Complex Formation between Heat Shock Protein 72 and Hepatitis B

Complex Formation between Heat Shock Protein 72 and Hepatitis B Virus X Protein in Hepatocellular Carcinoma Tissues Xiaoping Wang,*,† Yongxue Zhou,† Lijun Sun,† Wei Chen,‡ Xu Li,‡ Qiaoxia Wang,† and Huanping Lin† Laboratory of Molecular Pathology, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712046, People’s Republic of China, and Central of Molecular Medicine, the First Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, People’s Republic of China Received June 14, 2008

Hepatitis B virus (HBV) infection is a major factor contributing to the development of hepatocellular carcinoma (HCC) in the world. Hepatitis B virus X protein (HBx) has been verified to play an important role in hepatocarcinogenesis. Heat shock protein 72 (HSP72) as a molecular chaperone has been confirmed to overexpress in epithelial carcinoma cells. There may be a possible association between the expression of HSP72 and HBx during the growth and progression of hepatocellular carcinoma cells. The aim of the study was to investigate the relationship between heat shock protein 72 and HBx in human hepatocellular carcinomas. The localization of HSP72 and HBx in human hepatocellular carcinomas was determined by immunohistochemistry and confocal laser microscopy. The interaction between HSP72 and HBx in liver cancer cells was analyzed by immunoprecipitation and Western blot. Our results revealed that hepatocellular carcinoma synchronously co-expressed higher level of HSP72 and HBx than that in the adjacent tissues to hepatocellular carcinoma. HSP72 and HBx were mainly immunolocalized in the cytoplasm. On the basis of immunoprecipitation and Western blot, we found that HSP72 formed complex with HBx in human hepatocellular carcinoma cells. The association between HSP72 and HBx in human hepatocellular carcinoma cells may contribute to study the pathogenesis and immunotherapy of hepatocellular carcinoma. Keywords: Heat shock protein 72 (HSP72) • hepatitis B virus x protien (HBx) • immunohistochemistry • immunoprecipitation • hepatocellular carcinoma (HCC)

Introduction Hepatitis B virus (HBV) chronic infection is one of the major causes of hepatocellular carcinoma (HCC) in the world.1,2 Hepatitis B virus X protein (HBx), a small protein of 154 amino acids, has long been suspected to contribute to the liver carcinogenesis and the development of HCC. It is reported that the transcriptional activation triggered by HBx was implicated in regulating the expression of certain cellular genes that are crucialtohepatocyteproliferation,viability,andtransformation.3-5 Although many investigations for the function of HBx had been carried out, the molecular mechanisms involved in HBxmediated hepatocarcinogenesis remain obscure. Heat shock protein 72 (HSP72) belongs to the heat shock protein 70 family which are molecular chaperones emerging as biochemical regulators of cell growth, apoptosis, protein homeostasis and cellular targets of peptides.6 Up-regulated expression of HSP72 during the growth of cancer cells has a * To whom correspondence should be addressed. Wang Xiaoping, Laboratory of Molecular Pathology, Shaanxi University of Chinese Medicine, Xianyang, Shaanxi 712046, People’s Republic of China. Telephone: +86-2938185359. E-mail: [email protected]. † Laboratory of Molecular Pathology, Shaanxi University of Chinese Medicine. ‡ Central of Molecular Medicine, the First Affiliated Hospital, School of Medicine, Xi’an Jiaotong University. 10.1021/pr800435g CCC: $40.75

 2008 American Chemical Society

close relationship with the epithelial carcinoma proliferation and progression.7-9 Studies have showed that HBx plays a crucial role in initiating and promoting hepatocyte transformation, proliferation and progression.10,11 So there may be a possible correlation between the expression of HSP72 and HBx during the growth and development of hepatocellular carcinoma cells. In this study, by immunohistochemistry and immunoprecipitation, and Western blot, we observed that HSP72 was associated with HBx in hepatocellular carcinoma cell cytoplasm. The interaction between HSP72 and HBx in human hepatocellular carcinoma cells will provide a new route for studying the pathogenesis and immunity of HCC.

