dihydrofolate reductase gene amplification and possible ... · the journal. of biological chemistry...

THE JOURNAL. OF BIOLOGICAL CHEMISTRY Vol. 257, No. 24, Issue of December 25, pp. 15079-15086, 1982 Printed in U.S.A.

Dihydrofolate Reductase Gene Amplification and Possible Rearrangement in Estrogen-responsive Methotrexate-resistant Human Breast Cancer Cells*

(Received for publication, October 20, 1981)

Kenneth H. Cowan$& Merrill E. Goldsmith$, Richard M. Levinell, Susan C. AitkenJI, Edwin Douglassll, Neil ClendeninnS, Arthur W. Nienhuisl, and Marc E. Lippmanll From the $Clinical Pharmacology Branch and IlMedicine Branch, National Cancer Institute and YClinical Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20205

Methotrexate-resistant (MTXR) human breast cancer cells have been obtained which are 1000-fold less sensitive to this drug than the wild type MCF-7 cells from which they were derived. The resistant cells contain approximately a 25-fold increase in the activity of the target enzyme dihydrofolate (DHF) reductase. Enzyme inhibition studies and methotrexate affinity studies fail to reveal any difference in the DHF reductase present in the MTXR cells compared to wild type MCF-7 cells. Cytogenetic analysis demonstrates the presence of elongated marker chromosomes in the resistant cells which are not found in the parental cell line. Analysis of the DNA from MTXR and wild type MCF-7 cells using Southern blot hybridization indicates that the MTXR MCF-7 cells contain more copies of DHF reductase gene sequences than do wild type MCF-7 cells. These experiments also suggest that the amplified DHF reductase gene sequences in MTXR cells may have undergone a uniform structural rearrangement involving the 5’ flanking sequences during the process of amplification. MTXR MCF-7 cells respond to estradiol by increasing cell growth, and the level of DHF reductase in the MTXR cells is further induced following administration of estradiol. Radiolabeling studies demonstrate that estrogen stimulates the actual synthesis of DHF reductase in human breast cancer cells.

Methotrexate, a potent inhibitor of dihydrofolate reductase (EC 1.5.1.3), is an effective antineoplastic agent with activity in a wide variety of human malignancies. Its clinical useful- ness, however, is limited by the relative ease with which tumors develop resistance to this agent. Previous studies using methotrexate-resistant animal cell lines have described several mechanisms associated with resistance to this drug, including the following: 1) defective drug transport (1-4); 2) structurally altered dihydrofolate reductase with a reduced affiity for methotrexate (5-8); and 3) increased levels of dihydrofolate reductase (9-17). The latter mechanism appears to be a common mechanism by which animal cells become resistant to methotrexate following exposure to stepwise increases in drug concentration in vitro. Subsequent studies

* This work has been presented in part at the American Society of Clinical Investigation, April 26,1981, San Francisco, CA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

0 To whom reprints should be addressed at Clinical Pharmacology Branch, Building 10, Room 6N116, National Cancer Institute, Na- tional Institutes of Health, Bethesda, MD 20205.

have shown that in two methotrexate-resistant animal cell lines, each with elevated levels of DHF’ reductase, the increased concentration of enzyme results from an increased rate of enzyme synthesis (18, 19). Work by Schimke and co- workers has shown that this increased enzyme synthesis is associated not only with a corresponding increase in the concentration of DHF reductase messenger RNA (20) but also in the selective amplification of the gene which codes for the enzyme (21). Both of these findings have been confirmed in other resistant animal cell lines (22, 23).

Cytogenetic abnormalities associated with methotrexate-resistant hamster cells were fist reported by Biedler and Spen- gler (24). These workers noted elongated marker chromosomes with homogeneously staining regions in resistant cells. Other cytogenetic abnormalities found in methotrexate-resistant cells have included the appearance of double minute chromosomes which are small pairs of chromosomes lacking centromeres (25).

Little is known regarding the ways in which human tumors develop resistance to this drug. One study of patients with acute myelocytic leukemia suggested that the natural resistance of these patients to therapy with methotrexate is associated with the inherent ability of acute myelocytic leukemia cells to rapidly synthesize DHF reductase and accumulate intracellular methotrexate (26). However, few biochemical studies have been done in human neoplasms which have become resistant following treatment with methotrexate. This lack of understanding is due in large part to the technical difficulties inherent in the study of human tumor samples. In order to facilitate the study of the mechanisms of resistance to methotrexate in human cells, we developed methotrexate- resistant human breast cancer cells by serial passage of a human breast cancer cell line (MCF-7) in stepwise increasing concentrations of methotrexate. Eventually methotrexate-resistant MCF-7 cells emerged which are over 1000-fold less sensitive to methotrexate than the wild type MCF-7 cells.

As will be shown, these methotrexate-resistant human breast cancer cells contain increased concentrations of DHF reductase having the same apparent Ki and Kd for methotrexate as the enzyme present in the parental cell line. In addition, these cells have distinct elongated marker chromosomes and contain increased gene copies for DHF reductase relative to the wild type MCF-7 cells. However, in contrast to previous studies in murine cells containing amplified DHF reductase genes (27), restriction endonuclease analysis suggests that the amplified sequences in the MTXR MCF-7 cells have apparently undergone a structural alteration or rearrangement dur-

’ The abbreviations used are: DHF, dihydrofolate; MTXR, methotrexate-resistant; kb, kilobase pair.

