Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Transcription profile of a human breast cancer cell line expressing MMTV-like sequences

Transcription profile of a human breast cancer cell line expressing MMTV-like sequences Background: It has been postulated that inflammation caused by certain viruses might result in cancer. Recently, it was shown that childhood lymphoblastic leukemia, breast and ovarian cancers express an interferon-related signature, providing support for this notion. We have previously shown that 38% of the sporadic breast cancers contain MMTV-like env gene sequences. To find out if the presence and expression of MMTV-like sequences correlated with an inflammatory phenotype, we have compared the expression profile of two sublines of MCF-7 cells, one containing the MMTV-like sequences (env+), the other one lacking them (env-). Results: The results indicated that there were 47 differentially expressed genes between the two sublines. Among 27 upregulated genes in the env+ cells there were 7 interferon-related genes, 5 TNF-connected genes and 2 TGFβ-related genes. Conclusion: These results suggest that the env+ cells were most likely responding to an infectious agent, and support the hypothesis that a viral infection may play a role in breast cancer pathogenesis. terminal of human sag sequences, the cloned human sag Background We and others [1-4] have shown that 37 to 41% of spo- sequences expressed in human B lymphocytes can activate radic breast cancer samples contain MMTV-like env gene human T-cells, as can the mouse Sag, indicating that it can sequences. The sequences are expressed as RNA [5] and as be functional [9]. Moreover, viral particles with the mor- protein in breast cancers (Melana et al., submitted). They phological characteristics of betaretroviruses are observed are absent from the normal breasts of patients with env in primary cultures of human beast cancer [10]. Taken positive tumors [6] and are expressed as RNA exclusively together, these results suggest that an infectious agent is in the cancer cells [7]. The whole proviral structure, desig- present in some human breast cancers. nated human mammary tumor virus (HMTV), which has 95% homology to MMTV, can be detected in two tumors Chronic inflammation has been implicated in tumor pro- [8]. Although sequence variations are observed in the C- gression. New evidence suggests that the inflammation Page 1 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:7 http://www.infectagentscancer.com/content/1/1/7 caused by certain viruses results in cancer [Reviewed in to an interferon-related signature, we have compared the [11]]. Recently, it was reported that childhood lymphob- expression profiles of two sublines of MCF-7 [22], one lastic leukemia, as well as breast and ovarian cancers which contains the MMTV-like env gene sequence (env+) express an interferon-related signature, but not found in and one which lacks it (env-). other human cancers studied [12]. This finding provides molecular support for the role of inflammation or viral Results infection in cancer pathogenesis [12]. The presence of the Env protein was investigated in both sublines. In Fig. 1 the result of the immunoblotting exper- The established breast cancer cell line MCF-7 is widely iment is shown. The HMTV env+ cell line expressed a pro- used in research, and many subclones are available. Some tein of a MW of approximately 50 kD which reacted with of the original isolates produce retroviral-like particles mAbP2, a monoclonal antibody against a synthetic pep- [13]. Furthermore, May and Westerly [14] described the tide derived from human env sequences (Melana et al sub- presence of an MMTV-like 6.6 Kb EcoR1 fragment in some mitted). It was absent in the HMTV env- cells. Tubulin was of the MCF-7 cell lines, which was absent in other breast equally present in both extracts. cancer lines and in normal tissue. The results of the cDNA arrays are shown in Tables 1 and Continuous passage with subsequent chromosomal 2. Nineteen genes showed a > 2.5 fold difference in their change [15] may have eliminated viral sequences from adjusted intensity between HMTV env+ and env- cells, some of them. It has been reported that some sublines of while another eight genes were only expressed in the MCF-7 show biological differences [16] and significant HMTV env+ cells (Table 1). Twenty genes were downregu- genetic variation in RNA expression [17,18]. lated (Table 2) in HMTV env + cells. Taken together, there were 47 differentially expressed genes. Among the 27 We have previously reported that a subline of MCF-7 con- upregulated genes there were six interferon-inducible taining env and LTR sequences [19,20] and that it ones: IFI6, TRIM22, IFITM1, IFITM2+IFITM3, IFI27 and expressed the env gene as RNA [7], while other