Materials and Methods Immunohistochemical Reagents. Purified HSP72 and Mouse anti-human HSP72 monoclonal antibody was obtained from StressGen Biotechnologies (Victoria, BC, Canada). Mouse antiHBx monoclonal antibody, mouse anti-human β-actin monoclonal antibody, TRITC labeled goat anti-mouse antibody and FITC labeled goat anti-mouse antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). EnVisionTM kits and ProteinA/G-agarose beads were purchased from Dako Corp (Carpinteria, CA). Journal of Proteome Research 2008, 7, 5133–5137 5133 Published on Web 10/15/2008

research articles Tissue Samples. Patients. The investigation was approved by the Ethics Committee on Human Study at Shaanxi University of Chinese Medicine (2004-4B). Surgical specimens of 60 patients with primary hepatocellular cancer undergoing liver resection were collected from the Affiliated Hospital, Shaanxi University of Chinese Medicine, Xianyang, China from 2002 to 2007. The patients consist of 42 males and 18 females, with a mean age of 52.5 years, ranging from 37 to 66 years. Routine pathological diagnosis showed that all cases were primary hepatocellular carcinoma. Among them, 20 cases were well-differentiated type and 40 cases were poorly differentiated. All the patients were positive for HBsAg. The specimens were fixed in 10% buffered formalin and embedded in paraffin. Serial sections, 5-µm-thick, were cut and mounted on silane-coated glass slides. Immunohistochemical Labeling Methods. All sections were deparaffinized and rehydrated with graded alcohol. Endogenous peroxidase was then blocked with 0.3% H2O2 diluted in methanol for 30 min at room temperature. Antigen retrieval was performed by treating the slides in citrate buffer in a microwave for 10 min. The slides were incubated in a moist chamber with HSP72 mouse monoclonal antibody or HBx mouse monoclonal antibody, both at a dilution of 1:100, at 4 °C, overnight. After a complete wash in phosphate buffer solution (PBS), the slides were incubated with horseradish peroxidase-labeled goat anti-mouse antibody, diluted 1:100, for 45 min at 37 °C. After a complete wash in PBS, the slides were developed in 0.05% freshly prepared 3,3′-diaminobenzedine solution (DAB, Sigma Co.) with 0.03% hydrogen peroxide for 8 min, and then counterstained with hematoxylin, dehydrated, air-dried, and mounted in neutral resins. Normal mouse IgG was used to substitute for the primary antibody to serve as a negative control. No specific immunoreactivity was detected in these tissue sections. Assessment of Immunolabeling. Two of the observers initially examined the sections simultaneously using a dual-head light microscope. The evaluation of HSP72 and HBx positive cells was performed on high-power fields (×400) using a standard light microscope. The data were analyzed by two blind evaluations. Only distinctive intranuclear or intracytoplasmic immunoreactivity was considered positive. In each sample, more than 500 tumor cells were scored and the percentage of immunopositive cells was independently determined. When inter-observer differences were greater than 5%, the immunolabeled slides were reexamined simultaneously using a dual-head light microscope and the percentage of positive cells was determined by two observers working in concert. When inter-observer differences were less than 5%, the mean value was obtained as the positive rate. When more than 10% positive cells were detected, the case was considered positive. Immunoflurescence and Confocal Laser Microscopy. All sections were deparaffinized and rehydrated with graded alcohol. The tissues were blocked in 10 mg/L bovine serum albumin (BSA) for 30 min at room temperature, and then incubated with HSP72 mouse monoclonal antibody (1:100) or HBx mouse monoclonal antibody (1:100) at 4 °C overnight, respectively. After a complete wash in PBS, the cells were treated with TRITC labeled goat anti-mouse antibody or FITC labeled goat anti-mouse antibody (1:20) for 40 min at room temperature. After an extensive wash, the stained cells were observed under a laser scanner confocal microscope Bio-Rad MRC 1024ES equipped with a Nikon (Tokyo, Japan) Diaphot inverted microscope. Normal mouse IgG was used to substitute for the primary antibody as a negative control. 5134

