Differentiation therapy is potentiated by chemotherapy and hyperthermia in human and canine brain tumor cells in vitro.

Human glioblastoma (U-87MG) and canine glioma (canine brain tumor [CBT]) cell lines were tested in vitro for their therapeutic sensitivity to sequential treatment with differentiating agents and chemotherapy or hyperthermia. Both cell lines responded to the inducer combination dibutyryl adenosine-3',5'-cyclic monophosphate/sodium butyrate by the formation of cytoplasmic processes detectable within 7 hours and attained approximately 90% morphological differentiation within 2 days of exposure. The clonogenicity of CBT and U-87MG cells gradually decreased after 1 to 7 days of exposure to the inducer combination, but this treatment alone failed to kill the cells. After the removal of the inducers, both lines dedifferentiated and the rate of clonogenesis increased. 1,3-bis-(2-Chloroethyl)-1-nitrosourea administered to CBT and U-87MG cells before or after 3 days of treatment with inducers potentiated the antiproliferative effects of the differentiating agents. Cisplatin administered to U-87MG cells enhanced the antiproliferative effect of the differentiating agents to a greater extent when added before the inducers rather than after differentiation was stimulated. The sequential treatment of CBT cells with a 44 degrees C heat pulse for 30 minutes followed by differentiating agents produced an additive potentiation of cell killing, whereas the reverse sequence did not. Hyperthermia pretreatment at 44 degrees C for 15 minutes or at 42 degrees C for 30 minutes failed to enhance the antiproliferative effects of inducing agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Allografts of CNS tissue possess a blood-brain barrier: III. Neuropathological, methodological, and immunological considerations.

Development of a blood-brain barrier (BBB) within mammalian CNS grafts, placed either intracerebrally or peripherally, has been controversial. Published data from this laboratory have emphasized the presence or the absence of a BBB within solid mammalian tissue or cell suspension grafts is determined intrinsically by the graft and not by the surrounding host parenchyma (e.g., brain, kidney, testis, etc.). Nevertheless, correctly interpreting whether or not a BBB exists within brain grafts is manifested by methodologies employed to answer the question and by ensuing neuropathological and immunological consequences of intracerebral grafting. The present study addresses these issues and suggests misinterpretation for the absence of a BBB in brain grafts can be attributed to: (1) rupture of interendothelial tight junctional complexes in vessels of CNS grafts fixed by perfusion of the host; (2) damage to host vessels and BBB during the intracerebral grafting procedure; (3) graft placement in proximity to inherently permeable vessels (e.g., CNS sites lying outside the BBB) supplying the subarachnoid space/pial surface and circumventricular organs such as the median eminence, area postrema, and choroid plexus; and (4) graft rejection associated with antigen presenting cells and the host immune response. The latter is prevalent in xenogeneic grafts and exists in allogeneic grafts with donor-host mismatch in the major and/or minor histocompatibility complex. CNS grafts between non-immunosuppressed outbred donor and host rats of the same strain (e.g., Sprague Dawley or Wistar rats) can be rejected by the host; these grafts exhibit populations of immunohistochemically identifiable major histocompatibility complex class I+ and class EE+ cells (microglia, macrophages, etc.) and CD4+ T-helper and CD8+ T-cytotoxic lymphocytes. PC12 cell suspension grafts placed within the CNS of non-immunosuppressed Sprague Dawley rats are rejected similarly. Donor cells from solid CNS grafts placed intracerebrally and stained immunohistochemically for donor major histocompatibility complex (MHC) class I expression are identified within the host spleen and lymph nodes; these donor MHC expressing cells may initiate the host immune response subsequent to the cells entering the general circulation through host cerebral vessels damaged during graft placement. Rapid healing of damaged cerebral vessels is stimulated with exogenously applied basic fibroblast growth factor, which may prove helpful in reducing the potential entry of donor cells to the host circulation. These results have implication clinically for the intracerebral grafting of human fetal CNS cell suspensions.

In vitro response of human glioblastoma and canine glioma cells to hyperthermia, radiation, and chemotherapy.

