NALL-1, an acute lymphoblastic leukemia cell line with peroxidase activity

Summary All cells examined from the non-B, non-T acute lymphoblastic leukemia cell line, NALL-1, stained positive both for terminal deoxynucleotidyl transferase and for common ALL antigen. In addition, peroxidase activity was detected by light microscopy in 55 to 75% of cells and peroxidase-positive granules were detected ultrastructurally in >80% of cells. Peroxidase activity in NALL-1 may result from derepression of peroxidase genes or clonal proliferation of a biphenotypic precursor cell.     

NALL-1, an acute lymphoblastic leukemia cell line with peroxidase activity

All cells examined from the non-B, non-T acute lymphoblastic leukemia cell line, NALL-1, stained positive both for terminal deoxynucleotidyl transferase and for common ALL antigen. In addition, peroxidase activity was detected by light microscopy in 55 to 75% of cells and peroxidase-positive granules were detected ultrastructurally in greater than 80% of cells. Peroxidase activity in NALL-1 may result from derepression of peroxidase genes or clonal proliferation of a biphenotypic precursor cell.     

Prostaglandin D2, a potential antineoplastic agent

Cytotoxic actions of various prostaglandins were examined on L1210 mouse leukemia and several human leukemia cell lines, and prostaglandin D₂ (PGD₂) was found most active. PGD₂ exerted a dose dependent inhibition of L1210 cell growth over 3.6 μ$\text{M}$. At 14.3 μ$\text{M}$ growth was completely inhibited, and the number of viable cells remarkably decreased during culture. Microscopically the remaining cells showed degenerative changes with many vacuoles in their cytoplasm. The IC₅₀ value of PGD₂ on L1210 cell growth was calculated to be 6.9 μ$\text{M}$ (2.4 μg/ml), and at this concentration the DNA synthesis in 24 hr cultured cells was also decreased to a half of the level in the control cells. Such growth inhibition by PGD₂ was also found at similar concentrations with several human leukemia cell lines such as NALL-1, RPMI-8226, RPMI-8402, and Sk-Ly-16. Among other prostaglandins tested, PGA₂ showed a comparable, and PGE₂ a less but significant growth inhibitory activity, while PGB₂, PGF_2α and PGI₂ had no such effects on cell proliferation at 14.3 μ$\text{M}$ concentration. These results suggest a potential antineoplastic activity of PGD₂.     

Selective toxicity of neocarzinostatin-monoclonal antibody conjugates to the antigen-bearing human melanoma cell line in vitro

Summary Monoclonal antibodies (IgG₁) against high molecular weight antigen A-1-43 on human melanoma cell line A-375 were successfully linked to the anti-tumour protein neocarzinostatin (NCS) using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). The conjugate retained both the reactivity of the antibody and the toxicity of the drug. The antigen-bearing cell line A-375, antigen-lacking cell line MeWo and normal skin fibroblasts were exposed to NCS-monoclonal antibody conjugates. As negative control, cells were also treated with free NCS and NCS coupled to normal mouse IgG₁ antibodies. Inhibition of ³H-thymidine uptake after treatment was used to measure the biological activity of the cytotoxic drug complex or substance, respectively.Comparing the inhibition dose for 50% uptake (ID₅₀) it was found that the monoclonal antibody-drug complex is about 100 times more toxic for the antigen-bearing cell line than free NCS or normal mouse IgG₁-NCS. This high toxicity is due to a local increase of drug concentration on these cells. With the two cell lines lacking the appropriate antigen no significant differences in the ID₅₀ values were observed. A selectivity factor of 40–50 was obtained by comparing the cytotoxic effect of the monoclonal antibody-NCS conjugate upon the antigen-bearing as opposed to the antigen-lacking cell type. These data demonstrate, that the toxicity of NCS can be directed by monoclonal antibodies to human tumour cells carrying the corresponding surface antigen.     

