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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:246-255 (2007)
© 2007 The Gerontological Society of America

Stress Chaperones, Mortalin, and Pex19p Mediate 5-Aza-2' Deoxycytidine-Induced Senescence of Cancer Cells by DNA Methylation-Independent Pathway

Nashi Widodo, Custer C. Deocaris, Kamaljit Kaur, Kamrul Hasan, Tomoko Yaguchi, Kazuhiko Yamasaki, Takashi Sugihara, Tetsuro Ishii, Renu Wadhwa and Sunil C. Kaul

1 National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba, Japan.
2 Department of Molecular and Cellular Physiology, University of Tsukuba, Japan.
3 Department of Chemistry and Biotechnology, School of Engineering, University of Tokyo, Japan.
4 Cellular and Molecular Biology Group, Department of Radiobiology, Institute for Environmental Sciences, Aomori, Japan.

Address correspondence to Sunil C. Kaul, PhD, National Institute of Advanced Industrial Science & Technology (AIST), Central 4, 1-1-1, Higashi, Tsukuba, Ibaraki, 305-8562, Japan. E-mail: s-kaul{at}aist.go.jp


   Abstract
Top
 Abstract
Materials and Methods
 Results and Discussion
 References
 
DNA demethylating agents are used to reverse epigenetic silencing of tumor suppressors in cancer therapeutics. Understanding of the molecular and cellular factors involved in DNA demethylation-induced gene desilencing and senescence is still limited. We have tested the involvement of two stress chaperones, Pex19p and mortalin, in 5-Aza-2' deoxycytidine (5AZA-dC; DNA demethylating agent)-induced senescence. We found that the cells overexpressing these chaperones were highly sensitive to 5AZA-dC, and their partial silencing eliminated 5AZA-dC-induced senescence in human osteosarcoma cells. We demonstrate that these chaperones modulate the demethylation and chromatin remodeling-dependent (as accessed by p16INK4A expression) and remodeling-independent (such as activation of tumor suppressor p53 pathway) senescence response of cells. Furthermore, we found the direct interactions of 5AZA-dC with these chaperones that may alter their functions. We conclude that both mortalin and Pex19p are important mediators, prognostic indicators, and tailoring tools for 5AZA-dC-induced senescence in cancer cells.

5-METHYLCYTOSINE modification in the CpG islands of the 5'-upstream regulatory regions of genes imposes an important control on regulation of gene expression, DNA replication, and chromatin organization, and is one of the most consistent epigenetic changes in human cancers. Methylated DNA recruits histone deacetylase, which removes acetyl groups from histones, resulting in condensed chromatin structure blocking the basic transcriptional machinery access to the target gene leading to transcriptional repression (1). Hypermethylation of specific gene promoters that causes silencing of tumor suppressor genes has been most extensively studied and assigned as an important regulatory step in immortalization, tumor initiation, and progression of cancer (2–13). In contrast, global hypomethylation (reduction in genomic 5-methylcytosine content) has been recognized as a cause of oncogenesis that correlates with transformation and tumor progression (14–16). The DNA methyltransferase (DNMT) is a major enzyme that determines genomic methylation patterns. Consistent with the increased levels of many tumor suppressors in senescent cells, it was shown that the levels of DNMT enzyme activity decreases as normal fibroblasts were cultured to senescence (17,18). In contrast, simian virus 40 (SV40)–infected pre- or postcrisis cells or early transformed cells show high levels and activity of DNMT, implying that an ability to maintain DNMT level is acquired with SV40-induced escape from senescence (17,18). An increased level of DNMT has also been associated with the initiation and promotion of a variety of cancers including leukemia and colorectal, hepatic, gastric, breast, bladder, and lung-carcinomas (19–25).

