Malignant Transformation of Fanconi Anemia Complementation Group D2-deficient (Fancd2 -/-) Hematopoietic Progenitor Cells by a Single HPV16 Oncogene

Abstract

Aim: To demonstrate that Fanconi anemia complementation group D2-deficient (Fancd2^-/-) hematopoietic progenitor cell lines can be transformed by transfection with a plasmid containing either the E6 or E7 oncogene of human papillomavirus (HPV) to generate malignant plasmacytoma-inducing cell lines.

Materials and methods: In order to determine whether a single HPV type 16 (HPV16) oncogene induced malignant transformation, Fancd2^-/- and Fancd2^+/+ interleukin 3 (IL3)-dependent hematopoietic progenitor cell lines were transfected with plasmids containing E6 or E7 oncogene, or control empty plasmid.

Results: Fancd2^-/- but not Fancd2^+/+ cells were transformed into malignant IL3-independent cells by both E6, and E7 oncogenes, but not by empty plasmid. Hematopoietic cell lines and tumors induced by Fancd2^-/- E6 and Fancd2^-/- E7 cell lines were positive for each respective HPV RNA and protein.

Conclusion: A single HPV16 oncogene is adequate to produce malignant transformation of Fancd2^-/- hematopoietic cells.

Keywords: E6 and E7 oncogene; Fanconi anemia; Human Papillomavirus.

Figure 1. Murine stem cell virus (MSCV) plasmids used for transfection of Fanconi anemia complementation group D2 (Fancd2)−/− and Fancd2+/+ interleukin 3 (IL3)-dependent hematopoietic progenitor cell lines. A: pMSCV puro plasmid map; B: pMSCV puro plasmid showing E6 and E7 transgene insertion sites in plasmid.

Figure 2. Interleukin 3 (IL3)-dependent Fanconi anemia complementation group D2 (Fancd2)−/− cells, but not Fancd2+/+ cells are transformed by human papillomavirus type 16 (HPV16) E6 and E7 transgene insertion. IL3-dependent Fancd2+/+ (A) and Fancd2−/− (B) cells were transfected with pMSCV puro plasmid containing either HPV16 E6 or E7 transgene. IL3-dependent Fancd2−/− cells were transformed, but the IL3-dependent Fancd2+/+ cells were not.

Figure 3. Human papillomavirus type 16 E6- and E7-transfected interleukin 3 (IL3)-dependent Fanconi anemia complementation group D2 (Fancd2)−/− cells grown in the absence of IL3. IL3-dependent Fancd2−/− but not Fancd2+/+ cells transfected with either E6 or E7 become IL3- independent and malignant in appearance. Non-transfected Fancd2−/− cells (A) were dead by day 7 (magnification ×40). Fancd2−/− cells transfected with either E6 (B) or E7 (C) (but not control plasmid) transformed to adherent cells grown in the absence of IL3 and grew to high density. Fancd2−/− cells transfected with empty plasmid grown in the absence of IL3 were also dead by day 7 (magnification ×40).

Figure 4. Gross pathological appearance of tumors in athymic nude mice at day 30 after subcutaneous injection of 1×106 human papillomavirus type 16 E6-(A) or E7-(B) transfected interleukin 3 (IL3)-independent Fanconi anemia complementation group D2 (Fancd2)−/− cells (×10). Appearance of IL3-independent cells cultured from tumor in panel A (C) and tumor in panel B (D) (×40).

Figure 5. Detection of human papillomavirus type 16 E6 and E7 oncogenes by reverse transcriptase-polymerase chain reaction (RT-PCR) in 129/Sv mouse Fanconi anemia complementation group D2 (Fancd2)−/− cell lines transfected with human papillomavirus type 16 (HPV16) E6 or E7 oncogenes and in explanted tumors. Tumors that developed from the injection of Fancd2−/− E6- or Fancd2−/− E7-bearing cells into athymic nude mice were analyzed for the expression of E6 or E7 by RT-PCR. RNA was extracted from Fancd2−/− cells, E6- or E7-transfected Fancd2−/− cells grown in culture, and from tumors derived from these cell lines. CaSki cells, which are positive for E6 and E7, grown in culture and plasmid DNA containing the E6 or E7 transgene were used as positive controls. RT-PCR was performed using primers specific for HPV16 E6 and E7. N.C.: Empty plasmid DNA, negative control.

Figure 6. Detection of human papillomavirus type 16 (HPV16) E6 and E7 oncogene proteins in HPV16 E6- and E7-transfected Fanconi anemia complementation group D2 (Fancd2)−/− cell lines and derived tumors. Western blot analysis for FANCD2, E6 and E7 was performed on Fancd2−/− cells, E6- and E7-transfected Fancd2−/− cells grown in culture or from tumors. CaSki cells, which are positive for E6 and E7, were used as the positive control.

Figure 7. Human papillomavirus type 16 E6- and E7-transfected Fanconi anemia complementation group D2 (Fancd2)−/− cell lines expressed both T-cell (CD3) and B-cell (CD19) markers, and cell line-derived tumors also expressed B-cell-specific marker. Human papillomavirus type 16 E6- and E7-transfected interleukin 3-independent Fancd2−/− cells, grown in tissue culture or isolated from explanted tumors grown in athymic nude mice, were stained with an antibody to a T-cell-specific protein (CD3) or B-cell-specific protein (CD19) followed by a fluorescent secondary anti-mouse IgG or anti-rabbit IgG antibody. Cells were examined by fluorescent microscopy (×40).

Figure 8. Histopathological appearance of tumors formed by human papillomavirus type 16 E6- (A) and E7- (B) transfected interleukin 3- independent Fanconi anemia complementation group D2 (Fancd2)−/− cell lines stained with hematoxylin and eosin (×40).