Latexin: A Potential Tumor Suppressor Figure 1. Decrease or loss of Lxn expression in leukemic and lymphoma cell lines. (A) Lxn mRNA expression in leukemia cell lines. Lxn mRNA level was measured by quantitative real-time PCR and shown as mean (61 SD) (n = 12). Control is normal bone marrow (BM). (B) Lxn protein expression in leukemic cell lines measured by western blot. The blot (bottom) and their quantification (top) profiles demonstrate the absent or weak expression of LXN protein in leukemic and lymphoma cell lines. Actin was used as the internal control. doi:10.1371/journal.pone.0044979.g001 control vector-infected cells. Fig. 3C shows that at day 20 of culture neither uninfected A20 cells nor A20 cells infected with the control (empty) vector expressed detectable Lxn protein, whereas in the Western blot a strong Lxn band was evident in lysate of cells infected with the Lxn vector (upper band). LXN protein level at day 0 of culture in these cells is nearly identical to that at day 20 (data not shown). In a separate series of experiments, we have found a similar growth reduction in vitro when Lxn was ectopically expressed in WEHI231 lymphoma cells (data not shown). Fig. 3D shows a compilation of two independent experiments in which ectopic Lxn expression suppressed the growth of A20 cell in vivo. 100,000 GFP+ A20 cells infected with either the Lxn expression vector or the GFP only control vector, or uninfected A20 cells, were injected subcutaneously into the flanks of BALB/c mice. Beginning at day 12 when tumors were first palpable, the Lxn-expressing cells caused significantly smaller tumors (filled circles; P,0.005). By day 21 when the experiments were terminated, the tumors in the Lxn vector-injected group averaged only 40% of the volume of tumors in the other two control groups (P,0.05, n = 12 in each group). At day 21, we determined the fraction of tumor cells expressing GFP and Fig. 3F shows that virtually all of the tumor cells in the Lxn and control vector groups were GFP+. More to the point, Western blots confirmed strong expression of Lxn as in the cells analyzed after 20 days in culture (Fig. 3C). Thus, the reduction in tumor growth was due to durable Lxn expression in the tumor cells themselves. To explore the effects of cell dose on tumor size and latency before being palpable, we ejected graded doses of Lxn-expressing or control A20 cells (Fig. 3E) to BALB/c recipient (n = 4/group). At the 5,000 and 25,000 inoculum sizes, the Lxn-expressing cells not only resulted in more impressive suppression of the size of tumors than with the 100,000 cell inoculum in Fig. 3D, but resulted in delayed onset of measurable-sized tumors. At day 21, the reduction in tumor size caused by ectopic Lxn expression was 83% and 63% at the 5,000 and 25,000 cell doses, respectively. Host animals were necropsied for evidence of gross metastases to spleen, thymus and liver. No evident tumors were found in any of the treatment groups. Similarly, flow cytometry detected no GFP+ cells in these anatomical sites (data not shown). Lxn Inhibits Tumor Cell Growth through Increasing Apoptosis but not via its Canonical Function As we previously reported in our normal hematopoietic stem cell studies, high Lxn expression is associated with increased apoptosis and decreased proliferation. To determine if these mechanisms were applied to our results with tumor cells, we next measured these two parameters in vector-infected A20 cells throughout 21 days of culture. As shown in Fig. 4A, flow cytometric analysis of cells stained with BrdU and 7AAD allows for the discrimination of cell subsets that are apoptotic (A), necrotic (N) or reside in G0/G1, S and G2/M phase of cell cycle. By day 5 of culture, 10 fold more Lxn-expressing tumor cells were undergoing apoptosis than control cells (10.6% vs 1%). NO significant differences in the proliferation and the numbers of necrotic cells were observed between Lxn-overexpressing and control cells (data not shown). These results point to apoptosis as the major cellular mechanism in Lxn-mediated tumor suppression. We next investigated the molecular mechanisms underlying Lxn- induced apoptosis. We performed apoptosis pathway-specific PCR arrays in A20 cells infected with either empty or Lxn-expressing vectors. We found that 17 out of 84 apoptosis-related genes were differentially expressed in Lxn-overexpressing cells, and majority of them (15 genes) were down-regulated (data not shown). We specifically examined several well-known apoptotic genes, such as Bcl-2, Pim-2, Bcl-xl, Bax, and Bad, and plotted their expression in Fig. 4B. Two anti-apoptotic genes, Bcl-2 and Pim-2, were down- regulated by at least 3-fold in Lxn-overexpressing cells (left panel) as compared to the control, whereas pro-apoptotic genes, including Bax and Bad, did not show any change in their mRNA levels. Western blots of these proteins observed the similar reduction in Bcl-2 and Pim-2 (all three isoforms) but not in Bcl- xl, Bax and Bad in Lxn-overexpressing A20 cells (right panel), PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44979