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We previously identified BTBD1 and BTBD2 as novel topoisomerase I-interacting proteins that share 80% amino acid identity. Here we report the characterization of their subcellular localization. In a number of mouse and human cells, BTBD1 and BTBD2 (BTBD1/2) colocalized to punctate or elongated cytoplasmic bodies (< 5 microm long and several per cell) that were larger and more elongated in cancer cell lines than in fibroblasts and myoblasts. A search for potential colocalizing proteins identified TRIM family members that localize to morphologically similar cytoplasmic bodies, which were then tested for colocalization with BTBD1/2. TRIM5delta, expressed as a GFP fusion, colocalized with BTBD1/2 immunostaining and appeared to serve as a scaffold for the assembly of endogenous BTBD1/2 proteins. TRIM family members contain a RING domain, B-box(es), and coiled-coil regions, which have a characteristic order and spacing (RBCC domain). RING-dependent ubiquitin ligase activity and multimerization via the coiled-coil region may be defining properties of the RBCC/TRIM protein family. We found that TRIM5delta with a deleted coiled-coil region or a mutated RING domain failed to colocalize with BTBD1/2. Additionally, TRIM5delta ubiquitylated itself in a RING finger- and UbcH5B-dependent manner. BTBD1/2 each contain a PHR-similarity region, repeated twice on the putative ubiquitin ligases PAM, highwire and RPM-1, which also contain a RING and B-box. Thus, four protein modules found on each of these putative ubiquitin ligases, a RING, a B-box and two PHR repeats, are present on BTBD1/2 and TRIM5delta that are colocalized to cytoplasmic bodies.

The lifespan of human foreskin fibroblasts (HFF5), cultured under standard in vitro conditions (including ambient atmospheric oxygen tension), was extended slightly by expression of exogenous mortalin (mot-2)/mthsp70/Grp75, but not by the catalytic subunit of telomerase, hTERT. Together, mot-2 and hTERT permitted bypass of senescence, a substantial extension of lifespan, and possibly immortalization. This is the first demonstration that mot-2 and telomerase can cooperate in the immortalization process.

Hsp70 family member mot-2/mthsp70/GRP75/PBP74 was shown to bind to the tumor suppressor protein p53. In this study, by in vivo coimmunoprecipitation of mot-2 with p53 and its deletion mutants, the mot-2 binding site of p53 was mapped to its C-terminal amino acid residues 312-352, a region of p53 that includes its cytoplasmic sequestration domain. These data demonstrate that cytoplasmic sequestration and inactivation of p53 by mot-2 occurs by its binding to the cytoplasmic sequestration domain. Therefore, perturbation of mot-p53 interactions can be employed to abrogate cytoplasmic retention of wild-type p53 in tumors.

We have investigated the expression of mortalin in rat tissues by Northern analysis, RNA in situ hybridization, and immunohistochemical studies. By Northern assay, the highest level of expression was detected in brain, heart, and skeletal muscle followed by lung, liver, and kidney, and the least level of expression was detected in testis and spleen. RNA in situ and immunohistochemical studies showed that mortalin expression is significantly higher in upper nondividing layers than in the lower basal layers of skin, in neurons and nerve fibers than in surrounding glial cells in brain, in cardiomyocytes than in nonmyocytes in heart, and in interstitial secretory tissue than in germinating follicles in ovary. Such tissue- and cell-specific expression patterns of mortalin coordinates with its earlier reported antiproliferative function in normal cells. However, a deregulation of the expression is observed in rat brain tumor along with the detection of nonpancytosolic mortalin in rat glioma cell line C6. The study suggests that mortalin is involved in pathways that regulate division capacities of cells in vivo.

CARF is an ARF-binding protein that has been shown to regulate the p53–p21–HDM2 pathway. CARF overexpression was shown to cause growth arrest of human cancer cells and premature senescence of normal cells through activation of the p53 pathway. Because replicative senescence involves permanent withdrawal from the cell cycle in response to DNA damage response-mediated signaling, in the present study we investigated the relationship between CARF and the cell cycle and whether it is involved in the DNA damage response. We demonstrate that the half-life of CARF protein is less than 60 min, and that in cycling cells CARF levels are highest in G2 and early prophase. Serially passaged normal human skin and stromal fibroblasts showed upregulation of CARF during replicative senescence. Induction of G1 growth arrest and senescence by a variety of drugs was associated with increase in CARF expression at the transcriptional and translational level and was seen to correlate with increase in DNA damage response and checkpoint proteins, ATM, ATR, CHK1, CHK2, γH2AX, p53 and p21. Induction of growth arrest by oncogenic RAS and shRNA-mediated knockdown of TRF2 in cancer cells also caused upregulation of CARF. We conclude that CARF is associated with DNA damage response and checkpoint signaling pathways.

The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell-cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.

