Transport of 5-fluorouracil and uracil into human erythrocytes.

The transport of 5-fluorouracil (5-FU) and uracil into human erythrocytes has been investigated under initial velocity conditions with an "inhibitor-stop" assay using a cold papaverine solution to terminate influx. At 37 degrees and pH 7.3, 5-FU influx was nonconcentrative; was partially inhibited by adenine, hypoxanthine, thymine, and uracil; and was insensitive to inhibition by nucleosides or inhibitors of nucleoside transport. Inhibition of the influx of 5-FU or uracil by adenine (3.0 mM) did not increase when other pyrimidines or inhibitors of nucleoside transport were combined with adenine. 5-FU and uracil exhibited similar saturable (Km approximately 4 mM, Vmax approximately 500 pmol/sec/5 microL cells) and nonsaturable (rate constant approximately 80 pmol/sec/mM/5 microL cells) components of influx. 5-FU, uracil, adenine, and hypoxanthine were competitive inhibitors of each other's influx with Ki values matching their respective Km values for influx. We conclude that 5-FU and uracil enter human erythrocytes at similar rates via both nonfacilitated diffusion and the same carrier that transports adenine and hypoxanthine.

Membrane permeation mechanisms of 2',3'-dideoxynucleosides.

The mechanism of membrane permeation of several 2',3'-dideoxynucleosides was investigated at 37 degrees with human erythrocytes using an "inhibitor-stop" assay. Transport (per 5 microL cells) via the nucleoside and nucleobase carriers was assessed by inhibition of influx with dilazep and adenine, respectively. Mechanisms of cellular entry were highly individualized: 2',3'-dideoxyadenosine and 3'-deoxythymidin-2'-ene via nonfacilitated diffusion, with high rates; 2',3'-dideoxyguanosine mainly via the nucleobase carrier (Km = 390 microM, Vmax = 32 pmol/sec); 2',3'-dideoxyinosine by both nucleobase (Km = 850 microM, Vmax = 2.7 pmol/sec) and nucleoside (Km = 7.4 mM, Vmax = 16 pmol/sec) carriers, with a low rate of nonfacilitated diffusion; and 2',3'-dideoxycytidine, equally by the nucleoside carrier (Km = 23 mM, Vmax = 65 pmol/sec) and by nonfacilitated diffusion, with a low rate. These results demonstrate that the nucleobase carrier plays an important role in the influx of two of these dideoxynucleotides and that nonfacilitated diffusion is not necessarily the chief mode of membrane permeation of this class of drugs.

Inhibition of thymidine transport by 3'-azido-3'-deoxythymidine and its metabolites.

3'-Azido-3'-deoxythymidine (AZT) competitively inhibited the transport of thymidine (Km = 0.23 mM) into human erythrocytes with a Ki of 1.0 mM at 37 degrees C. The principal human metabolite of AZT in plasma, the 5'-glucuronide (GAZT), was a weak inhibitor of the nucleoside transporter (< 20% inhibition of the influx of 1.0 microM thymidine by 10 microM GAZT). The minor AZT metabolite, 3'-amino-3'-deoxythymidine (AMT), competitively inhibited thymidine transport with a Ki of 9.1 mM. The influx of AMT into human erythrocytes was found to be a saturable process (Km = 12 mM) that was largely inhibited by dilazep, thus indicating that AMT influx occurs via the nucleoside transporter. High extracellular concentrations of AZT may contribute to the synergistic cytotoxicity of AZT plus either 5-fluorouracil or methotrexate by inhibiting thymidine transport into cancer cells whose de novo biosynthesis of dTMP is impaired pharmacologically or by inhibiting efflux of 2'-deoxy-5-fluorouridine and/or 2'-deoxyuridine from these cells.

Enantiomeric selectivity of carbovir transport.

