Limits...
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.

Show MeSH

Related in: MedlinePlus

L-arginine, L-ornithine and L-glutamine levels. Tissue culture supernatants from CL-2, CL-19, and Renca cells were collected at 24, 48, and 72 hours. They were analyzed by HPLC after deproteinization with methanol and derivatization with OPA for (A) L-arginine and (B) L-ornithine and (C) L-glutamine. Standards of L-arginine, L-ornithine and L-glutamine in methanol were run with each experiment. Results are expressed as means ± SE of duplicate determinations from four independent experiments. (* P = 0.005. ** P < 0.0001 significant differences for CL-19 compared to the other cell lines).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2562378&req=5

Figure 2: L-arginine, L-ornithine and L-glutamine levels. Tissue culture supernatants from CL-2, CL-19, and Renca cells were collected at 24, 48, and 72 hours. They were analyzed by HPLC after deproteinization with methanol and derivatization with OPA for (A) L-arginine and (B) L-ornithine and (C) L-glutamine. Standards of L-arginine, L-ornithine and L-glutamine in methanol were run with each experiment. Results are expressed as means ± SE of duplicate determinations from four independent experiments. (* P = 0.005. ** P < 0.0001 significant differences for CL-19 compared to the other cell lines).

Mentions: The effect of arginase II on L-arginine, L-ornithine, and L-glutamine content in the conditioned culture medium was assessed by HPLC. The CL-19 cell line, which expressed high levels of arginase II, depleted the media L-arginine concentration by about 50% at 24 hours (P = 0.005, Figure 2A) and about 90% at 48 and 72 hours (P < 0.001) when compared to media controls. L-arginine levels remained unchanged in CL-2 and Renca cultures throughout the experimental time points. Concomitantly, there was a significant increase in L-ornithine production by CL-19 after 48 and 72 hours (P = 0.001 and P < 0.0001) compared to CL-2 and Renca cell lines in which the levels did not change significantly at any time point (Figure 2B). All cell lines also depleted the culture supernatants of L-glutamine at the same rate during the first 24 hours. However, at 72 hours, the depletion of L-glutamine was significantly higher in CL-2 and Renca (P = 0.001 and P = 0.016) than in CL-19 (Figure 2C).


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)

L-arginine, L-ornithine and L-glutamine levels. Tissue culture supernatants from CL-2, CL-19, and Renca cells were collected at 24, 48, and 72 hours. They were analyzed by HPLC after deproteinization with methanol and derivatization with OPA for (A) L-arginine and (B) L-ornithine and (C) L-glutamine. Standards of L-arginine, L-ornithine and L-glutamine in methanol were run with each experiment. Results are expressed as means ± SE of duplicate determinations from four independent experiments. (* P = 0.005. ** P < 0.0001 significant differences for CL-19 compared to the other cell lines).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: L-arginine, L-ornithine and L-glutamine levels. Tissue culture supernatants from CL-2, CL-19, and Renca cells were collected at 24, 48, and 72 hours. They were analyzed by HPLC after deproteinization with methanol and derivatization with OPA for (A) L-arginine and (B) L-ornithine and (C) L-glutamine. Standards of L-arginine, L-ornithine and L-glutamine in methanol were run with each experiment. Results are expressed as means ± SE of duplicate determinations from four independent experiments. (* P = 0.005. ** P < 0.0001 significant differences for CL-19 compared to the other cell lines).
Mentions: The effect of arginase II on L-arginine, L-ornithine, and L-glutamine content in the conditioned culture medium was assessed by HPLC. The CL-19 cell line, which expressed high levels of arginase II, depleted the media L-arginine concentration by about 50% at 24 hours (P = 0.005, Figure 2A) and about 90% at 48 and 72 hours (P < 0.001) when compared to media controls. L-arginine levels remained unchanged in CL-2 and Renca cultures throughout the experimental time points. Concomitantly, there was a significant increase in L-ornithine production by CL-19 after 48 and 72 hours (P = 0.001 and P < 0.0001) compared to CL-2 and Renca cell lines in which the levels did not change significantly at any time point (Figure 2B). All cell lines also depleted the culture supernatants of L-glutamine at the same rate during the first 24 hours. However, at 72 hours, the depletion of L-glutamine was significantly higher in CL-2 and Renca (P = 0.001 and P = 0.016) than in CL-19 (Figure 2C).

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