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Wang et al. Immunoprecipitation and Western Blot. The hepatocellular carcinoma tissues and normal liver tissues were dispersed through 100-well copper mesh. Then cells were treated with 0.5 g/L trypsin and 0.2 g/L EDTA. After washing with cold PBS, cells were centrifuged and harvested. Cells were then lysed with 500 µL of lysis buffer (10 mmol/L Tris-HCL, pH 7.4, 150 mmol/L NaCl, 1 mL/L Triton X-100, 10 g/L Na-deoxycholate) containing 1 µg/mL pepstatin and 1 mmol/L phenyl-methylsulfonyl fluoride (PMSF). Also, 5 units of apyrase (Sigma, Co.) were added to the lysate to deplete endogenous ATP. The cell lysates were sonicated and clarified by centrifugation. The supernatants were preabsorbed with 20 µL of protein A/G-agarose beads at 4 °C for 4 h. After centrifugation, the supernatants were incubated with 100 µL of HSP72 mouse monoclonal antibody (1:100) or 100 µL HBx mouse monoclonal antibody (1:100), respectively, at 4 °C for 60 min. Then 50 µL of protein A/G-agarose beads was added and incubated for 60 min at 4 °C. The immunoprecipitates were collected by centrifugation and washed five times with PBS. The precipitated protein complexes were released from the immunopellet with SDSsample buffer (62.5 mmol/L Tris-HCL, pH 6.8, 25 g/L SDS, 50 mL/L β-mercaptoethanol, 100 mL/L glycerol) at 100 °C for 5 min. The immunoprecipitates were then analyzed by 90 g/L SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose (NC) membrane (Bio-Rad) and detected by immunoblotting with anti-HBx monoclonal antibody or anti-HSP72 monoclonal antibody (1:100) at 4 °C overnight. After a complete wash in PBS, the membranes were treated with horseradish peroxidase-labeled goat anti-mouse antibody (1:100) for 45 min at 37 °C. After a complete wash in PBS, the membranes were developed in 0.5 g/L freshly prepared 3,3′-diaminobenzedine solution (DAB, Sigma Co.) for 8 min. Purified HSP72 was used as a positive control to determine the specificity of anti-HSP72 monoconal antibody. Anti-β-actin mouse monoclonal antibody was used as a negative control and internal reference to detect β-actin in hepatocellular carcinoma cells. Statistical Analysis. HSP72 and HBx expression differences between hepatocellular carcinoma and control groups were analyzed statistically using U test. P < 0.05 was considered statistically significant.

Results Immunolabeling of HSP72 and HBx in Hepatocellular Carcinoma Tissues. HSP72 immunopositivity was detected in 54 of 60 primary tumors (90.0%) and in 8 of 60 adjacent tissues to cancers (13.3%). HBx immunopositivity was detected in 95.0% (57/60) hepatocellular carcinomas and in 15.0% (9/60) adjacent tissues to cancer. There was a significant difference between carcinoma tissues and adjacent tissues to cancer P < 0.01. HSP72 were mainly immunolocalized in the cytoplasm, while HBx were immunolocalized in the nucleus and cytoplasm (Figure 1). Immunoflurescence and confocal laser microscopy showed that hepatocellular carcinoma synchronously co-expressed higher level of HSP72 and HBx. Both of them were mainly localized in cell cytoplasm (Figure 2). Complex Formation between HSP72 and HBx in Hepatocellular Carcinoma Cells. Immunoprecipitate and Western blot showed that there were two clear protein bands in the transferred NC membrane. When precipitated with antiHSP72 monoclonal antibody, there appeared a Mr 17 000 clear HBx band (lanes 3 and 4) and when precipitated with anti-

HSP72 Associated with HBx Protein in Hepatocellular Carcinoma

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Figure 1. Immunolocalization of HSP72 and HBx in hepatocellular carcinoma tissues by immunohistochemistry (counterstained with hematoxylin), ×200. (A) No specific immunoreactivity was detected in control group cancer cells. (B) HSP72 immunostained in tumor cell cytoplasm. (C) HBx positive expression in tumor cell nucleus and cytoplasm.

