Control by hyperthermia of ornithine decarboxylase in Ehrlich ascites tumor cells.

The effect of hyperthermia on the activity and the messenger RNA levels of ornithine decarboxylase (ODC), which has a rapid rate of turnover in cultured cells, was studied in Ehrlich ascites tumor cells. When the cells were incubated at 42 degrees C, elevation of ODC activity by a change of the medium was prevented. Total RNA was isolated from cells treated at 37 degrees C or 42 degrees C, and the relative abundance of the ODC mRNA was measured by Northern blot analysis. These levels in heat-treated cells were comparable to those in control cells. Inhibition by hyperthermia was reversible. The recovery was suppressed by cycloheximide but not by actinomycin D. In hyperthermic-treated cells, the biological half-life of ODC was 14 min, which was the same time as for cells cultured at 37 degrees C. These results suggest that hyperthermic treatment of Ehrlich ascites tumor cells suppressed ODC induction during translation, not during transcription or after translation.

Altered production of the active oxygen species is involved in enhanced cytotoxic action of acylated derivatives of ascorbate to tumor cells.

Our previous study shows that 6-O-acyl derivatives of L-ascorbic acid inhibits more markedly cell growth of mouse Ehrlich carcinoma than ascorbic acid. The present study shows that 6-O-palmitoyl ascorbic acid but not ascorbic acid prolongs the lifespan of mice into which tumors such as Meth A fibrosarcoma, MM46 mammary carcinoma, Ehrlich carcinoma and sarcoma 180 are implanted. The potentiated cytotoxicity of 6-O-palmitoyl ascorbic acid is not due to an increase in duration time of the cytotoxic action, because 6-O-palmitoyl ascorbic acid is gradually inactivated during contact with tumor cells and exhibits a similar action time curve to that of ascorbic acid as shown by clonal growth assay. Cytotoxicity of 6-O-palmitoyl ascorbic acid is markedly diminished by combined addition of catalase and superoxide dismutase (SOD), as shown by dye exclusion assay, whereas the cytotoxicity was slightly reduced by either enzyme alone but not by the specifically inactivated or heat-denatured enzymes. In contrast, cytotoxicity of ascorbic acid is abolished by catalyse but not SOD. Autooxidation of 6-O-palmitoyl ascorbic acid was not inhibited by catalase plus SOD. The results indicate that cytotoxicity of 6-O-palmitoyl ascorbic acid is attributed at least partly to both hydrogen peroxide (H2O2) and superoxide (O2-.) generated at the early stage. Cytotoxicity of 6-O-palmitoyl ascorbic acid is also appreciably attenuated by singlet oxygen (1O2) scavengers such as hydroquinone, 1,4-diazobicyclo-2,2,2-octane or sodium azide, but not by hydroxyl radical scavengers including butylated hydroxytoluene, D-mannitol, benzoic acid and ethanol. Thus, in contrast to cytotoxicity of ascorbic acid mediated entirely by H2O2 initially generated, acylated ascorbic acid produces a diversity of active oxygen species including H2O2, O2-. and other species secondarily generated via disproportion, which may be additively involved in the enhanced cytotoxic action.

Quantification of bone mineral content using dual-photon absorptiometry in a normal Japanese population.

The bone mineral content of lumbar spine and/or total body were quantified in 217 healthy Japanese (86 males and 131 females) using a Dichromatic Bone Densitometer (Norland Corp., Model 2600). The bone mineral density of the third lumbar vertebra (L3 BMD) decreased significantly after age 20 in males (r = -0.417, p less than 0.0002), with acceleration of the decrease after age 50 (r = -0.621, p less than 0.00002). A significant correlation was found between L3 BMD and age after age 40 (r = -0.747, p less than 0.0001) in females. L3 BMD correlated with both the body height (r = 0.335, p less than 0.0001) and the body weight (r = 0.340, p less than 0.0001). Total bone mineral content from the second to fourth lumbar vertebrae correlated significantly with total body bone mineral (r = 0.880, p less than 0.00001) in these normal subjects. Lumbar spine bone mineral as measured by dual-photon absorptiometry is lower in Japan than the bone mass in the United States, although not lower than in other parts of the world.

KIH-802: 2-nitroimidazole-1-acetohydroxamate as a hypoxic cell radiosensitizer.

