Mortalin, also known as mthsp70/GRP75/PBP74, interacts with the tumor suppressor protein p53 and inactivates its transcriptional activation and apoptotic functions. Here, we examined the level of mortalin expression in a large variety of tumor tissues, tumor-derived and in vitro immortalized human cells. It was elevated in many human tumors, and in all of the tumor-derived and in vitro immortalized cells. In human embryonic fibroblasts immortalized with an expression plasmid for hTERT, the telomerase catalytic subunit, with or without human papillomavirus E6 and E7 genes, we found that subclones with spontaneously increased mortalin expression levels became anchorage-independent and acquired the ability to form tumors in nude mice. Furthermore, overexpression of mortalin was sufficient to increase the malignancy of breast carcinoma cells. The study demonstrates that upregulation of mortalin contributes significantly to tumorigenesis, and thus is a good candidate target for cancer therapy. ' 2006 Wiley-Liss, Inc.

The mortalin genes, mot-1 and mot-2, are hsp70 family members that were originally cloned from normal and immortal murine cells, respectively. Their proteins differ by only two amino acid residues but exhibit different subcellular localizations, arise from two distinct genes, and have contrasting biological activities. We report here that the two proteins also differ in their interactions with the tumor suppressor protein p53. The pancytosolic mot-1 protein in normal cells did not show colocalization with p53; in contrast, nonpancytosolic mot-2 and p53 overlapped significantly in immortal cells. Transfection of mot-2 but not mot-1 resulted in the repression of p53-mediated transactivation in p53-responsive reporter assays. Inactivation of p53 by mot-2 was supported by the down-regulation of p53-responsive genes p21 WAF-1 and mdm-2 in mot-2-transfected cells only. Furthermore, NIH 3T3 cells transfected with expression plasmid encoding green fluorescent proteintagged mot-2 but not mot-1 showed an abrogation of nuclear translocation of wild-type p53. These results demonstrate a novel mechanism of p53 inactivation by mot-2 protein.Evidence has been accumulating that inactivation of p53, a tumor suppressor and cellular transcription factor (1), is involved in cellular transformation and immortalization (2-5). Extensive analyses of p53 have defined at least four functional domains, including an amino terminus transactivation domain (amino acids 1-44), a sequence-specific DNA-binding domain (amino acids 100 -300), a carboxyl terminus oligomerization domain, and a regulatory domain (amino acids Ref. 6), and shown that the conformation of p53 and its interactions with other proteins have key roles in its various cellular activities (7,8). Several cellular proteins, including some of the hsp70 family members, have been shown to interact with p53 (9 -12). Although mutational or mdm-2-mediated inactivation of p53 is a common event involved in cellular transformation (1), p53 is inactivated in a considerable number of tumors and transformed cells by an unknown mechanism(s).We initially cloned mortalins mot-1 and mot-2, which code for pancytosolically and perinuclearly distributed members of the hsp70 family of proteins, from normal and immortal murine cells, respectively (13,14). The open reading frames of the two types of murine mortalins differ in two nucleotides, encode proteins differing in two amino acids, arise from distinct genes, and have contrasting biological activities (13-16). RNA in situ hybridization and immunohistochemical studies on mortalin in normal murine tissues showed a higher level of expression in nondividing cell populations than in dividing cells. However, tumor tissues were seen to have a high intensity of mortalin staining by an antibody that reacts with both the mot-1 and mot-2 proteins (17, 18). Mortalin was also identified as PBP-74, mtHSP70, and Grp75 and has been assigned roles in antigen processing, in vivo nephrotoxicity, and radioresistance in independent studies from other groups (19,20)...

AcKNOwledgemeNTSWe are indebted to Prof. Mori M. (Kumamoto University, Japan) for generously providing pCAGGS-pOTC-GFP. We thank Ms. Nagami Yamashita for her supports. Prof. Ohkuma died on November 5, 2006 by myocardial infarction at his home. We greatly miss him as a scientist and a friend. We offer sincere thanks to all the friends, colleagues and former collaborators of Prof. Ohkuma who showed him kindness during his lifetime. AbSTrAcTAutophagy is the bulk degradation of cytoplasmic constituents in response to starvation and other environmental or intracellular cues. During this process, most of the cytoplasm is sequestered into autophagosomes, which then fuse with lysosomes where the degradation of the sequestered material proceeds. We investigated the relationship between autophagosome-lysosome fusion and the pH in acidic compartments by visualizing the fusion process using fluorescence in CHO cells. In this experiment, mitochondria were labeled with GFP by transfecting CHO cells with the presequence of ornithine transcarbamylase, and lysosomes were labeled with Texas Red Dextran; any fusion was identified by the colocalization of mitochondria (in autophagosomes) and lysosomes using fluorescence microscopy. When CHO cells were treated with rapamycin or starvation medium to induce autophagy, the colocalization of fluorescence was observed. Whereas when they were treated with 3-MA, an inhibitor of autophagy, the colocalization disappeared. We conclude that the colocalization reflects the fusion of autophagosomes and lysosomes. Moreover, when the CHO cells were treated with drugs that increase the pH of acidic compartments, the colocalization disappeared. This suggests that the autophagosome-lysosome fusion is inhibited by increasing pH in acidic compartments independently of V-ATPase activity in CHO cells.

The murine mortalin genes, mot-1 and mot-2, are members of the hsp70 family of proteins and di er from each other by only two amino acid residues. Mot-1 is expressed in normal cells and has pancytosolic cellular distribution whereas mot-2 is found in the perinuclear region of immortal cells. We report here that a high level of expression of mot-2 protein resulted in malignant transformation of cells as analysed by anchorage independent growth and nude mice assays. A high level of protein expression is attributed to the 900 bp 3' untranslated region of the cDNA which does not have any transforming activity per se. Mortalin cDNA clones isolated from human transformed cells were also found to have transforming activity in similar assays and a high level of expression was apparent in some of the human immortalized cells that showed non-pancytosolic mortalin immuno¯uorescence. Taken together, the data suggest that nonpancytosolic mortalin may have a role in tumorigenesis.

Mortalin/mtHsp70 (mitochondrial Hsp70) and HSP60 (heat-shock protein 60) are heat-shock proteins that reside in multiple subcellular compartments, with mitochondria being the predominant one. In the present study, we demonstrate that the two proteins interact both in vivo and in vitro, and that the N-terminal region of mortalin is involved in these interactions. Suppression of HSP60 expression by shRNA (short hairpin RNA) plasmids caused the growth arrest of cancer cells similar to that obtained by suppression of mortalin expression by ribozymes. An overexpression of mortalin, but not of HSP60, extended the in vitro lifespan of normal fibroblasts (TIG-1). Taken together, this study for the first time delineates: (i) molecular interactions of HSP60 with mortalin; (ii) their co- and exclusive localizations in vivo; (iii) their involvement in tumorigenesis; and (iv) their functional distinction in pathways involved in senescence.