sublines IP-30 and a receptor IFNGR2. In addition, there were five were negative for env gene [[21] and our own results]. To upregulated genes that have a connection with TNF or are find out whether the presence of viral sequences is related involved in its signaling, like LTBR, TRAF3, MMP17, Wester Figure 1 n blot of MCF-7 cells Western blot of MCF-7 cells. Experimental conditions as described in Materials and Methods. A: MCF-7 (+) cells; B: MCF-7(-) cells. Page 2 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:7 http://www.infectagentscancer.com/content/1/1/7 PKN1 and MAPK13. The cytokine TGFβ, and its down- sion profile of HMTV env+ cells suggests an increased stream effector early growth response protein 1 (EGR1), potential for cell growth, a fact that may be related to their were also upregulated in env+ cells. Twenty genes were more malignant phenotype as has been described in down regulated in HMTV env+ cells. breast cancer cells associated with HMTV [23,3,24]. It is remarkable that the alpha 7 and beta 4 integrins were sig- Discussion nificantly down regulated in env+ cells, as has been Comparison of the expression profiles of sublines derived reported in a set of finite life-span metastatic breast cancer from the same cell line provides an excellent model with cells which were also env+ [25]. minimal differences. Karyogenetic analysis revealed that the two sublines have similar complex chromosomal pat- Whether the HMTV works as initiator and/or as promoter terns (not shown). The comparison of expression profiles of malignant growth is uncertain. Molecular evidence that of MCF-7 env+ and env- cells indicated preferential expres- HMTV expression is responsible for the increase in inter- sion of interferon-related genes: 26% (7/27) of the up-reg- feron-related expression is being sought. ulated genes. These differences may indicate a trend. Einav et al. [12] have reported that 40% of clinical breast cancer Conclusion samples display an interferon-associated signature; 17 out The results clearly indicate that the transcriptional profile of 36 (47%) of the upregulated genes. Our results are con- of the cells expressing HMTV sequences is enriched in sistent with, but cannot be directly compared with those genes involved in inflammation process. This finding is of Einav's for several reasons: we used only one cell line significant because it was obtained comparing cells for analysis, the participation of stroma and surrounding derived from the same cell line that have similar genetic tissues has been eliminated from our study, and finally, background and minimal expressing differences. This sup- we used a different set of arrays. Nevertheless, our results ports the hypothesis that a viral infection may play a role strongly indicate that HMTV env+ MCF-7 cells express in breast cancer pathogenesis. more interferon-related genes than the HMTV env- MCF-7 cells, suggesting that they may be responding to an infec- tious agent as proposed by Einav et al. [12]. The expres- Table 1: Up-regulated genes in HMTV env+ cells Acc # Gene symbol Protein/gene Ratio Diff X02492 IFI6 Interferon-inducible protein 6 12.44 4108 J05633 ITGB5 Integrin beta 5 9.46 3401 X82200 TRIM22 Tripartite motif-containing 22 5.76 1495 L03840 FGFR4 Fibroblast growth factor receptor 4 5.72 1742 J04164 IFITM1 Interferon induced transmembrane protein 1 (9–27) 5.38 6776 M64595 small G protein 4.74 1720 X57351 IFITM2 + IFITM3 interferon induced transmembrane prot 2 (1–8D) + 3 (1–8U) 4.25 1082 X89576 MMP17 matrix metalloproteinase 17 4.20 1647 X52541 EGR1 early growth response protein 1 3.39 1327 L29220 CLK3 CDC-like kinase 3 3.38 1521 X66362 PCTK3 PCTAIRE protein kinase 3 3.34 1368 U33053 PKN1 protein kinase N1 2.85 1075 L04270 LTBR lymphotoxin beta receptor (TNFR superfamily, member3) 2.79 1406 U09579 CDKN1A cyclin-dependent kinase inhibitor 1A (p21, Cip1) 2.69 6193 U14966 RPL5 60S ribosomal protein L5 2.68 1641 M29039 JUNB jun-B 2.65 1313 M29971 MGMT 6-O-methylguanine-DNA methyltransferase 2.63 1971 M65199 EDN2 endothelin 2 2.58 1535 U57342 MLF2 myeloid leukemia factor 2 2.52 2897 X69398 CD47 CD47 glycoprotein; integrin-associated protein Up 4152 U12255 FCGRT Fc fragment of IgG, receptor, transporter Up 2960 AF004709 MAPK13 mitogen-activated protein kinase 13 Up 2049 X02812 TGFB transforming growth factor, beta 1 Up 1535 U21092 TRAF3 TNF receptor-associated factor 3 Up 1407 U05875 IFNGR2 interferon gamma receptor 2 Up 1372 X67325 IFI27 interferon, alpha-inducible protein 27 Up 1182 J03909 gamma-interferon-inducible protein; IP-30 Up 1076 Page 3 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:7 http://www.infectagentscancer.com/content/1/1/7 Table 2: Down-regulated genes in HMTV env+ cells Acc # Gene symbol Protein/gene Ratio Diff U02687 FLT3 fms-related tyrosine kinase 3 -6.20 -2143 X74295 IGA7B integrin alpha 7B -5.13 -2249 X53587 ITGB4 integrin beta 4 -4.80 -2384 M34671 CD59 CD59 molecule, complement regulatory protein -3.60 -2551 L25081 RHOC ras homolog gene family, member C -3.59 -2341 U89278 PHC2 polyhomeotic-like 2 -3.53 -1250 X16277 ODC1 ornithine decarboxylase 1 -2.87 -1295 AF029670 RAD51C RAD51 homolog C -2.61 -1713 M20430 HLA-DRB1 MHC class II HLA-DR-beta Down -4288 JUN c-jun proto-oncogene; transcription factor AP-1 Down -1745 J04111 U70310 FANCG DNA repair protein XRCC9 Down -1514 M59911 ITGA3 integrin alpha 3 Down -1393 M97934 STAT2 signal transducer and activator of transcription 2 Down -1248 L38518 SHH sonic hedgehog Down -1183 X51521 VIL2 ezrin; villin 2 Down -1177 L07515 CBX5 chromobox homolog 5; heterochromatin protein homolog 1 (HP1) Down -1153 M15400 RB1 retinoblastoma 1 Down -1109 M54995 PPBP pro-platelet basic protein Down -1100 X54199 GART trifunctional purine biosynthetic protein adenosine 3 Down -1092 U47686 STAT5 A +B signal transducer and activator of transcription 5 A+B Down -1072 relation coefficient based on the adjusted intensity of all Methods MCF-7 cells were obtained from American Type Culture genes spotted on the membrane. Collection (ATCC) and were propagated in vitro as rec- ommended by the provider and as described in previous Hybridizations with 30 μg of total RNA were performed publications (1, 5). To determine whether the viral pro- according to the manufacturer instructions. The hybrid- tein was expressed in our MCF-7 cells, western blotting ized membranes were exposed onto a phosphorimager was used. Protein lysates were prepared from approxi- screen and were read using a phosphorimager reader mately 1 × 10 cells. Equal amounts of protein from each (Molecular Dynamics). The scanned images were aligned sample were loaded onto an SDS-PAGE-10% polyacryla- and analyzed using AtlasImage 2.01 software (Clontech). mide gel, followed by transfer to PVDF membranes. West- When averaging or comparing samples, the adjusted ern blot analysis was performed using mAbP2 (a intensity signal was normalized using the global normali- monoclonal antibody against a peptide of the Env pro- zation mode featured in the software. We reported only tein), and mAb-tubulin as primary antibodies (Sigma those genes whose ratios of differential expression were Aldrich). Proteins were visualized using horseradish per- 2.5-fold or more, or genes that were undefined for one oxidase-labeled sheep anti-mouse IgG (GE Healthcare type of sample, but were detected on the other. (Unde- Bio-Sciences Corp.) as a secondary antibody followed by fined genes are those whose intensity were below the sig- enhanced chemiluminescence (GE Healthcare Bio-Sci- nal threshold) In the later event, when we lack a ences Corp.). numerical value for the ratio, it was defined as being "up" or "down". Furthermore, for each gene we stated the dif- The expression profile was studied using the Atlas Human ference (diff) in adjusted normalized intensity between Cancer 1.2 cDNA expression array; a nylon membrane the two cell lines. printed with 200–600 bp long fragments of 1176 charac- terized genes involved in cancer, 9 housekeeping genes Accession number (Acc#), gene symbol and protein or and 6 negative controls (Clontech, CA). These conditions gene name are according to GeneBank. were described in detail in a previous publication [25]. Briefly, RNA was extracted and labeled with Atlas pure Acknowledgements Grant support: The T.J. Martell Foundation for Leukemia, Cancer and AIDS total RNA labeling system and hybridized to an Atlas Research, The Jane Grinberg Memorial Fund, the Kash Family Foundation, Human Cancer 1.2 cDNA expression array (Clontech, CA) and the Ellen Block Memorial Fund. according to the manufacturer's instructions. Both cell sublines were probed twice in separate assays, and the We thank Cindy Hernandez for skillful technical assistance and Jennifer accuracy of each duplicate was assessed by Pearson's cor- Hasa and Julia Roboz for editorial work. Page 4 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:7 http://www.infectagentscancer.com/content/1/1/7 human breast carcinoma correlate with overexpression of References laminin receptor. Cl Cancer Res 1999, 5:2108-2111. 1. Wang Y, Holland JF, Bleiweiss IK, Melana SM, Xu D, Pogo BGT: 24. Wang Y, Melana SM, Baker B, Bleiweiss I, Fernandez-Cobo M, Mandeli Detection of mammary tumor virus env gene-like sequences J, Holland JF, Pogo BGT: Presence of MMTV-like env gene in human breast cancer. Cancer Res 1995, 55:5173-5179. sequences in gestational breast cancer. Med Oncol 2003, 2. Etkind P, Du J, Khan A, Pillitteri J, Wiernik P: Mouse mammary 20:233-236. tumor virus-like sequences in human breast tumors and in a 25. Fernandez-Cobo M, Holland JF, Pogo BGT: Transcription profile lymphoma of a breast cancer patient. Cl Cancer Res 2000, of non-immortalized breast cancer cell lines. BMC Cancer 6:1273-1278. 