Multinucleation-induced improvement of the spreading of transformed cells on the substratum.

Abstract

Multinucleation of various cultured cells was produced by polyethylene glycol-induced fusion or by cytochalasin-induced block of mitosis. It was found that multinucleation induced by both methods considerably improved deficient spreading of all the tested transformed fibroblastic lines; average substratum area occupied by one cell and divided per number of nuclei was 2.0-2. (12). The mouse embryo fibroblasts and L-cell derivatives were maintained in the mixture of Eagle's basal medium and 0.5% lactalbumin hydrolysate, 1:1 (vol/vol), with 10% bovine serum. All other cell types were cultivated as described in papers cited above. Cell culture media and foetal bovine serum were from Flow Laboratories. Cycloheximide (Actidione) and cytochalasin B were from Serva (Heidelberg) and Colcemid (demecolcine) was from Sigma. To measure protein synthesis, the cultures were labeled by the addition of 0.5 ,Ci/ml (1 Ci = 3.7x 1010 Bq) of [ 4C]alanine in Earle's solution for 30 min at 37°C and then were washed consecutively by saline and 5% trichloroacetic acid for 5 min. Acid-insoluble radioactivity was measured by liquid scintillation counting.Production of Multinucleated Cells. Two methods were used.Polethylene glycol fusion. The cells washed with serumfree medium were incubated for 70 sec in 50% solutions of polyethylene glycol (PEG) (Mr, 1000; Loba Chemia, Wien, Austria) at 37°C. Then PEG was washed off by the medium, cells were incubated for 2-3 hr at 37°C and seeded on the glass coverslips at a density of about 104 cells per cm2; after 24 hr of cultivation, the cells were fixed and used for examination.Cytochalasin-induced block of cytokinesis. Cytochalasin B (2.5 ,ug/ml) was added to the culture grown on coverslips. The cells were grown with cytochalasin B for 3-5 days until numerous multinucleated cells were formed and then were transferred into the medium without cytochalasin B, incubated for 24 hr, fixed, and used for examination.Methods of Examination. For indirect-immunofluorescence staining, cultures were extracted with buffered 1% Triton X-100 and fixed with formaldehyde. Details of the procedures were described earlier (13,14). The following first-step antibodies were used: rabbit antibodies to actin, Abbreviations: PEG, polyethylene glycol. tTo whom reprint requests should be addressed. 3098The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The HPV16 E5 oncogene inhibits endocytic trafficking

“…Thus the mechanisms and consequences of the reported disruption of the actin cytoskeleton caused by HPV16 E5 may be re¯ected in slow cell motility (Thomsen et al, 1999). Likewise, we observed multinucleation in the HPV16 E5 transfected C127 ®broblasts which may be caused by the disrupted actin cytoskeleton (Lyass et al, 1984). Both issues remain to be further explored.…”

Microtubule-dependent effect of phorbol ester on the contractility of cytoskeleton of cultured fibroblasts.

“…To score the degree of ATPinduced contraction, cultures incubated with and without ATP were photographed with phase-contrast optics and the projected areas of the cells were measured as described (15).…”

Circular Nuclear Alignment in Multinucleate PC12D Cells Produced by Cell Fusion with Polyethylene Glycol.

“…Watt et al (1980) observed after fusion of African green monkey kidney (Vero) cells that nuclei were often arranged around a large centrosome [26]. Lyass et al (1984) reported a wide nuclear ring in the syncytia of transformed mouse L cells [9]. On the fusion of viral infected baby hamster kidney fibroblasts (BHK21-F), nuclei migrated in the syncytial cytoplasm and aligned in tightly packed rows [24,25].…”

“…In contrast, multiple centrioles have been observed in the nucleus-free area of the cytoplasm of these giant cells [2,22]. An interesting feature of these cells was seen in the syncytia produced on cell fusion induced artificially by viral infection or PEG treatment [7,9,10,[24][25][26]. It has been shown that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes cell fusion and the formation of parallel rows of tightly packed nuclei in the syncytial cytoplasm [4].…”

“…Multinucleate giant cells have been studied in various tumor lesions, inflammatory lesions and experimentally induced foreign-body granulomas [9,13,19,22]. In these cells, nuclei were found to be concentrated in one area and occasionally arranged in a semicircular manner.…”