Insensitivity of stationary cultures of transformed mouse fibroblasts to agents stimulating DNA synthesis in normal cell cultures]

Stationary cultures of the mouse transformed cells L and S-40 sensitive to topoinhibition were found to be insensitive to the action of hyaluronidase, RNAase, and colcemid in doses known to stimulate multiplication of normal mouse fibroblasts. These cultures were still insensitive to the action of medium change and removal of a part of the monolayer.     

Adhesive specificity in normal and transformed mouse fibroblasts

Adhesive specificity was studied in normal and transformed $\text{Balb}\text{c}$ mouse fibroblasts by comparing the number of labeled cells collected from a suspension of these cells by aggregates of various cell types. Aggregates of the two malignant cells examined collected either very many cells (aggregates of SV3T3 cells) or very few cells (aggregates of 3T12 cells). In addition, the relative adhesive behavior of these two aggregate types did not vary according to the cell suspension in which they were circulated. These data make it unnecessary to assume that malignancy is always accompanied by a decrease in intercellular adhesion.The adhesive behavior of normal 3T3 cell aggregates, compared to the aggregates composed of either malignant cell type, varied according to the type of cells in the suspension. Aggregates of 3T3 cells collected an appreciable number of SV3T3 cells but few 3T12 cells. Collection of 3T3 cells by 3T3 aggregates was also low if the 3T3 cells of the suspension were harvested from confluent cultures. However, collection of 3T3 cells by 3T3 aggregates increased significantly, as compared to collection by SV3T3 and 3T12 aggregates in the same cell suspension, if the 3T3 suspension was prepared from sparse cultures.Flat-revertants of SV3T3 cells were also studied. These cells behave like nonmalignant 3T3 cells rather than like the SV3T3 cells from which they were derived.We suggest that malignancy may not be caused by decreased intercellular adhesion as compared to normal cells but, perhaps, by decreased intercellular recognition.     

Methionine biosynthesis in normal and transformed fibroblasts.

SV-40 transformed human fibroblasts show a growth requirement for methionine, whereas normal fibroblasts do not. Activities of the N⁵-methyltetrahydrofolate-homocysteine transmethylase and N^5–10-methylenetetrahydrofolate reductase in extracts of both cell lines are similar. These observations indicate that the absolute growth requirement for methionine observed in these transformed cells does not necessarily involve a deficiency in enzymes related to methionine synthesis.     

Repair replication in cultured normal and transformed human fibroblasts.

Repair replication in response to ultraviolet irradiation has been studied in normal human diploid fibroblast cultures, W138, and an SV40 transformant, VA13. Quantitative comparisons have been made using the combined isotopic and density labeling method for assaying repair replication. We find no significant difference in the amount of repair replication performed its dose response, or the time course between growing and confluent W138 cells, early passage and senescent cells, or normal W138 cells and the transformed VA13 cells. When [3H]dThd was employed as the isotopic label in the presence of a 30-200 fold excess of unlabelled BrdUrd, apparent differences in repair replication were seen between W138 cells shortly after subcultivation and cells which had been allowed to reach confluence. These differences were the same over a wide dose range and regardless of the passage number of the cells, but could be influenced by using different serum lots. The differences were not seen, however, when [3H]BrdUrd provided the isotopic label; thus they reflect either impurities in the [3H]dThd or a slight discrimination by some cellular process.     

Comparison of the mechanical properties of normal and transformed fibroblasts

In order to achieve coordinated migration through extracellular matrix and endothelial barriers during metastasis, cancer cells must be endowed with specific structural and adhesive properties. In this context, comparison of the mechanical properties of transformed versus normal cells, on which little quantitative information is available, was the focus of this study. Normal human dermal fibroblasts and their SV40-transformed counterparts were analyzed using various manipulations. First, micropipet aspiration of suspended cells allowed calculation of a cortical tension (similar for normal and transformed cells), and an apparent viscosity (30% lower for transformed than for normal fibroblasts); in addition, transformed fibroblasts exhibited a more fragile surface than their normal counterparts. Second, tangential ultracentrifugation of adherent cells demonstrated cellular elongation in the direction of the centrifugal field and the existence of critical forces for cell detachment, around 10⁻⁷ N: these were 1.6-fold greater for normal than for transformed cells. Finally, examination of the wrinkle patterns formed by cells plated on a deformable polydimethylsiloxane substrate, plus analysis of cell retraction caused by ATP treatment following detergent permeabilization showed that normal fibroblasts exhibited much more contractility than their transformed counterparts, which we characterized by a cell contraction rate. Such quantitative parameters which reveal differences in the mechanical behavior of normal and transformed cells may be used in the future as new markers; of oncogenic transformation.     

Spreading-independent growth of normal fibroblasts in three-dimensional cultures

Changes of cell shape resulting from cellular flattening on culture substratum have previously been demonstrated to correlate with mitotic activity of normal animal cells in monolayer cultures. Here, we compared the shapes and proliferation of chick embryo fibroblasts cultured either in multicellular, multilayered sheets extended between glass fibres, or in standard monolayers. Fibroblasts in sheets retained the mitotic activity characteristic of that observed in sparse monolayer cultures, i.e. considerably higher that in confluent monolayers. Morphometric analyses revealed, however, that the cells in sheets were considerably less flattened than in monolayer cultures. These observations indicate that the modulation of culture conditions resulting in multidirectional cell stretching leads to the dissociation of flattening and mitotic activity of normal animal cells, so long as an intracellular stress field, generated by contractile cytoskeleton and stabilised by intercellular contacts, is maintained.     