5-Aza-2' deoxycytidine (5AZA-dC) is a DNA demethylating agent. It causes global demethylation of CpG-rich promoters resulting in transcription of genes silenced by methylation, and is known to induce cellular senescence-like phenotype in a variety of human cells (26–30). The p16INK4a gene has a CpG-rich promoter, and is repressed by methylation in a large variety of in vitro–transformed and tumor-derived cell lines (31). Reactivation of p16INK4a gene promoter by demethylation or by exogenous expression is known to cause a senescence-like growth arrest (32–36). p16INK4a has been demonstrated to be a critical player in replicative and premature senescence of human cells (32–35). It is frequently inactivated during life-span extension or immortalization of human cells (37,38). Because of findings, DNMT inhibitor, 5AZA-dC, either alone or in synergistic combination with the inhibitors of histone deacetylase to achieve effective re-expression of tumor suppressors for cancer therapy has been through clinical trials (4,39–44). An alternative mechanism involving covalent binding and trapping of DNMT by 5-AZA-dC was proposed as its primary effect; demethylation of genomic DNA was demonstrated as a secondary event (45). To date, the molecular effects of 5AZA-dC have not been completely elucidated. Furthermore, characterization of cellular factors responsible for 5AZA-dC-induced senescence and the differential response of different cancers and cases are critical for its use in cancer therapy.

Molecular chaperones are the proteins that play an important role in cell survival. The activities of chaperones in both housekeeping and stress response are based on their ability to interact with unfolded and misfolded proteins and to assist in their proper folding to avoid the accumulation of aggregation-prone intermediates (46–48). Since 5AZA-dC evokes gene desilencing, we predicted that the 5AZA-dC would provoke the need of high chaperone activity in vivo and hence may constitute an essential component of 5AZA-dC response. We demonstrate that the two stress chaperones, Pex19p and mortalin, are critically involved in 5AZA-dC-induced senescence and have demethylation-independent effects.


   MATERIALS AND METHODS
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 Abstract
 Materials and Methods
Results and Discussion
 References
 
Plasmid Constructions
Full-length human Pex19p complementary DNA (cDNA) was cloned from human testis by reverse transcription–polymerase chain reaction (RT–PCR) using sense (5'-gaa ttc atg gcc gcc gct gag-3') and antisense (5'-gtc gac gca cct aga gag agg-3') Pex19p-specific primers with EcoRI and SalI sites, respectively. The PCR amplification (94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 3 minutes) product was purified and cloned into pEGFPC1 (mammalian expression vector for green fluorescent protein (GFP; Clontech, Mountain View, CA)-Pex19p fusion protein. For expression of hammerhead ribozymes, an expression plasmid (pPUR-KE) containing a chemically synthesized human RNA polymerase III (tRNAVal) promoter and a puromycin selection marker were used as described (49). Four target sites (with cleavage sites at nucleotides 40, 108, 122, or 168) for Pex19p were used. Integrity of all the plasmids was confirmed by sequencing. The efficacy of ribozymes was analyzed by detection of GFP-Pex19p protein in ribozyme-transfected cells by western blotting as described previously. For construction of retrovirus-driven expression of mortalin, pCX4neo (gift from Dr. Tsuyoshi Akagi, Osaka, Japan) was used. cDNA encoding for mortalin-myc protein was cloned into the BamHI site of the vector. RNA polymerase III-driven hammerhead ribozyme expression plasmids for mortalin were made as described (50). The empty vector containing the transfer RNA (tRNA) sequence, but without ribozyme, was used as a negative control.

