Different mechanisms between premitotic apoptosis and postmitotic apoptosis in X-irradiated U937 cells.

PURPOSE: Apoptosis is currently being evaluated for its importance as a pathway of radiation-induced cell death. However, the difference in the mechanisms between premitotic and postmitotic apoptosis following X-irradiation remains not well understood. We show here that the human monoblastoid cell line U937 can be induced to undergo these two different types of apoptosis. METHODS AND MATERIALS: U937 cells were irradiated at a dose of 5 or 20 Gy, and the DNA fragmentation rate was measured by both flow cytometric analysis and gel electrophoresis. Activation of caspase-3 was detected by Western blot analysis and fluorogenic assay using acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl-coumarin (Ac-DEVD-AMC). Detection of mitochondrial transmembrane potential (DeltaPsi) was performed by using Rho123. Chasing of S-phase fraction following X-irradiation was performed after labeling with 5-bromo-2'-deoxyuridine (BrdU). Thymidine was used for synchronization of the cells. Inhibition of caspase-3 activity was achieved by Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). RESULTS: Time courses of the apoptotic rates, caspase activation, and DeltaPsi indicated that two different types of cell death were induced by the different X-ray doses. High-dose X-ray (20 Gy) induced a rapid and strong apoptosis, whereas low-dose X-ray (5 Gy) induced a slow and mild apoptosis. Cell-cycle analyses revealed that there was cell death before cell division in the former apoptosis but the cells must be dying after cell division in the latter apoptosis. By means of cell-cycle synchronization, the S-phase cells proved to be the most sensitive fraction to premitotic apoptosis, but an obvious difference in the susceptibility to cell death among the cell-cycle phases was not observed in postmitotic apoptosis. Ac-DEVD-CHO treatment effectively blocked caspase activity and premitotic apoptosis, but it failed to block postmitotic apoptosis. CONCLUSIONS: Irradiation of U937 cells at different X-ray doses induced two different types of apoptotic cell death, premitotic apoptosis and postmitotic apoptosis, which are characterized by the time course and cell-cycle specificity. Decision concerning these two types of apoptotic cell death may be made by the difference in the magnitude of cell damage following X-irradiation.

Identification of the promoter region of human placental 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene.

The placenta-type isozyme of human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (HP2K) is expressed in several tissues such as placenta, brain, testis, liver, kidney, skeletal muscle, and primary blood mononuclear cells. To better understand the regulation of HP2K gene expression, we isolated and characterized its genomic DNA, which includes the promoter region. The results of oligo-capping analysis indicate that the transcription start point (tsp) is an adenine residue 329 bp upstream of the translational start codon. DNA sequence analysis of this gene shows that the promoter region that contains the TATA box sequence and the 5'-UTR is different from the other known PFK-2/F2, 6BPase genes. In addition, its 5'-flanking and 5'-UTR both have G + C-rich sequences containing Sp1 binding sites. To identify the promoter/enhancer region of HP2K gene, we performed transfection analyses of human choriocarcinoma BeWo cells with HP2K promoter-luciferase constructs. These experiments identified a promoter region 164 bp upstream from the tsp and an enhancer region between -1265 and -1329 on the 5'-flanking sequences. We also showed that Sp1 sites were not essential for HP2K transcription. Following transfection, stimulation experiments with serum, progesterone and phorbol 12-myristate 13-acetate showed that only the construct with the enhancer containing putative early growth response-1 binding motif was responsive to serum. We propose that the transcription of HP2K is strictly controlled by tissue-specific factors even though its genomic DNA contains several transcriptional elements.

Role of G1 phase in the UV-induced apoptosis of EL-4 mouse lymphoma cells.

