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We previously have found that 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) is a selective cytotoxin that enters cells via the extraneuronal transporter for monoamine transmitters (EMT). Both in vitro and in vivo studies demonstrated that SarCNU was more effective than BCNU against human gliomas. To clarify whether EMT expression correlates with antitumor efficacy of SarCNU, we determined human EMT (EMTh) and O(6)-methylguanine-DNA methyltransferase (MGMT) expression in nine human xenograft models using semiquantitative reverse-transcription polymerase chain reaction. These results were compared with the antitumor effects of SarCNU and the standard chloroethylnitrosourea antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). There was no significant correlation between EMTh expression and antitumor efficacy of SarCNU or BCNU. Also, there was no significant correlation between MGMT expression and SarCNU efficacy. However, a significant correlation was found between MGMT expression and BCNU antitumor efficacy. Interestingly, multiple regression analysis demonstrated a significant correlation between SarCNU efficacy and EMTh plus MGMT expression, whereas there was no correlation between BCNU efficacy and MGMT plus EMTh expression. Thus, the absence of a linear correlation between SarCNU efficacy and EMTh expression appears to be due, at least in part, to the presence of DNA repair, specifically, MGMT, in these xenograft models. These studies suggest that MGMT expression alone correlates with BCNU activity, whereas both EMTh and MGMT expression are important determinants of SarCNU activity against human tumor xenograft models. SarCNU is in clinical trials and these results may have important clinical implications.

We examined the O6-methylguanine-DNA methyltransferase (MGMT) protein as well as MGMT activity levels and the excision repair cross-complementing rodent repair deficiency gene, ERCC2 (XPD), protein levels in 14 human tumor cell lines not selected for chloroethylnitrosourea (CENU) resistance. These results were compared with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) cytotoxicity and UV light sensitivity. MGMT protein correlated significantly with MGMT activity (r = 0.9497, p = 0.0001). There was no significant linear correlation between BCNU cytotoxicity and MGMT content as determined by both Western analysis (r = 0.139, p = 0. 6348) and activity assay (r = 0.131, p = 0.6515). However, MGMT-rich cell lines were found to be more resistant than MGMT-poor cell lines to BCNU (t = 2.2375, p = 0.0225) but not to UV (t = 1.1734, p = 0.1317). Furthermore, the most BCNU-sensitive cell lines were all MGMT-poor. UV sensitivity was significantly correlated to BCNU cytotoxicity (r = 0.858, p = 0.0001). Significant correlations were found between ERCC2 protein levels and BCNU cytotoxicity (r = 0.786, p = 0.0009) or UV sensitivity (r = 0.874, p = 0.0001). Our results confirm that MGMT plays an important role in CENU resistance, but not in UV resistance. The correlation of UV sensitivity with BCNU cytotoxicity suggests that nucleotide excision repair is an important modifying factor of MGMT-mediated innate CENU resistance in human tumor cell lines, especially in highly resistant cell lines. ERCC2 may be implicated in this process.