We have identified potassium 2-nitroimidazole-1-acetohydroxamate (KIH-802) as a hypoxic cell radiosensitizer potentially superior to Miso. The water-soluble acetohydroxamates of 2-nitroimidazole (KIH-802; free acid 801) and 4-nitroimidazole (KIH-852) were designed, synthesized, and evaluated by in vitro and in vivo screening against EMT6 cells. Enhancement ratios of KIH-802 and 801 were 1.92 and 1.68, respectively, compared with 1.58 for MISO all at 1 mM. These acetohydroxamates are also expected to be more effective in vitro than SR-2508 based on our previous experiments. In vivo ERs of KIH-802, 801, and 852 were 1.75, 1.50, and 1.35, respectively, compared with 1.57 for MISO all at the same dose of 200 mg/kg. The data clearly show that the addition of an acetohydroxamic acid moiety to the 2-nitroimidazole skeleton can enhance radiosensitizing ability.

Radiosensitizing, toxicological, and pharmacokinetic properties of hydroxamate analogues of nitroimidazoles as bifunctional radiosensitizers/chemical modifiers.

We examined the pharmacokinetics of KIH-802, potassium 2-nitroimidazole-1-acetohydroxamate, using its radioisotope-labelled compound and the acute toxicity in mice. We discovered that the concentration of KIH-802 was very low in the brain and its LD50 was nearly half the value of that of MISO. We also present here new 2-nitroimidazole radiosensitizers/chemical modifiers (KIN-804, 811, 831, 841, 844, 821, 823 and 824) designed to enhance their sensitizing ability intensely by substituting various biologically active groups, such as hydroxamic acids and oximes, with moderate lipophilicity to the aromatic ring, if necessary, through some spacers. The sensitizing effects of all compounds were estimated to be almost equal to or better than that of MISO. The results of their toxicities shows that new hydroxamates KIN-804 and 831 are less toxic than KIH-802 and MISO. Their in vitro enhancement ratios are 2.00 and 1.75, respectively, compared with those of KIH-802, MISO and SR-2508, 1.77, 1.72 and 1.72, respectively, at each dose of 1 mM for EMT6/KU single cell. We concluded that they may be superior radiosensitizers of hydroxamic acid analogues to KIH-802.

In vivo radiosensitizing effect of nitroimidazole derivative KIN-804.

PURPOSE: In vivo characteristics of 2-nitroimidazole-1-methylacetohydroxamate (KIN-804), which is a newly developed hypoxic cell radiosensitizer, are presented. METHODS AND MATERIALS: The toxicity, pharmacokinetics, and radiosensitizing effect of KIN-804 were studied by in vivo experiments using C3H/He mice bearing the SCC-VII tumor. Results were compared with misonidazole (MISO). RESULTS: LD50(7) of KIN-804 and MISO were 3200 mg/kg and 2000 mg/kg, respectively. The peak of concentrations of KIN-804 in the tumor occurred 20 min after intraperitoneal injection and reached about 62% of the maximum concentration in the blood. The concentrations in brain and sciatic nerve were very low and clearance from sciatic nerve was rapid. Enhancement ratios of KIN-804 calculated using the growth delay method were 1.22, 1.50 and 1.71 at doses of 50, 100, and 200 mg/kg, respectively, compared with 1.36 for MISO at a dose of 100 mg/kg. In the TCD50 assay, enhancement ratios at a dose of 200 mg/kg were 1.69 for KIN-804 and 1.52 for MISO, respectively. CONCLUSION: KIN-804 is a promising radiosensitizer since it shows less toxicity and higher radiosensitizing activity than MISO.

Tumor imaging after administration of 99mTc-labeled bleomycin.

Bleomycin, an anticancer drug, was labeled with 99mTc using stannous chloride and ascorbic acid and specific activities of 1-3 mCi/mg-eq with labeling efficiencies of 50-75% were achieved. Very rapid excretion of 99mTc-bleomycin through the kidney and concomitant rapid decrease of radioactivity in blood, various tissues and organs, and whole body were observed after intravenous administration of the radiopharmaceutical into tumor-bearing mice. In such animals, approximately 1% of the label was found in a transplanted fibrosarcoma within 30 min while 0.58% was recovered in such lesions even after 24 hr. In patients positive tumor images were obtained by scintigraphy as early as 1 hr after intravenous administration of 3-5 mCi of 99mTc-bleomycin. A total of 142 cases were examined by scintigraphy after administration of 99mIc-bleomycin and/or 67Ga-citrate. In 93 cases with various malignant tumors, tumor was detected in 80% using 99mTc-bleomycin and in 63% using 67Ga-citrate. Technetium-99m-bleomycin scintigraphy successfully detected tumors of the thyroid, lung, face, breast, extremity, and digestive tract and was also useful in finding metastatic lesions and brain tumors. However, 67Ga scintigraphy gave superior results in detecting lesions in patients with malignant lymphomas. In patients with inflammatory diseases, accumulation in lesions was detected in 13% using 99mTc-bleomycin and in 48% using 67Ga-citrate. The further use of 99mTc-bleomycin scintigraphy for tumor detection in patients appears to be warranted.

