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Childhood cancer is on the rise in high-income countries. Epidemiological studies suggest that reduced exposure to common infections in early life is to blame. However, no specific infection responsible for protection against cancer has been identified, and the underlying mechanisms remain a matter of speculation. Recent findings that Epstein-Barr virus (EBV) can induce antitumor immunity lead us to hypothesize that the delay in EBV infection in such countries might contribute to the increase in childhood cancers.

Tumour-associated antigens (TAAs) comprise a large set of non-mutated cellular antigens recognized by T cells in human and murine cancers. Their potential as targets for immunotherapy has been explored for more than two decades1, yet the origins of TAA-specific T cells remain unclear. While tumour cells may be an important source of TAAs for T cell priming2, several recent studies suggest that infection with some viruses, including Epstein–Barr virus and influenza virus can elicit T cell responses against abnormally expressed cellular antigens that function as TAAs3,4. However, the cellular and molecular basis of such responses remains undefined. Here we show that expression of the Epstein–Barr virus signalling protein LMP1 in B cells provokes T cell responses to multiple TAAs. LMP1 signalling leads to overexpression of many cellular antigens previously shown to be TAAs, their presentation on major histocompatibility complex classes I (MHC-I) and II (MHC-II) (mainly through the endogenous pathway) and the upregulation of costimulatory ligands CD70 and OX40L, thereby inducing potent cytotoxic CD4+ and CD8+ T cell responses. These findings delineate a mechanism of infection-induced anti-tumour immunity. Furthermore, by ectopically expressing LMP1 in tumour B cells from patients with cancer and thereby enabling them to prime T cells, we develop a general approach for rapid production of autologous cytotoxic CD4+ T cells against a wide range of endogenous tumour antigens, such as TAAs and neoantigens, for treating B cell malignancies. This work stresses the need to revisit classical concepts concerning viral and tumour immunity, which will be critical to fully understand the impact of common infections on human health and to improve the rational design of immune approaches to treatment of cancers. Expression of the Epstein–Barr virus protein LMP1 in B cells increases expression of—and promotes T cell responses to—tumour-associated antigens, delineating a mechanism of infection-induced anti-tumour immunity, which could inform immune-based approaches to cancer treatment.

Epstein–Barr virus (EBV), the first identified human tumor virus, infects and takes up residency in almost every human. However, EBV genome–positive tumors arise in only a tiny minority of infected people, presumably when the virus-carrying tumor cells are able to evade immune surveillance. Traditional views regard viral antigens as the principal targets of host immune surveillance against virus-infected cells. However, recent findings indicate that EBV-infected/-transformed B cells elicit both cytotoxic CD8+ and CD4+ T-cell responses against a wide range of overexpressed cellular antigens known to function as tumor-associated antigens (TAA), in addition to various EBV-encoded antigens. This not only broadens the ways by which the immune system controls EBV infection and prevents it from causing cancers, but also potentially extends immune protection toward EBV-unrelated cancers by targeting shared TAAs. The goal of this review is to incorporate these new findings with literature data and discuss future directions for improved understanding of EBV-induced antitumor immunity, as well as the hopes for rational immune strategies for cancer prevention and therapy.

// Eonju Oh 1 , Il-Kyu Choi 2 , JinWoo Hong 1 , Chae-Ok Yun 1 1 Department of Bioengineering, College of Engineering, Hanyang University, Seongdong-gu, Seoul 133-791, Korea 2 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA Correspondence to: Chae-Ok Yun, email: chaeok@hanyang.ac.kr Keywords: oncolytic adenovirus, IL-12, decorin, TGF-β, Treg Received: July 25, 2016 Accepted: December 01, 2016 Published: December 16, 2016 ABSTRACT Interleukin (IL)-12 is a potent antitumor cytokine. However, immunosuppressive tumor microenvironments containing transforming growth factor-β (TGF-β) attenuate cytokine-mediated antitumor immune responses. To enhance the efficacy of IL-12-mediated cancer immunotherapy, decorin (DCN) was explored as an adjuvant for overcoming TGF-β-mediated immunosuppression. We designed and generated a novel oncolytic adenovirus (Ad) coexpressing IL-12 and DCN (RdB/IL12/DCN). RdB/IL12/DCN-treated tumors showed significantly greater levels of interferon (IFN)-γ, tumor necrosis factor-α, monocyte chemoattractant protein-1, and IFN-γ-secreting immune cells than tumors treated with cognate control oncolytic Ad expressing a single therapeutic gene (RdB/DCN or RdB/IL12). Moreover, RdB/IL12/DCN attenuated intratumoral TGF-β expression, which positively correlated with reduction of Treg cells in draining lymph nodes and tumor tissues. Furthermore, tumor tissue treated with RdB/IL12/DCN showed increases infiltration of CD8 + T cells and proficient viral spreading within tumor tissues. These results demonstrated that an oncolytic Ad co-expressing IL-12 and DCN induces a potent antitumor immune response via restoration of antitumor immune function in a weakly immunogenic murine 4T1 orthotopic breast cancer model. These findings provide new insights into the therapeutic mechanisms of IL-12 plus DCN, making it a promising cancer immunotherapeutic agent for overcoming tumor-induced immunosuppression.

