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This chapter focuses on the use of photosensitizing dyes, particularly merocyanine 540, as a means to rid autologous bone marrow grafts of tumor cells, enveloped viruses, and virally infected cells. Dye-sensitized photoirradiation is a recent addition to the arsenal of techniques used to manipulate bone marrow grafts, and the clinical experience with the technology is still limited. However, an initial comparison of MC 540 and Photofrin II shows that as marrow purging agents, MC 540 and Photofrin II are about equally ineffective against solid tumors. A preferential inhibition of glutathione biosynthesis or glutathione peroxidase activity in tumor cells should enhance the efficacy of MC 540 as a marrow purging agent. The dose of dye-sensitized photoirradiation was escalated by increasing the number of passages through the illumination cassette, or by reducing the concentration of serum in the illumination medium as detailed below. The dye-sensitized photoirradiation of a marrow graft could be accomplished as a batch procedure or in a flow apparatus.

Merocyanine 540-mediated photodynamic therapy (MC540-PDT) has been used in clinical trials for the purging of autologous hematopoietic stem cells grafts. When the same combinations of dye and light were applied to human peripheral blood lymphocytes, a broad range of T- and B-cell functions were impaired, prompting speculations about a potential role of MC540-PDT in the prophylaxis of graft-versus-host disease (GVHD). We here report on the effects of MC540-PDT on in vitro functions of murine lymphocytes as well as a preliminary evaluation of MC540-PDT for the prevention of GVHD in murine models of allogeneic bone marrow transplantation. Mixed lymphocyte reactions, proliferative responses to lectins, interleukin-2 and lipopolysaccharide, T-cell-mediated lysis, and NK activity were all inhibited by moderate doses of MC540-PDT. Whether MC540-PDT reduced the incidence and/or the severity of GVHD in murine models of allogeneic hematopoietic stem cell transplantation depended on the composition of the mismatched grafts and the intensity of the preparative regimen. MC540-PDT was only beneficial (i.e. reduced the incidence and/or severity of GVHD) when the spleen cell content of grafts was low and/or the radiation dose of the preparative regimen was not myeloablative, and, therefore, may have encouraged mixed chimerism.

Photodynamic therapy (PDT) with porphyrin precursors is an established therapy for certain tumors. This study aimed to explore the use of hexaminolevulinate (HAL), a porphyrin precursor, for photodynamic purging of BM grafts contaminated with cells of the 4T1 breast carcinoma cell line. The optimal PDT dose was not effective in eradicating 4T1 cells when the tumor cells were mixed with murine marrow cells in vitro. However, the number of pulmonary metastases was reduced, and the survival of experimental animals was prolonged substantially when they were subjected to TBI followed by transplantation of syngeneic BM containing metastasized 4T1 cells that had been treated ex vivo by HAL-PDT. Despite the failure of in vitro experiments, HAL-based photodynamic purging could be a useful modality for treating animals bearing an experimental breast carcinoma.

Photodynamic therapy (PDT) with porphyrin precursors has been established for tumor treatment. This study aimed at examining applicability of hexaminolevulinate (HAL) for photodynamic purging of leukemic cells from BM grafts and evaluating the clinical relevance of in vitro models. The PDT dose resulting in no colony formation by leukemic cells in vitro, in pure form or in a mixture with BM cells, was insufficient for complete killing of the leukemic cells ex vivo and for the treatment of the leukemia-bearing animals in vivo. The efficacy of HAL-PDT in cell lines in vitro should be verified in clinically relevant in vivo models.

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 microg/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.

Lung cancer has long been considered a disease that might benefit from the dose escalation of radio/chemotherapy afforded by a stem cell transplant. However, the clinical experience with high-dose chemotherapy and autologous bone marrow transplantation in lung cancer has been disappointing, with most trials showing little or no improvement in long-term survival. Unfortunately, lung cancer has a tendency to metastasize to the bone marrow, and lung cancer cells are known to circulate in the peripheral blood. Therefore, there is concern that autologous stem cell grafts from lung cancer patients may reinoculate recipients with live tumor cells. Photochemical purging of stem cell grafts with Merocyanine 540 (MC540) is highly effective against a wide range of leukemia and lymphoma cells and is well tolerated by normal hematopoietic stem and progenitor cells. Most solid tumor cells (including lung cancer cells), however, are only moderately sensitive or refractory to MC540-mediated photodynamic therapy (PDT). We report here that postirradiation hyperthermia (or = 42 degrees C, 3 h) potentiates the MC540-mediated photoinactivation of both wild-type (H69) and cisplatin-resistant mutant (H69/CDDP) small cell lung cancer cells by several orders of magnitude, while only minimally enhancing the depletion of normal human granulocyte/macrophage progenitor cells. Our data suggest that postirradiation hyperthermia provides a simple and effective means of extending the utility of MC540-PDT to the purging of stem cell grafts contaminated with lung cancer and possibly other solid tumor cells.

Elemental selenium generated by the photobleaching of selenomerocyanine dyes forms conjugates with serum albumin and serum lipoproteins that are toxic to leukemia and selected solid tumor cells but well tolerated by normal CD34-positive hematopoietic stem and progenitor cells. Serum albumin and lipoproteins act as Trojan horses that deliver the cytotoxic entity (elemental selenium) to tumor cells as part of a physiological process. They exploit the fact that many tumors have an increased demand for albumin and/or low-density lipoprotein. Se(0)-protein conjugates are more toxic than selenium dioxide, sodium selenite, selenomethionine, or selenocystine. They are only minimally affected by drug resistance mechanism, and they potentiate the cytotoxic effect of ionizing radiation and several standard chemotherapeutic agents. The cytotoxic mechanism of Se(0)-protein conjugates is not yet fully understood. Currently available data are consistent with the notion that Se(0)-protein conjugates act as air oxidation catalysts that cause a rapid depletion of intracellular glutathione and induce apoptosis. Drugs modeled after our Se(0)-protein conjugates may prove useful for the local and/or systemic therapy of cancer.