We have performed immunohistochemical studies of mortalin in normal and tumor human brain sections. In normal brain sections, the expression was seen mainly as being confined to neurons. Normal astrocytes showed undetectable expression of this unique member of the heat shock 70 protein family. Three grades of astrocyte tumors (low-grade astrocytoma, anaplastic astrocytoma, and glioblastoma), however, showed an increasing number of mortalin-positive cells. Other types of brain tumors, such as meningiomas, neurinomas, pituitary adenomas, and metastases, also showed elevated levels of mortalin expression compared to those in the normal brain. Mortalin has earlier been reported to have differential intracellular distribution in normal and transformed cellsin vitro.Therefore, we substantiated the present study with immunofluorescence localization of the protein in normal and glioblastoma cells. The observations indicated that the tumors might be expressing a nonpancytosolic mortalin. An increase in number of mortalin-positive cells with malignant progression of brain tumors and its correlation with Ki-67 (a cell proliferation marker)-positive cells further suggested an involvement of nonpancytosolic mortalin(s) in malignant transformation of cellsin vivo.

Introduction of human chromosome 7 by microcell-mediated chromosome transfer suppresses indefinite division of SUSM-1, anin vitroestablished human fibroblast line. This cell line has unusually long telomeric sequences although it lacks detectable telomerase activity. Thus, we examined whether such telomeric sequences change upon introduction of chromosome 7 or its segments. In the microcell hybrids that stopped dividing by introduction of chromosome 7, the telomeric sequences were found to be lost or markedly diminished. Introduction of various fragments (2–40 Mb) of chromosome 7 contained in radiation hybrids gave similar results. On the other hand, the telomeric sequences were not altered significantly in the unsuppressed hybrids, a revertant of one suppressed clone, or subclones of SUSM-1 used as controls. In the suppressed microcell hybrids, the distribution of a mortality marker, mortalin, was changed to the cytosolic type of mortal cells from the immortal type of perinuclear fibres. Also, senescence-associated β-galactosidase was induced to a level similar to that of normally senesced diploid fibroblasts. These results suggest that human chromosome 7 induces senescence in SUSM-1 by suppressing its telomere maintenance mechanism, which does not depend on telomerase.

To determine the genetic relation between the pancytosolic (uniformly distributed in cytoplasm-p66 mot-1 ) and the perinuclear (p66 mot-2 ) mortalin proteins found in normal and immortal mouse cells, respectively, we initiated the present study using PCR cloning, sequencing, and single nucleotide primer extension analyses. The results indicate that the difference between mot-1 and mot-2 at 2 bp in the open reading frame does not arise by mutations in the immortalized cells. Intriguingly, both mot-1 and mot-2 sequences were detected in genomic DNA from normal (CMEF) and immortal (RS-4) cells derived from the CD1-ICR mouse strain. We extended the analyses to four other strains of mouse and detected both mot-1 and mot-2 sequences in genomic DNA from the Balb/c mouse also. Swiss and C57BL/6 mice exhibited only mot-2 and the C3H He mouse only mot-1. Southern analyses on a mouse strain that showed the presence of only mot-2 (Swiss) and one that showed both mot-1 and mot-2 (CD1-ICR) by PCR revealed that there are structural differences between mot-1 and mot-2 loci. Furthermore, there appears to be a correlation between frequency of spontaneous immortalization of cultured mouse fibroblasts and the mot loci present in the mouse strain from which they were derived.

The dominance of cellular senescence over the immortal phenotype has been demonstrated by cell fusion experiments utilizing human and mouse cells. Mortalin, a novel 66-kDa member of the murine hsp70 family of proteins, has recently been identified as a marker of the mortal phenotype by virtue of its characteristic cytosolic distribution in mortal cells. Here we report the mortalin immunostaining observations on 21 human cell lines. These cell lines have previously been assigned by somatic cell hybridization analysis to one (18 lines) or more than one (3 lines) of the four complementation groups (A, B, C, and D) for immortalization. Four patterns of mortalin immunostaining were observed: granular-juxtanuclear cap, granular-gradient from nuclear to cell membrane, granular-juxtanuclear arch, and fibrous-perinuclear. In 17 of 18 cell lines assigned to a single complementation group, the mortalin staining corresponded with the complementation group. In two of the three cell lines previously assigned to multiple complementation groups, the mortalin staining corresponded to one of the assigned groups. Two cell lines, however, exhibited staining patterns which did not match to their assigned complementation groups. The basis of correlation between cellular distribution of mortalin and the complementation group remains unclear at present. However, the data (i) suggest that the intracellular distribution of mortalin can be used to distinguish mortal and immortal cells, confirming the association of mortalin with senescence; (ii) provide supportive evidence for the existence of at least four different pathways of immortalization in human cells; and (iii) indicate that mortalin is involved in processes that result in immortalization.

Mortalin, p66mol-1, from the cytosolic fractions of mouse embryonic fibroblasts (MEF) has been identified. We have cloned and characterized it as a novel member of mouse hsp70 family [R. Wadhwa, S.C. Kaul, Y. Ikawa, and Y. Sugimoto (1993) J. Biol. Chem. 268, 6615-6621]. We report that immortal clones from MEF such as NIH 3T3, RS4, and Balb/c 3T3 harbor the same or a very similar protein but in the perinuclear locale. Biochemical analysis revealed that the proteins from mortal (MEF) and immortal (NIH 3T3) fibroblasts do not show gross differences. However, the phenomenon of differential distribution of p66 protein in mortal and immortal fibroblasts is conserved in three different strains of mouse and, interestingly, in human fibroblasts, too. The data suggest that at least one common step is involved in immortalization of mouse and human fibroblasts and also point to the practical implication(s) of such easily detectable differences.