Effect of hyperthermia on tumor uptake of radiolabeled anti-neural cell adhesion molecule antibody in small-cell lung cancer xenografts.

UNLABELLED: This study was performed to examine the effect of hyperthermia on the intratumor accumulation of a monoclonal antibody (Mab) in an animal model. Mab NE150 (IgG1) recognizes the neural cell adhesion molecule (NCAM) expressed by human small-cell lung cancer (SCLC) cells. METHODS: Athymic mice inoculated with NCI-H69, an SCLC cell line, received an intravenous injection of 125I- and 111In-NE150 and the serial changes of the biodistribution were determined. Furthermore, athymic mice bearing NCI-H69 were either sham-treated or treated by a single hyperthermia at 42 degrees C or 43 degrees C for 1 hr, with the tumor-bearing leg in a water bath using pentobarbital anesthesia. Immediately after heating, the mice were given an intravenous injection of radiolabeled NE150, and the biodistribution was examined at 24 and 48 hr. RESULTS: NE150 localized well in the transplanted tumor when compared with a control Mab. In mice treated at 43 degrees C, there was a 1.34- to 1.67-fold increase in the tumor uptake of 125I- and 111In-NE150 compared to sham-treated mice at both 24 and 48 hr. In addition, a 1.84- to 2.22-fold increase of the tumor-to-blood ratio was demonstrated, since radiolabeled NE150 cleared faster from the circulation in the mice given hyperthermia. A histological study demonstrated the infiltration of neutrophils in the perivascular spaces, indicating an increase of tumor vascular permeability, which might be one of the main reasons for the enhancement of Mab uptake. CONCLUSION: Hyperthermia seems to be a potential method of achieving an increased tumor accumulation of Mab in the radioimmunotargeting of SCLC.

Promotive action of acylated ascorbate on cellular DNA synthesis and growth at low doses in contrast to inhibitory action at high doses or upon combination with hyperthermia.

Effects of 6-O-palmitoyl ascorbate (ascorbate) developed to increase the antitumour activity of ascorbic acid on DNA synthesis and proliferation of Ehrlich ascites tumour cells were investigated. Treatment of the cells with the acylated ascorbate at 25-50 microM for 1 h resulted in no effect on DNA synthesis, assayed by pulse incorporation of [3H]thymidine after a culture period of 20 h, but led to 49%-87% enhanced DNA synthesis after 4 days, suggesting that long-term culture is required for promotion by ascorbate to occur. At a dose as high as 75 microM acylated ascorbate, however, cellular DNA synthesis was 64% inhibited after 20 h and 99% after 4 days. The results suggest that acylated ascorbate exhibits a dual action on DNA synthesis: promotion at low doses and inhibition at high doses, both of which are potentiated in a time-dependent manner. In contrast to the above-mentioned results at 37 degrees C, acylated ascorbate at 25-75 microM inhibited but did not promote DNA synthesis at 42 degrees C whatever the culture period. Similar results were exhibited when proliferation of cells cultured for a long period was investigated. At 37 degrees C, 50 microM acylated ascorbate increased the number of the cells to 3.6 times the control values after 8 days and to 1.9 times after 11 days; in contrast, a 75-microM dose decreased the cell number considerably. Combination with hyperthermia (42 degrees C) suppressed the increase and cell growth was completely inhibited at 75 microM.

Effect of chymotryptic troponin T subfragments on the calcium ion-sensitivity of ATPase and superprecipitation of actomyosin.

One of the chymotryptic troponin T subfragments, troponin T2, reconstituted approximately 80% of the Ca2+-sensitizing action of troponin T on actomyosin in the presence of troponin C, troponin I, and tropomyosin. This indicates that the Ca2+-regulatory property of troponin T is mostly localized in the troponin T2 region of the C-terminal 101 residues.

