Photosensitization of leukemic cells and normal bone marrow cells by 514 nm laser light and effects of laser light on migration inhibition and lymphokine response

In a model for ex-vivo purging of bone marrow grafts, leukemic cells and normal bone marrow cells were treated with merocyanine 540 and exposed to 514 nm laser light. With this treatment, 99.9999% of leukemic cells were killed while 55% of the normal bone marrow cells survived. The deleterious effects of laser light alone in the absence of photosensitizer were not observed as determined by cell viability, cell migration, and response of target cells to human migration inhibition factor. These results indicate that laser light induced photodynamic therapy can be useful for ex-vivo autologous bone marrow purging without regard to the deleterious effects of laser light alone.     

Selective staining of immature hemopoietic cells with merocyanine 540 in flow cytometry

Merocyanine 540 (MC540) has been reported to bind hemopoietic cells specifically. In this study, MC540 was used as a probe for the cytofluorometric discrimination of hemopoietic cells. In PHA-stimulated lymphocytes or HL-60 cells induced to differentiate with DMSO, MC540 binding was increased in actively proliferating cells and undifferentiated cells as compared with the more differentiated cells of the same lineage. Mononuclear bone marrow cells exhibited a discrete distribution of MC fluorescence. Sorting a population with high MC540 fluorescence (MC+ population) produced a 9-14-fold enrichment of granulocyte-macrophage progenitors (CFU-GM), and a recovery of all S-G2M phase cells (BrdUrd/DNA analysis). Cytological examination of the sorted MC+ population confirmed the enrichment in immature cells from all lineages. Double-labeling experiments using MC540 and Hoechst 33342 on total bone marrow or peripheral blood cells confirmed that the MC+ population included all the cycling cells. The proportion of S-G2M phase cells in this MC+ population was 29.3 +/- 7.8 for 15 bone marrow samples and 16.3 +/- 6.8 for 10 blood samples. MC540 could therefore be used as a marker for human hemopoietic cells, and it represents a useful tool for investigation of hemopoiesis in normal or leukemic bone marrow.     

Effects of rare earth compounds on growth and apoptosis of leukemic cell lines

To explore a new agent for inhibiting leukemic cells, we investigated the effects of rare earth compounds (lanthanum chloride and cerium chloride) on the growth and apoptosis of HL-60 and NB4 cells. The growth of HL-60 and NB4 cells was tested by 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) colorimetric assay. The apoptosis was measured by light microscopy, flow cytometry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) method. The effect of LaCl(3) on normal bone marrow hematopoietic progenitor cells was evaluated by colony-forming unit-granulocyte-macrophage (CFU-GM) assay. Under our experimental conditions, MTT assay showed that 48-h treatment with 1, 2, and 3 mM LaCl(3) or 48- and 72-h treatments with 1 mM LaCl(3) could significantly inhibit the growth of HL-60 cells. Treatment with 2 and 4 mM CeCl(3) for 72 h could significantly inhibit the growth of NB4 cells. Apoptosis could be detected on treatment with 2 mM LaCl(3) for 24 h in HL-60 cells by light microscopic morphology examination, flow cytometric analysis, and TUNEL method. Apoptosis could be also detected on treatment with 2 mM CeCl(3) for 72 h in NB4 cells. Treatment with 1 mM LaCl(3) could arrest the transitions from G0/G1 to S phase. The granulocyte-macrophage colony formation of normal bone marrow cells was not significantly inhibited at lower concentrations of LaCl(3) (0.5 to 2 mM). Our results indicate that at certain concentrations, the rare earth compounds may inhibit the growth of leukemic cells, induce them to apoptosis, and have no significant inhibitory effects on normal bone marrow hematopoietic progenitor cells (CFU-GM). The mechanism needs to be further investigated.     

Merocyanine 540 as a fluorescent probe of membranes: selective staining of leukemic and immature hemopoietic cells.

We have reported (Easton, Valinsky and Reich, 1978) that merocyanine 540 (MC 540) specifically stains a variety of living excitable cells, but not nonexcitable cells. This paper describes the exceptional permeability to MC 540 of leukemic leukocytes and immature hemopoietic precursor cells. We have used fluorescence microscopy and uptake of radioactive dye to study MC 540 staining of peripheral blood leukocytes from 80 leukemic and 34 normal individuals; leukemic leukocytes stain, whereas normal leukcytes do not. The leukocyte staining reaction differs from that previously described for excitable cells since it is independent of the ionic composition of the staining medium, kinetically complex, enhanced by light, enhanced by oxygen and essentially irreversible. Virtually all circulating nucleated cells from leukemic individuals are stained to approximately the same extent, and there is no qualitative or quantitative distinction between the various forms of leukemia. We have also found that MC 540 interacts with granulopoietic colony-forming cells (CFU-C) and with spleen colony-forming cells derived from mouse bone marrow (CFU-S). We cannot as yet identify a specific property of leukocyte plasma membranes that determines MC 540 permeability; since changes in MC 540 uptake appear to be correlated with cellular maturation during normal hemopoiesis, the retention of staining by leukemic cells, some of which appear morphologically normal, may indicate of failure in membrane maturation during leukemic blood cell development.     

