Increased survival of normal cells during laser photodynamic therapy: implications for ex vivo autologous bone marrow purging

Laser light-induced, dye-mediated photolysis of leukemic cells was tested in an in vitro model for its efficacy in eliminating occult tumor cells for ex vivo autologous bone marrow purging. Merocyanine 540 (MC540) was mixed with acute promyelocytic leukemia (HL-60) cells in the presence of human albumin. This cell-dye mixture was irradiated with 514 nm argon laser light. Results show that in the presence of 0.1%, 0.25% and 0.5% albumin, laser light doses of 62.4 J/cm2, 93.6 J/cm2 and 109.2 J/cm2, respectively, were required for a 5 log reduction in the survival of leukemic cells. Under identical conditions, 80% to 84% of the normal bone marrow cells and 41% of the granulocyte-macrophage colony forming cells survived. The number of surviving stromal cells was reduced (1+) compared to the untreated control (4+). Mixing of irradiated bone marrow cells with equal number of HL-60 cells did not interfere with the killing of HL-60 cells treated with MC540 and laser light. The non-specific cytotoxicity of laser light alone was less than 6% for normal bone marrow cells. These results suggest that the concentration of human albumin plays an important role in laser light-induced phototoxicity. This laser light-induced selective photolysis of leukemic cells can be used in ex vivo purging of tumor cell-contaminated bone marrow grafts to achieve very high survival rates of normal bone marrow cells and granulocyte-macrophage colony forming cells.     

Influence of hematoporphyrin derivative concentration, incubation time, temperature during incubation and laser dose fractionation on photosensitivity of normal hemopoietic progenitors or leukemic cells

Photodynamic therapy represents a new approach for the local control of cancers. It has recently been claimed that photodynamic therapy mediated by hematoporphyrin derivative (HPD) is selectively more efficient for killing leukemic cells than normal progenitors. To improve this effect, we studied the influence of hematoporphyrin dose, temperature during incubation and/or treatment, hematoporphyrin derivative incubation time, and fractionation of the argon laser light (488-514 nm) used for hematoporphyrin stimulation. Plating efficiency calculated after a 7-day period of growth on collagen gel medium showed a dose-dependent phototoxicity of HPD reaching 0.01% for normal hemopoietic progenitors and 0.001% for leukemic cells (dose = 12.5 micrograms/ml). The 10:1 ratio of normal hemopoietic progenitors to leukemic cells was also found to be the same or increased when temperature was 37 degrees C during incubation and 4 degrees C during laser irradiation. Similar results were also found when incubation time was varied from 75-120 min, or when laser irradiation dose was fractionated into 2 or 3 periods. The ratio of normal progenitors to leukemic cells reached 100:1 when 75 J/cm2 were fractionated into 3 periods after an incubation time of 120 min with 10 micrograms/ml HPD. Selectivity in photodynamic treatment seems to occur between normal hemopoietic progenitors and leukemic cells. The mechanism of this selectivity remains unclear, but experiments with the fractionated irradiation dose suggest that as in radiotherapy, better potentially lethal damage repair in normal cells could be a factor for selectivity in photodynamic therapy. Our results obtained with leukemic cells are fully in agreement with data in the literature concerning similar experimental models.     

激光、血卟啉对肿瘤细胞DNA单链断裂及其重接的影响

用简化的Kohn氏碱洗脱法,观察了光敏剂血卟啉衍生物(HPD)对小鼠S-180肿瘤细胞DNA单链断裂及其重接修复的影响。激光HPD能导致S-180细胞DNA单链断裂明显增加,而且这种断裂随着保温时间的延长,继续增多。在本实验条件下没有观察到HPD对X线的增敏作用,HPD不能增加X线所致的DNA单链断裂,也不能影响其重接。单链断裂重接动力学的实验进一步证明了这个论点。     

