흰쥐에서 배란과정 동안 cell cycle을 조절하는 인자 : 가능한 sulfiredoxin의 역할

정락균 전남대학교 대학원 2010 국내박사

Although the regulation of cell cycle have been extensively studied in other tisses and immature follicle growth in the ovary, little information is available during the ovulation process. The present study was therefore designed to characterite ovarian local regulators involved in cell cycle progression during ovulation in the rat. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces ovulation rate in rats. This study was designed to investigate potential mechanisms of TCDD-mediated blockade of ovulation in gonadotropin-primed immature rats. Intact rats given TCDD (32 μg/kg) by gavage one day before PMSG (5 IU/rat) injection exhibited a reduced ovarian weight and ovulation rate after gonadotropin administration. Treatment of preantral follicles in vitro with TCDD retarded FSH-stimulated growth as well as estrogen production. Furthermore, treatment of granulosa cells with TCDD inhibited comitogenic actions of FSH and activin. The inhibitory effects of TCDD on follicle growth and proliferation were abolished by cotreatment with aryl hydrocarbon receptor (AhR) antagonist α-naphthoflavone. However, cell viability or cytochrome c content of granulosa cells collected from TCDD-treated rats was not different from that of control rats. AhR protein levels in granulosa cells remained elevated in TCDD-treated rats whereas it gradually declined in control rats following PMSG. Flow cytometric analysis demonstrated that the percentage of granulosa cells in S-phase peaked 24 h and declined to control levels 48 h following PMSG, and further declined below control levels 8 h following hCG. Interestingly, a reduction in the number of S-phase cells was observed in TCDD-treated rats at 24 h and 48 h following PMSG. In addition, TCDD inhibited mRNA levels of Cdk2 and cyclin D2 during 48-h post-PMSG. The present study indicates that TCDD attenuates the cell cycle progression during the developmental transition from small antral follicles to mature preovulatory follicles, and thus results in the reduced number of ovulating oocytes. Sulfiredoxin (Srx) is a low molecular weight, sulfur containing protein involved in the maintenance of cellular redox balance. The present study was designed to examine the gonadotropin regulation of Srx and to identity its interacting proteins during the ovulation process in immature rats. The levels of Srx mRNA were shortly stimulated by LH/human chorionic gonadotropin (hCG) within 1 h both in vivo and in vitro. The expression of Srx protein was also stimulated by LH within 1 h and maintained high until 6 h. Also, Srx was expressed by exogeneus H2O2. LH-induced Srx expression was suppressed by extracellular signal-regulated kinase (ERK) inhibitor U0126, the flavin inhibitor diphenyleneiodonium chloride (DPI) and the H2O2 scavenger 4,5-dihydroxy-1,3-benzenedisulfonic acid (tiron). Futhermore, LH treatment increased the production of total reactive oxygen species and superoxide anion within 15-30 min. Whereas, H2O2 was not phosphorlated Erk1/2. Intereastingly, six putative proteins interacting with Srx were identified by immunoprecipitation using His-tagged Srx. One of identified proteins, peroxiredoxin II (Prx II) was physically interacted with Srx. The present study demonstrates that LH/hCG stimulates Srx expression by activating ERK and H2O2 production in granulosa cells. The present study futher shows the physical association of Srx with Prx II, implicating the role of Srx in the reduction of Prx II during ovulation. Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. The study of my research revealed that the expression of cyclin G2 was regulated by gonadotropins during follicle development in the immature rat ovary. Notably, LH/hCG gradually reduced the expression of cyclin G2 mRNA within 6 hr. Morever, expression of cyclin G2 was induced by FSH and activin in cultured granulosa cells obtained from DES-treated rat ovary. The interating proteins with cyclin G2 were identified by yeast two hybrid screening. One of the identified proteins, prohibitin known as an inhibitor of cellular proliferation, was expressed in the same cell-type with that of cyclin G2 in the ovary. Therefore, the present study demonstrates the gonadotropin regulation of cyclin G2, suggesting it may play a role in the follicular growth and ovulatory process by regulating cell cycle progression.

Cell-Cycle Control in Aging, Cancer, and Homeostasis

Daigh, Leighton Harrison ProQuest Dissertations & Theses Stanford Universit 2020 해외박사(DDOD)

Precise regulation of cell-cycle progression is essential to mammalian homeostasis. Excess cellular proliferation occurs during tumorigenesis and causes cancer. Insufficient proliferation is associated with tissue dysfunction and organismal aging. The following studies investigate unique aspects of cell-cycle regulation and how they contribute to homeostasis, aging, and cancer.In the first study, we examined how dysregulation of central pathways involved in cellular proliferation drives cellular aging. The process of cellular aging, i.e. senescence, is characterized by irreversible cell-cycle arrest despite active growth factor signaling. We demonstrated that discordance between cell growth and cell proliferation pathways promoted a transition from quiescence to senescence. The induction of cell-cycle genes was necessary to repair DNA damage caused by growth factor signaling. Prolonged periods of growth factor signaling in the absence of cell-cycle progression caused the accumulation of DNA damage and activation of inflammatory signaling. Inflammation enforced irreversible cell-cycle arrest. These results highlight an interdependence between cell growth and cell proliferation pathways that can cause senescence when perturbed. Moreover, our results suggest that senescence is a self-reinforcing process that can occur via any stimulus causing sustained cell-cycle arrest despite persistent growth factor signaling.In our second study, we described a previously unrecognized mechanism underlying the efficacy of CDK4/6 inhibitors as cancer therapeutics. CDK4/6 inhibitors were developed to block cell-cycle entry by binding the active site of CDK4/6 and inhibiting kinase activity. However, we found that, in addition to a direct effect on CDK4/6 kinase activity, CDK4/6 inhibitors also indirectly inhibited the related kinase CDK2. This occurred via drug-mediated displacement of the CDK-inhibitory protein p21 from CDK4, causing p21 to bind and inhibit CDK2. This effect is important for ensuring robust suppression of cell-cycle entry. Importantly, p21 displacement did not occur from CDK6 upon drug binding. These results suggest that CDK4/6 inhibitors will be most efficacious inhibiting growth of cancers where p21 is present and CDK4 predominates over CDK6 as the primary G1 kinase initiating cell-cycle entry.The final study examined how cells navigate endogenous replication stress during S phase, which is an unavoidable hazard associated with genome duplication. We found that replication stress caused a transient activation of ATR activity. Once activated, ATR signaling inhibited CDK2 activity proportional to the degree of replication stress. Dynamic regulation of CDK2 activity allowed cells to tune the rate of DNA replication in response to the degree of replication stress. These results provide insight into how cells cope with stochastic stressors inherent to cell-cycle progression.Overall, these studies provide new insights into the fundamental processes regulating cellular proliferation. Moreover, the results presented here may contribute to the development of therapeutics targeting both aging and cancer. Future work will extend these findings into more physiological contexts to gain greater insight into how proper control of the cell cycle contributes to homeostasis and disease.