Carbovir (9-[4 alpha-(hydroxymethyl)cyclopent-2-ene-1 alpha-yl]guanine) (CBV) is a carbocyclic analogue of 2',3'-dideoxyguanosine that exhibits potent and selective in vitro activity against human immunodeficiency virus. Antiviral activity is associated with only the (-)-enantiomer. The transport characteristics of both (-)-CBV and (+)-CBV were investigated in human erythrocytes at 37 degrees C using a papaverine-stop assay. The influx of both enantiomers appeared saturable and was inhibited greater than 90% by a combination of adenine (a low Km permeant of the nucleobase carrier) and dilazep (a potent inhibitor of nucleoside transport). The influx of (-)-CBV and (+)-CBV proceeded primarily via the nucleobase carrier with Vmax (picomoles/second/5 microliters of cells)/Km (millimolar) values of 17/0.12 and 140/1.9, respectively. To a lesser extent, the influx of (-)-CBV and (+)-CBV also occurred via the nucleoside transporter. Although both compounds exhibited a similar low affinity for this latter carrier (Km approximately 2 mM), the Vmax for (-)-CBV influx was approximately 4-fold higher than the Vmax for (+)-CBV influx. We conclude that both CBV enantiomers enter human erythrocytes by two transporters that are enantiomerically selective.

Membrane permeation characteristics of 5'-modified thymidine analogs.

The membrane permeation characteristics of 5'-deoxythymidine (5'-ddThd) and 5'-azido-5'-deoxythymidine (5'-N3-5'-ddThd) were investigated in human erythrocytes, with an inhibitor-stop assay, at 20 degrees. Uptake of both nucleoside analogs occurred without metabolism, was nonconcentrative, and was partially inhibited by nucleosides or inhibitors of nucleoside transport at micromolar permeant concentrations. At higher permeant concentrations (greater than 1.0 mM), the influx rate of each analog was linearly dependent on concentration and insensitive to inhibition by nucleosides, inhibitors of nucleoside transport, and nucleobases. Kinetic analyses using nonlinear regression revealed that a saturable component of 5'-ddThd influx (Km = 200 microM) was competitively inhibited by thymidine (dThd) (Ki = 86 microM) or 5-iodo-2'-deoxyuridine (Ki = 84 microM). Similarly, a saturable component of 5'-N3-5'-ddThd influx (Km = 220 microM) was competitively inhibited by 2-chloroadenosine (Ki = 18 microM). The Ki values for these nucleoside inhibitors were similar to their reported Km values as permeants of the nucleoside transporter. Both 5'-ddThd and 5'-N3-5'-ddThd competitively inhibited the influx of dThd (Km = 60 microM), with similar Ki values (150 and 200 microM, respectively). We conclude that these two 5'-modified dThd analogs enter human erythrocytes both by nonfacilitated diffusion and by the nucleoside transporter. The absence of the 5'-hydroxyl group of dThd (5'-ddThd) resulted in a large increase in the octanol/buffer partition coefficient, in an ability to permeate human erythrocytes by nonfacilitated diffusion, and in a 3-fold diminished binding to the nucleoside transporter. The 5'-azido group (5'-N3-5'-ddThd) resulted in an additional 1.4-fold increase in the octanol/buffer partition coefficient and in a 2-fold increase in the rate of nonfacilitated diffusion.

Desciclovir permeation of the human erythrocyte membrane by nonfacilitated diffusion.

The mechanism of transport of desciclovir (DCV)--a structural analogue and prodrug of acyclovir (ACV) which provides an improved oral bioavailability of ACV--was investigated in human erythrocytes with a "papaverine-stop" assay. DCV influx was nonconcentrative, linearly dependent on DCV concentration (0.9 microM to 15 mM), insensitive (less than or equal to 20% inhibition) to nucleobases, nucleosides, or potent inhibitors of nucleoside transport, and occurred without permeant metabolism. However, DCV was a weak competitive inhibitor of the influx of adenine (Ki = 1.3 mM) and of 5-iodo-2'-deoxyuridine (Ki = 2.9 mM). permeants of the erythrocyte nucleobase and nucleoside carriers, respectively. This indicates that DCV has an affinity for both of these transporters, even though it appears not to be an effective permeant. We conclude that, in contrast to ACV which enters human erythrocytes primarily via the nucleobase carrier, DCV permeates these cells chiefly (greater than or equal to 80%) by nonfacilitated diffusion. This mechanistic difference in transport between ACV and DCV is attributed to differences in their desolvation energies and suggests an explanation for the differences in the oral bioavailability of ACV which is observed after the administration of these two "acyclic nucleosides."

Ganciclovir permeation of the human erythrocyte membrane.