15079

15080 Estrogen Response in DHF Reductase Gene-amplified MCF-7 Cells

ing the course of amplification. These MTX' human breast cancer cells also retain the

sensitivity to estrogens previously described in the wild type MCF-7 cells (28). While the level of DHF reductase in these MTX' cells is almost 25-fold higher than in the wild type MCF-7 cells, there is yet a further induction following the addition of estradiol.

MATERIALS AND METHODS*

RESULTS

Restriction Endonuclease Analysis-The finding of both increased concentrations of DHF reductase and elongated marker chromosomes in MTXH human breast cancer cells suggested that there might also be an increase in the number of gene copies of DHF reductase in these cells. DNA was isolated from both wild type and MTX' MCF-7 cells and treated in parallel with EcoRI restriction endonuclease. The DNA fragments were analyzed by Southern blot hybridization to a radiolabeled mouse DHF reductase probe containing the entire coding region. Fig. 6 depicts an autoradiograph from such an experiment in which 35 pg of wild type DNA (lane W ) was compared to an identical amount of MTX" DNA which was analyzed in parallel (lane A ) . The autoradiograph for the wild type DNA hybridization was developed after 7 days at -80 "C, while the MTX' DNA autoradiograph was developed after an overnight exposure. Two important conclusions result from this study. First, it should be noted that the intensity of hybridization of radiolabeled mouse DHF reductase probe to each EcoRI fragment produced following digestion of MTX' DNA is as great if not greater than the hybridization to the wild type DNA. Since the intensity of hybridization is proportional to the length of the time of exposure, these results indicate that there is indeed an amplification of DHF reductase gene sequences in the MTX" cells of approximately 10-fold over that which is present in the wild type MCF-7 cells.

Second, the results depicted in Fig. 6 (lanes W and A ) indicate that there are not only differences in the quantity of the DHF reductase gene sequences in the MTXH cells, but there are also qualitative differences as well. EcoRI digestion of wild type DNA results in three fragments of approximately equal intensity at 2.1 kb, 4.2 kb, and 6.6 kb, and a faint band which can occasionally be seen at 19 kb. In contrast, digestion of MTX" DNA produces two bands which are identical in size with that observed in the wild type DNA (6.6 kb and 4.2 kb) and a new band a t 1.8 kb which is not seen in the EcoRI digest of wild type DNA. In this figure, the 2.1 kb band seen in the wild type EcoRI digest is not visible in the MTX" DNA. In other experiments a faint 2.1 kb band is sometimes observed in the EcoRI digest of MTX' DNA which is clearly separate from the dense band appearing a t 1.8 kb and which always appears much lighter in intensity than the 1.8,4.2, and 6.6 kb bands. Thus, although some of the restriction endonuclease sites are retained during amplification of DHF reductase gene sequences in the MTX" cells, some new EcoRI sites are generated during the course of amplification. The results of Southern transfer experiments are consistent over a wide range of DNA concentrations. Furthermore, when the South-

Portions of this paper (including "Materials and Methods," part of "Results," Figs. 1-5, and Table I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 81M-2583, cite the authors, and include a check or money order for $5.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

A/W

6 - C

D

W A B C D

9.5- 6.6-

4.3-

2.1- 1.9-

DNA. Wild type DNA (lane W ) and MTX' DNA (lanes A to D) FIG. 6. Southern blot analysis of wild type and M T X R cell

were treated with EcoRI, separated by agarose gel electrophoresis, transferred to nitrocellulose paper, and hybridized with different radiolabeled probes containing varying amounts of the mouse DHF reductase coding region (see top for individual probes). In each case, identical amounts (35 pg) of cell DNA were analyzed. The autoradiograph depicted in lane W (wild type DNA) was developed following a 7-day exposure; lanes A-D were developed following an overnight exposure. Lanes W and A were probed with the entire coding sequence; lane B with a 5' end probe; lane C with a probe containing the 3' end of the coding region; and lane D with the middle coding region probe. The numbers at the left represent the sizes (kilobase pairs) of the Hind111 fragments of XDNA which were run in parallel. On top is a diagram of the mouse DHF reductase-cloned cDNA as reported by Chang et al. (37) and the fragments used in these experiments.

e m blot analysis is performed following digestion with other restriction enzymes, similar conclusions are reached, i.e. that there are both marked quantitative and significant qualitative changes in the structure of the DHF reductase gene sequences in the MTX" cells.

In order to identify the nature of the apparent rearrangement of the amplified DHF reductase sequences in the MTX' cells, EcoRI-digested MTX' DNA was electrophoresed and transferred to nitrocellulose paper as described above. In separate experiments, the digested DNA was then hybridized to radiolabeled probes corresponding to different portions of the mouse DHF reductase coding sequences (Fig. 6, lanes B, C, and D). The DNA in lane B was hybridized to a 5' end probe; lane C was hybridized to a 3' end probe; and lane D was hybridized to a probe containing the middle portion of the mouse DHF reductase coding sequence (see top, Fig. 6). The 4.2 kb EcoRI fragment of the MTX' genomic digest contains sequences homologous to the 3' end of the coding region (lane 0, whereas the middle region of the coding sequences hybridizes predominantly with the 6.6 kb EcoRI fragment and to a lesser extent to the 1.8 kb EcoRI fragment (lane D). Following hybridization with a radiolabeled fragment containing the 5' end of the mouse coding sequences,

Estrogen Response in DHF Reductase Gene-amplified MCF-7 Cells 15081

only the 1.8 kb EcoRI fragment of the digested MTXR DNA is observed (lane B). This is the only band which appears in the amplified DNA and not in the EcoRI digest of the wild type DNA. These data are consistent with the hypothesis that the MTXR DNA contains amplified DHF reductase sequences which have undergone an apparently uniform rearrangement in the upstream DNA sequences flanking the 5‘ end of the gene. Whether the original DHF reductase gene still exists in the MTXR cells is not answered by these studies but, since a 2.1 kb band is sometimes faintly visible in the MTXR DNA, it is possible that some of the original sequences may remain unaltered in the MTXR cells. These findings will be discussed in further detail below.

