J Korean Med Sci.  2010 Jul;25(7):1017-1023. 10.3346/jkms.2010.25.7.1017.

Ectopic Over-expression of Oncogene Pim-2 Induce Malignant Transformation of Nontumorous Human Liver Cell Line L02

Affiliations
  • 1Department of Hepatobiliary Surgery, the Second Affiliated Hospital of Chongqing Medical University, Chongqing, P.R. China. gongjianping11@126.com
  • 2Transplantation Immunity Laboratory, West China Hospital of Sichuan University, Chengdu, P.R. China.

Abstract

In order to prove that ectopic over-expression of Pim-2 could induce malignant transformation of human liver cell line L02, three groups of cells were set up including human liver cell line L02 (L02), L02 cells transfected with Pim-2 gene (L02/Pim-2) and L02 cells transfected with empty-vector (L02/Vector). Pim-2 expression levels were detected. The morphology, proliferation level, apoptosis rate and migration ability of the cells were detected respectively. Then the cells were subcutaneously inoculated into athymic mice and the microstructures of the neoplasm were observed. Compared with the controls, Pim-2 expression levels were significantly higher in L02/Pim-2 cells (P<0.05), and their morphology had obvious malignant changes. They also showed a significantly increased proliferation rate (P<0.05) and migration capacity (P<0.05), as well as a significantly decreased apoptosis rate (P<0.05). Only the athymic mice inoculated with L02/Pim-2 cells could generate neoplasm, and the morphology of the neoplasm coincided with that of the hepatoma. The results manifest that ectopic Pim-2 gene could be stably expressed in L02/Pim-2 cells. Both the morphological and biological changes of L02/Pim-2 cells demonstrate the trend of malignant transformation. L02/Pim-2 cells could generate hepatoma in athymic mice. In conclusion, Pim-2 could induce malignant transformation of human liver cell line L02.

Keyword

Pim-2; Malignant Transformation; Liver Cell; L02

MeSH Terms

Animals
Apoptosis
Cell Line
Cell Movement
Cell Proliferation
*Cell Transformation, Neoplastic
Humans
Liver/pathology/physiology
*Liver Neoplasms/genetics/pathology
Mice
Mice, Nude
Neoplasm Transplantation
*Oncogenes
Protein-Serine-Threonine Kinases/genetics/*metabolism
Proto-Oncogene Proteins/genetics/*metabolism

Figure

  • Fig. 1 Semi-quantitative RT-PCR of Pim-2 mRNA in the three groups of cells. The band gray value represents the mRNA level of the corresponding cell group. Pim-2 mRNA was expressed in all the three groups of cells but the expression level was significantly higher in L02/Pim-2 group (A) than that in L02/vector group (B) and L02 group (C). M, marker.

  • Fig. 2 Immunocytochemistry of Pim-2 protein in the three groups of cells. Buffy materials (arrow shows) are found in the cytoplasm and nucleus of the L02/Pim-2 group cells (A) but not in the L02/vector group (B) and L02 group (C). The buffy materials were regarded as Pim-2 protein. It indicated that the expression level of Pim-2 protein was significantly higher in L02/Pim-2 group than that in the A B C L02/vector group and the L02 group (×200).

  • Fig. 3 The morphology of the cells in the three groups. After culturing for 20 generations, the cells in the L02/Pim-2 group (A) are irregular fusiform shape of different size and many small protuberances are observed. The morphology of the cells in the Pim-2/vector group (B) and L02 group (C) are similar each other and they are all regular polygon or ellipse in concordant size (inverted microscope ×200).

  • Fig. 4 Migrated cells of the three groups in the Millicell room. (A-C) More cells in the L02/Pim-2 group (A) than in the L02/vector group (B) and L02 group (C) migrate through the microporous membrane. (D) There is significant difference between group A and group B, C, but no significant differences between group B and group C (*P<0.05 vs. Group A; †P>0.05 vs. Group B; inverted microscope ×200).

  • Fig. 5 Apoptosis rate of the three groups of cells. The data of flow cytometry results show that the apoptosis rate of group A is significantly lower than that in the other two groups (B, C) (*P<0.05 vs. Group A), and no significant differences are found between group B and group C (†P>0.05 vs. Group B). (Group A: L02/Pim-2; Group B: L02/vector; Group C: L02).

  • Fig. 6 The neoplastic mass found in the athymic mice which were inoculated with L02/Pim-2 cells. The first neoplasma was found in the 9th day after the inoculation, and in the 21st day, all the athymic mice in this group produced neoplasmas (A, B), with the average volume of 0.6×0.5×0.3 cm (C, D).

  • Fig. 7 The histology of the neoplastic tissue under light microscope. (A, B) The neoplastic tissue shows structurally disordered, with large areas of necrosis tissue (H&E, ×200); (C, D) The cells have marked cellular atypia, with giant nucleus, augmented karyoplasmic ratio, clear chromatospherite and prevalent mitosis, especially pathological mitosis (H&E, ×400).

  • Fig. 8 The ultrastructure of the neoplastic cells under TEM. (A) Much more smooth endoplasmic reticulum (arrow) are found (TEM ×5,000); (B) The cells have large and irregular nucleus with profound umbilication (white arrow). Few giant mitochondria are found in the cytoplasm. Big chromatospherite or even multiple nucleoli are also found (TEM ×7,000); (C) Multi-euchromatin (double arrows) are found in the nuclues (TEM ×8,000).


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