ODC antizyme 1 (AZ1) has been implicated as the primary regulator of ODC stability and activity, a regulator of growth-related proteins, a tumor suppressor gene, and as a mediator of sensitivity to the antitumor polyamine analogues (Kahana 2009
). Despite the fact that this unique protein potentially plays multiple critical roles, considerable work remains to fully elucidate the many functions of AZ1. In this study, siRNA-mediated knockdown of AZ1 expression produced a 10-fold increase in BENSpm sensitivity, with more than 2-fold greater analogue accumulation compared to SCR clones. Spermidine uptake was also elevated 50% in AZKD cells compared to SCR, suggesting a loss of uptake regulation and confirming that AZ1 plays a role in polyamine transport regulation.
It also has been previously stated that polyamine analogues such as BENSpm can actually limit their own uptake by their ability to induce antizyme and so downregulate additional polyamine uptake (Mitchell et al. 2007b
). In this case, the loss of AZ1 results in loss of uptake regulation, resulting in increased BENSpm uptake, accumulation, and subsequent toxicity in AZKD cells compared to SCR clones. A similar increase in polyamine (and presumably polyamine analogue) accumulation has been observed in cells with elevated levels of ODC, as more of the available AZ1 is utilized in an attempt to regulate the amount of ODC and, as such, less AZ1 is available for regulation of uptake (Sakata et al. 1997
The cellular effects observed in this model are all consistent with loss of AZ1. However, the significant decrease in ODC protein levels and enzyme activity after treatment with BENSpm in all clones demonstrates that ODC continues to be degraded, despite loss of AZ1. It is likely that there is a BENSpm-dependent decrease in the translation of ODC, as has been demonstrated for the natural polyamines (Kameji and Pegg 1987
; Shantz and Pegg 1999
). This phenomenon would further decrease the amount of ODC protein, but the significant loss over the time course measured suggests that there is also considerable degradation by some factor other than AZ1.
Clearly, the apparent stabilization of ODC in AZKD cells results from loss of AZ1, but these cells quickly compensated for this to decrease ODC activity and protein. It is important to note that the amount of ODC protein in AZKD clones is at least 1.5-fold that of SCR clones, and despite greater than 99 % knockdown of AZ1 (as in clone AZ 11), ODC protein is still decreased by over 50 % after 4 h BENSpm treatment. Although the decreased ODC can be accounted for by the combination of decreased protein synthesis and increased degradation (Kameji and Pegg 1987
), the significance of the decreases in ODC protein and activity in the AZKD cells strongly support the premise that AZ1 is not solely responsible for ODC degradation.
The essential role of antizyme in ODC degradation has been demonstrated in studies showing that its removal by immunoprecipitation resulted in an almost total loss of ODC degradation (Kanamoto et al. 1993
). It has been previously suggested that AZ1 is the major antizyme involved in ODC regulation, and that AZ2 is a poor regulator of ODC, but is involved in the regulation of polyamine transport (Zhu et al. 1999
). Our data suggest ODC regulation to be more complex than previously thought, since the loss of AZ1 results in no change in the growth rate of these cells, nor does it have any major effect on the changes in ODC protein or the suppression of ODC activity upon BENSpm treatment. We believe that the most likely explanation for these effects is that at least one other factor is functionally substituting for AZ1.
The most likely substitute candidate for AZ1 is AZ2, which is also found widely in cells and has been shown to bind to ODC monomers (Murakami et al. 2000
). AZ2 has previously demonstrated little ability to induce ODC degradation in a baculovirus expression system, but was able to inhibit ODC activity to a level comparable to AZ1 (Zhu et al. 1999
). Our data show that both ODC activity and protein were decreased comparably between the SCR and AZKD clones. The loss of ODC protein by 8 h strongly suggests another factor is targeting ODC for proteasomal degradation. The most likely explanation is that AZ2 is substituting for AZ1, although it is also possible that there are alterations in the translation of ODC resulting from treatment with BENSpm (Pegg et al. 1988
). To test this, we have completed a preliminary analysis of changes in AZ2 expression levels using real-time PCR. These preliminary results indicate that there is an upregulation of AZ2 mRNA of up to 4-fold in AZ1 knockdown clones. This is significant when one considers that the amount of AZ2 mRNA has been reported to be 16-fold less than that of AZ1 (Ivanov et al. 1998
Thus, the current belief that AZ2 can bind to ODC, but cannot target it for degradation may not be entirely accurate. If this were the case, it would result in a decrease in ODC activity, since AZ2-bound ODC cannot form homodimers, but this would not change the protein content, as AZ2 cannot target the ODC to the proteasome for degradation. This response is clearly not occurring in AZ1 knockdown model presented in this study. It therefore appears that AZ2 or other factors play a more important role in polyamine metabolism than has been previously considered, and thus warrants further investigation. Studies are currently in progress using an AZ1 and AZ2 double knockdown H157 cell line to determine if AZ2 does indeed have a more pronounced role in regulation of polyamine metabolism than has previously been suggested.
The elevated putrescine in AZKD clones is consistent with the higher basal levels of ODC activity, and has been detected in many cell types where ODC levels are elevated (Mitchell et al. 1996
; Gerner and Meyskens 2004
). The loss of AZ1 in the present cellular model is such that these cells can no longer maintain balanced polyamine pools due to the increased activity of ODC. Despite this, the cells showed only a moderate increase in polyamines and ODC protein. This could be due to the increased activity of another ODC regulator as a compensatory mechanism. Alternatively, these cells may increase their catabolic enzyme activities to increase export in an attempt to regain polyamine homeostasis. However, experiments to test effects of AZ1 knockdown on the polyamine catabolic pathway indicated that there is no significant change in either spermidine/spermine N1
-acetyltransferase or spermine oxidase, the two rate-limiting enzymes of polyamine catabolism (data not shown).
Another potentially important role attributed to AZ1 is its ability to modulate the degradation of cyclin D1, (Newman et al. 2004
). However, the role of AZ1 in cyclin D1 regulation has never been confirmed and the results in our H157 human non-small cell lung cancer cell system using stable silencing of AZ1 indicate that AZ1 has no role in cyclin D1 regulation. In contrast, it appears more likely that the observed modulation of cyclin D1 protein levels is a result of a decreased cellular growth rate generally.
AZ1 clearly has an important role to play in regulating polyamine homeostasis via its effects on ODC protein and activity, and on polyamine uptake. Removal of over 90% of AZ1 mRNA results in increased ODC activity and putrescine content, supporting AZ1 as the main ODC regulator. Likewise, AZKD cells exhibit increased uptake of spermidine and polyamine analogues, resulting in increased sensitivity to BENSpm. However, despite lower basal ODC levels in the AZKD clones, loss of AZ1 does not significantly alter the degradation of ODC protein in response to BENSpm over an 8 h time course, indicating the existence of additional regulatory mechanism(s) governing ODC levels. We hypothesize that this compensatory factor is AZ2, though further research will be needed to more completely clarify the complex and interconnected roles of the antizymes in regulating polyamine homeostasis.