Quantitative Measurement of the Cell Cycle: Insights into Differentiation and Basement Membrane Invasion

Kohrman, Abraham Wesley ProQuest Dissertations & Theses State University o 2020 해외박사(DDOD)

The cell cycle is critical for the existence of life on earth. Cell division is responsible for the production of the raw materials necessary to develop complex organisms. Regulation of the cell cycle is necessary for the processes of both development and disease. Many cell biological activities are regulated, directly or indirectly, by the cell cycle. One such behavior, basement membrane invasion, occurs exclusively in the G1/G0 stage of the cell cycle. In Caenorhabditis elegans, Anchor Cell (AC) invasion through the basement membrane is dependent on cell cycle arrest in G1/G0. The AC depends on both chromatin modification via the histone deacetylase hda-1 and the activity of cki-1(p21/p27) to arrest the cell cycle downstream of the nuclear hormone receptor nhr-67(TLX). Loss of nhr-67 results in ACs which divide, and consequently, fail to invade. G1/G0 cycle arrest is both necessary and sufficient to rescue the loss of nhr-67, indicating the critical importance of the cell cycle in basement membrane invasion. Direct measurement of cell cycle state is important to the furtherance of our understanding of cell biology. Understanding the interplay between cell cycle and cell biology has critical applications in the treatment of disease states. Therefore, I have developed a ratiometric cell cycle indicator for use in C. elegans, utilizing a portion of Human DNA Helicase B (DHB). Characterization of DHB in C. elegans demonstrates its ability to detect all four cell cycle states in various tissues across all developmental stages. I have used DHB probe the link between cell cycle and morphogenesis in C. elegans. DHB has allowed us to detect G0 phase for the first time in living animals, confirming the hypothesized association between cell cycle arrest cell differentiation.

(The) regulation of embryonic stem cell pluripotency by phosphorylation of Oct4 during the cell cycle

신지훈 서울대학교 대학원 2020 국내박사

Pluripotency transcription programs by core transcription factors (CTFs) might be reactivated during M/G1 transition to maintain the pluripotency of embryonic stem cells (ESCs). However, little is known about how CTFs are governed during cell cycle progression. Here, we demonstrate that the regulation of Oct4 by Aurora kinase b (Aurkb)/protein phosphatase 1 (PP1) during the cell cycle is important for resetting Oct4 to pluripotency and cell cycle genes in determining the identity of ESCs. Aurkb phosphorylates Oct4(S229) during G2/M phase, leading to the dissociation of Oct4 from chromatin, whereas PP1 binds Oct4 and dephosphorylates Oct4(S229) during M/G1 transition, which resets Oct4-driven transcription for pluripotency and the cell cycle. Aurkb phosphor-mimetic and PP1 binding-deficient mutations in Oct4 alter the cell cycle, effect the loss of pluripotency in ESCs, and decrease the efficiency of somatic cell reprogramming. Our findings provide evidence that the cell cycle is linked directly to pluripotency programs in ESCs. * This work is published in elife Journal (Shin et al., 2016). 배아 줄기세포가 전분화성을 유지하는 데 있어 주요 전사 인자들의 활성이 조절되는 메커니즘은 매우 중요하다. 특히 M/G1 세포주기 동안에는 주요 전사 인자들이 다시 타겟 유전자로 Reset되어야 할 것이다. 하지만 줄기세포의 세포주기 동안 핵심 전사 인자들이 어떻게 조절되는지에 대한 메커니즘은 알려진 바가 거의 없다. 우리는 마스터 전사인자인 Oct4의 활성이 세포주기동안 어떻게 조절되는지에 대해 주목하였다. G2/M 세포주기에서 Aurkb는 Oct4의 Serine 229번 잔기를 인산화하여 Oct4의 전사활성을 잃게 한다. 전사활성이 급격히 감소된 Oct4는 염색체에서 떨어지게 되고 타겟 유전자들의 발현이 감소하여 줄기세포 위계결정의 기회의 창을 만든다. 세포주기가 진행되어 다시 G1 세포주기로 들어갈 때 PP1에 의해 Serine 229번 인산화가 제거되고 전사활성이 회복된 Oct4는 본래의 타겟 유전자에 Reset되어 타겟 유전자들의 발현을 높인다. 우리는 세포주기에 따른 Oct4의 ChIP-seq을 통해 Genome-wide한 분석을 하였고 Oct4의 타겟 유전자들을 발굴하였다. 특히 세포주기에 따라 G2/M기에 줄어들었던 peak들이 G1기에 증가하는 것을 확인하였고 peak들의 density 또한 G1기에서 G2/M기에 비해 상대적으로 높은 것을 확인하였다. Oct4의 타겟 유전자들은 전분화성 유전자뿐만 아니라 세포주기 유전자와 관련되어 있으며 Oct4가 세포주기에 따라 조절될뿐만 아니라 줄기세포의 세포주기를 조절할 수 있음을 의미한다. 우리는 Oct4의 인산화 및 탈 인산화가 줄기세포에 미치는 영향을 확인하기 위해 인산화 유사 단백질인 S229D mutant와 PP1과의 결합능을 잃은 F271A mutant를 제작하여 줄기세포에 발현시킴과 동시에 세포내 Oct4는 결핍시켰다. 그 결과 두 mutant를 발현하는 줄기세포 모두 Oct4의 Resetting이 정상적으로 이루어지지 않았으며 세포주기는 분화한 세포와 유사하게 G1기가 길어지는 양상을 보였고 결과적으로 전분화성을 상실하였다. 본 연구에서는 배아 줄기세포의 마스터 전사인자인 Oct4가 세포주기동안 인산화에 의해 활성이 조절되는 것을 밝혔고 더 나아가 줄기세포의 세포주기와 전분화성의 연관성에 대해 제시하였다. * 본 내용은 eLife 학술지 (Shin et al., 2016)에 출판 완료된 내용임