MTXR MCF-7 Cells Are Estrogen Responsive-The parental MCF-7 cell line has been shown to have high affinity binding proteins for estrogens (as), progesterone (46), gluco- corticoids (46, 47), androgens (48, 49), and thyroid hormone (50). Furthermore, these cells respond to estradiol by an increase in cell growth (28), induction of DNA synthesis (28), and an induction of progesterone receptor (51).

In order to determine if the MTXR MCF-7 cells retain the sensitivity to estrogen observed in the wild type MCF-7 cells, cell growth was examined in the presence of either 1 nM estradiol or 1 IJM tamoxifen. As can be seen in Fig. 7, the growth of MTXR MCF-7 cells is markedly increased by estradiol and inhibited by the anti-estrogen tamoxifen. By 14 days there is almost a 300% stimulation of cell growth in the presence of estradiol and approximately an 80% inhibition in

Id -

10 20

DAYS

FIG. 7. Effects of estrogen and tamoxifen on growth of M T X R

MCF-7 cells. MTXR MCF-7 (20,000 cells/well) were plated in 47-mm Petri dishes in improved minimal essential medium containing 5% charcoal-stripped calf serum. The next day the medium was changed and included either 10 nM estradiol (A- - -A) or 1 ~ L M tamoxifen (W. . . .W). The control group consisted of MTXR cells (M) grown in the improved minimal essential medium containing 5% charcoal-stripped calf serum only. The medium was changed every 4 days and at various times the cells were harvested in phosphate- buffered saline + EDTA and counted in a Coulter counter. Each value represents the mean cell number of triplicate samples & 1 standard deviation.

24 Hours

+El T

FIG. 8. Effect of estrogen on DHF reductase activity. MTXR MCF-7 cells were plated in 47-mm Petri dishes in improved minimal essential medium containing 5% charcoal-stripped calf serum. When cells were nearly confluent the medium was changed to improved minimal essential medium without serum. Estradiol (10 nM) was added to half of the cells. At 24 h and 40 h the cells were harvested, sonicated, and the cytosol assayed for DHF reductase using radiolabeled folic acid. The results represent the specific activity of DHF reductase present in MTXR cells at various times after treatment and expressed as a fraction of the specific activity of DHF reductase present in cells at 24-h incubation in serum-free medium without estradiol. Each value represents the mean of triplicate samples & 1 standard deviation.

the presence of tamoxifen. These results are similar to those obtained with wild type MCF-7 (28).

Since DHF reductase is an important enzyme in the synthesis of DNA, we were interested in determining whether estrogen had any effect on the level of DHF reductase in the resistant cells. As can be seen in Fig. 8, following 24 h of incubation with estradiol there is a 50% increase in the specific activity of DHF reductase as compared to the basal level of activity present in these resistant cells incubated without hormone. Approximately a 100% increase in enzyme specific activity occurs after 40 h of incubation with estrogen. Thus, the base-line concentration of DHF reductase in the MTXR MCF-7 cells is not only vastly increased compared to the parental cell line but can be increased still further by incu- bating the cells in the presence of estrogen. A similar although somewhat smaller (30%) induction in DHF reductase activity by estradiol is found in the wild type MCF-7 cells (data not shown).

In order to identify the steps at which estrogen may regulate the level of DHF reductase in human breast cancer cells, parallel cultures of MTXR MCF-7 cells were incubated in the presence or absence of 1 X M estradiol. [35S]Methionine was added to the cultures 36 to 42 h after the addition of estradiol. At the end of that period the cells were harvested and the amount of newly synthesized DHF reductase was quantitated by affinity chromatography as described under “Materials and Methods.”

As shown in Table 11, following 42 h of incubation with estradiol there is a 61% increase in the level of DHF reductase as measured by [3H]methotrexate binding assay. During this period, estrogen increases total protein synthesis by 18% and there is a 34% increase in total soluble protein. These data are consistent with the observation that estrogen increases the


TABLE I1 Effect of estradiol on DHF reductase in MTXR MCF-7 cells

MTXR MCF-7 cells were incubated in improved minimal essential medium f 10 nM estradiol for 36 h. One set of plates was analyzed for actual DHF reductase level using r3H]methotrexate binding assay as described under “Materials and Methods.” Parallel cultures were incubated for 6 h with 100 pCi of [35S]methionine in methionine-free media beginning at 36 h. Incorporation of radiolabel into total cell protein and into DHF reductase was analyzed as described under “Materials and Methods.” Results are expressed as the median of tridicate samDles * 1 standard deviation.

[3HJMTx dpm/mgpro- dpm/mgpro- F$f%{ez tem/6 h x IO-’ tetn/6 h x

Control 14.41 & 0.45 2.29 & .I2 1.39 * 2 5 +10 rm estradiol 23.2 f 2.2 2.70 f .06 2.71 f .06 Increase 61% 18% 96%

rate of growth of MTXR MCF-7 cells. During this same period there is a 96% increase in the actual rate of DHF reductase synthesis as determined by [35S]methionine labeling and methotrexate-Sepharose affinity chromatography. Thus estrogen not only increases total protein synthesis and accumulation in these breast cancer cells, it also stimulates an even greater increase in the rate of synthesis and accumulation of DHF reductase.

