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Detection of Cyclin B1 Expression in G1-Phase Cancer Cell Lines and Cancer Tissues by Postsorting Western Blot Analysis

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Detection of Cyclin B1 Expression in G1-Phase Cancer Cell Lines and Cancer Tissues by Postsorting Western Blot Analysis [CANCER RESEARCH 64, 1607–1610, March 1, 2004] Advances in Brief Detection of Cyclin B1 Expression in G1-Phase Cancer Cell Lines and Cancer Tissues by Postsorting Western Blot Analysis Manli Shen,1 Yongdong Feng,1 Chun Gao,1 Deding Tao,1 Junbo Hu,1 Eddie Reed,2 Qingdi Q. Li,2 and Jianping Gong1 1Tongji Cancer Institute and Center for Molecular Medicine, Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, People’s Republic of China, and 2Mary Babb Randolph Cancer Center and Department of Microbiology, Immunology and Cell Biology, West Virginia University School of Medicine and Robert C. Byrd Health Sciences Center, Morgantown, West Virginia Abstract cyclin B1 can also be detected in the G1 phase under certain circum- stances. Pines et al. (6) reported that cyclin B1 mRNA and protein in Protein complex of cyclin B1 and cyclin-dependent protein kinase 1 HeLa cells were actually found to be present in G -phase cells after a induces phosphorylation of key substrates that mediate cell cycle transi- 1 thymidine-aphidicolin block; however, significant levels of cyclin A tion during the G2-M phase. It is believed that cyclin B1 accumulates in the S phase of the cell cycle and reaches the maximal level at mitosis but or cyclin B1 in the G1 phase were not detected by immunofluores- cence microscopy (7). Gong et al. (8, 9) found that the expression of is absent in G1-phase cells. In the present study, we demonstrated that cyclin B1 was expressed in the arrested G1-phase MOLT-4 lymphocyte cyclin B1 could be detected in synchronized cells arrested at the G1 leukemia cells and in G1 phase T-7 transitional tumor cells, as determined phase, as determined only by flow cytometry. Viallard et al. (10, 11) by flow cytometry. In addition, we showed that cyclin B1 was detected in also showed that cyclin B1 accumulated at the G1 phase in human the G1 phase in breast cancer cells from patient tissues and in lymphocytes leukemic cell lines. However, cyclin B1 expression in asynchronously from patients with leukemia. These findings were confirmed for the first growing subgroup cells was not studied at that time because of time by postsorting Western blot analysis and by confocal microscopy. technique limitations. Besides, they were not able to determine the Furthermore, by using postsorting Western blotting, we found that cyclin unscheduled mode of cyclin B1 in G1-phase cells or the maximal level B1 was expressed in different time-window sections of the G1 phase under different conditions. For the asynchronously growing T-7 cells, cyclin B1 of cyclin B1 expression in G1-phase cells. was detected in early G1 phase, whereas in MOLT-4 cells arrested in G1-S The current study was undertaken to investigate the unscheduled phase, cyclin B1 was mainly detected in late G1 phase. We propose that the cyclin B1 expression in different human cancer cells. We have estab- cyclin B1 expressed in the G1 phase may differ from that expressed in the lished a new method called postsorting Western blotting for the study G2-M phase, and that this unscheduled type of cyclin B1 may play an of unscheduled cyclin B1 expression in asynchronously growing cells. important role in tumorigenesis and apoptosis. We have confirmed, by using this new method, that the strictly conservative protein cyclin B1 in the G -M phase is expressed in Introduction 2 unscheduled mode in the G1 phase of the cell cycle. We have also The eukaryotic cell cycle consists of the following four discrete demonstrated that cyclin B1 is expressed in different time-window phases: G1,S,G2, and M. Cyclins and the cyclin-dependent protein sections of G1 in the G1-phase human tumor cells. kinases (CDKs) are two major classes of regulators for cell cycle progression in all eukaryotes (1–3). The CDKs are enzymes that Materials and Methods phosphorylate other proteins using ATP as the phosphate donor. The role of cyclins is to activate the appropriate CDK at the appropriate Isolation of the Cells from the Cancer Tissues of Patients. We first time in the cell cycle. Cyclin B1, an important member of cyclin isolated human breast cancer cells from the breast cancer tissues of patients. family, is known to form complex with CDK1, which phosphorylates For this, 17 fresh breast cancer tissues were placed in RPMI 1640 after being acquired from operation. Fatty, necrotic, and other extraneous tissues were their substrates to urge cells through the G -M phase (4). Cyclin B1 2 trimmed and minced. The trimmed tissues were filtered through 1-mesh cell has been classified in G2 cyclin because the accumulation of this sieves and rinsed. Finally, the sieved cells were centrifuged for 