| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
Departments of Medicine and Molecular Pharmacology, the Albert Einstein College of Medicine, and the Einstein Cancer Research Center, Bronx, New York
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-(-4-amino-4-deoxypteroyl)-N
-hemiphthaloyl-1-ornithine (PT523), respectively, compared with parental HeLa cells when grown with 2 µM folic acid. When folic acid was replaced with the more physiological 25 nM 5-formyltetrahydrofolate, R5 cells were 2-fold collaterally sensitive to PMX but still 40- and 200-fold resistant to ZD1694 and PT523, respectively. Sensitivity to PT523 and PMX could be completely restored, and sensitivity to ZD1694 nearly restored, by transfection of RFC cDNA into R5 cells, indicating that the defect in drug transport was the only, or major, factor in resistance. The preserved PMX activity in R5 cells could not be related to the very low expression of folate receptors. Rather, retained PMX activity in R5 cells was associated with residual transport by another process that exhibits good affinity for PMX (Kt = 12 µM) with much lower affinities for ZD1694, MTX, and PT523 (Kis of 
90, 100, and 250 µM, respectively). PMX transported by this route was rapidly converted to higher polyglutamates and, when grown with 25 nM 5-formyl-tetrahydrofolate, the rate of formation of these derivatives and their net accumulation in R5 cells was comparable to that of wild-type cells. These data suggest that selective preservation of PMX pharmacological activity in RFC-null R5 cells is due, in part, to partial preservation of transport by secondary process with a higher affinity for PMX than the other antifolates evaluated. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
There have been reports describing cells with primary resistance to PMX that have greater cross-resistance to raltitrexed (ZD1694; Tomudex) and cells with primary resistance to ZD1694 with lesser cross-resistance to PMX (7
, 8)
. This has been observed whether resistance was due to increased expression of TS or impaired drug accumulation (7
, 8)
. This is, however, not always the case (9)
. Polyglutamation of ZD1694 is also required for activity, and these derivatives of both agents have comparable TS inhibition constants (4)
. Likewise, both drugs are comparable, excellent substrates for folypolyglutamate synthase (0.8–1.4 µM), and have similar affinities for reduced folate carrier (RFC) and folate receptor- (FR-; Ref. 10
, 11
). One possible explanation for the preservation of PMX activity relative to ZD1694 is its inhibitory potential at the level of glycin-amide ribonucleotide formyltransferase because the site of ZD1694 action is solely at TS.
In a recent report from this laboratory, a methotrexate (MTX)-resistant HeLa cell line, R5, was characterized in which RFC was deleted from the genome (12)
. This resulted in a large, but incomplete, decrease in MTX influx compared with wild-type cells, consistent with a residual RFC-independent transport pathway. This is unlike what is observed in murine leukemia cells when RFC activity is lost, when virtually all influx ceases (13)
. The present report explores the cellular pharmacology of PMX in the R5 cell line, the extent to which it is transported by the RFC-independent pathway, and how this impacts on the activity of this agent in comparison to (a) the other inhibitors of TS, ZD1694 and AG331 (the latter diffuses into cells and does not undergo polyglutamation), and (b) the potent dihydrofolate reductase inhibitor N-(-4-amino-4-deoxypteroyl)-N-hemiphthaloyl-1-ornithine (PT523), which has a very high affinity for RFC and does not form polyglutamate derivatives.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cell Culture Conditions and Growth Inhibition Studies.
The HeLa, R5, and RFC-7 (RFC-transfected R5 derivative) cell lines were described in a previous report (12)
. Cells were maintained in RPMI 1640 (Hyclone) supplemented with 10% fetal bovine serum (Gemini Bio-Products), 2 mM glutamine, 20 µM 2-mercaptoethanol, penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere of 5% CO2. The RFC-7 transfectant was maintained in 600 µg/ml G418. HeLa and R5 cells were also maintained in folate-free RPMI medium (Hyclone) containing 25 nM 5-CHO-THF in addition to the supplements described above. Mycoplasma adherent to cells produce folate transport activity with very high affinity for PMX and other folates (15)
. This is particularly prominent in mesothelioma cells, but a low level of activity can also be detected in HeLa cells contaminated with this organism. (15)
. Because of this, cell cultures were monitored regularly with a Mycoplasma detection kit (American Type Culture Collection) and were shown to be free of this microorganism.