Little is known about the sensitivity of human glioblastoma cells to hyperthermia alone and in combination with other therapies. We carried out in vitro cell survival studies on the human glioblastoma cell line U-87MG and our model canine glioma canine brain tumor (CBT) cells after multimodality treatment. Ionizing radiation was administered to flasks of cells in logarithmic growth at 500 rads (5 Gy) with consecutive treatment by hyperthermia, 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), or cisplatin. Cells were treated with single doses of BCNU at 5 microM with sequentially added radiation or hyperthermia and at 1 to 2 micrograms/ml of cisplatin with hyperthermia. Hyperthermia was administered in a precision controlled water bath at 44 degrees C for 30 minutes in combination with chemotherapy or radiation. In general, the sensitivity of U-87MG and CBT cells was similar for all test regimens. For example, colony formation efficiency decreased by 64% in CBT cells and by 64.4% in U-87MG cells after hyperthermia alone at 44 degrees C for 60 minutes. All combinations of BCNU, hyperthermia, and radiation administered in vitro produced enhanced cell killing, but the effects of multiple modalities were generally additive in both cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Allografts of CNS tissue possess a blood-brain barrier. II. Angiogenesis in solid tissue and cell suspension grafts.

Angiogenesis and patency of blood vessels were analyzed qualitatively in solid CNS and peripheral tissue syngeneic, allogeneic, and xenogeneic grafts and in individual cell suspension grafts of astrocytes, fibroblasts, PC12, and three additional tumor cell lines placed intracerebrally in adult host mice. Postgrafting survival times were 1 day through 4 weeks. The patency of graft vessels was determined in sections from immersion-fixed tissues incubated to reveal the endogenous peroxidase activity of host red cells trapped within the lumen of blood vessels. Additionally, horseradish peroxidase (HRP) was administered intravenously to live hosts; HRP labels host brain and graft vessels on the luminal surface and reveals the presence or absence of a blood-brain barrier (BBB) within the grafts. The origins of blood vessels supplying solid tissue xenografts were identified immunohistochemically with primary antibodies against host (athymic AKR mice) and donor (fetal Lewis rats) major histocompatibility complex (MHC) class I. Blood vessels supplying solid CNS grafts at 1-7 days post-transplantation were identified ultrastructurally and possessed interendothelial tight junctional complexes; however, they were not perfused with either host blood or blood-borne HRP prior to 8 days. Graft vessels at 10 days were outlined consistently by peroxidase-positive red cells in immersion-fixed material and labeled with blood-borne HRP. These vessels provided a BBB to the circulating HRP and exhibited interendothelial tight junctions. Evidence of angiogenesis within solid anterior pituitary grafts and the variety of cell suspension grafts was obtained prior to 3 days post-transplantation in immersion-fixed preparations; the vessels, with the notable exception of those supplying astrocyte cell suspensions, failed to present a BBB to blood-borne peroxidase. Endothelia in the solid pituitary allografts and the PC12 cell grafts were highly fenestrated and exhibited open interendothelial junctions; those in the tumor and fibroblast cell grafts, for the most part, appeared nonfenestrated, and many possessed open interendothelial junctional complexes. Immunostaining for host and donor MHC class I revealed that donor blood vessels predominate over host vessels in CNS xenografts and supply pituitary xenografts exclusively; in both preparations, donor vessels were not identified within the host CNS. Because cell suspension grafts were derived from endothelia-free preparations grown in culture, blood vessels supplying these grafts were necessarily of host CNS origin and manifested a morphological transformation from a BBB to a non-BBB endothelium. The data suggest that angiogenesis in solid CNS grafts placed into the adult host CNS, compared to similarly placed solid peripheral tissue/cell suspension grafts, is not rapid.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of succinylacetone on growth and respiration of L1210 leukemia cells.

4,6-Dioxoheptanoic acid (succinylacetone, SA), a potent inhibitor of heme biosynthesis, suppressed growth and decreased respiration of L1210 leukemia cells in vitro. Growth of cells incubated in the presence of 2--4 mM SA for the first 2 days declined, and after 3 days virtually ceased. L1210 cells in the logarithmic growth phase exhibited active respiration (40 +/- 9.3 nanoatoms oxygen/min X 10(7) cells at 37 degrees C) which was inhibited by and released by uncouplers of oxidative phosphorylation. These and other inhibitors of mitochondrial function clearly demonstrate a mitochondrial basis for the cellular respiration in both control and SA-treated cells. L1210 cells in the stationary phase exhibited a marked decrease in oxygen consumption compared to cells in logarithmic growth. At the concentrations used in this study, SA was not immediately toxic to L1210 cells, but inhibited growth at 2 days without lowering levels of cellular heme. Thus, it appears unlikely that inhibition of growth of L1210 cells by SA can be ascribed either to heme depletion or to impairment of respiration.

Augmentation of hematoporphyrin uptake and in vitro-growth inhibition of L1210 leukemia cells by succinylacetone.