Tumor radiosensitivity prediction by the cytokinesis-block micronucleus assay

An in vivo to in vitro cytokinesis-block micronucleus assay technique using cytochalasin B (Cyt-B) was established in xenografted human and murine tumors, and the correlation between radiosensitivity measured by this assay and that measured by a colony-forming assay was investigated. Tumors were irradiated in situ, excised immediately, and disaggregated to single cells that were plated for the micronucleus and colony-forming assays. Some of the tumor cells were irradiated in vitro rather than in vivo. For the micronucleus assay, Cyt-B (0.5-3 micrograms/ml) was added to dishes soon after plating or in vitro irradiation and the cells were subsequently fixed and stained at intervals (12-144 h). The micronucleus frequency in binucleate cells was evaluated under conditions of maximum yield of the binucleate cells. The micronucleus frequency after irradiation was quite variable depending on the tumor type and the average number of micronuclei per single binucleate cell after 4 Gy ranged from 0.2 to 1.4. The results of in vitro irradiation were not significantly different from those of in vivo irradiation for all tumors. A good correlation was found between the radiosensitivity determined by the micronucleus assay and that found with the colony-forming assay in six human tumors (r = 0.94 approximately 0.98) but not in four murine tumors because of one exceptional tumor. When this tumor was excluded, a correlation was also found for the remaining nine tumors (r = 0.62 approximately 0.96). These results indicated that the cytokinesis-block micronucleus assay has some promise as a rapid predictive assay of radiosensitivity.     

Activity of tumor-associated lymphoid cells at short intervals after administration of irradiated syngeneic and allogeneic tumor cells.

After a single intraperitoneal injection of irradiated tumor cells, host cells capable of responding against syngeneic tumors were detected in peritoneal exudates of mice. Although irradiation of the injected tumor prevented its overgrowth, it did not significantly alter the antigenicity of the tumor. Immunologic activities of tumor-associated host cells in the peritoneal cavity were continuously monitored, starting 48 hr after tumor administration. In vitro cell-mediated lysis of syngeneic tumors appeared as early as 3 days after irradiated tumor administration. In addition, peritoneal exudate cells from inoculated mice were capable of adoptively transferring immunity. Purification of these peritoneal exudate cells on nylon wool columns yielded a nonadherent Ig-negative lymphocyte fraction whose cytolysis was tumor-specific and T cell-associated. The macrophage-free lymphocyte fraction exhibited a higher in vitro activity against tumors than unpurified peritoneal exudates. This tumor-host system allowed the study of cells which directly interact with the tumor cells in vivo, starting shortly after tumor administration. The results reported in this paper show that tumor-associated lymphoid cells capable of mounting anti-tumor response in vivo and in vitro can be demonstrated as early as 3 days after tumor inoculation.     

Microscopic instrumentation and analysis of laser-tissue interaction in a skin flap model

A dorsal skin flap model for microcirculatory studies has been modified for "in vivo" studies of laser-tissue interaction with microcirculation. An experimental apparatus has been built implementing a laser delivery system, video microscopy during irradiation, and thermal recordings. This model has been used to study irradiation effects on microcirculation using the argon laser (488 and 514.5 nm) and the argon pumped dye laser at 577 nm. The results include: measurements of the optical properties of the model; dosimetry measurements for the production of embolized and stationary coaguli in arterioles and venules; and focal vessel disappearance of venules irradiated with the argon or the argon pumped dye laser at 577 nm; a method to determine light attenuation in the model; a unique method for measurements of blood flow velocity in arterioles and venules and measurements obtained with this method; measurements of transient and steady state temperatures during irradiation and a study of laser induced photorelaxation phenomena in venules.     

激光、血卟啉对小鼠S-180V肿瘤细胞膜通透性的作用及其影响因素

通过Na_2~(51)CrO_4在肿瘤细胞膜内外的分布比值的测定,观察了激光血卟啉衍生物(简称HPD)对小鼠S-180V肿瘤细胞膜通透性的作用及其影响因素:(1)通过紫外吸收光谱的测定对肿瘤细胞摄取HPD的动态过程作了观察。选择了实验所需的合适HPD浓度和作用时间,并观察到细胞悬液中血清蛋白能阻抑细胞对HPD的摄取。(2)在氦氖激光照射后即可观察到含有HPD的肿瘤细胞膜外~(51)Cr/膜内~(51)Cr的比值明显增加,而单照激光或单加HPD两组的~(51)Cr比值与正常对照组相比无明显变化。(3)上述的~(51)Cr比值变化随着照后保温时间的延长而逐渐加大。与此同时细胞形态也发生相应的变化,细胞死亡率也逐渐增加。说明除了原初的光敏反应外,还有继发的细胞损伤。(4)细胞悬液中血清蛋白的存在虽然对激光血卟啉对肿瘤细胞的杀伤作用有所减弱,但在这样条件下的光敏反应比较接近临床上治疗肿瘤的实际情况。     

