- Research article
- Open Access
- Open Peer Review
Absence of annexin I expression in B-cell non-Hodgkin's lymphomas and cell lines
© Vishwanatha et al; licensee BioMed Central Ltd. 2004
- Received: 13 October 2003
- Accepted: 08 March 2004
- Published: 08 March 2004
Annexin I, one of the 20 members of the annexin family of calcium and phospholipid-binding proteins, has been implicated in diverse biological processes including signal transduction, mediation of apoptosis and immunosuppression. Previous studies have shown increased annexin I expression in pancreatic and breast cancers, while it is absent in prostate and esophageal cancers.
Data presented here show that annexin I mRNA and protein are undetectable in 10 out of 12 B-cell lymphoma cell lines examined. Southern blot analysis indicates that the annexin I gene is intact in B-cell lymphoma cell lines. Aberrant methylation was examined as a cause for lack of annexin I expression by treating cells 5-Aza-2-deoxycytidine. Reexpression of annexin I was observed after prolonged treatment with the demethylating agent indicating methylation may be one of the mechanisms of annexin I silencing. Treatment of Raji and OMA-BL-1 cells with lipopolysaccharide, an inflammation inducer, and with hydrogen peroxide, a promoter of oxidative stress, also failed to induce annexin I expression. Annexin I expression was examined in primary lymphoma tissues by immunohistochemistry and presence of annexin I in a subset of normal B-cells and absence of annexin I expression in the lymphoma tissues were observed. These results show that annexin I is expressed in normal B-cells, and its expression is lost in all primary B-cell lymphomas and 10 of 12 B-cell lymphoma cell lines.
Our results suggest that, similar to prostate and esophageal cancers, annexin I may be an endogenous suppressor of cancer development, and loss of annexin I may contribute to B-cell lymphoma development.
- Annexin I
- oxidative stress
- gene expression
The Annexins comprise a family of 20 calcium- and phospholipid-binding proteins. Expressed in organisms ranging from molds and plants to mammals, this family of proteins has proven evolutionarily conserved as well as functionally diverse. Structurally, annexins consist of a 70 amino acid core domain and an N-terminal domain, which is variable in both length and sequence, and imparts upon the family its functional diversity. Annexin I has been implicated to have a biological role in inhibition of phospholipase A2 , as a substrate for epidermal growth factor receptor  and intracellular calcium release , regulation of hepatocyte growth factor receptor signaling , and membrane trafficking . Substantial evidence suggests a role for annexin I in glucocorticoid-induced immunosuppression [6, 7] and MAPK/ERK pathway [7, 8]. Increased expression of intracellular annexin I is seen in bronchial epithelial cells grown in the presence of dexamethasone  and secreted annexin I appears to be proteolytically degraded by the human neutrophil elastase to an inactive form [10, 11].
Annexin I is a critical mediator of apoptosis [12–15]. While overexpression of annexin I has been observed in pancreatic , breast and gastric cancers , reduced or no expression of annexin I has been reported in prostate and esophageal cancers [18–21]. Thus differential regulation of annexin I in a tissue specific manner may be associated with the development of cancers in these sites.
Absence of annexin II expression has been reported in two B-cell lymphoma cell lines, Raji and OMA BL-1 . While annexin II is closely related to annexin I in amino acid identity, its cellular function is clearly different . Both annexins I and II are upregulated in pancreatic carcinoma , and recent reports have shown absence of both annexins I and II in prostate carcinoma [20, 21, 23, 24]. Thus, it appears that both annexins I and II may be coordinately regulated. In view of these observations, the expression of annexin I in human B-cell lymphomas and cell lines was investigated in this study.
