Statistical analysis

Data are analyzed as the mean ± SD. one-tailed Student’s t test was used to

analyze the difference between the means of the different groups. Differences with a p value of less than 0.05 were considered statistically significant.

3. Results

3.1 GRP78 Protein Is Critical for E1A–mediated inhibition of cell mobility

To investigate whether E1A affects the cancer metastatic activity, we transfected control vector or E1A expression vector into MDA-MB-231, HS578T and HBL100 cells. As expected, ectopic expression of E1A significantly reduce cell mobility and invasion activity in the vitro model (Fig.

1). To investigate the downstream target related to E1A-mediated anti-metastatic activity, we analyzed the differential expression of proteins in MDA-MB-231 breast cancer cells stably transfected with control vector (231/V) and E1A expression vector (231/E1A) using two-dimensional gel electrophoresis assay. We found 3 candidate proteins were increased and 9 candidate proteins were decreased in 231/E1A cells compared with 231/V cells (Table. 1). Expression of GRP78 protein relates to E1A-mediated anti-metastasis activity and GRP78, one of the downregulated protein in 231/E1A cells, make us interesting to understand which mechanism may involoved. Recent researches demonstrated that overexpression of GRP78 confers antiapoptosis and chemoresistant to promote tumor survival and has also been implicated in proliferation and cell motility in different types of tumors (55, 59). In the previous report, GRP78 is a key survival factor of cancer cells, and reduction of GRP78 expression inhibits tumor formation, growth, and suppresses cancer cell metastasis in xenografts models (45-46, 60).

In breast cancer patient, GRP78 expression is significantly higher in primary tumor compared with that in benign tissues (37). Because of the importance of GRP78 in cancer progression, we further determined whether GRP78 involves

in E1A-mediated tumor suppression activity. We examined the effects of E1A on GRP78 expression in various types of breast cancer cells and found that E1A suppresses the expression of GRP78 in protein levels but not mRNA level in E1A-transfected cells (Fig. 2). To define the role of GRP78 in breast cancer cells, we found that knockdown of GRP78 expression using GRP78 specific shRNA (shGRP78) significantly reduces migration and invasion ability compared with control shRNA in MDA-MB-231 and HS578T cells (Fig. 3).

To investigate the effects of GRP78 on E1A-mediated suppression of cell mobility, we stably transfected GRP78 or control vector (pcDNA6) in 231/E1A cells (Fig. 4A). Transfection of 231/E1A cells with GRP78 showed markedly promoted cell migration and invasion by transwell assay and cell tracing assays (Fig. 4B, 4C). However, cancer metastasis involves multiple steps including detachment of the metastatic cells from neighboring cells, the acquisition of motility and invasion to other tissue. Among these steps, reorganization of the actin cytoskeleton is an important for cell mobility. To investigate the effect of GRP78 on actin stress fiber formation, we examined whether GRP78 participates in actin remodeling to enhance metastasis. These data suggest that GRP78 is critical for E1A-mediated inhibition of cell mobility in breast cancer cells.

3.2 E1A Enhances Ub-dependent Proteolysis of GRP78

As shown in figure 2, E1A suppresses GRP78 in the protein level but not mRNA level. To determine the stability of GRP78 protein in response to E1A in MDA-MB-231, we treated with cycloheximide (CHX) for the indicated times in 231/V and 231/E1A cells. We found that the degradation rate of GRP78 protein is faster in 231/E1A cells than in 231/V cells (Fig. 5A). In

attempt to determine degradation of GRP78 protein in response to E1A, we analyzed expression of endogenous GRP78 protein in presence with proteasome inhibitor such as MG132. Our results showed that E1A-mediated GRP78 downregulation was rescued by MG132 (Fig. 5B), suggesting that E1A-mediated GRP78 degradation via ubiquitin proteasome process. To confirm that the ubiquitination of GRP78 is enhanced by E1A, we detected the level of GRP78 ubiquitination by immunoblotting (IB) after immunoprecipitation (IP) of GRP78 or ubiquitin in231/V and 231/E1A cells.