High-dose chemotherapy combined with autologous stem cell support has improved response rates in high-risk and metastatic breast cancer, but has failed to improve long-term survival. Breast cancer has a tendency to metastasize to the bone marrow, and live tumor cells are known to circulate in the peripheral blood of breast cancer patients. Sensitive immunohistochemical, culture-based, and reverse transcriptase polymerase chain reaction (RT-PCR)-based methods have shown that about 50% of histologically normal stem cell grafts from breast cancer patients are contaminated with occult tumor cells, which may cause or contribute to tumor recurrences. Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) inactivates a wide range of leukemia and lymphoma cells and is well tolerated by normal hematopoietic stem and progenitor cells. Unfortunately, most solid tumor cells (including breast cancer cells) are only moderately sensitive or refractory to MC540-PDT. We report here that if MC540-PDT is followed by a 1-h incubation with the alkyl-lysophospholipid, Edelfosine (ET-18-OCH 3 ), the depletion of murine and human breast cancer cells is greatly enhanced whereas the recovery of normal hematopoietic stem and progenitor cells is only minimally degraded. When used under conditions that reduce CD34-positive human bone marrow cells only 5.1-fold, and murine and human granulocyte/macrophage progenitors 6.8- and 3-fold, respectively, combination purging with MC540-PDT and Edelfosine depletes murine (Mm5MT) and human (MDA-MB-435S) breast cancer cells >17 000- and >125 000-fold, respectively. These data suggest that combination purging with MC540-PDT and Edelfosine may offer a simple, safe and effective method for the ex vivo purging of autologous stem cell grafts from breast cancer patients.

Toxicity of merocyanine 540 (MC540) was assessed in long-term (Dexter-type) bone marrow cultures and in a short-term in vitro clonal assay of fibroblast colony-forming cells (CFU-F). Exposure of freshly explanted mouse bone marrow cells to MC540 (15 micrograms/ml) and white light (fluence: 126 kJ/m2) reduced CFU-F by approximately 2 logs but did not abrogate the cells' capacity to establish and maintain long-term bone marrow cultures. Fat cells were rare or absence in adherent layers established with photosensitized bone marrow cells but the cultures' capacity to generate non-adherent cells, granulocyte/macrophage progenitors (CFU-GM), and early erythroid progenitors (BFU-E) was only moderately (28-36%) reduced.

Short-term exposure to the alkyl-lysophospholipid, rac-2-methyl-1-octadecyl-glycero-(3)-phosphocholine (ET-18-OCH3) or the photosensitizing dye, Merocyanine 540 (MC540) and light kills a wide range of leukemia and some solid tumor cells but is relatively well tolerated by normal pluripotent hematopoietic stem cells as well as certain classes of committed progenitor cells. Both ET-18-OCH3 and MC540-mediated photodynamic therapy (PDT) have been used as purging agents in preclinical models of autologous hematopoietic stem cell transplantation and are currently undergoing phase I/II clinical testing for the same purpose. We report here that ET-18-OCH3 synergistically enhances the MC540-mediated photoinactivation of leukemia cells but only minimally reduces the survival of normal granulocyte-macrophage progenitors. Therapeutic indices are most favorable when MC540-mediated PDT precedes incubation with ET-18-OCH3 and when purging is followed by cryopreservation. Taken together, these data suggest that combination purging with alkyl-lysophospholipid and MC540-mediated PDT may provide a simple, versatile, and effective means of eliminating large numbers of leukemia cells from autologous bone marrow grafts without causing excessive damage to normal hematopoietic stem cells.

— Merocyanine dyes with an oxygen in the electron donor heterocycle were rapidly degraded by plasma, serum and serum components. Replacement of the oxygen by a sulfur or selenium atom rendered the dyes refractory to degradation. The degradation of labile merocyanine dyes was temperature dependent and oxygen independent. The plasma component that was responsible for the degradation of merocyanine dyes was sensitive to heat and detergent, suggesting an enzymatic process. The identification of the structural requirements for sensitivity/resistance to degradation provides the experimenter with a simple means to manipulate the stability of mer-ocyanines in high serum or plasma environments and may expand the clinical utility of merocyanine photosen-sitizers beyond their traditional role in the extracorporeal purging of bone marrow grafts.

We recently reported that daily dietary supplementation with 100 μg selenium (a dose exceeding a rat’s nutritional requirement by about 33-fold) initiated immediately after total-body irradiation (TBI) and maintained for 21 weeks mitigates radiation nephropathy in a rat model as indicated by blood urea nitrogen (BUN) levels and histopathological criteria (Radiat Res. 2009; 17:368–73). In this follow-up study, we explored the risks and benefits of delaying the onset of supplementation, shortening periods of supplementation, and escalating selenium supplementation beyond 100 μg/day. Supplementation with 200 μg selenium/day (as selenite or seleno-L-methionine) substantially improved the mitigation of radiation nephropathy by lowering BUN levels at 4 months after TBI from 115 to as low as 34 mg/dl and by proportionally lowering the incidence of histopathological abnormalities. Shortening the period of supplementation to 3 or 2 months did not compromise efficacy. Delaying the onset of supplementation for 1 week reduced but did not abrogate the mitigation of radiation nephropathy. Supplementation with 300 μg/day mitigated radiation nephropathy less effectively than 200 μg and was poorly tolerated. Rats that had been given 10 Gy of TBI were less tolerant of high-dose selenium than nonirradiated rats. This reduced tolerance of high-dose selenium would need to be taken into consideration when selenium is used for the mitigation of radiation injury in victims of nuclear accidents or acts of radiological terrorism. The high dose requirements, the pronounced threshold effect, and the superior performance of selenite suggest that the mitigation of radiation nephropathy involves mechanisms that go beyond the induction of selenoproteins.