Inhibition of antigen-induced T-cell proliferation by antibodies to endogenous AKR-MuLV

Lymph node cells from BALB/c mice immunized with ovalbumin or human γ-globulin were restimulated in vitro with these antigens and assayed for antigen-induced proliferation. The proliferative response was shown to be antigen specific and T cell dependent. A rabbit antiserum to envelope and core proteins of AKR murine leukemia virus was found to inhibit antigen-induced T-cell proliferation. The IgG fraction and F(ab′)₂ fragments of the antiserum were also inhibitory. The inhibition occurred after the initial step of antigen-T cell interaction and viral absorption studies showed the inhibition to be specific for anti-AKR virus antibodies. A hypothesis for the mechanism of inhibition is discussed in relation to a functional role for endogenous murine leukemia virus.     

The use of antifibrin antibodies for the destruction of tumor cells

Summary The transplantable methylcholanthrene-induced sarcoma (MC-D) in strain 13 guinea-pigs was used to test the hypothesis that tumor cells growing within a fibrin matrix could be destroyed by an immunologically specific strategy involving an indirect cell-mediated immune reaction. The experimental design consisted of two steps: (1) in vivo fixation of anti-guinea pig fibrin antibodies (AGFA) on the fibrin matrix enmeshing the tumor cells, and (2) the reaction between AGFA fixed to the fibrin matrix and lymphoid cells from syngeneic animals sensitized to xenogeneic immunoglobulins isotypic with AGFA. Indeed, tests with ⁵¹Cr-labelled lymphoid cells yielded evidence for the localization of these sensitized lymphoid cells within the fibrin lattice when the latter was coated with AGFA. Moreover, significant tumor growth suppression (P<0.01) was achieved in guinea-pigs that had received rabbit or goat AGFA intravenously and lymphoid cells from syngeneic guinea-pigs sensitized to a state of cell-mediated immunity to rabbit or goat IgG by the subcutaneous route. On the other hand, the administration of the antibodies or of the sensitized cells alone did not affect the growth of the tumor. Preliminary results suggest that peritoneal exudate cells may have an important role in the success of this strategy for tumor cell destruction.     

Effects of anti-embryonal carcinoma serum on aggregation and metabolic cooperation between teratocarcinoma cells

Effects of rabbit anti-embryonal carcinoma IgG on embryonal carcinoma cells and their differentiated derivatives were studied at different levels of cell-cell interaction. Fab fragments of anti-EC IgG were found to inhibit aggregation of the majority of EC cell lines. Two, however, were insensitive. Anti-EC Fab fragments act also on the transfer of metabolites between EC cells: the rescue of HPRT^? EC cells by HPRT⁺ EC cells in selective medium is abolished. These findings are correlated with the disappearance of tight and gap junctions from the surface of EC cells (Dunia et al., 1979). The presence of the surface structure involved in the action of anti-EC Fab fragments was tested by absorption experiments followed by decompaction test on PCC4 Aza R1 cells. All EC cell lines and two embryonic cell lines—a trophectodermal and an endodermal line—were found to bear material absorbing the decompacting activity. The results are discussed in terms of state of differentiation of the cell lines and of complexity of aggregation of embyronic cells.     

Effects of 5-bromo-2′-deoxyuridine on arachidonic acid metabolism of neuroblastoma and leukemia cells in culture: A possible role of endogenous prostaglandins in tumor cell proliferation and differentiation

Effects of 5-bromo-2′-deoxyuridine (BrdU) were studied on two neuroblastoma and two leukemia cell lines, in terms of the relationship between prostaglandin (PG) synthesis and cell growth/differentiation. After treatment with BrdU (5 μg/ml), cell growth of the 4 cell lines was inhibited and one neuroblastoma cell line (GOTO) showed flattened morphology with positive S-100 protein, one of the differentiation markers for Schwann or glial cells. In the 4 cell lines, BrdU treatment reduced [1-¹⁴C]-arachidonic acid incorporation into phosphatidylinositol and phosphatidylethanolamine and was associated with an increase into phosphatidylcholine and triglyceride. BrdU treatment also increased fractions of 6-keto-PGF_1α and PGF_2α , with a decreased TXB₂ fraction. The decreased ratio of TXB₂ /6-keto-PGF_1α or increased 6-keto-PGF_1α fraction correlated significantly with cell growth inhibition, suggesting that the changes in the balance of endogenous PGs might be associated with BrdU-induced cell growth inhibition with or without differentiation of neuroblastoma and leukemia cells in culture.     