PLD repair in rat rhabdomyosarcoma tumor cells irradiated in vivo and in vitro with high-LET and low-LET radiation

Results are reported of studies to measure the extent of recovery of potentially lethal damage (PLD) in rat rhabdomyosarcoma tumor cells after irradiation both in vivo and in vitro with either high-LET or low-LET radiation. Stationary-phase cultures were found to exhibit repair of PLD following irradiation in vitro either with low-LET X rays or with high-LET neon ions in the extended-peak ionization region. Following a 9-Gy dose of 225-kVp X rays or a 3.5-Gy dose of peak neon ions, both of which reduced the initial cell survival to 6-8%, the maximum PLD recovery factors were 3.4 and 1.6, respectively. In contrast, the standard tumor excision assay procedure failed to reveal any recovery from PLD in tumors irradiated in situ with either X rays or peak neon ions. PLD repair by the in vivo tumor cells could be observed, however, when the excision assay procedure was altered by the addition of a known PLD repair inhibitor beta-arabinofuranosyladenine (beta-ara-A). When a noncytotoxic 50 microM concentration of beta-ara-A was added to the excised tumor cells immediately following a 14.5-Gy in situ dose of X rays, cell survival in the inhibitor-treated cells was lower than in the untreated cells (0.018 compared to 0.056), resulting in a PLD repair inhibition factor of 3.1. Delaying the addition of beta-ara-A for 1, 2, or 3 h following tumor excision reduced the PLD repair inhibition factor to 1.6, 1.5, and 0.9, respectively. Following tumor irradiation in situ with neon ions in the extended-peak ionization region (median LET = 145 keV/micron), less PLD repair was observed than after X irradiation. For 5.8 Gy of peak neon ions, the PLD repair inhibition factors were 2.1, 1.5, 1.3, and 1.1 at 0, 1, 2, and 3 h, respectively. We interpret the absence of measurable PLD repair using the standard tumor excision assay procedure as resulting from undetectable repair occurring during the long interval (about 2 h) required for the cell dissociation and plating procedures. We conclude that at least for our tumor system, PLD repair does occur after irradiation of tumors in situ, even though it is not detectable using the standard tumor excision assay procedure. Thus a failure to measure such repair by this assay in a given tumor system does not necessarily mean the cells are incapable of PLD repair.     

Micronuclei in human lymphocytes irradiated in vitro or in vivo

Venous blood from healthy donors or from patients with various lympho- and myeloproliferative diseases was incubated in vitro in the presence of cytochalasin B for the induction of binucleated lymphocytes. The time at which cytochalasin B was added depended on the proliferation rate of the lymphocytes. Proliferation was monitored using a semiautomatic microscope photometer/computer system. The background level of micronuclei in binucleated lymphocytes of the patients before radiotherapy was statistically indistinguishable from that of healthy persons. Blood from both groups was irradiated in vitro for the study of the dose-response relationship. The dose-response curves were very similar up to 3.75 Gy, and a somewhat lower micronucleus frequency was found in lymphocytes of patients after a 5-Gy exposure. These in vitro results were compared with in vivo exposure after total-body irradiation of leukemic patients. Due to heavy medication that accompanied radiation therapy, only two doses (1.25 and 2.5 Gy) could be checked after in vivo exposure. There was no statistically significant difference between in vitro and in vivo results after 1.25 Gy, but a slightly lower number of micronuclei was observed after in vivo exposure to 2.5 Gy.     

