Ken-ichi Yoshioka

The Plant Zinc Finger Protein ZPT2–2 Has a Unique Mode of DNA Interaction

ZPT2–2 is a DNA-binding protein of petunia that contains two canonical TFIIIA-type zinc finger motifs separated by a long linker. We previously reported that ZPT2–2 bound to two separate AGT core sites, with each zinc finger making contact with each core site. Here we present our further characterization of ZPT2–2 by using selected and amplified binding sequence imprinting and surface plasmon resonance analyses; together, these assays revealed some unusual features of the interaction between ZPT2–2 and DNA. These experiments allowed us to conclude that 1) the optimal binding sequence for the N-terminal zinc finger is AGC(T), and that of the C-terminal one is CAGT; 2) multiple arrangements of the two core sites accommodate binding; and 3) the spacing between the two core sites affects the binding affinity. In light of these observations, we propose a new model for the DNA-ZPT2–2 interaction. Further, consistent with this model, a high affinity binding site for ZPT2–2 was found in the promoter region of theZPT2–2 gene. This site may serve as acis-element for the autoregulation of ZPT2–2gene expression.

Induction of Cancerous Stem Cells during Embryonic Stem Cell Differentiation

Background: Cancer stem cells are responsible for tumorigenesis; however, the developmental process is poorly understood. Results: Differentiating stem cells in aberrant environments are subjected to carcinogenic stress, leading to genomic instabilization, mutation inductions, and cellular transformation with stemness characteristics. Conclusion: Aberrant environment for differentiation risks cancerous stem cell development. Significance: This is the first report showing normal stem cell transformation into malignant counterparts and their process. Stem cell maintenance depends on their surrounding microenvironment, and aberrancies in the environment have been associated with tumorigenesis. However, it remains to be elucidated whether an environmental aberrancy can act as a carcinogenic stress for cellular transformation of differentiating stem cells into cancer stem cells. Here, utilizing mouse embryonic stem cells as a model, it was illustrated that environmental aberrancy during differentiation leads to the emergence of pluripotent cells showing cancerous characteristics. Analogous to precancerous stages, DNA lesions were spontaneously accumulated during embryonic stem cell differentiation under aberrational environments, which activates barrier responses such as senescence and apoptosis. However, overwhelming such barrier responses, piled-up spheres were subsequently induced from the previously senescent cells. The sphere cells exhibit aneuploidy and dysfunction of the Arf-p53 module as well as enhanced tumorigenicity and a strong self-renewal capacity, suggesting development of cancerous stem cells. Our current study suggests that stem cells differentiating in an aberrational environment are at risk of cellular transformation into malignant counterparts.

Development of cancer-initiating cells and immortalized cells with genomic instability

Cancers that develop after middle age usually exhibit genomic instability and multiple mutations. This is in direct contrast to pediatric tumors that usually develop as a result of specific chromosomal translocations and epigenetic aberrations. The development of genomic instability is associated with mutations that contribute to cellular immortalization and transformation. Cancer occurs when cancer-initiating cells (CICs), also called cancer stem cells, develop as a result of these mutations. In this paper, we explore how CICs develop as a result of genomic instability, including looking at which cancer suppression mechanisms are abrogated. A recent in vitro study revealed the existence of a CIC induction pathway in differentiating stem cells. Under aberrant differentiation conditions, cells become senescent and develop genomic instabilities that lead to the development of CICs. The resulting CICs contain a mutation in the alternative reading frame of CDKN2A (ARF)/p53 module, i.e., in either ARF or p53. We summarize recently established knowledge of CIC development and cellular immortality, explore the role of the ARF/p53 module in protecting cells from transformation, and describe a risk factor for genomic destabilization that increases during the process of normal cell growth and differentiation and is associated with the downregulation of histone H2AX to levels representative of growth arrest in normal cells.

Gamma-irradiated quiescent cells repair directly induced double-strand breaks but accumulate persistent double-strand breaks during subsequent DNA replication.

H2AX is expressed at very low levels in quiescent normal cells in vivo and in vitro. Such cells repair DNA double‐strand breaks (DSBs) induced by γ‐irradiation through a transient stabilization of H2AX. However, the resultant cells accumulate small numbers of irreparable (or persistent) DSBs via an unknown mechanism. We found that quiescent cells that had repaired DSBs directly induced by γ‐rays were prone to accumulate DSBs during the subsequent DNA replication. Unlike directly induced DSBs, secondary DSBs were not efficiently repaired, although Rad51 and 53BP1 were recruited to these sites. H2AX was dramatically stabilized in response to DSBs directly caused by γ‐rays, enabling γH2AX foci formation and DSB repair, whereas H2AX was barely stabilized in response to secondary DSBs, in which γH2AX foci were small and DSBs were not efficiently repaired. Our results show a pathway that leads to the persistent DSB formation after γ‐irradiation.

Sensitization of Cancer Cells to Radiation and Topoisomerase I Inhibitor Camptothecin Using Inhibitors of PARP and Other Signaling Molecules

Radiation and certain anticancer drugs damage DNA, resulting in apoptosis induction in cancer cells. Currently, the major limitations on the efficacy of such therapies are development of resistance and adverse side effects. Sensitization is an important strategy for increasing therapeutic efficacy while minimizing adverse effects. In this manuscript, we review possible sensitization strategies for radiation and anticancer drugs that cause DNA damage, focusing especially on modulation of damage repair pathways and the associated reactions.

Genomic Destabilization Triggered by Replication Stress during Senescence

Most cancers develop after middle age, and are often associated with multiple mutations and genomic instability, implying that genomic destabilization is critical for age-related tumor development. In this manuscript, we review current knowledge regarding (1) the senescent cellular background, which is associated with a higher risk of genomic destabilization; and (2) the contributions of genomic destabilization to cancer development.