Although UV is known to induce apoptotic cell death to various animal cells, relationship between cell cycle and UV-induced apoptosis is still unclear. In this study, we investigated the role of G1 phase in UV-induced apoptosis by using EL-4 mouse lymphoma cells which have wild type p53. After 500 J/m2 UV irradiation, an increase of apoptotic fraction was accompanied by cell cycle accumulation in the G1 phase. Apoptotic fraction after UV-exposure was remarkably augmented by treatment with 2-AP, a G1 checkpoint inhibitor. In contrast, aphidicolin, an inhibitor of DNA polymerase-alpha, suppressed the rate of apoptotic fraction. These results suggest that mandatory cell cycle progression from G1 to S leaves the damaged DNA unrepaired and may increase the apoptotic fraction. To investigate the precise mechanism in the G1 phase, UV was exposed to the G1-synchronized cells and apoptotic fraction was serially observed. Synchronized EL-4 cells passed through the G1 phase in 8 h. Within the G1 phase, late-G1 cells (6 h after M) were more sensitive to UV-induced apoptosis than early-G1 cells (2 h after M) (49.7 +/- 9.0% vs. 41.5 +/- 8.5%, p < 0.05). In HL-60 cells, lacking in p53 expression, such a difference was not observed. Western blot analysis revealed that expression of p53 in synchronized EL-4 cells was increasingly enhanced during G1 phase. After UV-exposure, p53 expression gradually decreased in early-G1 cells, but it was kept at almost the same level in late-G1 cells. In addition, bcl-2 expression in early-G1 cells showed a more rapid and larger increase than that in late-G1 cells. These results suggest that susceptibility of the G1 cells to UV-induced apoptosis depends on their position within the G1 phase, and late-G1 is more sensitive than early-G1. Sensitivity to UV-induced apoptosis is closely related to the expression level of p53 and bcl-2 proteins. Early-G1 cells may be able to take enough time to repair damaged DNA until they reach the G1 checkpoint compared to the late-G1 cells.

Caffeine induces S-phase apoptosis in cis-diamminedichloroplatinum-treated cells, whereas cis-diamminedichloroplatinum induces a block in G2/M.

Caffeine overrides checkpoints in the G2 phase of the cell cycle by inhibiting DNA repair at this phase and increases the cytotoxicity of antitumor drugs, such as cis-diamminedichloroplatinum (CDDP). The enhanced cell death induced by caffeine is characterized by apoptosis. In this paper, we demonstrate that this apoptotic event occurs in S phase of the cell cycle, whereas CDDP induces a block in G2/M. DNA histogram analysis revealed that caffeine reduced G2 arrest in CDDP-treated EL-4 cells. In a synchronous population, the ratio of cyclin B:p34cdc2 was upregulated just before the cells went into the apoptotic pathway. A rapid increase in DNA fragmentation was detected at 12-24 h, when marked regression of G2/M phase was observed. Moreover, the degree of DNA fragmentation in CDDP + caffeine-treated cells was not reduced when the cell cycle was arrested at metaphase by exposure to the spindle-inhibitor nocodazole. It is possible that execution of the apoptotic program after treatment with caffeine did not require the EL-4 cells to reenter G1 phase. The apoptotic cell fraction in the group of CDDP + caffeine was recognized as an S population by bivariate analysis of apoptosis and DNA content. These results suggest that enhancement of the apoptotic activity of CDDP-treated cells by caffeine is not a G1-phase event but an S-phase-specific event, whereas cells were arrested in G2/M phase, and that it is regulated by G2 checkpoint-related proteins.

Effects of gonadotropin administration on follicular growth and in vitro fertilization in female hypogonadal mice.

Folliculogenesis in female hypogonadal (hpg) mice was examined after treatment with exogenous gonadotropins. The female mutant mice were characterized by a deficiency of hypothalamic gonadotropin-releasing hormone (GnRH), leading to the absence of estrus and ovulation. Gonadotropin administration induced resumption of gonadal development and vaginal opening. The follicles that developed with gonadotropin treatment were very similar to those in normal littermates. The oocytes from hpg mice showed the capacity for fertilization and development to produce viable young after in vitro fertilization and embryo transplantation. Thus, the combination of the exogenous gonadotropin administration, in vitro fertilization and embryo transfer method appear to be helpful to breed mutant hpg mice efficiently.

Proliferating cell nuclear antigen and Ki-67 in lung carcinoma. Correlation with DNA flow cytometric analysis.