Cell culture, drug treatment and reagents
The human B-cell lymphoma cell lines used in this study are: progenitor B-cell lines (Nalm-6, REH, HPB-Null, PBE-1), B-lymphoblast cell lines (WI-L2, TK-6, DW-10, DHL-16), Burkitt's lymphoma cell lines (Raji, Ramos, OMA-BL-1, Namalwa). TK-6 is a lymphoblast cell line that is heterozygote for thymidine kinase. TK-6 is a derivative of the WI-L2, a lymphoblast cell line. DW-10 and WI-L2 are EBV transformed mature B-cell lines. PBE-1 and NALM-6 are both precursor B cell acute lymphoblastic leukemia cell lines. NALM-6 is an established cell line and PBE1 is a line established short term from a patient with ALL at the University of Nebraska Medical Center [Please note that a DNA fingerprint analysis  of over 500 lymphoma-leukemia cell lines indicated that PBE-1 and NALM6 may be identical]. DHL-16 is a follicular B-cell lymphoma cell line . Human adenoids were used as a source of normal B-cells, and contained >80% B-cells as determined by cell sorting and flow cytometric analysis. In other experiments, normal B-cells were isolated from PBL of a healthy volunteer using the human B-cell isolation kit (Miltenyi Biotec Inc., Auburn, CA) as per manufacturer's guidelines. SW1116, HeLa and 293T cells were used as positive controls in the indicated experiments. Cells were grown in a growth medium consisting of Eagle's minimum essential medium (GIBCO-BRL, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 μg/ml). All cell lines were determined to be mycoplasma-free by a PCR-based mycoplasma detection assay. 5-aza-2'-deoxycytidine (deoxyC, Sigma Chemical Co., St. Louis, MO) was freshly prepared in distilled water. Raji and OMA BL-1 cells, growing in T25 flasks, were incubated in one of the following: 3 μM or 6 μM deoxyC for 3 days or 6 days; 2.5 to 10 μg/mL lipopolysaccharide from E. coli (Sigma) for 24 hours; or 100 μM H2O2 for 2 to 24 hours. Genomic DNA, total cellular RNA and protein were extracted from cells using previously published procedures [22, 27].
Formalin-fixed, paraffin-embedded tissues representing normal tonsil (n = 2), diffuse large cell non-cleaved B-cell lymphoma (n = 2), small lymphocytic B-cell lymphoma (n = 2) and follicular mixed B-cell lymphoma (n = 2) were generously made available for these studies by Dr. Dennis Weisenburger, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE. Sequential serial sections prepared from these specimens were used in immunohistochemical analysis.
Polyclonal rabbit anti-human annexin I antisera (α646) was a gift from Dr. Blake Pepinsky (Biogen, Boston, MA.). Polyclonal anti-human phosphoglycerate kinase (PGK) antiserum (α35) was prepared as described previously . The mouse monoclonal anti-CD 20 antibody was from Beckman/Coulter Inc. (Westbrook, MA).
Gel electrophoresis and immunoblotting
Protein extracts were prepared from cells using protein lysis buffer containing; 50 mM Tris-HCl pH 7.5, 2.0 mM PMSF, 5.0 mM iodoacetamide, 5.0 mM EDTA, 150 mM NaCl, 0.5% NP-40, and 0.5% Mega-9. Protease inhibitors were added just prior to use (leupeptin at 1 μg/ml and pepstatin at 2 μg/ml). Total amounts of protein extracts was quantitated using Bio-Rad protein assay (Pierce, Rockford, IL). A total of 50 μg of protein from each extract was separated on a 12% SDS-PAGE. After electrophoresis, proteins were transferred to PVDF membrane (Millipore, Bedford, MA). The membranes were blocked in 1X TTBS with 7% powdered milk overnight at 4°C and then probed for 1 hour with rabbit polyclonal anti-human annexin I (α646) at 1:500 dilution and rabbit polyclonal anti-human PGK (α35) at 1:1000 dilution. Anti-rabbit HRP (Promega, Madison, WI) at a dilution of 1:5000 was used for 1 hour at room temperature to detect antigen-antibody complexes. Membranes were developed using ECL+ (Amersham Pharmecia Biotech, Arlington Heights, IL). For quantitation of the immunoreactive band, the blot was scanned on a laser densitometer (Molecular Dynamics, Sunnyvale, CA).
