Our major objective was to assess whether arginase II was able to deplete L-arginine from the tissue culture supernatants of murine renal cell carcinoma cell lines and determine their effect on cell proliferation. In adult mammals, the majority of endogenous L-arginine is synthesized from citrulline in the kidney and released to systemic circulation where it is catabolized by arginase I or arginase II [4, 11]. Therefore, the study of the L-arginine metabolic pathway in mRCC cell lines provide us with a good model to better understand the biology of renal carcinoma.
Western blot and RT-PCR analyses confirmed that arginase activity from mRCC cell lines was attributable solely to arginase II and not to arginase I. This is an important finding, since most studies have demonstrated that only arginase I produced by tumor cells, macrophages, smooth muscle and endothelial cells [26–29] is capable of depleting L-arginine which results in the induction of T-cell dysfunction [19, 30]. The role of arginase II on L-arginine metabolism in disease and cancer has been quite underestimated, especially taking into account its wide tissue distribution and its role in polyamine production. Previous studies have shown that the expression of either arginase I or arginase II plays a key role in polyamine synthesis and cell proliferation . Although the three cell lines used in this study were all derived from kidney tumors, they had very different arginase II activities. CL-2 and CL-19, both derived from renal tumors induced by streptozotocin, had low and high arginase II activities, respectively. The Renca cell line, derived from a spontaneous renal tumor had intermediate activity. These three lines provide us with an ideal model to study the biology of RCC with regard to L-arginine consumption, L-ornithine production, and cell proliferation. We demonstrate for the first time that arginase II produced by the high arginase RCC cell line CL-19 dramatically depletes L-arginine from the tissue culture supernatants at 48 hours with a concomitant increase in L-ornithine production.
In contrast, the cell lines CL-2 and Renca, both of which expressed low levels of arginase II compared to CL-19 did not deplete L-arginine significantly; nor did they increase the levels of L-ornithine sufficiently to promote growth. Instead, we observed that CL-2 and Renca cells utilize L-glutamine at higher rates than CL-19, suggesting that this could be a possible mechanism used by these cells to convert L-glutamine to glutamate, bypassing arginase for the production of L-ornithine as described previously in murine macrophages and human monocytes . It is likely that CL-2 and Renca cells do not need arginase to make L-ornithine because they utilize L-glutamine to produce the necessary amount of L-ornithine needed for their cell growth. This observation indicates that arginase II is important for CL-19 growth but not for CL-2 and Renca cells due to the positive effect of nor-NOHA in suppressing cell growth in CL-19. We used nor-NOHA in our experiments because it has been demonstrated that nor-NOHA is a potent and selective inhibitor of arginase  in contrast to NOHA which is a key intermediate product in the biosynthesis of nitric oxide by L-arginine. We were expecting to have a greater arginase inhibition by nor-NOHA in our cultures similar to those observed when NOHA was used to inhibit cell proliferation in cell lines from breast, colon, prostate and endothelial cells as previously reported [16, 17, 34, 35]. This may be due to the fact that these cell lines can use L-arginine to synthesize NOHA from arginase, then increasing its inhibitory effect. In contrast, it is also possible the growth of renal cell carcinoma cells is arginase II independent resulting in the low inhibitory effect of nor-NOHA.
At 48 hours in culture, CL-19 cells significantly depleted L-arginine from the culture supernatant; however, these cells continued growing at the same rate up to 120 hours in the absence of the amino acid (data not shown). L-arginine deprivation should promote the death of CL-19 cells, as reported previously to occur in other cancer cells lines [36, 37], indicating that these cells are more adept at circumventing L-arginine deficiency by increasing the recycling efficiency from L-ornithine to citrulline to convert L-arginine fast enough to sustain relatively normal tumor cell growth rate as previously shown . Since RCC cells have a strong dependence for L-arginine , our laboratory is currently studying whether or not these cells are utilizing L-glutamine or citrulline as the source for L-arginine synthesis.
L-arginine is a non-essential amino acid that plays a central role in several biological systems including the immune response. Paradoxically, L-arginine deprivation can cause tumor cell death as well as T-cell dysfunction. The loss of CD3ζ is the only arginase-triggered mechanism described so far that has proven to have direct relevance to T-cell function [40, 41]. It has been previously shown that Jurkat T-cells cultured in medium lacking L-arginine showed decreased expression of CD3ζ and decreased cell proliferation . Similar results were obtained when stimulated normal human T-cell lymphocytes were cultured in the absence of L-arginine . In the current experiments, we found that after 48 hours in culture, depletion of L-arginine by CL-19 arginase II activity caused the decreased expression of CD3ζ in co-cultured Jurkat T-cells. Therefore, L-arginine availability can regulate the expression of CD3ζ, an essential component in T-lymphocyte signal transduction and function. L-arginine levels in the serum of normal individuals ranges from 115 μM to 210 μM . Our data show that at 48 hours, the levels of L-arginine in the trans-well tissue culture supernatant was 100 μM, a concentration sufficient to induce a decrease in CD3ζ expression.
Most tumor cells have a great demand for amino acids to support rapid proliferation and L-arginine is the first amino acid depleted faster than other nutrients by normal cell metabolism. Therefore, L-arginine could be a reasonable target of deprivation strategy for the type of tumors with a low recycling efficiency. Taken collectively, these findings, demonstrate that the availability of L-arginine and L-ornithine could be the limiting factor to control cell proliferation. We believe that treating RCC cells downstream from the L-arginine metabolic pathway by blocking polyamine production will have a major impact in suppressing tumor growth. This is supported by the use of DL-α-difluoromethylornithine, which completely blocks the proliferation of these cell lines independent of the presence of L-arginine, L-ornithine and arginase in addition to the promising anti-tumor effect in human tumors [43–45].
Renal cell carcinoma is a malignancy with poor prognosis due to its strong resistance to conventional cancer treatments and frequent metastases. With the standard immunotherapeutic treatment of IL-2 and IFNα for RCC, only 10–20% of the patients respond . This lack of response may be caused by the markedly impaired T-cell function associated with a decreased expression of the CD3ζ receptor. Therefore, it is still desirable to find better approaches to treat RCC. Modulating the L-arginine metabolic pathway by breaking down this amino acid required for tumor cell growth could be a novel approach to control it. The study of the mechanisms by which arginase II activity and L-arginine depletion affect tumor growth will help better understand the biology of RCC and its interaction with the immune system. The results of these studies may provide future therapeutic benefits.