Journal of Bone and Mineral Metabolism

, Volume 31, Issue 1, pp 64–70 | Cite as

Adenosine blocks aminopterin-induced suppression of osteoclast differentiation

  • Junpei Teramachi
  • Akiko Kukita
  • Pengfei Qu
  • Naohisa Wada
  • Yin-Ji Li
  • Seiji Nakamura
  • Toshio Kukita
Original Article


To search cell surface molecules involved in the regulation of osteoclastogenesis, especially in fusion process, it is one powerful approach to obtain monoclonal antibodies bearing ability to block formation of multinucleated osteoclasts. Ideally, direct bio-assay of hybridoma supernatants is quite convenient to screen monoclonal antibodies of interest from numerous culture wells. However, addition of hybridoma supernatant containing hypoxanthine–aminopterin–thymidine (HAT), components of the selection medium, to whole bone marrow cultures strikingly suppressed osteoclastogenesis. Here we clarified aminopterin is the responsible component in HAT medium to inhibit osteoclastogenesis. Methotrexate (MTX), mono-methylated aminopterin, showed similar suppressive effect on osteoclastogenesis. When bone marrow cells were cultured in the presence of all nucleosides, aminopterin and MTX-induced suppression of osteoclastogenesis was abrogated. Among four nucleosides only adenosine canceled aminopterin-induced suppression of osteoclastogenesis. Direct bio-assay of hybridoma supernatant containing HAT selection medium is now available to screen monoclonal antibodies if adenosine-containing culture medium was utilized for evaluating osteoclastogenesis.


Methotrexate HAT Monoclonal antibody Adenosine Osteoclastogenesis 



This work was partly supported by a Grant for Scientific Research from the Japanese Ministry of Education, Science, and Culture (project 19592116).

Conflict of interest

The authors have no conflicting financial interests.


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Copyright information

© The Japanese Society for Bone and Mineral Research and Springer Japan 2012

Authors and Affiliations

  • Junpei Teramachi
    • 1
  • Akiko Kukita
    • 2
  • Pengfei Qu
    • 1
  • Naohisa Wada
    • 3
  • Yin-Ji Li
    • 1
  • Seiji Nakamura
    • 4
  • Toshio Kukita
    • 1
  1. 1.Department of Molecular Cell Biology and Oral Anatomy, Faculty of Dental SciencesKyushu UniversityFukuokaJapan
  2. 2.Department of Pathology and Biodefense, Faculty of MedicineSaga UniversitySagaJapan
  3. 3.Department of Endodontology and Operative Dentistry, Faculty of Dental ScienceKyushu UniversityFukuokaJapan
  4. 4.Department of Oral and Maxillofacial Oncology, Faculty of Dental ScienceKyushu UniversityFukuokaJapan

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