Manganese superoxide dismutase in normal and transformed human embryonic lung fibroblasts

The manganese superoxide dismutase (MnSOD) activity of W138 human embryonic lung fibroblasts and SV40-transformed WI38 cells was measured. Under various growth conditions, the transformed cells always had lower MnSOD activity than their normal cell counterparts. The activity of MnSOD changes greatly with the growth conditions in the WI38 cells, while the MnSOD activity in the tumor cells remained more constant. The amount of immunoreactive MnSOD was measured by Western blotting. In all cases studied, the amount of immunoreactive MnSOD was lower in the transformed cells than in the normal cells.     

Eugenol from normal and transformed root cultures of Coluria geoides

Transformed root cultures of Coluria geoides Ledeb. were established with the use of Agrobacterium rhizogenes LBA 9402. Both normal and transformed root cultures were investigated for their growth and yield of eugenol. Normal roots were grown in B5 medium-supplemented with 0.2 mg l^-1 of kinetin and 0.2 mg l^-1 of 1-naphthaleneacetic acid (NAA). Hairy roots grew well in hormone-free B5 medium. Both hairy roots and normal roots produced glycosidic bound eugenol. as with the roots of intact plants, eugenol was the main component of the total essential oils obtained from hairy root and normal root cultures. The yield of eugenol from normal roots was 0.1–0.25% of the dry wt. and depended on the development stage of the culture. Yield of eugenol from hairy roots was 0.08–0.1% of the dry wt. NAA modified the hairy root morphology and influenced the yield of eugenol.Abbreviations NAA 1-naphthaleneacetic acid     

Sulfated mucopolysaccharides from normal and virus transformed rodent fibroblasts.

The sulfated mucopolysaccharide composition of normal and virus transformed Balb 3T3 and BHK21 cell lines is reported. It is shown that normal 3T3 cells contain mainly chondroitin sulfate B and heparitin sulfate. Relatively higher amounts of chondroitin sulface AC were observed in polyoma virus transformed 3T3 cells, besides an absolute increase of all the three sulfated mucopolysaccharides in the polyoma and SV 40 transformed cells. It is shown also that the three sulfated mucopolysaccharides are at least in part at the cell surface. Similar differences in sulfated mucopolysaccharide composition of normal and virus transformed BHK cell lines were also observed.     

The effect of CaCl2 and verapamil on the binding of cisplatin to cultures of normal and transformed human fibroblasts

Normal and transformed human fibroblasts were treated for either 1 sec or 1 h with the antitumor drug cis-dichlorodiamine platinum (cisplatin). The dose response of drug binding and cell survival was determined for cells treated with the drug in the presence or absence of 3.0 mM CaCl2. The levels of drug initially bound to both cell types was similar and was not affected by the presence of Ca2+. The dividing non-transformed cells were most sensitive to killing by short treatment with cisplatin compared to the transformed cells or the confluent non-transformed cultures. After 1 h of cisplatin treatment, the levels of drug bound to the cells were significantly less than that recovered after the shorter treatment. This time-dependent loss of cisplatin was inhibited both by CaCl2 and by the calcium channel blocking agent, verapamil. The higher levels of cisplatin bound after 1 h in the presence of these agents, however, did not in all cases result in decreased survival; the effects were dependent on cell type and on whether the cells were dividing or confluent. Analysis of cisplatin binding to cell cultures indicated that initially the cisplatin was weakly attached to the pericellular and substratum attached material but that with time, the drug bound to this material decreased. This time-dependent removal from the extracellular matrix was much less in the transformed cell cultures and was inhibited by calcium. We propose that the major site of interaction of cisplatin with these cells is in the extracellular matrix and with time the cultures alter their extracellular matrix to decrease this binding. This removal process appears to involve calcium or calcium transport since CaCl2 and verapamil both block these changes.     

Highly permeable intercellular junctions in normal and transformed fibroblast cultures]

Intercellular junctions permeable to ions and fluorescein Na were studied with the aid of intracellular microelectrodes in the cultures of normal and transformed mouse-embryo and hamster-embryo fibroblast-like cells. Normal cells were effectively coupled by the highly permeable junctions. In cultures of 7 types of transformed cells, 3 types of coupling were detected: effective, decreased, and fully reduced couplings. The degree of uncoupling was not correlated with morphological patterns of malignization and tumorogeneity of transformed cultures. The decrease of permeability of intercellular junctions to ions and small molecules is concluded not to be necessary for malignization.     

Lawsone accumulation in normal and transformed cultures of henna,Lawsonia inermis

The improvement of axillary shoot formation of Lawsonia inermis L. cultured in vitro depended on the iron concentration in the culture medium. Regenerated shoots were rooted on a hormone-free half-strength Murashige and Skoog medium (1/2 MS) before transfer to greenhouse conditions. Determination of lawsone in the plant material was investigated using a new HPLC method. The results showed that lawsone accumulation in vivo is restricted to the aerial part of the plant. In addition, the possibility of inducing lawsone biosynthesis in root cultures was studied. Hairy root cultures were established by a co-culture method using leaf segments of L. inermis and Agrobacterium rhizogenes NCIB 8196. Of several basal media tested, the production of lawsone (0.13% dry weight) was only observed in hairy roots tissues incubated in the dark and cultured in 1/2 MS or MS media. This revised version was published online in June 2006 with corrections to the Cover Date.