The transport of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU), an experimental anticancer compound, was investigated in the human glioma cell line SK-MG-1. The transport of [3H]SarCNU was examined in suspension. The uptake of [3H] SarCNU was found to be temperature dependent, with influx being linear to 4 sec at 37 degrees. Equilibrium was reached after 1 min at 22 degrees and 37 degrees, with accumulation slightly above unity. SarCNU was not significantly metabolized in the cells after a 60-min incubation at 37 degrees, as shown by thin layer chromatography. At 37 degrees, uptake of [3H]SarCNU was found to be saturable, sodium independent, and energy independent. Previous work demonstrated that SarCNU was able to inhibit the uptake of sarcosinamide, which is transported by the catecholamine uptake 2 system. This catecholamine system mediates the physiological transport of epinephrine. Epinephrine was able to significantly inhibit the uptake of [3H]SarCNU, at a concentration of 50 microM, by 40%. Additionally, several amino acids were unable to inhibit the uptake of SarCNU. The initial rate of SarCNU influx is mediated by both facilitated and nonfacilitated diffusion. The nonfacilitated diffusion rate could be estimated from the linear concentration dependence of the residual influx rate for SarCNU, which was not inhibited by the presence of excess co-permeant (epinephrine). Dixon plot analysis, corrected for nonfacilitated diffusion of SarCNU, revealed that epinephrine inhibited the uptake of SarCNU competitively, with a Ki of 163 +/- 15 microM, a value similar to the Km value for epinephrine influx in SK-MG-1 cells. Additionally, after appropriate corrections for nonfacilitated diffusion in the influx rates observed for SarCNU, it was revealed that SarCNU influx obeyed Michaelis-Menten kinetics over a 200-fold range of concentrations, with a Km of 2.39 +/- 0.37 mM and a Vmax of 236 +/- 53 pmol/microliters of intracellular water/sec. Metabolic poisons (2,4-dinitrophenol, iodoacetate, NaCN, NaF, or ouabain) were unable to inhibit the influx of SarCNU, suggesting that the carrier-mediated uptake of SarCNU is energy independent and mediated by facilitated diffusion. These findings indicate that SarCNU uptake in SK-MG-1 cells is mediated both by nonfacilitated diffusion and by facilitated diffusion via the catecholamine uptake 2 carrier system. SarCNU is the first chloroethylnitrosourea that has been demonstrated to have carrier-mediated uptake. Moreover, this carrier-mediated uptake may play a role in the increased cytotoxicity of SarCNU against gliomas, compared with that of 1,3-bis(2,-chloroethyl)-1-nitrosourea, which enters cells primarily by passive diffusion.

Resistance to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU), an experimental antitumor compound, was investigated in the sensitive SK-MG-1 cells and the 20-fold more resistant SKI-1 human glioma cells [which are 3-fold more resistant to 1,3,bis(2-chloroethyl)-1-nitrosourea (BCNU)]. The transport of SarCNU was examined by utilizing tritiated sarcosinamide. Sarcosinamide uptake into SK-MG-1 cells is via the catecholamine carrier that accommodates epinephrine. Dixon plot analysis of SarCNU inhibition of sarcosinamide uptake reveals that SarCNU is also accommodated by this carrier. The uptake of 0.5 mM [3H]sarcosinamide was temperature dependent, with similar levels of intracellular sarcosinamide accumulating at steady state in both cell lines. The uptake of sarcosinamide in SKI-1 cells obeyed Michaelis-Menten kinetics over a 200-fold range of concentrations with a Km of 1.52 +/- 0.151 mM and Vmax of 0.659 +/- 0.066 nmol/10(6) cells/min. This represents a more than 5-fold decrease in the uptake affinity and a more than 4-fold increase in the transport capacity compared with SK-MG-1 cells (Km = 0.282 +/- 0.041 mM; Vmax = 0.154 +/- 0.024 nmol/10(6) cells/min). The initial rate of sarcosinamide uptake is similar in both cell lines. Dixon plot analysis confirmed that SarCNU is a competitive inhibitor of sarcosinamide transport in SKI-1 cells with a Ki of 17.5 mM, which is more than 5-fold greater than the Ki obtained in SK-MG-1 cells. The steady state accumulation of SarCNU is significantly reduced by 47% in SKI-1 cells compared with the SK-MG-1 cells (cell to medium ratios of 0.65 +/- 0.11 and 1.22 +/- 0.08, respectively) (p less than 0.005). The accumulation of BCNU was comparable in the two cell lines. Since the Vmax of sarcosinamide (SarCNU) uptake is increased in the SKI-1 cells, the decrease in intracellular SarCNU is not related to decreased drug influx via the catecholamine carrier in SKI-1 cells. The efflux of tritiated sarcosinamide was temperature dependent and similar in both cell lines, with 54 and 58% of sarcosinamide being freely exchangeable in SKI-1 and SK-MG-1 cells, respectively. SarCNU efflux may or may not be altered. Since the expression of mdr is higher in the sensitive cells, it is unlikely that increased efflux of SarCNU mediated by the P-glycoprotein is responsible for drug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)