Serum is known to inhibit the merocyanine 540 (MC540)-sensitized photoinactivation of cells and enveloped viruses in a concentration-dependent manner. In diagnostic applications of MC540, a moderate amount of serum or serum albumin is frequently added to the staining solution because it enhances the contrast between intensely staining cells (e.g., electrically excitable cells or leukemia cells) and cells with a lower affinity for the dye (e.g., nonexcitable cells, red cells, normal leukocytes). In this communication we report on a quantitative analysis of the interactions of MC540 with serum and serum components. Human serum inhibited the MC540-sensitized photoinactivation of K562 leukemia cells most effectively, followed in order of decreasing potency by calf, newborn calf, horse, and fetal bovine serum. The photoprotective capacity of these five sera was directly proportional to their albumin content. Gel filtration experiments and differential spectroscopy showed that MC540 bound to serum albumin and lipoproteins. Both delipidated and lipidated albumin were capable of binding MC540. However, lipidated albumin had a considerably higher binding capacity and affinity for dye molecules.

The differential sensitivity to merocyanine 540 (MC540)-sensitized photoirradiation of leukemia cells, selected solid tumor cells, and normal pluripotent hematopoietic stem cells has been successfully exploited for the extracorporeal purging of simulated autologous remission bone marrow grafts. In this communication, we compare the effects of fractionated vs continuous irradiation upon the MC540-sensitized photoinactivation of L1210 and K562 leukemia cells. Exposure to MC540 (15 micrograms/mL) and fractionated doses of white light inactivated fewer in vitro clonogenic cells than exposure to an equivalent dose of continuous irradiation, provided the irradiation doses were small (8.1-16.2 kJ/m2) and spaced 1-2 h apart. The dye-sensitized photoinactivation of leukemia cells was enhanced when cells were stored at 4 degrees C instead of 37 degrees C between irradiation periods, most likely in part because the cells were unable to repair sublethal photodynamic damages at the lower temperature. These data suggest that cells can recover from sublethal damage inflicted by the plasma membrane-active photosensitizer, MC540.

L1210 leukemia cells were synchronized by a double thymidine block technique and then characterized with regard to their susceptibility to merocyanine 540 (MC540)-sensitized photoinactivation. Cells harvested 5 (G2/M phase) h after release from the second thymidine block were most susceptible to MC540-sensitized photoinactivation followed, in order of decreasing sensitivity, by cells harvested 2 (S phase) h and by cells harvested 7 (G1 phase) h after release from the second block. The expression of dye-binding sites changed very little during the cell cycle.

The purpose of this study was to evaluate the photosensitizing dye merocyanine 540 (MC540) as a means for extracorporeal purging of Plasmodium falciparum-infected erythrocytes from human blood. Parasitized red blood cells bound more dye than nonparasitized cells, and exposure to MC540 and light under conditions that are relatively well tolerated by normal erythrocytes and normal pluripotent hematopoietic stem cells reduced the concentration of parasitized cells by as much as 1,000-fold. Cells parasitized by the chloroquine- sensitive HB3 clone and the chloroquine-resistant Dd2 clone of P falciparum were equally susceptible to MC540-sensitized photolysis. These data suggest the potential usefulness of MC540 in the purging of P falciparum-infected blood.

The amphipathic dye, merocyanine 540 (MC540), which preferentially photosensitizes enveloped viruses and virus-infected cells, is currently being evaluated in preclinical models as a blood sterilizing agent. In this communication, we report on an initial analysis of the site and nature of MC540-mediated photodynamic damages to human herpes simplex virus type 1 and human cytomegalovirus. The comigration of dye molecules and virions on a gel filtration column, the red-shift of the fluorescence emission spectrum of virus-containing fractions, and the distribution of MC540-treated virions in an aqueous two-phase partition system were indicative of MC540 binding to the enveloped viruses and localizing in a lipophilic environment (most likely the viral envelope). Fluorescence quenching and fluorescence resonance energy transfer experiments suggested that both dye monomers and dimers were capable of partitioning into the lipid bilayer of the viral envelope. Adsorption and penetration assays and immunohistochemical analyses of viral antigen expression showed that MC540-sensitized irradiation interfered with early phases of the infectious process, the adhesion to the host cell, the penetration of the host cell, and the translocation of the virus into the nucleus of the host cell. The inactivation of viruses was inhibited if oxygen in the medium was displaced by argon, enhanced if air was displaced by pure oxygen or if water was replaced by deuterium oxide. This suggested that the MC540-sensitized photoinactivation of enveloped viruses is an oxygen-dependent process and that singlet oxygen is one but not necessarily the only mediator of the antiviral effects of MC540.

Structural analogs of Merocyanine 540 (MC540) were synthesized to probe biological mechanisms of action and to enhance viral inactivation. Novel pyrimidine-2- selone analogs of known and modified merocyanine sensitizers are described. A surprising result of placing selenium at the barbiturate site was the ∼ 100-fold enhancement of photogeneration efficiency for singlet oxygen (1O2). Conversely, selenium directly attached to the chromophore π-system was much less efficacious. The production of more 1O2 is reflected in greatly improved biological effectiveness of the new dyes. A puzzling high to low bioactivity isomer effect among molecules containing naphthoxazole or naphthothiazole components is shown by both thione and selone dyes.

Merocyanine 540 (MC 540), a photosensitizing dye, has been used in preclinical studies and in a phase I clinical trial for the purging of leukemia, lymphoma, and neuroblastoma cells from bone marrow grafts. We evaluated MC 540 as an agent for the inactivation of brain tumor cell lines of medulloblastoma or glioma origin. The U373 glioma and 74SA medulloblastoma demonstrated significantly reduced survival as determined by in vitro clonogenic assay compared to normal glial cells when exposed to MC 540 and light. U87 glioma and Daoy medulloblastoma, however, were less sensitive than normal glial cells to MC 540 photoinactivation. In vivo injection of MC 540 into mice with malignant brain tumors disclosed greater dye incorporation into the malignant tissue compared with normal control mice brains or normal tissue surrounding the brain tumor. Increased uptake of MC 540 was observed in mice injected with either photosensitive (U373 and 74SA) or photoresistant (Daoy) cell lines. These data suggest that MC 540 may be an effective agent against certain brain tumors and that dye uptake in vivo does not reflect photosensitivity.

