Transgenic expression of P1A induced thymic tumor: a role for onco-fetal antigens in tumorigenesis

Abstract

P1A is the first known tumor rejection antigen. It is expressed in embryonic stem cells and multiple tumors but is silent in adult tissues except for the testis and placenta. Therefore, P1A represents a prototype for onco-fetal antigens. To test the potential function of P1A in tumorigenesis, we used a transgenic mouse expressing P1A in lymphoid cells. We observed that immunodeficient host P1A transgenic mice developed thymic tumors after 7 months of age and had shorter survival rates compared to control groups. Most of the 7 examined tumors displayed B cell lineage markers. The P1A transgenic bone marrow cells had higher proliferation ability and more potential progenitors compared to control bone marrow cells. To our knowledge, our data provided the first example that onco-fetal antigen can promote tumorigenesis.

Figure 1. Expression of P1A.

A. Diagram of Eμmb-P1A transgene. P1A open reading frame (P1A-ORF) is under controlled by enhancer of immunoglobulin heavy chain (Eμ) and mb-1 promoter (P_mb-1), followed by ID2 gene and an SV40 polyadenylation signal sequence. B. Western blot analysis of P1A protein expression in J558 plasmacytoma cells, R1 murine ES cells, thymus, spleen, testis from P1A Tg mice and BALB/c WT mice; actin as internal control.

Figure 2. P1A Tg mice developed tumors.

A. Thymus weights of P1A⁺Rag2^−/− mice and Rag2^−/− mice. Enlarged thymus in P1A⁺Rag2⁻ mice after 7 months of age compared to P1A⁺Rag2⁻ mice. B. P1A⁺Rag2⁻ mice had shorter survival rates compared to P1A⁻Rag2⁻ groups. Only mice that died due to tumor formation were recorded. p = 0.0004 (Kaplan-Meier analysis). C. Although several P1A⁺Rag2⁺ mice died due to tumor formation, differences in the tumor incidences were not statistical significant. p = 0.1157 (Kaplan-Meier analysis).

Figure 3. Heterogeneity of tumors in individual mouse.

The thymic tumor from different mice was stained by the following monoclonal antibodies, CD4, CD8, B220, CD11c, Gr1, and CD11b. After antibody staining, thymic tumor cells were subsequently analyzed by flow cytometry. A. Mouse #931 had thymic tumors with B220⁺ and B220⁺CD11c⁺CD11b⁺ cell populations of tumor cells. B. Mouse #946 had thymic tumor of CD4⁺CD8⁺ cell population of tumor cells. C. Mouse #947 had B220⁺CD11c⁺ and B220⁺CD11c⁺Gr1⁺CD11b⁺ cell populations of tumor cells. D. Mouse #954 had B220⁺, CD8⁺B220⁺CD11c⁺, Gr1⁺CD11b⁺ cell populations of tumor cells. E. Mouse #956 thymic tumor had B220^lo cell population of tumor cells. F. Mouse #962 had tumor with B220⁺ cell population of tumor cells. G. Mouse #963 showed CD8⁺CD11c⁺CD11b⁺, B220⁺CD11c⁺ and CD11b⁺CD11c⁺ cell populations of tumor cells.

Figure 4. Enhanced proliferation and progenitor activity of P1A Tg bone marrow.

A. BrdU incorporation by flow cytometry; representative profiles of BrdU incorporation in bone marrow cells. B. The proportion of BrdU positive cells among bone marrow cells, showing mean ± SD of BrdU positive cells (n = 3, two-tailed unpaired Student's t-test). C. Colony-forming cell (CFC) assay. There was a higher colony number in P1A⁺Rag2⁻ compared to Rag2⁻ bone marrow cells. The cells cultured in second round were taken from the first round culture (n = 3, two-tailed unpaired Student's t-test). D. Higher colony number in all colony types, including burst-forming unit-erythroid (BFU-E), colony-forming unit granulocyte (CFU-G), colony-forming unit macrophage (CFU-M) and colony-forming unit granulocyte macrophage (CFU-GM), were seen in P1A⁺Rag2⁻ than in Rag2⁻ by inverted microscopy. (n = 3, two-tailed unpaired Student's t-test)