Cardiac endothelial cells isolated from mouse heart - a novel model for radiobiology

Cardiovascular disease is recognized as an important clinical problem in radiotherapy and radiation protection. However, only few radiobiological models relevant for assessment of cardiotoxic effects of ionizing radiation are available. Here we describe the isolation of mouse primary cardiac endothelial cells, a possible target for cardiotoxic effects of radiation. Cells isolated from hearts of juvenile mice were cultured and irradiated in vitro. In addition, cells isolated from hearts of locally irradiated adult animals (up to 6 days after irradiation) were tested. A dose-dependent formation of histone γH2A.X foci was observed after in vitro irradiation of cultured cells. However, such cells were resistant to radiation-induced apoptosis. Increased levels of actin stress fibres were observed in the cytoplasm of cardiac endothelial cells irradiated in vitro or isolated from irradiated animals. A high dose of 16 Gy did not increase permeability to Dextran in monolayers formed by endothelial cells. Up-regulated expression of Vcam1, Sele and Hsp70i genes was detected after irradiation in vitro and in cells isolated few days after irradiation in vivo. The increased level of actin stress fibres and enhanced expression of stress-response genes in irradiated endothelial cells are potentially involved in cardiotoxic effects of ionizing radiation.     

氦氖激光对离体小鼠腹腔巨噬细胞功能的影响

为探索低功率激光照射治疗的机理,本实验用氦氖激光照射离体小白鼠腹腔巨噬细胞观察其吞噬鸡红细胞折功能。当照射１５分钟时,巨噬细胞蚕噬功能达到最大值,以后开始下降,照射至４０分时,巨噬细胞吞噬功能下降至对照组以下,出现抑制现象。     

藻蓝蛋白色素肽光动力学抗肿瘤作用的实验研究

目的：探讨藻蓝蛋白色素肽的理化特性及光动力疗法（PDT）抗肿瘤效果。方法：用柱层析法从螺旋藻藻蛋白酶介产物中分离色素肽,并用MTT法及吖啶橙染色法检测色素肽对体外培养的小鼠肉瘤细胞S180及荷瘤小鼠PD。结果：分离出三种色素肽的分子量分别为17.4KD,7.72KD及6.6KD,它们的吸收光谱峰值分别位于618nm,580nm,605nm,荧光发射峰均在685nm处。MTT法检测证明,在浓度100μg/ml,用波长为580nm,600nm,照射剂量为28.8J/cm^2染料激光（氩离子为激光光源）照射,对肿瘤细胞的杀伤率达69．2％－80．2％;在色素肽CCP1,CCP3介导的PDT对小鼠移植瘤生长影响的实验发现,色素肽易被瘤细胞吸收,当激光照射剂量为120J/cm^2,肿瘤直径在0.5-0.7cm范围内,瘤体旁注射剂量为50μg的CPP1及CCP3光敏剂时,藻胆蛋白酶介产的的抑瘤率可达46％－81％,且被PDT作用后的细胞表现了典型的凋亡细胞特征。     

Infra-red laser irradiation enhances interleukin-1 receptor antagonist,increases 3H-thymidine incorporation and the release of [3H]arachidonic acid in human monocytes

The effect of infra-red laser irradiation has been experimented on various biological systems and particularly in human tissues, in vitro as well as in vivo. In order to examine the influence of laser irradiation on cells of the monocytic lineage we have irradiated human peripheral blood mononuclear cells with an infra-red laser at a wavelength of 904 nm, at 2000 Hz frequency and 15 mW for 2 min. Here, we report that laser irradiation for 2 min. at different preincubation times (T = 0 and T = 30 min) enhances LPS (10 g/ml or PHA (10 g/ml, suboptimal concentration) -stimulated monocytes by modifying cell proliferation, as judged by [3H] thymidine incorporation. IL-1 receptor antagonist (IL-1ra) along with an increased release of [3H] Arachidonic acid production, is also influenced by laser irradiated monocytes when treated for 2 min after 1 h incubation. IL-1RA production increased 4-5-fold after laser irradiation, while 3H-arachidonic acid incorporated from PMA-stimulated cellsased and the effect was significant at T = 0 and T = 30 min; while at T = 1 h the effect was negligible. These results may provide new information regarding the effect of laser irradiation on the immune system.     