HBx mAb, there existed a Mr 72 000 clear heat shock protein 72 band (lanes 1 and 2), demonstrating that HBx existed in the immunoprecipitate of anti-HSP72 monoclonal antibody and HSP72 existed in the immunoprecipitate of anti-HBx mAb. The results indicated that HSP72 was associated with HBx in hepatocellular carcinoma tissues (Figure 3).

Figure 2. Co-localization of HSP72 and HBx in hepatocellular carcinoma tissues by immunoflurescence and confocal microscopy, ×200. (A) HSP72 showed red immunofluorescence in cytoplasm. (B) HBx expressed green immunofluorescence in cytoplasm. (C) HSP72 and HBx showed yellow immunofluorescence in cytoplasm.

Discussion In this study, we examined the expressions of HSP72 and HBx in hepatocellular carcinoma tissues by immunohistochem-

istry, confocal microscopy and immunoprecipitate analysis. The results showed that most of hepatocellular carcinoma cells Journal of Proteome Research • Vol. 7, No. 12, 2008 5135

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Figure 3. Analysis of immunoprecipitate of anti-HSP72 mAb and anti-HBx mAb by SDS-PAGE and Western blot (lanes 1 and 2, precipitated with anti-HBx mAb in hepatocellular carcinoma tissues; lanes 3 and 4, precipitated with anti-HSP72 mAb in hepatocellular carcinoma tissues; lane 5, anti-β-actin mAb (β-actin was used as a negative control and internal reference detected in hepatocellular carcinoma); lane 6, anti-HSP72 mAb (purified HSP72 was used as a positive control to determine the specificity of anti-HSP72 mAb); lane 7, normal liver cells lysates supernatants; lane 8, hepatocellular carcinoma cells lysates supernatants; M, molecular mass markers).

expressed high level of HSP72 and HBx, which had a significant difference compared with adjacent tissues to cancer. HSP72 was associated with HBx mainly localizing in cell cytoplasm. HBV has been reported to be the prime cause for the development of HCC.12,13 Hepatitis B virus X protein (HBx), a small polypeptide (17 kDa) produced at very low levels during chronic and acute hepatitis, has been long suspected to be involved in hepatocarcinogenesis, although its oncogenic role remains controversial. It is well-established that HBx is a multifunctional regulator that modulates transcription, signal transduction, cell cycle progress, protein degradation pathways, apoptosis, and genetic stability by directly or indirectly interacting with host factors.14,15 In light of the presumed role of HBx for viral carcinogenesis, the major focus is the interference of HBx with signal transduction cascades that affect the control of the cell cycle, proliferation or apoptosis. The activation of these signaling cascades requires the presence of HBx in a certain compartment.16,17 The cellular distribution of HBx is still a matter of debate. There is evidence that a fraction of HBx is localized within the nucleus.18,19 On the other hand, there are reports providing evidence that HBx is localized in the cytoplasm as well as in the nucleus.16,20 The different localizations are associated with different functions. HBx localized in the nucleus is suggested to interfere directly with transcription factors or to exert a transcription factor-like function. While HBx localized in the cytoplasm is able to modulate intracellular signal transduction cascades, our results showed that HBx was co-localized in cell nucleus and cytoplasm, which further indicated that HBx probably simultaneously exerts its function in cell cytoplasm and nucleus. Heat shock protein 72 is a subtype of highly conserved heat shock protein 70 family synthesized under various stress. In nontransformed cells at normal conditions, HSP72 is expressed at very low levels. It is, however, present at elevated levels in the major fraction of tumors and in many transformed cell lines.21-23 It is commonly assumed that in tumor cells the expression of HSP72 at elevated levels is the consequence of oncogenic transformation and enhanced expression of HSP72 has a close relationship with the epithelial carcinoma cells growth.7-9 It is also found that high level expression of HBx 5136