Cell Culture, Transfections, and Infections
Transfections of expression plasmids encoding Pex19p, mortalin, or their specific ribozymes were performed as described earlier (49,50). Transfected cells were selected in a medium that contained puromycin (2 µg/mL; Sigma, St. Louis, MO) for 5 days and were then treated with 10 µM 5AZA-dC for 3–4 days. Empty vector-transfected cells were used as controls. For production of retroviruses, Plat-E, an ecotropic murine leukemia virus (MuLV)-packaging cell line was grown (1 day before the transfections) in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) at approximately 80% confluence. Cells were transfected with the pVPack-GP (gal and pol) and pVPack VSVG (env)-expressing vectors (Stratagene, La Jolla, CA) and retroviral vector or pCX4neo/mot myc using FuGENE6 (Roche Diagnostics K. K., Tokyo, Japan) following the manufacturer's instructions. After 48 hours, culture supernatants were collected, filtered through 0.45 µm filters, and used as viral stocks for infection. Cells were plated at the density of 2 x 105 cells/well in six-well dishes. After 24 hours, polybrene at 8 µg/mL was added to the cells and incubated at 37°C for 1 hour. Polybrene containing medium was then removed from the cells and infected with 300 µL of filtered viral stock, incubated at 37°C for 1 hour, and tilted after every 15 minutes to ensure the anchorage of viral particles to cells. After 1 hour, 2 mL of DMEM was added to the culture to dilute the viral stock, and then incubated at 37°C. At 48 hours postinfection, the cells were grown in selection medium containing G418 (at 1 mg/mL) until stable expressing cell lines were obtained. Selected populations were used for further experiments.

5-Aza-2' Deoxycytidine–Effector Assays
Human osteosarcoma (U2OS) cells transfected with indicated expression plasmids were examined for their growth by counting cell numbers, and then were assayed for azacytidine-induced p16INK4A expression by western blotting. Endogenous ß-galactosidase (ß-gal) staining of cells, an accepted marker for senescent cells (51), was used for detection of 5AZA-dC-induced senescence.

RT–PCR and Western Blot Analysis
Total RNA was prepared from 70% confluent cells using ISOGEN reagent (Nippon Gene, Toyama, Japan). RT–PCR for Pex19p was performed using specific primers for Pex19p (sense-5'-caa gat ggc cgc cgc tga gga a-3' and antisense 5'-tgt tgt gtt tca cat gat cag aca ct-3' primers). PCR product was visualized on 1% agarose gel.

For western blotting, the protein sample (10–20 µg) was separated on a sodium dodecyl sulfate–polyacrylamide gel, and electroblotted onto a nylon membrane (Millipore, Billerica, MA) using a semidry transfer blotter. Immunoassays were performed with polyclonal antimortalin antibody, a monoclonal anti-p16INK4a (#13251A; BD Pharmingen, San Diego, CA), anti p21WAF1 (sc-397; Santa Cruz Biotechnology, Santa Cruz, CA) or antiactin (MAB1501R; Chemicon, Temecula, CA) antibodies. The immunocomplexes formed were visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies using an enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech, Piscataway, NJ).

Immunostaining
Cells grown on glass coverslips placed in 35 mm plastic dishes were washed with cold phosphate-buffered saline (PBS) and fixed with a prechilled methanol/acetone (1:1, vol/vol) mixture for 5 minutes on ice. Fixed cells were washed with PBS, permeabilized with 0.2% Triton X-100 in PBS for 10 minutes, and blocked with 2% bovine serum albumin (BSA) in PBS for 20 minutes. Cells were stained with antimortalin antibody and were visualized by secondary staining with goat antirabbit immunoglobulin G (IgG) (Alexa-594-conjugated goat antirabbit or Alexa 488-conjugated goat antimouse; Molecular Probes, Carlsbad, CA). After six washings in PBS with 0.1% Triton X-100, cells were overlaid with a coverslip with Fluoromount (Difco/BD, Franklin Lakes, NJ). The cells were examined on a Carl Zeiss (Tokyo, Japan) microscope with epifluorescence optics. Images were saved as TIFF files and imported into Adobe Illustrator for labeling.

1-Anilinonapthalene-8-Sulfonic Acid Binding Assay
For 1-anilinonapthalene-8-sulfonic acid (1,8-ANS) (Sigma) fluorescence analysis, purified recombinant His-tagged mortalin protein was used. Protein (0.25 mg) in PBS was incubated with 0.5 and 1 µM 5AZA-dC at 25°C for 30 minutes. Ethanolic solution (1 mM) of 1,8-ANS was then added to a final concentration of 10 µM. After 30 minutes, fluorescence spectra were determined from 400 to 600 nm with a Shimadzu (Shimadzu Corporation, Kyoto, Japan) RF-5300PC spectrofluorometer at an excitation wavelength of 380 nm and emission and excitation band passes of 5 nm.

