Fritz Sieber

Cellular fibronectin promotes adrenergic differentiation of quail neural crest cells in vitro.

Abstract We have investigated the interaction of cellular fibronectin (CFN) with cultured quail neural crest cells and its possible role in crest cell migration and differentiation. In vitro, quail neural crest cells from the trunk region differentiate into at least two morphologically recognizable cell types, melanocytes and adrenergic nerve cells. The latter often aggregate spontaneously into ganglia-like structures. We found that neither melanocytes nor adrenergic nerve cells synthesize CFN. However, both cell types readily interacted with exogenous CFN: Melanocytes removed CFN from the substratum and accumulated it in an aggegated form on their upper cell surface, whereas unpigmented cells migrated on the CFN substratum, often rearranging it into a fibrillar network. The adsorption of CFN by melanocytes was apparently without further consequences. However, catecholamine-positive cells were substantially increased after treatment with exogeneous fibronectin. The stimulation of adrenergic differentiation of neural crest cells is the first evidence for a positive regulatory role of fibronectin in differentiation.

Suppression of hematopoietic-progenitor-cell proliferation by ethanol and acetaldehyde.

The effects of alcohol on bone marrow are not well understood. We measured the influence of ethanol and its metabolite, acetaldehyde, on the in vitro proliferation of hematopoietic progenitor cells from mice and human beings. Colony formation by both early and late erythroid progenitor cells was suppressed by concentrations of ethanol (0.05 to 0.2 per cent) that are easily achieved in vivo. The corresponding suppressing concentration of acetaldehyde was 0.001 per cent. In contrast, suppression of granulocyte/macrophage progenitor cells required 3.0 per cent ethanol or 0.03 per cent acetaldehyde. Spleen colony formation by pluripotent stem cells was resistant to concentrations of ethanol and acetaldehyde that suppressed in vitro colony formation of committed myeloid and erythroid progenitor cells by 50 per cent. The suppression of both myeloid and erythroid colony formation was partially reversed by supplementing the cultures with folinic acid or pyridoxine. These data provide an explanation for the preferential suppression of erythropoiesis observed clinically in ethanol abuse. They also suggest that acetaldehyde has a role in ethanol-mediated bone-marrow suppression.

Effect of Molecular Structure on the Performance of Triarylmethane Dyes as Therapeutic Agents for Photochemical Purging of Autologous Bone Marrow Grafts from Residual Tumor Cells

Extensively conjugated cationic molecules with appropriate structural features naturally accumulate into the mitochondria of living cells, a phenomenon typically more prominent in tumor than in normal cells. Because a variety of tumor cells also retain pertinent cationic structures for longer periods of time compared with normal cells, mitochondrial targeting has been proposed as a selective therapeutic strategy of relevance for both chemotherapy and photochemotherapy of neoplastic diseases. Here we report that the triarylmethane dye crystal violet stains cell mitochondria with efficiency and selectivity, and is a promising candidate for photochemotherapy applications. Crystal violet exhibits pronounced phototoxicity toward L1210 leukemia cells but comparatively small toxic effects toward normal hematopoietic cells (murine granulocyte-macrophage progenitors, CFU-GM). On the basis of a comparative examination of chemical, photochemical, and phototoxic properties of crystal violet and other triarylmethane dyes, we have identified interdependencies between molecular structure, and selective phototoxicity toward tumor cells. These structure-activity relationships represent useful guidelines for the development of novel purging protocols to promote selective elimination of residual tumor cells from autologous bone marrow grafts with minimum toxicity to normal hematopoietic stem cells.

Dye-mediated photolysis of normal and neoplastic hematopoietic cells.