The membrane permeation of ganciclovir (DHPG)--a structural analogue of acyclovir (ACV) with activity against cytomegalovirus--was investigated in human erythrocytes at 37 degrees with an "inhibitor-stop" assay. DHPG influx was nonconcentrative, occurred without permeant metabolism, and was rate-saturable. While substantial inhibition of the influx of 13 microM DHPG occurred only in the presence of permeants of the purine nucleobase carrier, nucleosides and inhibitors of nucleoside transport markedly inhibited DHPG influx at higher DHPG concentrations (greater than or equal to 200 microM). Adenine and dilazep (a potent inhibitor of the nucleoside carrier) each inhibited the influx of DHPG only partially; when present together, however, they inhibited DHPG permeation completely. DHPG permeation via the purine nucleobase carrier (Km = 0.89 mM) was characterized by assessing influx in the presence of 1.0 microM dilazep. Adenine and ACV were shown to competitively inhibit this process, while DHPG (Ki = 0.90 mM) was found to competitively inhibit adenine influx. DHPG influx via the nucleoside carrier (Km = 14 mM) was characterized by assessing influx in the presence of 2 mM adenine. DHPG (Ki = 10 mM) also appeared to competitively inhibit the influx of 5-iodo-2'-deoxyuridine. These results indicate that DHPG permeates the human erythrocyte membrane primarily by the purine nucleobase carrier and secondarily by the nucleoside transporter.

2',3'-Dideoxythymidine permeation of the human erythrocyte membrane by nonfacilitated diffusion.

The influx of 2',3'-dideoxythymidine into human erythrocytes was characterized to gain insight into the molecular properties of 3'-azido-3'-deoxythymidine which allow this latter nucleoside analog to permeate cell membranes by nonfacilitated diffusion (J. Biol. Chem. 262, 5748-5754 (1987]. The influx of 2',3'-dideoxythymidine was (1) nonconcentrative, (2) a linear function of permeant concentration (0.05 to 12 mM), and (3) insensitive to potent inhibitors of nucleoside transport and to permeants of either the nucleoside or nucleobase transporter. It is concluded that 2',3'-dideoxythymidine, like 3'-azido-3'-deoxythymidine, permeates the human erythrocyte membrane predominantly by nonfacilitated diffusion. This unusual characteristic of these two nucleoside analogs is attributed both to their lack of a 3'-hydroxyl moiety, a structural determinant which appears to be important for transport by the nucleoside carrier, and to their relatively high partition coefficients (greater than or equal to 0.2).

Purine nucleobase transport in human erythrocytes. Reinvestigation with a novel "inhibitor-stop" assay.

A novel "inhibitor-stop" method for the determination of initial rates of purine nucleobase transport in human erythrocytes has been developed, based on the addition of seven assay volumes of cold 19 mM papaverine to terminate influx. In view of our finding that the initial velocities of adenine, guanine, and hypoxanthine influx into human erythrocytes were linear for only 4-6 s at 37 degrees C, the present method has been used to reexamine the kinetics of purine nucleobase transport in these cells. Initial influx rates of all three purine nucleobases were shown to be the result of concurrent facilitated and nonfacilitated diffusion. The nonfacilitated influx rates could be estimated either from the linear concentration dependence of nucleobase influx at high concentrations of permeant or from residual influx rates which were not inhibited by the presence of co-permeants. Appropriate corrections for nonfacilitated diffusion were made to the influx rates observed at low nucleobase concentrations. Kinetic analyses indicated that adenine (Km = 13 +/- 1 microM, n = 7), guanine (Km = 37 +/- 2 microM, n = 5), and hypoxanthine (Km = 180 +/- 12 microM, n = 6) were mutually competitive substrates for transport. The Ki values obtained with each nucleobase as an inhibitor of the influx of the other nucleobases were similar to their respective Km values for influx. Furthermore, the transport of the purine nucleobases was not inhibited by nucleosides (uridine, inosine) or by inhibitors of nucleoside transport (6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, dilazep, dipyridamole). It is concluded that all three purine nucleobases share a common facilitated transport system in human erythrocytes which is functionally distinct from the nucleoside transporter.

Acyclovir transport into human erythrocytes.