2006, 6(1):99. 3. Ford CE, Tran D, Deng YM, Ta VT, Rawlinson WD, Lawson JS: Mouse Mammary Tumor Virus-like gene sequences in breast tumors of Australian and Vietnamese women. Clin Cancer Res 2003, 9:1118-1120. 4. Zammarchi F, Pistello M, Piersiguilli X, Murr R, Di Cristofano C, Nac- carato AG, Bevilacqua G: MMTV-like sequences in human breast cancer: a fluorescent PCR/laser microdissection approach. J Pathol 2006, 209:436-444. 5. Wang Y, Go V, Holland JF, Melana SM, Pogo BGT: Expression of MMTV-like env gene sequences in human breast cancer. Cl Cancer Res 1998, 4:2565-2568. 6. Melana SM, Holland JF, Pogo BGT: Search for MMTV-like env sequences in cancer and normal breast from the same indi- vidual. Cl Cancer Res 2001, 7:283-284. 7. Ford CE, Faedo M, Rawlinson WD: MMTV virus-like RNA tran- scripts and DNA found in affected cells of human breast can- cer. Cl Cancer Res 2004, 10:7284-7289. 8. Liu B, Wang Y, Melana SM, Pelisson I, Najfeld V, Holland JF, Pogo BGT: Identification of a proviral structure in human breast cancer. Cancer Res 2001, 61:1754-1759. 9. Wang Y, Jiang JD, Xu D, Li Y, Holland JF, Qu C, Pogo BGT: A MMTV-like LTR superantigen in human breast cancer. Can- cer Res 2004, 64:4105-4111. 10. Melana SM, Nepomnaschy I, Dales S, Rojowsky H, Abbott A, Jiang JD, Holland JF, Pogo BGT: A retrovirus in human berast cancer: Isolation and characterization of human mammary tumor virus [abstract]. Proc AACR 2006, 47:1043. 11. Coussins LM, Werb Z: Inflammation and cancer. Nature 2002, 20:860-867. 12. Einav U, Tabach Y, Getz G, Ytzhaky A, Ozbek U, Amariglio A, Israelí S, Rechavi G, Domany E: Gene expression analysis reveals a strong signature of an interferon-induced pathway in child- hood lymphoblastic leukemia as well as in breast and ovarian cancer. Oncogene 2005, 24:6367-6375. 13. McGrath CM, Grant PM, Soule HD, Glancy T, Rich MA: Replication of oncornavirus-like particle in human breast carcinoma cell line, MCF-7. Nature 1974, 252:247-250. 14. May EB, Westley BC: Characterization of sequences related to the mouse mammary tumor virus that are specific to MCF- 7 breast cancer cells. Cancer Res 1989, 49:3879-3883. 15. Bahia H, Ashman JNE, Cawkell L, Lind M, Monson JRT: Karyotypic variation between independently cultured strains of the cell line MCF-7 identified by multicolour fluorescence in situ hybridization. Int J Oncol 2002, 20:489-494. 16. Osborne CK, Hobbs K, Trent JM: Biological differences among MCF-7 human breast cancer 968 cell lines from different lab- oratories. Breast Cancer Res Treat 1987, 9:111-121. 17. Nugoli M, Chuchana P, Vendrell J, Orsetti B, Ursule L: Genetic var- iability in MCF-7 sublines: evidence of rapid genomic and RNA expression profile modifications. BMC Cancer 2003, 3:13. 18. Hiorns LR, Bradshaw TD, Skelton LA, Yu Q, Kelland LR, Leyland- Jones B: Variation in RNA expression and genomic DNA con- Publish with Bio Med Central and every tent acquired during cell culture. Brit J Cancer 2004, 90:476-482. 19. Wang Y, Pelisson I, Melana SM, Go V, Holland JF, Pogo BGT: MMTV- scientist can read your work free of charge like env gene sequences in human breast cancer. Arch Virol "BioMed Central will be the most significant development for 2001, 145:1-10. 20. Wang Y, Pelisson I, Melana SM, Holland JF, Pogo BGT: Detection of disseminating the results of biomedical researc h in our lifetime." MMTV-like LTR and LTR-env gene sequences in human Sir Paul Nurse, Cancer Research UK breast cancer. Int J Oncol 2001, 18:1041-1044. 21. Zangen R, Harden S, Cohen D, Parrella P, Sidransky D: Mouse Your research papers will be: mammary tumor-like env gene as a molecular marker for available free of charge to the entire biomedical community breast cancer. Int J Cancer 2002, 102:304-307. peer reviewed and published immediately upon acceptance 22. Soule HD, Vasquez J, Long A, Albert S, Brennan M: A human cell line from a pleural effusion derived from a breast carcinoma. cited in PubMed and archived on PubMed Central J Nat Cancer Inst 1973, 51:1409-1416. yours — you keep the copyright 23. Pogo BGT, Melana SM, Holland JF, Mandeli JF, Piloti S, Casalini P, Ménard S: Sequences homologous to the MMTV env gene in BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 5 of 5 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Infectious Agents and Cancer Springer Journals

Transcription profile of a human breast cancer cell line expressing MMTV-like sequences

Download PDF
5 pages

Loading next page...
 
/lp/springer-journals/transcription-profile-of-a-human-breast-cancer-cell-line-expressing-eZSt0M4wVc

References (30)

Publisher
Springer Journals
Copyright
Copyright © 2006 by Fernandez-Cobo et al; licensee BioMed Central Ltd.