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Wang et al. plays important roles in hepatocyte carcinogenesis.3-5 So, high level expression of HSP72 in carcinoma may be correlated with HBx in HCC development. Several researches have showed that a series of heat shock proteins chaperoned HBx in the celluar cytoplasm or nucleus,24,25 indicating that different heat shock proteins were likely to transmit HBx to different cellular compartment and exert its function. Previous studies showed that HBx protein can form a complex with mitochondrial HSP70 and HSP60 in human liver cells, which probably facilitates HBx-induced cell apoptosis.24,25 It is proved that upregulated expression of HSP72 in tumor cells may be required to serve as molecular chaperones in regulating and stabilizing oncofetal protein or mutant oncogene products during tumor growth process.26,27 Our results further verified that HSP72 can co-localize and form a complex with HBx in cytoplasm of liver cancer cells. From the above studies, it is presumed that various overexpressed HSPs, such as HSP60, HSP70, HSP72, may assist to stabilize HBx in cell cytoplasm, and transport it to cellular organelle or nucleus, which indirectly prompts and protects HBx function. Conversely, protected functional HBx itself via protein and protein interaction stimulates hepatocellular carcinoma cells proliferation and progression. This indicates that the proliferating hepatocellular carcinoma cells needs much more HSPs to maintain the formation of HBx activities. The observed fact that HSP72 was associated with HBx may be useful to further study the function of HSPs on the pathogenesis of human hepatocellular carcinoma. Numerous investigations have been verified that HSP72 is a better molecular chaperone and adjuvant which can process and present tumor antigen to MHC-I of host APCs, eliciting specific T-cell response and CTL reaction.28-30 HSP72-associated peptides can also anchor antigen on the cell membrane and directly present it to nature killer cells or γδ T cells as superantigen without being dependent on the stimulation of MHC-I molecules.31,32 Our data showed intense immunolabeling of HSP72 and HBx in hepatocellular carcinomas, and both HSP72 and HBx were mainly localized in cytoplasm. The complex that HBx formed with HSP72 in HCC may be helpful to study tumor immunity or design effective vaccine against hepatocellular carcinoma, and this aspect needs further study.

Conclusion Taken together, human hepatocellular carcinomas existed with high level of expression of HSP72 and HBx. HSP72 was associated with HBx mainly localizing in cell cytoplasm. Coexpression of HSP72 and HBx probably have some relationship with development of human hepatocellular carcinoma. The observed fact that HSP72 was associated with HBx may be useful to study the pathogenesis and tumor immunity against liver cancers.

Acknowledgment. This study was financially supported by the Key Project of Ministry of Education of China (205002) and the Research Program of Shaanxi Education Committee (07KJ233). References (1) Llovet, J. M.; Burroughs, A.; Bruix, J. Hepatocellular carcinoma. Lancet 2003, 362 (9399), 1907–1917. (2) Jepsen, P.; Vilstrup, H.; Tarone, R. E.; Friis, S.; Sørensen, H. T. Incidence rates of hepatocellular carcinoma in the U.S. and Denmark: recent trends. Int. J. Cancer 2007, 121 (7), 1624–1626. (3) Tu, H.; Bonura, C.; Giannini, C.; Mouly, H.; Soussan, P.; Kew, M.; Paterlini-Bre´chot, P.; Bre´chot, C.; Kremsdorf, D. Biological impact

research articles

HSP72 Associated with HBx Protein in Hepatocellular Carcinoma

(4) (5) (6) (7) (8)

(9)

(10)

(11)

(12) (13) (14) (15) (16)

(17)

(18)