The mechanism of transport of the antiviral agent acyclovir (ACV) into human erythrocytes has been investigated. Initial velocities of ACV influx were determined with an "inhibitor-stop" assay that used papaverine to inhibit ACV influx rapidly and completely. ACV influx was nonconcentrative and appeared to be rate-saturable with a Km of 260 +/- 20 microM (n = 8). However, two lines of evidence indicate that ACV permeates the erythrocyte membrane by means other than the nucleoside transport system: 1) potent inhibitors (1.0 microM) of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep) had little (less than 8% inhibition) or no effect upon the influx of 5.0 microM ACV; and 2) a 100-fold molar excess of several purine and pyrimidine nucleosides had no inhibitory effect upon the influx of 1.0 microM ACV. However, ACV transport was inhibited competitively by adenine (Ki = 9.5 microM), guanine (Ki = 25 microM), and hypoxanthine (Ki = 180 microM). Conversely, ACV was a competitive inhibitor (Ki = 240-280 microM) of the transport of adenine (Km = 13 microM), guanine (Km = 37 microM), and hypoxanthine (Km = 180 microM). Desciclovir and ganciclovir, two compounds related structurally to ACV, were also found to be competitive inhibitors of acyclovir influx (Ki = 1.7 and 1.5 mM, respectively). These results indicate that ACV enters human erythrocytes chiefly via the same nucleobase carrier that transports adenine, guanine, and hypoxanthine.

Species-dependent expression and induction of homologues of rabbit cytochrome P-450 isozyme 5 in liver and lung.

The presence of homologues of rabbit cytochrome P-450 isozyme 5 in pulmonary and hepatic microsomal preparations from guinea pig, mouse, monkey, hamster, and rat was examined by immunoblotting and inhibition of metabolism of 2-aminofluorene with antibodies to isozyme 5. Homologues to isozyme 5 were detected in pulmonary preparations from all five species. However, only hepatic preparations from hamster, in addition to those from rabbit, contained detectable levels of this isozyme. With the exception of induction by phenobarbital in rabbit liver, treatment of animals with phenobarbital or tetrachlorodibenzo-p-dioxin did not increase hepatic or pulmonary content of isozyme 5 homologues or the amount of 2-aminofluorene metabolism inhibited by antibodies to isozyme 5. Metabolism of 2-aminofluorene was measured both colorimetrically (formation of a reduced iron chelate from the N-hydroxyfluorene metabolite) and radiochemically (separation of 3H-metabolites by high performance liquid chromatography and quantitation by scintillation counting). A turnover number of 48 nmol of product X min-1 X nmol of enzyme-1 for isozyme 5-catalyzed metabolism of 2-aminofluorene was determined with incubations containing isozyme 5 purified from rabbit lung. A similar turnover number was calculated from the rabbit hepatic microsomal activity inhibited by antibodies to isozyme 5 and the microsomal isozyme 5 content measured by immunoquantitation. In other species, amounts of metabolism inhibited by antibodies to isozyme 5 agreed qualitatively with relative staining intensities on immunoblots. In all species except the hamster, rates of total and isozyme 5-catalyzed metabolism of 2-aminofluorene were greater with pulmonary than with hepatic microsomal preparations from untreated animals.

The cytochrome P-450 monooxygenase system of rabbit lung enzyme components, activities, and induction in the nonciliated bronchiolar epithelial (Clara) cell, alveolar type II cell, and alveolar macrophage.

Enzyme components and activities of the cytochrome P-450 monooxygenase system in microsomal preparations from the Clara cell, alveolar type II cell, and alveolar macrophage fractions isolated from lungs of untreated rabbits and rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin were examined. Results are compared to those obtained with microsomal preparations from whole lung. Concentrations of cytochrome P-450 isozymes 2 and 5 and NADPH-cytochrome P-450 reductase activities were higher in preparations from Clara cell fractions than in preparations from type II cell fractions or whole lung. For the most part, however, differences among these preparations were 2-fold or less. Microsomal preparations from the macrophage fraction contained low or undetectable levels of cytochrome P-450 isozymes but relatively high levels of cytochrome P-450 reductase activity. The concentration of cytochrome P-450 isozyme 6, in contrast to those of isozymes 2 and 5, was found to be highest in microsomal preparations from whole lung. Treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin increased the concentrations of isozyme 6 in preparations from the Clara and type II cell fractions and from whole lung about 20-fold. In contrast, the content of isozyme 6 in preparations from the macrophage fraction increased greater than 90-fold. In all cases, induction of isozyme 6 resulted in substantial increases in the O-deethylation of 7-ethoxyresorufin and only minor increases in the hydroxylation of benzo(a)pyrene. Activities per unit of isozyme 6, following induction, were similar in all preparations, and we estimate that less than 20% of the potential activity of isozyme 6 is expressed with benzo(a)pyrene and greater than 40% with 7-ethoxyresorufin. These similarities exist in spite of significant differences among the preparations from different fractions in the ratios of isozyme 6 to NADPH-cytochrome P-450 reductase.

