ICAM- melanoma cells are relatively resistant to CD3-mediated T-cell lysis

The primary activation pathway of T cells is via the T-cell receptor (TCR)/CD3 complex, which is functionally interrelated with various accessory molecules. We examined the contribution of the lymphocyte-function-associated antigen-I/intercellular adhesion molecule 1 (LFA-1/ICAM-1) interaction to CD3/TCR-mediated lysis by cytotoxic T lymphocytes (CTL). We used ICAM-I-or+ tumor cell lines as target cells and anti-CD3- or anti-LFA-1 containing hetero-cross-linked monoclonal antibody (MAb) to bridge CTL and target cells and simultaneously to activate CTL. The ICAM-1- melanoma-derived cell line IgR39 was relatively resistant to CD3-mediated lysis by both TCR alpha beta + and TCR gamma delta + CTL, when compared with ICAM-1+ cell lines. Induction of ICAM-1 on the membrane of IgR39 cells by tumor necrosis factor (TNF) rendered these cells more susceptible to CD3-mediated lysis. Anti-ICAM-1 MAb inhibited this TNF-enhanced susceptibility to lysis, directly demonstrating that the induction of ICAM-1 was critical in the TNF-induced increase in susceptibility to lysis of IgR39 cells. CTL formed less efficient conjugates with the ICAM-1- cells as compared to ICAM-1+ cells. Both spontaneous and CD3-induced conjugate formation as well as CD3-mediated lysis of ICAM-1- tumor cells by CTL were enhanced by the addition of anti-LFA-1 containing hetero-cross-linked MAb, thereby mimicking the LFA-1/ICAM-1 interaction between CTL and target cells. Soluble anti-CD18 MAb inhibited CD3-mediated lysis of ICAM-1- target cells by CTL without affecting their conjugate formation. Anti-LFA-1 MAb added after conjugate formation still inhibited lysis of both ICAM-1+or- tumor cells. Taken together, these findings suggest that the LFA-1/ICAM-1 interaction co-activates CD3/TCR-mediated lysis by CTL through both an enhanced CTL-target cell binding and the delivery of post-conjugate costimulatory signals.     

Sensitivity to EBV superinfection and IUdR inducibility of hybrid cells formed between a sensitive and a relatively resistant Burkitt lymphoma cell line

The sensitivity of Burkitt lymphoma-derived cell lines to Epstein-Barr virus (EBV) superinfection and the inducibility of the latent EBV genome in these lines were studied by somatic cell hybridization. Two non-EBV producer lines; AG R3, an adherent, 8-azaguanine-resistant variant of the Rajiline; and Namalwa, a non-adherent, 8-azaguanine-sensitive line, were fused with the aid of β-propiolactone-inactivated Sendai virus. The resident EBV genome in Raji is inducible with 5-iododeoxyuridine and the line is also sensitive to EBV superinfection. Namalwa is relatively resistant to both super-infection and induction, and synthetizes surface-associated IgM-lambda. Cytological studies showed that hybrid cells appear to be much larger than either parent and attach to culture plates less firmly than the adherent Raji variant parent. Karyological analyses showed that hybrids contain the expected sum of the parental chromosome markers. Membrane immunofluorescence tests also showed that hybrids synthetize IgM. All the hybrid cells appear to be non-EBV producers, but they are sensitive to both EBV super-infection and induction of latent EBV. These findings suggest the following explanations: (1) there is no evidence of any complementation between the two non-EBV producer lines (Raji and Namalwa) to elicit spontaneous EBV production in hybrid cells; (2) Namalwa is deficient in some factors required for the synthesis of EBV-specified early antigens after EBV superinfection and after induction of latent EBV by IUdR; these factors are supplied by the Raji parent in the hybrids; or (3) Raji, Namalwa and hybrid cells or EBV all produce a substance which inhibits activation of a productive viral cycle, but whose action is antagonized in Raji and hybrid cells to allow the synthesis of EBV-specific early antigens.     