IGF-IR determines the fates of BCR/ABL leukemia

Background

The tyrosine kinase receptor insulin-like growth factor 1 receptor (IGF-IR) contributes to the initiation and progression of many types of malignancies. We previously showed that IGF-2, which binds IGF-IR, is an extrinsic factor that supports the ex vivo expansion of hematopoietic stem cells (HSCs). We also demonstrated that IGF-IR is not required for HSC activity in vivo.

Methods and results

Here we investigated the role of IGF-IR in chronic myeloid leukemia (CML) using the retroviral BCR/ABL transplantation mouse model. Existing antibodies against IGF-IR are not suitable for flow cytometry; therefore, we generated a fusion of the human IgG Fc fragment with mutant IGF-2 that can bind to IGF-IR. We used this fusion protein to evaluate mouse primary hematopoietic populations. Through transplantation assays with IGF-IR⁺ and IGF-IR⁻ cells, we demonstrated that IGF-IR is expressed on all mouse HSCs. The expression of IGF-IR is much higher on CML cells than on acute lymphoblastic leukemia (ALL) cells. The depletion of IGF-IR expression in BCR/ABL⁺ cells led to the development of ALL (mostly T cell ALL) but not CML. Lack of IGF-IR resulted in decreased self-renewal of the BCR/ABL⁺ CML cells in the serial replating assay.

Conclusion

IGF-IR regulates the cell fate determination of BCR/ABL⁺ leukemia cells and supports the self-renewal of CML cells.     

Inhibition of calcium and glucose uptake by murine leukemia L5178Y cells treated with antiserum

Studies were performed to determine whether decreases in transport of calcium and glucose might be among the earliest changes triggered by the antigen-antibody reactions occurring on the cell surface of murine leukemia L5178Y cells after treatment with rabbit antisera. After treatment with antisera, in the absence of complement, these cells exhibited a decreased uptake of ⁴⁵Ca, 2-deoxy[³H]glucose, and 3-0-methyl[³H]glucose. These changes occurred rapidly, within 2 minutes after the addition of antiserum, in contrast to the previously reported inhibitory effects of antiserum on DNA, RNA, and protein synthesis, which became demonstrable only after 4 to 8 hours. The kinetics of uptake of the radioactive substrates was biphasic, with a very rapid initial uptake followed by less rapid linear uptake. The precise mechanism of cell growth inhibition remains to be elucidated, but one of the initial effects of antiserum treatment may be a perturbation at the cell membrane such that transport of specific nutrients is decreased, resulting in the observed effects on macromolecular synthesis.     

Photoreceptor calcium sensor proteins in detergent-resistant membrane rafts are regulated via binding to caveolin-1

Rod cell membranes contain cholesterol-rich detergent-resistant membrane (DRM) rafts, which accumulate visual cascade proteins as well as proteins involved in regulation of phototransduction such as rhodopsin kinase and guanylate cyclases. Caveolin-1 is the major integral component of DRMs, possessing scaffolding and regulatory activities towards various signaling proteins. In this study, photoreceptor Ca²⁺-binding proteins recoverin, NCS1, GCAP1, and GCAP2, belonging to neuronal calcium sensor (NCS) family, were recognized as novel caveolin-1 interacting partners. All four NCS proteins co-fractionate with caveolin-1 in DRMs, isolated from illuminated bovine rod outer segments. According to pull-down assay, surface plasmon resonance spectroscopy and isothermal titration calorimetry data, they are capable of high-affinity binding to either N-terminal fragment of caveolin-1 (1–101), or its short scaffolding domain (81–101) via a novel structural site. In recoverin this site is localized in C-terminal domain in proximity to the third EF-hand motif and composed of aromatic amino acids conserved among NCS proteins. Remarkably, the binding of NCS proteins to caveolin-1 occurs only in the absence of calcium, which is in agreement with higher accessibility of the caveolin-1 binding site in their Ca²⁺-free forms. Consistently, the presence of caveolin-1 produces no effect on regulatory activity of Ca²⁺-saturated recoverin or NCS1 towards rhodopsin kinase, but upregulates GCAP2, which potentiates guanylate cyclase activity being in Ca²⁺-free conformation. In addition, the interaction with caveolin-1 decreases cooperativity and augments affinity of Ca2 + binding to recoverin apparently by facilitating exposure of its myristoyl group. We suggest that at low calcium NCS proteins are compartmentalized in photoreceptor rafts via binding to caveolin-1, which may enhance their activity or ensure their faster responses on Ca²⁺-signals thereby maintaining efficient phototransduction recovery and light adaptation.     