The effect of substrate on the expression of activity catalyzed by cytochrome P-450: metabolism mediated by rabbit isozyme 6 in pulmonary microsomal and reconstituted monooxygenase systems.

The content of cytochrome P-450, isozyme 6, in the rabbit pulmonary microsomal fraction was estimated by immunochemical methods to be 1 to 3% of the total cytochrome P-450. Following treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin, the pulmonary microsomal concentration of isozyme 6 increased 16-fold. Isozyme 6 was also detected by immunochemical methods, but not by electrophoresis and staining for protein, in preparations of isozyme 5 isolated from the pulmonary microsomal fraction of untreated rabbits. The metabolism of benzo[a]pyrene in these preparations was found to be catalyzed by isozyme 6, not by isozyme 5 as previously concluded. Cytochrome P-450, isozyme 4, was not detected in the pulmonary microsomal fraction from untreated or 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rabbits. Although benzo[a]pyrene and 7-ethoxyresorufin are both substrates for isozyme 6, the pulmonary microsomal metabolism of these compounds was not increased to the same extent by treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin (about 13-fold for 7-ethoxyresorufin and less than 2-fold for BP). However, lack of agreement between increases in isozyme 6 content and activity, and between the relative increases of the activities with the two substrates, was overcome by the addition of purified NADPH-cytochrome P-450 reductase to the microsomal incubations. When alpha-naphthoflavone, at the minimum concentration required for greater than 90% inhibition of isozyme 6 catalysis, was present in the incubations, no increases in activity were obtained by the addition of purified reductase. The turnover numbers of isozyme 6 in microsomal preparations incubated with purified reductase were similar to those of the purified isozyme in a reconstituted monooxygenase system. The relevance of our results to determinations of the substrate specificities and the microsomal concentrations and activities of isozymes of cytochrome P-450 is discussed. In addition, these parameters are used to assess the extent to which the catalytic potential of isozyme 6 is expressed in the rabbit pulmonary microsomal fraction.

Correlation of placental microsomal activities with protein detected by antibodies to rabbit cytochrome P-450 isozyme 6 in preparations from humans exposed to polychlorinated biphenyls, quaterphenyls, and dibenzofurans.

Placental tissues were obtained from Chinese women in Taiwan who had been exposed to contaminated rice oils containing polychlorinated biphenyls and their thermal degradative products. Exposure via the diet occurred 4-5 years prior to pregnancy. Placental microsomal fractions from eight of the nine exposed subjects studied showed marked elevation of benzo(a)pyrene hydroxylation and 7-ethoxyresorufin O-deethylation activities related to control subjects. Placental microsomes from exposed subjects were found to contain a protein that cross-reacted with antibodies raised to rabbit cytochrome P-450 isozyme 6, an isozyme induced by polycyclic aromatic hydrocarbons. This protein was not observed with microsomal samples from control subjects. A significant correlation was found between the relative amounts of the immunoreactive protein and benzo(a)-pyrene hydroxylation and 7-ethoxyresorufin O-deethylation activities. The 7-ethoxyresorufin O-deethylation activities were inhibited by alpha-naphthoflavone, a compound known to inhibit activities of rabbit cytochrome P-450, isozyme 6.

Quantitation of rabbit cytochrome P-450, form 2, in microsomal preparations bound directly to nitrocellulose paper using a modified peroxidase-immunostaining procedure.