Far-UV Circular Dichroism Spectroscopy
Far UV circular dichroism spectra were recorded using a JASCO (Tokyo, Japan) spectropolarimeter. Experiments were performed with 0.5 µM mortalin protein in PBS buffer using a 1 cm path-length cuvette at 37°C. Azacytidine stock solution (1 mM) was added directly to the solution while stirring with a magnetic stirrer. All spectra reported are the average of eight accumulations.

In Vivo Chaperone Assay
Chaperone activities of mortalin and Pex19p in the presence of 5AZA-dC were evaluated by an in vivo luciferase refolding assay. Cells transfected with pGL3 firefly luciferase vector and empty vector (pCDNA3) and mortalin/Pex19p expression constructs were grown in 12-well dishes and incubated in complete DMEM containing cycloheximide at 10 mg/mL and 20 mM 4-morpholinepropanesulfonic acid (MOPS) (pH 7.0) 1 hour before heat treatment. Samples were lysed and assayed for luciferase activity in quadruplicate after 45°C heat-treatment for 30 minutes followed by 3-hour recovery. Relative chaperone activity was obtained by dividing the luciferase activity of heat-treated and heat-recovery cells by control (nonheat-shocked) cells. Chaperone modification indices (CMI) of 5AZA-dC were obtained by normalizing the relative chaperone activity of 5AZA-dC-treated cells with the nontreated cells.


   RESULTS AND DISCUSSION
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 Abstract
 Materials and Methods
 Results and Discussion
References
 
Normal cells can divide only for a limited number of times in vitro (Hayflick limit) and reach an irreversible nonproliferative state referred to as replicative cellular senescence (52–54). The most apparent characteristics of senescing cells include a large and flat morphology, a high frequency of nuclear abnormalities, and positive staining for ß-gal activity specifically at pH 6.0 (51). Because many tumor suppressor genes that are inactivated in cancers are indeed highly expressed in senescing cells, senescence is thought to be a tumor suppressor mechanism (52,53). Interestingly, (i) the rate of loss of 5-methylcytosine is inversely proportional to the Hayflick limit and to the life span of cell donor species (55), and (ii) immortal cancer cells can be induced to senesce by a DNMT inhibitor, 5AZA-dC, that causes demethylation of DNA and desilencing of gene expression. These studies have implied that replicative senescence can result from epigenetic changes in gene expression (30). We used human osteosarcoma (U2OS) cells in which p16INK4A is silenced by hypermethylation as a model for 5AZA-dC-induced cellular senescence. An induction of p16INK4A was used as a marker for demethylation as well as senescence as described (30,56,57). As shown in Figure 1A and B, 5AZA-dC-treated cells showed the expression of p16INK4A, enlarged cell size, growth arrest, and senescence-associated ß-gal staining.


Figure 01
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  		Figure 1. 5-Aza-2' deoxycytidine (5AZA-dC)-induced senescence in U2OS cells. Induction of p16INK4A expression (A), growth arrest (B, a and b), and induction of senescence-related ß-galactosidase staining (B, c and d). Chaperone activity of mortalin and Pex19p in in vivo luciferase folding assays. Both Pex19p and mortalin have significant chaperone activity in in vivo luciferase folding assays (C)

 
Although mortalin and Pex19p are categorized as chaperones, their chaperone activity has not been formally documented. Because we anticipated that chaperone activity of these proteins could be essential for increased gene expression evoked by demethylation, we first investigated their chaperone activity by an in vivo luciferase refolding assay. As expected, both proteins showed chaperone activity (Figure 1C). Involvement of Pex19p and mortalin in 5AZA-dC-induced senescence was examined by gene repression and overexpression studies as described below.