Clonogenicity of normal and malignant hematopoietic progenitor cells after exposure to synthetic alkyl-lymphospholipids

Alkyl-lysophospholipids (ALP) are synthetic analogues of natural lysophosphatidylcholine and represent a new class of anti-tumor agents. They are cytotoxic in vitro with a high selectivity for neoplastic cells which, in contrast to normal cells, lack an alkyl-cleavage enzyme to degrade the adsorbed ALP molecules. As ALP accumulates, it interferes with normal membrane phospholipid turnover and eventually causes disruption of membrane integrity. To evaluate the potential value of ALP in eliminating leukemic cells from remission marrows prior to autologous transplantation, we tested the effect of various ALPs on the clongenicity of normal human marrow cells and on promyelocytic leukemia HL-60. A remarkable difference in the dose response to ALP of normal marrow cells an HL-60 was observed. After an incubation period of 24 h, the same inhibition of clonogenicity in HL-60 occurred at ALP concentrations 4 times lower than in normal marrow cells. Reducing the exposure time to 6 h enhanced the selectivity further: whereas HL-60 colonies were nearly completely inhibited at 16 micrograms ALP/ml, more than 50% of normal CFU-c and BFU-E were recovered after incubation with 48 micrograms/ml. No further increase in selectivity was achieved by changing the incubation temperature. Both thioether- and alkyl-analogues were active and no difference was observed between methoxy- and acylamino-substituted ALPs. We conclude that this selective cytotoxicity makes ALP compounds worth further study as purging agents in autologous bone marrow transplantation programs.     

Elimination of clonogenic tumor cells from bone marrow using methylprednisolone (MP) and etoposide VP16: an in vitro pharmacologic study

The ability to eliminate malignant cells from bone marrow (BM) while retaining sufficient numbers of normal progenitors to ensure engraftment, may well establish the future of autologous BM transplantation (ABMT) for hematologic malignancies. In this study, we describe the effects of methylprednisolone (MP) and etoposide (VP16) alone or in combination on 5 tumor cell lines (HL-60, a promyelocytic cell line; Molt-4, a T cell leukemia; Daudi, a Burkitt's lymphoma and R10/8226 and R40/8226, doxorubicin-resistant myeloma cell lines). The tumor cell kill efficiency of the drugs was assayed using the limiting dilution assay. We determined the toxic effect on progenitor cells by assaying granulocyte-macrophage colony-forming units (CFU). With a combination of MP at 10(-3) M and VP16 at 75 microM, we observed the following log reduction in tumor cell clones: HL-60, 4.695 +/- 0.001; Molt-4, 3.626 +/- 0.036; Daudi, 5.633 +/- 0.001; R10/8226, 3.052 +/- 0.544; R40/8226, 3.126 +/- 0.080. CFU recovery was 24% +/- 5%. Mixing tumor cell lines with a 20-fold excess of normal irradiated BM cells did not eliminate the inhibitory effect of the drug combination. We propose that MP and VP16 used in concert produce effective purging of malignant hematopoietic cells from BM while sparing normal progenitors needed for engraftment.     

Use of merocyanine 540 for photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells

Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 mug of MC 540 per ml and a light dose of 600 J/cm2 or to 20 mug of MC 540 per ml and a light dose of 450 J/cm2. A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.     

Murine acute leukemia cell line with megakaryocytic differentiation (MK-8057) induced by whole-body irradiation in C3H/He mice: cytological properties and kinetics of its leukemic stem cells

