Resistance to induced apoptosis in the human neuroblastoma cell line SK-N-SH in relation to neuronal differentiation. Role of Bcl-2 protein family.

Much evidence suggests that apoptosis plays a crucial role in cell population homeostasis that depends on the expression of various genes implicated in the control of cell life and death. The sensitivity of human neuroblastoma cells SK-N-SH to undergo apoptosis induced by thapsigargin was examined. SK-N-SH were previously differentiated into neuronal cells by treatments with retinoic acid (RA), 4 beta-phorbol 12-myristate 13-acetate (PMA) which increases protein kinase C (PKC) activity, and staurosporine which decreases PKC activity. Neuronal differentiation was evaluated by gamma-enolase, microtubule associated protein 2 (MAP2) and synaptophysin immunocytochemistry. The sensitivity of the cells to thapsigargin-induced apoptosis was evaluated by cell viability and nuclear fragmentation (Hoechst 33258) and compared with pro-(Bcl-2, Bcl-x(L)) and anti-apoptotic (Bax, Bak) protein expression of the Bcl-2 family. Cells treated with RA and PMA were more resistant to apoptosis than controls. Conversely, the cells treated with staurosporine were more susceptible to apoptosis. In parallel with morphological modifications, the expression of inhibitors and activators of apoptosis was directly dependent upon the differentiating agent used. Bcl-2 expression was strongly increased by PMA and drastically decreased by staurosporine as was Bcl-x(L) expression. Bax and Bak expression were not significantly modified. These results demonstrate that drugs that modulate PKC activity may induce a modification of Bcl-2 expression as well as resistance to the apoptotic process. Furthermore, the expression of Bcl-2 was reduced by toxin B from Clostridium difficile and, to a lesser extent, by wortmannin suggesting a role of small G-protein RhoA and PtdIns3 kinase in the control of Bcl-2 expression. Our data demonstrate a relationship between the continuous activation of PKC, the expression of Bcl-2 protein family and the resistance of differentiated SK-N-SH to apoptosis.

Antiproliferative activity of a liposomal delivery system of mitoxantrone on rabbit subconjunctival fibroblasts in an ex-vivo model.

Wound healing is the main cause of the failure of filtering surgery in glaucoma. We developed a liposomal delivery system of mitoxantrone (MITX), an anthracyclin derivative, to allow a single adjuvant administration and to lessen ocular side-effects of the drug. In order to evaluate the antiproliferative activity of liposomal MITX, an ex vivo model consisting in the culture of subconjunctival tissue explants from rabbits pretreated with subconjunctival injections of free or liposomal MITX was used. We found that both forms of MITX decreased the growth rate as well as the explant proliferation surfaces 15 days or 1 month after a single administration of the drug in vivo. A morphometric analysis of the cells showed that the surface of the fibroblasts exposed to both forms of MITX was from 10 to 12 times as important as that of the control cells exposed to the empty liposomes and to the control buffer. A radioautographic study showed that more than 95% of the fibroblasts exposed to both forms of MITX were in the G1 phase of the cell cycle, while the control cell population was equally distributed among the different phases of the cell cycle.

Treatment of PC12 cells by nerve growth factor, dexamethasone, and forskolin. Effects on cell morphology and expression of neurotensin and tyrosine hydroxylase.

Several lines of anatomical, neurochemical, electrophysiological, and behavioral evidence suggest the existence of physiological interactions between neurotensin (NT) and the brain dopaminergic systems. Thus, NT has been shown to exert a neuroleptic-like action and could be implicated in the pathogenesis and treatment of schizophrenia. It is thus of particular importance to develop in vitro cell culture systems as models to study such interactions. Rat adrenal pheochromocytoma PC12 cells, which expressed high levels of tyrosine hydroxylase, were used in the present study. In contrast to rat brain cells in primary cultures, PC12 cells did not express functional NT receptors. However, they were able to express both NTmRNA and NT in response to NGF, forskolin, and dexamethasone. Those neurochemical modifications furthermore may be related to changes in the morphology of the PC12 cells in response to NGF, forskolin, and dexamethasone alone or in combination. These data suggest that PC12 cells may provide a useful model to study in vitro the regulation of both catecholamine and neurotensin phenotypes.