For assessment of growth inhibition by antifolates, cells were transferred to 96-well plates (500 cells/well) and exposed continuously to a spectrum of antifolate concentrations for 6 days. G418 was not included in the medium for the RFC-7 line in these experiments. Cell growth rate was quantified by sulforhodamine B staining (16) . For measurement of total PMX accumulation, cells were grown either in folic acid or 5-CHO-THF medium containing 50 nM [3H]PMX, 200 µM glycine, 100 µM adenine, and 10 µM thymidine for 1 week. Intracellular tritium was determined as in the transport studies described below.
Folic Acid-Binding Assay.
Cells near confluence were washed with ice-cold acid buffer [10 mM sodium acetate, 150 mM NaCl (pH 3.5)] followed by a wash with ice cold 20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 5 mM glucose (pH 7.4; HBS). Cells were incubated for 15 min in ice-cold HBS containing 5 nM [3H]folic acid in the presence and absence of 500 nM nonlabeled folic acid. The cells were then washed three times with ice-cold HBS. [3H]Folic acid bound to the cell surface was released with the acid buffer (0.5 ml) and measured on a liquid scintillation spectrometer. The difference in tritium released in the presence and absence of nonlabeled folic acid represented folic acid specifically bound to the cell surface.
Transport Studies.
Analysis of PMX transport followed a protocol designed for rapid uptake determinations on cells growing in monolayer cultures (17)
. Briefly, cells (3 x 105) were seeded into 20-ml Low Background Glass vials (Research Products International Corp, Prospect, IL) and grown for 3 days to reach early confluence. The cells were washed twice with HBS and incubated in this buffer at 37°C for 20 min. After removal of the incubation buffer, uptake was initiated by the addition of 0.5 ml of HBS containing radiolabeled folate at the desired concentrations. Uptake was terminated by injection of 5 ml of ice-cold HBS into the vials, after which the adherent cells were washed three times with 5 ml of ice-cold HBS. The cells were then dissolved by incubation in 0.2 M NaOH (0.5 ml) at 65°C for 45 min. Radioactivity in 0.4 ml of the lysate was determined, and 10 µl were processed for protein determination (BCA; Pierce, Rockford, IL). Cellular uptake is expressed in units of pmol/mg of protein.
HPLC Analysis of PMX Polyglutamates.
HeLa and R5 cells were grown in either folic acid or 5-CHO-THF medium in 100-mm plates. Cells were washed twice with HBS, exposed to HBS containing 0.5 µM [3H]PMX for 2 h at 37°C, and then washed three times with cold (0°C) HBS. Cells were then scraped mechanically in 1 ml of ice-cold 50 mM phosphate buffer (pH 6.0) containing 100 mM 2-mercaptoethanol. One portion of this suspension (50 µl) was used for determination of total PMX accumulation in cells as described above. In this case, protein was determined with the Bio-Rad protein assay because 2-mercaptoethanol was present. The remaining portion was boiled for 10 min to inactivate enzymes, and the supernatant containing radiolabeled PMX and its metabolites was separated by centrifugation. After nonlabeled PMX-monoglutamate, -triglutamate, and -pentaglutamate standards were added, the solution was injected onto a reversed-phase HPLC column (Waters Spherisorb; 5 µm ODS2; 4.6 x 250 mm) and processed as reported previously (18)
. The levels of PMX and its polyglutamate derivatives are expressed in units of pmol/mg of protein in the cells.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, R5 cells were 200-fold resistant to ZD1694 (Fig. 1B)
but only 2-fold resistant to PMX (Fig. 1A)
compared with the parental HeLa cells. R5 cells were not resistant at all to AG331 (Fig. 1C)
, consistent with a lack of change in the level or properties of TS. In addition, R5 cells were highly (400-fold) resistant to PT523 (Fig. 1D)
. Hence, R5 cells were cross-resistant to two antifolates with different enzyme targets, one of which does (ZD1694) and the other does not (PT523) form polyglutamate derivatives. Both, however, have high affinity for RFC.
