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Effect of arginase II on L-arginine depletion and cell growth in murine cell lines of renal cell carcinoma.

Tate DJ, Vonderhaar DJ, Caldas YA, Metoyer T, Patterson JR, Aviles DH, Zea AH - J Hematol Oncol (2008)

Bottom Line: Cell growth was independent of the amount of arginase activity expressed by the cells. nor-NOHA significantly (P = 0.01) reduced arginase II activity and suppressed cell growth in cells exhibiting high arginase activity.The depletion of L-arginine by mRCC induced the decrease expression of CD3zeta a key element for T-cell function.The results of this study show for the first time that arginase II produced by RCC cell lines depletes L-arginine resulting in decreased expression of CD3zeta.Understanding the interplay between arginase II and its interaction with the immune system may provide future therapeutic benefits to treat patients with RCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stanley S, Scott Cancer Center, LSUHSC, New Orleans, USA. dtate1@lsuhsc.edu

ABSTRACT

Background: L-arginine is the common substrate for the two isoforms of arginase. Arginase I, highly expressed in the liver and arginase II mainly expressed in the kidney. Arginase I-producing myeloid derived suppressor cells have been shown to inhibit T-cell function by the depletion of L-arginine. On the other hand, arginase II has been detected in patients with cancer and is thought to metabolize L-arginine to L-ornithine needed to sustain rapid tumor growth; however its role in L-arginine depletion is unclear. Thus, in tumor biology, L-arginine metabolism may play a dual role in tumor growth and in the induction of T cell dysfunction. Therefore, we studied in murine renal cell carcinoma (RCC) cell lines, the effect of arginase II on tumor cell proliferation and L-arginine depletion. The effect of arginase inhibitors on cell proliferation was also tested.

Methods: Three murine renal cell carcinoma (mRCC) cell lines were tested for the presence of arginase. nor-NOHA, an arginase inhibitor was used to substantiate the effect of arginase on cell growth and L-arginine depletion. Amino acid levels were tested by HPLC.

Results: Our results show that mRCC cell lines express only arginase II and were able to deplete L-arginine from the medium. Cell growth was independent of the amount of arginase activity expressed by the cells. nor-NOHA significantly (P = 0.01) reduced arginase II activity and suppressed cell growth in cells exhibiting high arginase activity.The depletion of L-arginine by mRCC induced the decrease expression of CD3zeta a key element for T-cell function.

Conclusion: The results of this study show for the first time that arginase II produced by RCC cell lines depletes L-arginine resulting in decreased expression of CD3zeta. These results indicate that RCC cell lines expressing arginase II can modulate the L-arginine metabolic pathway to regulate both cell growth and T-cell function. Blocking arginase may lead to a decrease in RCC cell growth and aid in restoring immune function by increasing L-arginine availability for T-cell use. Understanding the interplay between arginase II and its interaction with the immune system may provide future therapeutic benefits to treat patients with RCC.

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Effect of nor-NOHA on arginase activity and amino acid levels. (A) Significant arginase inhibition was observed in cell lysates of CL-19 cultures treated with nor-NOHA (2 mM) after 48 (*P = 0.002) and 72 hours (** P = 0.001) as compared to untreated cells. (B) Effect of nor-NOHA in inhibiting both L-arginine (μM) depletion (*P = 0.001) and L-ornithine (μM) production (**P < 0.0001) in the supernatants of CL-19 cultures, as compared to CL-19 untreated cultures. Results are expressed as means ± SE of duplicate determinations from four independent experiments.
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Figure 4: Effect of nor-NOHA on arginase activity and amino acid levels. (A) Significant arginase inhibition was observed in cell lysates of CL-19 cultures treated with nor-NOHA (2 mM) after 48 (*P = 0.002) and 72 hours (** P = 0.001) as compared to untreated cells. (B) Effect of nor-NOHA in inhibiting both L-arginine (μM) depletion (*P = 0.001) and L-ornithine (μM) production (**P < 0.0001) in the supernatants of CL-19 cultures, as compared to CL-19 untreated cultures. Results are expressed as means ± SE of duplicate determinations from four independent experiments.

Mentions: Since nor-NOHA significantly inhibited cell proliferation of CL-19, we wanted to test the effect of this inhibitor on arginase activity and L-ornithine production. Arginase activity increased in this cell line over the time of the experiments. When 2 mM of nor-NOHA was added to the cultures, significant reduction in arginase activity occurred at 48 and 72 hours (P = 0.002 and P = 0.001 respectively) (Figure 4A). Importantly, independent of the amount of arginase produced by this cell line, the effect of arginase inhibition by nor-NOHA was similar at all time points tested. The inhibition of arginase activity in CL-19 by nor-NOHA significantly blocked (P = 0.0001) the depletion of L-arginine as well as the accumulation of L-ornithine (P < 0.0001) after 48 hours compared to CL-19 cultures without the inhibitor (Figure 4B). We did not observe significant changes in arginase inhibition, L-arginine depletion and L-ornithine production when nor-NOHA was added to cultures with CL-2 and Renca cells (data not shown).


