Glen S. Germain

Imatinib Mesylate Is a Potent Inhibitor of the ABCG2 (BCRP) Transporter and Reverses Resistance to Topotecan and SN-38 in Vitro

Imatinib mesylate (Gleevec, STI571) is a kinase inhibitor selective for Bcr-Abl, activated c-Kit kinases, and platelet-derived growth factor receptor tyrosine kinase. Imatinib mesylate, similar to many other tyrosine kinase inhibitors (TKIs), such as members of the 4-anilinoquinazoline class, competes for ATP binding. Previously, 4-anilinoquinazoline TKIs have been shown to inhibit the function of the breast cancer resistance-associated drug transporter (ABCG2), reversing resistance to camptothecin derivatives topotecan and SN-38. However, the potential to inhibit ABCG2 for the 2-phenylamino-pyrimidine class of TKIs, exemplified by imatinib mesylate, has not been examined. Here, we show that imatinib mesylate potently reverses ABCG2-mediated resistance to topotecan and SN-38 and significantly increases accumulation of topotecan only in cells expressing functional ABCG2. However, overexpression of ABCG2 does not confer resistance to imatinib mesylate. Furthermore, accumulation and efflux of [14C]imatinib mesylate are unaltered between ABCG2-expressing and non-ABCG2-expressing cells or by ATP depletion. These results suggest that imatinib mesylate inhibits the function of ABCG2 but is not a substrate for this transporter.

Gefitinib enhances the antitumor activity and oral bioavailability of irinotecan in mice.

As a single agent the ERBB1 inhibitor, gefitinib (Iressa; ZD1839) showed minimal activity against a panel of 10 pediatric tumor xenografts that do not express the ERBB1 receptor. However, combined with irinotecan (CPT-11), significantly greater than additive activity was observed in four of eight models (P < 0.05), and the combination showed enhanced activity against three additional tumor lines. Breast cancer resistance protein (ABCG2), a transporter that confers resistance to SN-38 (the active metabolite of irinotecan), was readily detected in six of nine xenograft models examined by immunohistochemistry. In vitro gefitinib potently reversed resistance to SN-38 only in a cell line that overexpressed functional ABCG2. However, overexpression of ABCG2 did not decrease accumulation nor increase the rate of efflux of [14C]gefitinib. On the basis of these results and the distribution of Abcg2 in mouse tissues, we assessed the ability of gefitinib to modulate irinotecan pharmacokinetics. Oral gefitinib coadministration resulted in no change in clearance of intravenously administered irinotecan. However, gefitinib treatment dramatically increased the oral bioavailability of irinotecan after simultaneous oral administration. It is concluded that gefitinib may modulate SN-38 activity at the cellular level to reverse tumor resistance mediated by ABCG2 through inhibiting drug efflux and may be used potentially in humans to modulate the oral bioavailability of a poorly absorbed camptothecin such as irinotecan.

Cellular pharmacology of 5-fluorouracil in a human colon adenocarcinoma cell line selected for thymidine kinase deficiency

A human colon adenocarcinoma cell line (GC3TK-) was selected for thymidine kinase (TK) deficiency from cloned parental cells (GC3C1) by exposure to 5-bromodeoxyuridine (BrdUrd). The cellular pharmacology of 5-fluorouracil (FUra) and the influence of physiological concentrations of thymidine (dThd; 0.1 to 1 microM) on FUra cytotoxicity during brief exposure in both cell lines were examined. The uptake of FUra during a 1-hr drug exposure, its metabolism to ribo- and deoxyribonucleotides, incorporation into RNA, and inhibition of thymidylate synthase were similar in GC3C1 and GC3TK- cells as were the IC50 values for FUra (26 and 23 microM respectively). TK deficiency did not reduce the intracellular concentrations of FdUMP generated from FUra. In GC3C1, at FUra concentrations up to 100 microM, cytotoxicity was prevented by co-administration of dThd (0.1 to 20 microM). The relationship between cell survival and thymidylate synthase inhibition was close under these conditions. At higher drug concentrations, less dThd protection was observed, and none was detected in GC3TK- cells. Thus, the metabolism of FUra did not appear to be altered substantially in GC3C1 cells selected for TK deficiency. Also in these cells, at concentrations of FUra less than 100 microM, FUra cytotoxicity appeared to be mediated via the inhibition of thymidylate synthase.

Phosphorylation of nucleosides catalyzed by a mammalian enzyme other than uridine-cytidine kinase

Abstract Cells of a 3-deazauridine-resistant line of cultured lymphoblastoid cells, RPMI 6410, were found to be essentially devoid of uridine-cytidine kinase activity. The resistant cells tolerated high concentrations of 3-deazauridine in culture, but retained sensitivity, relative to the parent cells, to 6-azauridine and 5-azacytidine, compounds previously recognized as substrates for uridine-cytidine kinase. Intracellular formation of the 5′-monophosphate esters of these analogs is a necessary step in expression of their cytotoxicity. The sensitivity of 3-deazauridine-resistant cells to 6-azauridine and 5-azacytidine is interpreted as indicating that phosphorylation of these analogs by some enzymatic route other than uridine-cytidine kinase may occur in these cells.

Adenosine phosphorylase activity in mycoplasma-free growth media for mammalian cells.

Mammalian cells have enzymes that deaminate adenosine to inosine, which can readily be phosphorolysed to hypoxanthine. They do not, however, possess enzymes to form adenine by the cleavage of adenosine. For this reason, the release of adenine from adenosine by mammalian cell cultures has usually been interpreted as indicating the presence of mycoplasma, a frequent microbial contaminant that contains high levels of adenosine phosphorylase. We found that some human lymphoblast cultures free of mycoplasma showed high levels of adenosine cleavage and that this activity resulted from adenosine phosphorylase in the bovine serum used as the culture growth supplement. A survey of 13 serum supplements disclosed that fetal bovine serum (six lots) contains the highest adenosine phosphorylase activity, ranging from 9 to 648 nmol adenine produced per hour per ml serum; newborn calf serum (four lots) has much less activity, ranging from 0 to 5 nmol adenine produced per hour per ml serum; and donor horse serum (three lots) contains no detectable activity. These results suggest that mycoplasma tests dependent on the presence of adenosine phosphorylase or other enzyme activities may give false-positives with cultures containing fetal bovine serum supplements.

ANALYSIS OF THREE VARIABLE NUMBER TERMINAL REPEAT LOCI IS SUFFICIENT TO CHARACTERIZE THE DEOXYRIBONUCLEIC ACID FINGERPRINTS OF A PANEL OF HUMAN TUMOR CELL LINES

SummaryUsing primers for the MCT118, YNZ22, and COL2A1 loci in polymerase chain reaction analysis we could distinguish among the ∼20 cell lines routinely maintained in our laboratory. We also demonstrated that the cell line NB-1691 (a neuroblastoma) and its xenograft had an identical number of repeats at two loci. Rh30 (a rhabdomyosarcoma) made resistant to rapamycin was identical to its parent line and to a subline that had reverted to sensitivity after it was cultured without rapamycin in the medium.