Decorin is a natural transforming growth factor-β1 (TGF-β1) antagonist. Reduced decorin synthesis is associated with dermal scarring, and increased decorin expression appears to reduce scar tissue formation. To investigate the therapeutic potential of decorin for keloids, human dermal fibroblasts (HDFs) and keloid-derived fibroblasts (KFs) were transduced with decorin-expressing adenovirus (dE1-RGD/GFP/DCN), and we examined the therapeutic potential of decorin-expressing Ad for treating pathologic skin fibrosis. Decorin expression was examined by immunofluorescence assay on keloid tissues. HDFs and KFs were transduced with dE1-RGD/GFP/DCN or control virus, and protein levels of decorin, epidermal growth factor receptor (EGFR) and secreted TGF-β1 were assessed by Western blotting and ELISA. And type I and III collagen, and matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-3 (MMP-3) mRNA levels were measured by real-time RT-PCR. Additionally, we immunohistochemically investigated the expression levels of the major extracellular matrix (ECM) proteins in keloid spheroids transduced with dE1-RGD/GFP/DCN. Lower decorin expression was observed in the keloid region compared to adjacent normal tissues. After treatment with dE1-RGD/GFP/DCN, secreted TGF-β1 and EGFR protein expressions were decreased in TGF-β1-treated HDFs and KFs. Also, type I and III collagen mRNA levels were decreased, and the expression of MMP-1 and MMP-3 mRNA was strongly upregulated. In addition, the expression of type I and III collagen, fibronectin and elastin was significantly reduced in dE1-RGD/GFP/DCN-transduced keloid spheroids. These results support the utility of decorin-expressing adenovirus to reduce collagen synthesis in KFs and keloid spheroid, which may be highly beneficial in treating keloids.

Pancreatic cancer is a highly lethal disease for which limited therapeutic options are available. Pancreatic cancer exhibits a pronounced collagen-rich stromal reaction, which induces chemoresistance by inhibiting drug diffusion into the tumor. Complementary treatment with oncolytic virus such as an oncolytic adenovirus expressing relaxin (YDC002) is an innovative treatment option for combating chemoresistant pancreatic cancer. Here, we examined the ability of combined treatment with gemcitabine and YDC002, which degrades extracellular matrix (ECM), to efficiently treat chemoresistant and desmoplastic pancreatic cancer. Gemcitabine alone exhibited similarly low cytotoxicity toward pancreatic cancer cells throughout the concentration range (1-50 μM) used, whereas the combination of YDC002 and a subtherapeutic dose of gemcitabine (0.01-0.05 μM) resulted in potent anticancer effects through effective induction of apoptosis. Importantly, YDC002 combined with gemcitabine significantly attenuated the expression of major ECM components including collagens, fibronectin, and elastin in tumor spheroids and xenograft tumors compared with gemcitabine alone, resulting in potent induction of apoptosis, gemcitabine-mediated cytotoxicity, and an oncolytic effect through degradation of tumor ECM. Our results demonstrate that YDC002 can selectively degrade aberrant ECM and attenuate the ECM-induced chemoresistance observed in desmoplastic pancreatic tumor, resulting in a potent antitumor effect through effective induction of apoptosis.

