasma membrane origin. In transport-deficient L1210 cells, p118 and p50 were found to have a slightly higher molecular mass and were accompanied by a less distinct protein band (approximately 100 kDa). These findings indicate the presence of a polyamine binding protein at the surface of murine and human leukemia cells, which could be directly or indirectly related to the polyamine transport apparatus.