Abstract 1655: Expression of the HPV E7 oncogene in K14E7Fancd2-/- mouse long term bone marrow culture derived hematopoietic cells produces malignant plasmacytomas
Authors: Greenberger Joel S. , Rigatti Lora , Sivanathan Aranee , Cao Shaonan , Zhang Xichen , Shields Donna , Franicola Darcy , Epperly Michael 
Published: July 2016

Introduction: Transgenic FVB/n mice with the HPV E7 oncogene under the control of the CK14 promoter were bred with Fancd2+/- (129/Sv background) mice to obtain K14E7 Fancd2-/- mice Park, et al. Cancer Research, 70(23): 9959, 2010), in which delivery of 4-nitroquinoline N-oxide in drinking water produces oral and esophageal adenomas. The hematopoietic system, of these mice was studied, using long term bone marrow cultures (LTBMCs) from Fancd2-/- (SV129), K14E7 transgenic (FVB/n), and K14E7 Fancd2-/- mice.

Methods: LTBMCs were established by flushing bone marrow into T-25 flasks from which bone marrow stromal cell lines and IL3-dependent hematopoietic cell lines were developed. The cell lines were analyzed using irradiation survival curves, Western analysis for protein expression. K14E7 Fancd2-/- IL3 dependent clonal cell lines of the IL3-dependent cell lines were established by flow cytometry. Clonal lines were expanded and tested for tumorigenicity by injecting 1 × 106 cells of each of four clones into flanks of K14E7 Fancd2+/+ mice.

Results: K14E7 Fancd2-/- LTBMCs were similar to those from Sv/129 Fancd2-/-: 1) hematopoiesis was shortened when compared to K14E7 Fancd2+/+ or Fancd2+/+ LTBMCs, respectively. Stromal cell line irradiation survival curves with K14E7 Fancd2+/+ and Fancd2-/- cell lines showed (Do = 1.48 ± 0.05 and 1.52 ± 0.15 Gy) they were radiosensitive compared to those from K14E7 Fancd2+/+ or Sv/129 Fancd2+/+ cells (Do = 2.33 ± 0.11 and 2.23 ± 0.01 Gy, p = 0.0043 and 0.0368, respectively). Fancd2-/- stromal cells were more radiosensitive than Fancd2+/+ cells (decreased shoulder on the survival curve of n = 1.5 ± 0.5 compared to 4.7 ± 0.3 (p = 0.0126). K14E7Fancd2+/+ cells had an n of 1.9 ± 0.3 compared to 3.5 ± 0.1 (p = 0.0309) for Sv/129 cells. In contrast, hematopoietic IL-3 dependent K14E7Fancd2+/+ and Sv/129 Fancd2+/+ cell lines were similar (Do = 2.15 ± 0.4 and 1.92 ± 0.06, p = 0.6469, respectively). Confirming prior data, (Berhane et. al, Rad Res 182: 35, 2014) Sv/129 Fancd2-/- IL-3 dependent cell lines were radioresistant compared to Fancd2+/+ cell lines (Do = 2.12 ± 0.12 and 1.92 ± 0.06, respectively, p = 0.0236). In contrast, K14E7 Fancd2-/- cell lines were radiosensitive compared to K14E7 Fancd2+/+ cell lines (1.44 ± 0.13 and 2.15 ± 0.28, respectively, p = 0.0498). Hematopoietic and marrow stromal cell lines from K14E7Fancd2-/- marrow expressed cytokeratin 14 and E7 oncogene by Western analysis. K14E7 Fancd2-/- (but no other genotype derived) IL-3 dependent cell lines transformed in vitro to dense tumor cells lines. Cloned sublines were injected into K14E7 Fancd2+/- mice producing plasmacytomas.

Conclusions: Expression of the E7 oncogene in the K14/E7 Fancd2-/- mouse marrow derived IL-3 dependent cell lines leads to radiosensitivity and tumorgenicity.

Supported by NIAID/NIH U19-A 1068021 and the Fanconi Anemia Research Foundation.

Citation Format: Joel S. Greenberger, Lora Rigatti, Aranee Sivanathan, Shaonan Cao, Xichen Zhang, Donna Shields, Darcy Franicola, Michael Epperly. Expression of the HPV E7 oncogene in K14E7Fancd2-/- mouse long term bone marrow culture derived hematopoietic cells produces malignant plasmacytomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1655.