The Hsp70 family protein mortalin is an essential chaperone that is frequently enriched in cancer cells and exists in various subcellular sites, including the mitochondrion, plasma membrane, endoplasmic reticulum, and cytosol. Although the molecular mechanisms underlying its multiple subcellular localizations are not yet clear, their functional significance has been revealed by several studies. In this study, we examined the nuclear fractions of human cells and found that the malignantly transformed cells have more mortalin than the normal cells. We then generated a mortalin mutant that lacked a mitochondrial targeting signal peptide. It was largely localized in the nucleus, and, hence, is called nuclear mortalin (mot-N). Functional characterization of mot-N revealed that it efficiently protects cancer cells against endogenous and exogenous oxidative stress. Furthermore, compared with the full-length mortalin overexpressing cancer cells, mot-N derivatives showed increased malignant properties, including higher proliferation rate, colony forming efficacy, motility, and tumor forming capacity both in in vitro and in vivo assays. We demonstrate that mot-N promotes carcinogenesis and cancer cell metastasis by inactivation of tumor suppressor protein p53 functions and by interaction and functional activation of telomerase and heterogeneous ribonucleoprotein K (hnRNP-K) proteins.

Recurrent or metastatic cancer in most cases remains an incurable disease, and thus alternative treatment strategies, such as oncolytic virotherapy, are of great interest for clinical application. Oncolytic adenoviruses (Ads) have many advantages as virotherapeutic agents and have been safely employed in the clinics. However, the efficacy of oncolytic Ads is insufficient to eradicate tumors and current clinical applications are restricted to local administration against primary tumors because of immunological obstacles and poor tumor-cell targeting. Thus, alternative viable approaches are needed to establish therapies based on oncolytic Ad that will eliminate both primary and metastatic cancers. To this end, rational design of oncolytic Ads that express immunostimulatory genes has been employed. Even when restricted to local viral delivery, these oncolytic Ad-based immunotherapeutics have been shown to exert systemic antitumor immunity and result in eradication of both primary and metastatic cancers. Moreover, oncolytic Ad-based immunotherapeutics in combination with either dendritic cell-based vaccine or radiotherapy further strengthen the systemic tumor-specific immunity, resulting in complete suppression of both local and distant tumor metastatic growth. This review will focus on the most recent updates in strategies to develop potent oncolytic Ad-based immunotherapeutics for use in cancer gene therapy.

Intact p53 function is essential for responsiveness to cancer therapy. However, p53 activity is attenuated by the proto-oncoprotein Mdm2, the adenovirus protein E1B 55kD, and the p53 C-terminal domain. To confer resistance to Mdm2, E1B 55kD, and C-terminal negative regulation, we generated a p53 variant (p53VPΔ30) by deleting the N-terminal and C-terminal regions of wild-type p53 and inserting the transcriptional activation domain of herpes simplex virus VP16 protein. The oncolytic adenovirus vector Ad-mΔ19 expressing p53VPΔ30 (Ad-mΔ19/p53VPΔ30) showed greater cytotoxicity than Ad-mΔ19 expressing wild-type p53 or other p53 variants in human cancer cell lines. We found that Ad-mΔ19/p53VPΔ30 induced apoptosis through accumulation of p53VPΔ30, regardless of endogenous p53 and Mdm2 status. Moreover, Ad-mΔ19/p53VPΔ30 showed a greater antitumor effect and increased survival rates of mice with U343 brain cancer xenografts that expressed wild-type p53 and high Mdm2 levels. To our knowledge, this is the first study reporting a p53 variant modified at the N terminus and C terminus that shows resistance to degradation by Mdm2 and E1B 55kD, as well as negative regulation by the p53 C terminus, without decreased trans-activation activity. Taken together, these results indicate that Ad-mΔ19/p53VPΔ30 shows potential for improving p53-mediated cancer gene therapy.

Oncolytic viruses (OVs) are used to treat cancer, as they selectively replicate inside of and lyse tumor cells. The efficacy of this process is limited and new OVs are being designed to mediate tumor cell release of cytokines and co-stimulatory molecules, which attract cytotoxic T cells to target tumor cells, thus increasing the tumor-killing effects of OVs. To further promote treatment efficacy, OVs can be combined with other treatments, such as was done by Huang et al., who showed that combining OV injections with dendritic cell (DC) injections was a more effective treatment than either treatment alone. To further investigate this combination, we built a mathematical model consisting of a system of ordinary differential equations and fit the model to the hierarchical data provided from Huang et al. We used the model to determine the effect of varying doses of OV and DC injections and to test alternative treatment strategies. We found that the DC dose given in Huang et al. was near a bifurcation point and that a slightly larger dose could cause complete eradication of the tumor. Further, the model results suggest that it is more effective to treat a tumor with immunostimulatory oncolytic viruses first and then follow-up with a sequence of DCs than to alternate OV and DC injections. This protocol, which was not considered in the experiments of Huang et al., allows the infection to initially thrive before the immune response is enhanced. Taken together, our work shows how the ordering, temporal spacing, and dosage of OV and DC can be chosen to maximize efficacy and to potentially eliminate tumors altogether.

