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.     

The effect of calcium chelators on microsomal pyridine nucleotide-linked dehydrogenases of sugarbeet cells

Cell suspension cultures of Beta vulgaris L., treated with calciumchelators or untreated, were used to characterize pyndine nucleotide-dependentdiaphorases of microsomes. The microsomal activity of NADH-dependentduroquinone reductase from cultures treated with 10 mM Na₂EGTAfor 24 h increased by a factor of 1.8 with respect to controlmicrosomes, and was mainly associated with particles of d=1.17gml^–1. NADPH-duroquinone reductase and NADH-ferricyanidereductase activities showed smaller increases. Bacterial protein-lipopolysaccharidecomplexes (prLPS) also promoted the increase of microsomal diaphorases;CaEGTA was Ineffective. EGTA effects on enzymes of supernatantand mitochondria were negligible, although Na₂EGTA treatmentinduced cell aggregation and strong acidification of the medium. When microsomes from control cultures were solubilized with1% LPC and fractionated in high-efficiency gel permeation columns(FPLC) the diaphorase activities were found associated to threemajor proteins: (i) NADH-specific quinone reductase (NADH-QR)of 340 kDa; (ii) pyndine nucleotide-nonspecific quinone reductase(NAD(P)H-QR) of 85 kDa also having ferricyanide reductase activity;(iii) NADH-specific ferricyanide reductase (NADH-FCR) of 38kDa. The microsomes from EGTA-treated cells also showed a highlyactive NADH-QR having a larger molecular mass (440 kDa) thanin control cells. NAD(P)H-QR also showed increased activity.We conclude that external Ca²⁺ chelation induces changes indehydrogenase components in microsomes. Furthermore, prLPS probablyexert part of their effect on plants through Ca²⁺ chelation. Key words: Beta vulgaris, cell cultures, calcium chelators, diaphorase, NAD(P)H-dehydrogenase, lipopolysaccharide, EGTA, quinone reductase     

t(21q21q)/r[t(21q21q)] mosaic in two unrelated patients with mild stigmata of Down's syndrome

Two cases of t(21q21q)/r[t(21q21q)] mosaic in unrelated infants, 17 and 14 months old respectively are reported. The proportion of cells with the ring chromosome was 45% in the former, 80% in the latter. Both cases had mild manifestations of the Down's syndrome. The origin of this unusual mosaicism as well as the significance of the difference in the proportions of the ring chromosome in the two have been discussed.