(19)

of natural COOH-terminal deletions of hepatitis B virus x protein in hepatocellular carcinoma tissues. Cancer Res. 2001, 61 (21), 7803–7810. Tang, H.; Oishi, N.; Kaneko, S.; Murakami, S. Molecular functions and biological roles of hepatitis B virus x protein. Cancer Sci. 2006, 97 (10), 977–983. Lupberger, J.; Hildt, E. Hepatitis B virus-induced oncogenesis. World J. Gastroenterol. 2007, 13 (1), 74–81. Morimoto, R. I. Cells in stress: transcriptional activation of heat shock genes. Science 1993, 259 (5100), 1409–1410. Bausero, M. A.; Page, D. T.; Osinaga, E.; Asea, A. Surface expression of Hsp25 and Hsp72 differentially regulates tumor growth and metastasis. Tumour Biol 2004, 25 (5-6), 243–251. Wang, X. P.; Zhou, Y. X.; Ying, X. P.; Guo, L. S.; Zhao, Y. H.; Fang, Y. Interaction between heat shock protein 72 and alpha-fetoprotein in human hepatocellular carcinomas. Clin. Chim. Acta 2007, 279 (1-2), 158–162. Wang, X. P.; Wang, Q. X.; Ying, X. P. Correlation between clinicopathology and expression of heat shock protein 72 and glycoprotein 96 in human gastric adenocarcinoma. Tohoku J. Exp. Med. 2007, 212 (1), 35–41. Yu, D. Y.; Moon, H. B.; Son, J. K.; Jeong, S.; Yu, S. L.; Yoon, H.; Han, Y. M.; Lee, C. S.; Park, J. S.; Lee, C. H.; Hyun, B. H.; Murakami, S.; Lee, K. K. Incidence of hepatocellular carcinoma in transgenic mice expressing the hepatitis B virus X-protein. J. Hepatol. 1999, 31 (1), 123–132. Koike, K.; Moriya, K.; Yotsuyanagi, H.; Shintani, Y.; Fujie, H.; Tsutsumi, T.; Kimura, S. Compensatory apoptosis in preneoplastic liver of a transgenic mouse model for viral hepatocarcinogenesis. Cancer Lett. 1998, 134 (2), 181–186. Chan, H. L.; Sung, J. J. Hepatocellular carcinoma and hepatitis B virus. Semin. Liver Dis. 2006, 26 (2), 153–161. Waris, G.; Siddiqui, A. Regulatory mechanisms of viral hepatitis B and C. J. Biosci. 2003, 28 (3), 311–321. Zhang, X.; Zhang, H.; Ye, L. Effects of hepatitis B virus X protein on the development of liver cancer. J. Lab. Clin. Med. 2006, 147 (2), 58–66. Madden, C. R.; Slagle, B. L. Stimulation of cellular proliferation by hepatitis B virus X protein. Dis. Markers 2001, 17 (3), 153–157. Sirma, H.; Weil, R.; Rosmorduc, O.; Urban, S.; Israe¨l, A.; Kremsdorf, D.; Bre´chot, C. Cytosol is the prime compartment of hepatitis B virus X protein where it colocalizes with the proteasome. Oncogene 1998, 16 (16), 2051–2063. Hafner, A.; Brandenburg, B.; Hildt, E. Reconstitution of gene expression from a regulatory-protein-deficient hepatitis B virus genome by cell-permeable HBx protein. EMBO Rep. 2003, 4 (8), 767–773. Haviv, I.; Vaizel, D.; Shaul, Y. pX, the HBV-encoded coactivator, interacts with components of the transcription machinery and stimulates transcription in a TAF-independent manner. EMBO J. 1996, 15 (13), 3413–3420. Becker, S. A.; Lee, T. H.; Butel, J. S.; Slagle, B. L. Hepatitis B virus X protein interferes with cellular DNA repair. J. Virol. 1998, 72 (1), 266–272.