인간 암세포주에서 미토콘드리아 저해제들의 세포성장 억제에 대한 연구

한용환 전북대학교 대학원 2010 국내박사

미토콘드리아는 에너지 생성 장소이며 세포고사에 중요한 매개체를 함유하고 있다. 항암 기능의 목적으로 다양한 합성물들이 개발되었고 주로 미토콘드리아에 간접적으로 작용하여 세포고사를 일으킨다고 알려져 있다. 하지만 이러한 약물들의 효과는 몇몇 암세포에 국한되어 나타난다. 최근의 연구 결과들을 통하여, 암의 종류에 따라 유전적 변이가 다르기 때문에 종양의 생존에 있어서 필수적인 유전적 산물이나 단일 기작을 차단하는 것만으로 암세포를 줄일 수 없는 것으로 판단된다. 최근 들어 모든 암 세포에 존재하며 정상세포에 비해 적게 변이가 되는 것으로 밝혀진 미토콘드리아가 치료 표적으로 주목 받고 있다. 본 연구는 미토콘드리아 짝풀림 작용제인 (carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) 와 complex III 저해제인 antimycin A 의 인간 암세포주 (폐암과 자궁경부암)에서의 세포성장과 억제 효과를 규명하고, 그 작용 기전을 세포주기 조절, 세포고사 및 활성산소와 관련된 작용과정을 관련하여 밝히고자 하였다. FCCP는 폐암 세포주 Calu-6에 농도 의존적 세포 성장 억제를 보였으며, 약물 처리 후 50% 생존율을 보이는 농도는 6.64±1.84 μM로 관찰 되었다. FCCP에 의한 세포주기의 분석에서, 낮은 농도(<20 μM)의 FCCP는 G1 phase 정지를 보였으며 그 이상의 농도에서는 특별한 주기의 정지는 관찰되지 않았다. 세포주기와 관련된 단백질의 발현 양상을 관찰한 결과 사이클린 (cyclin)과 사이클린 의존적 키나제 (CDK)가 FCCP 처리군에서 농도 의전적으로 감소 하였다. CDK의 저해 단백질인 p27의 발현이 농도 의존적으로 증가하였으며 Rb 단백질의 인산화와 관련된 사이클린 의존적 키나제4와의 결합능이 FCCP 처리군에서 더욱더 증가됨을 관찰하였다. siRNA에 의한 키나제 저해 단백질 p27의 감소는 FCCP에 의한 G1 phase 정지를 정상으로 복귀 시켰으나 Rb 단백질의 인산화와 세포의 죽음을 오히려 더욱더 증가 시킴이 관찰되었다. 유세포 분석을 통한 세포고사 실험의 결과 FCCP는 농도 의존적으로 Bcl-2 단백질의 감소와 caspase 의 활성화에 따른 PARP 단백질의 분할에 의한 세포고사를 유도하였으며 각각의 caspase 저해제들은 유의성 있게 FCCP에 의한 세포고사를 억제하였다. 다음으로 FCCP의 세포고사에 있어 활성산소의 연관성을 연구하였다. FCCP 처리에 의한 세포내 초과산화이온과 글루타치온의 양은 처리 농도와 시간 의존적으로 감소하였으나 항산화제에 의한 활성산소 감소나 세포고사 억제는 관찰 되지 않았다. 하지만 티올기를 포함한 N-acetylcysteine (NAC)와 dithiothreitol (DTT)가 FCCP에 의한 효과들을 억제하고 글루타치온 합성 저해제인 buthionine sulfoximine (BSO)가 FCCP의 효과를 증폭 시키는 것으로 보아 글루타치온의 세포내 농도가 FCCP에 의한 세포고사 유도에 중요할 것이라 사료된다. 미토콘드리아 복합체 III의 저해제인 antimycin A는 폐암 세포주 Calu-6와 자궁경부암 세포주 HeLa 에서 농도와 시간 의존적 세포 성장 저해를 보였으며 약물 처리 후 50% 생존율을 보이는 농도는 약 50 μM로 관찰 되었다. Antimycin A에 의한 세포주기의 분설 결과, 폐암 세포주 Calu-6 에서는 G1 주기 정지가 관찰되었으며 자궁경부암 세포주 HeLa 에서는 S 주기 정지를 관찰할 수 있었다. Calu-6 세포주에서의 Antimycin A에 의한 G1 주기 정지는 사이클린 의존적 키나제 저해 단백질인 p27의 증가, 사이클린(cyclin) 단백질과 사이클린 의존적 키나제 (CDK) 발현의 감소에 의해 유도 되었고, HeLa 세포주는 p21, p27, CDK4, cdc2 발현의 감소와 더불어 CDK6, cyclin D1, cyclin E, cyclin A, cyclin B의 발현증가에 의해 S 주기 의 진행을 억제하는 것으로 사료된다. 세포주기 진행의 억제와 함께 antimycin A는 농도 의존적으로 세포고사를 유도함을 Calu-6 세포주를 통해 관찰 할 수 있었다. 세포고사가 유도될 때, Bax 단백질은 농도 의존적으로 증가하였고 미토콘드리아 막전위는 감소하였으며 caspase-3과 caspase-8이 활성화 됨을 알 수 있었다. 더 나아가 antimycin A를 처리한 세포주에 caspase 억제제인 Z-VAD-FMK를 같이 처리하여 배양 했을 때 세포고사를 억제할 수 있었다. 이를 통해서 antimycin A에 의한 세포고사는 여러 caspase 들에 의해서 조절 받고 있음을 확인 할 수 있었다. 결론적으로 미토콘드리아 저해제인 FCCP와 antimycin A는 암세포에서 세포주기 정지와 세포고사를 유도하여 세포성장을 억제하였다. 본 연구 결과를 통하여 미토콘드리아 저해제들이 암 치료의 후보 물질이 될 수 있음을 예측할 수 있다. Mitochondria are the powerhouse of the cell, providing it with energy, as well as the source of important mediators of apoptosis. Mitochondria have recently emerged as intriguing targets for anticancer drugs. A variety of compounds have been now identified that act via mitochondria. These compounds destabilize mitochondria and cause apoptosis, which is, at least in some cases, selective for cancer cells. Recent findings show that individual types of cancers are complex and can differ considerably in their array of DNA mutations, harboring different sets of genetic causes. This indicates that it will be very unlikely to cure cancer by drugs targeting only a few gene products or single pathways that are essential for tumor survival. Therefore, it is necessary to find common target to all cells, but which is predominantly only affected by drugs when delivered inside the cancer cells. Such targets appear to be mitochondria, with very rare mutations, and mitochondria-targeted drugs can be expected to be very efficient drugs of choice for a number of different types of cancer. Although mitochondria-targeted drugs induce cell death by deregulation of mitochondria functions and activating apoptosis signaling in cancer cells, little is known about the molecular mechanisms of growth inhibitory effects induced by mitochondria inhibitors. First, the in vitro effects of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) on the