Effect of starvation-induced autophagy on cell cycle of tumor cells

[Chinese Journal of Cancer 27:8, 102-108; August 2008]; 2008 Sun Yat-Sen University Cancer Center Basic Research Paper Effect of starvation-induced autophagy on cell cycle of tumor cells Jun-Na Ge, 1 Dan Huang, 1 Tian Xiao, 1 Zun Wang, 1 Xiao-Lan Li, 2 Hui Xiao, 2 De-Ding Tao 2 and Jian-Ping Gong 1, * 1 Department of Gastrointestinal Surgery; 2 Cancer Research Institute/Molecular Medical Center; Tongji Hospital; Tongji Medical College; Huazhong University of Science and Technology; Wuhan, Hubei, P.R. China Key words: starvation, autophagy, Cyclin, cell cycle, 3-methyladenine, flow cytometry Background and Objective: No serum starvation could induce autophagy and cell cycle arrest. Although autophagy and cell cycle have been widely explored, little is known about their relationship. This study was to observe the change of Cyclin expression during starvation-induced autophagy to discuss the effect of autophagy on cell cycle. Methods: In control group, HeLa cells were treated with d-hanks solution (a medium with no serum). In experiment group, HeLa cells were treated with d-hanks solution containing 3-methyladenine (3-MA, a specific inhibitor of autophagy). Cells were harvested after being starved for 0, 3, 6 and 12 h. Flow cytometry (FCM) and Weston blot were used to detect Cyclin and microtubule-associated protein 1 light chain 3 (LC-3) which marked autophagy specifically. Results: In control group, the expression of LC-3 protein was detected early after being starved for 3 h, and gradually increased along with starvation; the expression of Cyclin D3 and Cyclin E was decreased evidently after a short period of starvation (3 h) and descended to the minimum when cells were being staved for 6 h; the expression of Cyclin A and Cyclin B1 were apparently decreased after being staved for 6 h. In experiment group, LC-3 protein could not be detected during starvation when cells were exposed to 3-MA and the downregulation of Cyclins was suppressed. Conclusions: Autophagy is involved in starvation-induced hydrolysis of Cyclins. The hydrolysis of Cyclin D3 and Cyclin E is quicker than that of Cyclin A and Cyclin B1. Autophagy, a widespread phenomenon found in eukaryotic cells, is a biological process regulated by a group of autophagy-related genes (Atg). Autophagy provides raw materials for biosynthesis when cells are exposed to a harsh environment, for example when macromolecules cannot be acquired through extrinsic pathways. Autophagy plays *Correspondence to: Jian-Ping Gong; Department of Gastrointestinal Surgery; Tongji *Correspondence Hospital; Tongji Medical to: College; Huazhong University of Science and Technology; Wuhan, Hubei, 430030, P.R. China; Tel.: 86.27.83663301; Fax: 86.27.83662696; Submitted: XX/XX/XX; Revised: XX/XX/XX; Accepted: XX/XX/XX Email: jpong@tjh.tjmu.edu.cn This paper was translated into English from its original publication in Submitted: 11/12/07; Revised: 04/21/08; Accepted: 04/29/08 Chinese. Translated by: XXXXXXXXXXXXXXXX on XX/XX/XX. This paper was translated into English from its original publication in Chinese. The original Chinese version of this paper is published in: Ai Zheng(Chinese Translated by: Beijing Xinglin Meditrans Center and Wei Liu on 05/14/08. Journal of Cancer), 27(8); http://www.cjcsysu.cn/cn/article.asp?id=xxxxx The original Chinese version of this paper is published in: Ai Zheng(Chinese Previously published online as a Chinese Journal of Cancer E-publication: Journal of Cancer), 27(8); http://www.cjcsysu.cn/cn/article.asp?id=14333 http://www.landesbioscience.com/journals/cjc/article/xxxx Previously published online as a Chinese Journal of Cancer E-publication: Addendum to: http://www.landesbioscience.com/journals/cjc/article/6611 an irreplaceable role in early embryonic development. 1 Although in some eukaryotic cells, the proteins contained in cytoplasm and nuclei are degraded mainly through the ubiquitin-protease pathway, the hydrolytic process of proteins in most cells depends on the lysosomal pathway, particularly when there is lack of nutrition. 