Automatic cell culture quantitation with TRAKCELL: application to cell toxicology and differentiation.

An automated system, TRAKCELL, was developed for the quantitation of cells in culture. It enabled cell counting, classification according to morphological cell characteristics and measurement of cell proliferation and differentiation. The system was tested on the toxic effect of ascorbic acid on rat brain catecholaminergic neurons in primary culture. In parallel, the effects of nerve growth factor, dexamethasone and forskolin on cell differentiation were studied using rat pheochromocytoma PC12 cells. The results show that the system permits rapid and reproducible measurements of cell density and of the morphological changes observed following various drug treatments.

Evidence of non-deficient low-density lipoprotein receptor patients in a pool of subjects with clinical familial hypercholesterolemia profile.

For this study, we selected 41 adult patients with the classic clinical diagnosis of heterozygous familial hypercholesterolemia (FH), which is characterized by a low-density lipoprotein (LDL) cholesterol level above the 95th percentile, xanthomas, and/or personal or familial cardiovascular history. We used an indirect immunocytofluorimetric assay to classify these 41 subjects according to LDL receptor function on lymphocytes. We found that LDL receptor activity was normal in nine patients. A large study of plasma lipid, lipoprotein, and apolipoprotein levels found no significant difference between patients with and without LDL receptor defect. Familial defective apolipoprotein (apo) B-100 (FDB) and LDL-binding defects were not found in the nine patients without LDL receptor defect. These results suggest that other defects in the regulation of lipoprotein metabolism are capable of giving rise to a clinical and biochemical disorder indistinguishable from classic FH.

Characterization of five mouse monoclonal antibodies to apolipoprotein[a] from human Lp[a]: evidence for weak plasminogen reactivity.

We describe the development of five murine monoclonal antibodies (14A12, 39A1, 53A9, 73A7, and 128A6) specific to human apolipoprotein[a] (Mr approximately 570,000), and their characterization by a number of procedures including cotitration, competition and inhibition enzyme-linked immunosorbent assays (ELISA), immunoblotting of native lipoproteins and of SDS-solubilized apolipoproteins electrophoresed in polyacrylamide gels, and dot immunobinding assays. The patterns of immunoreactivity of these antibodies were similar. Each reacted in ELISA assays and upon electroimmunoblotting with purified apo[a], with apo[a] liberated by reduction of Lp[a], and with delipidated Lp[a] solubilized in SDS, but by contrast, they reacted with native Lp[a] to a significant degree only upon electroimmunoblotting. No reactivity was seen with LDL-apoB-100 or with other apolipoproteins. The cross-reactivity of these antibodies with the homologous protein, plasminogen, was examined by comparison of the amount of plasminogen or apo[a] required for 50% inhibition of antibody binding to apo[a], and by an ELISA assay. The inhibition assay showed reactivity with plasminogen to be 37- to 50-fold lower than with apo[a], while dot immunobinding showed the lower limit of detection of plasminogen and of apo[a] to be approximately 320 and 31 micrograms, respectively. In an ELISA sandwich assay based on monoclonal antibodies LHLP-1, 14A12, and 53A9, the lower limit of Lp[a] detection (approximately 1 ng/ml protein) was about 100-fold less than that of plasminogen. Chemical modification of apo[a] revealed a significant contribution of arginine residues to the epitopes of 14A12, 39A1, and 53A9. Modification of cysteine residues with iodoacetamide was without effect, thereby distinguishing these antibodies from LHLP-1. Each antibody reacted with the six major size forms of apo[a] (Mr approximately 450,000-750,000) in immunoblots of human sera electrophoresed in SDS-polyacrylamide gels. Marked heterogeneity in apo[a] phenotype was detected and both single and double band phenotypes were observed in a randomized study. Cotitration and competition binding studies showed varying degrees of interaction between all five epitopes, with the exception of 128A6 which appeared to be independent of 39A1 and 53A9 (and vice versa). These data suggest that our five monoclonal antibodies recognize epitopes on apolipoprotein[a] that are exposed and accessible on the native Lp[a] particle. We conclude that our monoclonal antibodies recognize a specific region of apo[a], and that this region undergoes a conformational change upon adsorption of Lp[a] to plastic thereby diminishing epitope recognition.(ABSTRACT TRUNCATED AT 400 WORDS)

