Motexafin Gadolinium (Mgd), a Redox Stress Inducer, Lowers p53 Protein but Not Message in Lymphoma Cell Lines: Mechanisms of Reactive Oxygen Species (ROS)-Mediated Apoptosis in Lymphoma.

Disruption of the tumor suppressor p53 is a frequent event in malignant disorders, including lymphomas. Loss of constitutive low-level p53-dependent expression of anti-oxidant proteins could render cells more susceptable to the acute oxidant stress induced by oxidant generating agents such as MGd, bortezomib and rituximab. Alternatively, loss of p53 would render cells more resistant to apoptosis normally seen after strong activation of p53 activity in response to DNA damage. We hypothesized that the ROS generating expanded porphyrin, MGd, which we have previously shown to cause apoptosis in lymphoma and myeloma cells, results in loss of low-level, basal p53 expression, making cells more susceptable to oxidant stress. In order to test this hypothesis, we incubated Ramos, HIF-1 and SUDHL-4 lymphoma cells for 30, 60, 120 and 300 min with MGd (0.1–100 μM) with or without zinc acetate (Zn) and ascorbate. Following incubation, whole cell lysates were collected, electrophoresed on SDS-PAGE gels and blotted with p53-DO1 antibody. GAPDH was used as an internal standard. We found that at 300 min incubation, there was a dose-dependent loss of p53 by immunoblotting in the presence of Mgd and either Zn or ascorbate in Ramos and SUDHL-4 compared with control, and a dramatic loss with MGd/Zn/ascorbate in each of the cell lines. Maximum loss of p53 was observed at 100 μM MGd with both Zn 100 μM and ascorbate 100 μM. MGd, Zn or ascorbate alone had no effect. To determine if p53 message was altered, Ramos cells were treated for 300 min with the same concentrations of MGd, ascorbate and Zn and total RNA 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 amplificatiion was performed at 94° C for 30 sec (denaturation), 58° C (annealing) and 72° C (extension) for 40 cycles. Anti-sense primers and sense primers (IDT Technologies, Coralville, IA) for p53 and GAPDH were used and DNA was identified by 1% agarose gel electrophoresis. We found that there was no loss of p53 message under these conditions in the Ramos cell line.

CONCLUSIONS: These results suggest that there is post-translational modification of p53 induced by the ROS-generating expanded porphyrin MGd, and that this effect depends on the presence of ascorbate or Zn, similar to the effects of MGd, ascorbate and Zn for lymphoma and myeloma cell apoptosis. These data suggest that oxidant generating agents can result in loss of low-level basal p53 protein expression in lymphoma cells and that this might be important in cellular apoptosis, perhaps through mechanisms that depend on downstream p53 targets.