The purpose of this study was to determine the sensitivity to merocyanine 540 (MC 540)-mediated photolysis of normal human hematopoietic progenitor cells and four leukemia cell lines (Daudi, Raji, K562 and HL-60). Late erythroid progenitors were the most sensitive normal cells. Early erythroid progenitors were of intermediate sensitivity. Granulocyte/macrophage progenitors and multipotent progenitors were the least sensitive normal marrow cells. A combination of dye concentration, serum concentration, and illumination that eliminated 50% of multipotent progenitor cells reduced the concentration of leukemic cells by greater than or equal to 4.5 log. It is conceivable that this difference in photosensitivity can be exploited for the extracorporeal purging of autologous remission marrow grafts.

Antiviral activity of merocyanine 540.

Abstract Simultaneous exposure to the lipophilic dye merocyanine 540 (MC 540) and white light inactivates several enveloped viruses. The same treatment appears to have little or no effect on pluripotent hematopoietic stem cells, mature red cells, and mature leukocytes. At least some components of the clotting system are spared, too. The molecular basis of the virucidal effect of MC 540 and light is not yet completely understood. Based on what is known about the interactions of MC 540 with cells and artificial membranes, it seems likely that MC 540 binds to and damages the viral envelope. MC 540‐mediated photosensitization may have implications for the sterilization of bone marrow and blood products, the preparation of vaccines, and selected areas of antiviral therapy.

Effect of ethanol on thrombopoiesis

Summary. Chronic ethanol abuse causes thrombocytopenia but the underlying mechanism is unknown. To determine the target cells involved, we examined the effects of the drug in vitro on both megakaryocyte progenitor cells (CFU‐Meg) and isolated, maturing megakaryocytes. In the presence of ethanol concentrations of 0.05–2.0 g/dl, megakaryocyte colony formation by mouse CFU‐Meg in soft agar was normal. At 5 g/dl ethanol, colony formation was reduced by 50%; with 7 g/dl ethanol, no megakaryocyte colonies were formed. Acetaldehyde did not inhibit colony formation unless very high concentrations (100 mg/dl) were employed. Isolated guinea‐pig megakaryocytes can maintain their viability and incorporate 3H‐leucine into TCA‐precipitable protein for at least 24 h. Incubation of these maturing megakaryocytes with ethanol did not affect their viability, but at concentrations greater than 120 mg/dl ethanol progressively inhibited protein synthesis. At 0.5 g/dl ethanol, protein synthesis was decreased by 23% while viability was still 93% of control. Like CFU‐Meg, maturing megakaryocytes were resistant to the toxic effects of acetaldehyde. To determine the in vivo correlates of these results, guinea‐pigs were fed 5 g/dl ethanol in a liquid diet. by 11 d, when blood ethanol levels were 20–150 mg/dl, platelet counts in the animals were reduced by 17–29%, while the number of marrow megakaryocytes was unaltered. These data indicate that the site of action of ethanol in suppressing thrombopoiesis is at the level of the maturing megakaryocyte.

Heterogeneity among early quail neural crest cells

In vitro analysis has shown that early migratory quail neural crest cells from the trunk region give rise to 3 types of clonal progeny: clones consisting entirely of melanocytes; clones containing both melanocytes and adrenergic neurons; and clones containing adrenergic neurons as the only differentiated phenotype. These 3 types of clonal progeny could either be the products of distinct subpopulations of progenitor cells or the descendants of one population of pluripotent progenitor cells which, for some reason, often express only one differentiative option. To distinguish between these two possibilities, we probed the plasma membranes of early migratory quail neural crest cells in sparse secondary cultures with the fluorescent dye, merocyanine 540. Progenitors of melanocytes were less sensitive to merocyanine 540-mediated photosensitization than cells giving rise to adrenergic neurons or mixed progeny. Minor differences in the response to merocyanine 540-mediated photosensitization were found between the progenitors of adrenergic neurons and mixed progeny. These results demonstrate that early migratory neural crest cells from the trunk region are a heterogeneous population and that the different types of clonal progeny are the descendants of distinct subpopulations of progenitor cells.

