Methods for improving medical therapies
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example 1
Preparation of P16-3MR Transgenic Mice
[0135]To examine the role of senescent cells in cancer, in the risk of developing cancer or in side effects arising after cancer treatment, a transgenic mouse comprising a p16Ink4a promoter operatively linked to a trimodal fusion protein was generated to allow for detection of senescent cells and for selective clearance of senescent cells in those transgenic mice.
[0136]The promoter, p16Ink4a, which is transcriptionally active in senescent cells but not in non-senescent cells (see, e.g., Wang et al., J. Biol. Chem. 276:48655-61 (2001); Baker et al., Nature, supra) was engineered into a nucleic acid construct. A fragment of the p16Ink4a gene promoter (see FIGS. 5 and 6 providing an exemplary vector and exemplary promoter sequence) was introduced upstream of a nucleotide sequence encoding a trimodal reporter fusion protein. The trimodal reporter protein is termed 3MR and consists of renilla luciferase (rLUC), monomeric red fluorescent protein (mRFP...
example 2
Senescent Cells can be Detected and Cleared in Transgenic P16-3MR Mice
[0137]Senescent cells can be detected using a variety of biomarkers, including the strongly upregulated p16-INK4a tumor suppressor protein (Campisi et al., Nature Rev. Molec. Cell Biol. 8:729-40 (2007)). Using such markers, it was shown that both normal and tumor cells undergo senescence, in mice and humans, after exposure to ionizing radiation or DNA-damaging chemotherapy (Coppe et al., PLoS Biol. 6:2853-68 (2008); Schmitt et al., Cell 109:335-46 (2002); to Poele et al., Canc. Res. 62:1876-83 (2002); Le et al., Aging Cell 9:398-409 (2010)). For example, p16-3MR transgenic mice will accumulate senescent cells when exposed to genotoxins (e.g., ionizing radiation, DNA damaging chemicals), epigenomic toxins (e.g., compounds that perturb histone modifications or DNA methylation), strong mitogenic signals (e.g., activated oncogenes, elevated levels of growth factors, certain hormones). But, as noted herein, one advanta...
example 3
Cellular Senescence Increases the Likelihood of Cancer and Metastasis
[0141]To examine the role of senescence in contributing to, inducing or increasing the likelihood of tumor formation or growth and metastasis, tumor engraftment was monitored in p16-3MR transgenic mice that were either depleted of senescent cells and in mice that had senescent cells (naturally developed or induced).
[0142]Briefly, 106 B16 mouse melanoma cells, a highly aggressive cell line that is syngeneic with p16-3MR transgenic mice (C57B16 background), that express firefly luciferase (fLUC, to enable their detection by bioluminescence) were injected into the tail vein of the p16-3MR transgenic mice approximately three months after being either mock irradiated or irradiated, as described in Example 2. Irradiated mice were treated daily with GCV (25 mg / kg) or vehicle only for 7 days, and then 3 days following the last GCV dose, B16 mouse melanoma cells were injected into the mice. B16 mouse melanoma cells first co...
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