Immunoglobulin synthesis and secretion by leukemic B cells from patients with chronic lymphocytic leukemia

We investigated the ability of purified E-rosette negative largely leukemic B cells from 15 patients with B-cell chronic lymphocytic leukemia (CLL) to synthesize and secrete IgM, IgA and IgG spontaneously or in the presence of purified autologous or allogeneic T4 cells from normal donors, in PWM-induced differentiation system. We observed moderate but significant IgM synthesis and secretion (19.7 +/- 8.9 micrograms/dl, n = 5) by leukemic B cells alone in 5 of 15 patients examined. These IgM concentrations were significantly higher (p less than 0.005) than those produced by purified E-rosette negative cells from normal donors (4.3 +/- 4.5 micrograms/dl; n = 6) in the absence of T cells. Purified E-rosette negative leukemic B cells alone from patients with CLL did not produce IgA or IgG. Addition of purified autologous or allogeneic T4 cells from normal donors resulted in significant increase of IgM production by leukemic B cells from certain patients or initiated IgM secretion in others. However, these IgM levels (73.9 +/- 56.6 micrograms/dl) were significantly lower (p less than 0.003) to those produced by mixtures of T4 cells and B cells form normal donors (211.6 +/- 58.0 micrograms/dl, n = 6). Addition of purified autologous or allogeneic T4 cells from normal donors to purified largely leukemic B cells from patients with CLL resulted in production of very small amounts of IgA in 4 of 15 patients (10.6 +/- 6.3 micrograms/dl vs 154.7 +/- 35.8 micrograms/dl produced by T4 and B cells from normal donors; n = 6), but did not support IgG synthesis and secretion. Purified T4 cells from certain patients with CLL exhibit defective helper function to immunoglobulin production by E-rosette negative cells from normal donors.     

Glycosyltransferase activities in leukemic cells from patients and human leukemic cell lines

The cell membrane fraction from c-ALL, B-ALL, Ph′+ ALL, B-CLL, T-CLL, AML, blastic-CML, normal leukocytes, PHA-stimulated lymphocytes and several T, B and myeloid human leukemic cell lines has been used in different cell types to demonstrate different patterns of glycosyltransferase activity. Both B- and T-CLL cell membranes have low fucosyltransferase B and A activity compared to acute leukemias; while sialyltransferase activity is higher in B- than in T-CLL. AML cell membranes and ML-1 human myeloblast cell line membranes have exceptionally high fucosyltransferase A activity compared to all other leukemic cells or cell lines. Human leukemic B cell lines expressed cell membrane sialyltransferase, fucosyltransferase B and probably fucosyltransferase A activity several times higher than T cell lines. Human myeloid cell lines ML-1 and HL-60 express 5- to 20-fold higher galactosyltransferase activity than human leukemic T and B cell lines. Both sialyltransferase and galactosyltransferase activity were higher in all leukemic cells than in normal leukocytes and PHA-stimulated normal lymphocytes. This is the first study carried out on glycosyltransferases using cells obtained from leukemic patients characterized immunologically. These results indicate that all glycosyltransferase activity, with the exception of fucosyl-transferase activity in CLL, were higher in leukemic cells than in normal cells. Moreover, large differences in these enzymes, e.g. very high galactosyltransferase activity in myeloid cell lines compared to B and T cell lines, of fucosyltransferase A in AML and myeloblast cell lines compared to all other cells, and of sialyltransferase in B-CLL or B cell lines compared to T-CLL or T cell lines, could be useful in characterizing certain leukemias and hematopoietic cell lines.     

Detection of functional platelet-activating factor receptors on leukemic B cells of chronic lymphocytic leukemic patients

Although platelet-activating factor receptors (PAF-R) are reported on normal B cells, few results are available concerning leukemic ones. We demonstrated functional PAF-R on cell and nuclear surfaces of leukemic B cells of chronic lymphocytic leukemic (CLL) patients. Analysis of 102 patients revealed dramatic differences for their membrane PAF-R expression, a result that might be related to their plasma IL-4 levels. In the light of the potent immunoregulatory role of PAF on B cell physiology, it is suggested that the presence or absence of PAF-R on leukemic B cells may profoundly affect their in vivo behavior.     

