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A Senescence Program Contolled by p53 and p16^INK4a Contribures to the Outcome of Cancer Therapy

Clemens A. Schmitt, Jordan S. Fridman, Meng Yang, Soyoung Lee, Eugene Baranov, Robert M. Hoffman, and Scott W. Lowe

Summary

p53 andINK4a/ARF mutations promote tumorogenesis and drug resistance, in part, by disabling apoptosis. We show that primary murine lympomas also respond to chemotherapy by engaging a senescence program controlled by p53 and p16^INK4a. Hence, tumors with p53 or INK4a/ARF mutations-but not those lacking ARF alone - respond poorly to cyclophosphamide therapy in vivo. Morover, tumors harboring a Bcl2-mediated apoptotic block undergo a drug-induced cytostasis involving the accumulation of p53, p16^INK4a, and senescence markers, and typically acquire p53 or INK4a mutations upon progression to a terminal stage. Finally, mice bearing tumors capable of drug-induced senescence have a much better prognosis following chemotherapy than those harboring tumors with senescence defects. Therefore, cellular senescence contributes to treatment outcome in vivo.

Figure 1. Contribution of p53 and Bcl2 to Treatment Responses

Mice harboring ctrl.-MSCV, p53 null-MSCV, and ctrl.-bcl2 lymphomas were treated at comparable tumor burdens (day 0) with a single dose of cyclophosphamide (CTX) and monitored by whole-body fluorescence imaging. Representative examples are shown.

Clemens A. Schmitt, Jordan S. Fridman, Meng Yang, Eugene Baranov, Robert M. Hoffman, and Scott W. Lowe

Whole body fluorescence imaging of lymphoma progression in live mice. The cover shows the temporal and spatial progression of Em-myc lymphoma cells tagged with green fluorescent protein in a live mouse (with time progression from top to bottom). Note that the lymphomas first expand within the lympoid compartments and bone. In the absence of p53 or following Bcl-2 overexpression, these lympomas readily disseminate into nonlympoid compartments. For details see Schmitt et al. (pp. 289-298) in this issue.

Summary: Although the p53 tumor supressor acts in plethora of processes that influence cellular proliferation and survival, it remains unclear which p53 functions are essential for tumor suppression and, as a consequence, are selected against during tumor development. Using a mouse model harbouring primary, genetically modified myc-driven lympomas, we show that disruption of apoptosis downstream of p53 by Bcl2 or a dominant-negative caspase 9 confers-like p53 loss-a selective advantage, and completely alleviates pressure to inactivate p53 during lymphomagenesis. Despite their p53-null-like aggressive phenotype, apoptosis-defective lymphomas that retain intact p53 genes do not display the checkpoint defects and gross aneuploidy that are charcteristic of p53 mutant tumors. Therefore, apoptosis is the only p53 function selected against during lymphoma development, whereas defective cell-cycle checkpoints and aneuploidy are mere byproducts of p53 loss.

Figure 4. Whole body fluorescence imaging allows visualisation of lymphoma dissemination.

Lymphomas with indicated genotypes and transduced with a GFP-coexpressing retrovirus were transplanted into recipients to monitor lymphoma dissemination in whole viable animals by GFP fluorescence.

Real-Time Optical Imaging of Primary Tumor Growth and Multiple Metastatic Events in a Pancreatic Cancer Orthotopic Model

Michael Bouvet, Jinwei Wang, Stephanie R. Nardin, Rounak Nassirpour, Meng Yang, Eugene Baranov, Ping Jang, A. R. Moosa and Robert M. Hoffman

ABSTRACT: We report here whole-body optical imaging, in real time, of genetically fluorescent pancreatic tumors growing and metastasizing to multiple sites in live mice. The whole-body optical imaging system is external and noninvasive. Human pancreatic tumor cell lines, BxPC-3 and MiaPaCa-2, were engineered to stably express high-levels of the Acqurea victoria green fluorescent protein (GFP). The GFP-expressing pancreatic tumor cell lines were surgically orthotopically implanted as tissue fragments in the body of the pancreas of nude mice. Whole-body optical images visualized real-time primary tumor growth and formation of metastatic lesions that developed in the spleen, bowel, portal lymph nodes, omentum, and liver. Intravital imaging was used for quatification of growth of micrometastasis on the liver and stomach. Whole-body imaging was carried out with either a trans-illuminated epi-fluorescence microscope or a fluorescent light box, both with a thermoelectrically cooled color CCD camera. The simple, noninvasive, and highly selective imaging made possible by the strong GFP fluorescence allowed detailed simultaneous quantitative imaging of tumor growth and multiple metastasis formation of pancreatic cancer. The GFP imaging affords unprecendented contionuous visual monitoring of malignant growth and spread within intact animals without the need for anesthesia, substrate injection, control agents, or restraint of animals required by the other imaging methods. The GFP imaging technology presented in this report will facilitate studies of modulators of pancreatic cancer growth, including inhibition by potential chemotherapeutic agents.

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The human pancreatic tumor cell line, BxPC-3, was engineered to stably express high-levels of the Aquorea victoria green fluorescent protein (GFP). The GFP-expressing pancreatic tumor cell line was surgically orthotopically implanted (SOI) as tissue fragments in the body of the pancreas of nude mice. Intravital imaging was used for quantification of growth metastasis on the liver. The diameters of the three micrometastases were 288 mm and 208 mm the right lobe and 344 mm on the left lobe as quantified by image analysis at day-70 post SOI.

ABSTRACT: We have developed mouse models of metastatic cancer with genetically fluorescent tumors that can be imaged in fresh tissue, in situ, as well as externally. To achieve this capability, we have transduced the green fluorescent protein (GFP) gene, cloned from the bioluminescent jellyfish Aequorea victoria, into a series of human and rodent cancer cell lines that were selected in vitro to stably express GFP in vivo after transplantation to metastatic rodent models. Techniques were also developed for transduction of tumors by GFP in vivo. With this fluorescent tool, we detected and visualized for the first time tumors and metastasis in fresh viable tissue or in situ in host organs down to the single cell level. GFP tumors on the colon, prostate, breast, brain, liver, lymph nodes, lung, pancreas, bone and other organs can also be visualized externally, transcutaneously by quantitative whole-body fluorescence optical imaging. Real-time tumor and metastatic growth and angiogenesis and inhibition by representative drugs can be imaged and quantified for rapid anti-tumor, anti-metastatic and anti-angiogenesis drug screening. The GFP-transfected tumor cells enabled a fundamental advance in the visualization of tumor growth and metastasis in real time in vivo.

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