Mechanism of Central Nervous System Leukemia Figure 5. The SCI-nu/nu mice model of CNS leukemia. (A) Clinical signs of CNS leukemia in mice. (A1, A2, arrow) The mouse developed paralysis in its left forelimb and cranial nerve with light loss. (A3, arrow) The mice developed paralysis in both fore limbs and hind limbs. (B, arrow) Neoplasms of different parts with or without intracranial hemorrhage in removed brains. (C) Infiltrations of SHI-1 examined with histopathology assay. (C1, C2, C3) Brain parenchyma pathobiology stained with H&E. (C4) Examination of bone marrow smears stained with Wright’s-Giemsa. (D) Organs with infiltrations of SHI-1 examined with RT-PCR analysis of human MLL/AF6 fusion gene transcription in mice with or without GM6001 treatment. (D1) RT-PCR analysis of MLL/AF6 fusion gene of brains and bone marrow from SCI nu/nu-mice. Lanes 1–5: Human MLL/AF6 fusion genegene amplified in SHI-1 cell and in brain, bone marrow, stomach and liver of mice with CNS leukemia. lane 6–9: Human MLL/AF6 fusion gene amplified in stomach, liver, brain and bone marrow of mice with GM6001 treatment. lane 10: negative control. (D2) The number of organs with infiltrations of SHI- 1 examined with RT-PCR analysis of human MLL/AF6 fusion gene transcription in mice with or without GM6001 treatment. Original magnification: (C1), 640; (C2), 6200; (C3), 61000; (C4), 61000. doi:10.1371/journal.pone.0020599.g005 CNS leukemia with brain parenchyma involvement was successfully established with the human acute monocytic leukemic cell line SHI-1. GM6001 protected mice against CNS leukemia Consistent with MLL/AF6, the human MMP-2 and -9 transcriptions were initially detected at day 21 after being challenged by SHI-1 and amplified in all killed mice with CNS leukemia (Fig. 6A). In order to examine the roles of MMP-2 and - 9, GM6001 was used to inject intraperitoneally into mice. We found that no mice developed CNS leukemia until they were killed when moribund at (41.5061.62) d (mean 6SD) after being challenged by SHI-1. The number of organs infiltrated by SHI-1 cells and the degree of infiltration also decreased (Fig. 5D). MMP- 2 and -9 transcripts were not detected in the brains of mice treated with GM6001. (Fig. 6A). Furthermore, we used in situ zymography to identify the regions with gelatinolytic activity. Bright fluorescent (green) spots were apparent in the areas of infiltration with leukemic cells and in the periphery of mouse brains with CNS leukemia (Fig. 6C). While after treatment with GM6001, gelatinolytic activities had not changed in all mice compared with PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e20599