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

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 circumstances. 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 microscopy. 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. C, cyclin B1 expression occurred in the G1-late phase of blocked MOLT-4 cells. As can be observed, the fluorescence of cyclin B1 is located in both nuclei of the two G1-phase cells (B and C).

MOLT-4 cells. In contrast, cyclin B1 in the G1-phase MOLT-4 cells was negative (Fig. 1A). However, analysis of cyclin B1 expression in

MOLT-4 cells blocked in G1-S phase and in T-7-transformed cells revealed that G1-phase cells had cyclin B1-positive fluorescence sig- nals (Fig. 1, C and D). These dot plots indicate that cyclin B1 is

expressed in cells residing not only in the G2-M phase but also in the G1 phase of the cell cycle. Cyclin B1 Expression in G1 Phase Cancer Cells Was Confirmed by Confocal Microscopy. Next, we confirmed that cyclin B1 expres-

sion occurred in G1-phase cells by confocal microscopy (Fig. 2). The cells sorted in exponentially growing G1-phase MOLT-4 cells were used as negative controls (Fig. 2A). As can be observed, the fluores-

cence of cyclin B1 in the G1-phase cells is located in the nuclei of both the T-7 cells (Fig. 2B) and the MOLT-4 cells (Fig. 2C).

The Time-Window Sections of G1 When Cyclin B1 Is Differ- entially Expressed in Synchronized and Asynchronously Growing Tumor Cells. Because the level of cyclin E is consecutively in-

creased during G1 phase, we used the fluorescence intensity of cyclin E as a marker for different time-window sections of cells in the G1 phase detected by flow cytometry. All G1-phase populations were divided into the following three groups: G0,G1-early, and G1-late- phase cells, based on the expression of cyclin E detected by immu- nofluorescence (Fig. 3, A and C). Western blot analysis of the three

sorted subgroups of the G1-phase cells showed that cyclin B1 expression was in G1-early phase in asynchronously growing T-7 cells (Fig. 3B). In contrast, cyclin B1 expression was mainly in G1-late phase in synchronized MOLT-4 cells (Fig. 3D).

Detection of Cyclin B1 Expression in G1-Phase Cancer Cells Fig. 1. Bivariate distributions of cell populations from untreated and arrested MOLT-4 from the Cancer Tissues of Patients. Unscheduled expression of cells and from T-7 cells. MOLT-4 cell blots incubated with the isotypic IgG instead of cyclin B1 antibody were used as a nonspecific antibody (IgG) control (A). Untreated cyclin B1 was also found in human primary cancer cells isolated from MOLT-4 cells were found to have the scheduled expression of cyclin B1 in populations patient cancer tissues, as described above. Fig. 4A is the contour map of G2-M cells but not in G1-S-phase cells (B). In contrast, T-7 cells were shown to be plot showing that cyclin B1 was expressed in the G1-phase breast cyclin B1-positive in G1-phase cells (C), and MOLT-4 cells arrested at the G1 phase had the same unscheduled cyclin B1 expression pattern of T-7 cells (D). cancer cells derived from patient breast cancer tissues. Similarly, Fig. 1608

analysis (cyclin versus DNA content) of multiparameter flow cytometry. That method allows one to assay the ectopic expression of cell cycle phase-specific protein (12). Because the DNA content was used to divide the different time-window sections of cells in the cell cycle,

the cells in the G1 phase with the same DNA content could not be divided into any sub-phases. Recently, we noticed that cyclin E is

expressed in G1-phase cells continuously and reaches peak level over a threshold at the time of entering into the S phase. Therefore, we selected the fluorescence intensity of cyclin E to be the marker (Fig.

3, A and C) as partition of early and late G1-phase cells (13). The expression of cyclin B1 in sorted cells can be assayed by a Western blot analysis. This method, which we call postsorting Western blotting, provides an approach to study more precisely the specific protein(s) for the cell cycles in subpopulations of asynchronously growing cells.

Unscheduled cyclin B1 expression in the G1 phase also occurs in primary cancer cells from clinical tumor tissues. Besides leukemia and breast cancer cells examined in the present study, we have shown that

unscheduled cyclin B1 was expressed in the G1 phase in other human cancer cells from tumor tissues of patients, including gastric carcinoma, colon cancer, nasopharyngeal carcinoma, and ovarian cancer (data not shown). However, the significance of unscheduled cyclin B1 expression in cancer biology is not clearly defined. Earlier studies reported that the overexpression of cyclins in human tumors was associated with tumor transformation and poor prognosis (15, 16). Because cyclin B1 normally forms a heterodimeric complex with

CDK1 during the G2 phase resulting in histone kinase activity, which is necessary for the initiation of mitosis, continuous expression of cyclin B1 in tumor cells may allow cyclin B1-CDK1 complex to

Fig. 3. Detection of cyclin B1 expression in G1-phase cells by postsorting Western blot analysis. A and C, cyclin E expression in T-7 cells and arrested MOLT-4 cells, respectively. The dot plots indicate that cyclin E expression intensity increases gradually in the G1 phase and peaks at the time when cells enter the S phase. The three windows created based on the fluorescence intensity of this protein represent G0,G1-early, and G1-late sub-phase cells, respectively. B and D, Western blot analysis of cyclin B1 expression in sorted three sub-phases of G1-untreated T-7 cells and blocked MOLT-4 cells. As seen, cyclin B1 can be measured in the G1-early phase of the asynchronously growing T-7 cells (B) and in the G1-late phase of the synchronized MOLT-4 cells (D).