The p53 gene plays a determinant role in radiation-induced cell death and its protein product is negatively regulated by MDM2. We investigated whether adenovirus-mediated modified p53 gene transfer, which blocks p53-MDM2 binding, is effective for radiation-induced cell death in hepatocellular carcinoma (HCC) at different MDM2 cellular levels. Human hepatocellular carcinoma cell lines expressing MDM2 at low levels (Huh7) and high levels (SK-Hep1) were used. Ad-p53 and Ad-p53vp are replication-deficient adenoviral vectors containing human wild-type or modified p53, respectively. The anti-tumor effect was highest for Ad-p53 + radiotherapy (RT) in the low-level MDM2 cells, whereas this effect was highest for Ad-p53vp + RT in the MDM2-overexpressing cells. In Huh-7 cells, Ad-p53 + RT decreased cell viability (32%) in vitro and inhibited tumor growth (enhancement factor, 1.86) in vivo. Additionally, p21 expression and apoptosis were increased. In contrast, in SK-Hep1 cells, Ad-p53vp + RT showed decreased cell viability (51%) in vitro and inhibition of tumor growth (enhancement factor, 3.07) in vivo. Caspase-3 expression and apoptosis were also increased. Adenovirus-expressing modified p53, which blocks p53-MDM2 binding, was effective in killing tumor cells overexpressing MDM2. Furthermore, the combination strategy for disruption of the p53-MDM2 interaction with RT demonstrated enhanced anti-tumor effects both in vitro and in vivo.

Purpose: We investigated whether an armed viral platform, where lytic property of a viral infection is coupled to viral-mediated delivery of therapeutic genes, could increase the therapeutic potential of a viral-based therapy.Experimental Design: We generated interleukin (IL)-12-expressing oncolytic adenovirus (YKL-IL-12) and IL-12- and B7-1-expressing (YKL-IL12/B7) oncolytic adenovirus. Therapeutic efficacy of these newly engineered adenoviruses was then evaluated in vivo using an immunocompetent mouse bearing murine melanoma B16-F10 tumors. Overall survival was assessed with the Kaplan-Meier method. The induction of immune cell cytotoxicity was assessed by CTL assay, IFN-γ enzyme-linked immunospot assay, and immunohistochemical studies.Results: YKL-IL12/B7 oncolytic adenovirus, expressing both IL-12 and B7-1, showed a higher incidence of complete tumor regression compared with the analogous oncolytic adenovirus, YKL-1, or IL-12-expressing, YKL-IL12. Significant survival advantage was also seen in response to YKL-IL12/B7. Moreover, IL-12 and IFN-γ levels produced in tumors treated with YKL-IL12/B7 was significantly greater than those treated with YKL-IL12. The enhanced survival advantage was mediated by the induction of immune cell cytotoxicity. In agreement with these results, massive infiltration of CD4+ and CD8+ T cells into tissues surrounding the necrotic area of the tumor was observed following in situ delivery of YKL-IL12/B7.Conclusion: Combination of oncolysis and the enhancement of antitumor immune response by oncolytic adenovirus expressing both IL-12 and B7-1 elicits potent antitumor effect and survival advantage.

Various ways to inhibit vascular endothelial growth factor (VEGF), a key facilitator in tumor angiogenesis, are being developed to treat cancer. The soluble VEGF decoy receptor (FP3), due to its high affinity to VEGF, is a highly effective and promising strategy to disrupt VEGF signaling pathway. Despite potential advantage and potent therapeutic efficacy, its employment has been limited by very poor in vivo pharmacokinetic properties. To address this challenge, we designed a novel oncolytic adenovirus (Ad) expressing FP3 (RdB/FP3). To demonstrate the VEGF-specific nature of RdB/FP3, replication-incompetent Ad expressing FP3 (dE1/FP3) was also generated. dE1/FP3 was highly effective in reducing VEGF expression and functionally elicited an antiangiogeneic effect. Furthermore, RdB/FP3 exhibited a potent antitumor effect compared with RdB or recombinant FP3. Consistent with these data, RdB/FP3 was shown to greatly decrease VEGF expression level and vessel density and increase apoptosis in both tumor endothelial and tumor cells, verifying potent suppressive effects of RdB/FP3 on VEGF-mediated tumor angiogenesis in vivo. Importantly, the therapeutic mechanism of antitumor effect mediated by RdB/FP3 is associated with prolonged VEGF silencing efficacy and enhanced oncolysis via cancer cell-specific replication of oncolytic Ad. Taken together, RdB/FP3 provides a new promising therapeutic approach in the treatment of cancer and angiogenesis-related diseases.