YE-SENSITIZED photoirradiation is a relatively reD cent addition to the arsenal of techniques used for the elimination of tumor cells from autologous bone marrow (BM) grafts.’ One photosensitizer, merocyanine 540 (MC 540), has been characterized extensively in preclinical and is now being used in a phase I clinical trial for extracorporeal purging of autologous marrow grafts in patients with acute lymphocytic or nonlymphocytic leukemia, Hodgkin’s or non-Hodgkin’s lymphoma, or stage IV neuroblastoma.’o~” The preclinical evaluation of MC 540 has shown that MC 540 has a low acute systemic toxicity” and low mutagenic potential.” It preferentially photosensitizes normal erythroid progenitor cells, leukemiabymphoma cell^,'^*^^-^ and neuroblastoma cell^.'^^ By contrast, pluripotent hematopoietic progenitor cells as measured by day 12 colony-forming unit-spleen (CFU-S) or CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM) are quite resistant to MC 540-sensitized ph~toirradiation~.’~ and substantial reductions of tumor cell concentrations can be achieved without causing excessive damage to the graft’s capacity to rescue lethally irradiated host~.’~~~~~~’~ In all of the reported animal experiments, survival was the end-point and the exact kinetics of hematopoietic and lymphopoietic reconstitution were not determined. The kinetics of the hematopoietic and lymphopoietic recovery may be influenced by T cells and accessory cells (ie, cells other than the pluripotent hematopoietic stem cell) and it is conceivable that the damage done to T cells and accessory cells by a given purging procedure may affect clinical transplantation. This report addresses the effects of MC 540-sensitized photoirradiation on the functions of T and B cells. Specifically, we ask whether treatment with MC 540sensitized photoirradiation: (1) modulates T-cell proliferative responses to mitogens; (2) alters the ability of T cells to respond to alloantigens; (3) modulates T-cell helper activity; and (4) affects the ability of B cells to produce polyclonal Ig. In this study, we show that treatment with MC 540 and white light is a potent inhibitor of T- and greater than 90%. In polyclonal lg synthesis, T-cell helper activity could be abrogated by 90 minutes of treatment in cocultures containing untreated B cells. Purified B cells treated for 90 minutes cocultured with normal T cells did not produce Ig. Treatment of B cells completely inhibited EpsteinBarr virus-stimulated lg synthesis. These data show that Tand B-cell immunity is suppressed by the MC 540-sensitized photoirradiation. Treatment of bone marrow with MC 540 and light may have profound effects on immune reconstitution in autologous marrow graft recipients. More provocative is the fact that the same immunomodulatory effects may be applicable to partially mismatched marrow transplant situations as a means of reducing graft-versus-host reactions. 0 1991 by The American Society of Hematology. B-lymphocyte functions at doses of dye and light comparable with or lower than those used in clinical trials. MATERIALS AND METHODS Viability after dye-mediatedphotoiradiation (DMP). Viability of the lymphocytes used for these assays was checked before culture and at 2, 6, and 24 hours after MC 540 DMP using trypan blue exclusion. Peripheral blood mononuclear cells (PBMC) were purified by Ficoll-hypaque density gradient centrifugation (F/H) of whole heparinized blood. T (E+) and non-T (E-) subpopulations were separated by rosetting with 2-aminoethylisothiuronium bromide hydrobromide-modified sheep red blood cells (RBC) as described.” The T-cell populations were greater than 95% E+ cells and the non-T-cell populations contained less than 5% E+ cells by rosetting. Unless otherwise mentioned, the initial lymphocytes placed into culture were viable based on trypan blue exclusion after treatment. RPMI 1640 supplemented with penicillinstreptomycin, 4 mmol/L glutamine, and 10% fetal bovine serum (FBS; Hyclone, Logan, UT) was used for culture except for the mixed lymphocyte cultures. Human AI3 serum (10%) was used in the mixed lymphocyte cultures. ’H-thymidine incorporation after Lymphocyte separation.

— 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.

Leukemia and lymphoma cells are much more sensitive to Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) than normal pluripotent hematopoietic stem cells and normal colony forming unit-granulocyte/macrophage progenitors (CFU-GM). By contrast, most solid tumor cells are only moderately sensitive to MC540-PDT. The limited activity against solid tumor cells has detracted from MC540's appeal as a broad-spectrum purging agent. We report here that noncytotoxic concentrations of amifostine (Ethyol, Ethiofos, WR-2721) and amphotericin B used either alone or in combination potentiate the MC540-sensitized photoinactivation of leukemia cells, wild-type small cell lung cancer cells and cisplatin-resistant small cell lung cancer cells. Amphotericin B also enhances the MC540-sensitized photoinactivation of normal CFU-GM, whereas amifostine protects CFU-GM against the cytotoxic action of MC540-PDT. The yield of CD34-positive normal hematopoietic stem and progenitor cells is only minimally diminished by pretreatment with amifostine, amphotericin B or combinations of amifostine plus amphotericin B. Purging protocols that combine MC540-PDT with amifostine or with amifostine plus amphotericin B could offer a simple and effective approach to the purging of autologous stem cell grafts that are contaminated with solid tumor cells or the purging of stem cell grafts from heavily pretreated leukemia patients that contain reduced numbers of normal stem and progenitor cells and, therefore, can ill afford additional losses caused by purging.