Succinylacetone (SA; 4,6-dioxoheptanoic acid), a specific inhibitor of delta-aminolevulinic acid dehydrase (ALAD) (the second enzyme of the heme biosynthetic pathway), was tested for its effect in L1210 cells from inbred DBA/2 mice. ALAD from broken L1210 cells was completely inhibited by 1 microM SA, but in whole cells activity was decreased only 83% after incubation of the cells with 2.5 mM SA for 3 days. When incubated with hematoporphyrin (HP), L1210 cells rapidly took up porphyrin from the medium, and this uptake could be augmented by pretreatment of the cells with SA; but this enhancement of porphyrin uptake occurred gradually over a period of days. When SA-treated and untreated L1210 cells were incubated with increasing concentrations of HP in the medium, SA-treated cells reached the saturation concentration of cellular porphyrin at lower medium HP concentrations than did untreated cells. Growth of L1210 cells could be inhibited by 2 mM SA or more. Addition of increasing amounts of serum to cultures of cells containing SA did not reverse the growth inhibition due to SA. Porphyrin uptake from HP in the medium in nonmalignant fibroblast line 3T3 was much lower than in L1210 cells and could not be enhanced by incubation of the cells with SA.

Comparative activation response of splenocytes oxidized by periodic acid and selenium dioxide.

Murine splenocytes were activated by selenium dioxide (SeO2) oxidation of cell membranes, as evidenced by increased tritiated thymidine (3H-TdR) incorporation. In contrast to splenocyte activation by periodic acid (H5IO6), the SeO2-induced response was not inhibited by neuraminidase degradation of cell membranes prior to oxidation, nor by the prior hydroxylamine (NH2OH) addition reaction. However, reduction by borohydride (NaBH4), as a preliminary step to the oxidation by SeO2 and H5IO6, inhibited the subsequent cell activation. Sequential oxidation by H5IO6 and SeO2 increased the cell stimulation index versus the response elicited after one step oxidation by SeO2 or H5IO6. The reverse order of the sequential oxidation depressed the stimulation index relative to oxidation solely by H5IO6, but not by SeO2. It is concluded that the activation of splenocytes by SeO2 is triggered primarily by the conversion of cell membrane carbonyls into corresponding dicarbonyls.

Growth inhibitory activity of succinylacetone: studies with Walker 256 carcinosarcoma, Novikoff hepatoma and L1210 leukemia.

Succinylacetone (SA, 4,6-dioxoheptanoic acid) inhibits d-aminolevulinic acid dehydrase, the second enzyme of the heme biosynthetic pathway and thereby inhibits heme biosynthesis. In the present study SA is shown to inhibit the growth of the Walker carcinosarcoma (W256) in vitro and in vivo, the Novikoff hepatoma in vivo, and L1210 leukemia in vitro, but only slightly in vivo. Rats can tolerate significantly larger doses of SA for at least twice as long as were administered in the present study without gross evidence of toxicity. In contrast to findings in previously published studies with murine erythroleukemia cells, the inhibition of growth of L1210 and W256 cells by SA in vitro is not accompanied by a decrease in cellular heme and is not reversed by addition of hematin to the medium. This suggests a second mechanism of growth inhibition of tumor cells unrelated to heme biosynthesis. Although the growth of both W256 and L1210 cells was markedly inhibited by the same concentration of SA in culture, there was a great difference in responsiveness in vivo, in that much greater inhibition of the growth of the Walker tumor was produced by SA.

Characteristics of hematin uptake in malignant, embryonic and normal cells.

The characteristics of hematin uptake were examined in three malignant cell lines [L1210 leukemia, 745 murine erythroleukemia (MEL) and Walker carcinoma (W256)], a cell line derived from normal rat liver (BRL-3A) and a normal embryonic cell, chick embryo fibroblasts (CEF). Uptake in the normal liver cell line was slight and occurred at a slow rate in contrast to the rapid uptake, which was more rapid and of greater magnitude in the three tumor cell lines, Saturation of the heme uptake mechanism was observed in MEL cells at an extra-cellular hematin concentration of 160 micro M and in L1210 cells at 300 micro M. At saturation L1210 cells achieved a cellular heme concentration nine times as high as MEL cells. Hematin uptake in MEL cells was markedly augmented by pretreatment with DMSO, procaine, detergent or proteolytic enzymes or by increases in the pH of the medium from 8 to 9.5. In contrast to MEL cells where SA inhibits growth by lowering cellular heme, the inhibition of growth of L1210 cells by SA appears to operate by a mechanism independent of heme. In gradual increase in hematin uptake capacity in MEL cells over a period of days. Afer exposure of MEL cells to a high concentration of hematin in the medium, the egress of heme was followed under various conditions. Of the various agents studied, only cyanide produced a loss of heme from MEL cells.