2 As we know, starvation could induce autophagy and could also retard cell cycles at G 0 phase. 3 Is there any connection between autophagy and cell cycle alteration after starvation? In this study, starvation model was used to determine the connection between autophagy and cell cycle, and the relationships between autophagy, Cyclins and cell synchronization, which were initially explored according to the principles of regulating cell cycle by Cyclins. Materials and Methods Cell lines and primary reagents. Human cervical cancer cell line HeLa was purchased from Wuhan University Typical Culture Center. LC-3 polyclonal antibody was purchased from Abgent Company, USA. FITC-labeled goat anti-rabbit IgG was purchased from Pierce Company, USA. Propidium iodide (PI) and 3-methyladenine (3-MA) were purchased from Sigma Company, USA. Cyclin A, Cyclin B1, Cyclin D3 and Cyclin E monoclonal antibodies, goat anti-mouse IgG and goat anti-rabbit IgG were all purchased from BD Company. Cell culture and treatment. HeLa cells were cultured in 5 ml of DMEM medium containing 10% fetal bovine serum, 100 u/ ml penicillin, 100 μg/ml streptomycin at 37 C in an incubator containing 5% CO 2. When the cells reached 70%-80% confluence, they were randomly divided into experiment group and control group. Cells in both groups were washed thrice with sterilized d-hanks balanced solution. Cells in control group were cultured in 5 ml of d-hanks balanced solution, while cells in experiment group were cultured in 5 ml of d-hanks balanced solution containing 900 μg/ml 3-MA. Cells were harvested at the 0, 3, 6 and 12-hour intervals, respectively. Two-parameter analysis of LC-3/DNA flow cytometry. A total of 5 10 5 HeLa cells were harvested, washed twice with pre-chilled phosphate buffer solution (PBS) at 4 C, fixed with 4% methanolfree formaldehyde on ice for 15 min, washed with PBS twice, and fixed with 80% iced ethanol at -20 C overnight. The fixed cells were washed with PBS twice, then added with 0.25% Triton-X 100 and placed on ice for 5 min. After washed with PBS twice, cells were treated with 1% BSA for 30 min and centrifuged at 1200 r/min (1335 g) for 5 min, and then resuspended in 100 μl 1% BSA. Rabbit antihuman LC-3 antibody (1:100) diluted in 1%BSA solution was added, 102 Chinese Journal of Cancer 2008; Vol. 27 Issue 8

Figure 1. Cell cycle of HeLa cells after being starved with d-hanks solution. The proportion of G0/G1 phase cells is increased along with starvation. and then cells were incubated at 4 C overnight. On the next day, cells were washed with PBS for three times, then added with FITC-labeled goat anti-rabbit IgG (diluted with 1% BSA at a ratio of 1:20) and incubated at room temperature for 30 min in the dark. Subsequently, cells were washed with PBS once again, and then 10 μg/ml PI and 0.1% RNase (Sigma Co.) were used for nuclear DNA staining at room temperature for 30 min. Cells were analyzed using flow tytometry (FCM). The data were analyzed with CellQuest software. Two-parameter analysis of Cyclin/DNA flow cytometry. The harvested cells were fixed in 80% iced ethanol at -20 C overnight. After being washed twice with PBS, cells were treated with 0.25% TritonX-100 on ice for 5 min, washed with PBS once, centrifuged and washed twice more with PBS. Mouse anti-human Cyclin antibody diluted with 1% BSA was added, and cells were incubated at 4 C overnight. On the next day, cells were washed with 5 ml PBS and centrifuged. Goat anti-mouse IgG antibody (Sigma Co., diluted with 1% BSA at a ratio of 1:40) was added, and cells were incubated at room temperature for 30 min in the dark. After washing with PBS once, 10 μg/ml of PI and 0.1% RNase (Sigma Co.) were used for nuclear DNA staining at room temperature for 20 min. Cells were then analyzed using FCM. For negative control group, isotype unspecific antibody (IgG) was used instead of mouse anti-human Cyclin monoclonal antibody. Western blot. Treated cells were harvested, washed with PBS, followed by cell protein extraction, electrophoresis, transfer, block, antibody incubation and ECI coloration, performed as routine