Rabbit hepatic microsomal suspensions were bound directly to nitrocellulose sheets using a "Hybridot" apparatus to ensure uniformity. Cytochrome P-450, form 2, was then detected by a modified immunochemical method wherein the nitrocellulose paper was incubated sequentially with antibody to form 2 for 1 h at 25 degrees C, rabbit anti-goat immunoglobulin G (IgG) at a 1:100 dilution for 15 min at 25 degrees C, goat peroxidase-antiperoxidase at a 1:2000 dilution for 15 min at 25 degrees C, and 3,3'-diaminobenzidine at 0.3 mg/ml plus 0.002% hydrogen peroxide for 30 min at 25 degrees C. These conditions, as opposed to those previously published, yielded less background staining. The density of the stain, scanned with a soft laser (Zeineh), increased linearly from 2 to 100 fmol for purified form 2. Cytochrome P-450, form 2, was detected and quantitated in microsomal samples containing 0.1 to 0.5 and 0.02 to 0.05 micrograms protein for preparations from untreated and phenobarbital-treated rabbits, respectively. The results agreed with those obtained by Western blotting and single radial immunodiffusion. This assay is more sensitive than either Western blotting or radial immunodiffusion and has significant advantages such as ease of operation, increased sample numbers, and reduced interference from extraneous proteins.

Establishment of dihydrofolate reductase-increased human cell lines and relationship between dihydrofolate reductase levels and gene copy.

A series of methotrexate (MTX)-resistant human sublines developed by step increases in selected MTX concentrations have been cloned and examined for dihydrofolate reductase (DHFR) content, relative DNA copy number, and sensitivity to MTX. These cloned sublines had increased DHFR levels which were dependent on the presence of MTX in the medium. The increased levels of DHFR in the absence of MTX were stable in all the clones examined for over a year. Antibody immunolocalization on Western blots showed good correlation of the intensity of the immunostained DHFR band with enzyme activities. Relative gene copy number in these sublines was low relative to the DHFR increases and was not dependent on the presence of MTX in the medium. The increase in gene copy number in these sublines did not correlate with either the levels of DHFR or the sensitivity to MTX.

Studies of amino-acid sequence in dihydrofolate reductase from a human methotrexate-resistant cell line KB/6b. Structural and kinetic comparison with mouse L1210 enzyme.

The partial amino acid sequence of dihydrofolate reductase (DHFR, EC 1.5.1.3) from human KB/6b cells has been determined by using 3.5 mg of protein. Peptides covering the entire polypeptide chain were recovered from preparative peptide maps generated by the combination of paper chromatography and electrophoresis at pH 4.4 Peptide maps from mouse L1210 DHFR were also generated for comparison. Amino acid sequence of 75% of the 186 amino acid residues in the polypeptide chain of human KB/6b DHFR was obtained from Edman degradations and the remaining sequence was deduced from the amino acid compositions, from electrophoretic mobilities of related peptides and from the sequence homologies with other known mammalian DHFR sequences. A comparison of the proposed human DHFR sequence with the previously known sequences of mouse enzyme [Stone, et al. (1979) J. Biol. Chem. 245, 480-488] indicates that 18 differences are located in the established sequence of 139 residues and that 5 additional differences are in the tentative sequence of the remaining 47 amino acids. Kinetic properties of human KB/6b and mouse L1210 DHFR, which were determined in parallel experiments, are also compared. The possible structural-functional relationships between human and mouse DHFR are discussed.

Quantitative correlation of homogeneously stained regions on chromosome 10 with dihydrofolate reductase enzyme in human cells.

Gene amplification may be visualized within a chromosome as a homogeneously stained region (HSR) and HSRs have rarely been reported in human tumor cells with identification of the amplified gene. A parental line and seven clones derived from KB cells resistant to methotrexate (MTX) contain dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; EC 1.5.1.3), ranging from 0.007 unit/mg in the parent to 0.369 unit/mg in clone 7A with a 13,000-fold increase in resistance to MTX. The enzyme is identical to DHFR from other human sources, including that from leukemic patients. A HSR localized to the long arm of chromosome 10(q26) is present in clones selected at or above 2.5 microM MTX. Increase in number of 10q per cell, increase in number of HSR, and increasing amounts of DHFR correlate well. The chromosome change is stable with time as is enzyme production even in the absence of selection by MTX. No clone has shown double minutes. The gene copy number is low. The stability and low gene copy in the presence of large HSRs differ from the pattern described for murine tumors. A human gene for DHFR may be associated with the long arm of chromosome 10.