Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression

When centrosomes are destroyed during prophase by laser microsurgery, vertebrate somatic cells form bipolar acentrosomal mitotic spindles (Khodjakov, A., R.W. Cole, B.R. Oakley, and C.L. Rieder. 2000. Curr. Biol. 10:59-67), but the fate of these cells is unknown. Here, we show that, although these cells lack the radial arrays of astral microtubules normally associated with each spindle pole, they undergo a normal anaphase and usually produce two acentrosomal daughter cells. Relative to controls, however, these cells exhibit a significantly higher (30-50%) failure rate in cytokinesis. This failure correlates with the inability of the spindle to properly reposition itself as the cell changes shape. Also, we destroyed just one centrosome during metaphase and followed the fate of the resultant acentrosomal and centrosomal daughter cells. Within 72 h, 100% of the centrosome-containing cells had either entered DNA synthesis or divided. By contrast, during this period, none of the acentrosomal cells had entered S phase. These data reveal that the primary role of the centrosome in somatic cells is not to form the spindle but instead to ensure cytokinesis and subsequent cell cycle progression.     

Basic studies on gene therapy of human malignant melanoma by use of the human interferon beta gene entrapped in cationic multilamellar liposomes. 1. Morphology and growth rate of six melanoma cell lines used in transfection experiments with the human interferon beta gene

Six cell lines of human malignant melanoma: A375, A375.2, G361, HMV-1, MM8.1 and WM115 were seeded at densities of 1 × 10⁴ cells/ml, 2 × 10⁴ cells/ml or 3 × 10⁴ cells/ml of RPMI medium supplemented with 10% fetal calf serum and antibiotics in a humidified atmosfere of 5% CO₂ at 37°C. A375 cells were also grown in Dulbecco's minimum Eagle's medium (DMEM medium). The morphology was studied by phase contrast light microscopy. At 4 days after seeding the colonies of A375 cells and HMV-1 cells were oval-shaped, the cells were polyhedrical and were making contact with each other regularly. The remaining cells were scattered, more elongated, and made contact randomly. G361 cells and MM8.1 cells tended to form superposed layers before 100% confluency was achieved. There were great differences in the growth rate and doubling time of melanoma cells. The doubling time in day 1 was short (around 6-12 h) in the case of A375, G361 and HMV-1 cells, longer (around 18h) in the case of MM8.1 cells and very long (ranging between 26 and 89 h) for A375.2 and WM115 cells. There were also differences in the doubling time of cells as a function of the cell density at seeding. On the other hand, except for MM8.1 cells, there were differences between the doubling time in day 2 compared to day 1.     

Endogenous Tumor Necrosis Factor α Unmasks the Binding Sites for N-Acetylglucosaminyl-(β1-4)-N-Acetylmuramyl-Alanyl-D-Isoglutamine on the Surface of Melanoma Cells

The surface of the melanoma BRO cells was shown to contain binding sites for N-acetylglucosaminyl-(1-4)-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP). Their number (1500 ± 200 per cell) and affinity (K _d= 10 ± 1.2 nM) were determined. The occurrence of these sites was found to correlate with the ability of the melanoma cells to react in vitrowith GMDP by increasing the expression of melanoma-associated antigens (MAA). An increased number of the GMDP binding sites (5200 ± 500 per cell) was observed upon treating the melanoma BRO cells with tumor necrosis factor (TNF-). The mechanism of the TNF- action most likely involves the unmasking of GMDP binding sites, initially expressed on the cell surface, by activating the endogenous protease that hydrolyzes surface proteins, in particular, highly glycosylated LAMP-2 protein exposed on the melanoma cell surface.     

Growth factor-dependent signaling and cell cycle progression

There are three central ideas contained within this review. Firstly, growth factor-stimulated signaling is not restricted to a 30–60 min window, but occurs at a much later time as well. Secondly, the second wave of signaling overlaps temporally with the cell cycle program and may be directly responsible for engaging it. Thirdly, the G1 to S interval appears to encompass two distinct phases of the cell cycle, during which the coordinated activation of distinct sets of signaling enzymes drives cell cycle progression. Each of these concepts is likely to initiate new investigation and hence provide additional insight into the fundamental question of how growth factors drive cell proliferation.     