The purpose of this award is to test in preclinical models the hypothesis that cytotoxic conjugates of elemental selenium and proteins are safe and effective for the systemic therapy of invasive breast cancer. The grant has three specific aims, 1) to evaluate the safety and efficacy of systemically administered Se(0)- protein conjugates in athymic nude mice bearing xenografts of human breast cancer cells, 2) to assess the functional integrity of conjugate-treated normal human hematopoietic stem cells, and 3) to determine by use of the combination index method how Se(0)-protein conjugates interact with standard chemotherapeutic agents that are commonly used in the treatment of invasive breast cancer. We report here on the preparation and evaluation of high potency cytotoxic Se(0)-protein conjugates, a quantitative in vitro analysis of the interactions of Se(0)-protein conjugates with other chemotherapeutic agents, the surprise finding that certain antibiotics can interfere with the generation of cytotoxic conjugates, and a simple and rapid spectroscopic method to detect such interferences.

Many Type II photosensitizers are substrates of the singlet oxygen they generate. They are, therefore, converted to photoproducts when exposed to light in the presence of oxygen. While most photoproducts appear to be biologically inert, one of the photoproducts generated by selenomerocyanine photosensitizers is a form of elemental selenium that is surprisingly toxic to tumor cells if allowed to form conjugates with serum albumin or lipoproteins. Albumin and lipoproteins act as delivery vehicles for the cytotoxic entity, exploiting the fact that many tumor cells have an increased capacity to bind and internalize albumin and/or lipoproteins. The cytotoxic mechanism of Se(0)-protein conjugates is not yet fully understood but is obviously quite different from the singlet oxygen-mediated mechanism of merocyanine-mediated photodynamic therapy (PDT). Whereas merocyanine-PDT is directed against the plasma membrane, is more effective at 4 °C than at room temperature, and is inhibited by excess albumin and lipoproteins, selenomerocyanine-derived cytotoxic photoproducts act on intracellular targets, are ineffective at low temperatures, and require albumin or lipoproteins as carrier molecules. The discovery of cytotoxic Se(0)-protein conjugates provides a rare example of photoproducts contributing substantially to the cytotoxic effect of PDT and challenges the widely held notion that elemental selenium is biologically inert.

Paediatric solid tumours exhibit steep dose-response curves to alkylating agents and are therefore considered candidates for high-dose chemotherapy and autologous stem cell support. There is growing evidence that autologous stem cell grafts from patients with solid tumours are frequently contaminated with live tumour cells. The objective of this study was to perform, in a preclinical purging model, an initial assessment of the safety and efficacy of a two-step purging procedure that combined Merocyanine 540-mediated photodynamic therapy (MC540-PDT) with a brief exposure to the alkyl-lysophospholipid, Edelfosine. Human and murine bone marrow cells and Neuro-2a murine neuroblastoma, SK-N-SH human neuroblastoma, SK-ES-1 and U-2 OS human osteosarcoma, G-401 and SK-NEP-1 human Wilms' tumour, and A-204 human rhabdomyosarcoma cells were exposed to a fixed dose of MC540-PDT followed by a brief incubation with graded concentrations of Edelfosine. Survival was subsequently assessed by in vitro clonal assay or, in the case of CD34-positive haematopoietic stem cells, by an immunohistochemical method. Combination purging with MC540-PDT and Edelfosine depleted all tumour cells by >4 log while preserving at least 15% of murine granulocyte/macrophage progenitors (CFU-GM), 34% of human CFU-GM, and 31% of human CD34-positive cells. The data suggest that combination purging with MC540-PDT and Edelfosine may be useful for the ex vivo purging of autologous stem cell grafts from patients with paediatric solid tumours.

Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) inactivates experimental leukemia, lymphoma, and neuroblastoma cells by a singlet oxygen-mediated mechanism but is relatively well tolerated by normal pluripotent hematopoietic stem cells and granulocyte/macrophage progenitors (CFU-GM). MC540 is currently undergoing phase I clinical testing for the extracorporeal purging of autologous bone marrow and peripheral blood stem cells. We report here that performing MC540-mediated PDT at 4.7 degrees C (hypothermia) instead of at ambient temperature enhanced the photoinactivation of L1210 cells and CFU-GM but left the photoinactivation of K562 cells unchanged. Hypothermia reduced dye binding in K562 but not in L1210 cells, whereas the photogeneration of lipid hydroperoxides (LOOH) was affected in neither cell line. Post-PDT incubation at 4 degrees C delayed the decay of LOOH and enhanced the photoinactivation of CFU-GM as well as L1210 and K562 cells. Taken together, these results suggest that hypothermia interfered with the repair of potentially lethal photodynamic damage. They stress the importance of temperature control during and immediately after the photochemical purging of autologous bone marrow and peripheral blood stem cells.

— Simultaneous exposure to merocyanine 540 (MC540) and light of a suitable wavelength kills leukemia, lymphoma and neuroblastoma cells but is relatively well tolerated by normal pluripotent hematopoietic stem cells. This differential phototoxic effect has been exploited in preclinical models and a phase I clinical trial for the extracorporeal purging of autologous bone marrow grafts. Salicylate is known to potentiate the MC540-mediated photokilling of tumor cells. Assuming that salicylate induces a change in the plasma membrane of tumor cells (but not normal hematopoietic stem cells) that enhances the binding of dye molecules it has been suggested that salicylate may provide a simple and effective means of improving the therapeutic index of MC540-mediated photodynamic therapy. We report here on a direct test of this hypothesis in a murine model of bone marrow transplantation as well as in clonal cultures of normal murine hematopoietic progenitor cells. In both systems, salicylate enhanced the MC540-sensitized photoinactivation of leukemia cells and normal bone marrow cells to a similar extent and thus failed to improve the therapeutic index of MC540 significantly. On the basis of a series of dye-binding studies, we offer an alternative explanation for the potentiating effect of salicylate. Rather than invoking a salicylate-induced change in the plasma membrane of tumor cells, we propose that salicylate displaces dye molecules from serum albumin, thereby enhancing the concentration of free (active) dye available for binding to tumor as well as normal hematopoietic stem cells.