Subject
Biomedicine; Cancer Research; Infectious Diseases; Oncology
eISSN
1750-9378
DOI
10.1186/1750-9378-1-7
pmid
17173685
Publisher site
See Article on Publisher Site

Abstract

Background: It has been postulated that inflammation caused by certain viruses might result in cancer. Recently, it was shown that childhood lymphoblastic leukemia, breast and ovarian cancers express an interferon-related signature, providing support for this notion. We have previously shown that 38% of the sporadic breast cancers contain MMTV-like env gene sequences. To find out if the presence and expression of MMTV-like sequences correlated with an inflammatory phenotype, we have compared the expression profile of two sublines of MCF-7 cells, one containing the MMTV-like sequences (env+), the other one lacking them (env-). Results: The results indicated that there were 47 differentially expressed genes between the two sublines. Among 27 upregulated genes in the env+ cells there were 7 interferon-related genes, 5 TNF-connected genes and 2 TGFβ-related genes. Conclusion: These results suggest that the env+ cells were most likely responding to an infectious agent, and support the hypothesis that a viral infection may play a role in breast cancer pathogenesis. terminal of human sag sequences, the cloned human sag Background We and others [1-4] have shown that 37 to 41% of spo- sequences expressed in human B lymphocytes can activate radic breast cancer samples contain MMTV-like env gene human T-cells, as can the mouse Sag, indicating that it can sequences. The sequences are expressed as RNA [5] and as be functional [9]. Moreover, viral particles with the mor- protein in breast cancers (Melana et al., submitted). They phological characteristics of betaretroviruses are observed are absent from the normal breasts of patients with env in primary cultures of human beast cancer [10]. Taken positive tumors [6] and are expressed as RNA exclusively together, these results suggest that an infectious agent is in the cancer cells [7]. The whole proviral structure, desig- present in some human breast cancers. nated human mammary tumor virus (HMTV), which has 95% homology to MMTV, can be detected in two tumors Chronic inflammation has been implicated in tumor pro- [8]. Although sequence variations are observed in the C- gression. New evidence suggests that the inflammation Page 1 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:7 http://www.infectagentscancer.com/content/1/1/7 caused by certain viruses results in cancer [Reviewed in to an interferon-related signature, we have compared the [11]]. Recently, it was reported that childhood lymphob- expression profiles of two sublines of MCF-7 [22], one lastic leukemia, as well as breast and ovarian cancers which contains the MMTV-like env gene sequence (env+) express an interferon-related signature, but not found in and one which lacks it (env-). other human cancers studied [12]. This finding provides molecular support for the role of inflammation or viral Results infection in cancer pathogenesis [12]. The presence of the Env protein was investigated in both sublines. In Fig. 1 the result of the immunoblotting exper- The established breast cancer cell line MCF-7 is widely iment is shown. The HMTV env+ cell line expressed a pro- used in research, and many subclones are available. Some tein of a MW of approximately 50 kD which reacted with of the original isolates produce retroviral-like particles mAbP2, a monoclonal antibody against a synthetic pep- [13]. Furthermore, May and Westerly [14] described the tide derived from human env sequences (Melana et al sub- presence of an MMTV-like 6.6 Kb EcoR1 fragment in some mitted). It was absent in the HMTV env- cells. Tubulin was of the MCF-7 cell lines, which was absent in other breast equally present in both extracts. cancer lines and in normal tissue. The results of the cDNA arrays are shown in Tables 1 and Continuous passage with subsequent chromosomal 2. Nineteen genes showed a > 2.5 fold difference in their change [15] may have eliminated viral sequences from adjusted intensity between HMTV env+ and env- cells, some of them. It has been reported that some sublines of while another eight genes were only expressed in the MCF-7 show biological differences [16] and significant HMTV env+ cells (Table 1). Twenty genes were downregu- genetic variation in RNA expression [17,18]. lated (Table 2) in HMTV env + cells. Taken together, there were 47 differentially expressed genes. Among the 27 We have previously reported that a subline of MCF-7 con- upregulated genes there were six interferon-inducible taining env and LTR sequences [19,20] and that it ones: IFI6, TRIM22, IFITM1, IFITM2+IFITM3, IFI27 and expressed