Stimulation of hemoglobin synthesis in murine erythro-leukemia cells by low molecular weight ketones, aldehydes, acids, alcohols and ethers.

The effects of short chain (C1-C5) aldehydes, ketones, acids alcohols and ethers on murine erythroleukemia (MEL) cells were examined to determine which particular chemical moieties and some of their combinations stimulated hemoglobin synthesis in these cells. The C4 series of compounds was active at lower concentrations than homologs of shorter chain lengths. Within an homologous series the potency and efficacy of the alcohol was always less then that of the acid and aldehyde compounds. Though hepanoic acid was found to be an inducer of hemoglobin synthesis in MEL cells, the 4,6-dioxoheptanoic acid analog is a potent inhibitor of hemoglobin synthesis. Analysis of porphyrin content of MEL cells incubated with the inducers 2-butanone, 2-methoxyethanol, acetone and methanol, showed that increased hemoglobin synthesis was always accompanied by the accumulation of porphyrins, most of which was protoporphyrin. These studies suggest that low molecular ketones, aldehydes, acids, ethers and alcohol can correct the defect in erythroid differentiation exhibited by MEL cells and they further suggest that the physiological trigger for inducing hemoglobin synthesis in these cells is less discriminating than previously recognized.

Uptake of hematin and growth of malignant murine erythroleukemia cells depleted of endogenous heme by succinylacetone.

Heme levels and growth of malignant murine erythroleukemia cells in heme-free medium are drastically reduced by incubation of these cells in the presence of 4,6-dioxoheptanoic acid [succinylacetone (SA)]. When hematin was added to the culture medium of heme-depleted cells, the intracellular heme levels returned to normal, and growth inhibition produced by SA was also reversed. However, when cells depleted of heme by growth in heme-free medium containing SA were placed in heme-free medium without SA, heme levels were restored to normal, and growth was resumed. Hematin uptake in both untreated and heme-depleted malignant murine erythroleukemia cells exhibited biphasic kinetics, with a rapid phase of about 2 min followed by a slower uptake. The rate of uptake of exogenous hematin was slightly greater at 37 degrees than at 20 degrees. Although supplementation of heme-free medium with exogenous hematin increased total cellular heme in both untreated and heme-depleted malignant murine erythroleukemia cells, the fraction of heme in the 20,000 X g sediment was unaffected. A nonmalignant fibroblastic cell line, 3T3, exhibited little or no capacity to take up exogenous hematin.

Evidence for multiple sites of regulation of heme synthesis in murine erythroleukemia cells.

Friend murine erythroleukemia (MEL) cells can be induced to differentiate along the erythroid pathway by such dissimilar agents as dimethyl sulfoxide (DMSO), butyrate, inhibitors of DNA synthesis, and certain highly polar agents. This study showed differential biochemical effects of the potent inducers DMSO and butyrate on the heme biosynthetic pathway in three clones of Friend MEL cells. When the cells were incubated with combinations of DMSO and butyrate, hemoglobin production was less than that amount produced when each inducer was incubated singly with the cells. Procaine, a local anesthetic that competes with Ca2+ and thus affects membrane permeability, slightly inhibited DMSO-mediated hemoglobin production but almost tripled the level stimulated by butyrate alone. Similarly, EDTA, which also can bind Ca2+ and which can modify the activity of heme biosynthetic enzymes, also inhibited hemoglobin production mediated by DMSO, whereas it stimulated hemoglobin production in cells exposed to butyrate. Total porphyrin accumulation was greater in DMSO-treated cells than in butyrate-treated cells, which suggests that butyrate induces the enzymes of the heme pathway more efficiently than does DMSO. DMSO and butyrate may affect the heme biosynthetic pathway by multiple mechanisms or, alternatively, they may affect the multistep pathway at various points, producing partial blocks or incomplete enzyme induction.

Viral reverse transcriptase suppression associated with erythroid differentiation of Friend leukemia cells.