|
, under these conditions R5 cells were 2-fold collaterally sensitive to PMX (Fig. 2A)
and AG331 (Fig. 2C)
, whereas they were still 40- and 250-fold resistant to ZD1694 (Fig. 2B)
and PT523 (Fig. 2D)
, respectively. Compared with cells grown with 2 µM folic acid, sensitivity of R5 cells to PMX, AG331, ZD1694, and PT523 was increased by factors of 4, 2, 5, and 2, respectively, in medium containing 25 nM 5-CHO-THF. Hence, the activity of PMX was completely preserved in R5 cells that lacked RFC expression under these physiologically relevant conditions, whereas resistance to ZD1694 and PT523 persisted, although all of these agents have high affinity for, and are transported by, this carrier.
|
, overexpression of RFC nearly fully restored sensitivity to ZD1694 (Fig. 3A)
and fully restored sensitivity to PT523 (Fig. 3B)
. The low level of PMX resistance in R5 cells was eliminated in the RFC-7 transfectant (Fig. 3C)
. Hence, impaired transport in R5 cells appears to be the major, if not sole, contributor to resistance to all these agents.
|
, FR expression in wild-type HeLa cells was quite low, and the level in R5 cells was 40% that of HeLa cells in the folic acid growth medium. When cells were grown in 25 nM 5-CHO-THF, folate acid binding in HeLa and R5 cells was increased by factors of 1.5 and 2.5, respectively, but FR expression in R5 cells was 60% that of HeLa cells. Hence, FR expression is lower in R5 than HeLa cells during growth in either folate.
|
, when 1 µM folic acid was added to the growth medium compared with growth in 1 or 10 nM 5-CHO-THF (19)
. The same approach was used to determine whether the low level of FR expression in R5 cells contributes to transport of, and sensitivity to, PMX, which has an affinity for FR- somewhat greater than that of folic acid (11)
. As indicated in Fig. 4
, when 1 µM folic acid was included in the growth medium, the PMX IC50 in R5 cells (20 nM) was increased by a factor of only 2, whereas the PMX IC50 in HeLa cells (40 nM) was unchanged. As a result, R5 and HeLa cells were equally sensitive to PMX. These concentrations of folic acid are 25- and 50-fold greater, respectively, than the IC50 for PMX in these cells and should markedly suppress any FR-mediated transport. The small increase in PMX IC50 in R5 cells was likely due to the increase in cellular folate cofactor pools. Hence, FR- mediated delivery of drug does not appear to play a role in PMX activity in these cells.
|
, uptake was constant over at least 1.5 min in both cell lines, but when the uptake slopes were extrapolated to time zero, there were high ordinate intercepts, consistent with a low level of PMX rapidly bound to the cell surface. On the basis of the uptake slopes, which represent initial uptake rates, PMX influx in R5 cells was decreased by only 55% compared with influx in HeLa cells, consistent with substantial RFC-independent residual transport. Co-addition of 20 µM folic acid with the [3H]PMX decreased the ordinate intercept in both lines, but there were no changes in initial uptake rates. This reflects inhibition of PMX binding to folate receptors that do not appear to make a measurable contribution to PMX transport across the cell membrane under these conditions. Inclusion of 5 µM PT632, which has a RFC influx Ki of 0.3 µM, one-tenth that of PMX (20)
and should therefore abolish RFC-mediated PMX influx, decreased PMX influx in HeLa cells to a rate comparable to that of R5 cells. Hence, there appears to be an uptake route for PMX independent of RFC in R5 cells, and this activity is also present in HeLa cells.