the env gene as RNA [7], while other sublines IP-30 and a receptor IFNGR2. In addition, there were five were negative for env gene [[21] and our own results]. To upregulated genes that have a connection with TNF or are find out whether the presence of viral sequences is related involved in its signaling, like LTBR, TRAF3, MMP17, Wester Figure 1 n blot of MCF-7 cells Western blot of MCF-7 cells. Experimental conditions as described in Materials and Methods. A: MCF-7 (+) cells; B: MCF-7(-) cells. Page 2 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:7 http://www.infectagentscancer.com/content/1/1/7 PKN1 and MAPK13. The cytokine TGFβ, and its down- sion profile of HMTV env+ cells suggests an increased stream effector early growth response protein 1 (EGR1), potential for cell growth, a fact that may be related to their were also upregulated in env+ cells. Twenty genes were more malignant phenotype as has been described in down regulated in HMTV env+ cells. breast cancer cells associated with HMTV [23,3,24]. It is remarkable that the alpha 7 and beta 4 integrins were sig- Discussion nificantly down regulated in env+ cells, as has been Comparison of the expression profiles of sublines derived reported in a set of finite life-span metastatic breast cancer from the same cell line provides an excellent model with cells which were also env+ [25]. minimal differences. Karyogenetic analysis revealed that the two sublines have similar complex chromosomal pat- Whether the HMTV works as initiator and/or as promoter terns (not shown). The comparison of expression profiles of malignant growth is uncertain. Molecular evidence that of MCF-7 env+ and env- cells indicated preferential expres- HMTV expression is responsible for the increase in inter- sion of interferon-related genes: 26% (7/27) of the up-reg- feron-related expression is being sought. ulated genes. These differences may indicate a trend. Einav et al. [12] have reported that 40% of clinical breast cancer Conclusion samples display an interferon-associated signature; 17 out The results clearly indicate that the transcriptional profile of 36 (47%) of the upregulated genes. Our results are con- of the cells expressing HMTV sequences is enriched in sistent with, but cannot be directly compared with those genes involved in inflammation process. This finding is of Einav's for several reasons: we used only one cell line significant because it was obtained comparing cells for analysis, the participation of stroma and surrounding derived from the same cell line that have similar genetic tissues has been eliminated from our study, and finally, background and minimal expressing differences. This sup- we used a different set of arrays. Nevertheless, our results ports the hypothesis that a viral infection may play a role strongly indicate that HMTV env+ MCF-7 cells express in breast cancer pathogenesis. more interferon-related genes than the HMTV env- MCF-7 cells, suggesting that they may be responding to an infec- tious agent as proposed by Einav et al. [12]. The expres- Table 1: Up-regulated genes in HMTV env+ cells Acc # Gene symbol Protein/gene Ratio Diff X02492 IFI6 Interferon-inducible protein 6 12.44 4108 J05633 ITGB5 Integrin beta 5 9.46 3401 X82200 TRIM22 Tripartite motif-containing 22 5.76 1495 L03840 FGFR4 Fibroblast growth factor receptor 4 5.72 1742 J04164 IFITM1 Interferon induced transmembrane protein 1 (9–27) 5.38 6776 M64595 small G protein 4.74 1720 X57351 IFITM2 + IFITM3 interferon induced transmembrane prot 2 (1–8D) + 3 (1–8U) 4.25 1082 X89576 MMP17 matrix metalloproteinase 17 4.20 1647 X52541 EGR1 early growth response protein 1 3.39 1327 L29220 CLK3 CDC-like kinase 3 3.38 1521 X66362 PCTK3 PCTAIRE protein kinase 3 3.34 1368 U33053 PKN1 protein kinase N1 2.85 1075 L04270 LTBR lymphotoxin beta receptor (TNFR superfamily, member3) 2.79 1406 U09579 CDKN1A cyclin-dependent kinase inhibitor 1A (p21, Cip1) 2.69 6193 U14966 RPL5 60S ribosomal protein L5 2.68 1641 M29039 JUNB jun-B 2.65 1313 M29971 MGMT 6-O-methylguanine-DNA methyltransferase 2.63 1971 M65199 EDN2 endothelin 2 2.58 1535 U57342 MLF2 myeloid leukemia factor 2 2.52 2897 X69398 CD47 CD47 glycoprotein; integrin-associated protein Up 4152 U12255 FCGRT Fc fragment of IgG, receptor, transporter Up 2960 AF004709 MAPK13 mitogen-activated protein kinase 13 Up 2049 X02812 TGFB transforming growth factor, beta 1 Up 1535 U21092 TRAF3 TNF receptor-associated factor 3 Up 1407 U05875 IFNGR2 interferon gamma receptor 2 Up 1372 X67325 IFI27 interferon, alpha-inducible protein 27 Up 1182 J03909 gamma-interferon-inducible protein; IP-30 Up 1076 Page 3 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:7 http://www.infectagentscancer.com/content/1/1/7 Table 2: Down-regulated genes in HMTV env+ cells Acc # Gene symbol Protein/gene Ratio Diff U02687 FLT3 fms-related tyrosine kinase 3 -6.20 -2143 X74295 IGA7B integrin alpha 7B -5.13 -2249 X53587 ITGB4 integrin beta 4 -4.80 -2384 M34671 CD59 CD59 molecule, complement regulatory protein -3.60 -2551 L25081 RHOC ras homolog gene family, member C -3.59 -2341 U89278 PHC2 polyhomeotic-like 2 -3.53 -1250 X16277 ODC1 ornithine decarboxylase 1 -2.87 -1295 AF029670 RAD51C RAD51 homolog C -2.61 -1713 M20430 HLA-DRB1 MHC class II HLA-DR-beta Down -4288 JUN c-jun proto-oncogene; transcription factor AP-1 Down -1745 J04111 U70310 FANCG DNA repair protein XRCC9 Down -1514 M59911 ITGA3 integrin alpha 3 Down -1393 M97934 STAT2 signal transducer and activator of transcription 2 Down -1248 L38518 SHH sonic hedgehog Down -1183 X51521 VIL2 ezrin; villin 2 Down -1177 L07515 CBX5 chromobox homolog 5; heterochromatin protein homolog 1 (HP1) Down -1153 M15400 RB1 retinoblastoma 1 Down -1109 M54995 PPBP pro-platelet basic protein Down -1100 X54199 GART trifunctional purine biosynthetic protein adenosine 3 Down -1092 U47686 STAT5 A +B signal transducer and activator of transcription 5 A+B Down -1072 relation coefficient based on the adjusted intensity of all Methods MCF-7 cells were obtained from American Type Culture genes spotted on the membrane. Collection (ATCC) and were propagated in vitro as rec- ommended by the provider and as described in previous Hybridizations with 30 μg of total RNA were performed publications (1, 5). To determine whether the viral pro- according to the manufacturer instructions. The hybrid- tein was expressed in our MCF-7 cells, western blotting ized membranes were exposed onto a phosphorimager was used. Protein lysates were prepared from approxi- screen and were read using a phosphorimager reader mately 1 × 10 cells. Equal amounts of protein from each (Molecular Dynamics). The scanned images were aligned sample were loaded onto an SDS-PAGE-10% polyacryla- and analyzed using AtlasImage 2.01 software (Clontech). mide gel, followed by transfer to PVDF membranes. West- When averaging or comparing samples, the adjusted ern blot analysis was performed using mAbP2 (a intensity signal was normalized using the global normali- monoclonal antibody against a peptide of the Env pro- zation mode featured in the software. We reported only tein), and mAb-tubulin as primary antibodies (Sigma those genes whose ratios of differential expression were Aldrich). Proteins were visualized using horseradish per- 2.5-fold or more, or genes that were undefined for one oxidase-labeled sheep anti-mouse IgG (GE Healthcare type of sample, but were detected on the other. (Unde- Bio-Sciences Corp.) as a secondary antibody followed by fined genes are those whose intensity were below the sig- enhanced chemiluminescence (GE Healthcare Bio-Sci- nal threshold) In the later event, when we lack a ences Corp.). numerical value for the ratio, it was defined as being "up" or "down". Furthermore, for each gene we stated the dif- The expression profile was studied using the Atlas Human ference (diff) in adjusted normalized intensity between Cancer 1.2 cDNA expression array; a nylon membrane the two cell lines. printed with 200–600 bp long fragments of 1176 charac- terized genes involved in cancer, 9 housekeeping genes Accession number (Acc#), gene symbol and protein or and 6 negative controls (Clontech, CA). These conditions gene name are according to GeneBank. were described in detail in a previous publication [25]. Briefly, RNA was extracted and labeled with Atlas pure Acknowledgements Grant support: The T.J. Martell Foundation for Leukemia, Cancer and AIDS total RNA labeling system and hybridized to an Atlas Research, The Jane Grinberg Memorial Fund, the Kash Family Foundation, Human Cancer 1.2 cDNA expression array (Clontech, CA) and the Ellen Block Memorial Fund. according to the manufacturer's instructions. Both cell sublines were probed twice in separate assays, and the We thank Cindy Hernandez for skillful technical assistance and Jennifer accuracy of each duplicate was assessed by Pearson's cor- Hasa and Julia Roboz for editorial work. Page 4 of 5 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:7 http://www.infectagentscancer.com/content/1/1/7 human breast carcinoma correlate with overexpression of References laminin receptor. Cl Cancer Res 1999, 5:2108-2111. 1. Wang Y, Holland JF, Bleiweiss IK, Melana SM, Xu D, Pogo BGT: 24. Wang Y, Melana SM, Baker B, Bleiweiss I, Fernandez-Cobo M, Mandeli Detection of mammary tumor virus env gene-like sequences J, Holland JF, Pogo BGT: Presence of MMTV-like env gene in human breast cancer. Cancer Res 1995, 55:5173-5179. sequences in gestational breast cancer. Med Oncol 2003, 2. Etkind P, Du J, Khan A, Pillitteri J, Wiernik P: Mouse mammary 20:233-236. tumor virus-like sequences in human breast tumors and in a 25. Fernandez-Cobo M, Holland JF, Pogo BGT: Transcription profile lymphoma of a breast cancer patient. Cl Cancer Res 2000, of non-immortalized breast cancer cell lines. BMC Cancer 6:1273-1278. 