Identification of a phosphoprotein that is downregulated in immortalized human fibroblasts Many lines of evidence indicate that the immortalization step is critical for the neoplas- tic transformation of normal human cells. Once normal human cells have been immor- talized, they are relatively easily transformed into neoplastic cells. In order to under- stand these phenomena, patterns of protein phosphorylation in proliferating normal human fibroblast cell strains and their immortalized cell lines were compared by using two-dimensional polyacrylamide gel electrophoresis. It was found that the expression and phosphorylation levels of the human heat shock protein 27 (HSP27) were predom- inantly downregulated in the immortalized cells compared with those in their normal counterparts. In the normal cells, HSP27 expression and phosphorylation were mark- edly increased by physiological and nonphysiological stresses, such as serum addi- tion, treatment with a carcinogenic agent like 4-nitroquinoline-1-oxide, and a high osmotic pressure. This may be a normal defense against acute changes of cellular environment and cytotoxic effects. However, these stresses had no effects on the expression and phosphorylation of HSP27 in the immortalized cells. These results sug- gest that an abnormal regulation of HSP27 expression and phosphorylation may be one of the reasons for easy neoplastic transformation of the immortalized cells by the treatment with carcinogenic agents. Keywords: Human fibroblasts / Immortalization / Heat shock protein 27 / Two-dimensional poly- acrylamide gel electrophoresis EL 4083 Masakiyo Sakaguchi Masahiro Miyazaki Tadashi Kondo Toshiya Tsuji Hirosuke Kouchi Masayoshi Namba Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University Medical School, Okayama, Japan 1 Introduction Many lines of evidence indicate that neoplastic transfor- mation of cells occurs by a multistep process, which is clearly determined by the successive stages of aging, immortalization, and neoplastic transformation [1, 2]. It is well known that normal human diploid cells are extremely refractory to in vitro neoplastic transformation because of the difficulty of immortalization. Once the cells have been immortalized, they can easily undergo neoplastic transfor- mation by treatment with carcinogenic agents, including chemicals, radiation, and oncogenes [1, 3]. These facts indicate that cellular immortalization is an important early step of human carcinogenesis. The molecular mechanism responsible for cellular immortalization is, however, still poorly understood. Expression of heat shock proteins (HSPs) rises in re- sponse to various stresses, though significant amounts of HSPs are present in unstressed cells, where they play important roles. High-molecular-weight HSPs 60, 70, and 90 have molecular chaperomin activities such as protein transduction, protein folding, and signal transduction [4]. However, functions of low-molecular-weight HSPs are still unclear. HSP27 is one of the small HSPs and is also called HSP25 or HSP28 [5, 6]. HSP27 is known as an early target of phosphorylation upon stimulation by serum, cytokines, and inducers of differentiation [7±13]. When quiescent cells are stimulated by serum, HSP27 is phosphorylated by MAP kinase-activated protein kinase II [14, 15], indicating that phosphorylation of HSP27 is regu- lated by an MAP kinase pathway (a major signal trans- duction pathway) [16]. HSP27 and its phosphorylated iso- forms are associated with numerous cellular events such as cell growth, morphological change, and differentiation [7±13]. In the present study, we used two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to ana- lyze the expression levels and phosphorylation of HSP27, and we found that, unlike in normal cells, HSP27 phos- phorylation in the immortalized human cells did not occur in response to various stresses. These results imply that abnormal regulation of cell growth and morphology in immortalized cells may be partly due to disregulation of HSP27. Correspondence: Prof. Masayoshi Namba, MD, PhD, Depart- ment of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University Medical School, 2-5-1- Shikata, Okayama 700-8558, Japan E-mail: mnamba@med.okayama-u.ac.jp Fax: +81-86-235-7400 Abbreviations: BNPS-scatole, 3-bromo-3-methyl-2-(2-nitrophe- nylmercapto)-3H-indole; DDW, double-distilled water; HSP27, heat shock protein 27; MM, molecular mass; 4-NQO, 4-nitroqui- noline-1-oxide Electrophoresis 2001, 22, 155±160 155  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001 0173-0835/01/0101-0155 $17.50+.50/0 Proteomics and 2-DE