Butyrate-induced differentiation in leukemic myeloid cells - in-vitro and in-vivo studies

A study of the effects of three differentiating agents, butyric acid, retinoic acid and cytosine arabinoside on proliferation and differentiation of primary cultures, obtained from sixteen patients with myelo-proliferative disorder was conducted. The results showed that BA was an effective inhibitor of cell proliferation and inducer of cytodifferentiation. An acute non-lymphoblastic leukemia patient was treated with sodium butyrate. A temporary increase in differentiation-associated parameters were noted. However, the effects of SB were short-lived. The lack of clinical response led to the development of a BA prodrug pivaloyloxymethylbutyrate (AN-9). This prodrug was more potent in vitro than BA in the induction of cytodifferentiation and inhibition of cell proliferation.     

Increased sensitivity to 1-beta-D-arabinofuranosylcytosine in P388 murine leukemic cells resistant to etoposide

A variant P388 murine leukemic cell resistant to 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidine)-beta-D-gl ucopyranoside (etoposide) (VP-16-213) was cloned. The variant P388/VP-16 cell line was 159-fold resistant to VP-16. We found that this variant P388/VP-16 cell line showed collateral drug sensitivity to 1-beta-D-arabinofuranosylcytosine(Ara-C), determined by comparing the 50% inhibitory concentrations in 48-h growth inhibition assay. To clarify the mechanism of this increased sensitivity to Ara-C, we quantified the deoxyribonucleoside triphosphate pools and 1-beta-D-arabinofuranosylcytosine triphosphate(Ara-CTP) using high-performance liquid chromatography in the parent and drug-resistant sublines of P388 cells. The analysis of deoxyribonucleoside triphosphate pools revealed that the pyrimidine triphosphate pools were significantly decreased in the P388/VP-16 cell line and the Ara-CTP concentration of two variant cell lines were not significantly different. The Ara-CTP/dCTP ratio was significantly increased in P388/VP-16 cells. These data suggest that the inhibition of the dCTP de-novo pathway and the preservation of the dCTP salvage pathway in P388/VP-16 cells might correlate with the increased sensitivity to Ara-C.     

Mesenchymal stem cells are resistant to carbon ion radiotherapy

Mesenchymal stem cells (MSCs) participate in regeneration of tissues damaged by ionizing radiation. However, radiation can damage MSCs themselves.Here we show that cellular morphology, adhesion and migration abilities were not measurably altered by photon or carbon ion irradiation. The potential for differentiation was unaffected by either form of radiation, and established MSC surface markers were found to be stably expressed irrespective of radiation treatment. MSCs were able to efficiently repair DNA double strand breaks induced by both high-dose photon and carbon ion radiation. We have shown for the first time that MSCs are relatively resistant to therapeutic carbon ion radiotherapy. Additionally, this form of radiation did not markedly alter the defining stem cell properties or the expression of established surface markers in MSCs.     

In-vivo and in-vitro association between leukemic cells and marrow stromal cells: a murine model

Several reports indicate that marrow stromal cells are conducive to development of both myeloid and lymphoid leukemic cell lines in vitro. We present data on an in-vitro/in-vivo murine model that establishes that: (i) murine LL cell lines (L5178Y, P388 and L1210) invade the bone marrow when injected via the tail vein; (ii) degree of marrow invasion by LL can be determined quantitatively; (iii) there is a close association or adherence between MSC and LL cells in vivo; and (iv) in-vitro adherence of murine LL cells to MSC can be quantitated. This in-vitro/in-vivo murine model will be helpful in deciphering the importance of heterotypic adherence between marrow stromal cells and leukemic cells in vivo.     