Five cases of murine leukemia with megakaryocytic differentiation were observed among the 417 cases of radiation-induced leukemias which developed in 30% of C3H/HeMs mice exposed at 8 to 10 weeks to 0.5 to 5 gy total body irradiation. Cells from individual leukemic colonies in the spleen of the irradiated mice, and cells from colonies in methylcellulose (MC) culture in vitro, derived from one of these leukemias, MK-8057, were injected into mice; both types of cells caused the deaths of the recipient mice by inducing the same type of leukemia. MK-8057 can be maintained in Dexter-type liquid culture with a feeder layer of irradiated bone marrow cells. There was a linear reciprocal relationship between the increasing number of MK-8057 cells injected versus the survival of the recipient mice. A reciprocal relationship also was seen between an increasing number of leukemic stem cells, corresponding to the number of MK-8057 cells, and the survival of mice injected with MK-8057. Giant nuclear megakaryocytes developed during the course of colony growth in the spleen as they did in the MC culture. Such megakaryocytes were acetylcholinesterase positive, whereas leukemic cells in the peripheral blood showed no sign of platelet production nor of a positive reaction to acetylcholinesterase. Cells maintained in culture were entirely positive in platelet glycoprotein IIb/IIIa when anti-human antibody was used. The larger cells in a splenic cell suspension derived from a moribund mouse were separated and enriched by velocity sedimentation using centrifugal elutriation (CE), and then subjected to flow cytometry using propidium iodide staining. Cells with up to 32N-DNA content were detected. After separating MK-8057 by counter-flow CE, the larger cell fraction (mode at 540 microns3) produced more leukemic colonies when injected into irradiated mice than did the small cell fraction (mode at 240 microns3). A higher percent of the larger cell fraction (61.9%) was killed by the addition of tritiated thymidine cytocide than in the smaller cell fraction (14.9%). Thus, the smaller cell fraction is considered to have more leukemic spleen colony-forming units (L-CFU-s) in the resting state.     

Plasma membrane lipid order of leukemic and normal immature avian erythroid cells

Mammalian erythroblasts and their leukemic counterparts contain characteristic disordered regions of plasma membrane identified as putative membrane protein collection sites. In order to determine whether erythroid cells which do not enucleate contain homologous membrane domains, immature avian erythroid precursor cells and avian erythroleukemic cells were examined using merocyanine 540 (MC540), a fluorescent dye whose binding is sensitive to the packing of membrane lipids. Results were found to contrast with previous studies of the murine equivalents of these cells. In birds, normal erythroid precursors, including basophilic erythroblasts from the bone marrow and spleen of anemic animals, contained no detectable (less than 0.1%) cells which were stained by the dye. But cells from chicks infected with avian erythroblastosis virus (AEV) did stain. Considering the pattern of staining observed on AEV-erythroblasts relative to other leukemic and normal phenotypes, however, we conclude that neither normal nor leukemic avian erythroid cells contain a functional equivalent to the membrane protein collection sites found on their mammalian counterparts.     

The response of Na+/K+ -ATPase of human erythrocytes to green laser light treatment

The objective of this study was to investigate the response of Na(+)/K(+)-ATPase of human erythrocytes to green laser irradiation. Effects of green laser light of fluences 9.5-63.3 J.cm(-2) and merocyanine 540-mediated laser light treatment were studied. Isolated erythrocyte membranes (protein concentration of 1 mg/ml) were irradiated by Nd:YAG laser (532 nm, 30 mW) and then incubated in a medium with 2 mM ATP for 30 min. Activity of ATPase was determined colorimetrically by measuring the colored reaction product of liberated inorganic phosphate and malachite green at 640 nm. Contribution of Na(+)/K(+)-ATPase to overall phosphate production was determined using ouabain. A positive effect of green laser light on Na(+)/K(+)-ATPase activity was observed. The dependence of enzymatically liberated inorganic phosphate on light fluence showed a linear correlation (R(2)=0.96, P=0.0005) for all fluences applied (9.5-63.3 J.cm(-2)). On the other hand, MC 540-mediated phototreatment caused a suppression of enzyme activity.     

人胎肝中低分子抑瘤物的分离纯化、结构鉴定及其对肿瘤细胞生长的选择性抑制作用

在胎儿组织中存在一类低分子肿瘤抑制物。胎肝细胞的甲醇－丙酮提取物中保留有大部分抑瘤活性。用体外液体培养条件下对人急性粒系白血病细胞系（ＨＬ－６０）的抑制作用为指标,跟踪指导分离过程。提取物经反相Ｃ１８中压液相色谱、ＳｅｐｈａｄｅｘＬＨ－２０凝胶色谱及氨基键合相高效液相色谱分离得到一纯活性物质,经ＮＭＲ和ＭＳ鉴定为７－酮基胆固醇（７－ｋｅｔｏｃｂｌｅｓｔｅｒｏｌ,７－ＫＣ）。体外琼脂培养条件下７－ＫＣ对小鼠ＷＥＨ１－３和Ｓ－１８０细胞较对正常小鼠骨髓粒一巨噬系祖细胞有更强的抑制增殖及集落生成作用。７－ＫＣ对ＨＬ－６０细胞增殖较对正常人骨髓ＣＦＵ－ＧＭ有更强的抑制作用。     

