Motexafin Gadolinium (MGD) Induces Oxidant Stress and Downregulates Both Pro and Anti-Oxidant Genes in the Ramos Lymphoma Cell Line: Signaling through p53 Mediated Pathways.

We have reported that MGd, an expanded porphyrin and redox stress inducer, lowers p53 protein but not message in lymphoma cell lines. These results suggested that there is post-translational modification of p53 induced by MGd, and that this effect depends on the presence of ascorbate (Asc) or zinc (Zn). To expand these earlier studies, we measured the expression of oxidant and antioxidant genes in Ramos (Burkitt’s) cells. We incubated Ramos cells for 5 hours with MGd 100μM with or without Zn acetate (Zn, 100μM), Asc 100μM, and Nutlin-3 15μM. Following incubation, total RNA was isolated by the Qiagen method and dissolved in RNase-free water. 1.0 μg RNA was used for reverse transcription using the Taq DNA polymerase system. PCR amplification was performed at 94°C for 30 sec (denaturation), 55°C (annealing) and 72°C (extension) for 35 cycles. This was followed by an extension at 72°C for 10 minutes. Anti-sense primers and sense primers for SESN1(T2), SESN2, GPX1, CDKN1A, and PP1A were used and DNA was identified by 1% agarose gel electrophoresis. The T2 transcript of SESN1 gene was absent in these cells. Co-treatment of MGd/Zn/Asc notably decreased GPX1, SESN2, and CDKN1A transcripts. To determine the role of p53 in the regulation of downstream redox targets, we added the MDM2 inhibitor, Nutlin-3. We found that GPX1, SESN2, and CDKN1A were restored with Nutlin-3. In addition, we measured another downstream target of p53, PUMA, in both Ramos and HF-1 cells (a follicular lymphoma cell line) by immunoblot after MGd, Zn, and Asc exposure in the presence or absence of Nutlin-3. PUMAα and PUMAβ isoforms returned to near baseline levels in the presence of Nutlin-3. The restoration of PUMA was more pronounced in HF-1 cells than in Ramos. In summary, then, we have shown that:

1. the MDM2 inhibitor, Nutlin-3 reversed the downregulation of SESN2, GPX1, and CDKN1A genes that occurred as a result of MGd/Zn/Asc in Ramos cells and

2. similarly, PUMAα and β isoforms were restored with Nutlin-3 after decreased expression following exposure to MGd, zinc, and ascorbate.

These data indicate that some MGd-mediated signaling events in lymphoma cells are mediated through p53-dependent pathways.