Ionic currents in multidrug resistant K562 human leukemic cells

In this study, the expression and functional characterization of currents through the CFTR (cystic fibrosis transmembrane regulator) and ORCC (outwardly rectifying chloride channels) were determined in wild-type K562 chronic human leukemia cells (K562-WT) and in its resistant counterpart, the vincristine resistant cell line (K562-Vinc). Expression of the CFTR and MDR1 (multidrug resistant) gene products was determined by a semi-quantitative RT-PCR protocol. The amplified products in K562-WT and K562-Vinc showed two bands corresponding to CFTR and MDR1. MDR1 mRNA increased by 20-fold in K562-Vinc whereas no change in CFTR mRNA levels was observed. CFTR and ORCC channel activity were measured with a whole cell configuration of the patch clamp technique. Forskolin (40 microM n activator of adenylate cyclase, added to the extracellular side increased the current in both cell lines. A fraction of the activated whole cell currents was inhibited by 500 microM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and subsequent addition of 500 microM diphenylamine-2-carboxylate (DPC plus DIDS) further inhibited the remaining currents. The levels of forskolin-activated currents and subsequent blockade were similar in both cell lines. The effect of forskolin was prevented in cells previously exposed to 500 microM DPC. The effects of DIDS and DPC on the forskolin-activated whole cell currents support the idea that both CFTR and ORCC are generating a significant fraction of these currents with DIDS inhibiting ORCC currents and DPC inhibiting CFTR currents when the blockers are added one after another to the extracellular side. Finally, we show that exposure of K562 cells to vincristine which results in the over expression of MDR1 is not accompanied by a significant down regulation of CFTR as in other cells.     

Monocyte-derived dendritic cells from HLA-matched allogeneic donors showed a greater ability to induce leukemic cell-specific T cells in comparison to leukemic cell-derived dendritic cells or monocyte-derived dendritic cells from AML patients

We investigated the usefulness of allogeneic monocyte-derived dendritic cells (allogeneic mDCs) pulsed with leukemic lysates in acute myeloid leukemia (AML). Allogeneic mDCs showed higher expressions of several molecules (HLA-DR, CD80, CD83 or CD86), higher production of IL-12 and higher capacity to stimulate allogeneic T cells compared to both leukemic DCs and autologous mDCs. Autologous T cells primed by allogeneic mDCs displayed a larger number of interferon-gamma-secreting cells against leukemic cells than those primed by either leukemic DCs or autologous mDCs. These results suggest that monocyte-derived DCs from HLA-matched allogeneic donors can be used as an alternative to generate leukemia-specific cytotoxic T cells and to overcome the limitation of leukemic DCs or autologous mDCs.     

Antineoplastic effect of new boron compounds against leukemic cell lines and cells from leukemic patients

Three new boron compounds, dihydroxy (oxybiguanido) boron (iii) hydrochloride monohydrate (HB), guanidine biboric acid adduct (GB) and hydroxosalicyl hydroxomato boron (iii) (SHB) were studied to observe their antineoplastic effect, if any. Leukemic cells isolated from acute lymphatic leukaemia (ALL) patients and chronic myeloid leukaemia patients (CML) and myeloid leukemia cell lines (HL 60 and U-937) showed cell growth inhibition after treatment with the boron compounds. MTT assay showed that the growth of metabolically active cells was inhibited by treatment with these drugs. The molecular mechanism by which SHB induced apoptosis in immature blast cells was also investigated by ladder formation in gel electrophoresis.     

Selective killing of malignant cells from leukemic patients by alkyl-lysophospholipid

We studied the effects of the alkyl-lysophospholipid, 1-octadecyl-2-methyl-sn-glycerol-3-phosphocholine (ALP), on human leukemia cells from 56 patients with various leukemias and on normal bone marrow progenitors in order to assess the application of ALP as an in vitro marrow-purging agent. The tumoricidal activity was analyzed by the elimination of clonogenic leukemia cells (leukemic colony-forming cells), by the inhibition of the proliferative capacity [( 3H]thymidine incorporation) of leukemia cells, and by the elimination of viable leukemia cells measured with flow cytometry. The tumoricidal activity of ALP was dose and incubation time related, as, although to a lesser extent, held true for normal marrow progenitors. For some leukemias the ALP dose necessary for the elimination of 100% of the leukemic colony-forming cells is probably too toxic for normal marrow cells. The results of this study strongly support the possibility that ALP is a promising purging agent in the majority of patients with leukemias.     