Discrepancies between patterns of potentially lethal damage repair in the RIF-1 tumor system in vitro and in vivo

Repair of potentially lethal damage (PLD) was studied in the RIF-1 tumor system in several different growth states in vivo and in vitro. Exponentially growing, fed plateau, and unfed plateau cells in cell culture as well as small and large subcutaneous or intramuscular tumors were investigated. Large single doses of radiation followed by variable repair times as well as graded doses of radiation to generate survival curves immediately after irradiation or after full repair were investigated. All repair-promoting conditions studied in vitro (delayed subculture, exposure of cells to depleted growth medium after irradiation) increased surviving fraction after a single dose. The D0 of the cell survival curve was also increased by these procedures. No PLD repair was observed for any tumors irradiated in vivo and maintained in the animal for varying times prior to assay in vitro. The nearly 100% cell yield obtained when this tumor is prepared as a single-cell suspension for colony formation, the representative cell sample obtained, and the constant cell yield per gram as a function of time postirradiation suggest that this discrepancy is not an artifact of the assay system. The most logical explanation of these data and information on radiocurability of this neoplasm is that PLD repair, which is so frequently demonstrated in vitro, may not be a major factor in the radioresponse of this tumor when left in situ.     

The participation of oxygen in the process of the interphase death of T-lymphocytes following irradiation in vivo

The ability of lymphocytes to inhibit proliferation of non-syngeneic stem cells decreases differently after exposure in vivo and in vitro. The causes of the observed differences and the mechanism of radiation impairment of this function under different irradiation conditions have been investigated. Cells exposed in vivo die in the interphase irreversibly. The newly formed lymphocytes start the repair process as late as one month after irradiation. The injury to in vivo exposed cells is severer due to the presence of oxygen in tissues. A definite time interval is needed for the damaging effect of oxygen radicals to be implemented: the effect is maximum as early as 4 h following irradiation. With in vivo exposure under hypoxic conditions the functional activity of lymphocytes is the same as that of lymphocytes irradiated in vitro with the same dose. In vitro irradiation of lymphocytes at a high oxygen content causes a decrease in the functional activity of cells.     

Proliferation and tumorigenity of murine hepatoma cells irradiated with polychromatic visible and infrared light

In experiments in vitro, the effects of polychromatic visible (VIS) light combined with polychromatic infrared light (VIS-IR, 480–3400 nm) and the effects of the entire spectrum of VIS radiation (385–750 nm) on viability and proliferative activity of the murine hepatoma cells MH22a were studied. In experiments in vivo, changes in the tumorigenic properties of cells MH22a were studied after the same kinds of light exposure. It was shown that irradiation of hepatoma cells with two kinds of polychromatic light at a wide range of doses (4.8–38.4 J/cm²) did not lead to an increase in the number of dead cells for 24–72 h of cultivation and did not cause deceleration of the hepatoma cell proliferation; moreover, the VIS-IR light at a dose of 4.8 J/cm² and the VIS light at a dose 38.4 J/cm² even promoted more intense cell proliferation after 24 h. In cells irradiated with VIS-IR and VIS light, the proliferation index rose by 1.6 and 1.4 times, respectively, and the time of the cells’ number doubling decreased as compared with control. Studying the tumorigenic properties of irradiated tumor cells has shown that, for 30 days after transplantation to syngenic mice C3HA of hepatoma cells 24 h after their irradiation with VIS-IR light at a dose of 4.8 J/cm², the tumor volume decreased significantly (2.6–4.1 times) at all periods of observation, while the incidence of tumor formation decreased, whereas the survival of the tumor-bearing mice did not change. Transplantation of cells irradiated with the same light at a dose of 9.6 J/cm² did not lead to significant changes in the tumor volume, the tumor formation incidence, and animal survival. The main contribution to the antitumor effect of VIS-IR light seems to be made by the VIS component, as transplantation into mice of cells irradiated with VIS light alone at a dose of 38.4 J/cm² also stimulating proliferation of hepatoma cells in vitro resulted in a decrease of their tumorigenic properties. However, the IR component in the combined VIS-IR radiation enhanced the antitumor effect of the VIS light; as a result, it was manifested after use of doses eight times lower (4.8 J/cm²) than in the case of VIS light alone (38.4 J/cm²). Mechanisms of the decrease of tumorigenic properties of hepatoma cells after irradiation with polychromatic light at doses stimulating their proliferation in vitro are studied.     