Inhibition of development of characteristic indices of myeloid differentiation by a factor released from a tetraploid variant of the human promyelocytic cell line, HL-60

HL-60TR, a tetraploid variant of the human promyeloid cell line HL-60, was obtained by culturing HL-60 cells for one week with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 400 pM. HL-60TR cells have lost the ability to develop several common markers of maturation in response to compounds that induce monocytoid or myelocytoid differentiation of HL-60 cells. In addition, they release a factor which inhibits induction of the same markers in HL-60 cells. Medium conditioned by HL-60TR cells also inhibits colony formation by normal mouse bone marrow cells. These properties have been maintained by HL-60TR cells through more than one year of constant subculture in the absence of TPA, a finding which suggests the possibility that TPA may promote tumor formation not only through direct effects on the phenotype of initiated cells but also through induction of continued production of factors that affect differentiation of normal stem cells.     

Enhancement of gamma-glutamyl transpeptidase expression in bone marrow cells by colony stimulating factors

Studies on blood serum from mammary carcinoma (MC) hosts, which promoted gamma-glutamyl transpeptidase (GGT) expression by normal rat bone marrow cells in liquid culture, were extended to various granulocyte-macrophage colony stimulating factors (CSFs). GGT concentration per cell was found to increase (without change in total cell number) by incubation for 48 h with purified CSF-2 gamma and CSF-1 (but not interleukin-3), with human giant cell elaborated GM-CSF and L-cell conditioned medium, as well as with the 3 MC preparations (host serum, MC extract and MC conditioned medium). GGT-inducing ability (per milligram protein) ranked the 7 preparations in the same order as did their proliferative effect (number of colonies per milligram protein) in the standard mouse bone marrow agar culture system. The quantitative correlation between these two kinds of activities (linear for their logarithmic values) was highly significant, r = 0.976, p less than 0.001. The alkaline phosphatase concentration of bone marrow cells in liquid culture was also increased in the presence of the same 7 preparations, and this again was proportional (r = 0.985, p less than 0.001) to their colony stimulating potential.     

人胎肝细胞分泌的低分子抑瘤物对白血病细胞的抑制作用

本工作证明了在胎儿组织中存在一类低分子天然肿瘤抑制物,它是胎儿组织细胞生成和分泌的,对瘤株细胞和原代白血病细胞有选择性抑制作用。初发期或复发期急性非淋巴细胞白血病患者的骨髓在体外液体培养条件下与肝细胞上清及其甲醇提取物共同孵育4d,可使所有病例的骨髓AML—CFU降低到不可检出的程度。因而,低分子天然抑瘤物是天然肿瘤免疫中的一个组成部分,它在肿瘤的诊断和治疗中有着深远的意义。     

Use of Merocyanine 540 for Photodynamic Inactivation of Staphylococcus aureus Planktonic and Biofilm Cells

Photodynamic inactivation of Staphylococcus aureus planktonic and biofilm cells by a phtotosensitizer, merocyanine 540 (MC 540), was investigated. For the planktonic experiments, MC 540 binding efficiency to bacterial cells was found to increase with both increasing MC 540 concentration and increasing incubation time, but the binding became saturated following 10 min of incubation. The antimicrobial activity was enhanced with an increasing light dose, but an increase in the light dose could not further improve the antimicrobial activity if the maximum excitation level attainable was less than the necessary minimum threshold level. Complete inactivation was achieved when the excitation level of MC 540 was somewhere above the threshold level. The relationship between antimicrobial activity and the excitation level of MC 540 revealed that the more MC 540 was excited, the more S. aureus cells were killed. For the biofilm experiments, the antimicrobial activity was enhanced with an increase in the light dose. No viable cells were detected when organisms were exposed to 15 μg of MC 540 per ml and a light dose of 600 J/cm² or to 20 μg of MC 540 per ml and a light dose of 450 J/cm². A quantitative analysis of MC 540 bound to biofilms was also performed, and the images from confocal laser scanning microscopy provided direct evidence that revealed the difference between the MC 540 remaining in the biofilms prior to irradiation and the MC 540 remaining in the biofilms after irradiation. The results of both the planktonic and biofilm experiments suggest that the antimicrobial activity of photodynamic inactivation of S. aureus is closely related to the excitation level of MC 540.     

Effects of two viral inactivation methods on platelets: laser-UV radiation and merocyanine 540-mediated photoinactivation.