Pex19p and 5AZA-dC Induced Senescence
U2OS cells overexpressing Pex19p were generated. Examination of 5AZA-dC response in Pex19p-overexpressing cells in comparison to the vector-transfected control cells revealed that the overexpression of Pex19p made the cells more sensitive to 5AZA-dC-induced senescence (Figure 2A). Furthermore, consistent with their stronger growth arrest (Figure 2A), Pex19p-overexpressing cells showed an enhanced induction of p16INK4A expression in response to 5AZA-dC treatment (Figure 2B). We also generated U2OS derivatives in which Pex19p expression was compromised by specific ribozymes. Choice of ribozymes in contrast to small interfering RNA (siRNA) pertained to their milder effect. Three independent target sites on Pex19p messenger RNA (mRNA) were used for ribozymes. Transfected cells were selected in puromycin-supplemented medium and then treated with 5AZA-dC. Induction of p16INK4A was used as an assay for induction of senescence. As shown in Figure 2C, each of the Pex19p specific ribozymes decreased the induction of p16INK4A subsequent to the 5AZA-dC treatment. Although the endogenous level of expression could not be determined due to the lack of anti-Pex19p antibody, the efficacy of Pex19p ribozymes was determined by their effect on GFP-Pex19p protein expressed by a cytomegalovirus (CMV) promoter-driven expression plasmid as described earlier (49 and data not shown). Of note, as compared to the untransfected control cells, the cells compromised for Pex19p showed less induction of p16INK4A in response to 5AZA-dC treatment (Figure 2C). These data demonstrated that the expression level of Pex19p influenced the 5AZA-dC response of cells. Because the normal and nondividing human cells are less responsive to 5AZA-dC, we investigated the correlation, if any, with Pex19p expression. In matched pairs of precrises and postcrisis SV40-transformed cells representing the mortal and the early stages of immortalization, respectively, postcrisis cells had an elevated level of Pex19p expression in 6/6 pairs (Figure 2D). Furthermore, normal cells at the nondividing confluent state showed less expression of Pex19p as compared to the dividing cells (Figure 2E). Cancer cells have been shown to be more sensitive to 5AZA-dC than normal cells as they incorporate more 5AZA-dC in their DNA due to a higher division rate. Although Pex19p expression was upregulated in postcrisis immortal cells and in dividing cells as compared to their precrisis and confluent nondividing counterparts, an overexpression of Pex19p, by itself, did not have significant proproliferation effect on cells (Figure 2A). From these data, it was suggested that Pex19p does not directly affect proliferation and the incorporation of 5AZA-dC (demethylation) into the DNA. Hence, the role of Pex19p in 5AZA-dC-induced senescence may involve DNA demethylation-independent pathway(s).


Figure 02
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  		Figure 2. Involvement of Pex19p in 5-Aza-2' deoxycytidine (5AZA-dC)-induced senescence. Cells overexpressing Pex19p showed stronger 5AZA-dC-induced growth arrest as compared to the control cells (A). Cells overexpressing green fluorescent protein (GFP)-tagged Pex19p showed much higher induction of p16INK4A than the control cells (B). Actin was used as a loading control. Induction of p16INK4A in response to 5AZA-dC in control and Pex19p-compromised cells. Note that the cells compromised for Pex19p expression showed less induction of p16INK4A (C). Human immortalized postcrisis cells showed a higher level of Pex19p expression as compared to their matched unimmortalized precrisis counterparts (D). Human normal lung fibroblasts at dividing stage showed higher level of expression of Pex19p than at density-arrested nondividing stage (E)

