Absence of Annexin I Expression in B-cell Non-Hodgkin's Lymphomas and Cell Lines

US Oncology Review, 2005;1(1):1-6

Abstract:

Background
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 its functional diversity on the family.

Annexin I has been implicated to have a biological role in inhibition of phospholipase A2,1 as a substrate for epidermal growth factor receptor2 and intracellular calcium release,3 regulation of hepatocyte growth factor receptor signaling,4 and membrane trafficking.5 Substantial evidence suggests a role for annexin I in glucocorticoid-induced immunosuppression6,7 and MAPK/ERK pathway.7,8

Citation US Oncology Review, 2005;1(1):1-6

Increased expression of intracellular annexin I is seen in bronchial epithelial cells grown in the presence of dexamethasone,9 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 over-expression of annexin I has been observed in pancreatic,16 breast, and gastric cancers,17 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 OMABL- 1.22 While annexin II is closely related to annexin I in amino acid identity, its cellular function is clearly different.9 Both annexins I and II are upregulated in pancreatic carcinoma,16 and recent reports have shown absence of both annexins I and II in prostate carcinoma.20 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.

Methods 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), and Burkitt s lymphoma cell lines (Raji, Ramos, OMA-BL-1, Namalwa). TK-6 is a lymphoblast cell line that is heterozygous 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 shortterm from a patient with acute lymphoblastic leukemia (ALL) at the University of Nebraska Medical Center (please note that a DNA fingerprint analysis25 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.26 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, NY) supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine (2mM), penicillin (100U/ml) and streptomycin (100µg/ml). All cell lines were determined to be mycoplasma-free by a polymerase chain reaction (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: 3µM or 6µM deoxyC for three days or six days; 2.5 to 10µg/ml lipopolysaccharide from E. coli (Sigma) for 24 hours; or 100µM H2O2 for two to 24 hours. Genomic DNA, total cellular ribonucleic acid (RNA), and protein were extracted from cells using previously published procedures.22,27 Absence of Annexin I Expression in B-cell Non-Hodgkin s Lymphomas and Cell Lines