The presence of lipocortin in human embryonic skin fibroblasts and its regulation by anti-inflammatory steroids.

Human embryonic skin fibroblasts in culture produce pro-inflammatory lipid mediators and all types of prostanoids. When these cells were treated with the anti-inflammatory steroid, dexamethasone, prostaglandin production was inhibited. This phenomenon required glucocorticoid receptor occupancy and mRNA and protein synthesis. The inhibitory effect was prevented by treating the cells with a monoclonal antibody, BF 26, raised against renocortin, a lipocortin-like protein formed in rat kidney medulla interstitial cells in culture. When the proteins present in the supernatants and the cell pellets derived from control and dexamethasone-treated cells were analyzed for their ability to inhibit phospholipase A2, four inhibitory peaks, at 45, 30, 15 kDa and one peak under 12 kDa, were found in the supernatants of control and dexamethasone-treated cells, whereas one single inhibitory peak at 15 kDa was found in the cell pellets. The antiphospholipase activity was much greater in dexamethasone-treated cells than in control cells. These results suggest that preformed lipocortin exists in human cells and that lipocortin is synthesized and released under glucocorticoid treatment.

Photochemotherapy using pyridopsoralens.

Aiming to decrease the acute side effects and genotoxic hazards of PUVA, pyrido (3,4-C) psoralen (PP) and 7-methyl pyrido (3,4-C) psoralen (MPP) were synthesized and studied. Their UVA maximum absorption lies at 325 and 330 nm, respectively. Their photostability is comparable to that of 8-MOP. They complex to DNA in the dark, and, in the presence of UVA, produce only monoadditions to DNA, as shown by fluorescence and DNA denaturation-renaturation studies. In diploid eukaryotic yeast they are more effective than 8-MOP for the induction of lethal effects and mitochondrial damage. Their mutagenic activity per unit dose of UVA is in the same range as that of 8-MOP. However, per viable cell they are clearly less mutagenic than 8-MOP. This difference is also observed for recombinogenic activity. No oxygen effect is observed. In mammalian cells the following ranges of effectiveness are found: inhibition of DNA synthesis in human fibroblasts: MPP greater than PP greater than 8-MOP; mutagenic activity in V79 Chinese hamster cells: MPP greater than PP greater than 8-MOP; cell transforming ability in C3H embryonic mouse cells: MPP greater than 8-MOP greater than PP as a function of UVA dose, and: 8-MOP greater than MPP greater than PP as a function of survival; induction of sister chromatic exchanges (SCE) per unit dose: MPP greater than PP greater than 8-MOP in the linear part of the induction curve, and : 8-MOP greater than PP greater than MPP at the maximum level of SCE obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction in human skin fibroblasts of sister-chromatid exchanges (SCEs) by photoaddition of two new monofunctional pyridopsoralens in comparison to 3-carbethoxypsoralen and 8-methoxypsoralen.

