Corticotrophin-releasing factor gene transcription is directly activated after deprivation of glucocorticoids in hypothalamic cells.

Corticotrophin-releasing factor (CRF) plays a central role in controlling the hypothalamic-pituitary-adrenal axis during stressful periods. CRF neurones are activated in the hypothalamic paraventricular nucleus (PVN) in response to stress, whereas the activated CRF neurones in the PVN are suppressed by glucocorticoids. Glucocorticoids may act directly on CRF neurones because glucocorticoid receptors are expressed highly on these neurones in the PVN. CRF expression levels in the PVN are also increased by adrenalectomy in vivo. The signalling pathways involved in the control of CRF gene transcription in the hypothalamus when negative feedback by glucocorticoids after adrenalectomy is lost remain undetermined. We investigated whether CRF gene transcription is regulated by both glucocorticoids and glucocorticoid withdrawal in hypothalamic cells. The present study demonstrates that CRF gene transcription activity and mRNA levels in the hypothalamic 4B cells were not modulated by incubation with dexamethasone for a short 2-h period, although they were stimulated by incubation for longer than 5 h. CRF gene transcription activity and mRNA levels were increased after 2 h of dexamethasone deprivation. The cAMP-response element (CRE) on the promoter was the main region that is regulated by both glucocorticoids and glucocorticoid withdrawal. We observed that the intracellular cAMP production levels were transiently increased 30 min after the removal of dexamethasone, whereas they were also increased 2.5 h after incubation with dexamethasone without the removal. Phosphorylated-CRE-binding protein (CREB)/CREB protein levels were also increased rapidly after the deprivation of glucocorticoids via an adenylate cyclase pathway. Therefore, the phosphorylation of CREB contributes to the activation of CRF gene transcription after the deprivation of glucocorticoids in hypothalamic cells.

Promotive effects of hyperthermia on the cytostatic activity to Ehrlich ascites tumor cells by diverse delta-alkyllactones.

AIM: To evaluate promotive effects of hyperthermia on antitumor activity of new delta-alkyllactones (DALs) of low molecular weight (184-254 Da), chemically synthesized, which are different from natural macrocyclic lactones of high molecular weight (348-439 Da), such as camptothecin and sultriecin. METHODS: A suspension of Ehrlich ascites tumor (EAT) cells was mixed with a DAL in a glass tube, heated at 37 or 42 degrees C for 30 min in a water bath, and cultured at 37 degrees C for 20 or 72 h. Cell viability was measured by the mitochondrial dehydrogenase- based WST-1 assay. DALs incorporated into EAT cells was extracted and measured by gas-liquid chromatography. RESULTS: The reduction of cell viability by DALs was markedly enhanced upon the treatment at 42 degrees C compared to that at 37 degrees C. At 37 degrees C, delta-hexadecalactone (DH16:0) and delta-tetradecalactone (DTe14:0) displayed cytostatic activity (at 100 microM survival level: 20.7%, 66.1%; at 50 microM--41.2%, 82.4%, respectively). Their activity was more marked at 42 degrees C (at 100 microM 10.6%, 27.6%; at 50 microM 30.6, 37.5%, ibid). The other DALs, delta-undecalactone (DU11:0), delta-dodecalactone (DD12:0), and delta-tridecanolactone (DTr13:0) were almost ineffective. Evaluation of survival rate in the cells treated for 30 min by DALs with the next culturing of EAT cells for 72 h resulted in the enhanced carcinostatic activity of DH16:0 and DTe14:0 even at concentrations as low as 25 microM at either 37 degrees C (18.5%, 78.5%, ibid) or 42 degrees C (5.0%, 42.0%, ibid), but the others exhibited slight activity or none. DH16:0 was effective at either 37 degrees C (36.0%) or 42 degrees C (23.0%) even at a lower dose of 10 microM. At the same time only the most cytostatic DH16:0 was incorporated into EAT cells and the rate of incorporation was more at 42 degrees C than at 37 degrees C. CONCLUSION: Delta-hexadecanolactone (DH16:0) exhibited the most cytostatic effect that was significantly enhanced by hyperthermia. It allows to consider it as a potent antitumor agent, especially in combination with hyperthermia.

Involvement of regulatory elements on corticotropin-releasing factor gene promoter in hypothalamic 4B cells.

