Transformed Phenotype of Bone Marrow Stromal Cell Lines Derived from K14E7 Fancd2^-/- mice

To determine whether cytokeratin 14 promoter linked expression of the Human Papilloma Virus (HPV) oncogene detectably influenced biologic parameters of cell phenotypes other than squamous epithelium, continuous bone marrow cultures were derived from K14E7 Fancd2^-/- mice (Park, et al., Cancer Research, 70(23): 9959-9968, 2010). Long-term bone marrow cultures derived from K14E7 Fancd2^-/-, control Fancd2^-/- (129/Sv), K14E7 (FVB/N), and wild type 129/Sv X FVB/N F1 mice were evaluated for longevity of hematopoiesis in long-term cultures and stromal cell lines were derived from each. Similar to Fancd2^-/- mouse, long-term marrow cultures, K14E7 Fancd2^-/- marrow cultures demonstrated decreased longevity of hematopoiesis with cessation of production of multi-lineage colony forming progenitor cells after 14 weeks. In contrast, wild type F1 and K14E7 long-term marrow cultures continued to produce hematopoietic cells for a significantly longer duration 25 weeks (p=0.0257). Bone marrow stromal and IL-3 dependent hematopoietic cell lines were derived from each genotype marrow culture. K14E7 Fancd2^-/- hematopoietic cells showed reversal of the radiation resistance of Fancd2^-/- IL-3 dependent cell lines (D₀ of 1.34 ± 0.197Gy, ñ 4.0 ± 0.9 compared to D₀2.213 ± 0.124 Gy (p = 0.0284), ñ 3.3 ± 0.8. Thus, one phenotypic difference associated with K14E7 oncogene expression was reversal of radioresistance of Fancd2^-/- hematopoietic cells. In contrast, bone marrow stromal cell lines from K14E7 Fancd2^-/- remained radiosensitive similar to those from Fancd2^-/- mice. K14E7 and wild type F1 marrow stromal cell lines showed intermediate radioresistance (p= 0.1759). To determine whether the E6/E7 oncogene had a biological effect in tissues other than squamous epithelium, tissue analysis for cytokeratins 13, 14, 6, and 10 was carried out. Cytokeratin 14 was detected only in squamous cells of the esophagus and oral cavity, not in bone marrow. E6/E7 oncogene was detected only in squamous cell lines expressing cytokeratin 14. However, bone marrow stromal cell lines from K14E7 Fancd2^-/- marrow cultures demonstrated a unique phenomenon of cellular density, piling up and formation of tumors in vitro. Each of 22 single cell derived clonal sub-lines of K14E7 Fancd2^-/- stromal cell lines demonstrated the same transformed phenotype. These data provide support for indirect effects of the E7 oncogene linked to the K14 promoter in Fancd2^-/- hematopoietic and mesenchymal stem cell tissues.

Supported by research grant NIAID/NIH, U19A168021.