 
Mortalin and 5AZA-dC-Induced Senescence
Mortalin is a stress protein, and its knockdown results in growth arrest of cancer cells (50,58). To elucidate its involvement in 5AZA-dC-induced senescence, we made derivatives of U2OS cells with high level of mortalin expression as described in the Materials and Methods section. As expected, overexpression of mortalin had a proproliferative effect (Figure 3A). Noticeably, mortalin-overexpressing cells were significantly more sensitive to 5AZA-dC (Figure 3A), and showed a stronger induction level of p16INK4A (Figure 3B). This finding might be due to the enhanced incorporation of 5AZA-dC in cells because of their higher proliferation rate. A higher level of mortalin expression has been reported in a variety of immortalized and tumor-derived human cells and tumor tissues (59–61). In a panel of normal and tumor tissues from the same individuals (Biochain, Hayward, CA), we detected a higher level of expression of mortalin in tumor tissues as compared to their normal counterparts (Figure 3C), suggesting that an upregulation of mortalin is a common event during carcinogenesis. The results, based on the data that the overexpression of mortalin sensitized the cells to 5AZA-dC (Figure 3A and B), suggest that an upregulated mortalin may contribute to the response of cells to 5AZA-dC. Together with the data on Pex19p, the study implied that a high level of expression of mortalin and Pex19p in transformed cells may be physiologically relevant to differential response of aging and nonaging cells to 5AZA-dC (62).


Figure 03
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  		Figure 3. Involvement of mortalin in 5-Aza-2' deoxycytidine (5AZA-dC)-induced senescence. Mortalin-overexpressing cells underwent stronger growth arrest (A) and showed higher induction of p16INK4A (B) in response to 5AZA-dC treatment. Human tumor tissues (T) showed higher levels of mortalin expression than did their matched normal tissues (N) (C)

 
We next investigated whether mortalin is a critical target of 5AZA-dC and what could be the molecular mechanism of its involvement in 5AZA-dC-induced senescence. The subcellular distribution of mortalin can distinguish normal cells from cancer cells (63,64). Induction of senescence in transformed cells, by addition of single chromosomes and chromosome fragments (65,66), BrdU (67), and MKT077 (68), resulted in changes in the subcellular localization (from perinuclear to the pancytoplasmic) of mortalin. Hence, we examined if intracellular distribution of mortalin was affected in response to 5AZA-dC treatment. As shown in Figure 4A, the perinuclear mortalin staining pattern of U2OS cells shifted to the pancytoplasmic type (typical of normal cells) in 5AZA-dC-treated cells. Perinuclear mortalin binds to p53 and inactivates its function in transformed cells (69,70). The shift of perinuclear mortalin to the pancytoplasmic type in 5AZA-dC-treated cells, therefore, suggested that the p53 activation might occur by abrogation of mortalin-p53 complexes. Of note, 5AZA-dC-treated cells indeed showed upregulation of p53 and its downstream effector, p21WAF1, in addition to p16INK4a (Figure 4B), and exhibited nuclear translocation of the p53 protein. Whereas only 10%–20% of control cells showed nuclear p53, 90% of the 5AZA-dC-treated cells showed strong p53 staining in the nucleus (Figure 4C). These results demonstrated that 5AZA-dC-induced growth arrest of U2OS cells involves not only the induction of p16INK4a that is mediated by its DNA-demethylation effect but also by activation of a second major senescence-associated tumor suppressor pathway, p53-p21WAF1 pathway (71). Time-course study revealed that the change in mortalin staining pattern from perinuclear to pancytoplasmic type occurs as early as 4 hours after 5AZA-dC treatment. Of note, p53 translocation to the nucleus occurred at about 8 hours after 5AZA-dC treatment, suggesting that it is an early event and is independent to that of the demethylation effects (Figure 4D and E). Consistent with this finding, an activation of p53 function precedes the induction of p16INK4A (Figure 4F). Whereas p53 function was activated (increase in the level of expression of p21WAF1 occurred within 6–12 hours of 5AZA-dC treatment), an induction of p16INK4A occurred between 24 and 48 hours of treatment. These results clearly demonstrate that p53 activation induced by a shift in subcellular distribution of mortalin is a major and early event in 5AZA-dC-induced senescence of cancer cells.