MODULATION BY THIOLS OF THE MEROCYANINE 540-SENSITIZED PHOTOLYSIS OF LEUKEMIA CELLS, RED CELLS, AND Herpes simplex VIRUS TYPE 1

Abstract— This paper reports on the role of endogenous and exogenous thiols in the merocyanine 540 (MC 540)‐sensitized photoirradiation of L1210 leukemia cells, human erythrocytes, and human Herpes simplex virus type 1. Several measures taken to decrease the intracellular content of glutathione enhanced the cells sensitivity to MC 540‐sensitized photoirradiation while stimulation of glutathione biosynthesis or supplementation of the extracellular or extraviral thiol content decreased the photosensitivity of cells and viruses. Taken together, these data suggest that endogenous and exogenous thiols can modulate the sensitivity of cells and enveloped viruses to MC 540‐sensitized photoirradiation. They also pose new questions as to the mechanism of MC 540‐sensitized photolysis.

Elimination of residual tumor cells from autologous bone marrow grafts by dye-mediated photolysis: preclinical data

Abstract MC540‐mediated photolysis has several features that make it potentially attractive as a clinical purging procedure. (1) The experience with experimental tumors suggests that MC 540‐mediated photolysis is effective against a broad range of leukemias and solid tumors, including drug‐resistant tumors (Sieber et al., 1984b). Drug‐resistant tumor cells are likely to occur in heavily pretreated patients. (2) MC540‐mediated photolysis is not cell‐cycle dependent (Manna and Sieber, 1985). It kills both resting and cycling cells. In this regard, MC 540‐mediated photolysis is a valuable complement to cell‐cycle specific cytotoxic drugs. (3) There is a large differential in sensitivity between normal pluripotent hematopoietic stem cells and leukemia and neuroblastoma cells. (4) The mechanism of action of MC 540‐mediated photolysis is different from that of lectins, antibodies and most cytotoxic drugs. MC 540 binds to the lipid portion of the plasma membrane and membrane lipids are probably a primary target of the toxic photoproducts. Antibodies and lectins react with proteins and carbohydrates and most drugs have intracellular targets (e.g. nuclear DNA). We would therefore expect little cross‐resistance if MC 540‐mediated photolysis were used in combination with other purging procedures. (5) The small amounts of dye that remain associated with the marrow graft and are infused into the patient are approximately 100 000‐fold less than the LD10 (in mice) and therefore unlikely to cause any harm. The outcome of the first clinical application of the technique supports this view (Sieber et al., 1986c).

High-Dose Selenium for the Mitigation of Radiation Injury: A Pilot Study in a Rat Model

Abstract Sieber, F., Muir, S. A., Cohen, E. P., North, P. E., Fish, B. L., Irving, A. A., Mäder, M. and Moulder, J. E. High-Dose Selenium for the Mitigation of Radiation Injury: A Pilot Study in a Rat Model. Radiat. Res. 171, 368–373 (2009). The purpose of this study was to evaluate in an animal model the safety and efficacy of dietary supplementation with high doses of selenium for the mitigation of the type of radiation injury that might be sustained during a nuclear accident or an act of radiological terrorism. Age-matched male rats were exposed to 10 Gy (single dose) of total-body irradiation (TBI) followed by a syngeneic bone marrow transplant, then randomized to standard drinking water or drinking water supplemented with sodium selenite or seleno-l-methionine. At 21 weeks after TBI, most rats on standard drinking water had severe renal failure with a mean blood urea nitrogen (BUN) level of 124 ± 29 mg/dl (geometric mean ± SE) whereas rats on selenium-supplemented drinking water (100 μg/day) had a mean BUN level of 67 ± 12 mg/dl. The mitigating effect of selenium was confirmed by histopathological analyses. None of the animals on high-dose selenium showed signs of selenium toxicity. Our results suggest that dietary supplementation with high-dose selenium may provide a safe, effective and practical way to mitigate radiation injury to kidneys.