RNA isolation and polymerase chain reaction analysis
Total RNA was isolated from the cell monolayers or tissue samples using RNeasy mini kit (Qiagen Inc., Valencia, CA) or by TRIzol method (GIBCO/BRL). Annexin I message was characterized by PCR using the enhanced Avian RT-PCR kit (Sigma). Total RNA (5 μg) was added to each RT reaction using random nonamer primers. The cDNA products were amplified using annexin I specific primers to give a 522 bp product. The primers designed for amplification were:
Annexin I forward: 5'-GCAAGAAGGTAGAGATAAAG-3',
Annexin I reverse: 5'-ATCTCTCTTCAGTTCCTCTC-3'
For verification of the integrity of the RNA samples and as a control in all the RT-PCR analyses, we examined the expression of the α-tubulin gene. The primers A-tu1 (5'-AAG AAA TCC AAG CTG GAG TTC-3') and A-tu2 (5'-GTT GGT CTG GAA TTC TGT CAG-3'), specific for the α-tubulin gene, generated a 300 bp PCR product corresponding to the α-tubulin gene.
Southern blot analysis
Genomic DNA was isolated from Raji, HPB-null, Nalm-6 and HeLa cell lines, and subjected to restriction endonuclease digestion. Southern blot analysis  of genomic DNA was performed using radiolabeled 522 bp annexin I cDNA product from the PCR reaction as described above.
Standard methods were used to prepare sequential 8 μM sections of paraffin-embedded tissues, which were mounted on polylysine-coated glass slides. Tissues were cleared and rehydrated with two 10-minute rinses in a ready-to-use tissue deparaffinization solution (Biogenex Inc. San Ramon, CA). Sections were then stained by the immunoperoxidase method using the ready-to-use Vectastain Quick kit (Vector Labs, Burlingame, CA). Primary antibodies used included rabbit polyclonal anti-human annexin I antibody and mouse monoclonal anti-human CD 20 antibody.
Expression of annexin I in human B-cell lymphoma cell lines
Annexin I is transcriptionally regulated in B-cell lymphoma cell lines
Effect of gene demethylation on annexin I expression
Methylation of the CpG dinucleotide has been shown to directly inhibit transcription or stabilize structural changes in chromatin that prevent transcription. The nucleotide analogue, deoxyC was used to inhibit DNA methylation in Raji cells to measure reexpression of annexin I protein. Exposure of Raji cells to either low (1 μM) or high (10 μM) deoxyC did not result in reexpression of annexin I protein after 3 days (lanes 3 and 4). However, after 6 days of treatment with deoxyC, as a weak protein band (lanes 5 and 6) that appeared at the position of annexin I was observed. There was no difference in annexin I reexpression at 1 μM or 10 μM deoxyC concentrations.
Raji cell line response to LPS and H2O2
Immunohistochemical analysis of annexin I expression in B-cell lymphoma
In this study, the expression of annexin I in B-cell lymphoma was examined. Annexin I is a pleotrophic, calcium and phospholipid binding protein whose proposed functions include anti-inflammatory activity, mediation of apoptosis, regulation of cell differentiation, and membrane trafficking [8, 15, 30]. Annexin I is expressed in the secretory bronchial epithelial cells  and its anti-inflammatory N-terminus is lost in the bronchoalveolar lavage fluids from healthy smokers  indicating the importance of this protein in human health and disease. Annexin I is frequently overexpressed in human cancers including pancreatic  and breast  cancers. However, recent reports indicate that annexin I expression is down-regulated in other human tumors, particularly esophageal and prostate tumors [19–21]. Annexin I is similar to the closely related protein annexin II, even though the two proteins are proposed to carry out distinct physiological functions. Curiously, both annexins I and II are lost in prostate cancers [20, 21, 23, 24]. Altered expression and loss of annexin II in B-cell lymphoma cell lines has been reported previously . In view of this, the expression of annexin I in B-cell lymphomas and cell lines were examined in the present study.