Electron microscopic study of troponin.

Troponin and its components or fragments were observed in an electron microscope by the use of the rotary shadowing technique. In freshly prepared troponin with low viscosity, globular particles were mainly observed. The size of the long axis of the particles was 13.2 +/- 1.3 nm and the size perpendicular to the long axis was 9.5 +/- 1.2 nm. The mean axial ratio was 1.4 +/- 0.3. Most of the particles observed in a stored troponin preparation, having a higher viscosity than that of fresh troponin, had a globular head with a thin tail, with the total length of 25.4 +/- 1.4 nm (head-tail type particles). The axial size of the globular portion was 8.3 +/- 1.2 nm and the tail length was 17.1 +/- 1.6 nm. Observation of various particles during the transitional stages indicated that, in the globular particles, the tail region of head-tail type particle was associated along the globular head region. Troponin T was a filamentous particle with 16.9 +/- 1.5 nm length. The 26K fragment of troponin T, which was devoid of the N-terminal 45 residues from troponin T, was a filamentous particle with the length of 14.4 +/- 1.3 nm. Troponin T1, one of two chymotryptic subfragments of troponin T, was a filamentous particle of 11.6 +/- 1.4 nm length. Troponin C.T in the presence of Ca2+ was a particle with a globular head (7 nm in size) and a tail of about 17 nm length. The Fab fragment of anti-troponin T1 formed regular transverse striations along the thin filament of rabbit skeletal muscle with a 38 nm period.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary structure of chymotryptic subfragments from rabbit skeletal troponin T.

The N-terminal and C-terminal residues as well as amino acid compositions were determined for the chymotryptic subfragments from rabbit skeletal troponin T. The positions of the subfragments in the sequence of troponin T were N-terminal acetylserine-tyrosine-158 for troponin T1 (m.w. 18,700), leucine-159-C-terminal lysine-259 for troponin T2 alpha (m.w. 11,900), and leucine-159-phenylalanine-242 for troponin T2 beta (m.w. 10,200), respectively.

Clinical evaluation of intermittent arterial infusion chemotherapy with an implanted reservoir for hepatocellular carcinoma.

A total of 45 patients with advanced hepatocellular carcinoma were treated at Osaka City University Hospital by intermittent arterial infusion chemotherapy with an implanted reservoir. The treatment consisted of intermittent infusion of doxorubicin (5-20 mg/body), mitomycin C (4-10 mg/body) or degradable starch microspheres (600-1200 mg/body) plus doxorubicin (30 mg/body). In all, 26% of the patients received this treatment for disease recurrence following transcatheter arterial embolization (TAE). Among 43 evaluable patients, 4 showed a complete remission (CR) and 16 showed a partial response (PR) on computed tomograms and angiograms. For all 45 patients, the 1-year survival value was 41% and the 2-year value was 14%. Of the 20 patients who showed a CR or PR, 77% survived for 1 year and 29% survived for 2 years. Tumor regression showed a close relationship with the duration of survival. Intermittent arterial infusion with an implanted reservoir caused the least adverse reactions and seems to be appropriate for use in patients with advanced tumor extension or stenosis of the hepatic artery caused by repeated TAE.

Adrenoleukodystrophy: new CT findings.

Three cases of adrenoleukodystrophy are presented with sequential findings by computed tomography (CT). In two cases, CT confirmed not only the findings of several previous reports of a caudocranial direction of low-density white matter and contrast enhancement at the periphery of affected regions during the active demyelinating process, but also two other unusual features: (1) calcification in the affected white matter along the trigones of the lateral ventricles and (2) development of a mass effect during the active demyelinating period. Calcification alone without low-density white matter was the initial CT presentation in one case. Periventricular calcification may be specific for adrenoleukodystrophy in the appropriate clinical setting.

Central giant cell granuloma of the temporal bone.

We present two cases of central giant cell granuloma in the temporal bone. CT showed an enhancing tumor causing a smoothly margined temporal squamosa and floor of the middle fossa. External carotid angiograms showed tumor stain mainly supplied by the temporal branches of the internal maxillary artery. In one case, MR images showed a hypointense mass on both T1- and T2-weighted images that was thought to reflect the pathologic character of this lesion.

Malignant fibrous histiocytoma of the temporal bone with endocranial extension.