Adoptive natural killer (NK) cell therapy may offer an effective treatment regimen for cancer patients whose disease is refractory to conventional therapy. NK cells can kill a wide range of tumor cells by patterned recognition of target ligands. We hypothesized that tumor targets sensitive to NK lysis would drive vigorous expansion of NK cells from human peripheral blood mononuclear cells (PBMC). Here, we provide the basis for developing a novel ex vivo expansion process. By screening class I–negative or –mismatched tumor cell lines we identified a Jurkat T-lymphoblast subline termed KL-1, which was highly effective in specifically expanding NK cells. KL-1 addition to PBMC cultures achieved approximately 100-fold expansion of NK cells with nearly 90% purity, accompanied by reciprocal inhibition of T-cell growth. Marked elevations in expression of activation receptors, natural cytotoxicity receptors (NKp30, NKp44), and adhesion molecules (CD11a, ICAM-1) were associated with high tumor-lytic capacity, in both in vitro and in vivo models. KL-1–mediated expansion of NK cells was contact dependent and required interactions with CD16, the Fcγ receptor on NK cells, with ligands that are expressed on B cells. Indeed, B-cell depletion during culture abrogated selective NK cell expansion, while addition of EBV-transformed B cells further augmented NK expansion to approximately 740-fold. Together, our studies define a novel method for efficient activation of human NK cells that employs KL-1–lysed tumor cells and cocultured B cells, which drive a robust expansion of potent antitumor effector cells that will be useful for clinical evaluation. Cancer Res; 73(8); 2598–607. ©2012 AACR.

The hormone relaxin has been shown to affect the extracellular matrix by inhibiting collagen synthesis and expression in fibroblasts stimulated with a profibrotic agent. It also increases matrix metalloproteinase (MMP) expression. To investigate its effect on expression of collagen and MMPs in keloid fibroblasts and human dermal fibroblasts, the authors introduced a relaxin-expressing adenovirus (dE1-RGD/lacZ/RLX) into a human dermal fibroblast cell line and keloid fibroblasts.Both fibroblasts were infected with dE1-RGD/lacZ/RLX or control virus, and protein levels of relaxin and secreted transforming growth factor (TGF)-β1 were assessed by enzyme-linked immunosorbent assay, and mRNA levels of collagen type I, collagen type III, MMP-1, and MMP-3 were assessed by real-time reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay. Expression of Smad3 and phosphorylated Smad3 was also examined, and relaxin's effect on Smad2/3 complex localization was evaluated.When human dermal fibroblasts and keloid fibroblasts were transduced with dE1-RGD/lacZ/RLX or dE1-RGD/lacZ (control), mRNA expression of type I and type III collagen was markedly decreased by relaxin regardless of TGF-β (10 ng/ml) treatment. Expression of Smad3 and phosphorylated Smad3 was reduced in keloid fibroblasts and decreased translocation of Smad 2/3 complex from cytosols to the nucleus of the human dermal fibroblasts with TGF-β after dE1-RGD/lacZ/RLX transduction, suggesting that relaxin reduces collagen synthesis by blocking TGF-β signaling. Analyses revealed that MMP-1 and MMP-3 expression were significantly increased in human dermal fibroblasts and keloid fibroblasts after dE1-RGD/lacZ/RLX transduction.These results suggest that the antifibrotic effect of relaxin-expressing adenovirus may have therapeutic effects on keloids.

We developed a three-dimensional organotypic multicellular spheroid scar model to mimic the microenvironment of human keloid tissues. Keloid tissues were cultured for 7 days. Changes in total cellularity and apoptotic index in the primary keloid spheroid cultures were evaluated histologically and with a TUNEL assay, respectively. The expression profiles of transforming growth factor-β (TGF-β), collagen I, collagen III, elastin, fibronectin, matrix metalloproteinase-2, and matrix metalloproteinase-9 were examined with immunohistochemistry. In addition, these expression profiles were investigated after treating primary keloid spheroids with triamcinolone acetonide. Cell viability and morphology of ex vivo cultured keloid spheroids were maintained, and the apoptotic index did not increase for up to 1 week in culture. Keloid spheroids cultivated ex vivo retained the major characteristics of keloids, such as high levels of collagen I and TGF-β expression for up to 7 days. The biological activity of keloids responding to TGF-β was also maintained during ex vivo culture. Moreover, ex vivo triamcinolone acetonide treatment of cultivated keloid spheroids significantly reduced collagen I, collagen III, elastin, and fibronectin expression levels, in accordance with clinical observations. The three-dimensional organotypic multicellular spheroid keloid culture will allow investigators to study keloid pathogenesis and test potential keloid therapeutic agents.

MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. To identify miRNAs that may regulate human cell immortalization and carcinogenesis, we performed comparative miRNA array profiling of human normal and SV40-T antigen immortalized cells. We found that miR-296 was upregulated in immortalized cells that also had activation of telomerase. By an independent experiment on genomic analysis of cancer cells we found that chromosome region (20q13.32), where miR-296 is located, was amplified in 28/36 cell lines, and most of these showed enriched miR-296 expression. Overexpression of miR-296 in human cancer cells, with and without telomerase activity, had no effect on their telomerase function. Instead, it suppressed p53 function that is frequently downregulated during human cell immortalization and carcinogenesis. By monitoring the activity of a luciferase reporter connected to p53 and p21(WAF1) (p21) untranslated regions (UTRs), we demonstrate that miR-296 interacts with the p21-3'UTR, and the Hu binding site of p21-3'UTR was identified as a potential miR-296 target site. We demonstrate for the first time that miR-296 is frequently upregulated during immortalization of human cells and contributes to carcinogenesis by downregulation of p53-p21(WAF1) pathway.

Recently, gene-based cytokine treatment has been actively pursued as a new promising approach in treating cancer. In an effort to augment the efficiency of antitumor effect by cytokine-mediated immunotherapy, we selected both interleukin (IL)-12 and 4-1BB ligand (4-1BBL) as suitable cytokines to fully activate the type-1 immune response. Coexpression of IL-12 and 4-1BBL mediated by oncolytic adenovirus (Ad) greatly enhanced the antitumor effect. Further, synergistic enhancement in interferon (IFN)-gamma levels were seen in mice treated with oncolytic Ad expressing both IL-12 and 4-1BBL. Next, to improve the overall antitumor immune response, we coadministered IL-12- and 4-1BBL-coexpressing oncolytic Ad with dendritic cells (DCs). Combination treatment of IL-12- and 4-1BBL-coexpressing oncolytic Ad and DCs elicited greater antitumor and antimetastatic effects than either treatment alone. Moreover, enhanced type-1 antitumor immune response and higher migratory abilities of DCs in tumors were also observed in the combination arms. The nature of the enhanced antitumor immune response seems to be mediated through the enhanced cytolytic activity of cytotoxic T lymphocytes (CTLs) and IFN-gamma-releasing immune cells. Taken together, these data highlight the potential therapeutic benefit of combining IL-12- and 4-1BBL-coexpressing oncolytic Ad with DCs and warrants further evaluation in the clinic.

Aging is a complex process accompanied by a decreased capacity of cells to cope with random damages induced by reactive oxygen species, the natural by-products of energy metabolism, leading to protein aggregation in various components of the cell. Chaperones are important players in the aging process as they prevent protein misfolding and aggregation. Small chaperones, such as small heat shock proteins, are involved in the refolding and/or disposal of protein aggregates, a feature of many age-associated diseases. In Drosophila melanogaster, mitochondrial Hsp22 (DmHsp22), is localized in the mitochondrial matrix and is preferentially up-regulated during aging. Its overexpression results in an extension of life span (30%) (Morrow, G., Samson, M., Michaud, S., and Tanguay, R. M. (2004) FASEB J. 18, 598-599 and Morrow, G., Battistini, S., Zhang, P., and Tanguay, R. M. (2004) J. Biol. Chem. 279, 43382-43385). Long lived flies expressing Hsp22 also have an increased resistance to oxidative stress and maintain locomotor activity longer. In the present study, the cross-species effects of Hsp22 expression were tested. DmHsp22 was found to be functionally active in human cells. It extended the life span of normal fibroblasts, slowing the aging process as evidenced by a lower level of the senescence associated beta-galactosidase. DmHsp22 expression in human cancer cells increased their malignant properties including anchorage-independent growth, tumor formation in nude mice, and resistance to a variety of anticancer drugs. We report that the DmHsp22 interacts and inactivates wild type tumor suppressor protein p53, which may be one possible way of its functioning in human cells.