The data presented in this manuscript indicate that annexin I is present in normal B-cells (Figure 1, panels A and B). Immunohistochemical examination of normal tonsil sections (Figure 6) indicates that a subset of cells within the germinal centers distinctly express annexin I. The germinal center normally harbors highly proliferative B-cells , however it also represents a highly dynamic environment that creates intense genomic instability among B-cells . It is unclear if the subset of B-cells that show annexin I expression represent proliferative cells or cells that are undergoing differentiation. Association of annexin I expression and cellular differentiation has been shown previously .
In contrast, most cells lines and neoplasms of pregerminal B-cells did not show detectable annexin I expression. Our data are consistent with the loss of annexin I observed in prostate and esophageal cancers . The etiology of reduced annexin I expression was studied by examining the possible mechanisms. Southern blot analysis indicated that annexin I gene was intact in the Raji and OMA-BL1 cells, indicating that genomic deletion of annexin I is not the cause for loss of annexin I. Gene silencing by hypermethylation of annexin I promoter was examined by culturing cells in the presence of a demethylating agent, and the data indicated reexpression of annexin I protein after prolonged exposure to 10 μM concentration of deoxyC. Thus, methylation of annexin I promoter could be one of the mechanisms for annexin I silencing in these cells. These results are similar to the reexpression of annexin II observed in a prostate cancer cell line after treatment with deoxyC , indicating methylation as a general mechanism for silencing annexins I and II in cancer tissues. Treatment of cells with a pro-inflammatory agent or an apoptosis inducing agent did not result in expression of annexin I.
The data presented in this paper show that the anti-inflammatory protein annexin I is expressed in normal B-cells, but not expressed in B-cell lymphomas and cell lines, similar to the absence of annexin I in prostate and esophageal cancers. Thus, annexin I may be an endogenous suppressor of cancer development, and loss of annexin I may contribute to B-cell lymphoma development. The additional mechanism(s) by which annexin I expression is down-regulated and the physiological consequences of annexin I loss in B-cell lymphomas need further investigation.
The author wishes to thank Dr. Dennis Weisenburger for providing tissue specimen, Dr. Sam Pirucello for providing the cell lines, and Dr. Abhijit Banerjee for consultation.
- Wallner BP, Mattaliano RJ, Hession C, Cate RL, Tizard R, Sinclair LK, Foeller C, Chow EP, Browing JL, Ramachandran KL, et al: Cloning and expression of human lipocortin, a phospholipase A2 inhibitor with potential anti-inflammatory activity. Nature. 1986, 320: 77-81.View ArticlePubMedGoogle Scholar
- Pepinsky RB, Sinclair LK: Epidermal growth factor-dependent phosphorylation of lipocortin. Nature. 1986, 321: 81-84.View ArticlePubMedGoogle Scholar
- Frey BM, Reber BF, Vishwanath BS, Escher G, Frey FJ: Annexin I modulates cell functions by controlling intracellular calcium release. FASEB J. 1999, 13: 2235-2245.PubMedGoogle Scholar
- Skouteris GG, Schroder CH: The hepatocyte growth factor receptor kinase-mediated phosphorylation of lipocortin-1 transduces the proliferating signal of the hepatocyte growth factor. J Biol Chem. 1996, 271: 27266-73. 10.1074/jbc.271.44.27266.View ArticlePubMedGoogle Scholar
