Involvement of KRAS G12A mutation in the IL-2-independent growth of a human T-LGL leukemia cell line, PLT-2

Abstract

Cytokine-dependent cell lines have been used to analyze the cytokine-induced cellular signaling and the mechanism of oncogenesis. In the current study, we analyzed MOTN-1 and PLT-2 cell lines established from different stages of a T-cell large granular lymphocyte leukemia patient (Daibata et al. 2004). MOTN-1 is IL-2-dependent derived from the chronic phase, whereas IL-2-independent PLT-2 is from the aggressive and terminal stage. They shared considerable chromosome abnormalities and the pattern of T-cell receptor rearrangement, presuming that the cytokine independence of PLT-2 was due to the additive genetic abnormality. Besides IL-2, IL-15 supported MOTN-1 cell growth, because these receptors share beta- and gamma-subunits. IL-2 activated ERK, AKT and STAT pathway of MOTN-1. STAT3 pathway of PLT-2 was also activated by IL-2, suggesting intact IL-2 induces signal transduction of PLT-2. However, ERK1/2 but not AKT, was continuously activated in PLT-2, consistent with the increased Ras-activity of PLT-2. Sequence analysis revealed KRAS G12A mutation but not NRAS and HRAS mutation of PLT-2 but not MOTN-1. Another signaling molecule affecting Ras-signaling pathway, SHP2, which has been frequently mutated in juvenile myelomonocytic leukemia (JMML), did not show mutation. Moreover, MEK inhibitor, PD98059, as well as farnesylation inhibitor inhibited PLT-2 cell growth. Using NIH3T3 and MOTN-1, ERK activation, increased cell proliferation and survival by KRAS G12A were shown, suggesting the important role of KRAS G12A in IL-2-independent growth of PLT-2. Taken together, KRAS G12A is important for IL-2-independent growth of PLT-2 cells and suggests the possibility of involvement of KRAS mutation with disease progression.

Fig. 1

Effects of IL-2 and IL-15 on cell proliferation of MOTN-1 and PLT-2 cells. (A) MOTN-1 and PLT-2 cells were cultured with or without IL-2 (100 U/ml). (B) IL-15 (1 or 10 ng/ml) was used to culture MOTN-1 cells. Viable cells were counted by trypan blue dye exclusion.

Fig. 2

Effect of IL-2 on cell cycle- and apoptosis-related proteins and cellular signaling pathways. (A) MOTN-1 and PLT-2 were cultured with or without IL-2 (50 U/ml) for 24 h. Cell cycle-related proteins were examined as described in the Materials and Methods. β-actin was shown as the internal control. (B) In similar ways shown in (A), the cellular signaling pathways were analyzed as described in the Materials and Methods.

Fig. 3

Ras activity, RAS mutation analysis, and effect of signaling inhibitors on PLT-2 cell growth. (A) MOTN-1 and PLT-2 were cultured with or without IL-2 (50 U/ml) for 24 h. Ras activity was examined as described in the Materials and Methods. (B) Mutation analysis of KRAS. Mutation of HRAS, KRAS, and NRAS was examined according to the Materials and Methods. Mutated G12A of PLT-2 was shown in italic. (C) Viable cell number of PLT-2 cells treated with or without signaling inhibitors on day 4 was demonstrated. Initial cell concentration of the control group was determined as 1.0. Concentrations of reagent used are: SB203580, 5 μM; JAK inhibitor I, 330 nM; LY294002, 10 μM; FTI277, 2 μM; PD98059, 100 μM; U0126, 20 μM.

Fig. 4

Effects of overexpression of wild- or mutated-KRAS in NIH3T3 cells. (A) NIH3T3 cells were transfected with mock-, KRAS-wild (WT), KRAS G12A or KRAS G12V, respectively according to the Materials and Methods. After G418 selection, ERK activation of established cells was determined by p-ERK protein level. β-actin was shown as the internal control. (B) Activated ERK was also demonstrated using PathDetect Elk1 Trans-Reporting System as described in the Materials and Methods. ERK activation was shown as the relative luciferase activity. MEK denotes the positive sample provided from the manufacturer. pFC2-dbd (negative control vector)-transfected Mock-NIH3T3 was regarded as the relative ratio of 1.0. The means +/- SD was calculated from the triplicate cultures. (C) Colony formation of KRAS-transfected cells. Colony formation assay of KRAS-transfected NIH3T3 cells was performed according to the Materials and Methods. Left part shows May-Giemsa staining of colonies, and the right illustrates the mean +/- SD of colony number/1.5×10³ cells calculated from triplicate culture.

Fig. 5

Effect of overexpressed wild or mutated KRAS in MOTN-1. (A) MOTN-1 was transfected with retrovirus vectors containing mock-, KRAS-wild (WT), KRAS G12A or KRAS G12V according to the Materials and Methods. After selecting highly GFP-expressing cells with FACS (data not shown), their ERK activation status was analyzed. β-actin was shown as the internal control. (B) Various KRAS-transfected cells were cultured without IL-2 for 24 h. Ras activity of respective cells was examined as described in the Materials and Methods. (C) Stably transfected cells were cultured in the medium containing 2.5% FCS but not IL-2. The initial cell concentration on day 0 was regarded as the relative ratio of 1.0. The means +/- was illustrated from triplicate cultures. (D) Viable cell number was counted after both serum and IL-2 depletion. The initial cell concentration on day 0 was regarded as the relative ratio of 1.0.