method. Primary antibodies were blocked at room temperature, including mouse anti-human Cyclin A, B1, D3, E antibodies (1:500), rabbit anti-human LC-3 antibody (1:500), and mouse anti-human β-actin antibody (1:1000). Horseradish peroxidase-conjugated goat anti-mouse IgG (1:5000), as secondary antibodies, was blocked by 5% defatted milk at room temperature for 90 min. The membrane was colorized with ECL in a dark room. Results Effects of 3-MA on cell cycle. When HeLa cells were cultured with d-hanks balance solution, instead of DMEM medium containing 10% fetal bovine serum, 100 u/ml penicillin and 100 μg/ml streptomycin, the cell proportion at G 0 phase was increased gradually from 50.07% at the time of changing culture medium to www.landesbioscience.com Chinese Journal of Cancer 103

Figure 2. (At left) Cell cycle of HeLa cells after being starved and autophagy being inhibited. The proportion of G0/G1 phase cells is not increased along with the inducement. Figure 3. Autophagy in HeLa cells analyzed by LC-3/DNA flow cytometry. The gate (R2 and R3) represents LC-3-positive cells in each figure. Autophagy is enhanced along with starvation but the enhancement is inhibited when cells 3-MA. 104 Chinese Journal of Cancer 2008; Vol. 27 Issue 8

Figure 4. Expression of Cyclins in HeLa cells during starvation-induced autophagy detected by flow cytometry. The gate (R6) represents Cyclin-positive cells in each figure. The proportion of Cyclin-positive cells is decreased along with starvation. www.landesbioscience.com Chinese Journal of Cancer 105

Figure 5. Expression of Cyclins in HeLa cells during inhibition of autophagy detected by flow cytometry. The gate (R2) represents Cyclin-positive cells in each figure. The proportion of Cyclin-positive cells is not decreased obviously when starved and exposed to 3-MA simultaneously. 106 Chinese Journal of Cancer 2008; Vol. 27 Issue 8

Figure 6. Cyclins and LC-3 detected by Western blot in control group and experiment group. Lanes 1 3: HeLa cells treated with d-hanks solution for 3, 6 and 12 h, respectively; lane C: control HeLa cells without treatment; lanes 4 6: HeLa cells treated with d-hanks solution and 3-MA for 3, 6 and 12 h, respectively. 62.30% at the 12-hour interval (Fig. 1). However, when autophagy specific inhibitor 3-MA was added during starvation, the change was not significant, from 50.35% to 53.68% (Fig. 2). Effects of 3-MA on starvation-induced autophagy of HeLa cells. In control group, when cultured with d-hanks balance solution, cell autophagy was induced, the expression of LC-3 was gradually increased along with the starvation, and this tendency was found in every phase in cell cycle. In experiment group, when cultured with d-hanks solution containing 3-MA, the expression of LC-3 in HeLa cells was very low, and did not increase along with the starvation, suggesting that 3-MA can specifically inhibit starvation-induced autophagy (Fig. 3). Effect of starvation on expression of Cyclins in HeLa cells. According to FCM results, the expression of Cyclin E and Cyclin D3 in HeLa cells started to decrease after 3-hour starvation in control group and descended to the minimum when the cells were starved for 6 h. While the expression of Cyclin A and Cyclin B1 started to decrease after 6-hour starvation, the decreasing velocities of Cyclin D3, Cyclin E, Cyclin A and Cyclin B1 expressions were slower in experiment group than in the control group (Figs. 4 and 5). Western blot results were consistent to FCM results (Fig. 6). Discussion Cyclins, cell cycle-specific proteins, drive cell cycle progression through phasic activation of Cyclin-dependant kinase (CDK). Researchers believe that the degradation of Cyclins under normal culturing condition is mainly through ubiquination pathway. 4 Recent studies revealed that, autophagy, a lysosomal degradation way, plays an important role in protein degradation and organelle renewal, especially in maintaining cell activity under malnutrition by catabolism, which is a defensive protection for cells. 