Effect of arginase II on L-arginine depletion and cell growth in murine cell lines of renal cell carcinoma.

Tate DJ, Vonderhaar DJ, Caldas YA, Metoyer T, Patterson JR, Aviles DH, Zea AH - J Hematol Oncol (2008)

Effect of nor-NOHA on arginase activity and amino acid levels. (A) Significant arginase inhibition was observed in cell lysates of CL-19 cultures treated with nor-NOHA (2 mM) after 48 (*P = 0.002) and 72 hours (** P = 0.001) as compared to untreated cells. (B) Effect of nor-NOHA in inhibiting both L-arginine (μM) depletion (*P = 0.001) and L-ornithine (μM) production (**P < 0.0001) in the supernatants of CL-19 cultures, as compared to CL-19 untreated cultures. Results are expressed as means ± SE of duplicate determinations from four independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562378&req=5

Figure 4: Effect of nor-NOHA on arginase activity and amino acid levels. (A) Significant arginase inhibition was observed in cell lysates of CL-19 cultures treated with nor-NOHA (2 mM) after 48 (*P = 0.002) and 72 hours (** P = 0.001) as compared to untreated cells. (B) Effect of nor-NOHA in inhibiting both L-arginine (μM) depletion (*P = 0.001) and L-ornithine (μM) production (**P < 0.0001) in the supernatants of CL-19 cultures, as compared to CL-19 untreated cultures. Results are expressed as means ± SE of duplicate determinations from four independent experiments.
Mentions: Since nor-NOHA significantly inhibited cell proliferation of CL-19, we wanted to test the effect of this inhibitor on arginase activity and L-ornithine production. Arginase activity increased in this cell line over the time of the experiments. When 2 mM of nor-NOHA was added to the cultures, significant reduction in arginase activity occurred at 48 and 72 hours (P = 0.002 and P = 0.001 respectively) (Figure 4A). Importantly, independent of the amount of arginase produced by this cell line, the effect of arginase inhibition by nor-NOHA was similar at all time points tested. The inhibition of arginase activity in CL-19 by nor-NOHA significantly blocked (P = 0.0001) the depletion of L-arginine as well as the accumulation of L-ornithine (P < 0.0001) after 48 hours compared to CL-19 cultures without the inhibitor (Figure 4B). We did not observe significant changes in arginase inhibition, L-arginine depletion and L-ornithine production when nor-NOHA was added to cultures with CL-2 and Renca cells (data not shown).

Bottom Line: Cell growth was independent of the amount of arginase activity expressed by the cells. nor-NOHA significantly (P = 0.01) reduced arginase II activity and suppressed cell growth in cells exhibiting high arginase activity.The depletion of L-arginine by mRCC induced the decrease expression of CD3zeta a key element for T-cell function.The results of this study show for the first time that arginase II produced by RCC cell lines depletes L-arginine resulting in decreased expression of CD3zeta.Understanding the interplay between arginase II and its interaction with the immune system may provide future therapeutic benefits to treat patients with RCC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stanley S, Scott Cancer Center, LSUHSC, New Orleans, USA. dtate1@lsuhsc.edu

ABSTRACT

Background: L-arginine is the common substrate for the two isoforms of arginase. Arginase I, highly expressed in the liver and arginase II mainly expressed in the kidney. Arginase I-producing myeloid derived suppressor cells have been shown to inhibit T-cell function by the depletion of L-arginine. On the other hand, arginase II has been detected in patients with cancer and is thought to metabolize L-arginine to L-ornithine needed to sustain rapid tumor growth; however its role in L-arginine depletion is unclear. Thus, in tumor biology, L-arginine metabolism may play a dual role in tumor growth and in the induction of T cell dysfunction. Therefore, we studied in murine renal cell carcinoma (RCC) cell lines, the effect of arginase II on tumor cell proliferation and L-arginine depletion. The effect of arginase inhibitors on cell proliferation was also tested.

Methods: Three murine renal cell carcinoma (mRCC) cell lines were tested for the presence of arginase. nor-NOHA, an arginase inhibitor was used to substantiate the effect of arginase on cell growth and L-arginine depletion. Amino acid levels were tested by HPLC.

Results: Our results show that mRCC cell lines express only arginase II and were able to deplete L-arginine from the medium. Cell growth was independent of the amount of arginase activity expressed by the cells. nor-NOHA significantly (P = 0.01) reduced arginase II activity and suppressed cell growth in cells exhibiting high arginase activity.The depletion of L-arginine by mRCC induced the decrease expression of CD3zeta a key element for T-cell function.

Conclusion: The results of this study show for the first time that arginase II produced by RCC cell lines depletes L-arginine resulting in decreased expression of CD3zeta. These results indicate that RCC cell lines expressing arginase II can modulate the L-arginine metabolic pathway to regulate both cell growth and T-cell function. Blocking arginase may lead to a decrease in RCC cell growth and aid in restoring immune function by increasing L-arginine availability for T-cell use. Understanding the interplay between arginase II and its interaction with the immune system may provide future therapeutic benefits to treat patients with RCC.

Show MeSH
Related in: MedlinePlus