DISCUSSION

In this report we describe the selection of methotrexate- resistant human breast cancer cells which are more than 1000- fold less sensitive to the drug than the parental cell line. Drug resistance in these MTXR MCF-7 cells is associated with increased levels of DHF reductase. Enzyme inhibition studies and methotrexate affinity studies fail to identify any major differences between the DHF reductase present in MTXR and wild type MCF-7 cells (see Figs. 1-4 and Table I in Miniprint). In addition, we have measured the transport of methotrexate in both cell lines and found no difference in drug uptake into either cell line (data not shown). Thus, resistance to methotrexate in these human breast cancer cells is associated pri- marily with the presence of increased concentrations of DHF reductase.

While the MTXR MCF-7 cells are more than 1000-fold less sensitive to the drug than the parental cell line, these cells contain only a 25-fold increase in DHF reductase concentration. This lack of correlation between the magnitude of resistance and the increase in enzyme level has also been found in methotrexate-resistant mouse and hamster cells (10, 11, 13, 16,51). Several explanations may account for this observation. First, although we have found no differences in methotrexate transport into either wild type or MTXR MCF-7 cells, drug uptake into wild type (45) and MTXR MCF-7 cells is nonlin- ear. Thus, a one log increase in extracellular methotrexate concentration does not result in a similar increase in intracellular methotrexate concentration. Secondly, other mechanisms besides increased DHF reductase might contribute to the lack of sensitivity to methotrexate in these resistant cells, including changes in other enzyme activities (i.e. thymidylate synthetase or thymidine kinase) (52). Furthermore, methotrexate undergoes a complex series of conversions within MCF-7 cells to poly-L-glutamyl derivatives (53,54). While the importance of these metabolites is not clear, it is conceivable that alteration in methotrexate metabolism including altera- tions in polyglutamate formation might contribute to resistance.

The cytogenetic abnormalities present in these MTXR MCF-7 cells (shown in Fig. 5) are similar to those noted previously in methotrexate-resistant animal cell lines (17,24). Elongated marker chromosomes have been previously shown to be associated with a drug-resistant phenotype which is relatively stable in animal cells in the absence of continuous selective pressure. This appears to be true in these resistant human cells as well, since we have detected essentially no loss in resistance over 7 months in the absence of selective pressure. In situ hybridization studies in methotrexate-resistant hamster and mouse cell lines containing elongated marker chromosomes have shown that the amplified DHF reductase genes are specifically localized, presumably as tandem repeats within the homogeneously staining region (55, 56). In murine L5178Y cells the homogeneously staining region was identifed as being part of the mouse number 2 chromosome (56). Unfortunately, numerous chromosomal translocations in the parental MCF-7 cells make it difficult to identify with certainty the origin of the marker chromosomes present in these human methotrexate-resistant cells. However, we have fre- quently been able to identify part of human chromosome number 7 and number 10 as part of the elongated marker chromosomes. Detailed cytogenetic studies on subclones of these MTXR MCF-7 cells and other methotrexate-resistant breast cancer cell lines which we have developed are currently in progress. It is anticipated that such studies might enable identification of the human chromosome(s) which is most often involved in the amplification of the DHF reductase gene.

The increase in size of these marker chromosomes is re- markable. Even if the human DHF reductase gene is as large as the estimated size of the mouse DHF reductase gene (>35 kb), the size of the elongated marker chromosomes in the MTXR MCF-7 cells is still several orders of magnitude larger than that needed to code for the increased DHF reductase gene copies. Similar conclusions have been made in MTXR animal cells (55-57). The significance of this additional DNA and whether it codes for any additional proteins remain un- known at present. Sodium dodecyl sulfate-gel electrophoresis demonstrates the presence of only one protein band which is in marked higher concentrations in the MTXR cells compared to the wild type MCF-7 cells and this band co-migrates with purified DHF reductase obtained from these cells.

Using Southern blotting hybridization techniques, we have shown that these methotrexate-resistant human breast cancer cells contain amplified DHF reductase DNA sequences. More- over, there apparently is a marked change in the pattern of restriction endonuclease sites present within the amplified DHF reductase genes or its flanking DNA sequences compared to the parental cell line. The Southern blot analysis depicted in Fig. 6 indicates that the alteration is quite uniform and occurred early in the process of gene amplification. As noted above, previous studies in methotrexate-resistant mouse cells containing amplified DHF reductase genes failed to detect any obvious difference in the restriction endonuclease sites present in the DHF reductase genes in parental and resistant cells (27). However, recent studies by Tyler-Smith and Alderson (57) have also noted DHF reductase gene rear- rangements in amplified mouse EL 4 lymphoma cells selected for in uitro resistance to methotrexate. Furthermore, studies by Hiscott et al. on revertants of temperature-sensitive SV40 tsA-transformed mouse embryo cells have also suggested that structural alteration or rearrangement may accompany specific gene amplification (58).

At the present time we are unable to identify the precise location of the apparent alteration in the amplified DNA

Estrogen Response in DHF Reductase Gene-amplified MCF-7 Cells 15083

sequences of the MTXR MCF-7 cells. Hybridization using different portions of mouse DHF reductase coding sequence has shown that the alteration or rearrangement has apparently occurred in the 5’ end of the DHF reductase gene or its immediate upstream flanking sequences. However, an alternative explanation for the difference in the wild type and MTXR genomic blotting patterns is possible. Recently, two highly conserved intronless pseudogenes for DHF reductase have been isolated from human recombinant DNA librarie~.~ It has also been suggested that at least in hamster cells there may be at least two DHF reductase genes coding for enzymes with different molecular weights (23). The presence of at least two human pseudogenes in addition to one or more normal human DHF reductase genes, all of which hybridize to the DHF reductase probe, make it difficult to interpret genomic blots. It is likely that not all the DHF reductase genes are amplified in our MTXR cells.