10 min at 1000 protein begins at the S phase, essentially restricted to the G2-M rpm, and cell pellets were resuspended in 1 ml of culture medium. In addition, transition, reaches the maximal level at mitosis, and then is rapidly lymphocytes were isolated by density gradient centrifugation from the speci- degraded at metaphase-anaphase transition (4, 5). Although the sched- mens of 15 patients with leukemia. Flow cytometry analysis and cell sorting uled expression mode of cyclin B1 has been reported to be strictly for the primary human breast cancer cells and lymphocytes were the same as conserved from yeast to mammalian, researchers have reported that those for the MOLT-4 and the T-7 cell lines, as described below. Cell Analysis by Flow Cytometry. Cell culture was performed as de- Received 10/22/03; revised 1/9/04; accepted 1/15/04. scribed previously (12, 13). Cell synchronization (double thymidine block) Grant support: Made possible by funds from the China State Key Basic Research was performed as described previously (8), and cell analysis by flow cytometry Program (G1998051212), the National Natural Science Foundation of China (Nos. was also performed as described previously (12). 39670265, 39730270, and 37725027), and the Science Foundation of Ministry of Health Cell Sorting. Cell sorting was performed by using a FACSVantage (Bec- (P7070239), and by an NIH/National Center for Research Resources grant (No. P20RR16440-010003). ton Dickinson, San Jose, CA). G1-phase cells were sorted based on DNA The costs of publication of this article were defrayed in part by the payment of page diploidy. To sort subgroups in the G1 phase, the cells were first labeled with charges. This article must therefore be hereby marked advertisement in accordance with cyclin E antibody, and then the labeled cells were sorted in tubes precoated 18 U.S.C. Section 1734 solely to indicate this fact. Note: M. Shen and Y. Feng contributed equally to this work. with FCS, containing 0.5 ml of FCS. The sorting windows were selected to Requests for reprints: Jianping Gong, Department of Surgery, Tongji Hospital, include the cells in G0,G1-early, and G1-late phase, respectively, based on the Tongji Medical College, Huazhong University of Science and Technology, Wuhan cyclin E expression threshold. Cyclin B1 expression in sorted cell groups was 430030, People’s Republic of China. E-mail: [email protected] or Qingdi Q. Li, then analyzed by Western blotting and confocal microscopy. MBR Cancer Center, West Virginia University School of Medicine and Health Sciences Center, P.O. Box 9300, Morgantown, WV 26506. Phone: (304) 293-6870; Fax: (304) 293- Confocal Fluorescence Microscopy after Sorting. Cell morphological 4667; E-mail: [email protected]. characters sorted in different time-window sections of the G1 phase were 1607 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2004 American Association for Cancer Research. DETECTION OF CYCLIN B1 IN G1-PHASE CANCER CELLS screened using a laser scanning confocal microscope (SPII Confocal System; Leica Microsystems, Inc., Exton, PA). Postsorting Western Blot Analysis. Cells collected by pipetting were lysed by Laemmli sample buffer [2% SDS, 62 mM Tris, 10% glycerol, 5% ␤-mercaptoethanol, 0.005% bromphenol blue (pH 6.8)]. Samples were boiled for 5 min and then supersonicated. Total cellular proteins from 0.5 ϫ 106 cells/sample in each lane were subjected to electrophoresis in 12% polyacryl- amide gel. The proteins in the gel were then transferred to polyvinylidene difluoride membrane in mini Vertical Electrophoresis (VE) systems (Amer- sham-Pharmacia, Piscataway, NJ). After the transfer, the membrane was saturated for 1 h with 5% nonfat dry milk in Tris-buffered saline (pH 7.6), with 0.1% Tween 20. The blots were then incubated with primary mouse antihuman cyclin B1 antibody (diluted to 1:1000), followed by alkaline phosphatase- conjugated horse antimouse IgG secondary antibody (Vector Laboratory, Inc., Burlingame, CA) diluted to 1:1000, and the results were detected using enzyme reaction. Molecular weights were determined by comparison to known markers. Results Detection of Cyclin B1 Expression in G1-Phase Cancer Cells Arrested in G1-S Phase. We first determined whether cyclin B1 could be detected in the G1 phase of U-937, T-7-transformed cells, and MOLT-4 leukemic cells blocked in G1-S phase. Fig. 1 compares the bivariate cyclin B1 versus DNA-content distribution of MOLT-4 cells with that of the arrested MOLT-4 cells and transformed T-7 cells. Scheduled expression of cyclin B1, believed to be positive Fig. 2. Confirmation of cyclin B1 expression in G1-phase cells by confocal micros- copy. We confirmed that cyclin B1 could be detected in G -phase cells by confocal strictly in G2-M-phase cells, was observed in exponentially growing 1 microscopy. A, the negative control of cyclin B1 expression. B, cyclin B1 expressed in the G1-early phase of transformed T-7 cells.
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