|
. Again, PMX influx in HeLa cells was approximately twice that in R5 cells; in neither cell line was the initial uptake rate altered by the folate growth sources (Fig. 6A
, inset). Net uptake of PMX beyond 10 min, which reflects synthesis and retention of polyglutamate derivatives [see below and as reported previously (18)
], continued at a constant rate that was similar for both cell lines over the period of observation. The absolute difference in the PMX level was constant so that by 90 min the difference between the two lines was negligible when cells were grown in 5-CHO-THF medium. A similar pattern was observed in cells grown in folic acid, but the absolute difference in total PMX uptake was much greater. Hence, although the PMX initial uptake rate in R5 cells was less than half that of HeLa cells, this appears to sustain a rate of PMX polyglutamation similar to that of wild-type cells irrespective of the folate growth source.
|
, no major radioactive peaks were detected other than PMX and its polyglutamate derivatives; these could be clearly distinguished and quantitated. Consistent with the data presented in Fig. 6A
, total PMX accumulation was greater in cells grown in 5-CHO-THF than in folic acid medium (Table 2)
. In 5-CHO-THF medium, 95% of total PMX represented polyglutamate derivatives, largely the higher derivatives, but in R5 cells grown in folic acid, only 75% of cell PMX was polyglutamates, and the level of derivatives at the triglutamate and higher was 50% that of wild-type cells. Under all conditions, long-chain polyglutamates (pentaglutamate and above) were the major metabolites of PMX along with smaller percentages of di-, tri-, and tetraglutamates.
|
. Hence, under conditions of growth in a physiological folate, there is only a negligible decrease in accumulation of PMX polyglutamates in R5 cells despite the loss of RFC function.
Kinetics of PMX Influx Mediated by a RFC-Independent Route.
PMX influx kinetics were determined in R5 cells grown in folic acid medium. Unlabeled folic acid (20 µM) was included to block PMX binding to FRs to minimize the ordinate intercept and thereby to increase the accuracy of influx measurements (see Fig. 5
). PMX initial uptake was saturable with a Kt of 12 ± 6 µM and a Vmax of 5.6 ± 1.4 pmol/mg protein/min determined from a nonlinear regression fit to the Michaelis–Menten equation (Fig. 7)
, or 18 µM and 6.9 pmol/mg protein/min, respectively, determined from a Lineweaver–Burk plot analysis (Fig. 7
, inset).
|
. On the basis of the measured PMX influx Kt of 12 µM and assuming that all of these agents inhibit PMX influx competitively, influx Kis were calculated and are indicated at the end of the columns in Fig. 8
. The PMX influx Ki determined in this way was comparable to the influx Kt determined directly with tritiated drug as indicated above. The folic acid Ki (92 µM) was consistent with a lack of appreciable inhibition of PMX influx by 20 µM folic acid (Fig. 5)
. This transport route also has a low affinity for ZD1694 and PT523 (7- and 14-fold less than for PMX, respectively), to which R5 cells were highly resistant in either folic acid or 5-CHO-THF medium. The affinities for 5-CHO-THF and 5-methyltetrahydrofolate (racemic), were 3 and 5 times lower, respectively, than that for PMX. Hence, this transport process favors PMX compared with the other folate and antifolate substrates tested.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Despite the loss of RFC, PMX activity in R5 cells was preserved; indeed, cells were collaterally sensitive to this agent when 5-CHO-THF was the folate growth source. This preservation of PMX activity could be attributed to two factors. First, there was an alternative transport route for this agent. Indeed, 45% of the initial PMX uptake rate was preserved in R5 cells despite the lack of RFC. This residual transport activity could not be attributed to expression of folate receptors in R5 cells because FR expression was very low, addition of folic acid did not alter PMX influx, and inclusion of 1 µM folic acid in the growth medium only minimally decreased PMX activity in R5 cells. This is in contrast to the 100-fold decrease in the activity of CB300638, a TS inhibitor that is an excellent substrate for FR- but not RFC in human epidermoid A430 cells that express high levels of FR- (19)
. Second, as indicated previously, when R5 cells are grown in 5-CHO-THF there is a contraction of cellular folate cofactor pools (12)
. This was shown in murine leukemia cells to augment PMX polyglutamation by decreasing feedback inhibition by cellular folate cofactors at the level of folypolyglutamate synthase, thereby compensating for the loss of PMX transport (18
, 21
, 22)
.