Figure 04
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  		Figure 4. Mortalin mediates 5-Aza-2' deoxycytidine (5AZA-dC)-induced senescence by a demethylation-independent pathway. Change in subcellular staining pattern of mortalin in response to 5AZA-dC treatment (A) and an activation of p53 function as seen by its stabilized amounts, p21WAF1 expression (B), and translocation into the nucleus (C). 5AZA-dC caused a shift in mortalin staining (D) and nuclear translocation of p53 (E) as early as 4 and 8 hours, respectively, leading to an activation of p53 function (6–12 hours) that preceded the induction of p16INK4A (24–48 hours) (F)

 
We next conducted an ANS binding assay to test the hypothesis that 5AZA-dC binds to mortalin and alters its physicochemical properties. ANS dye can bind to accessible hydrophobic regions in protein molecules and becomes highly fluorescent, a property that makes it a reliable reporter assay for studying structural changes in a protein. Purified recombinant mortalin protein was used. As shown in Figure 5A, 5AZA-dC caused a significant decrease in the amount of fluorescence in a dose-dependent manner ({Delta}F · I1/2 [the half-maximal loss of fluorescence intensity as a function of AZA concentration] = 5.3 µM). To further confirm the structural changes associated with 5AZA-dC and mortalin interaction, the circular dichroism spectra of mortalin was determined as shown in Figure 5B. The spectra of mortalin (with or without 5AZA-dC) exhibit a double minimum around 220 and 208, suggesting the presence of a significant amount of {alpha}-helical structure. With the addition of 5AZA-dC, the alteration in spectrum shape manifests lower {alpha}-helical content probably towards increased unordered structure. Molar ellipticity values of the samples at 215 nM were plotted to derive a dose-response analysis of the transitions. As shown in Figure 5C, at doses approximately > 7 µM of 5AZA-dC, mortalin dramatically showed loss in helicity reaching a plateau at the 12 µM level. Compared to the half-maximal value of loss in ANS binding, it seemed to require higher concentrations of 5AZA-dC ({Delta}{theta}1/2 = 6.4 µM). These findings confirmed that 5AZA-dC causes structural perturbations in mortalin with binding to its hydrophobic regions. Such interaction can also potentially explain the resulting abrogation of mortalin-p53 complexes and translocation of p53 to the nucleus as supported by the data in Figure 4C. Furthermore, we found that similar to the change in the staining pattern of mortalin, GFP-Pex19p undergoes alteration in its subcellular distribution (from diffuse cytoplasmic to granular cytoplasmic) in response to 5AZA-dC treatment (Figure 5D). Taken together, these data supported the hypothesis that 5AZA-dC-induced senescence involves structural changes in chaperone proteins, Pex19p and mortalin. A stronger growth arrest caused by 5AZA-dC in cells overexpressing either mortalin or Pex19p (Figures 2A and 3A) can be explained in two ways. First, following the 5AZA-dC-induced demethylation and gene expression, these chaperones may cause stabilization of tumor suppressor proteins such as p16INK4a as seen in the present study. Second, mortalin and Pex19p act as direct targets of 5AZA-dC and undergo structural changes (Figure 5) and functional decline contributing to growth arrest of cells. In this scenario, overexpression of these target proteins will further enhance the 5AZA-dC-caused damage to cell functions. These events are independent to the demethylation effect of 5AZA-dC. Because mortalin and Pex19p are chaperones (72,73) (Figure 1C), we suspected that the interactions with 5AZA-dC might abrogate their chaperone function leading to growth arrest of cells, independent of the demethylation effect. On the basis of this hypothesis, we analyzed chaperone activity of mortalin and Pex19p in response to 5AZA-dC treatment by an in vivo chaperone assay (as described in the Materials and Methods section). Notably, there was an induction of chaperone activity in 5AZA-dC (5 µM)-treated cell lysates. This result can be explained as an effect of demethylation and enhanced protein expression. It may also represent an adaptive or hormetic response, reported for several kinds of mild stresses (74,75), to accommodate increased protein synthesis associated with 5AZA-dC-induced desilencing. Noticeably, whereas overexpression of both mortalin and Pex19p resulted in enhanced chaperone function in response to 5 µM 5AZA-dC, a high dose (10 µM) that was consistent with the dose of 5AZA-dC required for structural perturbation of mortalin resulted in a decline in their chaperone function (Figure 5E). Taken together, these data suggest the possibility that these proteins are the direct targets of 5AZA-dC, as also supported by ANS fluorescence assay.


