Relationship between DNA replicon size and SCE induction in BALB/c and BALB/Mo mouse lymphocytes

We studied the DNA replicon size in BALB/c and BALB/Mo mouse lymphocytes by the method of bromodeoxyuridine photolysis. After treatment of the BALB/Mo lymphocytes in vitro with mitomycin C, the average DNA replicon size appeared to be significantly smaller than that observed in BALB/c lymphocytes treated similarly. In these conditions an increased susceptibility to SCE induction in BALB/Mo lymphocytes had been observed. In the presence of both mitomycin C and cordycepin (an antiviral drug), both the DNA replicon size and the SCE frequency returned to normal values.     

Phorbol 12-myristate 13-acetate induces resistance of human melanoma cells to natural-killer-and lymphokine-activated-killer-mediated cytotoxicity

Summary Human melanoma cells are sensitive to the lytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells in vitro. The events resulting in tumour cell killing by lymphocytic effectors have not been completely clarified, and the same target cell determinants regulating responsiveness to immune cytolysis have not yet been identified. Indeed, changes in the differentiative status of leukemia cells as well as in the expression of major histocompatibility complex (MHC) antigens have been described to modulate sensitivity to cytotoxic effectors; moreover surface expression of adhesion factors or extracellular matrix proteins by the cancer cells can promote the activation of the cytolytic effectors and has been described to correlate with tumour cell sensitivity to cytolytic cells. We reasoned that treatment with differentiation inducers could modulate melanoma cell sensitivity to NK and LAK cells. The present study demonstrates that human melanoma GLL-19 cells, when treated with the phorbol diester phorbol 12-myristate 13-acetate (PMA) in vitro, undergo growth inhibition and neuron-like differentiation. Moreover PMA treatment induces an evident inhibition of GLL-19 cell sensitivity to NK- and LAK-mediated cytotoxicity. GLL-19 cells express constitutively MHC class I antigens. PMA treatment, however, does not modify the expression of MHC class I and class II DR antigens in human melanoma GLL-19 cells. We have finally evaluated the effects of PMA on the expression at the cell surface of adhesion factors such as ICAM-1, and extracellular matrix proteins such as collagen IV, laminin and fibronectin; we have also studied the expression of the integrin vitronectin receptor, a membrane receptor for adhesive proteins. While adhesion factors and extracellular matrix proteins appear to play an important role in the interaction between immune effector and tumour target, it can be supposed that the modulation of such membrane-associated proteins or glycoproteins induces NK and LAK resistance in cancer cells. We indeed found that PMA treatment induced in GLL-19 a marked reduction of membrane expression of collagen IV and ICAM-1; moreover PMA reduced the cell membrane expression of the integrin vitronectin receptor. On the other hand, membrane expression of fibronectin and laminin was not affected by PMA. These data indicate that the acquisition of a NK- and LAK-resistant phenotype by GLL-19 cells occurs together with cell differentiation, down-regulation of membrane expression of collagen IV, ICAM-1 and vitronectin receptor, but in the absence of changes in MHC antigens.This work has been supported by the Italian Association for Cancer Research (A. I. R. C.) and by Istituto Superiore di Sanità, Italy-USA joint program on New Therapies on Neoplasia.     

Analysis of three-dimensional cell imaging obtained with optical microscopy techniques based on defocusing

The properties of an optical microscope are analyzed and analytically evaluated with a simple and effective model in order to understand the true meaning, limitations, and real capabilities of a defocusing technique. Major emphasis is given to the applications related to microscopic objects of biological interest using fluorescence and absorption light microscopy. A procedure for three-dimensional viewing is analyzed and discussed.     

Calf adrenocortical fasciculata cells secrete aldosterone when placed in primary culture

Aldosterone production occurs in the outer area of the adrenal cortex, the zona glomerulosa. The glucocortocoids cortisol and corticosterone, depending upon the species, are synthesized in the inner cortex, the zona fasciculata. Calf zona glomerulosa cells rapidly lose the ability to synthesize aldosterone when placed in primary culture unless they are incubated in the presence of the antioxidants butylated hydroxyanisol and selenous acid, the radioprotectant DMSO, and the cytochrome P-450 inhibitor metyrapone. In the presence of these additives, calf zona fasciculata cells in primary culture synthesize aldosterone at rates which can approach those from cells isolated from the zona glomerulosa. Calf zona glomerulosa and fasciculata cells both responded well to ACTH and angiotensin II, but the zona fasciculata cells respond very poorly compared to glomerulosa cells to increased potassium in the media. Rat zona fasciculata cells in primary culture under similar conditions did not synthesize aldesterone, suggesting that the regulation of the expression of the enzymes responsible for the biosynthesis of aldosterone in the two species is different. Two distinct cytochrome P-450 cDNAs which hydroxylate deoxycorticosterone at the 11β position have been described in the rat, human and mouse. Both cytochrome P-450 cDNAs have been cloned and expressed in non-steroidogenic cells, but only one is expressed in the zona glomerulosa and only this glomerulosa cytochrome P450 can further hydroxylate deoxycorticosterone to generate aldosterone. Two bovine adrenal cDNAs have been described with 11β-hydroxylase activity and their expression products in transiently transfected COS cells can convert deoxycorticosterone into aldosterone. Both enzymes are expressed in all zones of the adrenal cortex. Zonal regulation of aldosterone synthesis in the bovine adrenal gland may be due to an 11β-hydroxylase with aldosterone synthesizing capacity which has not yet been isolated. Alternatively, a single enzyme might be responsible for the several hydroxylations in the pathway between deoxycorticosterone and aldosterone and zonal synthesis might be controlled by unknown factors regulating the expression of C-18 hydroxylation. The incubation of zona fasciculata with antioxidants and metyrapone results in atypical expression of this activity by an unclear mechanism.     

Antiproliferative Activity and Ultrastructural Changes in Promastigote and Amastigote forms of Leishmania amazonensis Caused by Limonene-Acylthiosemicarbazide Hybrids

Leishmaniasis is a tropical zoonotic disease. It is found in 98 countries, with an estimated 1.3 million people being affected annually. During the life cycle, the Leishmania parasite alternates between promastigote and amastigote forms. The first line treatment for leishmaniasis are the pentavalent antimonials, such as N-methylglucamine antimoniate (Glucantime®) and sodium stibogluconate (Pentostam®). These drugs are commonly related to be associated with dangerous side effects such as cardiotoxicity, nephrotoxicity, hepatotoxicity, and pancreatitis. Considering these aspects, this work aimed to obtain a new series of limonene-acylthiosemicarbazides hybrids as an alternative for the treatment of leishmaniasis. For this, promastigotes, axenic amastigotes, and intracellular amastigotes of Leishmania amazonensis were used in the antiproliferative assay; J774-A1 macrophages for the cytotoxicity assay; and electron microscopy techniques were performed to analyze the morphology and ultrastructure of parasites. ATZ−S-04 compound showed the best result in both tests. Its IC₅₀, in promastigotes, axenic amastigotes and intracellular amastigotes was 0.35±0.08 μM, 0.49±0.06 μM, and 15.90±2.88 μM, respectively. Cytotoxicity assay determined a CC₅₀ of 16.10±1.76 μM for the same compound. By electron microscopy, it was observed that ATZ−S-04 affected mainly the Golgi complex, in addition to morphological changes in promastigote forms of L. amazonensis.