growth of Calu-6 lung cancer cells were evaluated. FCCP inhibited the growth of Calu-6 cells with an IC50 of 6.64±1.84 μM at 72 hours. DNA flow cytometric analysis indicated that FCCP induced G1 phase arrest below 20 μM of FCCP. Analysis of cell cycle regulatory proteins demonstrated that protein levels of cyclin dependent kinases (CDKs) and cyclins were decreased in FCCP-treated cells. In addition, FCCP not only increased the p27 level but also enhanced its binding with CDK4 compared to FCCP-untreated cells. While transfection of p27 siRNA inhibited G1 phase arrest in FCCP-treated cells, it did not enhance Retinoblastoma (Rb) protein phosphorylation and intensified FCCP-induced cell death. The apoptosis in Calu-6 cells exposed to FCCP was also detected by using flow cytometer. The apoptotic process was associated with down-regulation of Bcl-2 protein without change of Bax protein, activation of capases (3, 8 and 9) and PARP cleavage. All of the caspase inhibitors (caspase-3, -8, -9 and pan-caspase inhibitor) markedly rescued the Calu-6 cells from FCCP-induced cell death. Moreover, levels of intracellular O2&#8226;- were markedly increased depending on the concentrations (5 ~ 100 μM) of FCCP and the depletion of intracellular GSH content was observed after exposing cells to FCCP. Treatment with thiol antioxidants, N-acetylcystein (NAC) and dithiothreitol (DTT), showed the recovery of glutathione (GSH) depletion and the reduction of O2&#8226;- levels in FCCP-treated cells, which were accompanied by the inhibition of apoptosis. In contrast, L-buthionine sulfoximine (BSO), a well-known inhibitor of GSH synthesis, aggravated GSH depletion, oxidative stress of O2&#8226;- and cell death in FCCP-treated cells. These results suggest that FCCP, an uncoupling agent can inhibit cell growth via cell cycle arrest, apoptosis and depletion of intracellular GSH contents. Second, the effects of antimycin A (AMA) on the growth were evaluated in Calu-6 and HeLa cells. AMA significantly inhibited the growth of Calu-6 cells and HeLa cells in a dose dependent manner with IC50 of 50 μM. DNA flow cytometric analysis indicated that AMA induced a G1 phase arrest in Calu-6 cells and S phase arrest in HeLa cells at 72 hours, respectively. AMA increased a cyclin dependent kinase inhibitor (CDKI), p27 and decreased CDK2, CDK4, and CDK6, as well as cyclin D1 and cyclin E in Calu-6 cells. However, AMA decreased the expression of p21, p27, CDK4, and cdc2 proteins in HeLa cells. The expression of CDK6, cyclin D1, cyclin E, cyclin A, and cyclin B proteins was increased by 0.5 &#61549;M AMA, but was decreased by 2 and 10 &#61549;M AMA in HeLa cells. The phosphorylation of Rb on the Ser (780) residue was increased by 0.5 &#61549;M AMA. Furthermore, treatment with AMA caused the accumulation of cells expressing cyclin A, B, and D1 proteins at the S phase of the cell cycle in HeLa cells. AMA also induced apoptosis in Calu-6 cells. The apoptotic process in AMA-treated Calu-6 cells was accompanied by the up-regulation of Bax, the loss of mitochondrial membrane potential (MMP (&#61508;&#61529;m)), and the activation of caspase-3 and caspase-8. All of the tested caspase inhibitors, especially pan-caspase inhibitor (Z-VAD), markedly rescued Calu-6 cells from AMA-induced Calu-6 cell death. Inhibitors of pan-caspase and caspase-8 also prevented the loss of MMP (&#61508;&#61529;m). AMA decreased the intracellular reactive oxygen species (ROS) levels but increased the O2&#8226;- levels in Calu-6 cells. These results suggest that AMA as a mitochondrial electron transport inhibitor decreased the growth of lung cancer Calu-6 cell and cervical cancer HeLa cell via inducing the cell cycle arrest and apoptosis. In conclusion, FCCP and antimycin A induced growth inhibition via cell cycle arrest and apoptosis in lung and cervical cancer cell. These results suggest that mitochondria inhibitors can be a candidate for the treatment of cancers.