Identification of genes responsive to gamma radiation in rat hepatocytes and rat liver by cDNA array gene expression analysis

The mechanisms underlying hepatocellular damage after irradiation are obscure. We identified genes induced by radiation in isolated rat hepatocytes in vitro by cDNA array gene expression analysis and then screened in vivo experiments with those same genes using real-time PCR and Western blotting. Hepatocytes were irradiated and cDNA array analyses were performed 6 h after irradiation. The mRNA of differentially expressed genes was quantitatively analyzed by real-time PCR. cDNA array analyses showed an up-regulation of 10 genes in hepatocytes 6 h after irradiation; this was confirmed by real-time PCR. In vivo, rat livers were irradiated selectively. Treated and sham-irradiated controls were killed humanely 1, 3, 6, 12, 24 and 48 h after irradiation. Liver RNA was analyzed by real-time PCR; expression of in vivo altered genes was also analyzed at the protein level by Western blotting. Up-regulation was confirmed for three of the in vitro altered genes (multidrug resistance protein, proteasome component C3, eukaryotic translation initiation factor 2). Histologically, livers from irradiated animals were characterized by steatosis of hepatocytes. Thus we identified genes that may be involved in liver steatosis after irradiation. The methods shown in this work should help to further clarify the consequences of radiation exposure in the liver.     

Enhancement of monoclonal antibody production in CHO cells by exposure to He–Ne laser radiation

This study tested the effectiveness of laser biostimulation in small-scale cultures in vitro. We investigated the response of recombinant CHO cells, which are used for the production of monoclonal antibody, to low level laser radiation. The cells were irradiated using a 632.8 nm He–Ne laser in a continuous wave mode at different energy doses. We incubated the irradiated cells in small batch cultures and assessed their proliferation and productivity at various time intervals. Compared to untreated cells, the irradiated cells showed a significant increase in antibody production. Moreover, the results showed that laser irradiation did not affect viability and slightly enhanced proliferation rate.     

DNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light

DNA synthesis was followed in vivo and in permeable Escherichia coli after ultraviolet light irradiation, irradiation and incubation in a growth medium containing chloramphenicol and in unirradiated cells. In vitro, replicative type DNA synthesis was partially restored after incubation of cells in medium containing chloramphenicol, but not in vivo. The DNA was pulse-labeled in permeable cells in the presence of deoxyribonucleoside triphosphates and ribonucleoside triphosphates. dCTP was replaced by 5-Hg-dCTP as a substrate for DNA synthesis. Hg-DNA was separated from cellular nucleic acids on thiol-agarose affinity columns. The 5' termini of newly synthesized DNA were analyzed after treatment with alkaline phosphatase and rephosphorylation with polynucleotide kinase and [gamma-32P]ATP. DNA synthesis in unirradiated permeable E. coli represents a replicative process dependent on ATP and inhibited by novobiocin. About 70% of the nascent DNA carried terminally labeled RNA moiety at its 5' end. In vitro DNA synthesis in irradiated cells was suppressed and hardly influenced by the presence of ATP or novobiocin. The 5'-RNA content of this cell population was less than 5%.     

Rapid tumor formation of human T-cell leukemia virus type 1-infected cell lines in novel NOD-SCID/gammac(null) mice: suppression by an inhibitor against NF-kappaB

We established a novel experimental model for human T-cell leukemia virus type 1 (HTLV-1)-induced tumor using NOD-SCID/gammac(null) (NOG) mice. This model is very useful for investigating the mechanism of tumorigenesis and malignant cell growth of adult T-cell leukemia (ATL)/lymphoma, which still remains unclear. Nine HTLV-1-infected cell lines were inoculated subcutaneously in the postauricular region of NOG mice. As early as 2 to 3 weeks after inoculation, seven cell lines produced a visible tumor while two transformed cell lines failed to do so. Five of seven lines produced a progressively growing large tumor with leukemic infiltration of the cells in various organs that eventually killed the animals. Leukemic cell lines formed soft tumors, whereas some transformed cell lines developed into hemorrhagic hard tumors in NOG mice. One of the leukemic cell lines, ED-40515(-), was unable to produce visible tumors in NOD-SCID mice with a common gamma-chain after 2 weeks. In vivo NF-kappaB DNA binding activity of the ED-40515(-) cell line was higher and the NF-kappaB components were changed compared to cells in vitro. Bay 11-7082, a specific and effective NF-kappaB inhibitor, prevented tumor growth at the sites of the primary region and leukemic infiltration in various organs of NOG mice. This in vivo model of ATL could provide a novel system for use in clarifying the mechanism of growth of HTLV-1-infected cells as well as for the development of new drugs against ATL.