National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2017 Annual Report of the Division of Intramural Research

Regulation of Mammalian Cell Proliferation and Differentiation

Melvin DePamphilis
  • Melvin L. DePamphilis, PhD, Head, Section on Eukaryotic Gene Regulation
  • Alex Vassilev, PhD, Staff Scientist
  • Diane Adler-Wailes, MS, Senior Research Assistant
  • Xiaohong Zhang, BA, Technical Assistant
  • Gaurav Sharma, PhD, Visiting Fellow
  • Arup Chakraborty, PhD, Research Fellow
  • Sushil Jaiswal, PhD, Postdoctoral Fellow
  • Ajit Roy, PhD, Postdoctoral Fellow

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 the distance from 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 re-replicate 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. As we age, however, 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. (For a comprehensive description of genome duplication in all forms of life, refer to References 4,5,6.)

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 could manipulate these mechanisms to destroy cancer cells selectively.

Links between DNA replication, stem cells, and cancer

Cancers can be categorized into two groups: those whose frequency increases with age, and those resulting from errors during mammalian development. The first group is linked to DNA replication through the accumulation of genetic mutations that occur during proliferation of developmentally acquired stem cells that give rise to and maintain tissues and organs. These mutations, which result from DNA replication errors as well as environmental insults, fall into two categories: cancer-driver mutations that initiate carcinogenesis, and genome-destabilizing mutations that promote aneuploidy through excess genome duplication and chromatid mis-segregation. Increased genome instability results in accelerated clonal evolution, leading to the appearance of more aggressive clones with increased drug resistance. The second group of cancers, termed germ cell neoplasia, result from the mislocation of pluripotent stem cells during early development. During normal development, pluripotent stem cells that originate in early embryos give rise to all cell lineages in the embryo and adult, but when they mislocate to ectopic sites they produce tumors. Remarkably, pluripotent stem cells, like many cancer cells, depend on the geminin protein to prevent excess DNA replication from triggering DNA damage–dependent apoptosis. This link between the control of DNA replication during early development and germ cell neoplasia reveals geminin as a potential chemotherapeutic target in the eradication of cancer progenitor cells.

Genome duplication: the heartbeat of developing organisms

The mechanism that duplicates the nuclear genome during the trillions of cell divisions required to develop from zygote to adult is the same throughout the eukarya, but the mechanisms that determine where, when, and how much nuclear genome duplication occurs regulate development and differ among the eukarya. They allow organisms to change the rate of cell proliferation during development, to activate zygotic gene expression independently of DNA replication, and to restrict nuclear DNA replication to once per cell division. They allow specialized cells to exit their mitotic cell cycle and differentiate into polyploid cells, and in some cases, to amplify the number of copies of specific genes. It is genome duplication that drives evolution, by virtue of the errors that inevitably occur when the same process is repeated trillions of times. They are, unfortunately, the same errors that produce age-related genetic disorders such as cancer.

Genome duplication at the beginning of mammalian development

The basic mechanism for replicating DNA has been conserved throughout evolution, even though the magnitude of the problem became monumental. A human cell contains 670 times the DNA in an E. coli cell, and human development requires trillions of cell divisions that produce about 37 billion miles of DNA. But instead of increasing the speed of replication forks to compensate for increasing genome size and organism complexity, evolution simply increased the number of replication origins, which allowed mammals to regulate initiation of DNA replication during cell proliferation without interfering with the ever-changing demands of gene expression during cell differentiation. Moreover, it allowed developing tissues to complete genome duplication before beginning mitosis and to restrict genome duplication to once per cell division. However, to overproduce gene products during development, some cells are allowed to differentiate into nonproliferating polyploid cells. We are interested in the mechanisms that make these events possible. Ironically, aberrations in the mechanisms are linked to cancer. In fact, the pluripotent cells produced during preimplantation development not only share characteristics of cancer cells, but they can also initiate cancer.

Geminin is essential to prevent DNA re-replication–dependent apoptosis in pluripotent cells, but not in differentiated cells.