Encoding Cell Cycle Regulatory Information in the Genome

Buchert, Elli M University of Michigan ProQuest Dissertations & Th 2024 해외박사(DDOD)

The timing of cell cycle exit and initiation of terminal differentiation must be precisely coordinated to ensure proper development of many tissues. Additional cell cycles during development can disrupt, but not necessarily prevent, progression of terminal differentiation programs, leading to tissues with incorrect cell numbers and morphology. The mechanisms that coordinate the transition from a proliferative state to a fully differentiated post-mitotic state are not well understood, and even less is understood about how a non-cycling postmitotic state is maintained in terminally differentiated cells. The eyes and wings of the fruit fly Drosophila melanogaster are excellent tissues to study this phenomenon, as both tissues undergo a relatively synchronous final cell cycle before exiting the cell cycle permanently at 24h after the start of metamorphosis, coincident with visible progression of terminal differentiation programs.Cell cycle exit in Drosophila wings and eyes involves the transcriptional silencing of hundreds of cell cycle genes. However, maintaining cell cycle exit relies on preventing the re-activation of three rate-limiting cell cycle genes, the G1-S cyclin, Cyclin E, the cell cycle transcriptional activator, E2F1 and the regulator of mitotic entry, cdc25c, termed String in flies. Our prior work established that after cell cycle exit, chromatin accessibility is reduced at potential regulatory elements for these three genes, leading to a hypothesis that closing chromatin maintains cell cycle exit by preventing activation of the rate-limiting cell cycle genes. In this thesis I examine this hypothesis by developing new techniques to allow for more detailed assays of chromatin accessibility changes and chromatin modifications (Chapter 2). I also test and validate several regulatory elements for the e2f1 and string loci, to determine which elements are tissue specific vs. shared for the wing and eye and examine their shut off dynamics during chromatin accessibility changes after cell cycle exit (Chapter 3). Finally, I identify a chromatin remodeler responsible for the closing of chromatin accessibility at the string locus, and determine that it works together with a transcription factor expressed during metamorphosis to coordinate chromatin accessibility changes that decommission enhancers at cell cycle genes and early differentiation genes, to maintain cell cycle exit as terminal differentiation progresses (Chapter 4). Altogether this work examines how complex cell cycle regulatory events can be encoded in the genome, to ensure the proper coordination of cell cycle control with cellular differentiation.

Novel molecular approach to control the self-renewal and differentiation of human adipose-derived mesenchymal stem cells using microRNAs

박호 성균관대학교 일반대학원 2012 국내박사

The human body develops from a single fertilized egg, which grows and develops through continuous cell divisions. Cell division is established by a cell cycle, which is controlled by the catalytic reactions of cyclins and cyclin-dependent kinases (CDKs) that switch on and off at each step of the cell cycle. In stem cells, the cell cycle-regulating factors have close relationship with the differentiation of each tissue. Recent reports are revealing that microRNAs can regulate the cell cycle and differentiation of stem cells. However, there is a lack of research about the biological mechanisms of differentiation that is controlled by microRNAs in adult stem cells. The first part of this research investigated whether the cell cycle-regulating miRNAs might control the differentiation potency of human adipose-derived stem cells (hADSCs). After repeated spontaneous proliferation processes, the hADSCs showed the most decreased expression in CD44, a representative stem cell marker. As CD44 is known to be controlled by miRNA-34a, which is related to cell cycle regulation, the biological role of miRNA-34a in the biological behavior of hADSCs was examined. miR-34a treatment in hADSCs induced the decreased expression of CD44, and reduced the differentiation potency into adipogenesis by up to more than 50%. In addition, miR-34a decreased not only the expressions of cell cycle regulators, such as CDK2/4/6 and cyclin D/E, but also the expressions of stem cell maintenance factors, such as Oct4, Klf4, Sox2 and C-myc. These results present the importance of cell cycle-regulating miR-34a in the differentiation of hADSCs, as well as the importance of CD44 as an important indicator of the adipogenic potency of hADSCs. Cell cycle regulation is considered an important factor for neural cell development. Increasing reports that show that the cell cycle factors related to neurogenesis are regulated by microRNAs, indicate that miRNAs may influence the neurogenesis of stem cells. The second part of this study examined the potential role of miRNAs in the neurogenesis of hADSCs. Microarray for microRNA profiling was performed, using undifferentiated and neurally-differentiated hADSCs from 4 different donors, to identify the responsible miRNAs in neurogenesis of hADSCs. Compared with the undifferentiated control, 39~101 microRNAs with more than 2-fold higher expressions, and 3~9 microRNAs with 2-fold lower expressions, were identified. The identified microRNAs were further analyzed in terms of gene ontology (GO), in relation to neurogenesis based on their target mRNAs, as predicted by computational analysis. Among the over-expressed microRNAs after being differentiated into neural cells, 8 microRNAs (let7, miR-25, miR-34a, miR-124a, miR-146a, miR-125b, miR-126 and miR-149) relating to cell cycle were selected, and were further analyzed for their expressions, using real-time PCR, which showed the highest expression of miR-126. To investigate the influence of miR-126 on the trans-differentiation behavior of hADSCs, which proceeded from mesoderm to ectoderm, hADSCs were treated with miR-126, and then analyzed for trans-differentiation. RT-PCR results showed increased expressions of neural cell markers including GFAP, Olig2, NF-M, TUBB3, NSE and MAP2, and immunofluorescence staining results applied to GFAP, Olig2 MAP2 and NSE showed high fluorescence for each protein. In addition, miR-126 induced overall decreases in expressions of stem cell transcription factors (Sox2, KLF4, nanog and Oct4), cell cycle regulators (CDK2/4/6 and cyclin D/E), along with a decrease in expressions of PI3K and AKT. Taken together, these data suggest that miR-126 plays a key role in induction of trans-differentiation from mesoderm-derived stem cells to ectoderm-derived neural cells by down-regulating the important transcription factors for maintaining stem cell characteristics and cell cycle regulators.

