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Imaging studies of anti-tumorigenic effects of anti-oxidants in vivo

Director:
Ping Gao, Ph.D. (PI)
Chi Dang M.D., Ph.D. (Co-PI)

Brief Discription and findings:
We observed that MYC induction accompanies an increase in mitochondrial mass and function as well as cellular respiration and mtDNA copy number.  Conversely, loss of myc in Rat1 fibroblasts result in severely diminished numbers of mitochondria, which could be reconstituted by ectopic expression of human MYC.  Since the majority of cellular reactive oxidative species (ROS) emanate from mitochondria, we postulated that Myc induced mitochondrial biogenesis and metabolic pathways within mitochondria are accompanied by increased ROS production.  ROS has been well linked to double strand DNA breaks (DSBs).  Oxidative damage to DNA can result in DSBs which would then recruit ATM, ATR or DNA-PK to phosphorylate the histone H2AX (to form gH2AX) to initiate repair of the lesion.  Treatment with the oxidant scavenger N-acetyl-cysteine (NAC) remarkably reduced the level of gH2AX induced by Myc activation.  To determine the potential relevance of ROS in Myc induced tumorigenesis of P493 cells in vivo, we treated SCID mice with 40mM N-acetylcysteine in drinking water or control after injection with 30x106 cells.  P493 are human B cells bearing an EBV genome with a chimeric EBNA2-ER (estrogen receptor hormone binding domain) protein that enables P493 cells to grow in the presence of estradiol which activates EBNA2 activity.  In addition, a tetracycline-repressible MYC construct allows these cells to grow independently of EBNA2 when MYC is activated.  In the absence of tetracycline, exponentially growing P493 cells ectopically overexpress Myc, resulting in rapid cell proliferation and displaying a B cell lymphoma phenotype.  We found in a short-term experiment that NAC treatment reduces Myc induced P493 tumor formation as compared to control.  It is notable that mice treated with Doxycycline have markedly diminished tumor load (Fig. 4) and that NAC does not affect the ability of P493 cells to grow in vitro or inhibit luciferase activity.  These preliminary findings suggest that the P493 system is readily amenable to in vitro and in vivo studies relevant to tumorigenesis.

Figure 4. SCID mice were treated with control water (A), 40mM NAC in drinking water or with (B) 0.02% doxycyline for 7 days before inoculation with 30 million P493 cells. (C) Bioluminescent images were obtained using a Xenogen imaging system 10 days after inoculation and continued treatment through drinking water.

Main publication: Gao, P., Zhang, H., Dinavahi, R., Li, F., Raman, V., Bhujwalla, Z. M., Felsher, D., Cheng, L., Pevsner, J., Lee, L., Semenza, G. L., and Dang, C. V.  HIF-1-dependent Anti tumorigenic Effects of Anti-oxidants In Vivo, Cancer Cell. 2007 September; 12(3): 230–238..

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In Vivo Cellular Molecular Imaging Center (ICMIC)
Russell H. Morgan Department of Radiology and Radiological Science
Johns Hopkins University (JHU) School of Medicine
Baltimore, Maryland, USA