Unique to multicellular animals, geminin is a dual-function protein with roles in modulating gene expression and preventing DNA re-replication. We showed that geminin is essential at the beginning of mammalian development to prevent DNA re-replication in pluripotent cells, exemplified by embryonic stem cells as they undergo self-renewal and differentiation. Embryonic stem cells, embryonic fibroblasts, and immortalized fibroblasts were characterized before and after geminin was depleted either by gene ablation or siRNA. Depletion of geminin under conditions that promote either self-renewal or differentiation rapidly induced DNA re-replication, followed by DNA damage, then a DNA–damage response, and finally apoptosis. Once differentiation had occurred, geminin was no longer essential for viability, although it continued to contribute to preventing DNA re-replication–induced DNA damage. We detected no relationship between the expression of geminin and genes associated with either pluripotency or differentiation. Thus, the primary role of geminin at the beginning of mammalian development is to prevent DNA re-replication–dependent apoptosis, a role previously believed essential only in cancer cells. The results suggest that regulation of gene expression by geminin occurs only after pluripotent cells differentiate into cells in which geminin is not essential for viability.

Geminin is essential for pluripotent cell viability during teratoma formation, but not for differentiated cell viability during teratoma expansion.

Pluripotent embryonic stem cells (ESCs) are unusual in that geminin has been reported to be essential either to prevent differentiation by maintaining expression of pluripotency genes or to prevent DNA re-replication–dependent apoptosis. To distinguish between these two incompatible hypotheses, immune-compromised mice were inoculated subcutaneously with ESCs harboring conditional Gmnn alleles alone or together with a tamoxifen-dependent Cre recombinase gene. Mice were then injected with tamoxifen at various times during which the ESCs proliferated and differentiated into a teratoma. For comparison, the same ESCs were cultured in vitro in the presence of monohydroxytamoxifen. The results revealed that geminin is encoded by a haplosufficient gene that is essential for ESC viability before the cells differentiate into a teratoma, but once a teratoma is established, the differentiated cells can continue to proliferate in the absence of Gmnn alleles, geminin protein, or pluripotent stem cells. Thus, differentiated cells did not require geminin for efficient proliferation within the context of a solid tissue, although they did when teratoma cells were cultured in vitro. The results provide proof-of-principle that preventing geminin function could prevent malignancy in tumors derived from pluripotent cells by selectively eliminating the progenitor cells with little harm to normal cells.

Identification of genes that are essential to restrict genome duplication to once per cell division

Nuclear genome duplication is normally restricted to once per cell division, but aberrant events that allow excess DNA replication (EDR) promote genomic instability and aneuploidy, both of which are characteristics of cancer development. We provided the first comprehensive identification of genes that are essential to restrict genome duplication to once per cell division. An siRNA library of 21,584 human genes was screened for those that prevent EDR in cancer cells with undetectable chromosomal instability. We validated candidates by testing multiple siRNAs and chemical inhibitors on both TP53+ (also known as p53) and TP53 cells to reveal the relevance of this ubiquitous tumor suppressor to preventing EDR, and in the presence of an apoptosis inhibitor to reveal the full extent of EDR. The results revealed 42 genes that prevented either DNA re-replication or unscheduled endoreplication. They all participate in one or more of eight cell-cycle events. Seventeen of them had not been identified previously in this capacity. Remarkably, 14 of the 42 genes have been shown to prevent aneuploidy in mice. Moreover, suppressing a gene that prevents EDR increased the ability of the chemotherapeutic drug Paclitaxel to induce EDR, suggesting new opportunities for synthetic lethalities in the treatment of human cancers.

Publications

  1. Vassilev A, DePamphilis ML. Links between DNA replication, stem cells and cancer. Genes (Basel) 2017 8(2):E45.
  2. Adler-Wailes DC, Kramer JA, DePamphilis ML. Geminin is essential for pluripotent cell viability during teratoma formation, but not for differentiated cell viability during teratoma expansion. Stem Cells Dev 2017 26(4):285-302.
  3. Vassilev A, Lee CY, Vassilev B, Zhu W, Ormanoglu P, Martin SE, DePamphilis ML. Identification of genes that are essential to restrict genome duplication to once per cell division. Oncotarget 2016 7:34956-34976.
  4. DePamphilis ML. Genome duplication: the heartbeat of developing organisms. Curr Top Dev Biol 2016 116:201-229.
  5. DePamphilis ML. Genome duplication at the beginning of mammalian development. Curr Top Dev Biol 2016 120:55-102.
  6. DePamphilis, ML (Editor). Mammalian Preimplantation Development. Curr Top Dev Biol 2016 120:1-478.

Collaborators

  • Scott E. Martin, PhD, NIH Chemical Genomics Center, NHGRI, Rockville, MD
  • 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.

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