BACKGROUND: To study the prognosis of patients with lung carcinomas, the efficacy of proliferating cell nuclear antigen (PCNA) in ensuring both the proliferative activity defined using Ki-67 labeling and the cell cycle data obtained using flow cytometry was determined. METHODS: The authors used immunostaining to study frozen and paraffin embedded sections of 165 surgically resected lung carcinomas [squamous cell carcinoma, n = 84; adenocarcinoma, n = 62; large cell carcinoma, n = 15; small cell carcinoma, n = 4] for the presence of PCNA and Ki-67 antibodies. Also studied were two parameter flow cytometric analysis of fluorescein isothiocyanate conjugated PCNA/propidium iodide for 165 fresh frozen tissues. Clinicopathologic data (sex, age, tumor stage, survival period, histologic type, degree of cell differentiation, and cellularity) were evaluated using the Statistical Analysis System. RESULTS: The percentages of PCNA positive cells per 1000 nuclei were 52% of squamous cell carcinoma; 49% in adenocarcinomas; 76% of large cell carcinoma; and 63% of small cell carcinoma. Positive PCNA staining was significantly correlated with stage, cellularity, and DNA index. Calculation of logistic regression coefficients indicated an association between overall survivals and tumor cellularity (P < 0.0003), percentage of cells stained with PCNA antibody (P < 0.02), DNA pattern (aneuploid versus diploid) (P < 0.009), DNA index (P < 0.009), and percentage of cells in S-phase (P < 0.04). Both cellularity (P = 0.03) and DNA (P = 0.08) retained its independent level of significance by multivariate analysis. CONCLUSIONS: In addition to clinical stage and histologic differentiation, both cellularity and DNA content may help predict the course of lung carcinomas.

Enhancement of CDDP cytotoxicity by caffeine is characterized by apoptotic cell death.

The ability of caffeine, an agent that suppresses cell replication by inhibiting deoxyribonucleic acids (DNA) repair, to modulate the cytotoxicity of cis-diamminedichloroplatinum (CDDP) was investigated in murine lymphoma cell line EL-4. EL-4 cells were precultured with or without 20 micrograms/ml CDDP for 1 h and then cultured in the presence of 5 mM caffeine up to 48 h after reseeding. In CDDP-pretreated cells, suppression of cell growth and decrease in cell viability from 24 h were observed. Cell cycle arrest in G2 + M phase and a concomitant increase in both rhodamine 123 (R123) uptake and cell size (forward scatter) were observed in these cells. Treatment with caffeine alone suppressed growth rate, R123 uptake, cell size, and frequency of S phase fraction in the cell cycle. Combination of the two agents, CDDP+caffeine, strongly suppressed not only cell viability but also R123 uptake and cell size, compared with CDDP pretreatment alone. DNA histogram analysis by flow cytometry revealed that cultivation with caffeine hastened G2 + M arrest in CDDP-pretreated cells by reduction in the time of passing through S phase. DNA fragmentation was observed following incubation of CDDP-pretreated cells with caffeine for 16 h when marked accumulation in G2 + M phase was observed. The intensity of these ladder fragments increased in a time-related manner. These results demonstrate that enhancement of cytotoxic activity against CDDP-treated cells by caffeine is characterized by an acceleration of DNA degradation in G2 + M phase, namely apoptotic cell death. The fact that induction of DNA fragmentation during G2 + M phase by caffeine modulates the cytotoxicity of CDDP may give rise to a new combination regime of chemotherapy against malignant tumor cells.

Therapeutic effect of granulocyte colony-stimulating factor (G-CSF) on the protection against Listeria infection in SCID mice.

Anti-listerial activity in SCID mice as well as in control C.B-17 mice was augmented by granulocyte colony-stimulating factor (G-CSF). After 1 x 10(3) colony-forming units of Listeria monocytogenes (strain EGD) were intravenously inoculated, mice were intraperitoneally injected with G-CSF at a daily dose of 500 micrograms/kg for 5 days. The numbers of viable bacteria in the liver were significantly lower in G-CSF-treated SCID and C.B-17 mice than in non-treated mice. The surface marker analyses on gamma delta T-cell receptor (TcR), Mac-1 and F4/80, and dichlorofluorescein oxidative activity, showed a possible contribution of activated neutrophils, but not gamma/delta T cells nor activated macrophages, to the augmentation of anti-listerial activity in SCID mice. This study is one of the first reports on the anti-microbial effect of G-CSF in therapeutic use.

Therapeutic effect of granulocyte colony-stimulating factor and cephem antibiotics against experimental infections in neutropenic mice induced by cyclophosphamide.

Effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) against severe infections in cyclophosphamide (CY)-induced neutropenic mice were investigated by its single use or by its combination with cephem antibiotics. Treatment with rhG-CSF increased the number of peripheral blood leucocytes and strikingly shortened the duration of the neutropenic state. Most of the regained population in the peripheral blood leucocytes were neutrophils. Functions of neutrophils, such as phagocytic activity, superoxide release, and expression of Mac-1 molecules, were remarkably enhanced by administration of rhG-CSF. When rhG-CSF was administered to CY-induced neutropenic mice before infection, protective effects against various kinds of bacteria were remarkable. On the other hand, such remarkable effects were not observed when rhG-CSF was administered after infections. However, even in the case of post-infectious treatment, a combination therapy of rhG-CSF with cephem antibiotics, particularly with Cefodizime (CDZM), showed a significant improvement in the survival rate and a decrease in the number of viable bacteria in the liver. These results suggest that a combination therapy of rhG-CSF with cephem antibiotics, especially with CDZM, is an efficient regime against severe infections in patients with neutropenia induced by a heavy anti-tumour chemotherapy.

Granulocyte colony-stimulating factor enhances the extracellular emission of reactive oxygen from neutrophils stimulated with formylmethionylleucylphenylalanine.

Pretreatment of neutrophils with granulocyte colony-stimulating factor (G-CSF) enhanced luminol-dependent chemiluminescence emission by the stimulation of formylmethionylleucylphenylalanine (FMLP), opsonized zymosan, and zymosan. The intensity of the priming effects was in the following order: FMLP > zymosan > opsonized zymosan. However, the priming effect of G-CSF was not detected by the stimulation of phorbol myristate acetate. The reaction curve with the stimulation of FMLP showed a bimodal shape, and the elevation of the first peak by the priming effect was further prominent in comparison with the elevation of the second peak. This indicates that G-CSF mainly enhances the extracellular release of oxygen radicals in response to the stimulation with FMLP. This is the first report investigating intra- and extracellular events in chemiluminescence reaction of neutrophils primed with G-CSF.

Functional changes of alveolar macrophages in carragheenan-induced aspiration pneumonia model mice.

Alveolar macrophages (AMs) are cells with unique characteristics because of their localization in the aerobic environment and are receiving stimuli by inhalation. To estimate the functional changes of AMs induced by inflammation, a murine model of aspiration pneumonia was made by an intratracheal injection with carragheenan, whose mortality rate was approximately 25%. The cell component which increased predominantly in the inflammatory site was polymorphonuclear leukocytes, and the number of AMs did not show a remarkable increment. Control group showed a high level of intracellular oxidation of 2'7'-dichlorofluorescin (DCFH) by AMs, while that in carragheenan-treated mice decreased significantly (p < 0.05). There were two populations in AMs classified according to the oxidative activity of DCFH; the population showing high oxidative activity of DCFH was asialo GM1 positive, in contrast, that with lower oxidative activity was asialo GM1 negative. Decrease in DCFH-oxidative activity of AMs in control group was observed after a treatment with KCN or deferoxamine. But in the carragheenan-treated group, this decrease was not observed after treatment with KCN. These results show that both oxygen-derived radical produced in mitochondria, which is inhibited by KCN, and cytoplasmic OH radical, which is selectively inhibited by deferoxamine, are concerned with intracellular oxidation of DCFH by AMs, and that a decrease in DCFH-oxidative activity in the carragheenan-treated group was attributed to the depression of mitochondrial respiration. Nevertheless, increased expressions of Ia and F4/80 in AMs of the carragheenan-treated group were observed and phagocytic activity was well preserved at the control level. These results suggest that AMs may play a crucial role, as well as differentiated phagocytes possessing antigen-presenting ability and/or digestive activity against various types of foreign bodies despite showing an obvious decrement in oxidative activity. These results imply that AMs have a strong oxidative activity and deal with various types of antigens in normal states, but in case of acute inflammation they will change into mature type macrophages with high expression of class II molecules which correlates with an antigen-presenting capacity.

Effects of macrophage colony-stimulating factor on the activities of murine monocytes and peritoneal macrophages in vivo.

The ability of peripheral blood monocytes, granulocytes and resident peritoneal macrophages to generate hydrogen peroxide in response to concanavalin A was enhanced by a single intravenous injection of macrophage colony-stimulating factor (M-CSF) of recombinant human type in AKR mice. In response to phorbol myristate acetate, only granulocytes and resident peritoneal macrophages showed the enhanced ability. Phagocytosis by those cells was not stimulated by M-CSF. Surface marker analysis showed an increased expression of F4/80 and Mac1 on monocytes, Mac1 expression on granulocytes and LFA-1 expression on resident peritoneal macrophages. Ia antigen on resident peritoneal macrophages was suppressed by M-CSF. M-CSF can induce monocytes, granulocytes and resident peritoneal macrophages to generate hydrogen peroxide and enhances their maturation in vivo.