After IP with anti-GRP78 antibody and following by IB with anti-Ub, the ubiquitination of GRP78 was increased in the MG132-treated 231/E1A cells (Fig. 6). We showed that ubiquitin-dependent proteolysis of GRP78 is required for E1A-mediated GRP78 degradation, but which E3 ubiquitin ligase involves in this degradation process is unkown. Because of the ER localization of GRP78, we searched a series of ER-related E3 ubiquitin ligases such as gp78, Parkin, CHIP, Cul5 (61-63). Previous report showed that knockdown of gp78 increasedthe level of GRP78, and induced cell death in HEK 293 cells (64).

Here, we found that knockdown of gp78 could revert protein expression of GRP78 but not mRNA level in 231/E1A cells (Fig. 7A). To further examine whether the expression of GRP78 was also suppressed by the other E3 ubiquitin ligases, we used shRNA against the expression of CHIP , which play roles in the degradation of protein in ER and reduces chaperone efficiency (65-68). We found that knockdown of CHIP had slight effect on E1A-mediated GRP78 degradation (Fig. 7B). To further investigate the effects of gp78 on GRP78 E3 ubiquitin ligase, we transfected with gp78 or control vector (pcDNA3.1) in MDA-MB-468 and HS578T, and found that gp78 suppresses the expression of GRP78 (Fig. 8). Above data indicated that gp78 involves in

E1A-mediated suppression of cancer cell migration and invasion activity through degradation of GRP78 expression.

3.3 Gp78 Is Required for E1A-mediated Degradation of GRP78 and Suppression Cell Mobility

To further define the relationship between gp78 and GRP78, coimmunoprecipitation assays demonstrated that gp78 physically interacted with GRP78 (Fig. 9), indicting that gp78 might serve as a GRP78 E3 ubiquitin ligase for GRP78. To further the E3 ligase activity of gp78 is required for e1A-induced GRP78 degradation, a double mutation of gp78 RING finger (gp78R2m) which loss of E3 ligase activity is used (69-71). Transfected with gp78R2m into 231/E1A inhibits E1A-induced degradation of GRP78 was inhibited (Fig.10). As gp78 is a RING finger-domain-containing E3 ligase that regulates ubiquitin-dependent degradation of its substrates, we further transiently transfected with either wild-type gp78 or gp78/R2m along with haemagglutinin (HA)-tagged ubiquitin. The level of GRP78 ubiquitination detected by immunoblotting after immunoprecipitation of GRP78 shows that ubiquitination of GRP78 was enhanced by wild-type but not gp78/R2m (Fig.

11). Because gp78 functions as an E3 ligase for GRP78 protein, we attempted to determine whether knockdown of gp78 affect cell mobility. Our data showed that knockdown of gp78 in rescue the expression of GRP78, resulting in increasing cell migration and invasion in 231/E1A cells (Fig. 12). Furthermore, knockdown of GRP78 re-suppressed cell migration and invasion in gp78-silenced 231/E1A cells (Fig. 12), suggesting that gp78 is required for E1A-mediated inhibition of cell migration and invasion through degradation of GRP78 expression. Take together, these results suggest that gp78-mediated

GRP78 degradation is critical for E1A’s anti-metastatic activity.

4. Discussion

In current study, we showed that E1A suppresses cancer cell mobility and decreases expression of GRP78 protein in breast cancer cells. The previous data have been reported that GRP78 may enhance the activation of FAK which is correlation with mediating key signal transduction that regulation of actin remodeling (43, 72). We identified that GRP78 was degraded by E1A through the ubiquitin-proteasome system. Ubiquitin is covalently attached to target proteins through the action of three enzymes known as E1, E2, and E3. The ultimate outcome of this post-translational modification depends on the nature of the ubiquitin linkage and the extent of polyubiquitination. In most cases, ubiquitination results in degradation of the target proteins in the 26S proteasome. We show that the half-life of GRP78 protein expression was shorter in231/E1A cells compared with 231/V cells. GRP78 was rescued by treatment with MG132. Previous study showed that the GRP78 protein expression was increased in cells exposed to CHX (73), our data demonstrated that CHX indeed evaluate GRP78 expression in 231/V cells but not in 231/E1A cells, indicating that expression of E1A also attenuates GRP78 inducer. To investigate which E3 ubiquitin ligase involved in E1A-mediated degradation of GRP78, we silenced the expression of several ER-related E3 ubiquitin ligases by specific shRNA and found that knockdown of gp78 cause enhanced of GRP78 expression. However, we found that ectopic expression of E1A increased the association of gp78 with GRP78, following enhanced polyubiquitination and reduced GRP78 protein levels. Previous study indicated that both gp78 and CHIP E3 ligase effectively regulate hepatic CYP3A content (74). In our system, knockdown of CHIP expression had no effect on E1A-mediated GRP78 degradation, suggesting that gp78 serves as GRP78

