Somatic hybrids of normal and tumor Djzungarian hamster cells. I. Preferential elimination of chromosomes of the normal partner

Normal Djungarian hamster lymphoid cells were fused with SV40 transformed malignant fibroblasts. The resulting 11 hybrid clones were subjected to the chromosome analysis. The karyotype of hybrids proved to be unstable. In some cases the total tetraploid number of chromosomes in hybrids drastically decreased up to the near-diploid level close to that of the malignant parent cells. The G-band chromosome analysis showed that as a rule morphologically unchanged chromosomes were preferentially lost from the hybrid cells, the markers of the malignant partner being retained. On the basis of these data it is assumed than the hybrids between normal and tumour cells of Djungarian hamster preferentially lose the chromosomes of the normal parent cells during cultivation in vitro.     

Preferential induction of apoptosis of leukaemic cells by farnesol

Farnesol preferentially inhibits proliferation and induces apoptosis of tumour-derived but not non-transformed cell lines. We investigated whether farnesol induces apoptosis of blasts from patients with acute myeloid leukaemia (AML) and leukaemic cell lines, as compared with normal, human primary haemopoietic cells. We show that 30 microM farnesol causes apoptosis of leukaemic cell lines of T- and B-lymphocyte, myeloid or erythroid lineages and primary blasts obtained from patients with AML. However, the same concentration did not kill primary monocytes, or quiescent or proliferating T-lymphocytes. We conclude that farnesol selectively kills AML blasts and leukaemic cell lines in preference to primary haemopoietic cells.     

Preferential apoptosis of HIV-1-specific CD4+ T cells

In contrast to other viral infections such as CMV, circulating frequencies of HIV-1-specific CD4+ T cells in peripheral blood are quantitatively diminished in the majority of HIV-1-infected individuals. One mechanism for this quantitative defect is preferential infection of HIV-1-specific CD4+ T cells, although <10% of HIV-1-specific CD4+ T cells are infected. Apoptosis has been proposed as an important contributor to the pathogenesis of CD4+ T cell depletion in HIV/AIDS. We show here that, within HIV-1-infected individuals, a greater proportion of ex vivo HIV-1-specific CD4+ T cells undergo apoptosis compared with CMV-specific CD4+ T cells (45 vs 7.4%, respectively, p < 0.05, in chronic progressors). The degree of apoptosis within HIV-1-specific CD4+ T cells correlates with viral load and disease progression, and highly active antiretroviral therapy abrogates these differences. The data support a mechanism for apoptosis in these cells similar to that found in activation-induced apoptosis through the TCR, resulting in oxygen-free radical production, mitochondrial damage, and caspase-9 activation. That HIV-1 proteins can also directly enhance activation-induced apoptosis supports a mechanism for a preferential induction of apoptosis of HIV-1-specific CD4+ T cells, which contributes to a loss of immunological control of HIV-1 replication.     

Overexpressed LEF-1 proteins display different nuclear localization patterns of beta-catenin in normal versus tumor cells

Beta-catenin not only plays a role in cadherin-dependent cell adhesion, but also interacts with T-cell factor (TCF)/lymphoid enhancer factor-1 (LEF-1) to affect gene expression. In this report, we describe the effects of exogenous LEF-1 and of treatment with leptomycin B (LMB), a specific inhibitor of CRM1-medicated nuclear export, on the nuclear localization and export of beta-catenin. Normal epithelial cells overexpressing LEF-1 accumulate nuclear beta-catenin in a LEF-1 concentration-dependent manner. Nuclear beta-catenin, once imported from the cytoplasm, is rapidly removed from the nucleus. Treatment with LMB results in dramatic retention of nuclear beta-catenin in normal epithelial cells transfected with LEF-1, and this effect is intensified by treatment of N-Acetyl-leucyl-leucyl-norleucinal together with LMB. Colon carcinoma cells containing an adenomatous polyposis coli mutation retain significant amounts of LEF-1 induced nuclear beta-catenin considerably after the time-point when beta-catenin disappears from the nuclei of LEF-1 transfected normal epithelial cells. beta-Catenin binds directly to CRM1, and overexpression of CRM1 reduces nuclear beta-catenin-mediated transactivation function.     