An alternative explanation for the observation of a 2.1 kb EcoRI fragment in the wild type DNA hybridizations and a 1.8 kb fragment in the MTXR DNA is possible. Perhaps the wild type 2.1 kb Eco fragment represents a portion of a DHF reductase gene or pseudogene which is not amplified in the resistant cells. Hybridization to this fragment in the MTXR DNA would be faint or undetectable compared to the amplified sequences depending on the relative frequencies of the two DHF reductase genes in the MTXR DNA. It is also possible that the MTXR 1.8 kb fragment does not indicate a gene rearrangement in the amplified DNA sequences but may instead represent the true 5‘ end of the normal DHF reductase gene which is undetected by hybridization with wild type DNA. If exon 1 of the human DHF reductase gene contains only a small region of homology with the mouse gene probe, then these sequences may only be detected when they become amplified in the MTXK cells. In fact, the sequence of the recently isolated human DHF reductase cDNA is approximately 80% homologous to the first 72 nucleotides of the mouse coding sequences but the two sequences diverge signif- icantly just 12 nucleotides upstream from the initiation site (59). We have attempted to identify the 5’ end of the DHF reductase gene in the wild type DNA but, despite the use of high concentrations of DNA (up to 60 pg) and prolonged exposure times, these experiments have failed to determine with certainty this region of the wild type DNA. The use of a human cDNA containing a longer region of homology of the immediate 5’ flanking sequences should help to clarify the question regarding rearrangement of the amplified genes. Con- sidering the complexity of the DHF reductase gene locus, including the presence of more than one DHF reductase gene and pseudogenes, definitive analysis of the amplified gene sequences in the MTXR cells must await the isolation and structural characterization of cloned DHF reductase gene sequences from both wild type and MTXR cells.

Gene amplification as a mechanism of drug resistance is not unique to methotrexate, but has also been shown to occur in animal cells resistant to N-(phosphonacety1)-L-aspartate (60- 62). N-(phosphonacety1)-L-aspartate-resistant hamster cells contain increased levels of a multifunctional protein (CAD protein) which includes the enzyme activity inhibited by N- (phosphonacety1)-L-aspartate (aspartate transcarbamylase) (61). These resistant hamster cells have been shown to contain increased copies of messenger RNA coding for the CAD protein and amplified DNA sequences complementary to this mRNA (62). We have also isolated N-(phosphonacetyl)-L- aspartate-resistant human breast cancer cells in a fashion similar to that described for isolation of MTXR MCF-7 cells

Chen, J., Shimada, T., Moulton, A. D., Harrison, M., and Nien- huis, A. W. (1982) Proc. Natl. Acad. Sei. U. S. A, , in press.

(63). N-(phosphonacety1)-L-aspartate-resistant human breast cancer cells contain increased levels of aspartate transcarbamylase (63) and also contain amplified DNA sequences coding for this enzyme.4 However, in these drug-resistant human cells, Southern blot analysis fails to detect any differences in the pattern of restriction endonuclease sites in the amplified DNA sequences compared to the parental cell line. Thus, it appears that gene amplification may in fact be a common mechanism for development of drug resistance in human tumor cells, as has been demonstrated previously in animal cell lines. It is also apparent from these studies that structural alteration need not invariably accompany specific gene amplification in human cells.

Finally, we have shown that the MTXR MCF-7 cells retain the hormonal sensitivity previously noted in the wild type MCF-7 cells (28) and both parental and MTXR cells respond to estradiol with increases in cell growth and DNA synthesis. In addition, we found that there is an 1.5- to 2-fold induction of DHF reductase activity in MTXR MCF-7 cells in response to estradiol. Thus, even though MTXR MCF-7 cells contain a 25-fold increase in DHF reductase concentration relative to that present in wild type MCF-7 cells, these resistant cells respond to estrogen by a further induction in DHF reductase levels. This induction of DHF reductase has been noted using both a direct assay of enzyme activity and a radiolabeled methotrexate binding assay and indicates that there is an actual increase in the enzyme concentration and not simply an increase in activity secondary to a change in substrate concentration, pH, or salt concentration, all factors known to influence DHF reductase activity.

The radiolabeling studies shown in Table I1 demonstrate that estrogen increases total protein synthesis in these drug- resistant breast cancer cells. It is furthermore apparent from these studies that estrogen has an even greater stimulatory effect on the rate of synthesis of DHF reductase. We cannot exclude the possibility that estrogen may also have an effect on enzyme stability which may contribute to the overall increase in DHF reductase levels observed following incubation of these cells with estrogen. The increased rate of DHF reductase synthesis which follows the administration of estrogen suggests that the hormone or a hormone-inducible factor may be acting at the level of the amplified DHF reductase genes. Of course, it is possible that estrogen may increase DHF reductase synthesis through other mechanisms such as increased DHF reductase mRNA stability or increased effk ciency of translation. Mariani et al. (64) have recently demonstrated that DHF reductase synthesis increases during S phase. Since estrogen results in an increased rate of growth of the MTXR MCF-7 cells, it is possible that the effect of the hormone on DHF reductase synthesis does not represent a direct hormone receptor-mediated event at the level of the DHF reductase gene but instead reflects a series of events which occur secondarily during the estrogen stimulation of cell growth.