Effects of macrophage colony-stimulating factor on reduction of viable bacteria and survival of mice during Listeria monocytogenes infection: characteristics of monocyte subpopulations.

Mice could well tolerate infection with a lethal dose of Listeria monocytogenes after intraperitoneal preinjections with 250 micrograms of macrophage colony-stimulating factor (M-CSF) per kg of body weight for 5 days. The characteristic changes in the surface markers (Mac-1, LFA-1, and F4/80) of peripheral monocytes were also investigated in order to analyze the mechanism of protection by M-CSF. This investigation shows the excellent effect of intraperitoneal preinjections of M-CSF on the reduction of viable Listeria organisms and the improvement of survival after an intravenous Listeria infection.

[Augmentation of activities of peripheral granulocytes and of resistance against bacterial infection after administration of G-CSF into mice].

We evaluated the metabolic capability of murine peripheral granulocytes after administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) by quantitative flow cytometric assay for H2O2-dependent oxidative product formation. Intraperitoneal administration of a daily dose of 10 micrograms of rhG-CSF for 5 days induced doubling of the leukocyte population. Differential counting of peripheral leukocytes and scattergram by flow cytometry showed an increased mature granulocyte population. After stimulation with phorbol myristate acetate, the granulocytes of the rhG-CSF-administered mice demonstrated some hyperresponsive population and an increased H2O2 production. The hyperresponsive population showed H2O2 production 4-6 times higher than did normal cells. Granulocytes from the G-CSF-treated mice revealed an augmented phagocytic activity and an increased expression of Mac-1 molecules. Moreover, mice treated with G-CSF showed an enhanced resistance against intravenous infection with a lethal dose of E. coli. Granulocytes showing such markedly increased oxidative metabolism may be a significant component of the host defence to various infective organisms.

Adsorption and preparation of human viruses using hydroxyapatite column.

The adsorption and chromatographic properties of hydroxyapatite sorbents for application to different viruses have been investigated. The strong adsorption of viruses was observed on macroporous hydroxyapatite with hydrophilic properties of the sorbent surface. The viruses were purified on this sorbent without loss of biological activity. The column can be used for virus vaccine production.

Thymic depletion in pregnancy: kinetics of thymocytes and immunologic capacities of the hosts.

In the present study we observed a significant depression of thymocytes during pregnancy and investigated the influences of this thymic change on the immunologic capacities of peripheral lymphocytes. Thymocytes in pregnant mice began to decrease in number from Day 10 and reached about 0.1-fold of the nonpregnant level at Day 19, just before parturition. At late stage of pregnancy, thymocyte subpopulation expressing CD4+CD8+ and Thy1.2+PNA+ was selectively depressed. On the contrary, peripheral lymphocytes including splenocytes, peripheral blood lymphocytes and peripheral lymph node cells showed no depression. As to the immunologic capacities of the pregnant hosts, delayed footpad reaction and phagocytic activity of fixed liver macrophages in vivo were remarkably suppressed, but MLR reactivity and antibody response to SRBC or haptens were well preserved. Transfer of pregnant sera or administration with steroid hormones especially E3 into nonpregnant mice induced similar changes in the thymus and peripheral lymphocytes in number and subsets but this could not mimic the immunologic reactivities of the pregnant mice. These results suggest that sex steroid hormones such as E3 play an important role in the changes in cell populations of each lymphoid organ and the immune reactivities of the hosts during pregnancy. However, other factors also contribute to the immunologic capacities of the maternal hosts.

[Flow cytometric analysis of oxidative burst of phagocytes with small amount of peripheral blood].

We have developed a simple method for assessing the oxidative metabolic burst of peripheral blood leukocytes with a minute amount of whole peripheral blood by flow cytometry according to the method of Bass et al. with some modification. By this method, we can measure the H2O2 production by both granulocytes and monocytes in the same blood sample. The oxidative product formation by peripheral blood neutrophils can be monitored sequentially in the same mouse infected with E. coli. The mice infected intravenously with 0.1 LD50 of the bacteria showed increased basal activities from an early stage of infection; those infected intraperitoneally with the same dose of the bacteria showed a delayed enhancement. In case of infection with 0.01 LD50, the enhanced basal activities lasted for only a short period of time. The H2O2 production was correlated well with the clearance of the infected bacteria. These results demonstrated that the oxidative-product formation by peripheral blood neutrophils is affected by both the route and the dose of infection.