Screening for homologues of fission yeast cell cycle genes in plants

이원근 University of Cambridge 2000 해외박사

Several regulatory genes of the plant cell cycle have been identified over a decade. However, many parts of it are still missing, for example, no kinases or phosphatases that influence the activity of the CDKs have been isolated. Therefore, the objective of this work was to enlarge our basic knowledge of the control of the cell cycle in all organisms by extending the investigation of genes involved in cell cycle control to plants Four yeast genes were chosen for their strong potential for providing access to factors of particular importance in the regulation of the plant cell cycle: the CDC34 gene, encoding an ubiquitin conjugating enzyme essential for cyclin degradation; the rum1 gene, encoding a CDK inhibitor required to prevent mitosis during G1; the mik1 gene, encoding a cdc2-specific protein kinase for the phoshorylation of tyrosine residue in p34^(cdc2), and the cdc25 gene, encoding a protein phosphatase that removes the phosphate group on tyrosine 15 of p34^(cdc). Two approaches were employed to clone plant homologues of these 4 yeast genes. First, a low stringency hybridisation screen was carried out to detect shared nucleotide sequences. To identify a CDC34 homologue, an oilseed rape cDNA library was screened. To find a rum1 homologue, cDNA libraries of soybean and oilseed rape were used. Among 10 isolated clones, bc5, bc6 and bc9 had high identity to a Arabidopsis polyubiquitin gene, bc1 to a human ubiquitin-specific protease, and br11 to potato UDP-glucose pyrophosphryase. Other clones showed less identity or no noticeable identity to any known genes, at DNA and amino acid level. Bc5, bc6 and bc9 had 35% sequence identity to CDC34 sequence, gr2 and gr9 had 35% sequence identity to rum1. However, there was no convincing evidence that these clones are real counterparts for the CDC34 or the rum1. Secondly, complementation of yeast mutants was undertaken in attempts to detect plant functional counterparts for ruml, mikl and cdc25. For this approach, a new maize root tip cDNA library was constructed in the yeast expression vector pREP3X under the control of the inducible promoter nmt1. Then, the yeast mutant strains (diploid or temperature sensitive haploid mutant) defective in rum1, mik] and cdc25 were transformed with this cDNA library. Among 8 positives from the ruml screen, rd6 and rh13 had homology to Arabidopsis elongation factor, rh11 and rh12 to ATP synthase beta subunit, and rh53 to a rice chilling tolerance gene. The cells of the most clones showed slightly elongated cell shape. Four positive clones were identified from the mik1 screen. Mh5 had similarity to quinone oxidoreductase, mhl5 to Equine herpesvirus 4 thymidine kinase. The phenotype of mhl5 cell was fatter round-dumbbell-shaped at a restrictive temperature. Clone mh18 showed homology to the Drosophila pollux (plx) gene. Among three positive clones of the cdc25 complementation, ch3 had identity to an elongation factor and ch4 to histone H4. Although none of the clones appeared to have convincing identity to the coresponding target genes, some clones were indirectly associated with the cell cycle, including elongation factor, histone H4, ATP synthase beta subunit and ubiquitin-specific protease. The transgenic tobacco plants were generated to determine whether and how plant development is affected by over-expression of the rum1 yeast gene, and also to provide same clue, albeit indirect, to the possible existence of a plant functional counterpart of rum1. A chimaeric construct, caning the coding region of the rum1 cDNA in between the CaMV35s promoter and a nopaline synthase terminator in the binary vector pROK2, was introduced into the tobacco genome by Agrobacterium-mediated transformation of leaf discs. The rum1 transcripts in transgenic plants were detected by PCR. The results imply that rum1 expression does not affect the phenotype of the transgenic plants in terms of leaf morphology, cell size and flowering etc. Simultaneously with the above approaches, I set up a synchronisation system of the plant cell cycle for phase-specific research into gene expression. The concentrations of N-source, myoinositol and hormones were optimised so as to obtain the highest proportion of single cells, facilitating the efficiency of the synchronising treatment. Both an aphidicolin treatment and a double-phosphate-starvation-and-readdition technique were employed for synchronisation. Aphidicolin treatment produced higher synchrony (42% LI and 12% MI) than the phosphatestarvation-readdition method (21 % LI and 6% MI). In conclusion, as sufficient independent copies of the library were screened and a range of different approaches were made, these results may be considered as justifying the conclusion that there are no plant counterparts for the rum1, mik1 and cdc25 yeast genes. The results of this research imply that, unlike cdk and cyclins, the rum1, mik1 and cdc25 may well have diverged widely in evolution, at both structural and functional aspects. So, a radically different approach may be needed to find new plant cell cycle genes by the help of yeast genes, if it is other than those of the central core mechanism.