The data indicate the presence of a novel folate transporter in R5 cells with an influx Kt for PMX of 12 µM. Other antifolates had a much lower affinity for this transporter, with Kis of 340, 99, and 91 µM for PT523, MTX, and ZD1694, respectively. The affinity of this transporter for three naturally occurring folate substrates, folic acid, 5-CHO-THF, and 5-methyltetrahydrofolate was also lower with Kis of 92, 45, and 80 µM, respectively. This transporter does not distinguish among folic acid, ZD1694, and MTX as does RFC (with an affinity for folic acid two orders lower than for these antifolates) and does not distinguish MTX from folic acid, as does FR- (with a 100-fold higher affinity for folic acid; Ref. 11
). The data suggest that this transport activity is also present in wild-type HeLa cells because addition of folic acid, to abolish possible FR- mediated transport, along with PT632, to abolish RFC-mediated transport, left substantial residual transport activity that was comparable to PMX transport in R5 cells. It is not clear what role this transporter may play in PMX activity when RFC is intact. However, because this is a lower affinity process than RFC, it could contribute to the delivery of PMX and other antifolates into cells at the high initial concentrations that follow bolus administration of drugs in vivo, when RFC would be saturated.
The identity of this RFC-independent transport pathway in HeLa cells is unclear, nor is it clear as to the extent to which it might be expressed in other human solid tumor cell lines. There are a variety of organic anion transporters of the SLC21 and SLC22 superfamilies of facilitative carriers largely expressed in hepatic and renal tissues (23
, 24)
, some of which, such as OAT-K1-2 (25
, 26)
, LST-2 (27)
, and hOAT (28)
, transport MTX; and LST-2 has been detected in human gastrointestinal tumors (27)
. However, initial studies suggested that the secondary pathway in R5 cells is not related to these processes. This laboratory described a transport activity with high affinity (Kt = 30 nM) for PMX and low affinity for MTX (100 µM) in human mesothelioma cell lines. Recent studies, however, have clarified that this activity is associated with the presence of Mycoplasma in cell cultures and likely reflects transport into this microorganism (15)
. This activity is also present, although at a much lower level (<10-fold) in HeLa cell lines contaminated with Mycoplasma. However, in the present study, cells were tested regularly with a PCR-based Mycoplasma detection kit and were shown to be free from any contamination by this organism.
The high affinity of this secondary pathway for PMX in HeLa cells explains, in part, the preservation of PMX activity relative to ZD1694 and PT523 when RFC is deleted because the latter agents have much lower affinities for this process. In addition, previous studies have demonstrated that a decrease in cell folate cofactor pools has a much greater salutatory effect on the activity of PMX than ZD1694 and virtually no effect at all on the activity of PT523 (21)
. Relative preservation of PMX activity compared with that of ZD1694 has been observed previously under conditions in which transport is impaired. For example, 4-fold resistance to PMX was accompanied by 64-fold resistance to ZD1694 in ZR-75-1 MTXR breast cancer cells with impaired RFC function grown in 5-CHO-THF (4)
. Likewise, 41M:ZD1694 human ovarian cells with impaired MTX uptake were 7-fold resistant to PMX but 123-fold resistant to ZD1694 (8)
. In addition, in H630TDX cells in which "uptake" was impaired but TS activity was unaltered, there was 4-fold resistance to PMX but 7500-fold resistance to ZD1694 (7
, 29)
. Hence, preservation of PMX activity relative to ZD1694, despite impaired RFC-mediated transport, appears to have general relevance, although the extent to which this is due to the presence of a secondary transport pathway, as described in this report, is not clear.
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: I. David Goldman, Departments of Medicine and Molecular Pharmacology, the Albert Einstein College of Medicine, and the Einstein Cancer Research Center, Bronx, NY 10461.