specific E3 ligase. The domain of GRP78 for gp78 recognition and binding is unknown and some yet-to-be-identified protein candidate may be involved in the regulation of the gp78-GRP78 interaction. Based on our study, message RNAs and negatively regulate the gene expression by inhibiting their translation. In this case, it has not been reported miRNA regulate the expression of GRP78. We predicted miRNAs binding to 3’UTR of GRP78 mRNA.

However, we did not find miRNAs associated with regulation of GRP78 mRNA.

In addition to metastasis, E1A gene therapy has been reported to induce sensitization to multiple anticancer drugs (75-77). Recent study shows that GRP78 has antiapoptosis activity and associates with drug resistance such as etoposide and Temozolomide (78). Therefore, we investigated whether E1A-mediating anti-cancer drug sensitivity through regulation of GRP78 expression. GRP78 was not involved in E1A-mediated sensitization of taxol, suggesting that E1A-mediated taxol sensitivity is requiring for the other mechanisms. In the other hand, GRP78 also plays important role in the maintenance of cancer stem cells population (79). We hope that E1A-mediated downregulation of GRP78 might be a potential therapeutic target for cancer cells resulting in eliminating cancer stem cells.

In summary, we found that downregulation of GRP78 is critical for E1A-mediated inhibition metastasis. According to our finding, we provide a

model in which E1A represses GRP78 though induction of gp78-GRP78 interaction, which inhibits the breast cancer cells migration and invasion (Fig.

13).

5. Reference

1. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin. 2010 Sep-Oct;60(5):277-300.

2. Nguyen DX, Massague J. Genetic determinants of cancer metastasis. Nat Rev Genet. 2007 May;8(5):341-52.

3. Chalasani P, Downey L, Stopeck AT. Caring for the breast cancer survivor:

a guide for primary care physicians. Am J Med. 2010 Jun;123(6):489-95.

4. Nevins JR. Adenovirus E1A-dependent trans-activation of transcription.

Semin Cancer Biol. 1990 Feb;1(1):59-68.

5. Nevins JR, Raychaudhuri P, Yee AS, Rooney RJ, Kovesdi I, Reichel R.

Transactivation by the adenovirus E1A gene. Biochem Cell Biol. 1988 Jun;66(6):578-83.

6. Byrd PJ, Grand RJ, Gallimore PH. Differential transformation of primary human embryo retinal cells by adenovirus E1 regions and combinations of E1A + ras. Oncogene. 1988 May;2(5):477-84.

7. Ruley HE. Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature. 1983 Aug 18-24;304(5927):602-6.

8. Moran E, Zerler B, Harrison TM, Mathews MB. Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes. Mol Cell Biol. 1986 Oct;6(10):3470-80.

9. Shenk T, Flint J. Transcriptional and transforming activities of the adenovirus E1A proteins. Adv Cancer Res. 1991 Jan;57:47-85.

10. Shih JY, Tsai MF, Chang TH, Chang YL, Yuan A, Yu CJ, et al.

Transcription repressor slug promotes carcinoma invasion and predicts outcome of patients with lung adenocarcinoma. Clin Cancer Res. 2005 Nov

15;11(22):8070-8.

11. Frisch SM, Mymryk JS. Adenovirus-5 E1A: paradox and paradigm. Nat Rev Mol Cell Biol. 2002 Jun;3(6):441-52.

12. Yu DH, Scorsone K, Hung MC. Adenovirus type 5 E1A gene products act as transformation suppressors of the neu oncogene. Mol Cell Biol. 1991 Mar;11(3):1745-50.

13. Ueno NT, Yu D, Hung MC. E1A: tumor suppressor or oncogene?

Preclinical and clinical investigations of E1A gene therapy. Breast Cancer.