INTRODUCTION: Corticotropin-releasing factor (CRF) plays a central role in controlling the hypothalamic-pituitary-adrenal (HPA) axis during stressful periods. CRF is synthesized and secreted in the hypothalamic paraventricular nucleus (PVN) in response to stress, and stimulates ACTH in the pituitary corticotrophs. ACTH stimulates the release of glucocorticoids from the adrenal glands, and glucocorticoids sequentially inhibit hypothalamic PVN production of CRF and pituitary production of ACTH. The effects of glucocorticoids on CRF gene regulation, however, are possibly tissue-specific since glucocorticoids stimulate CRF gene expression in the placenta and the bed nucleus of the stria terminalis, while they inhibit it in the hypothalamus. METHODS AND RESULTS: In a hypothalamic cell line, 4B, we found that forskolin-stimulated CRF gene transcription was mediated by a functional cAMP-response element (CRE), which included -220 to -233 bp on the CRF 5'-promoter region. Protein kinase A, protein kinase C, and p38 mitogen-activated protein kinase pathways contributed to forskolin-induced transcriptional activity of CRF in hypothalamic 4B cells. Glucocorticoid-dependent repression of cAMP-stimulated transcriptional activity of CRF was localized to promoter sequences between -278 and -233 bp, which included a glucocorticoid regulatory element and a serum response element. CONCLUSION: Taken together, these findings indicate that the regulatory elements, including CRE, negative glucocorticoid regulatory element, and a serum response element on the promoter, contribute to the regulation of CRF gene transcription in hypothalamic 4B cells.

Abnormal cell morphology and cytotoxic effect are induced by 6-0-palmitol-ascorbate-2-0-phosphate, but not by ascorbic acid or hyperthermia alone.

A new derivative of ascorbic acid (AsA), 6-0-palmitoyl-ascorbate 2-0-phosphate (Asc2P6Pal) was developed to enhance the antitumor activity of AsA. When Ehrlich ascites tumor cells were treated with 50 microM Asc2P6Pal at 37 degrees C or 42 degrees C for 1 h and then cultured for 20 h, most of cells exhibited some morphological abnormalities, including exudation of intracellular granules together with other contents on the cell membrane surface, resulting in cell fragmentation. The abnormal features were further enhanced by a long term culture for 96 h and heat treatment at 42 degrees C. In contrast, no abnormality was detected for untreated cells or cells treated with AsA (free acid) at 37 degrees C or 42 degrees C. Cells cultured for 96 h after the treatment suffered from inhibition of DNA synthesis and proliferation. This inhibition was markedly enhanced by combination with the hyperthermic treatment at 42 degrees C, but not for a short-term culture of 20 h after the treatment. No effects were seen upon similar treatment with AsA. The abnormal cells produced during culture for 20 h after the treatment were evaluated to be viable, because they failed to be stained with trypan blue and retained most of the DNA synthesizing ability of Asc2P6Pal-untreated cells. However, they appeared and died after a continuous 76 h of culture (96 h).

Effect of lactic acid in tumours on antitumour activity of hyperthermia.

The change of lactic acid concentration in the tumour after intraperitoneal administration of 5 g/kg glucose, and the effect of the lactic acid concentration on antitumour activity of hyperthermia were studied in an experimental murine tumour. The lactic acid concentration in SCC VII tumours transplanted into the legs of C3H/HeJ male mice was measured by gas chromatography. Local hyperthermic treatment to the tumour was performed with a water bath at 43 degrees C for 40 min. Antitumour effects were evaluated by measuring tumour volume doubling time (DT) as an index. The mean concentration of lactic acid in the tumour was 10.4 mumol/g in the no-treatment group. The lactic acid concentration gradually increased after glucose administration, reaching a significantly high concentration of 20.0 mumol/g at 90 min later. The DTs in the no-treatment group and hyperthermia alone group were 2.4 and 3.7 days respectively. The DTs in the glucose administration groups shortly before, 30 and 60 min before the hyperthermia were 3.6, 3.6 and 5.6 days respectively. The DT in the 60 min group was significantly extended (p < 0.0001). Hyperthermia during the period of increased lactic acid concentration significantly prolonged the DT of the tumour. These results clearly showed that an increase of lactic acid concentration in the tumour improved the effect of local hyperthermia.

In vivo evidence that arginine vasopressin is involved in the adrenocorticotropin response induced by interleukin-6 but not by tumor necrosis factor-alpha in the rat.