[Basic study of chemo-embolization of the liver using hydroxyapatite granules].

The purpose of this study was to elucidate the availability of hydroxyapatite (HAp) granules as a chemoembolic agent in chemo embolization therapy. A mixture of adriamycin (ADM) and an embolic agent (HAp, Lipiodol) was injected via hepatic artery in normal Wistar rats. Then the concentration of ADM in the liver serum transaminase level were measured serially. The remaining ADM in the liver was higher in groups with HAp granules than the others. The serum transaminase, however, were lower in the HAp groups. There are some advantages of HAp using as a chemo embolic agent. (1) HAp is a physiological biomaterial and seem to be safe for human. (2) HAp granules injected into the liver are easily detectable by X ray and ultrasonography. (3) HAp granules have a large surface area and this characteristic is suitable for a carrier of drugs. It is concluded that HAp granules have some necessary prerequisites for a chemo embolic agent and the application to clinical practice may be expected.

Effects of magnesium on the tone of isolated human coronary arteries. Comparison with diltiazem and nitroglycerin.

To study the effects of magnesium (Mg2+) on human coronary arteries and to compare those effects with those of diltiazem and nitroglycerin, we measured the tension of ring segments from isolated human coronary arteries obtained at autopsy within 5 hours after death. Precontracted segments with 3 x 10(-6) M prostaglandin F2 alpha were studied after adding cumulative concentrations of these agents (1.0-8.0 mM, 10(-9)-10(-5) M, and 10(-10)-10(-6) M, respectively). Mg2+ significantly inhibited the tonic contraction compared with the time-matched controls at 1.0 and 2.0 mM (48.7 +/- 5.6% vs. 88.6 +/- 2.2%, p less than 0.01, 36.2 +/- 6.1% vs. 78.9 +/- 3.0%, p less than 0.01, respectively). 1.0 and 2.0 mM Mg2+ did not suppress, but actually increased, the amplitude of periodic contraction, but 8.0 mM Mg2+ reduced the amplitude compared with the controls (6.6 +/- 5.2% vs. 73.3 +/- 10.7%, p less than 0.01). Diltiazem at a concentration of 10(-5) M moderately inhibited the tonic contraction, and reduced the amplitude of periodic contraction almost completely. Nitroglycerin reduced the tonic contraction almost completely at a concentration of 10(-6) M but did not reduce the amplitude of periodic contraction at any concentration. We conclude that 1.0 and 2.0 mM Mg2+ inhibits the tonic contraction and that 8.0 mM Mg2+ inhibits the periodic as well as the tonic contraction of isolated human coronary arteries. Diltiazem inhibits the periodic contraction, whereas nitroglycerin suppresses tonic contraction without affecting the periodic contraction.

Denaturation and renaturation of a beta-1,6;1,3-glucan, lentinan, associated with expression of T-cell-mediated responses.

Correlation between the higher structure and biological functions of lentinan, a beta-1,6;1,3-glucan capable of potentiating T- and non-T-cell-mediated responses, were investigated by measurements of optical rotation and some biological responses. The addition of urea or dimethyl sulfoxide decreased specific rotation at 589 nm, [alpha]D, of lentinan in a concentration-dependent manner and the removal of these denaturants resulted in the recovery of [alpha]D values. Measurements of optical rotatory dispersion in the spectral region between 600 and 200 nm showed the change in the higher structure of lentinan more clearly. Denaturation and renaturation of lentinan using urea and dimethyl sulfoxide were associated with the decrease and the recovery of antitumor activity against P-815 mastocytoma and vascular dilation and hemorrhage-inducing activity, found to be T-cell-mediated responses. Lentinan was also denatured by NaOH and the transition of [alpha]D values and optical rotatory dispersion curves were seen in the manner of two concentration-dependent phases. Removal of NaOH led to the recovery of optical rotation of lentinan and its antitumor and vascular dilation and hemorrhage-inducing activity. However, recovery of these bioactivities was more difficult in the case of the higher concentrations of NaOH above 2% than the lower ones. During the process of renaturation of lentinan, random aggregation may take place. An increase of serum acute phase proteins, a non-T-cell-mediated response caused by lentinan, was not affected by the change of the higher structure of lentinan.