[Morphology, cell-cell interactions, and migratory activity of IAR-2 epithelial cells transformed with the RAS oncogene: contribution of cell adhesion protein E-cadherin].

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphologic and mi gration characteristics of epithelial cells of Iar1162 and IAR1170 clones derived from a mixed culture of on cogene N-RasV12-transformed cell line IAR-2. It was found that the mutant oncogene RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, formed E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the mutant oncogene RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR 1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of locomotor activity in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the locomotor activity of transformed epithelial cells and plays an important role in their collective migration.

[Sensitivity of the proliferation of normal and tumor cells to cytochalasin D]. / Chuvstvitel'nost' proliferatsii normal'nykh i opukholevykh kletok k tsitokhalazinu D.

The effect of cytochalasin D, which is known to disrupt specifically actin cytoskeleton, on DNA replication was studied. The incubation of cultured mouse embryonic fibroblasts (MEF), cells of Balb/3T3 line and cells of minimally transformed clones 12 MC and 6 st/T CAK-7 line with cytochalasin D leads to inhibition of DNA synthesis. A complete inhibition of labeled index in MEF culture was observed after an 8 day incubation in cytochalasin D. Part of cells of clones 12 MC and 6 st/T were insensitive to cytochalasin D and continued to enter to S-phase even after a 10 day incubation. The transfer of cells into a fresh medium leads to a rapid restoration of DNA synthesis. Strongly transformed L cells were almost insensitive to cytochalasin D. Thus, the reorganization of actin cytoskeleton caused by cytochalasin D can inhibit the cycle of normal and minimally transformed cells. In the course of neoplastic progression, in the transformed cells there is a loss of dependence of cell proliferation on microfilament system.

[Effect of cytochalasin D on DNA synthesis in cultured cells]. / Deistvie tsitokhalazina D na sintez DNK v kul'tiviruemykh kletkakh.

The effect of cytochalasin D (CD), an agent specifically destroying actin cytoskeleton, on DNA replication of cultured mouse embryonic fibroblasts (MEF) and BALB/3T3 strain cells was studied. Incubation of normal cells with CD resulted in progressive inhibition of DNA synthesis: in the first 16-20 h the percentage of cells pulse-labelled with 3H-thymidine was similar to that in control cultures, on day 8 the percentage of labelled cells was 7-8 times lower than in the control. The transfer of cells into fresh medium upon 8-day incubation in the presence of CD resulted in the recovery of DNA synthesis. Similar curves of DNA synthesis inhibition in the presence of CD and of DNA synthesis recovery in fresh medium were observed both in mononuclear and binuclear cells. Thus, CD-induced reorganization of actin cytoskeleton can have an abrupt but reversible disturbing effect on normal cell cycle.

Polarization of cytoplasmic fragments microsurgically detached from mouse fibroblasts.

We have studied the polarity of cytoplasm organization in tiny fragments of mouse embryo fibroblasts, produced by the microsurgical separation of long processes of cytochalasin-treated cells. In the cytochalasin-free medium fragments respread and developed small lamellas at one or both of their ends. Granules, visible at phase-contrast optics, were always collected in the central part of the fragment. Lamellas of the fragment, as well as lamellar cytoplasm of parent cells, were able to clear surface receptors patched by concanavalin A and an antibody to concanavalin A. Immunofluorescence microscopy showed that the fragments always contained actin microfilament bundles parallel to the long axis of the fragment, but microtubules were present not more than for 6 hrs after detachment of the fragments from the cell bodies. Fragments detached from the cells treated with colcemid and cytochalasin simultaneously and transferred into the drug-free medium never had any microtubules. In spite of that, their behaviour was similar to the behaviour of the fragments that were produced from the control cells treated only with cytochalasin. These results show that the small fragments of mouse embryo fibroblasts are able to maintain the polar organization of cytoplasm and the microtubules are not responsible for this organization.