Cancer Letters

Cancer Letters

Volume 191, Issue 2, 10 March 2003, Pages 165-170
Cancer Letters

Resistance to the anti-proliferative activity of recombinant arginine deiminase in cell culture correlates with the endogenous enzyme, argininosuccinate synthetase

https://doi.org/10.1016/S030-43835(02)00693-6Get rights and content

Abstract

Recombinant mycoplasma enzyme, arginine deiminase (rADI), has been proposed as a possible cancer treatment via arginine depletion. However, many cell lines are resistant to rADI-treatment, even though most require arginine for proliferation. We compared eight different cell lines for sensitivity in cell proliferation to the effect of either rADI or arginine deprivation. The activity of argininosuccinate synthetase (AS), the rate-limiting enzyme for converting citrulline to arginine, was also measured. Our results indicate that resistance to rADI-treatment may correlate with cellular AS activity, either constitutive or inducible, allowing cell survival by conversion of the product of the rADI reaction, i.e. citrulline to arginine.

Introduction

Limited success has been achieved using amino acid degrading enzymes in cancer therapy. To date, only l-asparaginase is in clinical use for treating acute lymphoblastic leukemia and a few sub-types of non-Hodgkin's lymphoma [1]. The therapeutic effectiveness of amino acid deprivation therapy in cancer treatment depends not only on the enzymatic properties, such as specific activity, Km, optimum pH and stability, but more importantly on the susceptibility of the target cells to the lack of the specific amino acid. For this reason, the unique and versatile amino acid l-arginine (l-arg) would seem to be an ideal candidate for therapeutic enzymatic amino acid degradation. Deprivation studies have demonstrated the unique requirement of l-arg for cultured transformed and malignant cells as well as human diploid fibroblasts [2], [3].

l-arg, although not classified as an essential amino acid for healthy adult humans, has evolved as an essential player in many cellular functions: as a precursor of protein and polyamine synthesis, a substrate of the novel arginine-dependent nitric oxide (NO) pathway, and a myriad of processes that are affected by NO in post-translational regulation [4].

Arginine deiminase (ADI) is a Mycoplasma enzyme that catalyzes the imine hydrolysis of l-arg to l-citrulline (l-cit) and ammonia. It has been reported that ADI can inhibit cell proliferation in vitro [5], [6], [7], [8] and tumor growth in vivo [9]. The anti-tumor activity of ADI was observed in tumor bearing mice with no apparent toxicity at 100 times the minimum effective dose [9]. The mechanism by which ADI exerts its anti-tumor effect has yet to be determined; however, it has been suggested that it may be due to simple amino acid depletion or inhibition of tumor angiogenesis [7], [10]. ADI was cloned by polymerase chain reaction (PCR) from mycoplasma genomic DNA [11] and the recombinant ADI (rADI) was purified to homogeneity in our laboratory [10]. In this report, we explore the effect of l-arg deprivation with arginine-free medium or rADI treatment in eight cell lines. The correlation of the cell proliferation and the argininosuccinate synthetase (AS) activity in these cell lines is investigated.

Section snippets

Materials

ADI was cloned by PCR from Mycoplasma genomic DNA [11] and the recombinant ADI was purified to homogeneity in our laboratory [10]. The specific activity of the renatured rADI was 32.7 U/mg protein, similar to the reported value for mycoplasmic-derived ADI (37 U/mg) [5]. [14C]Aspartic acid (200 mCi/mmol) was purchased from Moravek Biochemicals (Brea, CA). Dowex 1-X8-200-400 resin was obtained from Supelco (Bellefonte, PA). Micro BCA protein assay reagent kit was obtained from Pierce (Rockford,

Effect of rADI on cell growth

Experiments were performed to determine the range of rADI concentrations that would elicit growth inhibition. The in vitro growth-inhibitory activity of rADI in the different cell lines is shown in Fig. 1. rADI appreciably inhibited the growth of CHO, HUVE, MDCK, and A549 cells in a dose-response manner at concentrations between 0.01 mU/ml and 1.0 mU/ml. The rADI concentration required for 50% growth inhibition (IC50) was below 0.1 mU/ml for CHO, HUVE, and MDCK. The IC50 values for A549 and

Discussion

rADI has been suggested for use as an anti-tumor agent by several research groups, as it was assumed that rADI would be an effective inhibitor of proliferation for arginine-dependent tumor cells [5], [6], [7], [8], [9]. In this report, we found that in the eight cell lines tested, all required arginine for their growth (Fig. 2); however, sensitivity towards rADI-mediated growth inhibition varied extensively (Fig. 1). Most noteworthy is that L929 and HeLa cells are the most arginine-dependent

Acknowledgements

This study was supported by a grant, 6IB-0045, from the California Breast Cancer Research Program.

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    Citation Excerpt :

    ADI-based arginine deprivation therapy has been shown to effectively inhibit the growth of various kinds of ASS-deficient human cancer cells incapable of synthesizing arginine endogenously.9–14 Conversely, overexpression of ASS, or re-expression of ASS in ASS-deficient melanoma cells conferred resistance to ADI treatment.9,10,15,16 Immunological detection of tumor ASS status was thus proposed to be an useful method to identify patients who are likely to respond to arginine deprivation therapy.17

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