3′-Azidothymidine significantly alters glycosphingolipid synthesis in melanoma cells and decreases the shedding of gangliosides

In this report, we establish that 3-azido-3-deoxythymidine (AZT) treatment of melanoma cells greatly alters the pattern of glycosphingolipid biosynthesis. In SK-MEL-30 cells, synthesis of the gangliosides GM3 and GD3 was significantly inhibited (60% and 50% of control, respectively) and the production of their precursor, lactosylceramide, was stimulated by 2.5-fold. Control experiments established that phospholipid synthesis was not affected by AZT treatment, consistent with AZT treatment only affecting lipid biosynthetic reactions that involve glycosylation. Likely as a consequence of decreased rates of ganglioside synthesis, AZT treatment of SK-MEL-30 cells also significantly suppressed the amount of gangliosides shed from the membranes of these cells. Since shedding of gangliosides has been proposed to allow melanoma cells to avoid destruction by the immune system and alterations of glycosphingolipid levels are likely important for the malignant cell phenotype, these results may have important implications regarding the potential use of AZT or related glycosylation inhibitors as cancer chemotherapeutics.Abbreviations AZT 3-azido-3-deoxythymidine - Cer ceramide - Crbr cerebroside - Gal galactose - GalNAc N-acetylgalactosamine - Glc glucose - GlcNAc N-acetylglucosamine - GD3 disialyl lactosylceramide - GM3 sialyl lactosylceramide - HPTLC high-performance thin layer chromatography - LacCer lactosylceramide - MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide - NeuAc N-acetylneuraminic acid - PA phosphatidic acid - PBS phosphate-buffered saline - PC phosphatidylcholine - PDMP 1-phenyl-2-decanoylamino-3-morpholino-1-propanol - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SM sphingomyelin     

Genetic instability in human mismatch repair deficient cancers

Cancers showing microsatellite instability (MSI-H) are frequent tumors characterized by inactivating alterations of mismatch repair (MMR) genes that lead to an incapacity to recognize and repair errors that occur during DNA replication. These cancers can be inherited as in the human non-polyposis colorectal cancer syndrome, or can occur sporadically in 10-15% of colorectal, gastric and endometrial cancers. MSI-H tumors have different clinicopathological features compared to cancers without this phenotype, termed MSS, and the repertoire of genetic events involved in tumoral progression of both phenotypes is thought to be different. In MSI-H tumors, most of the genetic changes occur at both non-coding and coding microsatellites that are particularly prone to errors during replication due to their repetitive sequence. This mechanism appears to be the main "genetic pathway" by which functional changes with putative oncogenic effects are accumulated in these tumors.     

Reactive oxygen species,antioxidant mechanisms and serum cytokine levels in cancer patients: impact of an antioxidant treatment

Objective. So far, it is not well established whether oxidative stress found in cancer patients results from an increased production of oxidants in the body or from a failure of physiological antioxidant systems. To further investigate this question we have assessed the blood levels of reactive oxygen species as a marker of free radicals producing oxidative stress and the most relevant of the physiological body enzymes counteracting reactive oxygen species, namely glutathione peroxidase and superoxide dismutase. Serum levels of proinflammatory cytokines and IL‐2 were also investigated. All these parameters were studied in relation to the clinically most important index of disease progression, namely Performance Status (ECOG PS). We also tested the reducing ability of different antioxidant agents on reactive oxygen species levels by measuring the increase in glutathione peroxidase activity, and the reduction of serum levels of IL‐6 and TNF. Design, setting and subjects. We carried out an open non randomized study on 28 advanced stage cancer patients (stage III, 10.7%, and stage IV, 89.3%) with tumours at different (8) sites: all were hospitalized in the Medical Oncology Dept, University of Cagliari Interventions. The patients were divided into 5 groups and a different antioxidant treatment was administered to each group. The selected antioxidants were: alpha lipoic acid 200 mg/day orally, N‐acetylcysteine 1800 mg/day i.v. or carboxycysteine‐lysine salt 2.7 g/day orally, amifostine 375 mg/day i.v., reduced glutathione 600 mg/day i.v., vitamin A 30000 IU/day orally plus vitamin E 70 mg/day orally plus Vitamin C 500 mg/day orally. The antioxidant treatment was administered for 10 consecutive days. Results. Our results show that all but one of the antioxidants tested were effective in reducing reactive oxygen species levels and 2 of them (cysteine‐containing compounds and amifostine) had the additional effect of increasing glutathione peroxidase activity. Comprehensively, the “antioxidant treatment” was found to have an effect both on reactive oxygen species levels and glutathione peroxidase activity. The antioxidant treatment also reduced serum levels of IL‐6 and TNF. Patients in both ECOG PS 0‐1 and ECOG PS 2‐3 responded to antioxidant treatment.     

pH-sensitive liposomes are efficient carriers for endoplasmic reticulum-targeted drugs in mouse melanoma cells

Tyrosinase, the key enzyme of melanin biosynthesis, is inactivated in melanoma cells following the incubation with the imino-sugar N-butyldeoxynojirimycin, an inhibitor of the endoplasmic reticulum N-glycosylation processing. We have previously shown that tyrosinase inhibition requires high NB-DNJ concentrations, suggesting an inefficient cellular uptake of the drug. Here we show that the use of pH-sensitive liposomes composed of dioleoylphosphatidylethanolamine and cholesteryl hemisuccinate for the delivery of NB-DNJ reduced the required dose for tyrosinase inhibition by a factor of 1000. The results indicate that these pH-sensitive liposomes are efficient carriers for imino-sugars delivery in the endoplasmic reticulum of mammalian cells.