Requirement of phospholipase D1 activity in H-RasV12-induced transformation

Abstract

The ability of the Ras oncogene to transform normal cells has been well established. One downstream effector of Ras is the lipid hydrolyzing enzyme phospholipase D. Recent evidence has emerged indicating a role for phospholipase D in cell proliferation, membrane trafficking, and migration. To study the potential importance of phospholipase D in the oncogenic ability of Ras, we used Rat-2 fibroblasts with reduced phospholipase D1 activity (Rat-2V25). Here, we show that H-Ras transformation of Rat-2 fibroblasts requires normal phospholipase D1 activity. WT Rat-2 fibroblasts transfected with the H-RasV12 oncogene grew colonies in soft agar and tumors in nude mice. However, Rat-2V25 cells when transfected with the H-RasV12 oncogene did not form colonies in soft agar or produce tumors when xenografted onto nude mice. Interestingly, in the presence of phosphatidic acid, the product of phospholipase D, growth in soft agar and tumor formation was restored. We also observed a dramatic increase in the expression of phospholipase D1 in colorectal tumors when compared with adjacent normal mucosa. Our studies identify phospholipase D1 as a critical downstream mediator of H-Ras-induced tumor formation.

Fig. 1.

Evaluation of H-Ras^V12 and K-Ras^V12 transformation in WT Rat-2 and Rat-2V25 fibroblasts. (A) Protein expression levels of HA-tagged H-Ras^V12, HA-tagged K-Ras^V12, and total Ras in WT Rat-2 and Rat-2V25 cells. (B) Ras activation assay in WT Rat-2 and Rat-2V25 cells expressing H-Ras^V12 or K-Ras^V12(C) Growth of H-Ras^V12- or K-Ras^V12-transformed cells in soft agar. These results are representative of three independent experiments.

Fig. 2.

The reintroduction of PLD1 or its lipid product (PA) can restore H-Ras^V12-induced transformation of Rat-2V25 fibroblasts in soft agar. (A) Growth of WT Rat-2 H-Ras^V12-transformed cells in soft agar. Growth of Rat-2V25 H-Ras^V12-transformed cells (B) transfected with PLD1 (C), in the presence of 200 μM 12:0 PA (E), 200 μM 16:0 PA (F), 200 μM 1-oleoyl-2-acetyl-glycerol (G), or 200 μMPC(H). (D) PLD activation on WT Rat-2, Rat-2V25, and Rat-2V25 cells transfected with PLD1. The results are representative of three independent experiments performed in triplicate. Numbers at lower right are the number of colonies per field ± SEM.

Fig. 3.

Xenograft growth of H-Ras^V12-transformed WT Rat-2 and Rat-2V25 fibroblasts. (A and B) Xenograft growth of WT Rat-2 (A) and Rat-2V25 (B) cells transformed with H-Ras^V12 on day 10. (C–E) Effect of PA on xenograft growth of H-Ras^V12-transformed WT Rat-2 cells (C), H-Ras^V12-transformed Rat-2V25 cells (D), and vector-transfected Rat-2V25 cells (E). (F) Summary graph of tumor size from H-Ras^V12-transformed cells (*, P < 0.05). Data represents the average ± SEM of three independent experiments.

Fig. 4.

Comparison of PLD1 expression between colorectal carcinomas and adjacent normal mucosa. (A) Northern analysis of hPLD1 mRNA. (B) Western analysis of hPLD1 and hPLD2 protein expression.