Latexin: A Potential Tumor Suppressor regulation of Lxn expression by 5-aza-29-deoxycytidine. Lxn mRNA was measured by quantitative real-time PCR in leukemic and lymphoma cell lines exposed with or without 2 uM 5-aza-29-deoxycytidine for 4 days. Expression levels were normalized to endogenous control, Gapdh. Results shown are mean6 SD of 12 replicates from 3 independent experiments. doi:10.1371/journal.pone.0044979.g002 which is consistent with PCR array result. Thus our data indicate that Lxn-induced apoptosis in A20 cells is mainly through the down-regulation of anti-apoptotic regulators, such as Bcl-2 and Pim-2. A well-known function of Lxn is its role as the sole carboxypep- tidase A (CPA) inhibitor in mammalian cells. There are six Cpa genes (1–6) in this family. In order to test whether or not the suppressive effect of ectopic Lxn expression on the growth of A20 and WEHI231 cells was due to its canonical inhibitory activity, we carried out the following experiments. We first determined which Cpa genes are enriched in hematopoietic stem/progenitor cells. By searching our gene expression profile of hematopoietic stem/ progenitor cells (unpublished data), we found that Cpa3 is the only gene highly expressed in hematopoietic cells (Table S1). Next, we tested Cpa3 expression in A20 cells that were uninfected, or infected with empty or Lxn expression vectors, and found that it was not expressed in any type of these cells (Figure S1). Last, we treated A20 and WEHI cells with potato carboxypeptidase A inhibitor (PCPI), a 39 amino acid protein which strongly inhibits mammalian CPA, at a series of concentrations ranging from 5 to 60 ug/ml of culture medium. As seen in Fig. 4C, none of the concentrations had any effect on the growth patterns of the tumor lines, despite the continuous presence of PCPI for 12 days of culture. To rule out the possibility that PCPI failed to inhibit tumor growth because it did not enter the cells, we used fluorescein isothyiocyanate (FITC) to label PCPI in cells. Fig. 4D shows A20 cells following a 30 min incubation with labeled PCPI (30 ug/ml of culture medium). The three-color micrograph shows PCPI in green (FITC), the nucleus in blue (DAPI), and the B220 lymphoid cell surface marker in red (phycoerythrin, PE). It is apparent that PCPI is plentiful in the cytosol but is not found in the nucleus. The doses of PCPI chosen for the above experiments were taken directly from a study in which the inhibitor was shown to inhibit the growth of pancreatic adenocarcinoma cells by directly interfering with the epidermal growth factor signaling pathway [29]. Maximal growth inhibition was achieved at 30–50 ug/ml. Thus, the mechanisms by which Lxn regulates both stem cell population size and lymphoma growth inhibition reside in a novel pathway involving apoptosis. Down-regulation of Lxn in Primary Leukemia and Lymphoma Cells Based on the strong evidence for the tumor suppressive function of Lxn in leukemia and lymphoma cell lines, we asked whether or not Lxn expression is altered in primary cells derived from patients with malignancies. We isolated stem/progenitor-enriched CD34+ cells from bone marrow and blood cells in lymphoma and leukemia patients, as well as in normal donors. The patients included those with acute myeloid leukemia (AML), T cell pro- lymphocytic leukemia (T-PLL), plasma cell leukemia (PCL), acute T cell lymphoma (ATLL) and acute lymphoid leukemia (ALL, preB phenotype). The normal CD34+ cell samples were derived from cord blood (CB) and young (31 and 35 years) and old (85 and 97 years) adults. Lxn mRNA expression, quantified by real-time PCR, was decreased by at least two-thirds in primary malignant CD34+ cells (Fig. 5A). Quantification of LXN in CD34+ cells of all human normal and leukemic samples compiled to date is plotted in Fig. 5B. The result shows a significant decrease of LXN protein expression in malignant cells (P = 0.03) even though Lxn expression at the protein level is more variable than that at the mRNA level. Two additional old samples in the normal control group contribute to this variation, which is consistent with the report that the number and functionality of HSCs in old individuals, perhaps stem cell regulatory gene(s), showed more dramatic variations as compared to their young counterparts [30]. In summary, these data indicate that Lxn dysregulation may be involved in human hematological malignancies. In the future study, it will be interesting to examine the expression of two anti- apoptotic genes, Bcl-2 and Pim-2, in patient samples to see whether or not they are up-regulated. Discussion Recently we found that Lxn is a natural regulator of hematopoietic stem cell population in mice by influencing self- renewal and apoptosis [4]. Lxn expression in stem cells is inversely proportional to the size of the population in mice and thus acts as a negative regulator through cell-intrinsic mechanisms. The obser- vation that low Lxn expression in hematopoietic cells was associated with increased replication led us to hypothesize that Lxn was down-regulated in malignancies with high proliferative rates and/or low apoptosis. Here, we show that Lxn is either not expressed or is strongly down-regulated in a variety of leukemias and lymphomas. As a result of these findings and close linkage structurally and genetically with TIG1, we proposed that Lxn may similarly act as a tumor suppressor. Along with this proposal, there is expectation that manipulation of Lxn levels in malignant cells would alter their growth characteristics. Accordingly, two lymphoma cell lines in which Lxn expression was absent were infected with a Lxn expression vector. In one of the most significant findings of this study, we found that re-initiation of Lxn expression dramatically reduced their growth in vitro and in vivo. These results demonstrate that the level of Lxn expression is functionally related to both normal and malignant cell growth and, if the findings are true for human tumors, may provide an avenue for therapeutic intervention. Surprisingly, Lxn knock-out mice did not develop- ment leukemia nor lymphoma even though their hematopoietic stem cell pool was over-expanded and blast cells were detected in the peripheral blood [19]. These results suggest that Lxn may influence the crucial, step in carcinogenesis [31]. It thus will be of great interest to introduce additional carcinogenetic ‘‘hits’’, such as oncogenes or alkylating agents, to Lxn knockout stem cells to determine whether Lxn deletion will accelerate tumor develop- ment. In addition, we observed that re-initiation of Lxn expression did not completely suppress tumor cell growth, suggesting that it would at best be a part of a multi-drug regimen. We investigated the possible mechanisms by which ectopic Lxn expression may suppress growth of lymphoma cells and found increased apoptosis to be the major mechanism causing inhibition. Consistent with our study of normal hematopoietic cells, our recent findings indicate that high Lxn content is associated with more apoptotic cells, thus fewer stem cells [4]. Apoptosis is controlled by the balance of both positive (pro-apoptotic) and negative (anti-apoptotic) signals. We found that Lxn-induced apoptosis is mainly through the down-regulation of anti-apoptotic genes, such as Bcl-2 and Pim-2. Decreased level of Bcl-2 allows other pro-apoptotic proteins such as BAX and BAK to aggregate, PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e44979