J.E. Thompson

Correlation of (Na+K+)-ATPase activity with growth of normal and transformed cells

Abstract The (Na+K+)-stimulated Mg2+-dependent ATPase activities of 3T3 and SV40 transformed 3T3 cells were compared as a function of cell population density. For normal cells the enzyme activity remained relatively constant during exponential growth, but decreased sharply coincident with contact inhibition of growth at confluence. This decrease in activity could be reversed by stimulating contact-inhibited cultures to undertake renewed short-term growth either by adding fetal calf serum or changing the medium completely. Transformed cells did not experience a decrease in (Na+K+)-ATPase activity upon reaching confluence, but this is consistent with the fact that they were still growing exponentially at this stage. However, non-confluent cultures of both normal and transformed cells incurred a marked decrease in levels of the enzyme when growth was inhibited by serum depletion. The results have been interpreted as indicating that levels of (Na+K+)-ATPase in both normal and transformed cells are correlated with growth. Hence the different patterns of ATPase activity displayed by malignant cells and their normal counterparts with increase in cell number appear to be a reflection of their dissimilar growth behaviours rather than of any innate difference between them.

A scanning electron microscopic study of phagocytosis

Abstract Phagocytosis of latex beads by Acanthamoeba castellanii was examined by scanning electron microscopy. The detailed panoramic views of the cell surface provided by this technique have confirmed the formation of food cups designed to mediate ingestion and have also provided evidence for temporal changes in surface morphology that relate to the accumulation of internalized beads. In particular, the filopodia withdraw and convolutions in the plasma membrane characteristic of cells prior to the initiation of phagocytosis become less distinct. Ultimately the cells re-assume their pre-phagocytotic morphology. The observations suggest that there is surplus plasma membrane some of which is expended during phagocytosis, and that membrane replacement could, therefore, occur after rather than during the most active period of ingestion.

A scanning electron microscopic study of the excystment process of Acanthamoeba castellanii.

Abstract Excystment of Acanthamoeba castellanii has been examined by scanning electron microscopy with a veiw to establishing the manner in which the emerging trophozoite escapes from the cyst wall. The excystment process was initiated by placing mature cysts in the optimal growth medium used for routine culturing of the amoebae. Ostioles, identifiable by the presence of an operculum delimited by a circular ridge, were clearly evident on the surfaces of mature cysts. The first indication of emergence visible by scanning electron microscopy was the appearance of a cytoplasmic bud pushing through an ostiole from which the operculum had been removed. The emerging cytoplasmic bud did not possess the long filamentous filopodia characteristic of the trophozoite; these did not appear until the amoeba had completely excysted. The hole through which the amoeba had emerged was clearly apparent on the surfaces of the empty cyst walls. In other respects, however, the surfaces of the empty cyst walls were indistinguishable from those of mature cysts. Highly sculptured interconnecting ridges delimiting shallow craters were clearly apparent and in some cases intact ostioles were distinguishable.

An evaluation of lysosomal enzyme leakage as a factor influencing the behaviour of transformed cells

Abstract Commercially available fetal calf serum has been found to contain substantial amounts of protease, acid phosphatase, and glycosidase activity. All such activity can be inactivated by heating serum to 70 °C for 15 min. Moreover, this heat inactivation has no influence on the ability of serum to support growth of pyBHK cells, and only marginally affects the growth properties of BHK cells. Glycosidases and acid phosphatase were also detectable in medium containing heat-inactivated serum and conditioned by growth of either BHK or pyBHK cells. This latter activity can be attributed solely to enzymes derived from the growing cells and, for most of the enzymes monitored, was present to a greater extent in medium conditioned by transformed cells as compared to normal cells. In all cases, however, enzyme activity derived solely from cells was only 1 to 10% of that indigenous to commercial tissue culture medium by virtue of its fetal calf serum component. The relevance of this information to current speculation about a role for lysosomal enzymes as mediators of the uncontrolled growth characterizing transformed cells is discussed.

A scanning electron microscopic study of the encystment of Acanthamoeba castellanii

Abstract The sequential changes of the surface morphology during the induced encystment of Acanthamoeba castellanii were observed with the scanning electron microscope. The vegetative cell is covered with numerous long and slender filopodia, whereas the cyst completely lacks filopodia and has a surface composed of sculptured ridges that delimit irregular shallow craters. The initial stages of the transition from trophozoite to cyst are marked by a thickening and shortening of the filopodia. The recession of filopodia continues until the surface is covered with short stubby processes. In some cases, portions of the cell surface become quite smooth. At this stage of encystment, thin welts are also evident on the surface. These structures develop into the thick, interconnecting ridges that characterize the surface of the mature cyst.