Received 12/17/03. Revised 1/29/04. Accepted 2/13/04.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-glutamyl derivatives of methotrexate and their association with dihydrofolate reductase as assessed by high pressure liquid chromatography in the Ehrlich ascites tumor cell in vitro. J Biol Chem, 257: 1890-6, 1982.)-(4-amino-4- deoxypteroyl)-N()-hemiphthaloyl-L-ornithine (PT523) and its B-ring analogues. Biochem Pharmacol, 60: 41-6, 2000.[CrossRef][Medline]
-glutamate synthetase isoforms respond differently to feedback inhibition by folylpolyglutamate cofactors. Biochemistry, 41: 226-35, 2002.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  R. Zhao, A. Qiu, E. Tsai, M. Jansen, M. H. Akabas, and I. D. Goldman The Proton-Coupled Folate Transporter: Impact on Pemetrexed Transport and on Antifolates Activities Compared with the Reduced Folate Carrier Mol. Pharmacol., September 1, 2008; 74(3): 854 - 862. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Muller, R. Schibli, E. P. Krenning, and M. de Jong Pemetrexed Improves Tumor Selectivity of 111In-DTPA-Folate in Mice with Folate Receptor-Positive Ovarian Cancer J. Nucl. Med., April 1, 2008; 49(4): 623 - 629. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Yang, E. A. Kolb, J. Qin, A. Chou, R. Sowers, B. Hoang, J. H. Healey, A. G. Huvos, P. A. Meyers, and R. Gorlick The Folate Receptor {alpha} Is Frequently Overexpressed in Osteosarcoma Samples and Plays a Role in the Uptake of the Physiologic Substrate 5-Methyltetrahydrofolate Clin. Cancer Res., May 1, 2007; 13(9): 2557 - 2567. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Malempati, H. S. Nicholson, J. M. Reid, S. M. Blaney, A. M. Ingle, M. Krailo, L. C. Stork, A. S. Melemed, R. McGovern, S. Safgren, et al. Phase I Trial and Pharmacokinetic Study of Pemetrexed in Children With Refractory Solid Tumors: The Children's Oncology Group J. Clin. Oncol., April 20, 2007; 25(12): 1505 - 1511. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Chattopadhyay, R. G. Moran, and I. D. Goldman Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications Mol. Cancer Ther., February 1, 2007; 6(2): 404 - 417. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chattopadhyay, R. Zhao, S. A. Krupenko, N. Krupenko, and I. D. Goldman The inverse relationship between reduced folate carrier function and Pemetrexed activity in a human colon cancer cell line. Mol. Cancer Ther., February 1, 2006; 5(2): 438 - 449. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zhao, S. Zhang, M. Hanscom, S. Chattopadhyay, and I. D. Goldman Loss of Reduced Folate Carrier Function and Folate Depletion Result in Enhanced Pemetrexed Inhibition of Purine Synthesis Clin. Cancer Res., February 1, 2005; 11(3): 1294 - 1301. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Wang, A. Rajgopal, I. D. Goldman, and R. Zhao Preservation of folate transport activity with a low-pH optimum in rat IEC-6 intestinal epithelial cell lines that lack reduced folate carrier function Am J Physiol Cell Physiol, January 1, 2005; 288(1): C65 - C71. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zhao, S. Chattopadhyay, M. Hanscom, and I. D. Goldman Antifolate Resistance in a HeLa Cell Line Associated With Impaired Transport Independent of the Reduced Folate Carrier Clin. Cancer Res., December 15, 2004; 10(24): 8735 - 8742. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chattopadhyay, Y. Wang, R. Zhao, and I. D. Goldman Lack of Impact of the Loss of Constitutive Folate Receptor {alpha} Expression, Achieved by RNA Interference, on the Activity of the New Generation Antifolate Pemetrexed in HeLa Cells Clin. Cancer Res., December 1, 2004; 10(23): 7986 - 7993. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wang, R. Zhao, and I. D. Goldman Characterization of a Folate Transporter in HeLa Cells with a Low pH Optimum and High Affinity for Pemetrexed Distinct from the Reduced Folate Carrier Clin. Cancer Res., September 15, 2004; 10(18): 6256 - 6264. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Zhao and I. D. Goldman Enter Alimta(R): A New Generation Antifolate Oncologist, June 1, 2004; 9(3): 242 - 244. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