The estrogen stimulation of DHF reductase synthesis suggests that the amplified DHF reductase gene sequences present in the MTXR MCF-7 cells retain certain regulatory sequences. The fact that amplified genes do retain regulatory controls has been previously noted in studies of methotrexate- resistant animal cells, in which DHF reductase activity was further induced following addition of serum (65,66) and during viral infection (67). In contrast, recent studies by Mayo and Palmiter have demonstrated that amplified metallothionein I genes in cadmium-resistant mouse sarcoma cells may lose the

‘IC. H. Cowan, M. E. Goldsmith, R. M. Levine, S. C. Aitken, E. Douglas, N. Clendeninn, A. W. Nienhuis, and M. E. Lippman, unpublished observations.


responsiveness to glucocorticoid regulation while maintaining 1399-1397 the response to heavy metals observed in drug-sensitive ce& 33. Lowry, 0. H., Rosebrough, N. J., F m , A. L., and Randall, R. J. (68). Thus, amplified genes need not have the identical re- (1951) J. Biol. Chem. 193,265-275 sponse to control signals as that observed in the parental cells. 34. Myers, C. E., Lippman, M. E., Eliot, H. M., and Chabner, B. A.

(1975) Proc. Natl. Acad. Sei. U. S. A. 72,3683-3686

Acknowledgments"We thank J. Whang-Peng and E. Lee for tFir 36. Zaharko, D. S., Bruckner, H., and Oliverio, V. T. (1969) Science assistance in the cytogenetic analysis and L. Ulsh for expert technical assistance. We thank B. Chabner for helpful discussions and for 37. Chang, A. c, y,, Nunberg, J. H,, Kaufman, R. J,, H, A,, critically reviewing this manuscript. We also gratefully acknowledge the assistance of R. A. Rodbell and K. Moore in preparation of this Schimke, R. T., and Cohen, S. N. (1978) Nature (Lond.) 275,

manuscript. 38. Kretschmer. P. J.. Kaufman. R. E.. Coon. H. C.. Chen. M.-J. Y..

35. Oliverio, V. T. (1961) Anal. Chem. 33, 263-265

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2624

Estrogen Response in DHF Reductase Gene-amplified MCF-7 Cells 15085 Supplementary Materlal to:

DIHYDROFOLATE REDUC'IASE GENE AMPLIFICATION AND POSSIBLE REARRANGEMENT I N E S T R O G E Y - R E S P O N S I V E M E T H O T R E X A T E - R E S I S T A N T H U M M BREAST CANCER CELLS

Kenneth H . Cowan . Merrill E . Galdsmrth. Rlchard H. Levlne, Susan c . Artken, Ei iwln Douglass . Ne11 Clendenlnn.

bthur W. Ylenhuis. and Marc E. Llppman

W . T . HCF-7 or MTXR MCF-7 were plated in triplrcate ~n 6-well plastic Llnbro T w o mi of a cell suspenslon contalning 50,000 to 100,000 c e l l s of elther

dlshes 135 _n diameter) and incubated at 3 7 ° C under a humidified atmosphere cnntainlnq 5 % CO2. Twenty-four houra later the media was changed to INEM con- t a l n l n g 5% charcoal-strlpped calf serum 1 2 8 1 . 0 . 1 ufrn l of regular ~ n s u l i n ( E l i LlllY Co., Indianapolrs, I N 1 and varylng concentratlone Of mietharrexare. me charcoal treatment reduces the levels of nucleosides in the serum which are capable of rescuing cells from methotrexate toxicity and also reduces the l e v e l of Sterold hormones. Under these condltians the sensltivrty of MCF-7 cells to methotrexate Lnhlbltlon and to e s t r o g e n 4 t i m u l a r i o n o f c e l l growth are markedly enhanced. The rnedlurn was changed on day 5 and the cella harvested on day 8 using 2 rnl of mlbecca's phosphate buffered saline (HEM Research, Bethesda, Y D I contalnlng 0 . 0 2 % E D T A . The cells were diluted with Isoton I1 1 C o u l t e r

Hlnleah, FL) ElectronLC Inc.1 and counted i n a particle counter ( C o u l r e r E l e c t r o n i c Inc..

Preparation of C e l l Eltracts

Confluent c e l l monolayers verevashedvlth P B S I E D T A and the cells suspend- ed L" a small volume of the same s o l u t i o n by scrap~ng with a rubber policeman. Following centrlfugatlan at 600 x g for 10 mln, cells were resuspended ~n 1 m l o f 0 . 0 1 M T r l s . pH 7 . 5 . ,001 M E D T A , and sonicated Cvlce far 10 eec in a Bran-

natant fluld was then used i n bOth dihydrofolate reductase actlvlty assays and son SonLCator (Branson Son ic Power Co., Danbury, CT) for 15 set. m e super-

racl iolabeled methotrexate blndlng assays.

