Resistance to experimental tumorigenesis in cells of a long-lived mammal, the naked mole-rat (Heterocephalus glaber)

Abstract

The naked mole-rat (NMR, Heterocephalus glaber) is a long-lived mammal in which spontaneous cancer has not been observed. To investigate possible mechanisms for cancer resistance in this species, we studied the properties of skin fibroblasts from the NMR following transduction with oncogenes that cause cells of other mammalian species to form malignant tumors. Naked mole-rat fibroblasts were transduced with a retrovirus encoding SV40 large T antigen and oncogenic Ras(G12V). Following transplantation of transduced cells into immunodeficient mice, cells rapidly entered crisis, as evidenced by the presence of anaphase bridges, giant cells with enlarged nuclei, multinucleated cells, and cells with large number of chromosomes or abnormal chromatin material. In contrast, similarly transduced mouse and rat fibroblasts formed tumors that grew rapidly without crisis. Crisis was also observed after > 40 population doublings in SV40 TAg/Ras-expressing NMR cells in culture. Crisis in culture was prevented by additional infection of the cells with a retrovirus encoding hTERT (telomerase reverse transcriptase). SV40 TAg/Ras/hTERT-expressing NMR cells formed tumors that grew rapidly in immunodeficient mice without evidence of crisis. Crisis could also be induced in SV40 TAg/Ras-expressing NMR cells by loss of anchorage, but after hTERT transduction, cells were able to proliferate normally following loss of anchorage. Thus, rapid crisis is a response of oncogene-expressing NMR cells to growth in an in vivo environment, which requires anchorage independence, and hTERT permits cells to avoid crisis and to achieve malignant tumor growth. The unique reaction of NMR cells to oncogene expression may form part of the cancer resistance of this species.

Fig. 1

Characteristics of NMR fibroblasts after introduction of SV40 TAg and Ras. (A, A′) Phase contrast and fluorescence images of NMR cells 4 days after infection with a retrovirus encoding SV40 TAg, Ras and GFP. Below, presence of SV40 TAg and Ras in infected cells (+) shown by immunoblotting. (B, B′) A series of images of SV40 TAg/Ras-expressing NMR fibroblasts (phase contrast and fluorescence) obtained two weeks after retroviral infection, 1 to 4 days following plating of the cells. (C, C′, C″) Stages of crisis of SV40 TAg/Ras-expressing cells; 40, 45, and 50 population doublings following retroviral infection. (D) SV40 TAg/Ras-expressing cells that additionally had been infected with a retrovirus encoding hTERT, 50 population doublings following infection.

Fig. 2

DNA damage foci in NMR cells, detected by immunoreactivity with an antibody against γ-H2AX. The low power images on the left show γ-H2AX (immunofluorescence) and DNA (DNA-binding dye) in control NMR fibroblasts before transduction with SV40 TAg and Ras (con); in NMR cells following infection with a retrovirus encoding SV40 TAg and Ras (S/R); in late passage SV40 TAg/Ras-expressing NMR cells (S/R*); and in late passage SV40 TAg/Ras-expressing NMR cells further infected with a retrovirus encoding hTERT (S/R/hT*). Magnification × 100. The γ-H2AX immunofluorescence images were each obtained with a 2-second exposure; the DNA binding dye images were each obtained with a 0.5-second exposure. The fraction of γ-H2AX positive cells is indicated in each case. The high power images on the right (γ-H2AX immunofluorescence, DNA-binding dye fluorescence, and merged images) show an early-passage NMR cell expressing SV40 TAg and Ras (S/R); and two examples of late-passage SV40 TAg/Ras-expressing NMR cells (S/R*). The bottom image shows a cell in crisis with a large abnormal nuclear mass. Magnification × 4000.

Fig. 3

Tumorigenicity of NMR, mouse and rat cells expressing SV40 TAg, Ras, and hTERT. (top) NMR fibroblasts were infected with a retrovirus encoding SV40 TAg, Ras and GFP (S/R/G no hT) or were additionally infected with a retrovirus encoding hTERT (S/R/G + hT). Cells were injected subcutaneously in immunodeficient mice; site of injection was behind the right ear. After 60 days (no hTERT) or 45 days (+ hTERT), animals were sacrificed to allow examination of the nodules/tumors formed. (A, A′) Gross appearance and fluorescence of the nodules formed from no-hTERT cells; (B, B′) gross appearance and fluorescence of the tumors formed from hTERT⁺ cells. Additionally, the same retrovirus encoding SV40 TAg, Ras and GFP was used to infect primary mouse fibroblasts and primary rat fibroblasts. Tumor formation following subcutaneous injection in immunodeficient mice is shown for mouse cells at 25 days and for rat cells at 10 days.

Fig. 4

Nuclear abnormalities in SV40 TAg/Ras-expressing NMR cells in transplants. Cells were transplanted in immunodeficient mice. After 60 days, nodules were removed, sectioned, and examined for surviving NMR cells. Sections were stained with an anti-SV40 TAg antibody to locate NMR cells and adjacent serial sections were stained with DAPI to examine the nuclear morphology of NMR cells by fluorescence microscopy. (a) NMR cells were recognizable as large nuclei with prominent nucleoli. (b) Prometaphase rosettes observed en face and side. (c) Triradial. (d-g) Anaphases showing longer chromatin bridges (d) and various amounts of chromatin between the chromosome sets (e-g). (h-k) Examples of bizarre giant nuclear material showing multiple fused nuclei and briges between nuclei.

Fig. 5

Nuclear abnormalities in SV40 TAg/Ras-expressing NMR cells in vitro. Cells were cultured for 45 population doublings after infection by a retrovirus encoding SV40 TAg, Ras, and GFP. Cells were fixed and nuclear morphology was examined by staining with DAPI. (A) Example of multiple fused nuclei. (B) Complex of multiple fused nuclei linked by chromatin material. (C-F) Examples of nuclei bridged by various numbers of thin chromatin strings.

Fig. 6

Effect of hTERT on re-growth of NMR cells after being without anchorage. SV40 TAg/Ras-expressing NMR cells were held in suspension for 10 days and were then replated under regular growth conditions. Cells were photographed under phase-contrast at intervals following replating. Upper row, cells that were not additionally transduced with hTERT. Lower row, cells additionally expressing hTERT. The numbers indicate the number of days after replating following suspension.