Preferential target is mitochondria in alpha-mangostin-induced apoptosis in human leukemia HL60 cells

Our previous study has shown that alpha-mangostin, a xanthone from the pericarps of mangosteen, induces caspase-3-dependent apoptosis in HL60 cells. In the current study, we investigated the mechanism of apoptosis induced by alpha-mangostin in HL60 cells. Alpha-mangostin-treated HL60 cells demonstrated caspase-9 and -3 activation but not -8, which leads us to assume that alpha-mangostin may mediate the mitochondrial pathway in the apoptosis. Parameters of mitochondrial dysfunction including swelling, loss of membrane potential (deltapsim), decrease in intracellular ATP, ROS accumulation, and cytochrome c/AIF release, were observed within 1 or 2 h after the treatment. On the other hand, alpha-mangostin-treatment did not affect expression of bcl-2 family proteins and activation of MAP kinases. These findings indicate that alpha-mangostin preferentially targets mitochondria in the early phase, resulting in indication of apoptosis in HL60 cells. Furthermore, we examined the structure-activity relationship between xanthone derivatives including alpha-mangostin and the potency of deltapsim-loss in HL60 cells. Interestingly, replacement of hydroxyl group by methoxy group remarkably decreased its potency. It was also shown that the cytotoxicity substantially correlated with deltapsim decrease. These results indicate that alpha-mangostin and its analogs would be candidates for preventive and therapeutic application for cancer treatment.     

Distinct signaling pathways in TRAIL- versus tumor necrosis factor-induced apoptosis

Trimeric tumor necrosis factor (TNF) binding leads to recruitment of TRADD to TNFR1. In current models, TRADD recruits RIP, TRAF2, and FADD to activate NF-kappaB, Jun N-terminal protein kinase (JNK), and apoptosis. Using stable short-hairpin RNA (shRNA) knockdown (KD) cells targeting these adaptors, TNF death-inducing signaling complex immunoprecipitation demonstrates competitive binding of TRADD and RIP to TNFR1, whereas TRAF2 recruitment requires TRADD. Analysis of KD cells indicates that FADD is necessary for Fas-L- or TRAIL- but not TNF-induced apoptosis. Interestingly, TRADD is dispensable, while RIP is required for TNF-induced apoptosis in human tumor cells. TRADD is required for c-Jun phosphorylation upon TNF exposure. RIP KD abrogates formation of complex II following TNF exposure, whereas TRADD KD allows efficient RIP-caspase 8 association. Treatment with TRAIL also induces formation of a complex II containing FADD, RIP, IKKalpha, and caspase 8 and 10, leading to activation of caspase 8. Our data suggest that TNF triggers apoptosis in a manner distinct from that of Fas-L or TRAIL.     

Centrosome function in normal and tumor cells

Centrosomes nucleate microtubules that form the mitotic spindle and regulate the equal division of chromosomes during cell division. In cancer, centrosomes are often found amplified to greater than two per cell, and these tumor cells frequently have aneuploid genomes. In this review, we will discuss the cellular factors that regulate the proper duplication of the centrosome and how these regulatory steps can lead to abnormal centrosome numbers and abnormal mitoses. In particular, we highlight the newly emerging role of the Breast Cancer 1 (BRCA1) ubiquitin ligase in this process.     

Signalling pathway of tumor necrosis factor in normal and tumor cells

Summary Several aspects of the activity and effects of tumor necrosis factor (TNF) were investigated to gain further insight into its cytotoxic mechanism. The relation between number of TNF receptors and TNF susceptibility of both tumor cells and normal cells was studied, utilizing a specific binding assay. Among the tumor cells, a fairly close correlation (r=0.855) was observed between receptor number and sensitivity to TNF. No cytotoxic effect by TNF was observed on any of the normal cells tested, even though TNF receptors were shown to be present, and cell proliferation was apparently stimulated by TNF in some cases. TNF internalization and intracellular distribution were studied by pulse-labelling and Percoll density gradient centrifugation. In L-M (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) cells and HEL (human embryonic lung cells, non-sensitive to TNF cytotoxicity) cells, receptor-bound ¹²⁵I-labelled recombinant human TNF was rapidly internalized and delivered to lysosomes within 15–30 min, and this was followed by degradation and release into the culture medium. The presence of either a cytoskeletal disrupting agent or a lysosomotropic agent was observed to inhibit the cytotoxic effect of TNF, thus also indicating that TNF internalization, followed by delivery to lysosomes, is essential in the cytolytic mechanism of TNF.As observed by [³H]uridine incorporation, TNF did not affect RNA synthesis in L-R cells (TNF-resistant cell lines derived from L-M cells) and HEL cells, but markedly stimulated (by 3.5 times) RNA synthesis in L-M cells.     