- Ernst JD, Hoye E, Blackwood RA, Mok TL: Identification of a domain that mediates vesicle aggregation reveals functional diversity of annexin repeats. Journal of Biological Chemistry. 1991, 266: 6670-6673.PubMedGoogle Scholar
- Goulding NJ, Guyre PM: Glucocorticoids, lipocortins and the immune response. Current Opinion in Immunology. 1993, 5: 108-113. 10.1016/0952-7915(93)90089-B.View ArticlePubMedGoogle Scholar
- Croxtall JD, Choudhury Q, Flower RJ: Glucocorticoids act within minutes to inhibit recruitment of signalling factors to activated EGF receptors through a receptor-dependent, transcription-independent mechanism. Br J Pharmacol. 2000, 130: 289-298.View ArticlePubMedPubMed CentralGoogle Scholar
- Alldridge LC, Harris HJ, Plevin R, Hannon R, Bryant CE: The annexin protein lipocortin 1 regulates the MAPK/ERK pathway. J Biol Chem. 1999, 274: 37620-37628. 10.1074/jbc.274.53.37620.View ArticlePubMedGoogle Scholar
- Vishwanatha JK, Muns G, Beckmann JD, Davis RG, Rubinstein I: Differential expression of annexins I and II in bovine bronchial epithelial cells. American Journal of Respiratory Cell and Molecular Biology. 1995, 12: 280-286.View ArticlePubMedGoogle Scholar
- Smith SF, Tetley TD, Guz A, Flower RJ: Detection of lipocortin 1 in human lung lavage fluid: lipocortin degradation as a possible proteolytic mechanism in the control of inflammatory mediators and inflammation. Environ Health Perspect. 1990, 85: 135-144.View ArticlePubMedPubMed CentralGoogle Scholar
- Vishwanatha JK, Davis RG, Rubinstein I, Floreani A: Annexin I degradation in bronchoalveolar lavage fluids from healthy smokers: a possible mechanism of inflammation. Clin Cancer Res. 1998, 4: 2559-2564.PubMedGoogle Scholar
- McKanna JA: Lipocortin 1 in apoptosis: Mammary regression. Anatomical Record. 1995, 242: 1-10.View ArticlePubMedGoogle Scholar
- Sakamoto T, Repasky WT, Uchida K, Hirata A, Hirata F: Modulation of cell death pathways to apoptosis and necrosis of H2O2-treated rat thymocytes by lipocortin I. Biochem Biophys Res Commun. 1996, 220: 643-647. 10.1006/bbrc.1996.0457.View ArticlePubMedGoogle Scholar
- Solito E, de Coupade C, Canaider S, Goulding NJ, Perretti M: Transfection of annexin 1 in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase-3 activation. Br J Pharmacol. 2001, 133: 217-228.View ArticlePubMedPubMed CentralGoogle Scholar
- Rothwell NJ, Flower R: Lipocortin-1 exhibits novel actions, providing clinical opportunities. Trends in Pharmacological Sciences. 1992, 13: 45-46. 10.1016/0165-6147(92)90019-3.View ArticlePubMedGoogle Scholar
- Kumble KD, Hirota M, Pour PM, Vishwanatha JK: Enhanced levels of annexins in pancreatic carcinoma cells of Syrian hamsters and their intrapancreatic allografts. Cancer Research. 1992, 52: 163-167.PubMedGoogle Scholar
- Ahn SH, Sawada H, Ro JY, Nicolson GL: Differential expression of annexin I in human mammary ductal epithelial cells in normal and benign and malignant breast tissues. Clin Exp Metastasis. 1997, 15: 151-156. 10.1023/A:1018452810915.View ArticlePubMedGoogle Scholar
- Cole KA, Krizman DB, Emmert-Buck MR: The genetics of cancer--a 3D model. Nat Genet. 1999, 21: 38-41. 10.1038/4466.View ArticlePubMedGoogle Scholar
- Zhi H, Zhang J, Hu G, Lu J, Wang X, Zhou C, Wu M, Liu Z: The deregulation of arachidonic acid metabolism-related genes in human esophageal squamous cell carcinoma. Int J Cancer. 2003, 106: 327-333. 10.1002/ijc.11225.View ArticlePubMedGoogle Scholar