The Friend erythroleukemia cell line T3-C12, which produces Friend murine leukemia virus (F-MuLV) and can be induced to synthesize hemoglobin by dimethyl sulfoxide (DMSO), was monitored for viral RNA-dependent DNA polymerase reverse transcriptase (RT) activity. The amount of viral 60-70S RNA released from DMSO-treated cells was unaffected or increased compared to that from control cells, while RT activity from treated cells was decreased. Accordingly, the specific activity in F-MuLV from DMSO-treated cells expressed as RT/70S RNA was decreased to 8% of the control activity. The 5-bromo-2'-deoxyuridine added to cultures containing DMSO reversed the differentiation process, and the F-MuLV thus treated did not exhibit the reduced RT activity normally observed in DMSO-treated virus. Cell-free F-MuLV incubated with and without DMSO showed the same RT activity, indicating that DMSO itself did not inhibit RT activity. However, when F-MuLV-containing pellets from control and DMSO-treated culture fluids were mixed, there was marked inhibition of the control RT activity, suggesting that RNase hybrid activity was stimulated or that an inhibitor was produced. Assays of F-MuLV-RNase hybrid released from control and DMSO-treated cells showed no difference in activity, indicating that a specific inhibitor of RT was produced or activated. Additions of certain nucleotide triphosphates to RT incubation mixtures did not result in any stimulation of RT activity in DMSO-treated F-MuLV, suggesting that phosphatase was not responsible for the observed inhibition. The results suggested that DMSO treatment of T3-C12 cells caused a reduction in viral RT activity by stimulating the production of an inhibitor, the nature of which is unknown.

Inheritance in protoporphyria. Comparison of haem synthetase activity in skin fibroblasts with clinical features.

The activity of haem synthetase, the enzyme which chelates iron to protoporphyrin to form haem, was measured in cultured skin fibroblasts of children with protoporphyria and their parents from three families. In each family, one parent had deficient haem synthetase activity (3-0-11-1 pmol protohaem formed/mg protein/h) when compared to values in eight non-porphyric controls (means 24-9, range 13-7-51-5). The level of activity in the three parents was similar to that in their affected children. In two families the parent with deficient activity was also thought to be the carrier of the abnormal gene, as judged from a history of photosensitivity and analysis of erythrocyte protoporphyrin concentrations, but in the third family the pattern of inheritance could not be determined from these criteria. The activity of delta-aminolaevulinic acid synthetase was normal in cultured fibroblasts from the protoporphyric children and their parents, excluding a generalised defect in haem-pathway enzymes. These results support the premise that deficient haem synthetase activity, inherited in an autosomal dominant patter, is the primary defect in protoporphyria.

Erythroid differentiation in cultured Friend leukemia cells treated with metabolic inhibitors.

The induction of erythroid differentiation in the T3-C12 clone of Friend leukemia cells by dimethyl sulfoxide is accompanied by reduction in viral RNA-dependent DNA polymerase activity with increased cellular delta-aminolevulinic acid synthetase activity and hemoglobin synthesis. These cells were treated with a variety of compounds to determine whether other durgs are capable on inducing erythroid differentiation. While several hormones, inhibitors of RNA synthesis, organic solvents, inhibitors of DNA polymerase, sulfhydryl inhibitors, and inducers of delta-aminolevulinic acid synthetase administered singly did not stimulate hemoglobin synthesis like dimethyl sulfoxide, inhibitors of DNA and RNA synthesis such as adriamycin, mitomycin C, and hydroxyurea:mithramycin were synergistic in stimulating erythroid differentiation.

Heme synthetase deficiency in human protoporphyria. Demonstration of the defect in liver and cultured skin fibroblasts.

The final step in heme biosynthesis is chelation of porphyrin with Fe++ catalyzed by the mitochondrial enzyme heme synthetase. We have employed a sensitive radiochemical assay for this enzyme, using 59Fe and deuteroporphyrin or protoporphyrin as substrates. In this method iron is maintained in the ferrous state, oxygen is excluded from the incubation system, and labeled heme product is extracted into ethyl acetate. This assay has been used to measure the activity of heme synthetase in homogenates of liver, obtained by needle biopsy, and in sonicates of human skin fibroblasts, cultured in vitro. In addition, activity of the first enzyme of the heme synthetic pathway, delta-aminolevulinic acid synthetase, has been measured in fibroblast lysates. Lysates of fibroblasts from eight patients with protoporphyria had activities of delta-aminolevulinic acid synthetase which did not differ significantly from those of eight normal fibroblast lines, whereas activity of heme synthetase, with either deuteroporphyrin or protoporphyrin as substrate, was markedly decreased in sonicates of skin fibroblasts from these patients, the mean being 8% of control with deuteroporphyrin and 14% with protoporphyrin as substrate. In homogenates of liver from five patients with protoporphyria, activity of heme synthetase was also significantly less than that found in six patients without prophyria, the mean being 13% of control with protoporphyrin as substrate. These results provide evidence that decreased activity of heme synthetase is the basic defect in the heme synthetic pathway in protoporphyria. This deficiency is probably responsible for protoporphyrin accumulation and hence the biochemical and clinical features observed in protoporphyria.