cytochalasin-d and Leukemia--Erythroblastic--Acute

The cytotoxic interaction between cloned human Natural Killer (NK) cells and K562 target cells was studied using confocal laser scanning microscopy (CLSM) and conventional fluorescence microscopy. We observed, using fixed as well as living cells, the occurrence of (pseudo) emperipolesis during the interaction. About 30% of conjugated NK cells penetrated, partly or completely, into the target cells (in-conjugation). Virtually all in-conjugated target cells exhibited polymerized actin. Killer cells of in-conjugates were frequently seen approaching the target cell nucleus or aligning along it. If the cytotoxic process was inhibited by the absence of calcium neither actin polymerization nor in-conjugation were observed. A kinetic study showed that in-conjugation starts somewhat later than actin polymerization but still within a few minutes after addition of calcium to conjugates previously formed in the absence of calcium. The presence of cytochalasin D (an inhibitor of actin polymerization) completely inhibited in-conjugation and partly reduced the cytotoxic activity. Zinc ions (endonuclease inhibition) inhibited in-conjugation and decreased the total number of target cells with polymerized actin in a concentration dependent manner. Cytotoxic activity was also reduced but not as efficiently as in-conjugation. Our study demonstrates that in-conjugation represents a significant fraction of the cytotoxic interaction. The results indicate that it may be a consequence of an actin polymerization and endonuclease activity dependent part of a cytotoxic mechanism.

Topics: Actins; Apoptosis; Biopolymers; Calcium; Cell Adhesion; Cell Fusion; Cytochalasin D; Cytotoxicity, Immunologic; Endonucleases; Humans; Killer Cells, Natural; Leukemia, Erythroblastic, Acute; Microscopy, Confocal; Microscopy, Fluorescence; Sulfates; Tumor Cells, Cultured; Zinc Compounds; Zinc Sulfate

There is a dynamic equilibrium between monomeric G-actin and polymeric F-actin microfilaments (MFs) in eucaryotic cells. We have previously shown that disruption of MFs with cytochalasin D (CD) induced beta-actin gene transcription, resulting in elevated levels of beta-actin mRNA and protein synthesis. CD also inhibited cell growth by arresting progression through the S phase of the cell cycle. These CD-induced responses were reversible since recovering cells progressed through the G2 phase and resumed normal growth while beta-actin mRNA and protein synthesis rapidly returned to control levels. In the present study, we show that the response of beta- and gamma-actin genes is due to the synthesis of a protein(s) acting at a 5' regulatory element that may be independent of or require sequences in addition to the serum response element (SRE). CD induces beta- and gamma-actin mRNA in a dose-dependent manner, reaching a maximum of 20-fold over control mRNA levels at 30 microM. beta- and gamma-Actin gene expression was also induced 5-fold by serum stimulation of quiescent murine erythroleukemia (MEL) cells, while combined treatment with serum and CD had an additive effect. Two protein synthesis inhibitors, cycloheximide and puromycin, blocked the CD-induced increase in beta-actin mRNA, in contrast to the serum-induced increase which is insensitive to inhibitors of protein synthesis. The rapid return of beta-actin mRNA to basal levels following CD removal did not require protein synthesis nor did it require progression through the G2 phase of the cell cycle. A vector containing the 5' end of the beta-actin gene linked to a CAT reporter responded to CD when transfected into MEL cells, localizing the responsive element to the 5' portion of the beta-actin gene. By contrast, a minimal 99-bp actin promoter-CAT construct containing a functional SRE did not respond to CD.

Topics: Actins; Animals; Cytochalasin D; Gene Expression; In Vitro Techniques; Leukemia, Erythroblastic, Acute; Mice; Protein Synthesis Inhibitors; RNA, Messenger; RNA, Neoplasm; S Phase; Tumor Cells, Cultured

The expression of cytoskeletal protein genes may be linked to both cell growth and the status of the cytoskeleton. Actin gene expression was examined in murine erythroleukemia cells treated with the microfilament disrupting agent, cytochalasin D (CD), at a concentration which was determined to inhibit cell growth and arrest cells in the S and G1 phase of the cell cycle. Levels of actin mRNA and protein synthesis were elevated eight- and sixfold, respectively, after 9 h in CD. This increase was reflected in levels of nuclear run-on actin transcripts and prevented by actinomycin D, suggesting that enhanced transcription of the actin gene was responsible for the increase. Removal of CD resulted in immediate resumption of cell cycle progression with the accumulation of a G2-phase-enriched population and a rapid return of actin mRNA and protein synthesis to control levels (half-life 4.8 h). These results are consistent with a model linking actin gene expression to cell growth by regulating transcription during the G1 and mRNA decay during the G2 phase of the cell cycle.

Topics: Actin Cytoskeleton; Actins; Animals; Cell Division; Cytochalasin D; Cytoskeleton; Gene Expression; Interphase; Leukemia, Erythroblastic, Acute; Mice; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured