Mechanisms of resistance to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) in sensitive and resistant human glioma cells.
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)
Skalski
V
V
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada.
Yarosh
D B
DB
Batist
G
G
Gros
P
P
Feindel
W
W
Kopriva
D
D
Panasci
L C
LC
D000123:Acetyltransferases / Q000032:analysis; D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D001692:Biological Transport; D002330:Carmustine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases; D004351:Drug Resistance; D005910:Glioma / Q000473:pathology*; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000032:analysis; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D004247:DNA; C044565:2-((((2-chloroethyl)nitrosoamino)carbonyl)amino)propanamide; D000123:Acetyltransferases; C020646:glutamate acetyltransferase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D002330:Carmustine