Preferential infection of immature dendritic cells and B cells by mouse mammary tumor virus

Until now it was thought that the retrovirus mouse mammary tumor virus preferentially infects B cells, which thereafter proliferate and differentiate due to superantigen-mediated T cell help. We describe in this study that dendritic cells are infectable at levels comparable to B cells in the first days after virus injection. Moreover, IgM knockout mice have chronically deleted superantigen-reactive T cells after MMTV injection, indicating that superantigen presentation by dendritic cells is sufficient for T cell deletion. In both subsets initially only few cells were infected, but there was an exponential increase in numbers of infected B cells due to superantigen-mediated T cell help, explaining that at the peak of the response infection is almost exclusively found in B cells. The level of infection in vivo was below 1 in 1000 dendritic cells or B cells. Infection levels in freshly isolated dendritic cells from spleen, Langerhans cells from skin, or bone marrow-derived dendritic cells were compared in an in vitro infection assay. Immature dendritic cells such as Langerhans cells or bone marrow-derived dendritic cells were infected 10- to 30-fold more efficiently than mature splenic dendritic cells. Bone marrow-derived dendritic cells carrying an endogenous mouse mammary tumor virus superantigen were highly efficient at inducing a superantigen response in vivo. These results highlight the importance of professional APC and efficient T cell priming for the establishment of a persistent infection by mouse mammary tumor virus.     

Antibody targeting of camptothecin-loaded PLGA nanoparticles to tumor cells

Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immuno-nanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactide-co-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 microg coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC 50 of 21.8 ng mL (-1). Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC 50 of 0.37 ng mL (-1). Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.     

Pristimerin induces apoptosis by targeting the proteasome in prostate cancer cells

Pristimerin is a natural product derived from the Celastraceae and Hippocrateaceae families that were used as folk medicines for anti inflammation in ancient times. Although it has been shown that pristimerin induces apoptosis in breast cancer cells, the involved mechanism of action is unknown. The purpose of the current study is to investigate the primary target of pristimerin in human cancer cells, using prostate cancer cells as a working model. Nucleophilic susceptibility and in silico docking studies show that C6 of pristimerin is highly susceptible towards a nucleophilic attack by the hydroxyl group of N-terminal threonine of the proteasomal chymotrypsin subunit. Consistently, pristimerin potently inhibits the chymotrypsin-like activity of a purified rabbit 20S proteasome (IC50 2.2 micromol/L) and human prostate cancer 26S proteasome (IC50 3.0 micromol/L). The accumulation of ubiquitinated proteins and three proteasome target proteins, Bax, p27 and I kappa B-alpha, in androgen receptor (AR)-negative PC-3 prostate cancer cells supports the conclusion that proteasome inhibition by pristimerin is physiologically functional. This observed proteasome inhibition subsequently led to the induction of apoptotic cell death in a dose- and kinetic-dependent manner. Furthermore, in AR-positive, androgen-dependent LNCaP and AR-positive, androgen-independent C4-2B prostate cancer cells, proteasome inhibition by pristimerin results in suppression of AR protein prior to apoptosis. Our data demonstrate, for the first time, that the proteasome is a primary target of pristimerin in prostate cancer cells and inhibition of the proteasomal chymotrypsin-like activity by pristimerin is responsible for its cancer cell death-inducing property.     