CD3AK细胞对耐药白血病细胞的体外净化作用

目的 :观察CD3 AK细胞对耐药的白血病细胞系及慢性髓细胞白血病 (chronicmyelogenousleukemia ,CML)急变患者原代肿瘤细胞的体外净化作用。方法 :采用固化的抗CD3单克隆抗体联合小剂量IL 2诱导CD3 AK细胞 ;MTT法观察CD3 AK细胞对K5 6 2、HL6 0及其耐药株的细胞毒活性 ;肿瘤细胞集落培养 (tumorcolonyassay ,TCA)观察CD3 AK细胞对K5 6 2、HL6 0及其耐药株集落形成的抑制作用 ;流式细胞仪 (flowcytometry ,FCM)检测耐药的CML急变患者原代细胞经CD3 AK细胞净化后Pgp阳性细胞的比例变化。结果 :MT法显示CD3 AK细胞在体外对K5 6 2细胞、HL6 0细胞及其耐药株有相似的杀伤作用 ;集落培养观察CD3 AK细胞对HL6 0细胞株及其耐药株的集落形成均有较强的抑制作用 ;FCM结果显示耐药CML原代细胞经CD3 AK细胞净化后Pgp阳性细胞比例下降 2 3 2 0 %。结论 :CD3 AK细胞在体外对耐药白血病细胞株及耐药白血病原代细胞均有较强的净化作用。     

Comparison ofin vitro assays for detecting subpopulations of P388 leukemic cells resistant to AraC

The kinetics of cytosine arabinoside (AraC) were studiedin vitro using P388 murine leukemia lines sensitive (P388-S) and resistant (P388-R) to the drug. Using P388-S cells, the³H-thymidine index (L.I.) was paralleled by the³H-AraC index, i.e. any cell in the S-phase of the cycle also incorporated the³H-AraC into DNA. With the resistant line, the³H-AraC index was zero in spite of high L.I. This discrepancy between, the L.I. and³H-AraC index could be used to predict the percentage of resistant cells when known mixtures of sensitive and resistant populations were used. The reason for lack of incorporation into DNA by the resistant cells was shown to be the presence of very low levels of the enzyme deoxycytidine kinase in P388-R cells resulting in an inability to phosphorylate AraC to its active form AraCTP. Immediate inhibition of DNA synthesis caused by AraC was directly proportional to the number of sensitive cells present in the population, but was not as accurate a predictor of sensitivity as the³H-AraC index. Cloning in methylcellulose was found to be the least sensitive predictor of the percentage of sensitive and resistant cells, most likely related to the difference in cloning efficiency of the sensitive and resistant lines.     

Migratory behavior of leukemic cells from acute myeloid leukemia patients.

The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 contribute to stem cell homing and may play a role in the trafficking of leukemic cells. Therefore, we analyzed migration across Transwell filters of cells derived from bone marrow (BM) or peripheral blood (PB) from 26 acute myeloid leukemia (AML) patients. The presence of the extracellular matrix protein fibronectin (FN) strongly enhanced the spontaneous and SDF-1-induced migration of leukemic PB and BM cells. No differences in spontaneous, SDF-1-induced migration or CXCR-4 expression were observed between the different AML subtypes. Subsequently, it was determined whether SDF-1 preferentially promoted migration of subsets of leukemic cells. Leukemic cells expressing CD34, CD38 and HLA-DR were preferentially migrating, whereas cells expressing CD14 and CD36 showed diminished migration. Analysis of paired PB and BM samples indicated that significantly higher SDF-1-induced migration was observed in AML for CD34(+) BM-derived cells compared to CD34(+) PB-derived cells, suggesting a role for SDF-1 in the anchoring of leukemic cells in the BM or other organs. The lower percentage of circulating leukemic blasts in patients with a relatively high level of SDF-1-induced migration also supports this hypothesis. In conclusion, we have shown that primary AML cells are migrating towards SDF-1 independent of the AML subtypes.