Figure 05
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  		Figure 5. 5-Aza-2' deoxycytidine (5AZA-dC) binds to mortalin directly and leads to its structural changes. 1-Anilinonapthalene-8-sulfonic acid (ANS) fluorescence intensity of mortalin in the presence of 5AZA-dC. ANS fluorescence emission from 400 to 600 nm was plotted for 0.5 mM mortalin–AZA complexes under different concentrations of the drug. Excitation wavelength was 365 nm, with excitation and emission slits of 3 and 5 nm, respectively. {Delta}F · I1/2 is the half-maximal loss of fluorescence intensity as a function of AZA concentration. The purified protein, buffer, or 5AZA-dC did not show any fluorescence (A). Far-UV circular dichroism spectra of mortalin–AZA complex. Spectra were recorded at a mortalin concentration of 0.5 mM using a 1 cm path length at 37°C. Altered secondary structures are observed at concentrations > 7.5 mM (inset). a = buffer, b = 2.5 mM; c = 5 mM; d = 7.5 mM; e = 10 mM; f = 25 mM (B). Plot of molar ellipticity at 215 nM depicting concentration of 5AZA-dC at half-maximal change (6.4 mM) in helical structures that reaches a saturation value at > 7.5 mM (C). Subcellular distribution of green fluorescence protein (GFP)-Pex19p changed from the diffuse pancytoplasmic to granular cytoplasmic in 5AZA-dC-treated cells (D). Changes in in vivo chaperone activity of mortalin and Pex19 in U2OS cells cotransfected with PGL3, a firefly luciferase, and mortalin expression constructs. Two days after transfections, the cells were incubated in medium containing 5AZA-dC for 6–8 hours. After incubation, the set-ups were subjected to 45°C heat treatment for 1 hour. In vivo chaperoning activity of 5AZA-dC-treated mortalin was evaluated by comparing luciferase activity immediately after heat shock and after 3 hours postrecovery at 37°C. Control cells were incubated at 37°C. 5AZA-dC caused moderate improvement in the chaperone function of both mortalin and Pex19 at low dose (5 µM). Of note, at a higher dose (10 µM), there was a reduction in chaperone function of both mortalin and Pex19p (E)

 
It has been demonstrated earlier that the covalent trapping of DNMT is a primary event in 5AZA-dC-induced desilencing of genes, and the level of DNMT serves as a good prognostic marker (45). Interestingly, despite normal or even high levels of DNMT expression, methyl-donor limiting conditions have been proposed to arise in early stages of tumor development, leading to global hypomethylation of tumor cell DNA (76). In this regard and on the basis of the present study, we propose that a shift of pancytoplasmic mortalin to perinuclear type that occurs in all immortalized cells, so far tested, may contribute to a hypomethylated environment for DNA. Mortalin complexes located in the perinuclear region of cancer cells may trap or serve as a barrier for entry of methyl donors to the nucleus or inactivate enzymes involved in DNA methylation causing global hypomethylation. The direct proof of the proposal warrants further studies.

Taken together, we have demonstrated, for the first time, that 5AZA-dC has DNA demethylation-independent effects that critically mediate its senescence-inducing function. These effects are mediated by stress chaperones, Pex19p and mortalin, that may act as novel prognostic factors for cancer therapy and may also be used for tailoring the response of tumor cells to 5AZA-dC-induced senescence.


   Acknowledgments
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 Abstract
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 Results and Discussion
 References
 
We thank Roger R Reddel, Australia, for providing a panel of precrisis and postcrisis cells.


   Footnotes
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 Materials and Methods
 Results and Discussion
 References
 
Decision Editor: Huber R. Warner, PhD

Received March 8, 2006

Accepted September 21, 2006


   References
Top
 Abstract
 Materials and Methods
 Results and Discussion
 References
 

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