Single amino acid (arginine) deprivation: rapid and selective death of cultured transformed and malignant cells

Abstract

The effects of arginine deprivation (–Arg) has been examined in 26 cell lines. Less than 10% of those with transformed or malignant phenotype survived for > 5 days, and many died more rapidly, notably leukaemic cells. Bivariate flow cytometry confirmed that vulnerable cell lines failed to move out of cell cycle into a quiescent state (G0), but reinitiated DNA synthesis. Many cells remained in S-phase, and/or had difficulty progressing through to G2 and M. Two tumour lines proved relatively ‘resistant’, A549 and MCF7. Although considerable cell loss occurred initially, both lines showed a ‘cell cycle freeze’, in which cells survived for > 10 days. These cells recovered their proliferative activity in +Arg medium, but behaved in the same manner to a second –Arg episode as they did to the first episode. In contrast, normal cells entered G0 and survived in –Arg medium for several weeks, with the majority of cells recovering with predictable kinetics in +Arg medium. In general, cells from a wide range of tumours and established lines die quickly in vitro following –Arg treatment, because of defective cell cycle checkpoint stringency, the efficacy of the treatment being most clearly demonstrated in co-cultures in which only the normal cells survived. The findings demonstrate a potentially simple, effective and non-genotoxic strategy for the treatment of a wide range of cancers. © 2000 Cancer Research Campaign

Arginine deprivation, growth inhibition and tumour cell death: 2. Enzymatic degradation of arginine in normal and malignant cell cultures

“…Almost identical data were recorded for cultures in complete medium treated with either bovine arginase or E. coli arginine decarboxylase (hereafter ETM1 and ETM2, respectively) in situ ( Figure 1A). Arrested cells entered G1/G0, as previously described in AFM by Scott et al (2000). In terms of cell growth inhibition kinetics sampled after 3 days treatment, arginase was active at 0.25 -0.5 units ml À1 (IC 50 B0.3 units ml À1 ), the inhibition becoming increasingly felt with time.…”

“…For experiments, they were set out in 2 ml of culture medium at 5 Â 10 4 cells ml À1 in 12-well plates. HeLa cells (cervical carcinoma) and human diploid fibroblasts (the latter being early passage numbers from primary cultures grown from human skin taken at breast reduction) were seeded at 50 000 ml À1 in flasks or plates in a similar manner previously described in Scott et al (2000). The cells were used at the lowest passage numbers that would provide adequate stocks of cells, they were checked as mycoplasm free, and they were authenticated from source, as required by the UKCCCR 'Guidelines on the use of cell lines in cancer research' (ohttp://ukcccr.icnet.uk4).…”

“…[Note: RPMI 1640 normally has an arginine concentration of 1.0 or 1.1 mM. However, in order to compare with most of our previous studies where arginine was kept at 0.4 mM (see Scott et al, 2000), this same (more critical) level was used in the present studies. )…”

Arginine deprivation, growth inhibition and tumour cell death: 3. Deficient utilisation of citrulline by malignant cells

“…This potential Achilles' heel in tumour metabolism deserves further exploitation in view of our previous findings (Scott et al, 2000;Campbell, 2001, 2002;Philip et al, 2003). Although melanomas do not exclusively show this argininosuccinate synthetase deficiency, the findings show that most cases of this dangerous and aggressive tumour can be appropriately and quickly selected for treatment.…”

“…creatine production, polyamine synthesis and nitric oxide (NO) generation). Its removal from culture medium by medium formulation or arginase treatment quickly leads to death in B80% of tumour cell lines (Scott et al, 2000). The addition of citrulline can often circumvent this deficiency, but few cells are capable of its biosynthesis in culture.…”