MAST4 regulates spermatogonial stem cell self-renewal controlling cell cycle via the FGF2/ERM pathway

이승준 Graduate School, Yonsei University 2024 국내박사

Spermatogenesis is an important cellular differentiation process through which the male gametes are produced, and it remains active throughout an individual’s lifespan. Sertoli cell-only syndrome (SCO), including infertility, refers to the dysfunction of the male reproductive system. The self-renewal of spermatogonial stem cells (SSCs) must be accurate to prevent SCO syndrome. This study investigated the role of microtubule-associated serine/threonine kinase family member 4 (MAST4) in spermatogenesis in mice. Results revealed that MAST4 was localized in Sertoli cells before puberty, providing a somatic niche for spermatogenesis in mice. The size of Mast4 knockout testes was reduced compared with that of wild-type testes, and germ cell depletion associated with an increase in apoptosis and subsequent loss of tubular structure were similar to the SCO phenotype. MAST4 phosphorylated the ETS-related molecule (ERM), specifically the serine 367 residue. ERM phosphorylation ultimately controlled the transcription of ERM target genes related to the self-renewal of SSCs. Mast4 deletion led to the decreased promyelocytic leukemia zinc finger (PLZF) expression and cell cycle progression in the testes. MAST4 also induced cyclin-dependent kinase 2 to phosphorylate PLZF, and the activated PLZF suppressed the transcriptional levels of genes related to cell cycle arrest; consequently, SSCs retained their stem cell state. Therefore, MAST4 is associated with the fibroblast growth factor 2 (FGF2)/ERM pathway, and this association helps us explore the ability of SSCs to maintain a vertebrate stem cell niche. 정자형성과정은 남성의 수명 전반에 걸쳐 배우자를 생성하고 활성 상태를 유지하는 중요한 세포 분화 과정이다. 세르톨리세포단독증후군은 불임을 포함한 남성 생식 기관의 기능 장애를 말하고, 이를 예방하려면 정원줄기세포의 정확한 자가재생이 필수적이다. 본 연구는 생쥐의 정자형성과정에서 MAST4의 역할에 대해 제시하였다. MAST4는 사춘기 이전 세르톨리세포에 발현되어 생쥐의 정자형성과정을 위한 미세환경을 제공했다. Mast4 녹아웃 정소는 야생형 정소에 비해 크기가 감소했으며, 세포사멸의 증가 및 그에 따른 정세관 구조의 손실과 관련된 생식세포의 고갈은 세르톨리세포단독증후군의 표현형과 유사했다. 기존에 세르톨리세포에 발현되는 ERM 단백질은 FGF2 신호전달경로에 의해 조절되어 정원줄기세포의 자가재생을 유도하였다. 본 연구에서 MAST4 또한 FGF2의 조절을 받으며 ERM의 세린 367 잔기를 인산화했다. ERM의 인산화는 궁극적으로 정원줄기세포의 자가재생과 관련된 ERM 타겟 유전자의 전사를 조절했다. Mast4 유전자의 결실은 정소에서 PLZF의 발현 및 정원줄기세포의 세포주기 진행을 감소시켰다. MAST4는 또한 CDK2를 유도하여 PLZF를 인산화시켰고, 활성화된 PLZF는 세포주기 정지와 관련된 유전자의 전사를 억제하여 정원줄기세포가 줄기세포 상태를 유지할 수 있도록 유도했다. 따라서 MAST4는 FGF2/ERM 신호전달경로와 연관되어 있으며 이 연관은 척추동물의 줄기세포 미세환경을 유지하는 정원줄기세포의 능력을 탐구하는 데 도움이 된다. 또한 MAST4를 통한 불임 예방과 다른 상피성 기관의 줄기세포 자가재생에도 중요한 역할을 할 것을 시사한다.

