Regulation of Mammalian Cell Proliferation and Differentiation
- Melvin L. DePamphilis, PhD, Head, Section on Eukaryotic Gene Regulation
- Alex Vassilev, PhD, Staff Scientist
- Xiaohong Zhang, BA, Technical Assistant
- Arup Chakraborty, PhD, Research Fellow
- Sushil Jaiswal, PhD, Postdoctoral Fellow
- Ajit Roy, PhD, Postdoctoral Fellow
- Constandina O'Connell, BS, Postbaccalaureate Fellow
- John Oh, BS, Postbaccalaureate Fellow
- Jack Ahrens, Summer Student
Nothing is more fundamental to living organisms than the ability to reproduce. Each time a human cell divides, it must duplicate its genome, a problem of biblical proportions. A single fertilized human egg contains 2.1 meters of DNA. An adult of about 75 kg (165 lb) consists of about 29 trillion cells containing a total of about 60 trillion meters of DNA, a distance equal to 400 times that of Earth to sun. Not only must the genome be duplicated trillions of times during human development, but it must be duplicated once and only once each time a cell divides (termed mitotic cell cycles). If we interfere with this process by artificially inducing cells to rereplicate their nuclear genome before cell division, the result is DNA damage, mitotic catastrophe, and programmed cell death (apoptosis). On rare occasions, specialized cells can duplicate their genome several times without undergoing cell division (termed endocycles), but when this occurs, it generally results in terminally differentiated polyploid cells, which are viable but no longer proliferate. However, as we age, the ability to regulate genome duplication diminishes, resulting in genome instability, which allows genetic alterations that can result in promiscuous cell division, better known as cancer.
Our research program focuses on three questions: the nature of the mechanisms that restrict genome duplication to once per cell division; how these mechanisms are circumvented to allow developmentally programmed induction of polyploidy in terminally differentiated cells; and how we can manipulate these mechanisms to destroy cancer cells selectively.
CDK1 inhibition facilitates formation of syncytiotrophoblasts and expression of human chorionic gonadotropin [Reference 4].
Human placental syncytiotrophoblast (STB) cells play essential roles in embryo implantation and nutrient exchange between the mother and the fetus. STBs are polyploid, formed by fusion of diploid cytotrophoblast (CTB) cells. Abnormality in STBs formation can result in pregnancy-related disorders. While several genes have been associated with CTB fusion, the initial events that trigger cell fusion are not well understood. The primary objective of this study was to enhance our understanding of the molecular mechanism of placental cell fusion.
FACS (fluorescence-activated cell sorting) and microscopic analysis were used to optimize Forskolin-induced fusion of BeWo cells (surrogate of CTBs) and subsequently, changes in the expression of different cell-cycle regulator genes were analyzed through Western blotting and qPCR (quantitative polymerase chain reaction). Immunohistochemistry was performed on first-trimester placental tissue sections to validate the results in the context of placental tissue. We studied the effect of the cyclin-dependent kinase 1 (CDK1) inhibitor RO3306 on BeWo cell fusion by microscopy and FACS, and by monitoring the expression of human chorionic gonadotropin (hCG) by Western blotting and qPCR.
The data showed that the placental cell fusion was associated with down-regulation of CDK1 and its associated cyclin B, as well as a significant reduction in DNA replication. Moreover, inhibition of CDK1 by an exogenous inhibitor induced placental cell fusion and expression of hCG. Thus, the placental cell fusion can be induced by inhibiting CDK1. The study has a high therapeutic significance for the management of pregnancy-related abnormalities.
DHS (4,4′-dihydroxy-trans-stilbene) suppresses DNA replication and tumor growth by inhibiting RRM2 (ribonucleotide reductase regulatory subunit M2) [Reference 3].
DNA replication machinery is responsible for accurate and efficient duplication of the chromosome. Given that inhibition of DNA replication can lead to replication fork stalling, resulting in DNA damage and apoptotic death, inhibitors of DNA replication are commonly used in cancer chemotherapy. Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the biosynthesis of deoxyribonucleoside triphosphates (dNTPs), which are essential for DNA replication and DNA–damage repair. Gemcitabine, a nucleotide analog that inhibits RNR, has been used to treat various cancers. However, patients often develop resistance to this drug during treatment. Thus, the development of new drugs that inhibit RNR is needed. We identified a synthetic analog of resveratrol (3,5,4′-trihydroxy-trans-stilbene), termed DHS (4,4′-dihydroxy-trans-stilbene), that acts as a potent inhibitor of DNA replication. Molecular docking analysis identified the RRM2 (ribonucleotide reductase regulatory subunit M2) of RNR as a direct target of DHS. At the molecular level, DHS induced cyclin F–mediated down-regulation of RRM2 by the proteasome. Thus, treatment of cells with DHS reduced RNR activity and consequently decreased synthesis of dNTPs with concomitant inhibition of DNA replication, arrest of cells at S-phase, DNA damage, and finally apoptosis. In mouse models of tumor xenografts, DHS was efficacious against pancreatic, ovarian, and colorectal cancer cells. Moreover, DHS overcame both gemcitabine resistance in pancreatic cancer and cisplatin resistance in ovarian cancer. Thus, DHS is a novel anticancer agent that targets RRM2 with therapeutic potential either alone or in combination with other agents to arrest cancer development.