2001 Jan;8(4):285-93.

14. Song CZ, Loewenstein PM, Toth K, Green M. Transcription factor TFIID is a direct functional target of the adenovirus E1A transcription-repression domain. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10330-3.

15. Chen H, Hung MC. Involvement of co-activator p300 in the transcriptional regulation of the HER-2/neu gene. J Biol Chem. 1997 Mar 7;272(10):6101-4.

16. Geisberg JV, Chen JL, Ricciardi RP. Subregions of the adenovirus E1A transactivation domain target multiple components of the TFIID complex. Mol Cell Biol. 1995 Nov;15(11):6283-90.

17. Mazzarelli JM, Atkins GB, Geisberg JV, Ricciardi RP. The viral oncoproteins Ad5 E1A, HPV16 E7 and SV40 TAg bind a common region of the TBP-associated factor-110. Oncogene. 1995 Nov 2;11(9):1859-64.

18. Maguire K, Shi XP, Horikoshi N, Rappaport J, Rosenberg M, Weinmann R. Interactions between adenovirus E1A and members of the AP-1 family of cellular transcription factors. Oncogene. 1991 Aug;6(8):1417-22.

19. Yu D, Wolf JK, Scanlon M, Price JE, Hung MC. Enhanced c-erbB-2/neu expression in human ovarian cancer cells correlates with more severe malignancy that can be suppressed by E1A. Cancer Res. 1993 Feb

15;53(4):891-8.

20. Yu D, Suen TC, Yan DH, Chang LS, Hung MC. Transcriptional repression of the neu protooncogene by the adenovirus 5 E1A gene products.

Proc Natl Acad Sci U S A. 1990 Jun;87(12):4499-503.

21. Shao R, Tsai EM, Wei K, von Lindern R, Chen YH, Makino K, et al. E1A inhibition of radiation-induced NF-kappaB activity through suppression of IKK activity and IkappaB degradation, independent of Akt activation. Cancer Res.

2001 Oct 15;61(20):7413-6.

22. de Stanchina E, McCurrach ME, Zindy F, Shieh SY, Ferbeyre G, Samuelson AV, et al. E1A signaling to p53 involves the p19(ARF) tumor suppressor. Genes Dev. 1998 Aug 1;12(15):2434-42.

23. Timofeev OV, Pospelov VA. [The p21(WAF1) cyclin-kinase inhibitor: in vivo interaction with E1A adenoviral Ad2 and Ad12 oncoproteins]. Tsitologiia.

2003 May;45(11):1109-18.

24. Grooteclaes ML, Frisch SM. Evidence for a function of CtBP in epithelial gene regulation and anoikis. Oncogene. 2000 Aug 3;19(33):3823-8.

25. Bernhard EJ, Hagner B, Wong C, Lubenski I, Muschel RJ. The effect of E1A transfection on MMP-9 expression and metastatic potential. Int J Cancer.

1995 Mar 3;60(5):718-24. epithelial ovarian cancer that overexpresses HER-2/neu or epithelial ovarian

cancer. Hum Gene Ther. 1998 Aug 10;9(12):1775-98.

28. Hortobagyi GN, Ueno NT, Xia W, Zhang S, Wolf JK, Putnam JB, et al.

Cationic liposome-mediated E1A gene transfer to human breast and ovarian cancer cells and its biologic effects: a phase I clinical trial. J Clin Oncol. 2001 Jul 15;19(14):3422-33.

29. Villaret D, Glisson B, Kenady D, Hanna E, Carey M, Gleich L, et al. A multicenter phase II study of tgDCC-E1A for the intratumoral treatment of patients with recurrent head and neck squamous cell carcinoma. Head Neck.

2002 Jul;24(7):661-9.

30. Madhusudan S, Tamir A, Bates N, Flanagan E, Gore ME, Barton DP, et al.

A multicenter Phase I gene therapy clinical trial involving intraperitoneal administration of E1A-lipid complex in patients with recurrent epithelial ovarian cancer overexpressing HER-2/neu oncogene. Clin Cancer Res. 2004 May 1;10(9):2986-96.