The two-cross immunodiffusion technique: diffusion coefficients and precipitating titers of IgG in human serum and rabbit serum antibodies.

The two-cross technique, a new two-dimensional double-diffusion technique in gelplates, has been applied for simultaneous determination of precipitating titers and diffusion coeffients of antigen and antibody in body fluids. The advantage of this technique is that it works without using any standard solution and ensures conditions of “time-invariant sink”. The theory of the technique has been verified by experimental results on the precipitating system human serum-rabbit anti-human IgG in phosphate-buffered saline solution at pH 7.4. The results obtained using several modes of calculations from experimental parameters have been compared and found satisfactory. The accuracy and reproducibility of the results have been confirmed. It has been found that at 20°C the diffusion coefficient of human IgG in 10-times-diluted serum is (4.4 ± 0.2) × 10^?7 cm² s^?1, while the diffusion coefficient of rabbit anti-human IgG in a purified preparation is (2.9 ± 0.2) × 10^?7 cm² s^?1. The critical precipitating concentration of human IgG against rabbit anti-human IgG is invariable to concentration and amounts to 0.174 ± 0.03 mg/100 ml at pH 7.4.     

Differential effect of D-penicillamine on the cell kinetic parameters of various normal and transformed cellular types

The cell-growth-inhibitory and phase-specific effects of D-penicillamine on cell-cycle progression were investigated using cell-proliferation patterns, quantitative cell-cycle analysis by flow cytometry, and determination of the mitotic index and binucleate cell fraction of normal (rabbit articular chondrocytes, L 809, rabbit fibroblasts) and transformed (HeLa, L 929) cells. D-penicillamine treatment resulted in an inhibition of growth within a dose range of 5 × 10^?4 M to 7.5 × 10^?3 M. Examination of DNA by flow cytometric analysis revealed that rabbit articular chondrocytes were preferentially arrested in the G0/1 phase of the cell cycle, whereas the other cell lines were blocked in the G2 + M phase; the increase in the proportion of cells with G2 + M DNA content was partially due to an enhancement of binucleate cells, resulting in a cytokinesis perturbation for HeLa and L 929 cells. These results showed that D-penicillamine affects cell proliferation through different events according to cell type.     

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin

Aims: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. Methods and Results: Polyclonal and Mabs against the intimin‐conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti‐intimin IgG‐enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1·3 × 10^?8 mol l^?1, failed in the detection of some of these isolates. Conclusion: All employed antibodies showed 100% specificity, not reacting with any of the eae‐negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti‐intimin IgG‐enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti‐intimin IgG‐enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.     

A novel diquinolonium displays preclinical anti-cancer activity and induces caspase-independent cell death

Quinolines are a class of chemical compounds with emerging anti-cancer properties. Here, we tested the activity of series of quinolines and quinoline-like molecules for anti-cancer activity and identified a novel diquinoline, 1-methyl-2-[3-(1-methyl-1,2-dihydroquinolin-2-yliden)prop-1-enyl]quinolinium iodide (Q²). Q² induced cell death in leukemia, myeloma, and solid tumor cell lines with LD50s in the low to submicromolar range. Moreover, Q² induced cell death in primary acute myeloid leukemia (AML) cells preferentially over normal hematopoietic cells. In a mouse model of leukemia, Q² delayed tumor growth. Mechanistically, Q² induced cell death through caspase independent mechanisms. By electron microscopy, Q² increased cytoplasmic vacuolization and mitochondrial swelling. Potentially consistent with the induction of autophagic cell death, Q² treatment led to a punctate distribution of LC3 and increased MDC staining. Thus, Q² is a novel quinolinium with preclinical activity in malignancies such as leukemia and myeloma and warrants further investigation.     