Recent preclinical and clinical investigations indicate that phototherapy and photochemotherapy may have applications that go far beyond their "traditional" roles in the treatment of skin disorders, selected solid tumors, and neonatal hyperbilirubinemia. Bone marrow transplantation is one area that may benefit substantially from these new developments. This review focuses on new applications of phototherapy and photochemotherapy that pertain to the inactivation of tumor cells in autologous bone marrow grafts, the prevention and treatment of acute and chronic graft-versus-host disease, the prevention of transfusion-induced allosensitization and graft rejection, and the inactivation of pathogenic viruses and parasites in bone marrow grafts and blood products.

This paper examines the relationship between lipid composition, plasma membrane fluidity, expression of dye binding sites, and susceptibility to merocyanine 540 (MC540)-sensitized irradiation in L1210 leukemia cells. Reducing the cells' cholesterol content by exchange diffusion with phosphatidylcholine liposomes or by inhibiting its biosynthesis with 25-hydroxycholesterol enhanced plasma membrane fluidity, the expression of dye binding sites, and the cells' susceptibility to MC540-sensitized irradiation. Conversely, if the cholesterol content was enhanced by exchange diffusion with cholesterol:phosphatidylcholine liposomes, the cells' susceptibility to MC540-sensitized irradiation was decreased. However, contrary to expectations, dye-binding was slightly enhanced and plasma membrane fluidity remained unchanged. Growing the cells in fatty acid-supplemented medium had profound effects on their lipid composition. Cells enriched in polyunsaturated fatty acids had more fluid plasma membranes. However, dye-binding was not significantly affected and photosensitivity was slightly reduced. These results suggest that cholesterol is one, but probably not the only, determinant of the expression of cellular dye binding sites and, consequently, the cell's susceptibility to MC540-sensitized irradiation. By contrast, plasma membrane fluidity does not appear to play a major role in the regulation of dye-binding site expression.

Action spectra of the antileukemic and antiviral activities of merocyanine 540 (MC540) were determined using L1210 leukemia cells and human Herpes simplex virus type 1. The major peak of both action spectra aligned closely with the absorption spectrum of membrane-bound dye monomer, and by implication, the action spectrum of 1O2 generation. These results are compatible with the notion that the antileukemic and antiviral activities of MC540 are primarily attributable to membrane-bound monomer and at least in part mediated by 1O2.

The photosensitizing dye merocyanine 540 (MC 540) was evaluated as a means for purging malarially infected red cells from murine blood using the rodent malarial pathogens, Plasmodium yoelii and Plasmodium berghei, as models of human malaria. Malarially infected red cells bound more MC 540 and were more sensitive to MC 540-sensitized photoirradiation than were noninfected erythroid cells. Extracorporeal exposure of infected red cells to the dye and white light prevented the transmission of the disease in a transfusion model. P. berghei-infected red cells were more resistant to the antimalarial activity of MC 540 than were P. yoelii-infected cells, presumably because P. berghei preferentially infects reticulocytes whereas P. yoelii infects mature red cells. The possibility of using photoirradiation sensitized by MC 540 or related dyes to purge malarially infected donor blood is discussed.

This review discusses various opportunities for applications of photodynamic therapy in the field of bone marrow trans- plantation. These include the elimination of tumor cells from autologous bone marrow grafts (marrow purging), the pro- phylaxis and treatment of graft-versus-host disease, the prevention of graft rejection, and the inactivation of viruses in bone marrow grafts and blood products.

Most animal cells are insensitive to visible light because they lack chromophores that absorb at these wavelengths. However, cells can be made susceptible to visible light by staining them with a photosensitizing dye. Photosensitizers that bind preferentially to neoplastic cells offer interesting opportunities for new approaches to cancer therapy. The preferential retention of intravenously administered hematoporphyrin derivative (HPD) by tumor tissue is now exploited with encouraging results in the treatment of head and neck, lung, breast and bladder cancer, and several other solid tumors [1, 2].

There are four major motives behind current efforts to develop sterilization procedures for blood products. 1) There is concern about the small number of false negative results caused by currently used donor tests. 2) There is concern about the transmission of viruses for which donor tests have not yet been developed. 3) There is concern about the mounting costs of donor screening. 4) In certain geographical areas, the pool of seronegative donors is too small to meet the growing demand for virus-free blood products. Soluble blood products such as factor VIII or albumin can be adequately sterilized by exposure to dry or wet heat, solvents, or detergents [1]. None of these techniques is, of course, applicable to cellular blood products. In this paper, we review our preclinical data on the use of merocyanine 540-sensitized photoirradiation as a means to inactivate enveloped viruses in cellular and soluble blood products.

Elemental selenium generated by the photobleaching of selone photosensitizers combined with serum albumin and lipoproteins to generate conjugates that were highly cytotoxic to leukemia and selected solid tumor cells but well tolerated by normal CD34-positive hematopoietic cells. Selenium-protein conjugates were internalized by an endocytotic mechanism and localized in lysosomes. Internalized conjugates caused an extremely rapid and extensive (up to 80% in 1 hour) depletion of intracellular glutathione (GSH), the oxidation of 2′,7′-dichlorofluorescin, a loss of plasma membrane asymmetry, a breakdown of mitochondrial potential, and the activation of several caspases. Additive or synergistic anti-tumor effects were obtained when Se-protein conjugates were used in combination with ionizing radiation, melphalan, amifostine, 4-hydroperoxycyclophosphamide, edelfosine, buthionine sulfoximine, or moderate doses of arsenic (III) oxide. For example, pretreatment with a nontoxic dose of amifostine enhanced the depletion of L1210 leukemia cells from 7 log to 12 log (depletions in excess of 6 log estimated from linear extrapolations of survival curves) while the yield of normal CD34-positive cells remained at 100%. Antagonistic interactions were obtained with simultaneously applied cisplatin or very high doses of arsenic (III) oxide. The antagonistic interaction with cisplatin probably reflected a competition of cisplatin and selenium for the same binding site at or near cysteine-34 of albumin. Melphalan-resistant L1210/L-PAM1 leukemia cells (elevated intracellular GSH) were more sensitive to Se-protein conjugates than wild-type cells because of an unexpected increase in conjugate uptake by mutant cells. Cisplatin-resistant H69/CDDP cells (elevated GSH, metallothioneine, glutathione-S-transferase-pi) were slightly more sensitive than wild-type cells. Cisplatin-resistant PC14/CDDP cells (reduced drug uptake) and adriamycin-resistant HL-60/ADR cells (MRP-mediated efflux) were slightly less sensitive than the corresponding wild-type cells. In the latter cases, the reduced sensitivity of mutant tumor cells correlated with reduced conjugate uptake and/or higher intracellular GSH levels. Taken together, the above results suggest that compounds modeled after our Se-protein conjugates may prove useful for the systemic therapy of leukemia and selected solid tumors. Supported by CA77387, MACC Fund, Children’s Hospital Foundation, and Wisconsin Breast Cancer Showhouse for a Cure.