The induction of SCEs in human fibroblasts by photoaddition of a pyrido[3,4-c]psoralen (PyPs) and its 7-methyl derivative (MePyPs), two newly synthesized monofunctional agents proposed for photochemotherapeutic use, was compared to that of another monofunctional agent, 3-carbethoxypsoralen (3-CPs) and to the bifunctional compound, 8-methoxypsoralen (8-MOP). The yield of SCEs/cell and of SCEs/chromosome was determined at equimolar concentrations (10(-6) M) of all the drugs with increasing doses of 365 nm radiation (UVA). In the dark, the drugs alone had either no effect (8-MOP, PyPs) or a very slight effect (3-CPs, MePyPs). Nor did UVA alone demonstrate at inducing action (14.4 kJ/m2). With all the agents the average frequencies of SCE increased with increasing UVA doses, reaching a plateau level for the monofunctional compounds. The order of effectiveness for the linear part of the induction curves was MePyPs greater than PyPs greater than 8-MOP much greater than 3-CPs, whereas at the maximal level the order was 8-MOP greater than PyPs greater than MePyPs greater than 3-CPs. Determination of the frequencies of 2nd generation mitosis indicates that MePyPs is the most cytotoxic. The results focus the attention on the importance of the structure of psoralen monoadducts which, for certain genetic endpoints, might be as efficient as cross-links.

Induction of cytogenetic effects in human fibroblast cultures after exposure to formaldehyde or X-rays.

Cytogenetic changes induced by formaldehyde (FA) in exponentially growing human fibroblasts were compared with those produced by X-rays. Chromosome aberrations of different types were detected. Exposure to 2 mM FA for 15 min resulted, quantitatively and qualitatively, in effects comparable to those produced by an X-ray dose of 100 rad. X-Rays at higher exposures produced types of chromosome aberrations that differed from those induced by higher exposures to FA.

Comparison of effects of leukocyte and fibroblast interferon on immunological parameters in cancer patients.

Patients with metastatic cancer were given single intramuscular injections of 10(7) units of partially purified preparations of either leukocyte or fibroblast IFN. Serum levels of inteferon, of beta 2-microglobulin and of carcino-embryonic antigen (CEA), as well as NK activity of circulating lymphocyte, were followed over a period of 96 hr post injection. In confirmation of previous studies, levels of circulating IFN were lower after injection of fibroblast IFN than after injection of leukocyte IFN. Despite this difference in pharmacokinetics, the natural killer activity of circulating lymphocytes was enhanced with both IFNs. Levels of DEA were not influenced by the IFN injections. Leukocyte but not fibroblast IFN caused an increase in serum levels of beta 2-microglobulin in the circulation. A similar difference between leukocyte and fibroblast IFN in their ability to influence the beta 2-microglubulin system was observed in experiments on cell cultures. Only leukocyte IFN was able to cause release of beta 2-microglobulin by either leukocytes or fibroblasts.

Effect of various interferons on the spontaneous cytotoxicity exerted by lymphocytes from normal and tumor-bearing patients.

Natural killer cell activity, which represents the spontaneous cytotoxicity of lymphocytes toward tumor cells, has been measured in 173 tumor-bearing patients and 25 healthy volunteers; no significant difference was found in mean natural killer cell activity between the two groups. The parameters of interferon-induced activation of natural killer cells were studied in order to provide a suitable test for monitoring the effect of interferon in clinical trials. The three interferons tested (leukocyte, lymphoblastoid, and fibroblast) were equally active in inducing spontaneous cytotoxicity of lymphocytes from all healthy individuals and tumor-bearing patients studied. Incubation for one hr with 100 units of interferon was sufficient to increase spontaneous cytotoxicity activity, the maximum effect being obtained when lymphocytes were incubated with 1000 units of any of the interferons used. This effect was blocked with the appropriate antiinterferon sera. The target cells for interferon seem to be positive Fc gamma receptor lymphocytes.

Lack of DNA synthesis inhibitory activity in an immunosuppressor obtained from spleen.

A tissue extract derived from bovine spleen which is an immunosuppressor in vivo inhibits the incorporation of the two DNA pyrimidine nucleosides but does not inhibit the incorporation of purine nucleosides. The results indicate that the immunosuppressive action of the spleen extract is not mediated via inhibition of cell division.