31. Lee AS. The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem Sci. 2001 Aug;26(8):504-10.

34. Quinones QJ, de Ridder GG, Pizzo SV. GRP78: a chaperone with diverse roles beyond the endoplasmic reticulum. Histol Histopathol. 2008 Nov;23(11):1409-16.

35. Lee AS. GRP78 induction in cancer: therapeutic and prognostic

implications. Cancer Res. 2007 Apr 15;67(8):3496-9.

36. Wang Q, He Z, Zhang J, Wang Y, Wang T, Tong S, et al. Overexpression of endoplasmic reticulum molecular chaperone GRP94 and GRP78 in human lung cancer tissues and its significance. Cancer Detect Prev. 2005

38. Gazit G, Lu J, Lee AS. De-regulation of GRP stress protein expression in human breast cancer cell lines. Breast Cancer Res Treat. 1999 Mar;54(2):135-46.

39. Zhang J, Jiang Y, Jia Z, Li Q, Gong W, Wang L, et al. Association of elevated GRP78 expression with increased lymph node metastasis and poor prognosis in patients with gastric cancer. Clin Exp Metastasis. 2006 Dec;23(7-8):401-10.

40. Pootrakul L, Datar RH, Shi SR, Cai J, Hawes D, Groshen SG, et al.

Expression of stress response protein Grp78 is associated with the development of castration-resistant prostate cancer. Clin Cancer Res. 2006 Oct 15;12(20 Pt 1):5987-93.

hepatocarcinogenesis. J Hepatol. 2003 May;38(5):605-14.

43. Su R, Li Z, Li H, Song H, Bao C, Wei J, et al. Grp78 promotes the invasion of hepatocellular carcinoma. BMC Cancer. 2010 Jan;10:20.

44. Fu Y, Wey S, Wang M, Ye R, Liao CP, Roy-Burman P, et al. Pten null prostate tumorigenesis and AKT activation are blocked by targeted knockout of ER chaperone GRP78/BiP in prostate epithelium. Proc Natl Acad Sci U S A.

2008 Dec 9;105(49):19444-9.

45. Fu Y, Lee AS. Glucose regulated proteins in cancer progression, drug resistance and immunotherapy. Cancer Biol Ther. 2006 Jul;5(7):741-4.

46. Jamora C, Dennert G, Lee AS. Inhibition of tumor progression by suppression of stress protein GRP78/BiP induction in fibrosarcoma B/C10ME.

Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7690-4.

47. Misra UK, Deedwania R, Pizzo SV. Activation and cross-talk between Akt, NF-kappaB, and unfolded protein response signaling in 1-LN prostate cancer cells consequent to ligation of cell surface-associated GRP78. J Biol Chem.

2006 May 12;281(19):13694-707.

48. Finley D. Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem. 2009 Jun;78:477-513.

49. Crews CM. Feeding the machine: mechanisms of proteasome-catalyzed degradation of ubiquitinated proteins. Curr Opin Chem Biol. 2003 Oct;7(5):534-9.

50. Jung T, Catalgol B, Grune T. The proteasomal system. Mol Aspects Med.

2009 Aug;30(4):191-296.

51. Sorokin AV, Kim ER, Ovchinnikov LP. Proteasome system of protein degradation and processing. Biochemistry (Mosc). 2009 Dec;74(13):1411-42.

52. Bouska A, Lushnikova T, Plaza S, Eischen CM. Mdm2 promotes genetic

instability and transformation independent of p53. Mol Cell Biol. 2008

Targeting heat shock proteins on cancer cells: selection, characterization, and cell-penetrating properties of a peptidic GRP78 ligand. Biochemistry. 2006 Aug 8;45(31):9434-44.

55. Pyrko P, Schonthal AH, Hofman FM, Chen TC, Lee AS. The unfolded protein response regulator GRP78/BiP as a novel target for increasing chemosensitivity in malignant gliomas. Cancer Res. 2007 Oct 15;67(20):9809-16.

56. Martin S, Hill DS, Paton JC, Paton AW, Birch-Machin MA, Lovat PE, et al. Targeting GRP78 to enhance melanoma cell death. Pigment Cell Melanoma Res. 2010 Oct;23(5):675-82.