The photosensitizer, Merocyanine 540 (MC540) is currently undergoing phase I/II clinical evaluation as a purging agent for autologous stem cell grafts from patients with AML, ALL, CML, HD, and NHL. The preclinical data that provide the basis for these trials were mostly conducted in or with cells from B6D2F1 and B6AF1 mice. These experiments indicated that combinations of MC540 and light that deplete leukemia cells by 5–8 log deplete granulocyte/macrophage progenitors (CFU-GM) and the radio-protective capacity of bone marrow cells by about 1 log. We have recently shown that hematopoietic cells from 129 mice are substantially less sensitive to MC540-purging than cells from B6D2F1, B6AF1, and C57BL/6 mice. For instance, a combination of MC540 and light that depletes CFU-GM from C57BL/6mice 7.9 fold, depletes CFU-GM from 129 and B6129F1 mice merely 1.4-fold and 2-fold, respectively. This marked difference in photosensitivity cannot be explained by strain-specific differences in the ratio of photosensitive granulocye (CFU-G to photoresistant macrophage (CFU-M) progenitors, as the same rank order of sensitivity is also observed among unipotent CFU-G and CFU-GM as well as among erythroid progenitors from 129, B6129F1, and C57BL/6 mice.Flow cytometric binding studies show increased binding of MC540 to bone marrow cells from C57BL/6 mice, suggesting that the greater photosensitivity of C57BL/6 cells is at least in part attributable to a greater expression of MC540 binding sites in C57BL/6 marrow cells. These findings have obvious implications for the preclinical and clinical evaluation of merocyanines and related purging agents. They also suggest new experimental approches for investigations into the genetic control of photosensitivity.

Normal hematopoietic progenitor cells from 129S6/SvEv mice are substantially less sensitive to Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) than hematopoietic progenitors from sex- and age-matched C57BL/6 mice. When exposed to a combination of MC540 and light commonly used for the extracorporeal purging of hematopoietic stem cells, granulocyte/macrophage progenitors (CFU-GM) from C57BL/6 mice are depleted 7.9-fold whereas CFU-GM from 129S6/SvEv and (C57BL/6 x 129S6/SvEv) F1 mice are depleted 1.4- and 2-fold, respectively. The same rank order of sensitivity is also found with regard to unipotent progenitors of granulocytes and macrophages and with regard to early and late erythroid progenitors. The resistance of hematopoietic progenitors from 129S6/SvEv mice to MC540-PDT appears to be the result of reduced dye binding rather than the result of high levels of intracellular glutathione. These findings have practical implications for the design of preclinical tests of PDT in animal models. They may also provide a useful tool for future investigations into the molecular determinants of sensitivity to MC540-PDT.

As we have shown previously, cocultures of bone marrow cells with large numbers of syngeneic thymocytes enhance erythroid colony formation in plasma clots, whereas cocultures with low numbers of thymocytes suppress erythroid colony formation. In this article, we present evidence that the enhancing and suppressing functions of thymocytes are most likely mediated by at least two separate subpopulations. When thymocytes were fractionated on the basis of cell surface density of the theta antigen, enhancing cells were limited to the high theta density fraction, whereas suppressing cells were accumulated in the low theta density fraction. When thymocytes were fractionated on the basis of negative surface charge, the enhancing cells were recovered among the more negatively charged cells. A short in vitro incubation with polyamino acids selectively abrogated the suppressor function.

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.

Cells from three different human neuroblastoma cell lines and normal human bone marrow cells were exposed to the lipophilic fluorescent dye, merocyanine 540 (MC 540), and white light. In vitro clonogenic tumor cells were inactivated up to 25,000 times more rapidly than multipotent hematopoietic progenitor cells (CFU-GEMM). It is conceivable that this pronounced difference in sensitivity to MC 540-mediated photolysis can be exploited for the selective killing of residual neuroblastoma cells in autologous remission marrow grafts.

The photochemistry of merocyanine 540 (MC 540), a sensitizing dye that binds preferentially to leukemia and electrically excitable cells, has been investigated. MC 540-mediated photooxidation of histidine, arachidonate, and unsaturated phospholipid vesicles was assessed by spin label oximetry and shown to involve type II (singlet oxygen) chemistry. The dye was also shown to be a potent sensitizer of lipid peroxidation in a natural cell membrane, the erythrocyte ghost. Inhibition by azide, stimulation by 2H2O, and identification of the cholesterol product 5 alpha-cholest-6-ene-3 beta,5-diol in this system, all were consistent with singlet oxygen intermediacy. Finally, MC 540 was found to be considerably more phototoxic to K-562 leukemia cells in 2H2O than in H2O. We conclude that singlet oxygen plays a major role in the phototherapeutic effects of this dye.

Assay of red cell progenitors by colony formation in culture is expected to allow study of early events in erythroid differentiation in animal models and in man. In this communication, a simplification of the culture method for mouse bone marrow cells originally reported by Stephenson et al. ('71) is described. In the modified procedure, plasma clot is replaced by methyl cellulose, and scoring of erythroid colonies is done directly in the plates without staining. With additional modification the system proved applicable to the culture of erythroid colonies from human bone marrow as well. The development of both mouse and human erythroid colonies was dependent on “erythroid colony stimulating activity” (E-CSA) supplied by extracts of anemic sheep plasma, or by extracts of urine from anemic patients. Over a certain range, the number of colonies was a function of dose of E-CSA. In addition to E-CSA, these extracts also possessed erythropoietin activity as measured in plethoric mice. The possible chemical equivalence of urinary E-CSA and erythropoietin was suggested by their similar behavior on both gel filtration and affinity chromatography on agarose-concanavalin A. The finding further suggests that the culture method might prove useful for the bioassay of erythropoietin. Granulocyte colony stimulating activity (G-CSA) was also present in the urine extracts, as detected in cultures of mouse bone marrow. Virtually all of this activity was bound on agarose-con A, whereas only a small fraction of E-CSA was retained on this material. Agarose-con A may thus be useful for the purification of erythropoietin.

A new method is presented for the quantitative analysis of intercellular adhesive specificity. In this assay, two cell types are mixed, one unlabeled and the other labeled with the fluorescent dye, fluorescamine [4-phenylspiro(feran-2[3H],1'-phthalan)-3,3'-dione]. The resulting aggregates are analyzed by fluorescence microscopy to determine the number of labeled and unlabeled cells per aggregate. Random (nonspecific) aggregation was characterized by a binomial distribution, and adhesive specificity was accordingly quantified by the deviation (as determined by a chi-square test) from the calculated binomial distribution. The labeling procedure was simple and rapid, and experiments with 18 different cell types showed that it did not affect cell viability, morphology, rate and extent of adhesion, plating efficiency, and the capability of myogenic cells to undergo terminal differentiation. Most important, assays with morphologically identifiable cell pairs indicated that the fluorescent label neither induced apparent nor destroyed existing adhesive specificity. The most pronounced adhesive specificities were observed with freshly explanted cells from adult tissues and also with mixtures of simian virus 40-transformed and nontransformed BALB/c 3T3 cells. A glucosamine-6-phosphate N-acetylase-deficient mutant 3T3 line (AD6), however, aggregated randomly with parental 3T3 cells. Lectin-resistant mutant Chinese hamster ovary (CHO) cells displayed marginal adhesive specificity when mixed with normal CHO cells.

Human urinary erythropoietin was adsorbed to phytohaemagglutinin coupled to agarose or porous glass and quantitatively eluted by a saturated solution of MgCl2. This method provides a means of separating erythropoietin from several of its contaminants, presumably on the basis of its carbohydrate side chains. Erythropoietin which had been purified by chromatography on insolubilized phytohaemagglutinin was sufficiently free of toxicity to be assayable in tissue culture even when crude urine was used as a starting material.

The Friend virus complex was used as a model to study the effects of merocyanine 540 (MC 540)-mediated photosensitization on enveloped viruses. Simultaneous exposure to the lipophilic dye MC 540 and white light inactivated cell-free virus, cell-associated virus, and virus- transformed cells. When used under experimental conditions that are known to preserve most mature blood cells, at least some coagulation factors, and a significant portion of the pluripotent hematopoietic stem cell compartment, MC 540-mediated photosensitization reduced virus titers by greater than or equal to 4 log and the concentration of in vitro clonogenic erythroleukemia cells by greater than or equal to 5 log. Animals that received a single intravenous injection of photosensitized virus were resistant to a subsequent challenge with live virus. High sensitivity to MC 540-mediated photosensitization appears to be a property that is shared by other enveloped viruses. Thus, photosensitization mediated by MC 540 may be of benefit in the sterilization of blood products (in particular, cellular products), the production of vaccines, and selected areas of antiviral therapy.

Tumor-promoting phorbol esters were used to manipulate the in vitro development of neural crest cells. When plated at clonal density in secondary culture, quail neural crest cells from the trunk region gave rise to three types of colonies, pigmented, unpigmented, and mixed. Pigmented colonies consisted exclusively of melanocytes; up to 50% of the unpigmented mixed colonies contained adrenergic nerve cells which could be identified by a catecholamine-specific histofluorescence method. Addition of potent tumor promoters to the culture medium shortened the doubling time of neural crest cells and altered their morphologic appearance. It also delayed the onset of pigmentation, prevented the expression of the adrenergic phenotype, reduced the number of unpigmented and mixed colonies, and increased the number of pigmented colonies, most likely by directing progenitor cells preferentially to the melanogenic pathway. There was a clear correlation between the ability of phorbol esters to promote skin tumors in mice and their ability to interfere with the in vitro development of quail neural crest cells. The potent promoters 12-0-tetradecanoyl phorbol 13-acetate (TPA) and phorbol 12,13-didecanoate (PDD) were most effective, phorbol 12,13-diacetate (PDA) was considerably less effective, the nonpromoting analogues 4-0-methyl 12-0-tetradecanoyl phorbol 13-acetate (4-0-Me-TPA) and 4 alpha-phorbol 12,13-didecanoate (4alpha-PDD) and the parent alcohol phorbol (PHR) had little or no effect.

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.

In this article, we present evidence that sialic acid-containing surface components play a role in the regulation of erythropoiesis. A 1- hr exposure of mouse bone marrow cells to high concentrations of neuraminidase reduced erythroid colony formation. Coculture of 10(6) untreated thymocytes with neuraminidase-treated bone marrow cells restored erythroid colony growth. Neuraminidase-treated thymocytes retained their ability to suppress erythroid colony formation by untreated marrow cells, but lost their ability to enhance erythroid colony formation. Continuous exposure to low concentrations of neuraminidase enhanced erythroid bone marrow cell colony growth in response to a suboptimal dose of erythropoietin.