title: Adenosine blocks aminopterin-induced suppression of osteoclast differentiation.
abstract: 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.
authors.affiliation: Department of Molecular Cell Biology and Oral Anatomy, Faculty of Dental Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan.
authors.forename: Junpei
authors.initials: J
authors.lastname: Teramachi
authors.forename: Akiko
authors.initials: A
authors.lastname: Kukita
authors.forename: Pengfei
authors.initials: P
authors.lastname: Qu
authors.forename: Naohisa
authors.initials: N
authors.lastname: Wada
authors.forename: Yin-Ji
authors.initials: YJ
authors.lastname: Li
authors.forename: Seiji
authors.initials: S
authors.lastname: Nakamura
authors.forename: Toshio
authors.initials: T
authors.lastname: Kukita
mesh_terms: D000241:Adenosine / Q000494:pharmacology*; D000630:Aminopterin / Q000494:pharmacology*; D000700:Analgesics / Q000494:pharmacology*; D000818:Animals; D002454:Cell Differentiation; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006825:Hybridomas / Q000166:cytology / Q000378:metabolism; D008297:Male; D051379:Mice; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000700:Analgesics; D005493:Folic Acid Antagonists; D000630:Aminopterin; D000241:Adenosine