57. Dong D, Ni M, Li J, Xiong S, Ye W, Virrey JJ, et al. Critical role of the stress chaperone GRP78/BiP in tumor proliferation, survival, and tumor angiogenesis in transgene-induced mammary tumor development. Cancer Res.

2008 Jan 15;68(2):498-505.

predictor for breast cancer response to adjuvant taxane therapy. Int J Cancer.

2011 Feb 1;128(3):726-31.

60. Wang M, Wey S, Zhang Y, Ye R, Lee AS. Role of the unfolded protein response regulator GRP78/BiP in development, cancer, and neurological disorders. Antioxid Redox Signal. 2009 Sep;11(9):2307-16.

61. Chen C, Seth AK, Aplin AE. Genetic and expression aberrations of E3 ubiquitin ligases in human breast cancer. Mol Cancer Res. 2006 Oct;4(10):695-707.

62. Cyr DM, Hohfeld J, Patterson C. Protein quality control: U-box-containing E3 ubiquitin ligases join the fold. Trends Biochem Sci. 2002 Jul;27(7):368-75.

63. Soucy TA, Smith PG, Rolfe M. Targeting NEDD8-activated cullin-RING ligases for the treatment of cancer. Clin Cancer Res. 2009 Jun

Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions.

Mol Cell Biol. 1999 Jun;19(6):4535-45.

66. Murata S, Minami Y, Minami M, Chiba T, Tanaka K. CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein. EMBO Rep.

2001 Dec;2(12):1133-8.

67. Connell P, Ballinger CA, Jiang J, Wu Y, Thompson LJ, Hohfeld J, et al.

The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nat Cell Biol. 2001 Jan;3(1):93-6.

68. McDonough H, Patterson C. CHIP: a link between the chaperone and proteasome systems. Cell Stress Chaperones. 2004 Apr;8(4):303-8.

69. Fang S, Ferrone M, Yang C, Jensen JP, Tiwari S, Weissman AM. The tumor autocrine motility factor receptor, gp78, is a ubiquitin protein ligase implicated in degradation from the endoplasmic reticulum. Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14422-7.

70. Ballar P, Shen Y, Yang H, Fang S. The role of a novel p97/valosin-containing protein-interacting motif of gp78 in endoplasmic reticulum-associated degradation. J Biol Chem. 2006 Nov 17;281(46):35359-68.

71. Chen B, Mariano J, Tsai YC, Chan AH, Cohen M, Weissman AM. The activity of a human endoplasmic reticulum-associated degradation E3, gp78, requires its Cue domain, RING finger, and an E2-binding site. Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):341-6.

72. Gerthoffer WT, Gunst SJ. Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle. J Appl Physiol. 2001 Aug;91(2):963-72.

73. Faria G, Cardoso CR, Larson RE, Silva JS, Rossi MA.

Chlorhexidine-induced apoptosis or necrosis in L929 fibroblasts: A role for endoplasmic reticulum stress. Toxicol Appl Pharmacol. 2009 Jan 15;234(2):256-65.

74. Kim SM, Acharya P, Engel JC, Correia MA. Liver cytochrome P450 3A ubiquitination in vivo by gp78/autocrine motility factor receptor and C terminus of Hsp70-interacting protein (CHIP) E3 ubiquitin ligases:

physiological and pharmacological relevance. J Biol Chem. 2010 Nov 12;285(46):35866-77.

75. Ueno NT, Yu D, Hung MC. Chemosensitization of HER-2/neu-overexpressing human breast cancer cells to paclitaxel (Taxol) by adenovirus type 5 E1A. Oncogene. 1997 Aug 18;15(8):953-60.

76. Liao Y, Zou YY, Xia WY, Hung MC. Enhanced paclitaxel cytotoxicity and prolonged animal survival rate by a nonviral-mediated systemic delivery of E1A gene in orthotopic xenograft human breast cancer. Cancer Gene Ther.

2004 Sep;11(9):594-602.

77. Sakakibara A, Tsukuda M, Kondo N, Ishiguro Y, Kimura M, Fujita K, et al. Examination of the optimal condition on the in vitro sensitivity to

77. Sakakibara A, Tsukuda M, Kondo N, Ishiguro Y, Kimura M, Fujita K, et al. Examination of the optimal condition on the in vitro sensitivity to