Growth imbalance and altered expression of cyclins B1, A, E, and D3 in MOLT-4 cells synchronized in the cell cycle by inhibitors of DNA replication.
Expression of cyclins at the translational level is generally studied by immunoblotting lysates of cells synchronized in the cycle. Most methods used to synchronize transformed cells induce growth imbalance. The aim of the present study was to analyze levels of cyclins B1, A, E, and D3 in the respective phases of the cycle in synchronized human leukemic MOLT-4 cells, correlate them with total cellular protein content (reflecting growth imbalance), and compare the synchronized cells with cells from unperturbed, asynchronous cultures. Expression of cyclins detected immunocytochemically in individual permeabilized cells was analyzed by multiparameter flow cytometry, which made it possible to relate position of the cell in the cell cycle with cyclin expression. Cells synchronized at the G1-S boundary by thymidine, mimosine, or aphidicolin had about 40% increased total protein and 4-5 fold higher levels of cyclins E and B1 compared to their G1 counterparts from unperturbed cultures. Expression of cyclin A in synchronized cells was 2-fold higher, while expression of cyclin D3 was essentially unaltered. The synchronized cells traversing S phase after release from the block had elevated but decreasing levels of cyclins E, B1, and A. Although the cyclin expression of cells reentering G1 was similar to that of their counterparts from asynchronous cultures, the total protein content was still elevated by about 30%. The data indicate that due to different degrees of imbalance in total protein and individual cyclin content, levels of cyclins detected by immunoblotting of cell lysates from synchronized cultures may not be representative of their expression in unperturbed cells. The elevated level of cyclin B1 in the cells arrested at the G1-S boundary may reflect the increased half-life of this protein, stabilized as the result of the overexpression of cyclin E.
Gong
J
J
Cancer Research Institute, New York Medical College, Valhalla 10595, USA.
Traganos
F
F
Darzynkiewicz
Z
Z
D002453:Cell Cycle / Q000235:genetics*; D002455:Cell Division / Q000502:physiology; D019926:Cyclin B; D056744:Cyclin B1; D056743:Cyclin D3; D016213:Cyclins / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D016193:G1 Phase / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D007938:Leukemia; D008938:Mitosis / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D016196:S Phase / Q000502:physiology; D013936:Thymidine / Q000037:antagonists & inhibitors; D014407:Tumor Cells, Cultured / Q000166:cytology
C534699:CCNB1 protein, human; C534696:CCND3 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D056743:Cyclin D3; D016213:Cyclins; D011506:Proteins; D004247:DNA; D013936:Thymidine