Proc. Nati. Acad. Sci. USA Vol. 75, No. 12, pp. 6064-6068, December 1978

Cell Biology

G2 phase-specific proteins of HeLa cells (two-dimensional gel electrophoresis/cell fusion/alkylating agents/G2 arrest)

ABDULLATIF A. AL-BADER*t, ANTONIO ORENGO*, AND POTU N. RAO*§ Departments of *Developmental Therapeutics and *Biochemistry, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030

Communicated by Daniel Mazia, September 13,1978

ABSTRACT The objective of this study was to determine if HeLa cells irreversibly arrested in G2 phase of the cell cycle by a brief exposure to a nitrosourea compound were deficient in certain proteins when compared with G2-synchronized cells. Total cellular proteins of G2-synchronized, G2-arrested, and S phase-synchronized cells were compared by two-dimensional polyacrylamide gel electrophoresis. The S phase cells differed from the G2-synchronized and G2-arrested cells by the absence of about 35 and 25 protein spots, respectively, of a total of nearly 150. At least nine protein spots in the molecular weight range of 4-5 X 104 that were present in the G2-synchronized cells were absent in both the G2-arrested and the S phase cells. Thus, these studies suggest that the missing proteins are probably necessary for the transition of cells from G2 phase to mitosis. Supplying the missing proteins to the G2-arrested cells by fusion with G2-synchronized cells facilitated the entry of the former into mitosis. Mammalian cells can be blocked in the post-DNA synthetic (G2) period by a brief exposure to certain anticancer drugs such as cis-4-[[[(2-chloroethyl)-nitrosoaminolcarbonyllamino]cyclohexane carboxylic acid (cis-acid), 1,3-cis-(2-chloroethyl)-l-nitrosourea (CCNU), and neocarzinostatin (1, 2). This G2 block, however, is irreversible and is associated with extensive chromosome damage (2). Rao and Rao (2) suggested that clastogenic agents causing DNA damage may induce a loss or modification of a gene(s) necessary for the synthesis of specific proteins required for the transition of cells through G2 to mitosis. The objective of the present study was to test this hypothesis-i.e., to determine (i) if the drug-arrested G2 cells are deficient in some specific proteins compared with cells synchronized in the G2 phase and (ii) if the G2-arrested cells are deficient in certain proteins, could these cells be induced to proceed into mitosis by supplying these proteins through fusion with G2-synchronized cells.

MATERIALS AND METHODS Cells and Cell Synchrony. HeLa cells were grown in suspension culture in Eagle's minimal essential medium as described (3). Cells in a spinner culture were synchronized in S phase by the excess (2.5 mM) deoxythymidine (dThd) double-block technique (4). A high degree of G2 synchrony was obtained by incubating monolayer cultures of synchronized S phase cells with Colcemid (0.05 Ag/ml) between 6 and 8 hr after reversal of the second dThd block and removing mitotic cells by selective detachment. In such G2 populations, the labeling and mitotic indices were less than 5%. Drug Treatment. cis-Acid (NSC-153174), one of the nitrosourea compounds, was selected for this study because it was shown to be relatively more effective in inducing G2 arrest than other agents (2). A stock solution (1000 ,Ag/ml) of cis-acid was prepared just before use by dissolving the drug in 1 ml of abThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

solute ethanol and then bringing the total volume to 10 ml by adding serum-free medium. Induction of G2 Arrest. To induce G2 arrest, spinner cultures of HeLa cells in exponential growth (or those synchronized in S phase) were treated with cis-acid at an optimal concentration (75 ,g/ml) for 60 min. The drug was then removed by centrifuging the cells and washing them twice with fresh drug-free medium. The duration of post-treatment incubation in regular medium was 24 hr for random cultures and 16 hr for cells synchronized in S phase. The percentage of cells arrested in G2 phase was determined by the prematurely condensed chromosome (PCC) method of cell cycle analysis in which drugarrested cells are fused with mitotic HeLa cells by the use of UV-inactivated Sendai virus as described (3). Cell Cycle Kinetics. To test if the drug-induced G2 block could be reversed, the unlabeled G2-arrested cells were fused with G2-synchronized cells prelabeled with [3H]dThd. After completion of cell fusion as described (5), the fused cells were plated in 35-mm Falcon plastic dishes with fresh medium containing Colcemid (0.05 ;tg/ml). Cell samples were taken at regular intervals over a period of 22 hr by trypsinizing the cells in one of the dishes for each sampling. The trypsinized cells were deposited directly on clean slides by the use of the cytocentrifuge. The cells were then fixed in absolute methanol/ glacial acetic acid, 3:1 (vol/vol), extracted with cold 5% (wt/vol) trichloroacetic acid three times, processed for autoradiography, stained with Giemsa, and scored for the percentage of cells in mitosis. Mitotic indices were determined separately for mononucleate and each of the three classes (LL, LU, UU) of binucleate and four classes (LLL, LLU, LUU, UUU) of trinucleate cells (L = labeled nucleus, U = unlabeled nucleus). Among the binucleate and trinucleate cells, there was almost perfect mitotic synchrony among the nuclei-i.e., all the nuclei within a cell were either in mitosis or in interphase. Determination of the Effects of cis-Acid on Cells That Were Not in S Phase. Monolayer cultures of HeLa cells were synchronized into S phase by the dThd double-block technique. Eight hours after the reversal of the second dThd block, when almost all of the cells have completed S phase (LI =

G2 phase-specific proteins of HeLa cells.

Proc. Nati. Acad. Sci. USA Vol. 75, No. 12, pp. 6064-6068, December 1978 Cell Biology G2 phase-specific proteins of HeLa cells (two-dimensional gel...
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