Two viral inactivation methods suggested for use with cellular blood products have been evaluated as to their effects on platelets. In the first study, it was proposed that pulsed laser-ultraviolet radiation (UVB) at 308 nm could favor photodamage to UVB-sensitive viral nucleic acid with minimal effects on blood platelets. A "window of efficacy" was observed with UVB doses of 10.5-21.5 J/cm2 at which 4-6 log10 poliovirus were inactivated while platelets were relatively tolerant. However, this "window" occurred only with low-intensity UVB radiation (less than or equal to 0.25 MW/cm2). Damage to platelet proteins, evident at high laser intensities, was probably due to multiple photon excitation of amino acids. In the second study, platelets and viruses were treated with the photosensitizer, merocyanine 540 (MC 540) (less than or equal to 24 micrograms/ml), and visible light (450-600 nm) (less than or equal to J/cm2). Activation of washed platelets by dye/light treatment resulted in a spontaneous release of serotonin, spontaneous aggregation, and marked morphological changes. Increasing concentrations of albumin in the suspension medium protected against dye-mediated photodamage to platelets, but also significantly reduced the antiviral activity of MC 540 and light. These results illustrate the relative sensitivities of platelets and viruses to two inactivation methods and the difficulty in optimizing inactivation of viruses and preservation of platelet function in a protein-rich medium.     

Properties of a nuclear protein marker of human myeloid cell differentiation

The Mr 55,000 nuclear antigen present in the human promyelocytic cell line HL-60 is a basic protein that is extracted from nuclei or chromatin by 0.35 M NaCl. The antigen is confined to the nucleus of the interphase HL-60 cell as judged by immunocytochemical localization but disperses throughout the cell during mitosis. The antigen was not detected in leukemic cell lines with blast cell properties or in cell lines representing other lineages. Additional cell lines (ML-1, ML-2, and U937) with myeloid cell characteristics similar to those of the HL-60 cells, which also differentiate in vitro, express the antigen. The presence of antigen in normal human myeloid cells in peripheral blood and bone marrow is consistent with its proposed role in nuclear events associated with normal human myeloid cell differentiation.     

Growth of normal versus leukemic bone marrow cells in long term culture from acute lymphoblastic and myeloblastic leukemias

The ability of the in vitro long-term bone marrow culture (LTBMC) system to impair the survival of leukemic cells and to enhance the growth of normal progenitors has been studied. Bone marrow cells from 19 acute lymphoblastic leukemia (ALL) and 30 acute myeloid leukemia (AML) patients at diagnosis were grown in LTBMC for 4-10 weeks. In half of the cases the leukemic population declined down to undetectable levels and was replaced by putative normal hemopoietic precursors, both in ALL and in AML. In the remaining cases, leukemic cells persisted throughout the culture time and few if any normal hemopoietic cells were detected. These data led us to extend to the lymphoid compartment the previous observation of decreasing leukemic myeloid blasts in LTBMC. The potential of such cultures as an in vitro purging system for autologous bone marrow transplantation in selected poor-prognosis lymphoid malignancies should be explored, as has been done for acute and chronic myeloid leukemias.     

Thymosin beta4 and AcSDKP inhibit the proliferation of HL-60 cells and induce their differentiation and apoptosis

Our previous works have shown that bone marrow stromal cells secrete thymosin beta4 (Tbeta4) and AcSDKP. Tbeta4 and AcSDKP are existed in the conditioned medium of bone marrow endothelial cells. They exerted inhibitory effects on hematopoietic cells and then had protective effect on the early hematopoietic cells, which were cultured in the presence of hematopoietic stimulators. Thymosin beta4 consists of 43 peptides with a molecular weight of 4963. It contains at its N-terminal end the sequence of the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP). This study was performed to evaluate the effect of Tbeta4 and AcSDKP on the growth of HL-60 cells. It was showed that Tbeta4 (10(-11)-10(-7)mol/L) and AcSDKP (10(-11)-10(-7)mol/L) had the dose-dependent inhibitory effect on the proliferation of HL-60 cells. Based on cell morphology and NBT reduction, Tbeta4 and AcSDKP induced differentiation of HL-60 cells. Morphologic and DNA fragment analysis proved that Tbeta4 and AcSDKP induced apoptosis of HL-60 cells. In order to analyze the mechanism of the effects of Tbeta4 and AcSDKP, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of HL-60 leukemic cells was tested and Atlas cDNA Expression Array was performed. The results showed that Tbeta4 and AcSDKP could increased [Ca(2+)](i) by stimulating the release of Ca(2+) from intracellular Ca(2+) pool. Moreover, AcSDKP could also elicit a potent extracelluar calcium influx in HL-60 cells. Tbeta4 could also change apoptotic-related gene expression in leukemic cells, and resulted in the inhibition of proliferation and induction of differentiation and apoptosis of leukemic cells.