Immunochemical distinctions among histones and their variants in a solid-phase radioimmunoassay

A solid-phase radioimmunoassay allows detection of small structural differences in histones. In this assay, histones are absorbed to plastic tubes; the coated tubes bind antibody from the IgG fraction of antihistone rabbit antiserum, and the bound rabbit IgG is detected with ¹²⁵I-labeled purified goat anti-rabbit IgG. H2a-anti-H2a and H2b-anti-H2b systems showed no cross-reactivity with each other. H2a variants (H2a.1 and H2a.2) showed some cross-reaction but were distinguished reciprocally by corresponding antisera even though they differ in only two amino acids. This quantitative distinction was detected with a wide range of IgG concentrations, whereas only low concentrations revealed the differences in complement fixation assays. Histones in solution competed with the tube-absorbed histone for binding of the IgG.     

Deamination rates of 1-beta-D-arabinofuranosylcytosine, deoxycytidine and 5-methyl-2'-deoxycytidine in seven hematopoietic cell lines

Enzymatic deamination activity was determined with tritium-labelled substrates in seven established hematopoietic cell lines, in order to compare deamination rates in intact vs. broken cells with cytosine arabinoside, deoxycytidine and 5-methyldeoxycytidine. Deaminase activity was found in all the cell lines, although it was very low in mouse leukemia L1210 cells. The deamination activity of intact cells varied from 1.0 to 38.3 pmoles/micrograms protein/30 min, being highest in the human null-cell ALL line (NALL-1), the human promyelocytic leukaemia line (HL-60) and the human T-ALL line (JM). The variation in specific activities in the broken cells was between 0.9 and 30.2 pmoles/micrograms protein/30 min. The deamination rate of deoxycytidine was in general higher than that of 5-methyldeoxycytidine or cytosine arabinoside.     

UPTAKE AND INTRACELLULAR PROCESSING OF PEG-LIPOSOMES AND PEG-IMMUNOLIPOSOMES BY KUPFFER CELLS IN VITRO*

Specific targeting of drugs to for instance tumors or sites of inflammation may be achieved by means of immunoliposomes carrying site-specific antibodies on their surface. The presence of these antibodies may adversely affect the circulation kinetics of such liposomes as a result of interactions with cells of the mononuclear phagocyte system (MPS), mainly represented by macrophages in liver and spleen. The additional insertion of poly(ethylene glycol) chains on the surface of the immunoliposomes may, however, attenuate this effect.

We investigated the influence of surface-coupled rat or rabbit antibodies and of PEG on the uptake of liposomes by rat Kupffer cells in culture with ³H-cholesteryloleyl ether as a metabolically stable marker. Additionally, we assessed the effects of surface-bound IgG and PEG on the intracellular processing of the liposomes by the Kupffer cells, based on a double-label assay using the ³H-cholesteryl ether as an absolute measure for liposome uptake and the hydrolysis of the degradable marker cholesteryl-¹⁴C-oleate as relative measure of degradation.

Attachment of both rat and rabbit antibodies to PEG-free liposomes caused a several-fold increase in apparent size. The uptake by Kupffer cells, however, was 3–4 fold higher for the rat than for the rabbit IgG liposomes. The presence of PEG drastically reduced the difference between these liposome types. Uptake of liposomes without antibodies amounted to only about 10% (non-PEGylated) or less (PEGylated) of that of the immunoliposomes.

In contrast to the marked effects of IgG and PEG on Kupffer cell uptake, the rate of intracellular processing of the liposomes remained virtually unaffected by the presence of these substances on the liposomal surface.

These observations are discussed with respect to the design of optimally formulated liposomal drug preparations, combining maximal therapeutic efficacy with minimal toxicity.     

In vitro block of murine L 1210 leukemia cell growth by amiloride,an inhibitor of passive Na+ influx

The present study was aimed to decide whether Na⁺ influx can be involved in regulation of murine L 1210 leukemia cell growth. Cells were cultivated in the presence of different concentrations of amiloride and cellular growth was monitored by ³H-thymidine incorporation/10⁵ cells. This drug inhibited cell growth in concentrations ranging from 1 x 10^?5 to 1 x 10^?3 mmol/ml. Even short time treatments with amiloride caused irreversible alterations: the cells, although survived, lost their ability to divide. The results support the hypothesis that Na⁺ influx is necessary for the duplication of tumor cells.