Dlhydrofolate Reductase Rssay

'ng to a procedure described by Rothenberg 131) and modlfied by Nakamura and Dihydrofolate reductaee acilvlty l a 3 measured using C3Hlfolic a c i d *-cord-

Llttlefleld ( 3 2 1 except that the incubatlon mixture was increased to 2 0 0 u 1

obialned without addltion of cell extract were subtracted from each sample. and the speclfxc actlvity of the [3illfolic acid was 10 m c i f m m o l . Blank va lues

P r o t e l n was #measured by the method of Lowry uslnq bovine serum albunln as a standard ( 3 2 1 .

for D H f R previously descrlbed (121. The assay mixture consisted of 0 . 2 "mol M e t h o t r e x a t e lnhihltton rtudles were done Ysinga spectrophotometric assay

drhydrofolate. 0 . 2 ~ l m o l N A D P H . 0.1 m r n o l e potasslum phosphate, pH 6.8. 0 . 4 mmol K C I . and 1 " m o l of dithlothrertol l n a totdl volume of 1 ml. The reaction was run at 25'C with continuous measurement of the decrease ln absorbance at 340 nrn for 10 mlnukes in a double beam Beckman DU spectrophotometer. One u n l t of actlvlry 18 defined as the amount of enzyme which reduces 1 nmale of dlhydro- f"1ate I" 1 minute at 25.c.

'ng to a procedure described by Rothenberg 131) and modlfied by Nakamura and Dihydrofolate reductaee acilvlty l a 3 measured using C3Hlfolic a c i d *-cord-

Llttlefleld ( 3 2 1 except that the incubatlon mixture was increased to 2 0 0 u 1

obialned without addltion of cell extract were subtracted from each sample. and the speclfxc actlvity of the [3illfolic acid was 10 m c i f m m o l . Blank va lues

P r o t e l n was #measured by the method of Lowry uslnq bovine serum albunln as a standard ( 3 2 1 .

for D H f R previously descrlbed (121. The assay mixture consisted of 0 . 2 "mol M e t h o t r e x a t e lnhihltton rtudles were done Ysinga spectrophotometric assay

drhydrofolate. 0 . 2 ~ l m o l N A D P H . 0.1 m r n o l e potasslum phosphate, pH 6.8. 0 . 4 mmol K C I . and 1 " m o l of dithlothrertol l n a totdl volume of 1 ml. The reaction was run at 25'C with continuous measurement of the decrease ln absorbance at 340 nrn for 10 mlnukes in a double beam Beckman DU spectrophotometer. One u n l t of actlvlry 18 defined as the amount of enzyme which reduces 1 nmale of dlhydro- f"1ate I" 1 minute at 25.c.

C3H1Methotrerate Blndlng i issay

The speciflc blndrng of radlolabeled methotrexate to cell extracts was measured using a procedure dercrlbedby Myers et a1 134). The reaction mixture contalnei l ~n a final volume of 450 VI: 10 pmol of C3Hlmerhotrerate (specrfic actlvlty 9.5 c ~ f m o l e l , 0.075 una1 potassium phosphate, pH 5.2, 0.24 " m o l of freshly prepared NADP11 (Slgrna Chemlcal C o . , St. LOULS, KO1 and various allquots of cell extracts. Following incubation at room temperature f o r 5 mln, 2 5 PI nf Charcoal slurry was a4de.i. The charcoal slurry Consisted of 10 g/100 ml N O r l t A ( S i g m a ] : 2 . 5 g f 1 0 0 m l bovine serum a l b u m i n Fractlon V, IRehels Chemical

The pH of the resulting slurry was adlusted to 6 . 2 . The solution was mzxed Company. Phoenix, h21, and 0.1 g/lOO ml hlqh molecular weight dextran (Sigma).

hy vortexrnq and centrifuged at 6 0 0 x 9 for 30 m L n to remove the charcoal. i t l lquotr I200 "1) of the supernatant Solutron were placed in glass scint~llatlon vial4 and counted In a llquld scintlllation spectrophotometer Wlrh a couniioq effxcieocyof 33% for t r i f i l i m . Radiolabeled methotrexate was purified hy chromatography over D E A E Sephacel (Pharrnacla. Uppsala. Sweden1 as descrlbed prev~ously 1 3 5 . 3 6 1 .

Cytoqenetlc Studles

M r X R MCF-7 cells were prepared and stained wrth Giemsa as previously clescrrbed Chromosome preparatrons from ColChrcrne 1S1gma) treated W.T. MCP-7 and

(29).

Growth of Bacterla and Preparatlon of Plasmid DNA

. _. plier. DNl i fragments were separated by electrophoresis ~n 1% agdrose slab

were ldentlfled by Southern transfer 143) of the DNA from agarose gels to gels a s described previously ( 4 0 ) . DNA fragments contalning DHFR sequences

that the transfer buffer was 6 x standard saline cltrdte ( 1 1 S S C equals 0.15 M n l t r ~ c e i l u l o ~ e filters uslng a procedure descrcbed by Lawn et ai ( 4 4 1 , except

NaCl and .015 M S o d l u m citrate, pH 7 . 0 ) . Nlck translation was performed using reagents and cOndlt1ons supplied by Bekhesda Research Laboratories. The DNA probe used was the Pet 1-691 11 fragment of the plasmid pDHFR 11 which contains the 5' end and a l l Of the coding sequences of the c D N l ( 3 7 1 . Previous thermal denaturation studies have suggested a high degree Of homology between mouse and human DHFR gene sequencee 1 2 7 ) . I n fact. recomblnani hacterlophage Con- talnlng human DHFR gene sequences have been rsalated and the coding ~equenues are 8 5 to 90% hOmOloqOUS to mouse DHFR codrng sequences IJ. Chen and A . D a v l s .

purlfled by chromatography over Sephadex GlOO prior to use in hybrldiratlon personal communicationl. The radlolabeled D N l i was preclpltdted I n ethanol and

experiments. Follovrng hybridlratian the fkIters w e r e wa-hed four LLmeS In

RUtOradlOqraDhS were ~reuared usinq Kodak XR5 fllm with Dupont Llghtrng p l u s 100 m l of 0.1 r S S C containing 0.18 Si>S and ".I% sodrum pyrophosphate at 55'C.