- Kang JS, Calvo BF, Maygarden SJ, Caskey LS, Mohler JL, Ornstein DK: Dysregulation of annexin I protein expression in high-grade prostatic intraepithelial neoplasia and prostate cancer. Clin Cancer Res. 2002, 8: 117-123.PubMedGoogle Scholar
- Paweletz CP, Ornstein DK, Roth MJ, Bichsel VE, Gillespie JW, Calvert VS, Vocke CD, Hewitt SM, Duray PH, Herring J, Wang QH, Hu N, Linehan WM, Taylor PR, Liotta LA, Emmert-Buck MR, Petricoin EF,III: Loss of annexin 1 correlates with early onset of tumorigenesis in esophageal and prostate carcinoma. Cancer Res. 2000, 60: 6293-6297.PubMedGoogle Scholar
- Chiang Y, Davis RG, Vishwanatha JK: Altered expression of annexin II in human B-cell lymphoma cell lines. Biochim Biophys Acta. 1996, 1313: 295-301. 10.1016/0167-4889(96)00103-6.View ArticlePubMedGoogle Scholar
- Chetcuti A, Margan SH, Russell P, Mann S, Millar DS, Clark SJ, Rogers J, Handelsman DJ, Dong Q: Loss of annexin ii heavy and light chains in prostate cancer and its precursors. Cancer Res. 2001, 61: 6331-6334.PubMedGoogle Scholar
- Vaarala MH, Porvari K, Kyllonen A, Vihko P: Differentially expressed genes in two LNCaP prostate cancer cell lines reflecting changes during prostate cancer progression. Lab Invest. 2000, 80: 1259-1268.View ArticlePubMedGoogle Scholar
- Drexler HG, Dirks WG, Matsuo Y, MacLeod RA: False leukemia-lymphoma cell lines: an update on over 500 cell lines. Leukemia. 2003, 17: 416-426. 10.1038/sj.leu.2402799.View ArticlePubMedGoogle Scholar
- Pan Z, Shen Y, Du C, Zhou G, Rosenwald A, Staudt LM, Greiner TC, McKeithan TW, Chan WC: Two newly characterized germinal center B-cell-associated genes, GCET1 and GCET2, have differential expression in normal and neoplastic B cells. Am J Pathol. 2003, 163: 135-144.View ArticlePubMedPubMed CentralGoogle Scholar
- Chiang Y, Rizzino A, Sibenaller ZA, Wold MS, Vishwanatha JK: Specific down-regulation of annexin II expression in human cells interferes with cell proliferation. Mol Cell Biochem. 1999, 199: 139-147. 10.1023/A:1006942128672.View ArticlePubMedGoogle Scholar
- Kumble KD, Vishwanatha JK: Immunoelectron microscopic analysis of the intracellular distribution of primer recognition proteins, annexin 2 and phosphoglycerate kinase, in normal and transformed cells. Journal of Cell Science. 1991, 99: 751-758.PubMedGoogle Scholar
- Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975, 98: 503-517.View ArticlePubMedGoogle Scholar
- Seemann J, Weber K, Osborn M, Parton RG, Gerke V: The association of annexin I with early endosomes is regulated by Ca2+ and requires an intact N-terminal domain. Mol Biol Cell. 1996, 7: 1359-74.View ArticlePubMedPubMed CentralGoogle Scholar
- Kuppers R, Zhao M, Hansmann ML, Rajewsky K: Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections. EMBO J. 1993, 12: 4955-4967.PubMedPubMed CentralGoogle Scholar
- Pasqualucci L, Migliazza A, Fracchiolla N, William C, Neri A, Baldini L, Chaganti RS, Klein U, Kuppers R, Rajewsky K, Dalla-Favera R: BCL-6 mutations in normal germinal center B cells: evidence of somatic hypermutation acting outside Ig loci. Proc Natl Acad Sci U S A. 1998, 95: 11816-11821. 10.1073/pnas.95.20.11816.View ArticlePubMedPubMed CentralGoogle Scholar
- Solito E, de Coupade C, Parente L, Flower RJ, Russo-Marie F: Human annexin 1 is highly expressed during the differentiation of the epithelial cell line A 549: involvement of nuclear factor interleukin 6 in phorbol ester induction of annexin 1. Cell Growth Differ. 1998, 9: 327-336.PubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://0-www.biomedcentral.com.brum.beds.ac.uk/1471-2407/4/8/prepub
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