We examined whether arginine vasopressin (AVP) is involved in the adrenocorticotropin (ACTH) response induced by interleukin (IL)-6 or tumor necrosis factor (TNF)-alpha in the rat. To accomplish this, we employed immunoneutralization of brain AVP by injecting anti-AVP antiserum intracerebroventricularly (i.c.v., 3rd ventricle). For comparison, we also tested the effect of immunoneutralization of corticotropin-releasing hormone (CRH) in the brain. Anti-CRH antibody, anti-AVP antibody, or normal rabbit serum (control) was given i.c.v. 15 min before an i.c.v. administration of human recombinant IL-6 (100 ng) or TNF-alpha (100 ng). Both IL-6 and TNF-alpha significantly elevated plasma ACTH levels. The IL-6-induced ACTH response was significantly suppressed by both anti-CRH and anti-AVP antibodies. On the other hand, the TNF-alpha-induced ACTH response was not significantly affected by anti-AVP antibody, although anti-CRH antibody could suppress the response. These results suggest that the IL-6-induced ACTH response may be mediated by both CRH and AVP, whereas the ACTH response to TNF-alpha is only via CRH.

Enhanced inhibitory effects of hyperthermia combined with ascorbic acid on DNA synthesis in Ehrlich ascites tumor cells grown at a low cell density.

Effects of hyperthermia and cell densities on inhibitory activity of ascorbic acid on DNA synthesis in Ehrlich ascites tumor cells were studied. When cells at a low density of 5 x 10(3)/ml were treated with 75 microM ascorbic acid for 1 h, DNA synthesis was inhibited after treatment at 37 degrees C and the inhibition was significantly enhanced at 42 degrees C. At a cell density as high as 1 x 10(5)/ml, however, inhibition did not occur at 37 degrees C or 42 degrees C. In contrast, dehydroascorbic acid was inactive even at a low cell density under similar conditions. Inhibitory effects of ascorbic acid on DNA synthesis were also markedly enhanced by treatment at 40 degrees C. DNA synthesis was not inhibited in the absence of the drug. Furthermore, mice transplanted with cells treated with a combination of 75 microM ascorbic acid and hyperthermia at 42 degrees C, considerably prolonged their survival time in comparison with untreated cells. Addition of ascorbic acid to hyperthermia is suggested to be an advantageous treatment for cancer.

Effect of hyperthermia and protein kinase C inhibitors on DNA synthesis and cell proliferation on Ehrlich ascites tumour cells in vitro.

Effect of hyperthermia and/or protein kinase inhibitors on DNA synthesis and cell proliferation was investigated in Ehrlich ascites tumour cells in vitro. Both H-7 and H-8, potent inhibitors of protein kinase C, suppressed DNA synthesis significantly, but HA1004, an inhibitor of cAMP- and cGMP-dependent protein kinase, did not. Hyperthermia increased greatly the suppressive activity of H-7 and H-8 but not that of HA1004. H-7 also inhibited cell growth. These results suggest that the inhibition of protein kinase C enhances the suppression of DNA synthesis and the proliferation of tumour cells by hyperthermia.

Rapid and simple gas chromatographic measurement of lactic acid in red blood cells, plasma, and tumor cells after hyperthermia.

The content of lactic acid in red blood cells, plasma, and Ehrlich ascites tumor cells were measured by a gas-liquid chromatography using a column with a terephtalic acid support coated with polyethylene glycol-6000. The lactic acid contents were directly determined in aqueous samples, because they were converted to a volatile derivative in the column. The method was rapid and simple, compared with previous methods which need time-consuming conversion of lactic acid to volatile derivatives. Our measurements showed the increase in the contents of intra- and extracellular lactic acid after hyperthermia.

[Combined effects of gamma rays, cis-diamminedichloroplatinum (CDDP) and systemic hyperthermia mastocytoma transplanted into muscle in the mouse]. / Etude des effets combinés des rayons gamma, du cis-diamminedichloroplatinium (CDDP) et de l'hyperthermie systémique sur le mastocytome transplanté dans le muscle chez la souris.

Comparison was made between the effects of local irradiation of gamma rays, s. c. injection of cis-diamminedichloroplatinum (II) (CDDP), systemic hyperthermia and their combinations on the i. m. transplanted murine mastocytoma. Increase of the mean survival time (M. S. T.) by a factor of 1.72 and of 1.68 was achieved by a single irradiation at 20 Gy, given on day 5 after transplantation, and by injections of CDDP at 2 mg/kg, given s. c. on days 5 and 12 after transplantation, respectively. Increase of M. S. T. by a factor of 1.10 which was achieved with systemic hyperthermia of 41.8 degrees C of the core body temperature for 5 min, given twice, on days 5 and 12 after transplantation, was not statistically significant. The most effective one among all possible combinations within the 3 modalities was that of radiation and CDDP. Increase of M. S. T. was by a factor of 4.01.