미토콘드리아의 형태 변화가 세포 분열 주기에 미치는 영향

이승민 아주대학교 대학원 2005 국내석사

목적 : 세포는 분열을 통해 모세포가 두개의 딸세포로 나누어지면서 균등하게 유전 물질을 배분한다. 그때 각각의 딸세포는 DNA와 더불어 미토콘드리아, centrosome, 소포체, 골지체 등과 같은 소기관들을 고르게 배분받아야 한다. 이 중 DNA의 분열 과정과 그 과정에 문제가 생겼을 때 그것을 인지하고 복구하는 체계에 대해서는 비교적 잘 알려져 있다. 그러나 세포 내 소기관들이 어떻게 두 개의 딸세포로 배분되는지는 지에 대해서는 아직 정확하게 밝혀져 있지 않다. 그 중에서 골지체가 세포 분열이 진행되는 동안에 소포체에 완전히 흡수되었다가 분열이 끝난 후 다시 재조합되는데, 이것이 저해될 경우 세포가 사멸한다는 내용이 보고 된바있다. 이러한 사실을 통해 세포가 분열을 할 때, 세포 내 소기관들의 분포 또한 일종의 점검 요소로 작용하고 있음을 예상할 수 있다. 본 연구에서는 미토콘드리아를 대상으로 첫째, 세포 분열 주기 동안 미토콘드리아 biogenesis의 변화를 알아보기 위해 미토콘드리아 DNA copy number의 변화, 미토콘드리아의 mass 변화, 미토콘드리아의 biogenesis를 조절하는 전사 인자들의 발현, 그리고 세포 분열 주기 동안 미토콘드리아의 형태 변화를 알아보았다. 둘째, 미토콘드리아의 형태 변화를 유도하였을 때 세포 분열 주기 진행에 어떠한 영향을 줄 것인가를 규명하고자 하였다. 재료 및 방법 : HeLa 세포에서 세포 동일화 방법을 이용하여 세포 분열 주기 진행에 따른 미토콘드리아 mass를 미토콘드리아에 특이적으로 염색되는 MitoTracker와 NAO(10-nonylacridine orange) 형광물질로 염색하여 유세포분석법(FACS analysis)을 통해 알아보았다. 이때 미토콘드리아 DNA copy number를 real-time PCR을 통해 측정하였고, 또한 미토콘드리아의 biogenesis를 조절하는 인자들의 mRNA 발현정도를 역전사-중합효소 연쇄반응(reverse-transcription PCR)을 통해 확인하였다. 미토콘드리아의 형태와 분포를 MitoTracker 형광물질을 염색하여 알아보았고, 세포 분열 주기 진행에 따라 미토콘드리아의 형태를 조절하는 단백질들의 발현을 western blotting을 통해 조사하였다. 미토콘드리아 형태 변화를 유도하였을 때 세포 분열 주기 진행에 어떠한 영향을 것인가를 알아보기 위해 미토콘드리아 분열(fission)과 융합(fusion)을 조절하는 인자들을 과 발현시켜 미토콘드리아 형태가 변화된 세포주를 제작하였고, 이 세포주에서 세포분열주기를 관찰하였다. 결과 : 세포 분열 진행 과정 중 미토콘드리아의 mass는 G₁/S기에서 G₂기, M기로 가면서 2배로 증가했다가 다시 G₁기로 돌아오면서 감소하는 현상을 보였으며, mtDNA copy number는 G₁/S기에서 G₂기로 가면서 증가하지만 미토콘드리아 mass와는 달리 세포 분열을 마치고 다시 G₁기로 돌아와도 G₁/S기에 비해 상대적으로 높게 유지되는 것을 관찰하였다. 이때 미토콘드리아의 biogenesis를 조절하는 인자들인 mtTFA, NRF-1, PRC의 mRNA 발현에는 변화가 없는 것을 확인할 수 있었다. 그리고 미토콘드리아의 형태를 관찰하던 중 interphase와 mitosis에서 미토콘드리아의 형태가 차이가 남을 알게 되었다. Interphase에서는 미토콘드리아가 연결 관상구조(interconnected tubular structure)를 이루고 있는데 비해 mitosis로 가면서 세포 골격구조가 변함에 따라 세포 형태가 변하게 되는데 미토콘드리아 형태 또한 재구성되어 연결 관상구조가 감소하고 막대기 형태(rod type)의 미토콘드리아가 현저하게 증가하는 현상을 관찰하였다. 그리고 interphase에서는 미토콘드리아의 연결 관상구조 이외에 미토콘드리아 길이가 3 ㎛미만의 작은 길이, 3-10 ㎛의 중간 길이, 10 ㎛이상의 긴 길이의 미토콘드리아 분포가 다양한데 비해서, mitosis에서는 3-10 ㎛의 중간 길이의 미토콘드리아가 76 %까지 증가하여 미토콘드리아 개체군이 동질성을 나타냄을 확인할 수 있었다. 세포 분열이 진행되는 동안 미토콘드리아 형태를 조절하는 인자들의 단백질 발현정도를 알아보았는데 미토콘드리아의 분열(fission)을 조절하는 Drp1, hFis1의 단백질 발현정도는 차이를 나타내지 않았으나, 미토콘드리아의 융합(fusion)을 조절하는 Mfn1이 G₂ 시기에 유의하게 증가되는 현상을 관찰하였다. 또한 세포내에서 미토콘드리아의 분열을 조절하는 hFis1을 과 발현시킨 세포주와 분열을 억제하는 변이형 Drp1(Drp1^(K38A))을 과 발현시켜 미토콘드리아의 elongation을 유도한 세포주를 확립하였다. 이들 세포주의 세포 분열 주기를 조사하였을 때 미토콘드리아의 elongation이 유도된 세포주에서 G₂기에서 M기로의 전이가 지연되는 것을 관찰하였다. 결론 : 본 연구에서는 세포 분열 주기 진행에 맞추어 미토콘드리아의 mass와 mtDNA copy number가 변하며, 미토콘드리아의 biogenesis를 조절하는 인자들에는 변화가 없다는 결과를 얻었다. 또한 interphase에서 나타나던 연결 관상구조가 mitosis로 가면서 감소하였고 미토콘드리아 개체군이 동질성을 나타냄을 알 수 있었다. 또한 세포내에서 미토콘드리아의 분열을 억제하여 elongation을 유도한 세포주에서 세포 분열 주기, 특히 G₂->M 전이가 지연되는 현상을 관찰하였다. 따라서 본 연구 결과로 첫째 세포 분열과 coupling되어 미토콘드리아 mass와 mtDNA copy number가 증가하며, 둘째 mitosis로 진행되면서 미토콘드리아의 형태의 재구성이 필요하다는 것을 알 수 있다. 셋째 이러한 미토콘드리아의 형태의 재구성을 억제하여 elongation을 과도하게 유도하였더니 세포 분열 주기 진행이 지연되는 것으로 보아 미토콘드리아 또한 일종의 점검 요소로 작용하고 있다는 증거로서 세포내에서 이를 인식하고 조절하는 장치의 연구가 앞으로 진행되어야 할 것이다. Chromosome duplication and segregation is the most critical event during cell cycle progression and appears to be precisely controlled by several defined machineries. On the other hand, the mechanisms by which cellular organelles duplicate and partition into two daughter cells during cell division have been barely studied. Here, we investigated how mitochondria duplicate and partition during cell cycle progression. Using cell synchronization techniques, different phases of cells in cell cycle were collected and analyzed for the changes in mitochondrial DNA copy number and mitochondrial mass. We found that mitochondria mass gradually increased from G1 phase to mitotic phase and reduced back at the returning G1 phase. However, mtTFA, NRF-1, PRC, known as transcriptional factors regulating mitochondria biogenesis, did not significantly change during cell cycle progression. Interestingly, we found that heterogeneous mitochondrial population such as tubular network and fragmented small mitochondria found in interphase no longer exist in mitotic cells, suggesting that the mitochondrial reorganization may occur during G2 to M transition. When expression levels of hFis1, Mfn and Drp1 regulating mitochondrial fusion and fission were compared during cell cycle progression, we found that Mfn1 was specifically increased at G2 phase. Finally, we established the HeLa cell lines overexpressing hFis, Mfn or Drp1 genes, which accompanied with elongated or fragmented mitochondria. Interestingly, we observed that HeLa cells overexpressing the dominant-negative mutant of Drp1 gene showed delayed cell cycle progression, especially at G2/M phase. We are currently investigating whether the alteration in mitochondrial dynamics affects cell cycle progression in mammalian cells.