themes in carcinogenesis. [21] Both often occur through aberrant DNA methylation that is accentuated during aging. [22,23,24,25,26,27] A CpG-enriched region was identified in Lxn promoter and its hypermethylation was found in a variety of human gastric carcinoma cell lines. [18] Promoter hypermethyla- tion of TIG1 was also associated with its silencing in tumor cells. [13,16] However, it is not known whether Lxn expression is regulated by promoter methylation in hematopoietic malignancy. Molecular mechanisms underlying the role of Lxn in regulating hematopoiesis has not been determined yet. Lxn protein binds to carboxypeptidase A (Cpa) and inhibits its enzymatic activity, indicating that Lxn regulates protein degradation and metabolism. [19] Lxn was highly expressed in mast cells and its expression was further enhanced by LPS, indicating its potential function in inflammation. [11] Proteomic analysis on Lxn knock-out hemato- poietic cells revealed that Lxn deletion reduced the abundance of multiple cellular proteins, especially those involving cell-stroma interaction, such as N-cadherin, Tie2, and Roundabout 4. [19] Ectopic expression of Lxn in gastric cancer cell lines led to the differential expressions of several cancer-related genes, including Maspin, WFDC1, SLPI, S100P, and PDGEFB [18], although none of them are overlapped with previously mentioned proteomic results. A recent study revealed ATP/GTP binding protein-like 2 (AGBL2) as a novel binding partner of TIG1. The interaction between TIG1 and AGBL2 regulates the microtubule tyrosination cycle, which is implicated in tumorigenesis, stem cell differentia- tion and development. [28] Lxn thus may have the similar mechanisms as TIG1 to regulate hematopoiesis. Here, we provide evidence obtained from a variety of lymphoma and leukemia cell lines, as well as from primary cells from patients with these diseases, that Lxn expression is almost universally absent or significantly reduced from that of normal stem and progenitor cells. Moreover, treatment with a de- methylating agent at least partially restores Lxn expression in a variety of tumor cell lines, and methylation level of the CpG island in the Lxn promoter region is inversely associated with its expression. Perhaps most importantly, we show that when Lxn expression was re-initiated ectopically in two lymphoma cell lines using a retroviral expression vector, their growth, both in vitro and in vivo, was significantly blunted. We further show that Lxn inhibits lymphoma cell growth by significantly increasing apoptosis through the down-regulation of anti-apoptotic genes and that its anti-tumor activity is mediated via mechanisms unique from its canonical inhibition of carboxypeptidase A. These results demon- strate that Lxn plays a functional role in tumor cell growth and introduces an unexplored pathway potentially important to cancer treatment in patients. Results Loss of Lxn Expression in Malignant Cells We first determined Lxn mRNA abundance in tumor cell lines by quantitative real-time PCR (Fig. 1A). Compared with normal primitive hematopoietic cells, Lxn mRNA expression was com- pletely absent in a majority of tested leukemic lines, including K562, Molt4, CRF-CEM, J45.01, Jurkat and U937, and was significantly diminished in HL-60, KG-1 and SupB15 cell lines. LXN protein expression was also tested in all samples using Western blotting and nearly identical results were obtained in tested cell lines (Fig. 1B). Our results are consistent with those previously reported [19] and include additional leukemia and lymphoma cell lines. Latexin: A Potential Tumor Suppressor Aberrant Promoter Hypermethylation of Lxn in Hematopoietic Malignancy To assess whether the loss or decrease of Lxn expression in malignant cell lines resulted from promoter hypermethylation, methylation of the 59 CpG island of Lxn gene surrounding its transcriptional start site was determined by genomic bisulfite sequencing. The CpG island spans from within the canonical 59 promoter (2208 nt) to the transcription start site (+1 nt), and extends through the entire first exon (+44 nt). There are 15 CpG dinucleotides within this region (Fig. 2A). The CpG island identified here is not exactly same as the one reported previously, this may be due to the different criteria we used for defining this region. Figure 2B depicts the quantitative variation in methylation for each CpG site among nine tumor cell lines. Almost complete methylation was seen in J45.01, U937, Jurkat, Molt4 and CCRF- CEM (.90%) lines, which commensurately showed an absence of Lxn expression. Scatteredly methylated CpG sites were found in K562, KG-1 and SupB15 lines, which were linked to the weak expression of Lxn. Surprisingly, although HL-60 had very low Lxn expression, we found that none of the CpG sites were methylated. This is probably due to other epigenetic mechanisms, such as histone deacetylation of latexin promoter in HL 60 cells. Reactivation of Lxn Expression with Demethylating Reagent Treatment To test the hypothesis that Lxn promoter hypermethylation might be involved in the loss of expression in leukemia and lymphoma cell lines, we studied the effect of 5-aza-29-deoxycyti- dine, a DNA demethylating reagent, on Lxn expression (Fig. 2C). After treatment with 2 uM 5-aza-29-deoxycytidine for 4 days, Lxn gene expression was reactivated in cell lines completely lacking Lxn expression prior to treatment (K562, Molt4, CCRF-CEM, J45.01, Jurkat and U937), and was significantly up-regulated in HL-60, KG-1 and SupB15 lines. The up-regulation of Lxn by 5-aza-29- deoxycytidine in HL60 cells, which shows no hypermethylation of CpG sties (Fig. 2B), might be because the demethylation of other DNA sequences induces chromatin remodeling, thus exposing Lxn promoter for the transcriptional initiation. Suppression of Growth of Mouse Lymphoma Cell Lines in vitro and in vivo Following Ectopic Lxn Expression To determine a correlative or causative relationship between Lxn expression and tumor development, we next asked whether or not the re-initiation of Lxn expression affected the growth rate of malignant cells in vitro and in vivo. To that end, we ectopically expressed Lxn, using a retroviral expression vector containing green fluorescent protein (GFP) marker, in the mouse BALB/c- derived A20 B lymphoma cell line, which lacks Lxn expression (Fig. 3C). The controls are A20 cells either un-infected or infected with empty vector. The GFP positive A20 cells were purified by flow cytometric cell sorting, and their growth was determined in vitro and in vivo. Fig. 3 shows the potent effects of Lxn expression on inhibiting tumor growth in vitro and in vivo. Fig. 3A shows that cultures of A20 cells infected with the Lxn expression vector contained only about half the number of cells at day 3 as compared to A20 cells infected with the control (empty) vector or uninfected control cells (A20 control). The growth suppression by Lxn overexpression was exponentially amplified during subsequent days of culture and by day 20 nearly 16 fold less tumor cells were present in Lxn- overexpressing group than in control cultures (right panel). Fig. 3B shows that the fraction of GFP+ cells remained at 90–100% throughout the 20 days of culture in both the Lxn vector- or PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44979