5,6 As a special experimental condition, serum-free starvation may induce autophagy, and meanwhile increase the proportion of cells at G 0 phase. This study used starvation models and proved that autophagy is connected with Cyclin degradation, and the degradation of different Cyclins is in an unsynchronized pattern. Autophagy mediates Cyclin degradation during starvation. Using established FCM technique in simultaneous assay of autophagy and cell cycle in our laboratory, 7 we have proven in this study that starvation could induce gradual increase of autophagic vacuole-specific protein LC-3 expression, which is unspecific in the cell cycle. 3-MA, a specific inhibitor of autophagy, 8 could inhibit the expression of LC-3. Short-term starvation could obviously downregulate the expression of Cyclin D3 and Cyclin E; when autophagy is inhibited by 3-MA, the downregulation of Cyclin expression is not significant. Therefore, we claim that autophagy during short-term starvation could mediate Cyclin degradation based on the coincidences of high LC-3 expression accompanied with Cyclin degradation and low or no LC-3 expression, accompanied with no Cyclin degradation, including our discovery of unaltered cellular total protein during short-term starvation. Short-term starvation could induce Cyclin degradation sooner and faster in G1/S phase than in G2/M phase. Cyclins regulate the progression of cell cycle. Our results showed that short-term starvation in tumor cells could alter Cyclin expression. The expression of Cyclin D3 and Cyclin E in G 1 /S phase, especially Cyclin D3, was started to decrease after 3 hours of starvation and decreased to the minimum when cells were being starved for 6 h, while the expression of Cyclin A and Cyclin B1 in G 2 /M phase only started to reduce after 6 hours of starvation. Comparing with that in G 2 /M phase, the earlier degradation of Cyclins in G 1 /S phase ensures that cells prior to G 1 -phase checkpoint not to progress into the DNA replication phase, but stop in G 1 phase; it also ensures sufficient time for cells in G 2 /M phase go back to G 1 phase, therefore, cells are synchronized at G 1 phase. Serum-free starvation induces G 0 phase synchronization of cells, and lead to cell death through bcl-2 pathway mediated by G 0 asynchronous phase-activated CDK1 (unpublished data). The most prominent features of tumor cells are uncontrolled growth and undifferentiation. Unbalance of cell cycle regulation resulting from multiple factors may cause uncontrolled cell growth and result in tumorigenesis. Overexpression of Cyclins has been found in many tumor cells. Cyclin overexpression and cell cycle deregulation has recently become a new target for cancer therapy. The researches on gene therapy targeting Cyclins, as well as development of new anti- Cyclin antibody and CDK drugs have shown encouraging results. 9,10 Autophagy, as a cell defensive mechanism, has been gradually elicited of its inhibitory effect on tumor formation and tumor growth. 11 Our study has proven that autophagy lysosomal pathway is involved in the hydrolysis of Cyclins. From long-term perspective, the regulation of autophagy on cell cycle of tumor cells may be a new orientation for tumor treatment, bearing significant importance. Acknowledgements Grants: National Natural Science Foundation of China (No. 30570908); Clinical Key Subject Foundation of Health Ministry of China References [1] Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism [J]. Oncogene, 2004,23(16):2891-2906. [2] Liang XH, Jackson S, Seaman M, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1 [J]. Nature, 1999,402(6762):672-676. [3] Liang XH, Anger M, Carnwath JW, et al. Cell cycle synchronization of porcine fetal fibroblasts: effects of serum deprivation and reversible cell cycle inhibitors [J]. Biol Reprod, 2000,62(3):412-419. [4] King RW, Deshaies RJ, Peters JM, et al. How proteolysis drives the cell cycle [J]. Science, 1996,274(5293):1652-1659. [5] Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy [J]. Dev Cell, 2004,6(4):463-477. [6] Mizushima N. The pleiotropic role of autophagy: from protein metabolism to bactericide [J]. 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