Cranial malignant fibrous histiocytomas are rare tumors. Most are hypervascular, destructive masses that are similar to other malignant lesions and to malignant fibrous histiocytomas found elsewhere in the body. We describe a myxoid malignant fibrous histiocytoma of the temporal bone, possibly of dural origin, with features that more closely resembled a meningioma at CT, MR imaging, and angiography.

The maximum and minimum water content and cell volume of human erythrocytes in vitro.

The maximum and minimum water contents of human erythrocytes were measured after exposure to various osmotic pressures. Within a range of osmolarities, at which no haemolysis occurred, the water content reached its maximum, 78.1%, at 180 mosM and its minimum, 54.8%, at 800 mosM. Simultaneously, the mean cell volume increased to 98.5 microns 3 at 180 mosM and decreased to 77.2 microns 3 at 800 mosM.

Radiation enhancing effect of pentoxifylline.

In order to find radiosensitizers with low toxicity, radiation enhancing effects of the pentoxifylline (PENTO), which is known to bear so little toxicity as to be widely used for clinical purpose to improve peripheral blood circulation for vascular disorders, was compared with the nitroimidazole derivative SR 2508 (SR), which is an effective radiosensitizer but has severe neurotoxicity as do other nitroimidazole derivatives. We used an experimental tumor in mice and calculated an-enhancement ratio (ER) using a growth delay method. ER of PENTO and SR were 1.6 and 1.4, respectively, showing PENTO has stronger radiosensitizing effect than SR. Also combined effects with nicotinamide (NA) were compared between PENTO and SR. When combined with NA, the enhancing effects of PENTO was enhanced especially at lower radiation doses, whereas that of SR was decreased. Together with the fact that both PENTO and NA have very low toxicity, the usefulness of PENTO as a radiosensitizer either alone or combined with NA is suggested.

Correlation of DNA synthesis-inhibiting activity and the extent of transmembrane permeation into tumor cells by unsaturated or saturated fatty alcohols of graded chain-length upon hyperthermia.

The carcinostatic activity has been studied for fatty acids of diverse species but scarcely for fatty alcohols. Three unsaturated fatty alcohols at 35-50 microM inhibited DNA synthesis and the proliferation of tumor cells by a combination with hyperthermia to greater extents in the order: oleyl (C18:1)-> linoleyl (C18:2)-> alpha-linolenyl (C18:3) alcohol, which is an order inverse to that known for the corresponding fatty acids (4). In contrast, two saturated fatty alcohols, palmityl (C16:0)- and stearyl (C18:0) alcohols, did not inhibit at the same concentrations. At 100 microM, palmityl alcohol inhibited, whereas stearyl alcohol did not. The effective fatty alcohols appreciably permeated the cells. The inhibition of the unsaturated fatty alcohols on DNA synthesis and proliferation was nearly proportional to the amount of their intercellular accumulation at 37 degrees C or 42 degrees C; the most inhibitory, oleyl alcohol, was the most membrane-permeable, whilst inversely the least inhibitory, alpha-linolenyl alcohol, was the least permeable. A proportional correlation was not observed for saturated fatty alcohols; palmityl alcohol underwent an approximate 2-fold more abundant accumulation than other fatty alcohols, but was weakly inhibitory. Thus, oleyl alcohol may exert an antitumor action via appropriately efficient transmembrane permeation and a combination with hyperthermia.

Cytotoxicity of docosahexaenoic acid and eicosapentaenoic acid in tumor cells and the dependence on binding to serum proteins and incorporation into intracellular lipids.

Docosahexaenoic acid (DHA) inhibited the DNA synthesis in Ehrlich ascites tumor cells more markedly than eicosapentaenoic acid (EPA), which was more inhibitory than oleic-, linoleic-, linolenic-, and arachidonic acids. Their activities augmented according to the increase of number of double bonds in the molecule. To correlate the cytotoxicity with lipid syntheses in the cells, distribution of EPA and DHA incorporated into cellular lipids was assessed. EPA was incorporated into triglycerides (TG) and DHA into phosphatidylcholine (PC). These synthesis into TG and PC etc., which shattered from cytotoxicity, may be involved in tumor-cellular protecting actions against EPA or DHA. EPA and DHA involved in cytotoxicity exhibition are their free acid forms. Thus, as an anticancer reaction, intracellular accumulation in the free acid form of DHA was more marked than that of EPA.