Cytosol counterparts of plasma membrane proteins and their incorporation into the plasmalemma of normal and transformed cells

Abstract Isolated plasma membranes from BHK and pyBHK cells incorporated radioactivity when incubated in corresponding cytosol fractions which had been labelled in vivo by growing the cells in 14C-labelled protein hydrolysate or in vitro by lactoperoxidase-catalysed iodination. The incorporation increased linearly with time and reached saturation at high cytosol : membrane protein ratios in the incubation mixture. Polyacrylamide gel electrophoresis of treated membranes showed that for plasmalemma from both BHK and pyBHK cells four major membrane proteins were clearly labelled, suggesting that they had been exchanged for counterparts in the cytosol. The same proteins were labelled in each type of membrane. These proteins were also discernible in polypeptide profiles of labelled cytosol and showed higher specific radioactivities in the cytosol than in the treated membranes. Inasmuch as these proteins were not detached from the membrane by sonication, high ionic strength or chelation, they appear to be integral rather than peripheral. The data thus indicate that integral proteins native to the plasma membrane are present in the cytosol and can incorporate into pre-existing plasmalemma.

Enzymatic properties of microsomal membranes from the protozoan Acanthamoeba castellanii

Analyses of microsomes from Acanthamoeba castellanii have revealed certain fundamental similarities in enzymatic organization between microsomal membranes from this cell, a protozoan, and corresponding membranes from mammalian cells. Separation of the microsomes into their rough and smooth subfractions was achieved by density gradient centrifugation of a microsomal suspension. The subfractions were identified by chemical analyses. Enzymatic determinations showed that the rough and smooth membranes possess certain enzymes in common but distinctive differences in enzyme activities were also apparent. Substantial levels of two phosphatases, glucose-6-phosphatase and 5′-nucleotidase, were present in both rough and smooth subfractions. 5′-Nucleotidase was approximately equally distributed between the two, but the specific activities of glucose-6-phosphatase were consistently 2–3 fold higher in the rough membranes than in the smooth. On the other hand, NADPH-cytochrome c reductase and rotenone-insensitive NADH-cytochrome c reductase displayed a distinctly non-random distribution in that they were primarily associated with smooth surfaced membranes and showed only low activities in the rough subfraction. Since the major component of a microsomal fraction is fragmented endoplasmic reticulum, it would appear that in Acanthamoeba the rough and smooth membranes of this organelle have diverse functions.

Changes in glycosidase enzyme activity during growth of normal and transformed cells

Abstract Homogenate specific activities of four glycosidases—N-acetyl-β- d -galactosaminidase, N-acetyl-β- d -glucosaminidase, β- d -galactosidase and α- d -mannosidase—were determined at daily intervals during preconfluent growth and after confluence for BHK and pyBHK cells. Regression lines were determined and the regressions were subjected to analysis of covariance in order to compare slopes and elevations. The results indicate that levels of glycosidase activity are correlated with growth; activity increased in growing cells but remained essentially constant in quiescent cells. Thus by extrapolation, in the in vivo state tumour cells would tend to have higher levels of glycosidase activity than their normal counterparts by reason of their inability to achieve growth control. The data are consistent with previous reports of elevated glycosidase and protease activity in oncogenically transformed cells and provide further evidence for a role of lysosomal enzymes in mediating the uncontrolled growth of transformed cells.

Contact-mediated changes in cycloheximide-induced agglutinability of BHK cells.

Abstract Suspension cultures of BHK cells grow in MEM supplemented with 10% fetal calf serum at about 50% the rate of corresponding monolayer cultures. If the serum supplement is reduced to 2% no increase in cell number is observed. When 10% serum is used small spheroids comprising 3–4 cells form within a 24 h period, but in 2 % serum the cells remain single over the same period. The addition of cycloheximide to contact-inhibited monolayer cultures induces high levels of ConA agglutinability within 6 h, yet growing non-confluent cells are rendered only about half as agglutinable by the same treatment. Cycloheximide treatment of suspension cultures causes growing cells to become agglutinable, but non-growing cells, which do not form spheroids, remain non-agglutinable even after 24 h of treatment. This suggests that the pronounced effect of cycloheximide on the agglutinability of contact-inhibited cells in monolayer culture reflects their confluence rather than suspended growth, and that the turnover rate of surface molecules determining the agglutinability state of cells is enhanced as cell-to-cell contact increases.