A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis [Reference 2].
High-throughput screening identified five chemical analogs (termed the WX8-family) that disrupted three events in lysosome homeostasis: (1) lysosome fission via tubulation without preventing homotypic lysosome fusion; (2) trafficking of molecules into lysosomes without altering lysosomal acidity; and (3) heterotypic fusion between lysosomes and autophagosomes. Remarkably, the compounds did not prevent homotypic fusion between lysosomes, despite the fact that homotypic fusion required some of the same machinery essential for heterotypic fusion. These effects varied 400-fold among WX8–family members, were time- and concentration-dependent, reversible, and resulted primarily from their ability to bind specifically to the PIKFYVE phosphoinositide kinase. The ability of the WX8 family to prevent lysosomes from participating in macroautophagy/autophagy suggested that they have therapeutic potential in treating autophagy-dependent diseases. In fact, the most potent WX8 family member was 100 times more lethal to 'autophagy-addicted' melanoma A375 cells than the lysosomal inhibitors hydroxychloroquine and chloroquine. In contrast, cells that were insensitive to hydroxychloroquine and chloroquine were also insensitive to the WX8 family. Therefore, the WX8 family of PIKFYVE inhibitors provides a basis for developing drugs that could selectively kill autophagy-dependent cancer cells, as well as for increasing the effectiveness of established anticancer therapies through combinatorial treatments.
The Cdk2-c-Myc-miR-571 axis regulates DNA replication and genomic stability by targeting geminin [Reference 1].
DNA rereplication leads to genomic instability and has been implicated in the pathology of a variety of human cancers. Eukaryotic DNA replication is tightly controlled to ensure that it occurs only once during each cell cycle. Geminin is a critical component of this control: it prevents DNA rereplication from occurring during S, G2, and early M phases by preventing MCM helicases (essential for genomic DNA replication) from forming prereplication complexes. Geminin is targeted for degradation by the anaphase-promoting complex (APC/C) from anaphase through G1 phase. However, accumulating evidence indicates that Geminin is downregulated in late S-phase owing to an unknown mechanism. We used a high-throughput screen to identify miRNAs that can induce excess DNA replication, and we found that the microRNA miR-571 could reduce the protein level of Geminin in late S-phase independently of the APC/C. Furthermore, miR-571 regulated efficient DNA replication and S-phase cell-cycle progression. Strikingly, the transcription factor c-Myc suppressed miR-571 expression by binding directly to the miR-571 promoter. At the beginning of S-phase, the cell cycle regulator Cdk2 (cyclin-dependent kinase 2) phosphorylated c-Myc at Serine 62, promoting its association with the miR-571 promoter region. Collectively, we identified miR-571 as the first miRNA that prevents aberrant DNA replication and the Cdk2–c-Myc–miR-571 axis as a new pathway for regulating DNA replication, the cell cycle, and genomic stability in cancer cells. The significance of these finding is that they identify a novel regulatory mechanism critical for maintaining genome integrity by regulating DNA replication and cell-cycle progression.
Publications
- Zhang Y, Li Z, Hao Q, Tan W, Sun J, Li J, Chen CW, Li Z, Meng Y, Zhou Y, Han Z, Pei H, DePamphilis ML, Zhu W. The Cdk2-c-Myc-miR-571 axis regulates DNA replication and genomic stability by targeting Geminin. Cancer Res 2019;79:4896-4910.
- Sharma G, Guardia CM, Roy A, Vassilev A, Saric A, Griner LN, Marugan J, Ferrer M, Bonifacino JS, DePamphilis ML. A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis. Autophagy 2019;15:1694-1718.
- Chen CW, Li Y, Hu S, Zhou W, Meng Y, Li Z, Zhang Y, Sun J, Bo Z, DePamphilis ML, Yen Y, Han Z, Zhu W. DHS (trans-4,4'-dihydroxystilbene) suppresses DNA replication and tumor growth by inhibiting RRM2 (ribonucleotide reductase regulatory subunit M2). Oncogene 2019;38:2364-2379.
- Ullah R, Dar S, Ahmad T, de Renty C, Usman M, DePamphilis ML, Faisal A, Shahzad-Ul-Hussan S, Ullah Z. CDK1 inhibition facilitates formation of syncytiotrophoblasts and expression of human Chorionic Gonadotropin. Placenta 2018;66:57-64.
Collaborators
- Juan Bonifacino, PhD, Section on Intracellular Protein Trafficking, Bethesda, MD
- Marc Ferrer, PhD, Chemical Genomics Center, NCATS, Bethesda, MD
- Juan Marugan, PhD, Division of Pre-Clinical Innovation, NCATS, Bethesda, MD
- Zakir Ullah, PhD, Lahore University of Management Sciences, Lahore, Pakistan
- Wenge Zhu, PhD, George Washington University Medical School, Washington, DC
Contact
For more information, email depamphm@mail.nih.gov or visit http://depamphilislab.nichd.nih.gov.