4B shows that cyclin B1 expression was also detected in the G1-phase lymphocytes derived from the patients with leukemia. Fig. 4D is a representative result of the Western blot analysis of the sorted G1- phase breast cancer cells. As seen, cyclin B1 was expressed in both whole cells and the sorted G1-phase cells, suggesting that these in vivo uncontrolled proliferating tumor cells exhibited expression of cyclin

B1 not only in the G2-M phase but also in the G1 phase of the cell cycle.

Discussion It is generally believed that scheduled cyclin B1 is expressed in eukaryotic cells, i.e., synthesized first at late S phase, peaked at G2-M phase, and then rapidly degraded by ubiquitin-dependent proteolysis, which was taken for granted to be conservative from yeast to mammalian (1–3). However, in recent years some studies have shown that cyclin B1 could be detected in some G1-phase tumor cell lines Fig. 4. Cyclin B1 expression in G1-phase cancer cells from cancer tissues of patients. (10–11, 14). Gong et al. (12) studied the expression of cyclin B1 in Human primary breast cancer cells from clinical breast cancer tissues (A) and lymphocytes from the patients with leukemia (B) allowed for the observation of unscheduled expression MOLT-4 lymphocyte leukemia cells synchronized in the G1 phase of the cell cycle and found unscheduled expression of cyclin B1 in the of cyclin B1 occurring in the G1 phase in these cells. C, the sorting window from a DNA histogram of leukemia cells. D, cyclin B1 was expressed in the sorted G1-phase lympho- G1 phase in this cancer cell line, as determined by the bivariate cytes of leukemia patients, as determined by Western blot analysis. 1609

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2004 American Association for Cancer Research. DETECTION OF CYCLIN B1 IN G1-PHASE CANCER CELLS phosphorylate a series of substrates regardless of the phase in the cell expressed in early G1 phase in asynchronously growing T-7 cells and cycle and lead to out of control cell cycle progression. Therefore, the in late G1 phase in synchronized MOLT-4 cells. Although the role and ectopic expression of cyclin B1 in the G1 phase may be one of the importance of G1-phase cyclin B1 in the tumorigenesis and apoptosis mechanisms of carcinogenesis for some cancer types. of human malignancy are becoming increasingly apparent, the precise The mechanism accounting for unscheduled expression of cyclin mechanisms of unscheduled expression of cyclin B1 have not been B1 in certain tumor cells is not understood at this time. However, completely understood. Studies are in progress to further investigate there may exist two possibilities, as follows: (a) the synthesis of cyclin the different patterns of unscheduled cyclin B1 expression in different B1 in these cells is not limited to the strict period in the cell cycle, and tumor cells and to elucidate the role and mechanism of this protein in (b) the proteolytic degradation of this protein is impaired. Therefore, cell proliferation and cell death. low levels of cyclin B1 can be detected during the G1 phase of the cell cycle in some tumor cell lines. No matter what reason may lead to the References unscheduled expression of cyclin B1, the presence of cyclin B1 in 1. Wikson, E. B. The cell in development and heredity, 3rd Ed. Macmillan, New York, G1-phase cells may suggest a possible dysfunction or misregulation of 1995. the machinery for cell cycle progression. 2. Nurse, P. Ordering S phase and M phase in the cell cycle. Cell, 79: 547–550, 1994. 3. Morgan, D. O. 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Okadaic acid, a potent inhibitor of type 1 and type cycle progression and lead to uncontrolled cell proliferation, whereas 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a prema- a transient expression of cyclin B1 in the G1 phase may lead to ture mitosis-like state in BHK21 cells. EMBO J., 9: 4331–4338, 1990. activation of the apoptosis pathways that cause cell death. 15. Keyonarsi, K., and Pardee, A. P. Redundant cyclin overexpression and gene ampli- fication in breast cancer cells. Proc. Natl. Acad. Sci. USA, 90: 1112–1116, 1993 In summary, we showed in this study that cyclin B1 was expressed 16. Ito, Y., Takeda, T., Sakon, M., Monden, M., Tsujimoto, M., and Matsuura, N. in the G1 phase in the synchronized MOLT-4 cells and asynchro- Expression and prognostic role of cyclin-dependent protein kinase 1 (cdc2) in nously growing T-7 cells, as accessed by flow cytometry. Addition- hepatocellular carcinoma. Oncology 59: 68–74, 2000. 17. Shi, L., Nishioka, W. 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Manli Shen, Yongdong Feng, Chun Gao, et al.

Cancer Res 2004;64:1607-1610.

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