2006, 6(1):99. 3. Ford CE, Tran D, Deng YM, Ta VT, Rawlinson WD, Lawson JS: Mouse Mammary Tumor Virus-like gene sequences in breast tumors of Australian and Vietnamese women. Clin Cancer Res 2003, 9:1118-1120. 4. Zammarchi F, Pistello M, Piersiguilli X, Murr R, Di Cristofano C, Nac- carato AG, Bevilacqua G: MMTV-like sequences in human breast cancer: a fluorescent PCR/laser microdissection approach. J Pathol 2006, 209:436-444. 5. Wang Y, Go V, Holland JF, Melana SM, Pogo BGT: Expression of MMTV-like env gene sequences in human breast cancer. Cl Cancer Res 1998, 4:2565-2568. 6. Melana SM, Holland JF, Pogo BGT: Search for MMTV-like env sequences in cancer and normal breast from the same indi- vidual. Cl Cancer Res 2001, 7:283-284. 7. Ford CE, Faedo M, Rawlinson WD: MMTV virus-like RNA tran- scripts and DNA found in affected cells of human breast can- cer. Cl Cancer Res 2004, 10:7284-7289. 8. Liu B, Wang Y, Melana SM, Pelisson I, Najfeld V, Holland JF, Pogo BGT: Identification of a proviral structure in human breast cancer. Cancer Res 2001, 61:1754-1759. 9. Wang Y, Jiang JD, Xu D, Li Y, Holland JF, Qu C, Pogo BGT: A MMTV-like LTR superantigen in human breast cancer. Can- cer Res 2004, 64:4105-4111. 10. Melana SM, Nepomnaschy I, Dales S, Rojowsky H, Abbott A, Jiang JD, Holland JF, Pogo BGT: A retrovirus in human berast cancer: Isolation and characterization of human mammary tumor virus [abstract]. Proc AACR 2006, 47:1043. 11. Coussins LM, Werb Z: Inflammation and cancer. Nature 2002, 20:860-867. 12. Einav U, Tabach Y, Getz G, Ytzhaky A, Ozbek U, Amariglio A, Israelí S, Rechavi G, Domany E: Gene expression analysis reveals a strong signature of an interferon-induced pathway in child- hood lymphoblastic leukemia as well as in breast and ovarian cancer. Oncogene 2005, 24:6367-6375. 13. McGrath CM, Grant PM, Soule HD, Glancy T, Rich MA: Replication of oncornavirus-like particle in human breast carcinoma cell line, MCF-7. Nature 1974, 252:247-250. 14. May EB, Westley BC: Characterization of sequences related to the mouse mammary tumor virus that are specific to MCF- 7 breast cancer cells. Cancer Res 1989, 49:3879-3883. 15. Bahia H, Ashman JNE, Cawkell L, Lind M, Monson JRT: Karyotypic variation between independently cultured strains of the cell line MCF-7 identified by multicolour fluorescence in situ hybridization. Int J Oncol 2002, 20:489-494. 16. Osborne CK, Hobbs K, Trent JM: Biological differences among MCF-7 human breast cancer 968 cell lines from different lab- oratories. Breast Cancer Res Treat 1987, 9:111-121. 17. Nugoli M, Chuchana P, Vendrell J, Orsetti B, Ursule L: Genetic var- iability in MCF-7 sublines: evidence of rapid genomic and RNA expression profile modifications. BMC Cancer 2003, 3:13. 18. Hiorns LR, Bradshaw TD, Skelton LA, Yu Q, Kelland LR, Leyland- Jones B: Variation in RNA expression and genomic DNA con- Publish with Bio Med Central and every tent acquired during cell culture. Brit J Cancer 2004, 90:476-482. 19. Wang Y, Pelisson I, Melana SM, Go V, Holland JF, Pogo BGT: MMTV- scientist can read your work free of charge like env gene sequences in human breast cancer. Arch Virol "BioMed Central will be the most significant development for 2001, 145:1-10. 20. Wang Y, Pelisson I, Melana SM, Holland JF, Pogo BGT: Detection of disseminating the results of biomedical researc h in our lifetime." MMTV-like LTR and LTR-env gene sequences in human Sir Paul Nurse, Cancer Research UK breast cancer. Int J Oncol 2001, 18:1041-1044. 21. Zangen R, Harden S, Cohen D, Parrella P, Sidransky D: Mouse Your research papers will be: mammary tumor-like env gene as a molecular marker for available free of charge to the entire biomedical community breast cancer. Int J Cancer 2002, 102:304-307. peer reviewed and published immediately upon acceptance 22. Soule HD, Vasquez J, Long A, Albert S, Brennan M: A human cell line from a pleural effusion derived from a breast carcinoma. cited in PubMed and archived on PubMed Central J Nat Cancer Inst 1973, 51:1409-1416. yours — you keep the copyright 23. Pogo BGT, Melana SM, Holland JF, Mandeli JF, Piloti S, Casalini P, Ménard S: Sequences homologous to the MMTV env gene in BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 5 of 5 (page number not for citation purposes)

Journal

Infectious Agents and Cancer – Springer Journals

Published: Dec 15, 2006

There are no references for this article.