(20) Lee, Y. I.; Hwang, J. M.; Im, J. H.; Lee, Y. I.; Kim, N. S.; Kim, D. G.; Yu, D. Y.; Moon, H. B.; Park, S. K. Human hepatitis B virus-X protein alters mitochondrial function and physiology in human liver cells. J. Biol. Chem. 2004, 279 (15), 15460–15471. (21) Kato, K.; Yamanaka, K.; Nakano, M.; Hasegawa, A.; Okada, S. 72kDa stress protein (hsp72) induced by administration of dimethylarsinic acid to mice accumulates in alveolar flat cells of lung, a target organ for arsenic carcinogenesis. Biol. Pharm. Bull. 2000, 23 (10), 1212–1215. (22) Lo´pez-Cotarelo, C.; Sellhaus, B.; Baba, H. A.; Manegold, E.; Luka, J.; Handt, S.; Mittermayer, C.; Klosterhalfen, B.; Tietze, L. Expression of heat shock proteins 72/73 in human peritoneal mesothelial cells in vivo and in vitro. Nephron. 2000, 85 (2), 148–155. (23) Luk, J. M.; Lam, C. T.; Siu, A. F.; Lam, B. Y.; Ng, I. O.; Hu, M. Y.; Che, C. M.; Fan, S. T. Proteomic profiling of hepatocellular carcinoma in Chinese cohort reveals heat-shock proteins (Hsp27, Hsp70, GRP78) up-regulation and their associated prognostic values. Proteomics 2006, 6 (3), 1049–1057. (24) Zhang, S. M.; Sun, D. C.; Lou, S.; Bo, X. C.; Lu, Z.; Qian, X. H.; Wang, S. Q. HBx protein of hepatitis B virus (HBV) can form complex with mitochondrial HSP60 and HSP70. Arch. Virol. 2005, 150 (8), 1579–1590. (25) Tanaka, Y.; Kanai, F.; Kawakami, T.; Tateishi, K.; Ijichi, H.; Kawabe, T.; Arakawa, Y.; Kawakami, T.; Nishimura, T.; Shirakata, Y.; Koike, K.; Omata, M. Interaction of the hepatitis B virus X protein (HBx) with heat shock protein 60 enhances HBx-mediated apoptosis. Biochem. Biophys. Res. Commun. 2004, 318 (2), 461–469. (26) Dorsey, W. C.; Tchounwou, P. B. CYP1a1, HSP70, P53, and c-fos expression in human liver carcinoma cells (HepG2) exposed to pentachlorophenol. Biomed. Sci. Instrum. 2003, 39, 389–396. (27) Hwang, T. S.; Han, H. S.; Choi, H. K.; Lee, Y. J.; Kim, Y. J.; Han, M. Y.; Park, Y. M. Differential, stage-dependent expression of Hsp70, Hsp110 and Bcl-2 in colorectal cancer. J. Gastroenterol. Hepatol. 2003, 18 (6), 690–700. (28) Wells, A. D.; Rai, S. K.; Salvato, M. S.; Band, H.; Malkovsky, M. Hsp72-mediated augmentation of MHC class I surface expression and endogenous antigen presentation. Int. Immunol. 1998, 10 (5), 609–617. (29) Faure, O.; Graff-Dubois, S.; Bretaudeau, L.; Derre, L.; Gross, D. A.; Alves, P. M. Inducible Hsp70 as target of anticancer immunotherapy: Identification of HLA-A/0201-restricted epitopes. Int. J. Cancer 2004, 108 (6), 863–870. (30) Wang, X. P.; Liu, G. Z.; Song, A. L.; Li, H. Y.; Liu, Y. Antitumor immunity induced by DNA vaccine encoding alpha-fetoprotein/ heat shock protein 70. World J. Gastroenterol. 2004, 10 (21), 3197– 3200. (31) Multhoff, G.; Botzler, C.; Jennen, L.; Schmidt, J.; Ellwart, J.; Issels, R. Heat shock protein 72 on tumor cells: a recognition structure for natural killer cells. J. Immunol. 1997, 158 (9), 4341–4350. (32) Zhang, H.; Hu, H.; Jiang, X.; He, H.; Cui, L.; He, W. Membrane HSP70: the molecule triggering gammadelta T cells in the early stage of tumorigenesis. Immunol. Invest. 2005, 34 (4), 453–468.

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