Mitochondrial targeting drug lonidamine triggered apoptosis in doxorubicin-resistant HepG2 cells

Mitochondria play a crucial role in the induction and execution of apoptosis. Accordingly, recent suggestions have been made to use agents that directly act on mitochondria to trigger apoptosis so that drug-sensitive and-resistant tumour cells can be eliminated. To test this hypothesis, human hepatocarcinoma HepG2 and its derivative R-HepG2 with doxorubicin (Dox) resistance as a result of expression of P-glycoprotein were used to investigate the effect of lonidamine (LND), a new mitochondrial targeting drug, on the induction of apoptosis. Results from our study indicate that R-HepG2 cells were more sensitive to LND than parental cells in terms of cytotoxicity determined by alamar blue assay. Cell death induced by LND was associated with the hallmarks of apoptosis such as mitochondrial membrane depolarization, release of cytochrome c, phosphatidyl-serine externalization and DNA fragmentation. Moreover, combined treatment of cells with Dox and LND elicited more cell death. Taken together, our results suggest a potential use of LND as an anti-cancer drug to bypass drug resistance and to trigger tumour destruction through apoptosis in HepG2 and R-HepG2 cells.     

Quantitative imaging of apoptosis commitment in colorectal tumor cells

We have studied caspase-3 activation by combined DNA damage induction and EGFR kinase inhibition in order to identify potential EGFR-mediated survival signals conferring resistance to apoptosis in human colorectal tumor cells. The onset of apoptosis was microscopically imaged with a newly developed caspase-3 substrate sensor based on EGFP and tHcred1, enabling us to monitor caspase-3 activation in cells by fluorescence lifetime imaging microscopy or fluorescence correlation spectroscopy. Both optical approaches provide parameters quantitatively reporting the ratio between cleaved and uncleaved sensor, thereby facilitating the comparison of caspase-3 activation between different cells. Using these methods, we show that EGFR kinase inhibitors sensitize colorectal SW-480 tumor cells for 5-fluorouracil-induced apoptosis, indicating that EGFR-mediated survival signaling contributes to apoptosis resistance via its intrinsic kinase activity.     

Induction of apoptosis of tumor cells by binase

An induction of apoptosis by RNase from Bacillus intermedius (binase) and its mutants characterized with low catalytic activity (Lys26Ala and His101Glu) in human myelogenic erythroleukemia K562 cells, human lung carcinoma A549 cells and human peripheral blood mononuclear cells was studied. For the first time selective apoptogenic effects of binase toward leukemic blood cells was determined. Neither antiproliferative nor apoptotic effects of binase were detected in normal human peripheral blood mononuclear cells. Formation of low molecular weight oligonucleosomal DNA fragments (less than 50 Kb) which are an early marks of apoptosis was registered in solid tumor cells treated by binase. Using mutant RNases it was shown that decrease of catalytic activity to 2.5% of wild type enzyme activity leads to the loss of apoptogenic properties of enzyme. Selective apoptogenicity of binase found towards malignant cells confirmed that antitumor agents based on bacterial RNases could be considered as an alternative to standard chemotherapeutic drugs.     

NMR exposure sensitizes tumor cells to apoptosis

NMR technology has dramatically contributed to the revolution of image diagnostic. NMR apparatuses use combinations of microwaves over a homogeneous strong (1 Tesla) static magnetic field. We had previously shown that low intensity (0.3–66 mT) static magnetic fields deeply affect apoptosis in a Ca²⁺ dependent fashion (Fanelli et al., 1999 FASEBJ., 13;95–102). The rationale of the present study is to examine whether exposure to the static magnetic fields of NMR can affect apoptosis induced on reporter tumor cells of haematopoietic origin. The impressive result was the strong increase (1.8–2.5 fold) of damage-induced apoptosis by NMR. This potentiation is due to cytosolic Ca²⁺ overload consequent to NMR-promoted Ca²⁺ influx, since it is prevented by intracellular (BAPTA-AM) and extracellular (EGTA) Ca²⁺ chelation or by inhibition of plasma membrane L-type Ca²⁺ channels. Three-days follow up of treated cultures shows that NMR decrease long term cell survival, thus increasing the efficiency of cytocidal treatments. Importantly, mononuclear white blood cells are not sensitised to apoptosis by NMR, showing that NMR may increase the differential cytotoxicity of antitumor drugs on tumor vs normal cells. This strong, differential potentiating effect of NMR on tumor cell apoptosis may have important implications, being in fact a possible adjuvant for antitumor therapies.