Lntenslfi;r ;creens a t -SO'C.

DHFR Synthesis Studies

serum for two pas4ages and t h e n plated ~n rr~plicate i n 35 mm Llrnbra dishes nrxn MCF-7 c e l l s were qrown ~n IWEM conraining 5 % charcoal-stripped calf

at a cell density of 400,000 cells per well. TWO days later the medla were changed to serum-free IMEM. Half of the cells were xncubated wlth 1 X IO-'M

placed w i t h two m l of methlonlne-free media Contalnlnq 100 U C l of C35S1meth- estradiol. Following an 18-hour incubation the media In each well Were re-

lonine with a speciflc Of 1175 C i / m o l ( N e w England Nuclear. Boston. M A ) . After 6 hours the cells were washed with PBS, harvested by scraprnq. centrl- fuged at 1000 x g for 5 minutes, and the cell pellets frozen and stored at -20'C.

described above. Following centrifugation at 1000 g x 15 minutes, an aliquot Cells were resuspended i n 1.0 ml Of PBS and sonlcated far 20 seconda as

of the supernatant bovine serum dlbumln was added to a flnal ConCentraLlon of 100 uq/ rn l and the nurture was precipitated ~n 10% trlchloraceClc acld. An aliquot of the supernatant I300 "11 was mlred wlrh 600 1 ' 1 of 1.0 M potasslum phosphate buffer. pH 6.2, and 100 p l of i i n l a b e l e d rnethionlne (10 mg/ml) and passed through d me2hot=eiare-=eph~ro4e afflnlty column (0.5 x 1.0 C m ) 5 L l m e 4 . The methotrerate-sepharose was kindly provlded by Dr. Bernard Kaufman. The column was then washed wlth 1 M potasslum phospllate buffer until the radio- acrivlry eluted from the column was approrlmarely background. The [35Sllabele0 DHFR was then eluted with 10 m l of 1.0 W potasslum phosphate buffer conra~ning 1 mH methotrexate. me eluate was collected ~n 2 m l fractions and allquots of each fraction were counted in a liquid scint~llatlon counter.

R E S U L T S

The sensitivity of W.T. and MTXR WCF-7 cells to methotrexate 1s shown

methotrexate concentrations as low a3 4 x lo-%, there is essentlally no rnhl- i n Figure 1. While the growth of W . T . MCF-7 c e l l s i s markedly lnhiblted by

bition of growth of MTXR MCF-7 cells untll the concentcation of methotrexate in the nedlum z e greater than 1 x IO-%. The MTX concehtrailons re ulred for 50% inhibitlon of cell growth i s Over 1000-fold greater ~n <he MTX8 than the W . T . MCF-7 Cells. In the absence of any drug. MTXR MCF-7 c e l l s grow Somewhat

24 hours far the W.T. MCF-7 cells. We have grown MTXR MCF-7 cells for 7 months slower than W.T. cells with a doubllng time of 32 hours for the MTXR cells and

(more than 100 c e l l doublingsl In the absence of any methotrexate and have noted essentially no loss in the l e v e l of drug res14tance (data not shown). Thus, the resistant phenotype Of these merhotrexate-re619tant human breast cancer cells is relatively stable, even ~n the absence of selective pressure.

DHFR Activity ln W . T . and MTXR MCF-7 C e l l s

Studies in mauve and hamster cells have ruaaebred Chat one of the more frequent mechanisms by which cells acqulre methotrexate resrstance 13 the development Of increased amounts of the target enzyme dihydrofolate reductase. We therefore measured the actlvlry of thls enzyme I n both W . T . and MTXR human breast cancer cells lF14. 2 1 . MTXR UCF-7 cells contain a D o i ~ x l m a r e l v 25-fold higher levels Of DHFR dCtlVitY compared to the parental ceil line.

In orller to determine whether the DHFR present I" increased cnncenrratlon in the MTXR MCP-7 cells was qualltatlvely the same a8 that rn W . T . MCP-7 cells. enzyme inhibition studles were performed. The effect of MTX On the activity of DHFR ~n cell extracts obtained from MTXR and W . T . MCF-7 cells la shown ln Flgure 3 . There Ls essentlally DO drfference i n the lnhihitlan of DHFR by MTX i n either cell l i n e .

blnding assay Using radiolabeled MTX was previouPly developed to measljre the Slnce MTX blnds tightly. specifically and stolchiometrlcally to DHFR, a

to DHFR present In NTXR and W.T. MCF-7 as well as provldlng an alternative enzyme (111. This binding a4say was uaed to compare both the affinity of MTX

means tO quantitate the level of 0HFR I" sensltlve and resistant MCB-7 cells. Scatchard analysis of the binding of radrolabeled MTX to extracts of W . T . and MTXR c e l l s is shown ~n F ~ q u r e 4. The 3traight llnes wlth nearly parallel slopes Indicate the presence of a single class of hlgh affinity blnding sites

FIG. 1 .

f B o -

g " 40-

20-


//' M T X ~ MCF 7

2 v d : W.T. MCF 7

OO 1 0 0 m 300 ug Rotein

F I G . 1.

1 0- I

1 0 ' 10"

METHOTREXATE CONCENTRATION ImolalL)

dihydrofolate reductase gene amplification and possible ... · the journal. of biological chemistry...

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