Development of an O(6)-alkylguanine-DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene-3H]O(6)-benzylguanine to the protein

Abstract

Although it is known that (i) O(6)-alkylguanine-DNA alkyltransferase (AGT) confers tumor cell resistance to guanine O(6)-targeting drugs such as cloretazine, carmustine, and temozolomide and that (ii) AGT levels in tumors are highly variable, measurement of AGT activity in tumors before treatment is not a routine clinical practice. This derives in part from the lack of a reliable clinical AGT assay; therefore, a simple AGT assay was devised based on transfer of radioactive benzyl residues from [benzene-3H]O(6)-benzylguanine ([3H]BG) to AGT. The assay involves incubation of intact cells or cell homogenates with [3H]BG and measurement of radioactivity in a 70% methanol precipitable fraction. Approximately 85% of AGT in intact cells was recovered in cell homogenates. Accuracy of the AGT assay was confirmed by examination of AGT levels by Western blot analysis with the exception of false-positive results in melanin-containing cells due to [3H]BG binding to melanin. Second-order kinetic constants for human and murine AGT were 1100 and 380 M(-1)s(-1), respectively. AGT levels in various human cell lines ranged from less than 500 molecules/cell (detection limit) to 45,000 molecules/cell. Rodent cell lines frequently lacked AGT expression, and AGT levels in rodent cells were much lower than in human cells.

Fig. 1

Structure of [³H]BG. Counting efficiency was determined as described in Materials and methods. Stoichiometry was derived from the counting efficiency and definitions; Avogadro’s number = 6.022 × 10²³ mol^-1 and 1 μCi = 2.22 × 10⁶ dpm. Because mass spectrometry indicated that [³H]BG consisted of a mixture of unlabeled, ³H-mono-labeled, ³H-di-labeled, and ³H-tri-labeled materials, ³H was placed in the center of the benzene ring to indicate that ³H was located in the benzene ring of O⁶-BG.

Fig. 2

Rate of [³H]benzyl transfer to AGT in intact HL-60 and L1210/AGT1 cells. Cells (2 × 10⁶ cells/100 μl) were incubated with 1 μCi of [³H]BG in the absence or presence of 20 μM MG132 or 20 μM CHX for the indicated periods of time, and radioactivity in a 70% methanol-insoluble fraction was determined. Radioactivity in the presence of excess unlabeled O⁶-BG was subtracted from the total radioactivity. The bottom panels show the plots used to determine the second-order kinetic constant (k).

Fig. 3

Effects of various purines on the reaction of O⁶-BG with AGT. (A) HL-60 cells were incubated with [³H]BG in the absence or presence of 150 μM various purines for 2 h. TMG was synthesized in our laboratory and dissolved in Me₂SO at a concentration of 20 mM. (B) HL-60 cells were exposed to O⁶-BG or TMG for 1 h, followed by the AGT assay for 1 h, to measure remaining AGT activity.

Fig. 4

Inability of O⁶-BG to serve as a substrate for hypoxanthine—guanine phosphoribosyltransferase. Cytoplasmic extracts (120 μg of protein) from wild-type (WT) and HGPRT^–F-MEL cells were incubated with 0.2 mM [¹⁴C]6-MP or [³H]BG at a radioactive concentration of 2 μCi/ml and 2 mM 5-phosphoribosyl-1-pyrophosphate in a volume of 200 μl. NMP, nucleoside monophosphate.

Fig. 5

Optimal temperature for AGT activity and temperature-dependent increase in nonspecific binding. Standard AGT assays using intact HL-60 cells were conducted at various temperatures (Temp) for 30 min.

Fig. 6

AGT assay using cell homogenates. (A) Effects of various ingredients on AGT activity were measured using HL-60 cell homogenates prepared in H₂O(5 × 10⁶ cells/ 100 μl). PMSF, phenylmethylsulfonylfluoride. (B) Kinetics of [³H]benzyl transfer using HL-60 cell homogenates (5 × 10⁶ cells/100 μl) prepared in a buffer (pH 7.5) containing 1 mM DTT and plots to determine the second-order kinetic constant (k).

Fig. 7

AGT assays conducted for various melanoma cell lines and the correlation between AGT levels measured by the AGT assay and Western blot analysis. (A) Standard AGT assays were performed for various human melanoma cell lines and for intact B₁₆F₁₀ cells in the presence of HQ or L-DOPA. HQ was dissolved in Me₂SO at a concentration of 20 mM. L-DOPA was dissolved in 0.001 N HCl at a concentration of 5 mM. (B) Whole cell extracts were prepared from various human cell lines and subjected to Western blot analysis for AGT using HSC 70 as a loading control. K, thousands (000) (far right of panel B).